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Graduate Studies The Vault: Electronic Theses and Dissertations

2019-03-12

Computer Simulation of Biomolecular Systems of

Interest in Bionanotechnology

Ramezanpour, Mohsen

Ramezanpour, M. (2019). Computer simulation of biomolecular systems of interest in

bionanotechnology (Unpublished doctoral thesis). University of Calgary, Calgary, AB.
http://hdl.handle.net/1880/110059
doctoral thesis

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 UNIVERSITY OF CALGARY

Computer Simulation of Biomolecular Systems of Interest in Bionanotechnology

by

Mohsen Ramezanpour

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

GRADUATE PROGRAM IN BIOLOGICAL SCIENCES

CALGARY, ALBERTA

MARCH, 2019

© Mohsen Ramezanpour 2019

 Abstract

Biomolecules, including lipids and enzymes, are of special interest in biotechnology and nano-medicine

applications. A knowledge of structure, dynamics, and function of these biomolecular systems is required

for development and improvement of drugs and drug delivery systems. Computer simulation, as a

complementary technique to experiments, could assist in such an understanding by providing atomistic

details on the molecular systems. In this thesis, molecular dynamics (MD) simulation technique was used

to study three molecular systems of interest in biotechnological applications: 1) Lamellar phases

composed of zwitterionic and ionizable cationic lipids, 2) inverted hexagonal (HII) phases composed of

cone-shaped lipids, and 3) a membrane enzyme called CTP:phosphocholine cytidylyltransferase (CCT).

First, lipid bilayers composed of POPC and DLin-KC2-DMA (also known as KC2) ionizable cationic

lipids were studied as a function of pH, temperature, and mixing ratio. Simulations suggest that neutral

KC2 are segregated in the presence of POPC. This segregation was proposed to affect both the internal

structure and drug release efficacy of lipid nanoparticles containing KC2.

Next, DOPE and POPE HII phases were constructed, simulated, and their structural properties as a

function of hydration level and temperature were studied using MD simulations. HII systems are usually

challenging to study experimentally, especially at low hydration regimes. Our findings suggest that MD

simulation could successfully reproduce structural properties of HII phase in a good agreement with

experimental data. Furthermore, a computational protocol for construction of HII molecular systems

equivalent to the corresponding HII systems in experiments was proposed.

Finally, the auto-inhibition and activation mechanism of CCT enzyme upon attachment or detachment of

two autoinhibitory helices was studied. Attachment of autoinhibitory helices were shown to affect the

dynamics and consequently the catalytic function of the CCT enzyme. Accordingly, a novel two-part

autoinhibition mechanism for CCT enzyme was proposed.

ii
 Preface

This thesis is organized in seven chapters:

Chapter 1 provides the reader with a general introduction required for understanding the results

presented in the following chapters. The questions of interest in this study and their importance are also

briefly discussed.

In Chapter 2, molecular dynamics (MD) simulation technique and its underlying algorithms are

discussed. Next, the concept of biomolecular force field, the protocol used to develop force field

parameters for KC2 and its protonated state (KC2H), as well as the protocols used to construct the

molecular systems of interest are explained.

Chapter 3 is reproduced with permission from a review article published in the Biochimica et Biophysica

Acta (BBA)-Biomembranes journal in 2016. This chapter is a comprehensive review of computational

studies on nanoparticle-based drug delivery systems.

In Chapter 4, lipid bilayers composed of POPC and KC2 were studied using MD simulations. KC2 is an

ionizable cationic lipid of interest in lipid nanoparticle formulations developed for drug and gene delivery

applications.

In Chapter 5, the structural properties of DOPE and POPE HII phases were studied as a function of

hydration level and temperature. HII systems are of interest in nano-medicine field of study.

Chapter 6 is reproduced with permission from a research article published in the Journal of Biological

Chemistry (JBC) in 2018. In this chapter, the autoinhibition mechanism of CTP:phosphocholine

cytidylyltransferase (CCT) enzyme is studied.

Finally, in Chapter 7 the general conclusions are made, and directions for future studies are discussed.

iii
Chapters 3 and 6 are based on the following publications reproduced with permission from the

publishing journals.

Chapter 3:

Ramezanpour, M., Leung, S. S. W., Delgado-Magnero, K. H., Bashe, B. Y. M., Thewalt, J., &

Tieleman, D. P. (2016). Computational and experimental approaches for investigating nanoparticle-based

drug delivery systems. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1858(7), 1688-1709.

Copyright © 2018 Elsevier.

Chapter 6:

Ramezanpour, M.*, Lee, J.*, Taneva, S. G., Tieleman, D. P., & Cornell, R. B. (2018). An auto-

inhibitory helix in CTP:phosphocholine cytidylyltransferase hi-jacks the catalytic residue and constrains a

pliable, domain-bridging helix pair. Journal of Biological Chemistry, 293(18), 7070 –7084. (*

contributed to work equally). Copyright © the American Society for Biochemistry and Molecular

Biology.

Contributions to the chapters:

Chapters 3, 4, 5, and 6 were conducted in collaboration with several experimental groups. My

contributions on these chapters, as well as the names of co-authors/collaborators are briefly described at

the beginning of each corresponding chapter. Briefly, I designed, ran, analyzed, and interpreted all the

simulations, and developed the computational protocols and models in this study, although

acknowledging tremendous help and guidance from my supervisor and collaborators.

iv
 Acknowledgements

Special thanks to my supervisor, Dr. Peter Tieleman for his support and mentoring. Peter, it was an

honour to be a Ph.D. student in your research lab. I have learned a great deal from you in all scientific,

management and mentoring aspects. Thanks for providing me with all the resources, collaborations, and

travels needed for conducting my research. None of these achievements would have been possible

without your mentoring and support.

Thanks to my supervisory committee, Dr. Justin MacCallum and Dr. Vanina Zaremberg, for their

instructive comments, feedbacks, and guidance during last four years.

I would like to thank the external examiners, Dr. Peter Kusalik and Dr. Michel Lafleur, for agreeing to be

in the examining committee, and for reviewing and commenting on my thesis.

The last four years was an honour for me to work on several projects in collaboration with Dr. Pieter

Cullis’s research group at the University of British Columbia, Dr. Jenifer Thewalt’s and Dr. Rosemary

Cornell’s research groups at the Simon Fraser University, and Dr. Paul Harper’s research group at Calvin

College. I enjoyed every minute of our collaborations and learned a lot through these collaborations.

Thank-you all.

Thank-you Dr. Elmar Prenner, Dr. Gregory Moorhead, and Dr. Hamid Habibi for your career advices and

support.

v
Thanks to all the former and current lab members, as well as other members of the Centre for Molecular

Simulation (CMS) for creating an active, scientific and friendly environment suitable for learning.

Thanks to the support staff at the department of biological sciences for providing a friendly environment

for graduate students and assisting them in any possible way they could. I would also like to thank the

Faculty of Graduate Studies (FGS) at the University of Calgary for providing all the resources a graduate

student might need to complete his/her study successfully.

I would like to thank both the funding agencies, including NSERC, and Compute Canada for their

financial and technical supports, respectively.

Last but not least, a great thanks to my parents who dedicated their whole lives to help me succeed.

Without any doubt, I would not be able to achieve these without your support. I would also like to thank

my lovely wife for her support throughout my Ph.D. study.

vi
 Dedication

To my lovely wife and beloved parents

vii
 Table of Contents

Abstract........................................................................................................................................................ ii

Preface......................................................................................................................................................... iii

Acknowledgements ..................................................................................................................................... v

Dedication .................................................................................................................................................. vii

Table of Contents ..................................................................................................................................... viii

List of Tables ............................................................................................................................................. xv

List of Figures........................................................................................................................................... xvi

Chapter One: General Introduction and Thesis Outline ............................................................................. 1

1.1 Abstract .............................................................................................................................................. 1

1.2 Introduction ........................................................................................................................................ 2

1.2.1 Biomolecules as drug targets. ........................................................................................... 2

1.2.1.1 Messenger RNAs (mRNAs) as drug targets. .................................................................... 3

1.2.1.2 Enzymes as drug targets. .................................................................................................. 3

1.2.2 Lipids and lipid polymorphism. ........................................................................................ 4

1.2.2.1 Lamellar phase. ................................................................................................................. 7

1.2.2.2 Inverted hexagonal (HII) phase. ....................................................................................... 8

1.2.3 Lipid nanoparticles for drug delivery. ............................................................................ 11

1.2.3.1 Lipid nanoparticles containing ionizable cationic lipids. ............................................... 11

1.2.4 Lipids of interest in this study......................................................................................... 14

1.2.4.1 POPC. ............................................................................................................................. 15

viii
 1.2.4.2 DOPE and POPE. ........................................................................................................... 15

1.2.4.3 KC2 and KC2H............................................................................................................... 16

1.3 Thesis Outline................................................................................................................................... 16

1.4 Bibliography ..................................................................................................................................... 20

Chapter Two: Methods ............................................................................................................................. 28

2.1 Abstract ............................................................................................................................................ 28

2.2 Molecular Dynamics (MD) Simulation ............................................................................................ 29

2.2.1 Isothermal-isobaric (NPT) and canonical (NVT) ensembles.......................................... 31

2.2.2 Temperature coupling. .................................................................................................... 32

2.2.3 Pressure coupling. ........................................................................................................... 33

2.2.4 Periodic boundary condition. .......................................................................................... 36

2.2.5 Non-bonded interactions and cut-offs............................................................................. 37

2.2.6 Integration algorithms. .................................................................................................... 38

2.3 Biomolecular Force Fields ............................................................................................................... 39

2.3.1 CHARMM36 FF for the simulation of lipid aggregates. ................................................ 43

2.3.2 AMBER99SB-ILDN FF for the simulation of CCT enzyme. ........................................ 43

2.4 Force Field Development for KC2 and KC2H ................................................................................. 44

2.4.1 KC2 and KC2H parameterization in CHARMM36 FF. ................................................. 45

2.4.2 KC2 and KC2H model validation. .................................................................................. 48

2.5 Construction of Binary and Ternary Mixtures in Lamellar and HII Phase ...................................... 51

2.5.1 Bilayers. .......................................................................................................................... 51

2.5.2 Inverted hexagonal (HII) phase. ..................................................................................... 53

2.6 Bibliography ..................................................................................................................................... 55

ix
Chapter Three: Computational and Experimental Approaches for Investigating Nanoparticle-Based

Drug Delivery Systems ........................................................................................................................ 62

3.1 Abstract ............................................................................................................................................ 63

3.2 Introduction ...................................................................................................................................... 64

3.3 Commonly Used Experimental Techniques for Nanoparticle Characterization .............................. 69

3.4 Computational Approaches to Study Nanoparticles ......................................................................... 77

3.5 Computational Studies on Drug and Gene Delivery Systems .......................................................... 81

3.5.1 Dendrimers and dendrons. .............................................................................................. 84

3.5.2 Polymer-based delivery systems. .................................................................................... 90

3.5.3 Peptide- and nucleic acid-based delivery systems. ......................................................... 96

3.5.4 Carbon-based delivery systems. ................................................................................... 102

3.5.5 Lipid-based delivery systems. ....................................................................................... 105

3.5.6 Gold-nanoparticles. ....................................................................................................... 111

3.6 Conclusions and Future Perspectives ............................................................................................. 112

3.7 Bibliography ................................................................................................................................... 114

Chapter Four: Ionizable Amino Lipid Interactions with POPC: Implications for Lipid Nanoparticle

Function .............................................................................................................................................. 157

4.1 Abstract .......................................................................................................................................... 157

4.2 Introduction .................................................................................................................................... 158

4.3 Methods .......................................................................................................................................... 160

4.3.1 Approach....................................................................................................................... 160

4.3.2 Computational method.................................................................................................. 161

4.3.2.1 System construction. ..................................................................................................... 161

x
 4.3.2.2 Molecular dynamics simulations. ................................................................................. 162

4.3.2.3 Simulation Analysis. ..................................................................................................... 163

4.3.3 Experimental methods. ................................................................................................. 165

4.3.3.1 Materials and sample preparation. ................................................................................ 165

4.3.3.2 Deuterium NMR (2H NMR). ........................................................................................ 166

4.3.3.3 Small angle X-ray scattering (SAXS). .......................................................................... 168

4.3.3.4 Cryo-TEM of LNPs. ..................................................................................................... 169

4.4 Results and Discussion ................................................................................................................... 169

4.5 Conclusions .................................................................................................................................... 178

4.6 Bibliography ................................................................................................................................... 178

Chapter Five: Structural Properties of Inverted Hexagonal Phase: A Hybrid Computational and

Experimental Approach ...................................................................................................................... 185

5.1 Abstract .......................................................................................................................................... 185

5.2 Introduction .................................................................................................................................... 187

5.3 Methods .......................................................................................................................................... 192

5.3.1 Computational method.................................................................................................. 192

5.3.1.1 System construction. ..................................................................................................... 192

5.3.1.2 Molecular dynamics simulation. ................................................................................... 194

5.3.1.3 Simulation analysis. ...................................................................................................... 195

5.3.2 Experimental methods. ................................................................................................. 202

5.3.2.1 Sample preparation. ...................................................................................................... 202

5.3.2.2 Small angle X-ray scattering (SAXS). .......................................................................... 203

5.3.2.3 Electron density reconstruction and water core radius estimation. ............................... 203

xi
 5.3.2.4 Deuterium NMR (2H NMR). ........................................................................................ 204

5.4 Results ............................................................................................................................................ 206

5.4.1 HII structural parameters as a function of hydration. ................................................... 206

5.4.2 Structural differences between DOPE and POPE HII systems. ................................... 209

5.4.3 Effect of hydration level on shape of lipid cylinders. ................................................... 212

5.4.4 SCD parameters as a function of hydration and temperature. ........................................ 215

5.4.5 Estimation of the maximum hydration in HII phase. .................................................... 218

5.4.6 Temperature effect on DOPE HII lattice distance and maximum hydration. ............... 220

5.5 Discussion....................................................................................................................................... 223

5.5.1 MD predicts the HII structural parameters as a function of hydration. ........................ 223

5.5.2 HII water channels are not straight and cylindrical at low hydrations.......................... 225

5.5.3 MD reproduces the differences between DOPE and POPE in HII. .............................. 226

5.5.4 Both dehydration and raising temperature reduces SCD in HII. .................................... 226

5.5.5 Temperature affects the maximum hydration and lattice distances in HII. .................. 227

5.5.6 Protocol for construction of equivalent HII systems to experiments. ........................... 228

5.6 Conclusions .................................................................................................................................... 230

5.7 Bibliography ................................................................................................................................... 231

Chapter Six: An Auto-Inhibitory Helix in CTP:Phosphocholine Cytidylyltransferase Hijacks the

Catalytic Residue and Constrains a Pliable, Domain-Bridging Helix Pair ........................................ 240

6.1 Abstract .......................................................................................................................................... 241

6.2 Introduction .................................................................................................................................... 241

6.3 Results ............................................................................................................................................ 246

6.3.1 Optimal catalysis requires a flexible Loop L2 at Lys-122, enabled by Gly-123. ......... 246

xii
 6.3.2 The AI helices selectively repress the dynamics of helix αE........................................ 249

6.3.3 The AI helix supplies H-bonding partners for Lys-122 that compete with CTP. ......... 253

6.3.4 Removal of the constraining AI helices allows bending of the E helices. ................. 256

6.3.5 Membrane binding reorganizes the E helices, a pre-requisite for activation. ............ 260

6.4 Discussion....................................................................................................................................... 264

6.4.1 AI restriction of L2 dynamics. ...................................................................................... 265

6.4.2 Novel backbone competition for the catalytic lysine. ................................................... 266

6.4.3 Silencing by constraining the E helices...................................................................... 267

6.5 Materials and Methods ................................................................................................................... 272

6.5.1 Molecular dynamics simulations. ................................................................................. 272

6.5.2 Construction of CCT mutants. ...................................................................................... 274

6.5.3 pET14b-CCT constructs. .............................................................................................. 274

6.5.4 pET24a-CCT312 constructs. ........................................................................................ 275

6.5.5 Expression and purification of CCT constructs. ........................................................... 276

6.5.6 Preparation of lipid vesicles or micelles. ...................................................................... 277

6.5.7 Tryptophan fluorescence anisotropy. ............................................................................ 277

6.5.8 CCT activity assay. ....................................................................................................... 277

6.5.9 Bis-maleimide cross-linking. ........................................................................................ 278

6.5.10 Disulfide bond formation by air oxidation. .................................................................. 278

6.6 Bibliography ................................................................................................................................... 279

Chapter Seven: Conclusions and Future Directions................................................................................ 287

7.1 Conclusions .................................................................................................................................... 287

xiii
 7.2 Future Directions ............................................................................................................................ 289

7.3 Bibliography ................................................................................................................................... 294

References ................................................................................................................................................ 298

Appendix A: Parameters for the Parameterized Segment in the KC2 and KC2H ................................... 376

Appendix B: Supplementary Information for Chapter Four .................................................................... 390

B.1 KC2 and KC2H Parameterization in the CHARMM36 Force Field .............................................. 390

B.1.1 KC2 and KC2H model validation. ................................................................................ 390

B.2 KC2(H) Effects on POPC as a Function of Temperature and Mixing Ratio .................................. 393

B.3 KC2(H) Effect on POPC Lamellar Repeat Spacing ....................................................................... 397

B.4 KC2H has Little to no Effect on the POPC Bilayer Thickness ...................................................... 398

B.5 KC2H Repels Na Ions from and Attracts Cl Ions to the Lipid-Water Interface ............................. 400

B.6 KC2H Affects the POPC Headgroup Distribution ......................................................................... 402

B.7 Bibliography ................................................................................................................................... 404

Appendix C: Supplementary Information for Chapter Five .................................................................... 407

C.1 Semi-isotropic versus Anisotropic Pressure Coupling ................................................................... 407

C.2 Bibliography ................................................................................................................................... 410

Appendix D: Supplementary Information for Chapter Six ...................................................................... 411

Appendix E: Copyright Permissions ....................................................................................................... 424

xiv
 List of Tables

Table 3-1: Questions relevant to drug delivery system design that can be answered by computational

simulations. ........................................................................................................................................ 67

Table 3-2: Experimental methods used to study drug delivery systems. .................................................... 69

Table 4-1: Summary of simulations. ......................................................................................................... 163

Table 5-1: Simulated HII systems. ............................................................................................................ 193

Table 5-2: Maximum hydrations for DOPE and POPE HII systems at several temperatures. ................. 220

Table A-1: Parameters for the ring segment in KC2 and KC2H. ............................................................. 378

Table A-2: Parameters for the amino segments in KC2 and KC2H. ........................................................ 378

Table A-3: Parameters for the hydrogen atoms in KC2 and KC2H. ........................................................ 379

Table A-4: Parameters for several bonds defined in KC2 and KC2H. ..................................................... 379

Table A-5: Parameters for several angles defined in KC2 and KC2H...................................................... 380

Table A-6: Parameters for several dihedrals defined in KC2 and KC2H. ................................................ 381

Table A-7: Dihedral parameters specific to KC2. ..................................................................................... 383

Table A-8: Dihedral parameters specific to KC2H. .................................................................................. 386

xv
 List of Figures

Figure 1.1: Lipid phase as a function of lipid shape. .................................................................................... 6

Figure 1.2: Phosphate-to-phosphate (P-P) thickness for POPC bilayer. ....................................................... 8

Figure 1.3: The two-dimensional structure of KC2 and its protonated state, KC2H. ................................. 12

Figure 1.4: Models for a typical siRNA-LNP containing KC2. ................................................................. 13

Figure 1.5: Two-dimensional structure of the lipids studied in this work. ................................................. 14

Figure 2.1: Lennard-Jones potential energy. ............................................................................................... 41

Figure 2.2: KC2(H) breakdown into two segments for parameterization. .................................................. 45

Figure 2.3: KC2(H) acyl chain parameters. ................................................................................................ 46

Figure 2.4: KC2(H) headgroup breakdown into two segments. ................................................................. 47

Figure 2.5: Validation of developed models for KC2 and KC2H............................................................... 50

Figure 2.6: Construction of the POPC/KC2H (80/20) bilayer. ................................................................... 52

Figure 2.7: Construction of the DSPS/KC2H/cholesterol (35/35/30) HII system. ..................................... 54

Figure 3.1: Drug delivery pathway for a drug-nanocarrier system. ............................................................ 65

Figure 3.2: Examples of nanoparticulate DDS. .......................................................................................... 66

Figure 3.3: Categories of simulation methods and their respective spatio-temporal domains of

applicability. ....................................................................................................................................... 79

Figure 3.4: PAMAM dendrimer-DNA interaction and deformation as a function of generation number. . 85

Figure 3.5: Proposed mechanism of endosomal escape for pH-responsive dendrimers. ............................ 88

Figure 3.6: Time evolution of nanoparticle-polymer complex endocytosis as a function of pH. ............... 93

Figure 3.7: Solubilization of bexarotene in sterically stabilized micelles (SSM). ...................................... 95

Figure 3.8: DNA nanocage with temperature-controlled conformational transition capability.................. 98

Figure 3.9: Effect of pH on microstructure of self-assembled micelles.................................................... 101

xvi
Figure 3.10: Effect of PEGylation method, and PEG chain size and grafting density on the conformation

of PEG layer on carbon nanotube..................................................................................................... 104

Figure 3.11: Distribution and orientation of hypericin within liposome................................................... 109

Figure 3.12: Lipid nanoparticle................................................................................................................. 111

Figure 4.1: KC2 segregation from POPC as a function of pH. ................................................................. 171

Figure 4.2: Systematic increase in POPC inter-leaflet distance upon rising the KC2 concentration at basic

pH level. ........................................................................................................................................... 174

Figure 4.3: KC2 confinement as a function of mixing ratio. .................................................................... 176

Figure 5.1: HII phase in experiments and simulation. .............................................................................. 189

Figure 5.2: Definitions for several structural parameters calculated from MD simulations. .................... 198

Figure 5.3: Structural parameters for DOPE HII system as a function of hydration. ............................... 207

Figure 5.4: Radius of water core from reconstructed electron density profiles and MD simulation. ....... 209

Figure 5.5: Structural parameters of DOPE and POPE HII systems as a function of hydration. ............. 210

Figure 5.6: Cylinder length in DOPE and POPE HII systems as a function of hydration. ....................... 212

Figure 5.7: Effect of hydration level on lipid tubules shape in DOPE HII systems. ................................ 214

Figure 5.8: Deuterium order parameter (SCD) for DOPE and POPE in HII phase. ................................... 217

Figure 5.9: Estimation of maximum hydration in HII systems at several temperatures. .......................... 219

Figure 5.10: Lattice distance as a function of temperature for DOPE HII systems. ................................. 222

Figure 5.11: A proposed protocol for molecule parameterization in HII phase. ...................................... 230

Figure 6.1: Structure of mammalian CCT and its active site. ................................................................... 245

Figure 6.2: CCT relies on a single lysine followed by glycine in loop L2 for catalysis. .......................... 248

Figure 6.3: Effect of the AI helix and CTP on the dynamics of active-site loops and side chains. .......... 251

Figure 6.4: Backbone trap for Lys-122. .................................................................................................... 255

Figure 6.5: αE conformational change triggered by removal of the AI helices. ....................................... 259

xvii
Figure 6.6: Lipid vesicles reduce interchain cross-linking efficiency between the two αEc helices. ....... 261

Figure 6.7: A disulfide bond linking the αE helices at Cys-217 inhibits CCT activity. ........................... 263

Figure 6.8: New model for CCT activation. ............................................................................................. 271

Figure 7.1: Two-dimensional structure of KC2 and MC3. ....................................................................... 290

Figure 7.2: HII phase corresponding to DSPS/KC2H/cholesterol (35/35/30). ......................................... 293

Figure A.1: Atom index numbers for the parameterized segment of KC2(H). ......................................... 377

Figure B.1: Validation of developed models for KC2 and KC2H. ........................................................... 392

Figure B.2: SCD parameters for the palmitoyl chain (sn-1) of POPC at 288 K and 293 K. ...................... 394

Figure B.3: SCD parameters for the palmitoyl chain (sn-1) of POPC at 303 K and 313 K. ...................... 396

Figure B.4: SAXS curves for POPC/KC2 multilamellar vesicles at several basic pH values. ................. 398

Figure B.5: Effect of adding KC2H on POPC inter-leaflet distance. ....................................................... 399

Figure B.6: Effect of KC2H on ion distribution. ...................................................................................... 401

Figure B.7: Effect of KC2 and KC2H on POPC P→N vector angle distribution. .................................... 403

Figure C.1: Lattice distance as a function of hydration for DOPE HII systems (semi-isotropic versus

anisotropic pressure coupling).......................................................................................................... 409

Figure D.1: Dynamics of active site loops and side chains based on the 20 simulations with positional

restraints (PR) on residues 216-223 of the EC helices. .................................................................. 411

Figure D.2: Dynamics of Loop L2 in vitro. .............................................................................................. 413

Figure D.3: Correlations between K122 backbone dihedrals and H-bonding partners............................. 416

Figure D.4: Stability of 4-helix bundle during 1000 ns simulation time. ................................................. 417

Figure D.5: Alpha-E hinge dynamics enables contacts with the active site. ............................................ 418

Figure D.6: Alpha-E configuration 3. ....................................................................................................... 419

Figure D.7: Lipid vesicles reduce inter-chain cross-linking between αEc................................................ 420

xviii
Figure D.8: CCT(A217C) binding to lipid vesicles via domain M is not impaired by oxidative disulfide

formation. ......................................................................................................................................... 421

Figure D.9: PR vs NOE restraints. ............................................................................................................ 422

Figure D.10: Detailed description of CCT mutant constructs................................................................... 423

xix
 Chapter One: General Introduction and Thesis Outline

1.1 Abstract

Biological molecules, including lipids, proteins and nucleic acids, are of special interest for their

therapeutic applications. They can be used as targets for drugs, as therapeutics, or as delivery systems for

targeting drugs to their site of action [1-4].

Rational design of new therapeutics and development of effective drug/gene delivery systems

could benefit from deeper insights on the role, structure, dynamics, function, and regulatory mechanisms

of these biomolecular systems. The current knowledge in this field of research has been gained mainly

through extensive experimental research. Over the last few decades, however, there have been many

studies demonstrating how computer simulations can lead to a deeper understanding of molecular

systems, such as drug delivery systems [5]. Computational techniques can provide insights into the nano-

system in an atomic resolution and a femtosecond time scales, a spatiotemporal resolution which is not

easily achievable otherwise. Among all the computational techniques, molecular dynamics (MD)

simulation is the most commonly used technique for investigating the structure and dynamics of

biomolecular systems [6].

In this thesis, MD simulations were used to study the structure of lipid nano-aggregates in both

lamellar and inverted/reverse hexagonal (HII) phases, and to investigate the structure and dynamics of a

membrane enzyme called CTP:phosphocholine cytidylyltransferase (CCT).

This chapter provides the reader with the background information on the problems studied and

the results presented in Chapters 3-6. In the last section of this chapter, i.e. thesis outline, both the goals

and their importance will be briefly explained for each chapter. This chapter is mainly focused on the

1
parts which either were not discussed or briefly discussed in the following chapters. For more information

on each topic, the reader is referred to the articles cited in this and corresponding chapters.

1.2 Introduction

1.2.1 Biomolecules as drug targets.

Cellular membranes are among the most common targets for many drugs [1, 4]. Most drugs either interact

with cell membranes as their target or require passing across them to reach their site of action, i.e.

cytoplasm and biomolecules in it.

Biomembranes are mainly composed of lipids and proteins [7]. There are many different lipid

types found in cellular membranes, and the roles of these lipid polymorphisms still remain to be

determined [8, 9]. Phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylserines

(PSs), and sphingomyelins are the common lipids found in the plasma membrane of eukaryotic cells.

These lipids span a wide variety of lengths and unsaturation degrees in their acyl chains. Cell membranes

also include sterols, with cholesterol the major sterol in mammalian cell membranes. Membrane proteins

are the proteins which interact with the lipid membrane. These proteins could be part of the membrane,

e.g. integral proteins, or interact with the membrane and its integral proteins temporarily. Lipid content

and organization, lipid-protein and protein-protein interactions, as well as the proper function of

membrane proteins are known to be critical for a correct cell function and its survival. These make

cellular membranes an attractive target for drug development [1, 4].

Drugs, which their biomolecular target is inside the cell, need to reach and be internalized to the

target cells. Therapeutics with a high molecular weight and/or being highly charged, e.g. short interfering

RNA (siRNA), are not stable in the circulation and cannot readily get into the cell. As a solution, drug

delivery systems (DDSs) can be used to protect such drugs in the bloodstream and pass them across the

2
cell membranes [3]. When inside the cell, drugs could target a variety of biomolecules, including

messenger RNAs (mRNA) and enzymes, and result in therapeutic effects.

1.2.1.1 Messenger RNAs (mRNAs) as drug targets.

Targeting and degrading mRNAs, and consequently preventing the expression of disease-causing genes is

a promising therapeutic approach called RNA-interference [2]. Short interfering RNAs (siRNAs) are post

transcriptional silencing agents experimentally designed to prevent the expression of problematic genes

by degrading the corresponding mRNAs. siRNAs have a great potential in treatment of a wide range of

diseases, including cancer [10]. This class of therapeutics are highly selective, and capable of affecting

targets which are undruggable otherwise [2].

Despite their promising applications, siRNAs have difficulty to reach the cytoplasm of target cells

[11]. Given their large size (~22 nucleotides), high molecular weight (~14 kDa in mass), and negatively

charged structure, naked siRNAs must overcome several barriers on their way to the cytoplasm. They are

rapidly degraded by RNases and cleared from the bloodstream. They activate the immune responses in the

body and cannot be internalized into the cell via passive diffusion.

There are many techniques developed to assist in siRNA delivery, such as chemical modifications

of siRNA strands, conjugating them with receptor-binding ligands, conjugation with peptides, and loading

siRNAs into/on the nanoparticles [12]. Lipid nanoparticles (LNPs) are among the clinically successful

DDSs for both drugs and gene materials, e.g. siRNA (see Section 1.2.3).

1.2.1.2 Enzymes as drug targets.

Enzymes are one of the most commonly targeted biomolecules, which affecting their catalytic activities

could have therapeutic effects [1]. As an example of interest in this study, inhibiting lipid biosynthesis

3
pathways via inhibiting CCT enzyme, a critical enzyme which catalyzes the rate limiting step in PC

biosynthesis, is known to be correlated with apoptosis [13]. Furthermore, mutations in this enzyme are

known to cause several disorders related to bone growth, vision, and lipid metabolism [14-17]. Finally,

inhibiting the PC biosynthesis pathway by blocking the CCT enzyme in Plasmodium falciparum is

considered a potential target for drug development against malaria [18, 19]. Therefore, to develop

effective drugs and inhibitors, or to reveal the underlying reasons by which these mutations cause genetic

disorders, a deeper knowledge of the CCT activation and autoinhibition is required. For instance, when

the catalytic activity of CCT enzyme is a valuable target, the underlying structural factors playing roles in

its catalytic activity, activation and inhibition mechanisms are important factors to be studied and

determined.

1.2.2 Lipids and lipid polymorphism.

Lipids are amphiphilic molecules, i.e. molecules with both hydrophobic and hydrophilic segments, which

are well known for their presence and vital roles in living cells [8]. Hydrated lipids are capable of self-

assembly into a variety of nano-structures [20, 21]. The phase diagram, i.e. the structure of a molecular

system at different temperatures, concentrations, and mixing ratios, for a variety of lipid-water systems

has been constructed experimentally [22, 23]. These lipid phases are usually classified according to the

lattice type and symmetry, and conformation of lipids hydrocarbon chains in the systems. Lamellar (L),

hexagonal (H), and cubic phases (Q) are among the commonly studied structural phases, and of a high

interest in nanotechnology, including nano-medicine [24-26]. Except the lamellar phase, other phases

could exist in either normal (type I or oil-in-water) or inverted (type II or water-in-oil) types. For instance,

there are two types of hexagonal phases: HI (normal) and HII (inverted) phases. In the normal phase,

lipids hydrocarbon chains form hydrophobic rods which are separated form the outside solvent by the

lipid-water interface. For the inverted hexagonal phase [Figure 1.1-right], water is trapped inside the

4
lipid cylinders while the lipid hydrocarbons fill the space between the hexagonally packed cylinders [22,

23].

The amphiphilic nature of the lipids results in their specific packing in solvent so that the free

energy of the system is minimized [27]. For instance, in a lipid bilayer, the polar headgroups face the

aqueous media, while the hydrophobic hydrocarbon chains pack together in the bilayer interior to

minimize their exposure to the solvent.

There are a wide variety of factors which could favor a specific structural phase or promote the

transitions from one to another [28, 29]. Lipid composition, lipid type, hydration level, ionic strength of

the media, and temperature are among the main determining factors.

Lipid type is of great importance on its own because it includes other factors such as headgroup

size, charge, hydrocarbon chain length and tail unsaturation degree. All the above-mentioned factors can

affect the “lipid shape” [28, 30], a concept upon which the observed polymorphism preference of the

lipids could be explained. The effective shape of a lipid molecule can be quantified by the “critical

packing parameter”, 𝑆, defined as the Equation 1.1 [31]:

𝑆 = 𝑣 ⁄𝑙𝑐 𝑎0 Equation 1.1

where the 𝑣 is the hydrocarbon volume, 𝑙𝑐 is the critical chain length (i.e. the length of the fully extended

acyl chain), and 𝑎0 is the optimal area per lipid molecule (i.e. the area per headgroup which the

interaction energy between the lipids is at its minimum). The molecules with a packing parameter of S ~ 1

have an effective cylindrical shape and prefer the lamellar phase. The lipids with a S > 1 and S < 1 are

referred to as “cone” and “inverse cone” shaped lipids, respectively [28]. The cone-shaped lipids form

inverted phases [Figure 1.1-right], whereas the inverse cone-shaped lipids prefer normal phases.

5
Figure 1.1: Lipid phase as a function of lipid shape.

Lipid shapes and corresponding S parameters are shown (Middle). Lipids with S ~ 1 have a cylindrical

shape and support a lamellar phase (Left), whereas the lipids with S > 1 are cone-shaped and support the

HII phase (Right). Spheres represent the lipid headgroups. Blue areas represent the solvents while the

grey areas are representative of hydrophobic parts formed by lipid acyl chains. In the HII phase, unit cell

spacing (or d-spacing) and radius of water core are shown as parameters 𝑎 and 𝑅𝑤 , respectively. Inkscape

was used to generate the figure [32].

A change in the effective molecular shape could result in a phase preference for the lipidic system

and subsequent phase transition to the more favorable phase [28]. Taking the lamellar-to-HII phase

transition as an example, several factors are known to influence this transition [33]. An increase in

temperature, unsaturation degree and length of the lipids’ acyl chains will push the system towards the

6
HII phase, whereas an increase in hydration, headgroup size and charge favors the lamellar phase.

Charged headgroups have a larger effective 𝑎0 compared to their neutral states due to the electrostatic

repulsions between the charged headgroups. For instance, phosphatidylserines (PSs) with a net charge of

-e have an effective cylindrical shape which favors lamellar phase. However, lowering the pH to less than

3 results in the carboxyl group protonation which reduces the electrostatic repulsions, and consequently

reduces the optimal area per lipid. This change in the shape makes the HII phase as the phase of

preference for PS lipids in this pH regime [34]. Adding cations to other anionic lipids, e.g. cardiolipins,

has been also shown to promote the HII phase due to screening the headgroup charges and reducing the

inter-lipid electrostatic repulsions.

1.2.2.1 Lamellar phase.

Lamellar phase is referred to a system with several lipid bilayers stacked on the top of each other where

solvent separates each of two adjacent bilayers [23, 35]. Lipid bilayers, themselves, are membranes

composed of two layers of lipids tightly bound to each other, in which the lipid headgroups are facing the

solvent while the acyl chains form a hydrophobic layer in the middle [Figure 1.1]. This hydrophobic

layer acts like a barrier and blocks the water-soluble molecules to pass through it. Assuming the bilayer is

in the XY-plane, the lipids are in average parallel to the normal vector to the membrane, i.e. the Z axis.

Lipid bilayers have been extensively studied, both experimentally and computationally, and much is

known about both their structure and dynamics [36-39]. Given an average length of ca. 2 𝑛𝑚 for a

phospholipid, a typical phospholipid bilayer has a phosphate-to-phosphate (P-P) thickness of

approximately 4.0 𝑛𝑚 [Figure 1.2].

7
Figure 1.2: Phosphate-to-phosphate (P-P) thickness for POPC bilayer.

Right) A snapshot taken from POPC bilayer simulation. All lipids are colored in orange, and the

phosphorous atom of each lipid is shown as a red sphere. The blue box represents the simulation box.

Water and ions on both sides of the bilayer are not shown for clarity. Left) The corresponding electron

density of the phosphorous atoms along the bilayer normal, with respect to the bilayer center, is shown.

1.2.2.2 Inverted hexagonal (HII) phase.

Inverted/reverse hexagonal phase is simply made of several lipid cylinders packed in a two-dimensional

hexagonal geometry [Figure 1.1]. Each cylinder has a hydrophobic shell and an aqueous core. Lipids are

oriented perpendicular to the cylinder surface with their headgroups point inward to the solvent core,

while the lipid tails point outward. The structure of HII and other non-lamellar phases, as well as the

dynamics of the comprising lipids have been extensively studied experimentally, and to a much lesser

extent, computationally [21, 23, 40-42]. The lipid components of HII phase assume an effective cone

shape, and the radius for the aqueous core in HII phase is more than 1 𝑛𝑚 [28]. One of the structural

8
parameters of interest in the HII phase is 𝑎, the d-spacing or the unit cell spacing, defined as the distance

between the centers of two adjacent water cores [43]. This parameter can both be measured using small

angle X-ray scattering (SAXS) experiments and be directly calculated from the simulations by ensemble

averaging of the simulation box size. Given the hexagonal arrangement of the cylinders, the 𝑎 parameter

can be converted to the HII lattice distance (𝑑ℎ𝑒𝑥 ) according to the Equation 1.2:

𝜋 Equation 1.2
𝑑ℎ𝑒𝑥 = 𝑎(𝑐𝑜𝑠 ( ))
6

Assuming a cylindrical geometry for the water cores in HII phase, the d-spacing parameter, the

radius of water core (𝑅𝑤 ), and the water (core water) volume fraction (𝜑𝑤 ) are related through the

Equation 1.3 (see Figure 1.1 for definitions) [44]:

Equation 1.3
𝑅𝑤 = 𝑎 √𝜑𝑤 ( √3 / 2 𝜋)

Therefore, the approximate (𝑅𝑤 ) could be calculated if both (𝑎) and (𝜑𝑤 ) are provided. Although (𝑎) can

be obtained from SAXS experiments, it is not easy to estimate (𝜑𝑤 ) for a given HII phase when

experiments are done in the presence of excess water. In simulations, however, this quantity can be

measured easily, which makes the calculation of 𝑅𝑤 possible.

To measure 𝜑𝑤 in simulations, the volume of simulation box (𝑉𝑡𝑜𝑡𝑎𝑙 ) should be measured first.

The volume taken by the water molecules inside the lipid cylinder, i.e. 𝑉𝑐𝑜𝑟𝑒−𝑤𝑎𝑡𝑒𝑟 , can also be

calculated based on Equation 1.4:

9
𝑉𝑐𝑜𝑟𝑒−𝑤𝑎𝑡𝑒𝑟 = 𝑁𝑤𝑎𝑡𝑒𝑟 𝑉𝑤 Equation 1.4

with 𝑁𝑤𝑎𝑡𝑒𝑟 and 𝑉𝑤 be the number of water molecules inside the lipid cylinder and volume of one single

water molecule at the temperature at which simulations were conducted, respectively. Thus, 𝜑𝑤 can be

calculated as:

𝜑𝑤 = 𝑉𝑐𝑜𝑟𝑒−𝑤𝑎𝑡𝑒𝑟 / 𝑉𝑡𝑜𝑡𝑎𝑙 Equation 1.5

Since the systems are composed of water and lipids, the lipid volume fraction (𝜑𝑙 ) in the sample can be

calculated using the Equation 1.6.

𝜑𝑙 = 1 − 𝜑𝑤 Equation 1.6

Also, the volume taken by a single lipid in the system is equal to:

𝑉𝑙 = (𝑉𝑡𝑜𝑡𝑎𝑙 − 𝑉𝑐𝑜𝑟𝑒−𝑤𝑎𝑡𝑒𝑟 )/𝑁𝑙𝑖𝑝𝑖𝑑𝑠 Equation 1.7

with 𝑁𝑙𝑖𝑝𝑖𝑑𝑠 as the total number of lipids in the simulation box.

Finally, given that all these parameters (i.e. 𝑅𝑤 , 𝜑𝑙 , 𝑑ℎ𝑒𝑥 , 𝑉𝑙 ) are known, the area per lipid (APL)

on the cylinder surface of radius 𝑅𝑤 can be calculated using the Equation 1.8 [45].

10
𝐴PL = (√3 𝜋 𝑅𝑤 𝑉𝑙 )⁄(𝑑ℎ𝑒𝑥 2 𝜑𝑙 ) Equation 1.8

1.2.3 Lipid nanoparticles for drug delivery.

Nanoparticles based on lipids have a wide range of applications in nanotechnology, including DDSs [46,

47]. Lipid nanoparticles (LNPs) can be used for delivery of different types of therapeutics, to the target

cells, e.g. cancer cells. Indeed, LNPs containing optimized ionizable cationic lipids (ICLs) are currently

the most potent systems and the lead carriers for siRNAs [48-50].

1.2.3.1 Lipid nanoparticles containing ionizable cationic lipids.

LNPs containing optimized ICLs are currently the most effective in liver-related diseases, e.g. cancer,

hypercholesterolemia, and transthyretin-induced amyloidosis [48-50]. ICLs with an apparent pKa of 7 or

lower are one of the main components of LNPs with key roles in siRNA encapsulation, siRNA-LNP

structure and stability, and endosomal escape [51]. This pKa range is known to be critical so that it

ensures that ICLs are neutral in the physiological pH levels inside the bloodstream, while are protonated

and positively charged inside the endosomes where the pH is more acidic. This way, these ionizable lipids

assist in LNP stability and prolong its circulation time in bloodstream, while assisting in the endosomal

membrane destabilization and siRNA release into the cytoplasm, respectively [50, 52].

DLin-KC2-DMA (2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane) or KC2 [Figure 1.3]

with an apparent pKa of ca. 6.7 is an optimized and highly efficient ICL in silencing hepatic genes [51,

53]. Despite the importance, its role in LNP size, internal structure, stability, and siRNA release is not

known. The role of KC2 in each of the above-mentioned aspects could be determined by studying its

interactions with other components of LNP formulations.

11
Figure 1.3: The two-dimensional structure of KC2 and its protonated state, KC2H.

Top) KC2, Bottom) KC2H. Hydrogen atoms, except the protonation one, are not shown for clarity.

Avogadro [54] and Chimera [55] were used to generate the figure.

A schematic view of the internal structure for a typical siRNA-LNP containing KC2 is shown in

Figure 1.4. These siRNA-LNPs have a diameter of about 100 nm, mainly composed of KC2,

phosphatidylcholines (PCs), cholesterol, polyethylene glycol (PEG)-lipids, targeting ligands, and siRNAs

[56]. Although the exact distribution of lipids and siRNAs inside the LNPs is not known, these siRNA-

LNPs are shown to have an electron dense core [57-61]. An initial model for these systems was proposed

based on the results from simulations and observed electron dense cores in the experiments [Figure 1.4-

Left] [56, 59]. According to that model, this electron dense core was due to the siRNAs entrapped inside

inverted micelles comprised of protonated KC2 (KC2H). However, the recent experiments [57] suggest

otherwise. According to this study, the neutral KC2 molecules are segregated from other components in

12
the siRNA-LNPs and form an oil droplet in the core of LNPs, whereas the siRNAs are packed between

lipid bilayers surrounding this oily core. Based on these results, a new model for the siRNA-LNPs has

been proposed [Figure 1.4-Right].

Figure 1.4: Models for a typical siRNA-LNP containing KC2.

Left) Previous model. Figure adapted from (Tam, Yuen, Sam Chen, and Pieter Cullis. Advances in lipid

nanoparticles for siRNA delivery. Pharmaceutics 2013, 5(3): 498-507) with permission from the Authors

owing the copyrights. Right) Updated model. Reprinted (adapted) with permission from (Kulkarni,

Jayesh A., Maria M. Darjuan, Joanne E. Mercer, Sam Chen, Roy van der Meel, Jenifer L. Thewalt, Yuen

Yi C. Tam, and Pieter R. Cullis. On the Formation and Morphology of Lipid Nanoparticles Containing

Ionizable Cationic Lipids and siRNA. ACS nano. 2018, 12(5): 4787-4795). Copyright (2018) American

Chemical Society. Inkscape was used to generate or modify the models [32].

13
1.2.4 Lipids of interest in this study.

Chapters 4 and 5 are based upon the results from simulation of bilayers and HII structures, respectively.

These lipid aggregates are composed of one or more of the lipids shown in Figure 1.5. This group of

lipids contains both neutral and positively charged lipids.

Figure 1.5: Two-dimensional structure of the lipids studied in this work.

14
From left to right: DOPE, POPE, POPC, KC2H, and KC2. Except the KC2H which are positively

charged, the other lipids have a net charge of zero. Oxygen, nitrogen, phosphorus, carbon, and hydrogen

atoms are represented in red, blue, orange, light yellow, and white, respectively. Most of the hydrogen

atoms are not shown for clarity. A few of the hydrogen atoms in the lipid tails are shown to locate the

C=C bonds in them. Avogadro [54] and Chimera [55] were used to generate the figure.

1.2.4.1 POPC.

Phosphatidylcholines (PCs) are the major component of the mammalian plasma membranes [8].

Moreover, given to their bilayer forming preference, they are one of the main components in the siRNA-

LNP formulations [56]. Therefore, PCs are both of a biological importance and of interest for practical

use in LNP nano-formulations.

From a computational point of view, the parameters for these lipids are optimized and well-tested

in CHARMM36 force field (C36 FF) (see Section 2.3) and many other force fields [62]. POPC bilayers

are also one of the most frequently studied systems in both simulations and experiments, providing a

considerable amount of data in the literature to compare our simulations with. Taken together, POPC is a

good candidate for KC2 and KC2H parameterization and model validation, and for looking at interactions

between KC2 and KC2H with PCs.

1.2.4.2 DOPE and POPE.

Phosphatidylethanolamine (PE) bilayers are commonly used to model the inner leaflet of the plasma

membrane because PEs are the major lipids in the cell membrane inner (cytoplasmic) leaflet [63]. DOPE

and POPE are both capable of forming the HII phase at high temperatures due to their cone-shaped

15
structures [20, 64]. These lipids are also known to promote the lamellar-to-HII transition as helper lipids

in mixed lipid systems [52]. These neutral lipids are well studied in HII phases, especially using

experimental techniques [44, 65-67], and the parameters for them are well optimized and validated

through many computational studies [62]. These factors make them good candidates to study the HII

structures and validate the system setup and protocols developed in this study.

1.2.4.3 KC2 and KC2H.

KC2 is another lipid of interest in this thesis. This ICL is one of the critical components of the siRNA-

LNPs [51]. At the physiological pH level, the amino group in the KC2 headgroup is deprotonated and

KC2 has a net charge of zero. Upon a drop in the pH, corresponding to the acidic condition inside the

endosomes, this amino group is protonated and KC2H with a net charge of +e forms. Where KC2 is

localized in the nano-formulation and how it interacts with PC lipids as a function of pH and mixing ratio

is one of the aims in this thesis. Force field parameters for these lipids, however, were not available and

were developed as part of this study.

1.3 Thesis Outline

Following are brief descriptions of what will be discussed in each chapter and the importance of each

study.

Chapter 2: Methods

MD simulation using atomistic force fields (FFs) was used for all the simulations conducted in this thesis.

In this chapter, the theory of molecular dynamics simulation, as well as the underlying concepts and

algorithms implemented within will be briefly introduced first. Next, common biomolecular FFs, e.g.

16
CHARMM36 FF [62] and AMBER99SB-ILDN FF [68], and the reason for choosing each FF will be

briefly discussed. The protocol which was developed and used for parameterization of KC2 and KC2H in

CHARMM36 FF, will be presented next. In the last section of this chapter, construction of molecular

systems, e.g. bilayers and HII phases, composed of two or three molecule types will be explained.

Chapter 3: Computational and experimental approaches for investigating nanoparticle-based drug

delivery systems

Nanoparticles are of special interest in several fields of studies, including nanomedicine [69]. To achieve

the maximum potential of these systems as drug/gene delivery systems, molecular structures and

molecule-molecule interactions should be known. This deeper understanding could be achieved by

combining both experiments and computer simulations as complementary techniques.

This chapter is a comprehensive review of the previous studies which successfully implemented

computational techniques, either alone or in combination with experimental techniques, to characterize a

variety of nano-particles commonly used for drug/gene delivery purposes. Reading through this review

article, the reader will find out the type of questions in the nano-medicine field of research which could be

investigated using computational techniques. We have further discussed the experimental techniques

which are commonly used in nanomedicine field of research, and how computer simulations could

potentially assist experimentalists in the rational design of nano-particulate drug delivery systems [5].

Chapter 4: Ionizable amino lipid interactions with POPC: Implications for lipid nanoparticle

function

Lipid nanoparticles (LNPs) containing ICLs are one of the molecular systems of interest in this thesis.

These systems are clinically validated non-viral delivery systems [51, 57], with ONPATTROTM

(Patisiran) as the first siRNA-based drug recently approved by the Food and Drug Administration (FDA).

17
KC2 is one of the most efficacious ICLs which is known to play critical roles in determining the internal

structure and size of this class of LNPs, their stability in the bloodstream, and drug release from

endosomes. However, the KC2 distribution in LNPs and its effect on the structure, stability and drug

release profile of the LNPs is not currently well understood.

The main goal of this study was to look at the interactions between KC2 and its protonated state

(KC2H) with other lipids (e.g. PCs) in the LNPs. Investigation of these molecular interactions could

provide deeper insight into the KC2 distribution in the LNPs, and the molecular nature of the electron-

dense core observed in the structure of this class of LNPs. These interactions could be studied using MD

simulation technique. However, computational study of these systems requires the parameters for the

amino lipids. Thus, the FF parameters for these synthetic lipids were developed and validated in this

study. Given these parameters, computer simulation of POPC/KC2 binary mixtures as a function of pH

and mixing ratio were conducted.

Chapter 5: Structural properties of inverted hexagonal phase: A hybrid computational and

experimental approach

Inverted/reverse hexagonal phase is a non-lamellar phase of interest in the field of nanomedicine [24].

The current knowledge in this phase is mainly due to extensive experimental research, and a limited

number of computational studies. Experiments on HII phase are usually conducted in presence of excess

water. In these situations, the number of water molecules taken up by the HII lattice is difficult to be

determined. Therefore, determination of internal dimensions of HII phase in these conditions are

challenging. Conducting experiments in low hydration regimes are also challenging and could be time

and cost consuming [44].

The main goal of this study was to develop computational protocols for investigation of HII

phases composed of amphiphilic molecules e.g. lipids, and to examine the effect of hydration level and

18
temperature on the structural properties of HII systems. Inverted hexagonal phases composed of DOPE

and POPE were constructed and simulated for several hydration levels and temperatures. Structural

properties, including the internal dimensions, of HII systems were calculated from simulations and

compared with experiments.

Chapter 6: An auto-inhibitory helix in CTP:phosphocholine cytidylyltransferase hijacks

the catalytic residue and constrains a pliable, domain-bridging helix pair

CCT enzyme is a key enzyme in phosphatidylcholine (PC) synthesis. Catalytic activity of CCT enzyme is

critical for the PC homeostasis and cell proliferation [70]. Inhibition of PC biosynthesis by antitumor

phospholipid analogues have been shown to cause apoptosis, with some of these inhibitors directly

targeting the CCT enzyme [13]. Moreover, mutations in CCT enzyme, e.g. Arg223Ser, have been

previously reported to result in genetic disorders [14-17], although the underlying reason is not known.

CCT is known to be activated only when it is bond to a PC-deficient membrane. This activation is

known to be due to disassociation of two auto-inhibitory (AI) helices in this enzyme. The underlying

mechanism by which these AI helices can suppress the catalytic activity of CCT enzyme is not known

either. The main goal of this study was to investigate the effect of AI helices on the structure and

dynamics of CCT enzyme.

Chapter 7: Conclusions and future directions

This chapter will summarize the main findings of this study. The results reported, the constructed

molecular systems, as well as the developed methods and force field parameters can be used for future

studies. The last section of this chapter provides directions for future studies.

19
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27
 Chapter Two: Methods

2.1 Abstract

Molecular dynamics (MD) simulation is a powerful technique for study of molecular systems. This

technique was used to study both the structure and dynamics of biomolecules of interest: lipid aggregates

in Chapters 4 and 5, and CCT enzyme in Chapter 6. In Section 2.2, the MD simulation methodology

and some of its underlying algorithms will be explained. The material presented in that section are mainly

based on the GROMACS user manual [1] and a book written by Andrew R. Leach on molecular

modelling principles and applications [2].

Classical MD simulations are based on classical descriptions of atoms in the system via so-called

classical force fields (FF). Thus, in Section 2.3, the main concepts in the biomolecular force fields will be

discussed, and their functional forms as well as different functional terms used in the common force fields

will be explained. In this thesis, CHARMM36 FF was used for simulations in Chapters 4 and 5, and

AMBER99SB-ILDN for simulations in Chapter 6. A short description of each of these two FFs, and the

reason why these FFs were chosen for each study will be provided next.

MD simulations require force field parameters for all the molecules in the system to be well

defined. Since these parameters for KC2 and KC2H did not exist in any force field, the parameters for

these two key lipids were developed and validated. The protocol developed and used for parameterization

of these cationic lipids, as well as the discussions on parameter validation will be explained in detail in

Section 2.4.

Finally, in Section 2.5, construction of molecular systems simulated in this study will be

explained.

28
2.2 Molecular Dynamics (MD) Simulation

Molecular dynamics (MD) simulation is a well-known computational method for studying molecular

systems [3]. This technique can provide insights into structural, dynamic, and thermodynamic properties

of biomolecular systems.

MD simulation works by numerical integration of the Newton’s equations of motion [Equation

2.1] for each atom in the system [1, 2].

𝑚𝑖 (𝑑 2 𝒓𝒊 / 𝑑𝑡 2 ) = 𝑭𝒊 , i = 1 . . . N Equation 2.1

Where, 𝑁 is the number of particles in the system, and 𝑭𝑖 is the force applied to an atom 𝑖 in the system

by other particles in the system. The forces are given by the negative derivative of the potential energy

function (𝑈(𝑹)) - which (𝑹) refers to ( 𝒓1 , 𝒓2 , … , 𝒓𝑁 ) - with respect to the atomic coordinates [Equation

2.2].

𝑭𝒊 = − 𝑑 𝑈(𝑹) / 𝑑 𝒓𝒊 Equation 2.2

See Equation 2.10 for 𝑈(𝑹) definition.

The total simulation time (𝑡) - ranging from a few nanoseconds to tens of microseconds or even

milliseconds, depending on the question asked – is broken into a number of time steps (𝑛) with a step size

of 𝛥𝑡. At a given position in space, each atom experiences a net force (𝑭𝑖 ) because of its interactions with

other surrounding atoms in the system, although the net force on the whole system will be zero. A change

in the system configuration after a time step results in a change in the net force on each atom. Therefore,

29
at each time step, the net force on each atom (𝑖) is calculated and subsequently used to update both their

positions (𝒓𝑖 ) and velocities (𝒗𝑖 ).

In a classical MD simulation, the potential energy function 𝑈(𝑹) is a classical mechanic

description of the interaction energy of the system and is a function of atomic positions (𝒓𝑖 ). These

classical potential functions are relying on the Born-Oppenheimer approximation which allows the

separation of motions of electrons from nuclei in a molecule. This approximation is valid due to the mass

difference between nuclei and electrons because it causes the electrons to move much faster than atomic

nuclei. Both the function and the parameters required to calculate the potential energy of a molecular

system is provided as so-called force fields (FF) (see Section 2.3).

To start a MD simulation, an initial structure (preferably close to the equilibrium state of interest)

is needed. This initial structure could be either taken from the ones determined by experiments (e.g. X-ray

or NMR), or be constructed using molecular modeling packages, e.g. GROMACS [4]. In addition, initial

velocities corresponding to the desired temperature should be assigned to each particle in the system.

Starting from this state (𝒓𝑖 and 𝒗𝑖 , for 𝑖 = 1,2,3, . . . , 𝑁 with 𝑁 as the number of atoms in the system),

atoms interact with each other and their positions and velocities are updated over time producing a so-

called trajectory. This trajectory can be further analysed by statistical mechanics methods to extract the

macroscopic properties of interest from these atomic motions (microscopic data). This task is possible

based on the ergodic hypothesis. According to this hypothesis, when a system is simulated for a long

time, it will span all the accessible microstates in the phase state in an equiprobable way. Thus, the

trajectory will be equivalent to an ensemble of microstates for the system of interest. This indicates that

the time average over trajectory will be equivalent to the ensemble average. This equivalency, however,

is valid when the system is simulated for enough time so that the system can sample all the corresponding

microstates to that specific total energy [2]. The lack of sampling is one of the major limitations and could

30
result in inaccurately averaged thermodynamic properties calculated from computer simulation

techniques, including MD simulations [5].

There are several molecular simulation packages such as GROMACS [4], CHARMM [6], LAMMPS [7],

AMBER [8], and NAMD [9], which support MD simulations. GROMACS is a fast, flexible, efficient,

scalable, and free package for molecular simulations and trajectory analysis. It is one of the most popular

packages for simulations of biomolecules, and supports the use of several major FFs. The reported studies

in this thesis were conducted using several versions of the GROMACS MD packages - the version

numbers are mentioned in each chapter.

2.2.1 Isothermal-isobaric (NPT) and canonical (NVT) ensembles.

All the simulations in this study were conducted under constant number of particle (𝑁), temperature (𝑇),

and pressure (𝑃) conditions. These conditions were corresponding to the experimental conditions in the

laboratory where the temperature is the room temperature and the pressure is equal to atmospheric

pressure. The thermodynamic ensemble, i.e. the set of all the microstates accessible to and sampled by the

system - produced under such a thermodynamic condition - is referred to as the isothermal-isobaric or

𝑁𝑃𝑇 ensemble in computer simulations. In some cases, such as the first equilibration phase of the

simulations or when the experiments/simulations in constant volume (𝑉) is required, the canonical (𝑁𝑉𝑇)

ensemble is used. In principle, in the thermodynamic or macroscopic limit, i.e. where the systems are

composed of a large/infinite number of particles, all the ensembles are expected to result in the same

thermodynamic properties for the system. However, this is not the case in the simulations because only a

finite number of particles are usually simulated in practice.

There are several algorithms in the MD simulation which can be used to control the temperature

and pressure of the molecular system. In the following section, these algorithms will be briefly explained.

31
For the pressure coupling, different types of coupling such as isotropic, semi-isotropic, and anisotropic,

and their importance in different molecular systems will also be discussed.

2.2.2 Temperature coupling.

Temperature of the system (consist of 𝑁 particles) is calculated from the ensemble average (shown as

angle brackets) of the total kinetic energy < 𝐸𝑘𝑖𝑛 >, and the total number of degrees of freedom 𝑁𝑑𝑓 in

the system [Equation 2.3]. In this equation 𝑘𝐵 represents the Boltzmann’s constant, 𝑚𝑖 the atomic mass

for atom 𝑖, and 𝑣𝑖 stands for the velocity of atom 𝑖. The left part of this equation is relying on the

equipartition theorem which states that each degree of freedom in the system contributes as 1/2 𝑘𝐵 and

1/2 𝑘𝐵 𝑇 to the system’s heat capacity and total kinetic energy, respectively. For a system consisting of 𝑁

atoms, 3𝑁 degrees of freedom exist. However, 𝑁𝑑𝑓 is usually less than 3𝑁 due to the applied constraints

(𝑁𝑐 ), e.g. applied constraints on bonds and angles, and center-of-mass (COM) motion removal (𝑁𝐶𝑂𝑀 = 3)

of the system [Equation 2.4].

𝑁 Equation 2.3
< 𝐸𝑘𝑖𝑛 >𝑁𝑉𝑇 = (1/2) 𝑁𝑑𝑓 𝑘𝐵 𝑇 = (1/2) ∑ 𝑚𝑖 𝑣𝑖2
𝑖=1

𝑁𝑑𝑓 = 3𝑁 − 𝑁𝑐 − 𝑁𝐶𝑂𝑀 Equation 2.4

There are several ways to maintain a constant temperature during the simulation course (required

for 𝑁𝑉𝑇 and 𝑁𝑃𝑇 simulations), including Berendsen [10], velocity-rescaling [11], and extended Nose-

Hoover [12, 13] schemes. In these methods, the system is coupled to a heat bath with a given temperature

32
of 𝑇𝑏𝑎𝑡ℎ which is the reference temperature desired for the system. This way, systems can exchange heat

(thermal energy) with the bath.

Berendsen coupling is a weak coupling method based on rescaling the atomic velocities at each

time step. The scaling factor (𝜆 𝑇 ) is calculated from the difference between the temperature of the system

and the reference temperature at each time step [Equation 2.5], where 𝛥𝑡 and 𝜏 are the time step and the

coupling constant in picoseconds.

𝜆2𝑇 = 1 + (𝛥𝑡/𝜏) ((𝑇𝑏𝑎𝑡ℎ /𝑇(𝑡)) − 1 ) Equation 2.5

Although the Berendsen algorithm is a good choice for the equilibration phase to adjust the

system temperature to the reference temperature, it is known to fail to generate a correct canonical

ensemble. Therefore, for the production runs, other algorithms with the ability to sample a correct

canonical ensemble are recommended. Velocity-rescaling and Nose-Hoover algorithms are two

temperature coupling algorithms used for the production runs in the simulations conducted in this thesis.

Velocity rescaling algorithm is based on stochastic terms added to the kinetic energy calculations. In

Nose-Hoover algorithm, the bath is considered as part of the system (extended systems) and is included in

the system Hamiltonian and consequently into the particles’ equations of motion.

2.2.3 Pressure coupling.

For a molecular system, the pressure is defined as the average perpendicular force applied on the

container walls caused by random particle collisions with those walls. In simulations, the macroscopic

pressure tensor 𝑃 is the ensemble/time average of the microscopic (or instantaneous) pressure tensor Ƥ

given by Equation 2.6, where 𝑉 is the total volume of the system.

33
𝑃 = < Ƥ >𝑒𝑛𝑠𝑒𝑚𝑏𝑙𝑒 Equation 2.6

Which Ƥ = 2/𝑉 ((1/2) ∑𝑁 𝑁 𝑁

𝑖=1 𝑚𝑖 𝒗𝑖 ⊗ 𝒗𝑖 − (−1/2) ∑𝑖 =1 ∑ 𝑗 > 𝑖 𝒓𝑖𝑗 ⊗ 𝑭𝑖𝑗 )

In this equation, the ⊗ stands for direct product of two vectors, whereas 𝒓𝑖𝑗 and 𝑭𝑖𝑗 are the interatomic

distance and force vectors, respectively.

Like temperature coupling, there are several types of barostats which can maintain the pressure of

the system close to a reference value of Ƥ0 . The pressure can be controlled by rescaling the simulation

box volume, and consequently the atomic coordinates. Berendsen [10] and Parrinello-Rahman [14, 15]

algorithms are the most frequently used methods for controlling the system pressure in the MD

simulations. In both algorithms, the system is coupled to a pressure bath. Berendsen algorithm is a weak

coupling algorithm, where the box volume and atomic coordinates are scaled by a scaling matrix which is

a function of (𝑃 − Ƥ0 ). Parrinello-Rahman algorithm, however, is an extended pressure system method

where the pressure bath is considered as a piston acting on the system and is part of the system.

Therefore, the particles’ equations of motion are modified as in the case of the Nose-Hoover algorithm.

Herein again, the Berendsen weak coupling can be used for the equilibration phase, while for the

production run or when the box vector fluctuations are important, Parrinello-Rahman coupling is

recommended. This is because the Berendsen coupling is not capable of generating the accurate ensemble

and therefore, will affect the ensemble averaged thermodynamic properties extracted from the

simulations.

The pressure of the molecular system can be controlled in three different ways; isotropic, semi-

isotropic and anisotropic schemes.

34
 In the isotropic scheme, all three box vectors are scaled with the same scaling factor, and the

system pressure is an average over all three directions X, Y, and Z. The isotropic pressure coupling is

usually used for the simulation of proteins or other solutes in solution [5]. In Chapter 6 we used isotropic

pressure coupling for the simulation of CCT enzyme solvated in water.

In the semi-isotropic scheme, box size in two directions (e.g. X and Y) are scaled together but

treated independently from the third direction (i.e. Z). When lipid membranes or other interfaces are of

interest, the symmetry of the system suggests that the semi-isotropic pressure coupling is the best choice

for pressure control. This way, both the height and the surface area of the simulation box will be better

controlled by the coupling algorithm. Thus, as recommended in the literature [5], this scheme was used

for all the simulations conducted on lipid bilayers in this thesis (e.g. Chapter 4).

In addition to the lipid bilayer, the HII systems should also be simulated using the semi-isotropic

scheme. This is mainly because HII is not homogenous in all three directions (thus, the isotropic scheme

is not appropriate for this case) and there is a hexagonal symmetry in the way lipid cylinders are arranged

in space. Assuming lipid cylinders are parallel to the Z axis, the pressure in XY-plane could be treated

separately from the pressure in Z direction [Figure 1.1]. This allows the lipid cylinder height and

diameter be freely adjusted to optimize the system free energy. In Chapter 5, several PE HII systems

were simulated using semi-isotropic pressure coupling.

The third scheme is the anisotropic pressure coupling in which pressure in each direction is

treated independently. The anisotropic coupling is not usually recommended because like semi-isotropic

coupling it does not create the physical ensemble [5]. Furthermore, it allows large deformations of the box

size in each direction, and the box shape. However, this suggests that anisotropic pressure coupling might

be a choice where phase transitions or system deformations from their equilibrium states are expected to

happen or are desired (see [16-18] as examples). For instance, a lamellar-to-HII phase transition in a lipid

system requires special conditions such as water/lipid ratio, box size and shape, to be fulfilled so that a

35
HII phase can form. Given the lipid density and temperature in the system, the box should be able to

change in all three directions to form the unit cell suitable for HII phase formation. In Chapter 5 of this

thesis, an anisotropic pressure coupling was used to study DOPE and POPE HII structures. The results

were also compared with simulations conducted using a semi-isotropic coupling method.

Both semi-isotropic and anisotropic pressure couplings do not correspond to any experimental

ensemble [19]. This could result in an incorrect ensemble and affect the comparisons between

experimental and computational data.

2.2.4 Periodic boundary condition.

Periodic boundary condition (PBC) is often applied in MD simulations to approximate an infinite/bulk

system. This will, indeed, minimize the artifacts caused by a finite-size molecular system (the

surface/edge effect), although PBC itself may cause periodicity artifacts.

To apply the PBC, a space-filling box enclosing all the molecules in the system is constructed

first. Next, this unit cell (or the original box) is replicated in space (in the desired directions) for an

infinite number of times (these copies are also called images) to mimic the large system. According to the

minimum-image convention, each particle in the unit cell will only interact with the nearest image of other

particles (could be in either the original box or one of the adjacent cells) when short-range non-bonded

interactions (e.g. van der Waals interactions) are calculated. For the long-range electrostatic interactions,

however, the effect of particles in other image cells might be critical to consider. As a solution to this,

there are a few algorithms, e.g. Particle Mesh Ewald (PME), which could take advantage of this

periodicity and consider these long-range non-bonded pair interactions in an efficient way (see Section

2.2.5).

36
 There are several box types, e.g. triclinic, cubic, and dodecahedron, supported in GROMACS

MD package. Depending on the biomolecule to be simulated and the geometry/symmetry of the system (if

any), one of these box types might be preferred.

Simulation of solute proteins are often conducted in rhombic dodecahedron box shape. This is

mainly because this box is the smallest unit cell capable of fully covering the protein and its solvation

shell, while still saving a lot of computational time by minimizing the required number of water

molecules to fill the box. Thus, in Chapter 6 of this thesis, where the simulation of CCT enzyme was of

interest, the enzyme-substrate complex was enclosed and simulated in this box type.

For the simulation of lipid bilayers (e.g. simulations represented in Chapter 4), a rectangular box

type is the most appropriate and the recommended box type. Using this simulation box, the bilayer is

usually oriented on XY-plane and the vector normal to the bilayer surface is parallel to the Z axis.

For the simulation of lipid aggregates in HII phase (e.g. simulations in Chapter 5) the situation is

a bit complicated. As discussed earlier, in HII phase the lipid cylinders are arranged in a hexagonal

geometry. So, when the original box is copied in the space by PBCs, the infinite system must have this

hexagonal symmetry. Unfortunately, a hexagonal box shape is not supported in GROMACS yet.

However, simulation of HII phase is still possible using either rectangular or triclinic box types.

2.2.5 Non-bonded interactions and cut-offs.

Both the electrostatic (∝ 1/𝑟𝑖𝑗 ) and the dispersion part of the van der Waals (∝ 1/𝑟𝑖𝑗6 ) interactions are

long-range interactions. In principle, these nonbonded interactions between each pair of atoms (except the

ones separated by 1, 2, or 3 bonds) in the system (i.e. particles in both the original and image boxes)

should be considered in the potential energy and atomic force calculations. However, this is too time-

consuming in practice. Assuming the system is constituted of 𝑁 particles, the number of pair interactions

37
to be calculated is an order of 𝑁 2 . Therefore, to save computational time, the interactions between the

pairs are cut-off at a certain distance (usually between 1 to 2 𝑛𝑚 ). This sudden truncation of the

interactions results in a discontinuity in the potential energy, force, and its derivative, and consequently

cause significant artifacts. For the Lennard-Jones potentials, modifying the potential energy or the force

functions using either “switch” or “shift” functions could greatly reduce these artifacts. These methods,

however, are not recommended for treating the coulomb potential. For electrostatic part, the interactions

are cut-off at the given distance and the effect of other particles beyond this cut-off is considered using

other algorithms such as reaction field [20] or Particle Mesh Ewald (PME) [21, 22]. The PME method is

the recommended algorithm for treating the long-range electrostatic interactions, especially for the highly

charged systems such as DNA, and nonhomogeneous systems such as lipid bilayers [5, 23]. Therefore,

the PME method was used in all the simulations presented in this thesis. For more details on reaction field

and PME methods, the reader is referred to [24] and other references within.

2.2.6 Integration algorithms.

MD simulation works by the numerical integration of Newton’s equations of motion for each particle in

the system at each time step. There are several well-established integrators, including leap-frog and

velocity Verlet algorithms, used in MD simulations which are available and supported in GROMACS

package. The leap-frog algorithm is the default integrator in GROMACS and is the algorithm used in all

the simulations presented in this thesis. In this method, the positions of atoms at time 𝑡 + 𝛥𝑡 are

calculated from the atomic positions at time 𝑡 and and their velocities at 𝑡 + (𝛥𝑡/2):

𝒓𝒊 (𝑡 + 𝛥𝑡) = 𝒓𝒊 (𝑡) + 𝛥𝑡 𝒗𝒊 (𝑡 + (𝛥𝑡/2)) Equation 2.7

38
Therefore, the velocities at 𝑡 + (𝛥𝑡/2) need to be calculated first. The 𝒗𝑖 (𝑡 + (𝛥𝑡/2)) are calculated

from the 𝒗𝑖 (𝑡 − (𝛥𝑡/2)) and accelerations at time 𝑡 as shown in Equation 2.8.

𝒗𝒊 (𝑡 + (𝛥𝑡/2)) = 𝒗𝒊 (𝑡 − (𝛥𝑡/2)) + 𝛥𝑡 𝒂𝒊 (𝑡) Equation 2.8

Now, given both 𝒗𝑖 (𝑡 + (𝛥𝑡/2)) and 𝒗𝑖 (𝑡 − (𝛥𝑡/2)), the velocities at time 𝑡 are calculated as:

𝒗𝒊 (𝑡) = (1/2) [𝒗𝒊 (𝑡 + (𝛥𝑡/2)) + 𝒗𝒊 (𝑡 − (𝛥𝑡/2)) ] Equation 2.9

This process is done for each time step. The accelerations at time 𝑡 are calculated as 𝒂𝑖 (𝑡) =

𝑭𝑖 (𝑡)/𝑚𝑖 , i.e. the force applied on particle 𝑖 at that time step divided by particle mass. The 𝑭𝑖 (𝑡) can also

be calculated from the atomic positions at time 𝑡 and the potential functions 𝑈(𝑹) provided in force fields

(Equation 2.2 in Section 2.2).

2.3 Biomolecular Force Fields

Force fields (FF) are referred to a set of functions and associated parameters required to model

all the comprising molecules, and the way each atom in the system interacts with other atoms in

the system [2, 23, 25]. Molecular mechanic techniques, including MD simulation, make use of

the classical FFs; the FFs which are based on classical description of the potential energy of the

system. There are several FFs such as CHARMM [26], AMBER [8, 27], OPLS [28] and

GROMOS [29] which are used for simulation of biological molecules. These FFs include all the

parameters required for the simulation of main classes of biological molecules, including

proteins and lipids. There are other FFs, e.g. Berger lipids [30], Slipids [31, 32], and Lipid14

39
[33], which are usually modified/combined versions of the FFs mentioned above and are

developed for simulation of specific class of these biomolecules, e.g. lipids. Irrespective of slight

differences between these FFs, they are similar in their functional forms [5, 6] [Equation 2.10].

𝑈(𝑹)= Equation 2.10

∑𝑏𝑜𝑛𝑑𝑠 𝑘𝑏 (𝑏 − 𝑏0 )2 + ∑𝑎𝑛𝑔𝑙𝑒𝑠 𝑘𝜃 (𝜃 − 𝜃0 )2 +

∑𝑑𝑖ℎ𝑒𝑑𝑟𝑎𝑙𝑠 𝑘𝜑 (1 + 𝑐𝑜𝑠 (𝑛𝜑 − 𝛿 )) + ∑𝑖𝑚𝑝𝑟𝑜𝑝𝑒𝑟𝑠 𝑘𝜔 (𝜔 − 𝜔0 )2 +

𝑚𝑖𝑛 𝑚𝑖𝑛 𝑚𝑖𝑛

∑𝑛𝑜𝑛−𝑏𝑜𝑛𝑑𝑒𝑑 𝑝𝑎𝑖𝑟𝑠 (𝜀𝑖𝑗 [(𝑅𝑖𝑗 /𝑟𝑖𝑗 )12 − 2 (𝑅𝑖𝑗 /𝑟𝑖𝑗 )6 ] + 𝑞𝑖 𝑞𝑗 /4𝜋𝜀0 𝜀 𝑟𝑖𝑗 )

This function is a sum over six terms, each describing either a bonded (terms 1-4) or non-bonded (terms

5, 6) interaction contribution to the potential energy of the system.

The first and second terms are the energy contributions for bond and angle deformations from

their reference values (𝑑0 , 𝜃0), respectively. These are harmonic functions with force constants of (𝑘𝑏 )

and ( 𝑘𝜃 ) for bonds and angles, respectively.

The third and fourth terms are the energy contributions for the proper and improper dihedrals in

the system. The proper dihedrals (term 3) are sinusoidal expansions - usually are expanded up to six terms

for each individual dihedral angle (torsion) about a certain bond - where (𝑘𝜑 ), (𝑛), and (𝛿) are the force

constant, multiplicity, and phase shift, respectively. The improper dihedral term (term 4) is a harmonic

function with a force constant of (𝑘𝜔 ) and a reference value of (𝜔0), which is a required term in the

energy function to maintain the planarity of the rings, and to keep the chirality of chiral atoms the same

throughout the simulation.

Finally, the last two terms in Equation 2.10 are the contributions for the van der Waals (term 5)

and electrostatic (term 6) non-bonded interactions, using Lennard-Jones (L-J) and columbic potential

40
functions, respectively. 𝑖 and 𝑗 subscriptions are the atomic indices for the interacting atoms. In L-J term,

𝑚𝑖𝑛 𝑚𝑖𝑛
the 𝜀𝑖𝑗 is the well depth, 𝑅𝑖𝑗 is the distance between two interacting atoms in which the L-J energy is

minimum, and 𝑟𝑖𝑗 is the interatomic distance [Figure 2.1]. In the coulombic term, 𝑞𝑖 and 𝑞𝑗 are the point

charges assigned to atoms 𝑖 and 𝑗, whereas 𝜀0 and 𝜀 are the permittivity of vacuum and relative dielectric

constants, respectively.

Figure 2.1: Lennard-Jones potential energy.

Lennard-Jones potential energy (𝑉 (𝑟𝑖𝑗 )) as a function of interatomic distances 𝑟𝑖𝑗 . Inkscape was used to

generate the figure [34].

41
 Most FFs consist of these six terms, although there are some which have additional terms,

including Urey-Bradley and torsional correction terms (CMAP) [Equation 2.11].

∑𝑈𝑟𝑒𝑦−𝐵𝑟𝑎𝑑𝑙𝑒𝑦 𝑘𝑈𝐵 (𝑆 − 𝑆0 )2 + ∑𝑟𝑒𝑠𝑖𝑑𝑢𝑒𝑠 𝑈𝐶𝑀𝐴𝑃 (𝜑, 𝜓) Equation 2.11

Urey-Bradley is also a harmonic function of distance between 1-3 atoms in a three-bond atom group

forming an angle. This term is additional to the angle term (term 2 in Equation 2.10) and is a rarely used

term except that vibrational spectra be of interest. Similar to the bond and angle terms, the 𝑆0 and 𝑘𝑈𝐵 are

the reference 1-3 distance and the force constant, respectively. The CMAP term is a correction term

applied to the protein mainchain torsion energies resulting in a significantly improved reproduction of

both structure and dynamics of proteins.

Each FF, regardless of its functional form and parameter set, has been developed and validated

based on a certain number of quantities, and using a limited group of model small molecules. Extensive

computational studies and comparison with corresponding experimental data available in the literature

suggests that some FFs are more successful in reproduction of certain structural, dynamic, or

thermodynamic properties of molecular systems [35]. As mentioned earlier, there are also FFs, e.g.

Slipids [31, 32], which have been optimized for a specific class of biomolecules which makes them

capable of reproducing experimental data in a good to excellent level. Moreover, each FF was originally

developed in a MD package, e.g. GROMACS or NAMD, with a specific model for the water molecules,

e.g. SPC/E or TIP3P, and specific choices for other parameters such as cut-offs and algorithms for

treatment of interactions [36]. Therefore, choosing the most appropriate FF in each simulation is highly

dependent to the molecular system under study and the questions of interest in that study. Depending on

the type of biomolecules present in the system, as well as the physical and thermodynamic properties of

interest to be extracted from simulations, different FFs might be the best candidate. Finally, depending on

42
the spatio-temporal scale and the level of resolution necessary to get the properties of interest from

simulations, either all-atom, united-atom, or coarse-grained FFs might be chosen [37].

2.3.1 CHARMM36 FF for the simulation of lipid aggregates.

The behavior of a lipid aggregate is highly sensitive to the assigned parameters of the comprising lipids

[38, 39]. There are several atomistic force fields (FF) which could be used in computer simulations of

biomembranes [5]. There are studies which suggest C36 FF is among the best atomistic FFs at

reproducing the structural data for lipids [40]. For instance, C36 is known to yield improved lipid volume

and bilayer thickness for PC lipids [35]. C36 was also shown to reproduce the lipid diffusion with very

good agreement to experimental data.

For simulations conducted in Chapters 4 and 5, the systems are merely composed of either one

or more lipid molecules [Figure 1.5] dispersed in water, making the C36 FF as one the best FF choices

for this study. The parameters for PC and PE are currently available and highly optimised in the C36 FF

[41]. These parameters can successfully reproduce several corresponding experimental data sets,

including deuterium order parameters (𝑆𝐶𝐷 ), in reasonable agreement. However, there are no parameters

available for both KC2 and KC2H in C36 FF.

2.3.2 AMBER99SB-ILDN FF for the simulation of CCT enzyme.

Protein parameters are available in several FFs, including CHARMM [26] and AMBER FFs [8, 27]. The

performance of both FFs have been slightly improved by incorporating so-called CMAP backbone

corrections - corrections to the protein “mainchain” dihedral energy [42, 43]. Further improvements were

implemented into AMBER FF in 2010, resulting in a new version of AMBER FF called AMBER99SB-

ILDN [44]. In this modified version, the torsion energy of four amino acids (isoleucine, leucine, aspartate,

43
and asparagine) “side chains” were optimized. Due to these corrections and improvements compared to

other FFs, the use of AMBER99SB-ILDN has been recommended for the simulation of proteins in the

long (micro-second and beyond) simulations [44], where the side chain rotations are possible.

In Chapter 6 of this thesis, the structure and dynamics of CCT enzyme, as well as its interactions

with its substrate cytidine triphosphate (CTP) was studied using MD simulations. Since the stability and

dynamics of the enzyme, as well as the dynamics of the loops and its side chains were critical, and the

required time-scale was estimated to be 1 microsecond, AMBER99SB-ILDN was used for the

simulations in that study.

2.4 Force Field Development for KC2 and KC2H

CHARMM36 FF is the chosen FF to study the lipid aggregates in this thesis (Chapters 4 and 5).

Although the parameters for most of the lipids of interest in this thesis [Figure 1.5] are available,

optimized and well-tested in C36 FF [41], the parameters for KC2 and KC2H are not present and need to

be developed. This section briefly describes the process by which these lipids were parameterized, as well

as provides the reader with the parameters for future simulations using these models.

Parameterization of novel molecules in a parent FF could be too time-consuming, especially for

lipids which their bulk properties are highly sensitive to the assigned parameters. Such a parameterization

for novel molecules is usually done by analogy to other parameterized molecules in the chosen FF.

Unfortunately, there are no functional groups analogous to the KC2 and KC2H headgroups, and the linker

segment connecting the headgroup and the acyl chains, which are already parameterized in C36 FF.

There are several computational tools, e.g. CHARMM-GUI [45], GAAMP [46], and ParamChem

[47, 48], which could assist in parameterization of novel molecules in C36 FF. Although these tools are

not designed and meant to parameterize the lipid molecules, they can facilitate the parameterization

process to a high degree. It is the user experience and chemical intuition to make efficient use of these

44
tools, adjust the parameters if required, and combine them properly to achieve a final set of parameters.

These parameters should generate data with an acceptable level of agreement with corresponding

experimental data. The protocol described in the next section could be used as a guide for

parameterization of new lipids in C36 FF in a time efficient way using freely available tools.

2.4.1 KC2 and KC2H parameterization in CHARMM36 FF.

The KC2H molecule was first divided into two main segments: lipid tails and lipid headgroup; the

headgroup includes the first two CH2 atoms in each acyl chain to consider the effect of headgroup on the

beginning of the acyl chains [Figure 2.2].

The parameters for the lipid tails were simply taken from DUPC lipid downloaded from

CHARMM-GUI [45] [Figure 2.3].

Figure 2.2: KC2(H) breakdown into two segments for parameterization.

45
Figure 2.3: KC2(H) acyl chain parameters.

The parameters for the tails are assigned based on available parameters for DUPC lipid on CHARMM-

GUI [45].

Next, the headgroup segment was parameterized using GAAMP server [46] as follows [Figure

2.4]. The headgroup itself was divided into two smaller segments: the ring segment and the amino

segment. The amino segment was parameterized twice, once for neutral amino group and once for the

protonated case. Parameters for each segment were calculated separately. To do so, a few constraints were

applied to, I) identify the equivalent atoms in the segment and II) assign a partial charge of 0.090 to each

non-polar hydrogen atom in that segment. The 0.090 value is the standard value for the partial charge of

aliphatic hydrogen atoms in C36 FF, and was required to be consistent with the C36 FF charge

assignment rules [48]. These constraints were included in the input file submitted to the GAAMP server.

46
GAAMP uses the previously parameterized molecules to automatically assign the initial parameters to

each atom in a small molecule, while applying all the given constraints [46]. Starting from this initial

parameter set, it then optimizes the parameters using the ab initio quantum mechanical calculations as the

target data. We used these optimized parameters given by GAAMP as the initial parameters for each

segment. To obtain the optimized model for each segment, these parameters were further modified and

tested in an iterative manner.

Figure 2.4: KC2(H) headgroup breakdown into two segments.

These fragments had some overlapping CH2 and CH3 groups (shown as orange and pink spheres,

respectively) which made the process of merging them together easier.

47
 The parameters developed for the segments were subsequently merged to give the parameter set

for the whole headgroups of KC2 and KC2H. To facilitate the merging process of segments in the next

step, extra CH2 and CH3 groups were added to the segments. The partial charges for these extra CH2 and

CH3 groups were taken to be the same as the aliphatic CH2 and CH3 groups of lipids available in C36

FF.

So far, all the atom types for the acyl chains are taken from the parent FF, i.e. C36FF [41],

whereas the ones for the headgroup are taken from CHARMM General force field (CGenFF) [47-49]. The

last step, therefore, is to assign missing bonded parameters for the parts where atom types from CGenFF

and C36FF are combined through bonded interactions, e.g. the dihedral of HGA2-CG321-CTL2-HAL2.

This step could be done by analogy between the atom type definitions in CGenFF and C36FF and taking

the parameters from either C36FF or CGenFF, while the preference was with C36FF. Based on these

definitions, CG321, HGA2, CG331, and HGA3 in CGenFF are equivalent to the CTL2, HAL2, CTL3,

and HAL3 in C36FF, respectively. As an example, to assign parameter to the HGA2-CG321-CTL2-

HAL2 dihedral, C36FF was searched for HAL2-CTL2-CTL2-HAL2 first. Since there was no such

combination in C36FF, HGA2-CG321-CG321-HGA2 was searched instead, found, and was assigned to

the dihedral of interest.

The final force field parameters (atom types, partial charges, and the bonded parameters) for the

parameterized segment [Figure A.1] in KC2 and KC2H are provided in Appendix A as reference.

2.4.2 KC2 and KC2H model validation.

There are several physicochemical properties of lipids and lipid bilayers which could be targeted for

parameterization purposes. Deuterium order parameter (SCD), area per lipid, and bilayer thickness are

48
examples of such experimental data which could be potentially used for lipid model validation [39]. The

amount of target data to validate parameters against are limited for the case of KC2 and KC2H.

In this study, the effect of adding KC2(H) on POPC palmitoyl chain order parameters in

POPC/KC2(H) mixtures at 313 K was used for KC2(H) model validation.

The SCD profiles for the POPC palmitoyl chain extracted from 2H NMR spectra 1 and

calculated from simulations of POPC/KC2(H) binary mixtures are shown in Figure 2.5. In

simulations, systems with KC2 and KC2H correspond to the 2H NMR experiments conducted at

pH 8.1 and 4.4, respectively.

The SCD parameters from simulations are in good agreement with the experimental data.

The overall trend in SCD profiles is similar between simulations and experiments. The acyl chain

order gradually decreases along the acyl chain towards the terminal methyl group. Also, in both

simulations and experiments, the pH seems to have no or little effect on acyl chain order

parameter at 313 K. The only difference was observed between pure POPC bilayers at two pH

values. We note that the experimental POPC palmitoyl chain order parameters are smaller at pH

4.4 than at pH 8.1. This is likely due to the higher interfacial surface tension observed in POPC

membranes at pH 4.4 [50]. In simulations, however, the SCD parameters for palmitoyl chain in

pure POPC systems are the same between two pH levels. That is mainly because the effect of

buffer and the higher surface tension was not considered in our simulations.

Finally, the SCD parameters calculated from simulations are higher than the corresponding

experimental values. This is also true for POPC which the parameters are well optimized in C36

1
Data provided by Dr. Jenifer Thewalt research group at the Simon Fraser University

49
FF. To conclude, the models developed here are valid and can be used for the purpose of simulations in

Chapter 4.

Figure 2.5: Validation of developed models for KC2 and KC2H.

Smoothed deuterium order parameter (SCD) for the palmitoyl chain of POPC in POPC/KC2(H) binary

mixtures were measured using 2H NMR experiments (Left column). The same parameters were

calculated from simulations and smoothed (Right column). This comparison was done for two pH levels

of 4.4 (a and b) and 8.1 (c and d) at temperature of 313 K.

50
2.5 Construction of Binary and Ternary Mixtures in Lamellar and HII Phase

Construction of a molecular system is the first, and usually a time-consuming step, in computer

simulations. This process usually takes more time for complicated structures; i.e. the systems composed

of arbitrary molecules, with a desired mixing ratio, and with complex architectures such as HII and cubic

phases. Given that the force field parameters for all the comprising molecules in the system are available

and validated, reaching thermodynamically stable and equilibrated systems in the desired state is the next

challenge to overcome [51]. In this study, both lamellar (bilayers) and non-lamellar (HII) phases are of

interest. Systems with several mixing ratios, at several water per lipid regimes and ion concentrations

were required for answering the questions of interest in this study.

There are several computational tools [52-55] designed for construction of molecular systems

required for doing molecular simulations. However, there are shortcomings in each one of these tools

when complicated structures are of interest. Therefore, GROMACS modules [4] (see below) were used to

generate the molecular systems according to the protocol described in the next two sections.

2.5.1 Bilayers.

Construction of the bilayers was relatively straightforward. The only challenge was to make bilayers

composed of KC2(H) with other lipids such as POPC, at several mixing ratios. Consider the POPC/KC2H

system for example. To do so, two monolayers each composed of only one lipid type are built first

[Figure 2.6]. These two monolayers both must be composed of the same number of lipids, e.g. 100,

which are distributed on a two-dimensional grid (10 by 10) on a square-shaped surface with the same total

area. Two GROMACS modules, gmx editconf and gmx genconf, were used to translate and rotate the

lipids on each grid point, to build these pure monolayers. Each grid point on these surfaces will be

51
assigned a number in the range of 1 to 100, which is equal to the residue number of each lipid. The next

step is to generate a set of random numbers from 1 to 100 so that they satisfy the desired mixing ratio. For

instance, if POPC/KC2H (80/20) is of interest, 20 random numbers need to be generated. These 20

numbers will be used to both keep the KC2H in these grid points, and to delete the POPC in the same grid

points. Finally, the coordinate files from two modified monolayers are merged together resulting in one

mixed monolayer with a desired mixing ratio; which was taken as the upper leaflet [Figure 2.6].

Assuming that this monolayer is in the XY-plane, its rotation (using gmx editconf module) around either

the X or Y axis gives the lower leaflet.

Adjusting the center of both leaflets to (0,0,0) and subsequently merging the coordinate files for

these two leaflets results in the POPC/KC2H (80/20) bilayer. Waters and ions were subsequently added

on the top and bottom of the system. The same approach can be applied to make bilayers composed of

three or more lipids as well.

Figure 2.6: Construction of the POPC/KC2H (80/20) bilayer.

52
2.5.2 Inverted hexagonal (HII) phase.

Considering the DSPS/KC2H/cholesterol (35/35/30) system as an example, first, a single lipid tube for

each lipid type (DSPS, KC2H, and cholesterol) was built and oriented in the Z direction [56]. Each lipid

tube was composed of 360 lipid molecules distributed on a cylinder with a radius of ca. 2.5 𝑛𝑚 and a

height of ca. 17 𝑛𝑚. Lipids were distributed on 15 rings, with each ring composed of 24 lipids molecules.

Two GROMCS modules, gmx editconf and gmx genconf, were used to translate and rotate the lipids. The

angular distance between lipids on a ring was 15 degrees, equals to 360 degrees divided by the number of

lipid molecules per ring (i.e. 24). The distance between the adjacent rings were 1.2 nm, to prevent bad

contacts in the initial structures.

Given these pure cylinders for each lipid type, the lipid tube with the desired mixing ratio was

constructed as described for bilayers in Section 2.5.1. Next, a water cylinder of the same dimensions and

orientation was built using CHARMM-GUI Solvator module [45]. These two systems, i.e. the mixed lipid

tube and the water cylinder, were subsequently merged to give a water cylinder covered by lipids

[Figure 2.7]. Water molecules were gradually deleted so that the systems had 30 or fewer water per

lipids (nw) when desired. For the systems with hydration levels less than 20 and 10, the relaxed HII

systems with 20 and 10 water per lipid were used to remove the water from, respectively. A triclinic box

was employed for the simulations and periodic boundary conditions (PBC) were applied in all three

directions [Figure 2.7].

This system was energy minimized using the steepest descent algorithm and used for

equilibration and production runs using either semi-isotropic or anisotropic pressure couplings. Using

these pressure coupling methods, each system could further adjust its height and diameter, as

energetically favourable. When systems with more than one lipid cylinder was of interest, the lipid

cylinder [Figure 2.7-left] was replicated in X and Y directions as required.

53
Figure 2.7: Construction of the DSPS/KC2H/cholesterol (35/35/30) HII system.

Left) a single lipid tube composed of DSPS, KC2H and cholesterols, filled with water molecules. Red,

dark blue, and green spheres represent the nitrogen atom of DSPS, nitrogen atom of KC2H, and oxygen

atom in cholesterol molecules, respectively. Orange and light blue lines represent the DSPS and KC2H

tails, whereas the cholesterol body are colored in tan. Water represented as the cyan surface. Hydrogens

are not shown for clarity. Right) a HII system composed of such single water channel. Lipid cylinders are

arranged in a hexagonal geometry. Blue box represents the triclinic simulation box. System is periodic in

all X, Y, and Z directions. There are empty spaces (or voids) in the corners in the initial structures. During

the equilibration step, both the box size and lipid tails are adjusted in a way to fill these gaps. VMD [57]

was used to make the figures.

54
 Given the background information and methodology provided in Chapters 1 and 2, the next

chapter presents a comprehensive review on computational nanomedicine field of study (Chapter 3).

Next, the results for three research studies are presented in Chapters 4, 5, and 6. Finally, the conclusions

and future studies are discussed in Chapter 7.

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Structural Biology, 16(2), 142-151.

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issues. Journal of Computational Chemistry, 25(13), 1584-1604.

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M.J., Fischer, S., Gao, J., Guo, H., Ha, S. & Joseph-McCarthy, D. (1998). All-atom empirical

potential for molecular modeling and dynamics studies of proteins. The Journal of Physical

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OPLS all-atom force field on conformational energetics and properties of organic liquids. Journal

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Huber, T., Krüger, P. & van Gunsteren, W. F. (1999). The GROMOS biomolecular simulation

program package. The Journal of Physical Chemistry A, 103(19), 3596-3607.

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dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature.

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atom force field for phosphatidylcholine lipids. The Journal of Physical Chemistry B, 116(10),

3164-3179.

[32]. Jämbeck, J. P., & Lyubartsev, A. P. (2012). An extension and further validation of an all-

atomistic force field for biological membranes. Journal of Chemical Theory and Computation,

8(8), 2938-2948.

[33]. Dickson, C. J., Madej, B. D., Skjevik, Å. A., Betz, R. M., Teigen, K., Gould, I. R., & Walker, R.

C. (2014). Lipid14: The Amber lipid force field. Journal of Chemical Theory and Computation,

10(2), 865-879.

[34]. Team, I. (2004). Inkscape: A vector drawing tool. URL http://www. inkscape. org.

[35]. Pluhackova, K., Kirsch, S. A., Han, J., Sun, L., Jiang, Z., Unruh, T., & Böckmann, R. A. (2016).

A critical comparison of biomembrane force fields: Structure and dynamics of model DMPC,

POPC, and POPE bilayers. The Journal of Physical Chemistry B, 120(16), 3888-3903.

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[36]. Lee, J., Cheng, X., Swails, J.M., Yeom, M.S., Eastman, P.K., Lemkul, J.A., Wei, S., Buckner, J.,

Jeong, J.C., Qi, Y. & Jo, S. (2015). CHARMM-GUI input generator for NAMD, GROMACS,

AMBER, OpenMM, and CHARMM/OpenMM simulations using the CHARMM36 additive

force field. Journal of Chemical Theory and Computation, 12(1), 405-413.

[37]. Ingólfsson, H. I., Arnarez, C., Periole, X., & Marrink, S. J. (2016). Computational ‘microscopy’of

cellular membranes. Journal of Cell Science, 129(2), 257-268.

[38]. Lyubartsev, A. P., & Rabinovich, A. L. (2016). Force field development for lipid membrane

simulations. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1858(10), 2483-2497.

[39]. Poger, D., Caron, B., & Mark, A. E. (2016). Validating lipid force fields against experimental

data: Progress, challenges and perspectives. Biochimica et Biophysica Acta (BBA)-

Biomembranes, 1858(7), 1556-1565.

[40]. Zhuang, X., Dávila-Contreras, E. M., Beaven, A. H., Im, W., & Klauda, J. B. (2016). An

extensive simulation study of lipid bilayer properties with different head groups, acyl chain

lengths, and chain saturations. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1858(12),

3093-3104.

[41]. Klauda, J.B., Venable, R.M., Freites, J.A., O’Connor, J.W., Tobias, D.J., Mondragon-Ramirez,

C., Vorobyov, I., MacKerell Jr, A.D. & Pastor, R. W. (2010). Update of the CHARMM all-atom

additive force field for lipids: Validation on six lipid types. The Journal of Physical Chemistry B,

114(23), 7830-7843.

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Comparison of multiple Amber force fields and development of improved protein backbone

parameters. Proteins: Structure, Function, and Bioinformatics, 65(3), 712-725.

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energetics in protein force fields: Limitations of gas‐phase quantum mechanics in reproducing

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E. (2010). Improved side‐chain torsion potentials for the Amber ff99SB protein force field.

Proteins: Structure, Function, and Bioinformatics, 78(8), 1950-1958.

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interface for CHARMM. Journal of Computational Chemistry, 29(11), 1859-1865.

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polarizable atomic models based on ab initio target data. Journal of Chemical Theory and

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[48]. Vanommeslaeghe, K., Raman, E. P., & MacKerell Jr, A. D. (2012). Automation of the

CHARMM General Force Field (CGenFF) II: Assignment of bonded parameters and partial

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Guvench, O., Lopes, P., Vorobyov, I. & Mackerell Jr, A. D. (2010). CHARMM general force

field: A force field for drug‐like molecules compatible with the CHARMM all‐atom additive

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 Chapter Three: Computational and Experimental Approaches for Investigating

Nanoparticle-Based Drug Delivery Systems

Copyrights:

This research was originally published in the Journal of Biochimica et Biophysica Acta (BBA)-

Biomembranes. Ramezanpour, M., Leung, S. S. W., Delgado-Magnero, K. H., Bashe, B. Y. M.,

Thewalt, J., & Tieleman, D. P. (2016). Computational and experimental approaches for investigating

nanoparticle-based drug delivery systems. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1858(7),

1688-1709. Copyright © 2018 Elsevier.

Contributions:

I had a major contribution in writing this review article: most of the computational sections, as well as the

abstract and part of the introduction. I created all the figures in this manuscript, except the ones taken

(with permission) from cited publications. The section on experimental techniques and studies, as well as

part of the introduction section were provided by Dr. Jenifer L. Thewalt, Dr. Sherry S.W. Leung, and

Bashe Y.M. Bashe at the Simon Fraser University. The section on lipid-based delivery systems was

provided by Karelia H. Delgado-Magnero at the University of Calgary.

Abbreviations:

5-FU, 5-fluorouracil; AuNPs, gold nanoparticles; BSA, bovine serum albumin; CD, cyclodextrin; CG,

coarse-grained; CNP, carbon-based nanoparticle; CNT, carbon nanotube; CPe, inverse-

phosphatidylcholine; CPNT, cyclic peptide based nanotube; CPP, cell penetrating peptide; DDS, drug

delivery system; DLS, dynamic light scattering; DOX, doxorubicin; DPD, Dissipative Particle Dynamics;

62
DPPC, dipalmitoylphosphatidylcholine; DSC, differential scanning calorimetry; ESR, electron spin

resonance spectroscopy; FT-IR, Fourier transform-infrared spectroscopy; gamma-PGA, poly(gamma-

glutamic acid); HSA, human serum albumin; IDP, intrinsically disordered proteins; IFN, interferon alpha-

1b; IR, infrared; ITC, isothermal titration calorimetry; LD, laser diffraction; LNP, lipid nanoparticle; MD,

Molecular Dynamics; NMR, nuclear magnetic resonance spectroscopy; NP, nanoparticle; p-THPP,

5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin; PAMAM, poly(amido amine); PEI, polyethylenimine;

PPI, poly(propylene imine); QD, quantum dot; SAXS, small angle X-ray scattering; SCPs, star

copolymers; SEM, scanning electron microscopy; SSMs, sterically stabilized micelles; TAT, trans-

activator of transcription; TEM, transmission electron microscopy; VIP, vasoactive intestinal peptide;

WAXS, wide angle X-ray scattering; XRD, X-ray diffraction.

3.1 Abstract

Most therapeutic agents suffer from poor solubility, rapid clearance from the bloodstream, a lack of

targeting, and often poor translocation ability across cell membranes. Drug/gene delivery systems (DDSs)

are capable of overcoming some of these barriers to enhance delivery of drugs to their right place of

action, e.g. inside cancer cells.

In this review, we focus on nanoparticles as DDSs. Complementary experimental and

computational studies have enhanced our understanding of the mechanism of action of nanocarriers and

their underlying interactions with drugs, biomembranes and other biological molecules. We review key

biophysical aspects of DDSs and discuss how computer modeling can assist in rational design of DDSs

with improved and optimized properties. We summarize commonly used experimental techniques for the

study of DDSs. Then we review computational studies for several major categories of nanocarriers,

including dendrimers and dendrons, polymer-, peptide-, nucleic acid-, lipid-, and carbon-based DDSs, and

gold nanoparticles.

63
3.2 Introduction

In medicine, the delivery of a drug can be as important as the drug itself. Physiology poses key challenges

to effective drug delivery; an administered drug must penetrate obstacles such as endo- or epithelial

membranes and also survive the host's defenses in order to be effective. Addressing such challenges

requires some form of drug encapsulation, and the entity forming the capsule, which has a defined

molecular architecture, is known as a "drug delivery system (DDS)." From a functional point of view, an

ideal DDS is easy to administer, non-toxic, carries the drug to its desired destination, and then releases it

[Figure 3.1] [1, 2]. Practically, an ideal DDS is cheap and straightforward to make, as well as stable

prior to its administration. Intense research into DDS design is providing increasingly effective disease

treatment.

A central aim for specialized DDS development is the optimization of tiny drug encapsulation

vehicles known as nanoparticles (NPs). NPs typically have diameters in the range of 10 to 100 nm [3].

These small DDS can circulate freely even in capillaries [4], and are intrinsically better at traversing

biological barriers than larger DDS. It is worth mentioning that nanomedicines, however, have a more

complicated and time-consuming FDA approval process than parent unimolecular therapeutics [5]. In

addition to efficacy and side effect evaluations common between both small molecules and

nanomedicines, there are other concerns about nanocarriers which need further evaluation. Nanocarrier

aggregation in vivo and their drug release, as well as evaluation of each component of the formulation are

some of those concerns. FDA regulations for nanomedicines, the complexity of nanocarriers, and the

prospect of only a small increase in performance upon reformulating small drugs in nanocarriers can

discourage pharmaceutical companies from investing in these systems. In fact, it seems that investigations

into DDS are much more successful in generating papers than in developing new treatment methods [5].

64
However, considering the number of approved drugs and current ones in clinical trials [6, 7], as well as

clearer guidelines for nanomedicine approval by the FDA, there is growing momentum in transferring

these systems from publication to clinical development [5].

Figure 3.1: Drug delivery pathway for a drug-nanocarrier system.

After the DDSs enter the bloodstream (1), drug (green circle) leakage might occur if the drug-DDS

complex is unstable (2). Depending on the surface chemistry, DDSs might interact with biomolecules (3)

and ions (4) in circulation. Eventually, they will extravasate to the extracellular space (5). After reaching

the target cell, they interact with the cell membrane (6) and can be internalized either via direct

penetration (7) or endocytosis (8). In the latter case, the nanocarrier will be entrapped in endosomes and

must be released (9) before endosomal maturation. Drug-DDS dissociation (10) is necessary for drugs to

65
be effective in the cytoplasm or nucleus (11). This dissociation can be mediated by interactions with

endogenous biomolecules (12). Not drawn to scale.

Many different types of NPs exist, including liposomes [7, 8], dendrimers [9-11], carbon

nanotubes (CNTs) [12-14], inorganic [15-20], and polymer-based [21, 22] NPs, with each having its own

unique properties [Figure 3.2]. Physicochemical properties including size, shape, deformability, surface

charge and chemical composition affect how well they evade phagocytosis and how they interact with

vasculature, traverse cell membranes and escape from endosomes prior to drug degradation [1]. Thus,

physical characterization of NPs is an important step in the DDS design process.

Figure 3.2: Examples of nanoparticulate DDS.

From top-left to bottom-right, monolayer-protected gold nanoparticle, polymeric micelle, DNA cage,

carbon nanotube, solid lipid nanoparticle, polymer, dendrimer and dendron, liposome, and protein-based

66
DDS. For dendrimer: orange, green, red, and blue represents G0, G1, G2, and G3, respectively. Dendron

has also been shown as black branch. Not drawn to scale.

Despite the increasing sophistication of experimental efforts to measure, design and optimize the

structure and dynamics of NPs, such research inevitably faces intrinsic and practical limitations.

Resolution of structural details can be difficult or impossible, and systematic variation of properties such

as composition, size and surface charge can be prohibitively time consuming and expensive. As a result,

general biophysical principles connecting the effectiveness of a DDS with its composition are difficult to

elucidate. Predicting optimum DDS design solely on the basis of experimental research is therefore

unlikely. Another challenge in DDS development is that many DDSs show promise in vitro but fail in

vivo [6]. This is mainly because of the lack of mechanistic insight obtainable by experiments which are

based on trial and error. Theoretical methods, both analytical and computational, can allow primary

screening of variables in order to predict suitable conditions for further experiments [23]. Computer

modeling techniques provide detailed information about molecular interactions and other

physicochemical properties of drugs and carriers [Table 3-1] and have found broad applications in

biology, biochemistry, and biophysics [24].

Table 3-1: Questions relevant to drug delivery system design that can be answered by

computational simulations.

Drug Delivery Step Properties of Interest

Physicochemical characterization Self-assembly of DDS components

67
 Structure and dynamics of drug-DDS complex

Drug loading

Drug distribution

Drug-DDS interactions

Functionalization

Drug leakage

Aggregation

Circulation Interaction with biomolecules

Stability

Interaction with ions

Cell-membrane Interactions with membrane

Cellular internalization

Interaction with membrane proteins

Intracellular Drug-DDS dissociation

Drug release from endosome

Interaction with endogenous molecules

Nucleus translocation

Computer simulation is capable of complementing experiments and assisting in the rational

design of new formulations with improved efficacies. In this chapter, we describe the main experimental

approaches to characterize NP-based DDSs and focus in more detail on current applications of computer

simulations aimed at understanding molecular aspects of DDSs.

68
 In the next sections, we first give a brief overview of commonly used experimental techniques,

followed by a section that describes several common computational approaches. Section 3.5 highlights

recent computational studies of what currently are the major classes of NP-based DDSs. We conclude

with a brief outlook on the role of computer simulations in drug delivery technology.

3.3 Commonly Used Experimental Techniques for Nanoparticle Characterization

Here we provide a brief summary of techniques regularly used to characterize NP size, zeta potential,

surface morphology, and the existence of colloidal structures [Table 3-2]. We first describe the

importance of each of these properties and the experimental techniques used to measure them. Next, we

describe some sophisticated physical chemistry tools that are used in DDS development to provide data

that can parameterize and validate computational simulations. Experiments used to visualize cellular

uptake, NP-cell interactions, as well as those used to determine cell viability will be omitted, even though

they are vital to pharmaceutical development and are also commonly performed, since details on these

can be found elsewhere (e.g. [25]).

Table 3-2: Experimental methods used to study drug delivery systems.

Experimental Abbreviation Properties

Technique

Dynamic light scattering DLS Particle size, aggregation

Laser diffraction LD Particle size, aggregation

Transmission electron TEM Particle size, dispersity, shape, surface morphology

microscopy

69
Scanning electron SEM Particle size, dispersity, shape, surface morphology

microscopy

Atomic force microscopy AFM Particle size, dispersity, shape, surface morphology

Fluorescence FLIM Drug release

Zeta potential - Surface charge

Gel electrophoresis - Encapsulation efficiency of nucleic acids

Ultraviolet-visible light UV-vis Encapsulation efficiency

spectroscopy

Fourier transform-infrared FT-IR Bond vibrations

light spectroscopy

X-ray scattering SAXS and WAXS Repeat spacing, chemical connectivity

Neutron scattering - Crystallinity

High performance liquid HPLC Separate, identify and quantify components in mixture

chromatography

Nuclear magnetic NMR Crystallinity, molecular mobility, chemical

resonance spectroscopy connectivity

Electron spin resonance ESR Carrier fluidity, gadolinium concentration,

spectroscopy microviscosity, micropolarity

Fluorescence spectroscopy Viscosity, polarity, concentration, drug release

Differential scanning DSC Phase transitions

calorimetry

Isothermal calorimetry ITC Phase transitions

70
 Particle size is an important property because the size of the carrier can affect circulation time,

encapsulation efficiency, and cellular uptake [1, 26, 27]. For example, in the case of cancer treatment,

nano-drug carriers of the appropriate sizes can extravasate from the bloodstream to tumor tissues (380 –

780 nm) [1, 28], but not from the tighter capillaries (50 – 200 nm) in healthy tissues [29]. This contributes

to the well-known enhanced permeability and retention effect [30-33]. Light scattering techniques such as

laser diffraction (LD) and dynamic light scattering (DLS) can be used to measure NP size [34]. In both of

these experiments, a laser beam is directed at a dilute sample (e.g. a suspension or solution of NPs). In

LD, the diffraction angle is used to determine the particle size [35]. In DLS, the time dependence of the

scattered light intensity is detected at a known scattering angle. The Brownian motion of suspended

particles causes the scattered light intensity to fluctuate. These fluctuations are correlated with the

particle’s velocity and can be used to determine particle size via an appropriate theoretical model (e.g.

Stokes-Einstein equation). Most models assume the particles are spherical and monodisperse; artifacts

may arise if they are not. DLS can be used to measure the size of particles in the 2 nm – 3 μm range, and

LD can be used in the 20 nm – 2 mm range [34, 36]. Resolution of small differences in particle size can

be improved by combining light scattering techniques with a separation technique, such as field flow

fractionation [37, 38].

Microscopy techniques such as transmission electron microscopy (TEM), scanning electron

microscopy (SEM), and atomic force microscopy can be used to measure NP size, as well as size

distribution (dispersity), shape, and surface morphology. Dispersity – also known as polydispersity in

older literature – in a formulation can result in varying body-residence time and immunogenicity [39, 40].

Particle shape can affect cellular uptake mechanisms and kinetics [41-45] as well as margination

dynamics – the lateral drift of NPs to endothelial walls [1]. The particle surface is the first point of contact

between the NP and its environment, and surface morphology is known to affect drug release kinetics

[46]. In electron microscopy, an electron beam is directed at the sample, and differences in electron

71
density in the sample gives contrast to structures. Since electron microscopy can give high-resolution

images approaching molecular resolution, all motions on the supramolecular scale have to be halted. For

SEM, the NPs are deposited onto a carbon conductive tape, dried, and covered with a thin layer of metal

[47]. In cryo-TEM, samples are applied onto an electron microscopy grid, plunge-frozen rapidly in ethane

to prevent the formation of damaging cubic ice and then imaged under cryogenic conditions to prevent

radiation damage to the sample during imaging [48]. Magnifications on the order of 50,000 ×, with a

resolution of 0.3 nm, are possible [49]. Atomic force microscopy probes surface terrain using a nanoscale

mechanical probe with a vertical resolution as small as 0.01 nm [35].

A NP’s shape can influence the physiological fate of its cargo [1]. Particle shape can be an

indication of undesirable aggregate structures. Aggregate structures can also cause inaccurate particle size

determination by LD or DLS [34]. Light microscopy can be used for determining if microparticles are

aggregates of smaller particles [36, 50], even though it cannot be used for directly observing NP structure

due to limitations in optical resolution (~ 200 nm). Isothermal titration calorimetry (ITC), which will be

described in greater detail below, has been used to study aggregate formation in polycationic nucleic acid

carriers [51]. Aggregation of NPs can be prevented via electrostatic repulsion [31, 52]. Surface charge

measurements can be a predictor of aggregation behavior, and consequently a predictor of colloidal

dispersion stability during storage.

Surface charge can affect cellular uptake, cytotoxicity, and circulation times [1, 31, 53, 54]. Zeta

potential is used to characterize the surface charge of NPs. When a particle moves in a liquid, a thin layer

of liquid moves with it. The zeta potential is defined as the electrical potential at the boundary of this

moving liquid, defined as the shear plane. It is a function of surface charge density, shear plane location,

and surface structure [55]. Zeta potential is determined by measuring light scattering caused by particle

motion in an applied electric field. Charged particles with larger zeta potential (|zeta| > 25 mV) are less

likely to form aggregates but neutral particles generally have longer circulation times [56]. Surface charge

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effects are very complex; positive and negative zeta potentials correlate with higher cellular uptake in

non-phagocytic and phagocytic cells, respectively [54]. Future DDS designs may require NPs to switch

zeta potential at the target site to maximize circulation time while targeting a particular type of cell [1]. It

should be noted that protein adsorption in cellular media can change the surface charge [52]. Conclusions

on surface-charge effects, therefore, are only valid when comparing functionalized or non-functionalized

particles of similar sizes [54].

Encapsulation efficiency – the ratio of encapsulated drug to the amount of drug used during

formulation preparation – is an important parameter [46]. Low encapsulation efficiency implies that more

carrier material is needed, heightening the risk of carrier material toxicity [2, 57]. Encapsulation

efficiency can be determined in a number of ways, depending on the particular type of NP. Drug loading

can be determined by weight [16]. Gel electrophoresis has been used to determine encapsulation

efficiency by analyzing protection from RNase degradation in siRNA lipid nanoparticles

(LNPs). Physical chemistry techniques can also be used to identify and quantify the degree to which

components have been encapsulated. For example, fluorescence spectroscopy can be used if the drug is

fluorescent [16]. Ultraviolet-visible (UV-Vis) light spectroscopy can be used to determine the amount of

cargo (e.g. siRNA, anticancer drug doxorubicin (DOX)) present [58, 59]. High performance liquid

chromatography can be used to separate, identify, and quantify the components of a mixture [46, 60].

Novel single molecule measurements have been applied to measure encapsulation efficiency in individual

lipid vesicles [61].

An array of physical chemistry techniques has been instrumental in evaluating the chemical and

physical properties of NP components. Dendrimer structures have been simulated based on experimental

data from nuclear magnetic resonance spectroscopy (NMR) and X-ray [62]. LNP matrix state,

polymorphism, and phase behavior have been characterized by differential scanning calorimetry (DSC),

X-ray diffraction (XRD), and neutron scattering [63]. Lipid crystallinity of solid LNPs has been studied

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using X-ray scattering, DSC, NMR, and electron spin resonance spectroscopy (ESR) [64]. CNTs have

been characterized using XRD, UV-Vis spectroscopy, Fourier transform-infrared spectroscopy (FT-IR),

ESR, SEM, and energy dispersive X-ray diffraction [65]. Iron oxide NPs, which are magnetic, can be

studied using magnetometry, NMR relaxation dispersion profiles [65], and magnetic field flow

fractionation [66]. Some more common physical chemistry tools will be briefly described here.

In optical spectroscopy, the absorption of electromagnetic radiation by NPs occurs at frequencies

characteristic of the molecular structures present. UV-Vis light is absorbed if it supplies the right amount

of energy to excite valence electrons, while infrared (IR) light is absorbed if it excites bond vibrations.

FT-IR is a more efficient variant of IR spectroscopy where all frequencies are sampled simultaneously. It

has been used to monitor cholesterol-induced changes in liposomal membrane structure [67] and to verify

conjugation of block copolymers onto iron oxide NPs [68]. UV-Vis spectroscopy has been used

extensively in NP characterization. For example it was used to confirm the presence of cleavable disulfide

bond in CNT-nucleic acid conjugates [69]; monitor the metal to ligand charge transfer band (5d(Pt11) to

π*(diimine)) in dendrimer synthesis [70]; measure dye and drug encapsulation in poly(lactic-co-glycolic

acid) polymeric carriers [71, 72] and study drug release kinetics of liposomal formulations [67].

NMR and ESR both rely on spin manipulations in the presence of an applied external magnetic

field. NMR monitors the nuclear spin of nuclei with non-zero nuclear spins, and ESR monitors the

electron spin of excited unpaired electrons [73]. NMR can provide a wealth of information on molecular

arrangements. NMR is sensitive to small changes in the local environment: it can be used to differentiate

between various layers up to the fourth generation in dendrimers [74], and between neighboring carbon

atoms in lipids [75]. 2D NMR techniques can be used to ascertain chemical connectivity (e.g. COSY,

HMQC, HSQC) and distances (e.g. NOESY and ROESY) between specific atoms [74, 76, 77]. Dynamic

information is also accessible to NMR. Pulsed field-gradient spin echo 1H NMR and diffusion-ordered

spectroscopy have been used to determine diffusion coefficients in aliphatic polyester and poly(propylene

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 31
imine) (PPI) dendrimers [74, 78]. P NMR has been used to determine the mobility of siRNA

encapsulated in LNPs [79]. NMR relaxation was used to monitor molecular tumbling activities [80] and

this information, in turn, was used to determine the location of a spin-labeled anticancer drug in micelles

formed by PEG modified with long alkyl groups and of paclitaxel in cyclodextrin (CD) vesicles [77, 80].

Most DDSs are invisible to ESR, requiring the addition of paramagnetic spin probes [81]. Lipophilic ESR

probes have been used as model lipophilic drugs to help determine where these drugs localized in solid

LNPs [35]. In gadolinium poly(amido amine) (PAMAM) dendrimer containing spin probes, ESR was

used to determine the location and concentration of the magnetic resonance imaging contrast agent

gadolinium [78]. ESR has also been used to prove that the terminal interbranch H-bonded groups preclude

backfolding in PAMAM dendrimers [78]. Since ESR can probe membrane fluidity, ESR has been used to

study interactions between polymeric micelles and lipid membranes, which are models for cell

membranes [68]. ESR can also give microviscosity and micropolarity information [81]. Viscoelastic

properties of dispersions can affect DDS interactions with the body. For example, capillary blockage can

occur with solid LNPs, which are less deformable than nano-emulsions [64].

Scattering techniques can be used to study the structure of colloidal systems. In scattering studies,

a monochromatic beam of light, neutrons, or X-ray is focused on the sample, and the intensity of the

scattered beam is measured. Light scattering was covered earlier in this section. X-ray scattering results

from variations in electron density and neutron scattering results from variations in the spatial distribution

of atomic nuclei [82]. The interference pattern formed by the scattered beam can be used to determine

characteristic lengths using Bragg’s Law. Larger angle measurements contain information for shorter

length scales: small and wide-angle X-ray scattering (SAXS and WAXS) are used to look at liquids and

solids with structures on the length scale of 1-200 nm and sub-nm, respectively. For example, SAXS is

used to monitor monolayer and bilayer repeat spacing, and hydrophobic thickness in lipid membranes,

while WAXS is used to determine chain packing and extent of lipid motion [83, 84]. SAXS has been used

75
to study NPs with cubosome structures [85, 86], to provide micelle shape, size and density of nonsteroidal

anti-inflammatory drug celecoxib-loaded protein micelles [50], and to observe in situ shell growth of

polymeric nanocapsules [87]. WAXS has been used to study lipid polymorphic transformations in solid

LNPs and crystallinity of encapsulated drugs [88], and crystallinity of polyelectrolyte complexes made

with polysaccharides [89]. SANS, useful for studying structures of 1-100 nm [90], has been used to

determine molecular weight and end group locations of dendrimers [74]. Neutron scattering can also be

used to study drug diffusion and internal molecular motions in LNPs [63].

Fluorescence comprises a family of spectroscopy and microscopy techniques relying on

excitation of fluorescent species. An extrinsic fluorescent probe is added when no fluorescent moieties are

present in the material of interest. Absorption of a photon excites the fluorophore and fluorescence occurs

when a photon is later emitted. Between excitation and emission, the fluorophore’s motions and local

environment (e.g. viscosity, polarity, temperature, pH, ion concentration) influence the fluorophore’s

spectrum [91]. Polarity sensitive fluorescent probes are commonly used to determine the critical

association concentration of polymeric micelles – analogous to the critical micellar concentration of

surfactant micelles [92]. Fluorescence quenching assays have been done to evaluate nucleic acid-

polycation binding in polycationic nucleic acid carriers [51] and dye leakage assays have been used to

evaluate drug release characteristics [93].

Thermal analysis is a versatile tool for DDS characterization [94, 95]. DSC measures enthalpy

changes – the heat required for a sample to undergo a physical phase transition (e.g. glass transition,

melting, decomposition, isomerization or heats of solution, water sorption-desorption) [95, 96]. DSC was

used to monitor the physical state of the antineoplastic drug paclitaxel inside polymeric microspheres, for

example [46]. More specific to DDS design, DSC has been used to measure the gel-to-liquid crystalline

transition temperature of lipids in liposomal formulations, which can be used to predict release rates of

encapsulated drugs [95, 97]. In a closely related technique, ITC, the evolved heat is measured as

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concentrated aliquots of one substance are added to a solution of a second substance. The released heat is

directly proportional to the amount of substance added, and can be used to measure enthalpy changes,

stoichiometry, and binding constants. ITC has been used to measure cyclodextran-guest molecule

interactions, micelle-drug interactions, and polyelectrolyte aggregation [98].

NP DDSs are complex systems that can evolve over time. Dynamic processes cannot be captured

in steady-state measurements and can pose as a challenge to characterization [35]. For example,

PEGylated NPs are known to lose their PEG coating before reaching target cells [99-101]. Time-resolved

DLS combined with TEM was used to investigate morphology transition kinetics of multiblock

copolymer micelles [102]. Time-resolved fluorescence lifetime measurements have been used to follow

the release of fluorescent compounds from polymeric carriers [71], and fluorescence lifetime imaging

microscopy could extend drug release studies to cellular systems [103, 104].

3.4 Computational Approaches to Study Nanoparticles

Computer simulations in general describe the physics of materials at a suitable level of detail for a

particular application. A simulation is described by the level of detail in the physics used to model the

system of interest, and the algorithms used to generate enough simulation data to draw statistically valid

conclusions about the behavior of the system of interest. NPs can be described at different levels of detail

[Figure 3.3], but for most of the applications in this review the appropriate levels of detail are limited to

atomistic and semi-atomistic, slightly coarser, levels.

At the highest level of detail, a system is described by quantum mechanics in one of its

approximations. Quantum mechanical calculations have been widely applied to CNTs and play a role in

optimizing less detailed simulations, but in practice they are only useful for small systems with limited

degrees of freedom and generally not in solution. They are essential to correctly model electronic

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properties and can be used to calculate electrical, optical, magnetic and mechanical properties of CNTs,

quantum dots (QDs), magnetic NPs and similar systems. For soft-condensed matter systems such as

liposomes or dendrimers in solution their usefulness is currently limited.

If we average over electronic properties by representing electrons as partial charges on atoms

rather than taking them into account explicitly, we significantly simplify calculations. At this level of

detail, atoms are described as point charges that interact through simplified empirical potential terms

including Coulomb interactions, Van der Waals interactions, and simplified terms describing bonds,

angles, and dihedrals as harmonic terms and cosine expansions [105]. The resulting technique is called

Molecular Dynamics (MD) and is widely used for biological and soft-condensed matter systems. The vast

majority of the papers reviewed below use this approach, which accounts for a substantial fraction of

super computer time on the worlds’ major academic computer centers. MD is a mature technique that is

implemented in a number of widely-used software packages, including GROMACS [106], NAMD [107],

LAMPPS [108], CHARMM [109], and AMBER [110]. MD simulations essentially generate a movie of a

set of molecules over time, incorporating every degree or nearly every degree of freedom, from which

through statistical mechanics thermodynamic properties can be calculated. Its main limitations include the

computational cost and the corresponding limited time scale (realistically, microseconds in most cases)

and length scale (~10 nm per dimension) and the limitations implicit in the physics that describes the

interactions between atoms: point charges mean electronic effects like polarization, pi-electron clouds

possibly important for CNTs, and charge transfer are typically ignored. In addition, the simplified

interaction function sometimes imposes other limitations.

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Figure 3.3: Categories of simulation methods and their respective spatio-temporal domains of

applicability.

The question of interest dictates the level of resolution required. Briefly, if electronic motion play an

important role in the property we are studying, quantum mechanics (QM) level is appropriate. However,

if there is no bond formation and cleavage, all atom (AA) level works well; we can safely ignore

electronic motion by assigning point charges to each atom. If electrostatic and hydrophobic interactions

are the dominant contributors, coarse grain models (CG) can be used: individual atoms can be ignored

and a group of atoms (e.g. 4 heavy atoms in MARTINI) can be treated as one interaction point/bead. CG

models allow the exploration of a larger area in phase space, at the expense of losing atomistic details.

Coarser levels of simulations (e.g. DPD and continuum models) are appropriate for systems and processes

which require longer times and lengths.

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 For many properties of interest, especially in mesoscopic-scale systems like NPs with a size of

20-200 nm, atomistic detail may not always be necessary. Many papers below use coarse-grained (CG)

simulations, often based on the MARTINI model [111]. Here CG means that several atoms have been

grouped into interaction sites, which are no longer recognizable as atoms but instead are ‘beads’ that

represent molecular fragments. Depending on the level of coarse-graining, these beads can still maintain a

significant amount of chemical specificity, or they can represent very generic properties such as those that

represent a lipid by one head group bead and two tail beads. The same physics applies as in atomistic

simulations, and sufficient conformations of a system of interest have to be generated by a sampling

algorithm to accurately calculate thermodynamic and structural properties. This can be done by MD,

Monte Carlo, or various other algorithms including Langevin dynamics or Dissipative Particle Dynamics

(DPD) [105].

At the CG level, individual molecules and usually fragments of molecules are still clearly

recognizable. In material science, there exists a whole hierarchy of additional models. Beyond molecules,

DPD solves hydrodynamics equations for materials by representing materials as discrete particles that can

be much larger than individual molecules. DPD blurs the line with CG MD a little by its choice of particle

volume: if the volume is chosen to coincide with molecules or molecular fragments its resolution is

similar to CG MD, although it typically treats problems that are more generic. Beyond DPD there are

many other possibilities, including field-based treatments where a system is described by density fields.

Simulations coarser than DPD are beyond the scope of this review.

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3.5 Computational Studies on Drug and Gene Delivery Systems

Computer modeling, as complementary tools to experiments, is excellent in shedding light into the

structural and dynamical properties of systems of interest at atomistic or molecular levels of detail [112,

113]. Applied to DDSs, computer simulations have been used to address a broad range of questions

[Table 3-1]. Computer simulations can be used to study self-assembly [114-116], the structural and

dynamical characteristics of the resulting aggregates [117, 118], drug loading capacity, mechanism and

rate [119-121], drug distribution/localization in DDS [121], complex stability [118, 122], drug retention,

release mechanism and release rate [123-125], dominant drug-DDS interactions [119, 126, 127], and to

design or optimize DDSs targeting capabilities [128, 129]. Environmental conditions, e.g. pH,

temperature, salt type and concentration, counterions [126, 130], and external stimulus such as external

magnetic fields [131-133], as well as interactions with other biomolecules (e.g. serum proteins, miRNA,

heparin), all might affect the aforementioned aspects of DDSs [126, 134-137] and can be studied

computationally.

The way DDSs interact with cell membranes is one of the most commonly studied steps in drug

delivery by computational studies [138, 139]. Computer modeling can investigate the driving forces for

NP-membrane interactions, as well as how factors such as design parameters and environmental

conditions affect these. These parameters include size, shape, surface chemistry, and concentration of

NPs, as well as mechanical and elastic properties of both NPs and membranes [138-143]. Surface

chemistry refers to NPs hydrophobicity/hydrophilicity, charge density and distribution, as well as coating

ligand’s length, grafting density and distribution, rigidity, and their affinity to receptors [144-147]. Since,

in real systems the membrane often interacts with more than one NP simultaneously, NP aggregation and

interaction of multiple NPs with the membrane also is a relevant factor [138, 139]. Membrane properties

such as lipid phase, membrane composition, surface tension, charge density, receptor types and density

81
also play important roles [148-150]. In addition to all of these, external macromolecules, e.g. proteins in

the bloodstream, and different environmental conditions in different cell types [134], as well as

differences between external and internal cellular environments, are also of great importance and worth

further investigation. Of course, many of these factors are coupled and have to be taken into account

together in the design of NPs [138, 139].

NPs are usually functionalized by coating with polymers, lipids, or ligands, to increase their

stability, targeting capability, cellular uptake efficiency, and also to reduce their toxicity [131]. Both the

type of coating molecule and the coating pattern and density strongly affect the physicochemical

properties of these NPs, and as a result their interactions with their cargo and target lipid membranes

[147, 151-155]. Simulations can be used to study these coatings, how they affect NP properties including

their interactions with other molecules [152, 156], and can be used to optimize these coatings towards

DDS with improved delivery capabilities [151, 154]. Functionalization with PEG polymers, a process

called PEGylation, is a good example of functionalization [157, 158]. Drugs/proteins/nucleic acids and

NPs’ surfaces are usually PEGylated to improve their water solubility, stability, and circulation lifetime,

and to reduce their aggregation, toxicity, and immune response [157, 159].

Considering the importance of PEG and its wide usage in drug delivery applications, PEG is

worth further discussion. PEG is the most commonly used non-ionic polymer with stealth behavior for

drug delivery purposes [160]. These biocompatible hydrophilic polymers can solubilize hydrophobic

molecules and reduce renal clearance and toxicity of drugs and nanoparticles. They also inhibit the

aggregation of PEGylated agents by steric stabilization, as well as mask therapeutic agents from the host

immune system and consequently suppress immunogenicity and antigenicity. This protective polymer

layer, which is known as stealth sheath, can reduce the fast recognition by the immune system and reduce

the non-specific interactions with blood components, resulting in longer blood circulation times. These

properties have made it the gold standard polymer for drug delivery and other biomedical applications

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and have been the topic of several computational modeling studies reviewed in more detail below.

Despite the widespread use of PEG, there are several drawback to PEG including immunogenicity and

antigenicity, e.g. hypersensitivity reactions and the development of antibodies against PEGs [160], which

adversely affects pharmacokinetics [161], and the search for alternatives, e.g. poly(amino acids),

continues, in part aided by computer simulations.

In addition to different aspects of DDS mechanism, different types of DDS have been simulated.

There are many types of nanoparticulate DDS. DDSs based on polymers, peptides, nucleic acids, lipids,

carbon, dendrimers and dendrons, and gold have been investigated via computer modeling and are

described in more detail below. Combined, these applications show that computer simulation has become

a powerful technique for rational design and optimization of DDS and demonstrates the growing

importance of computational pharmaceutics.

For the rest of this chapter, we will present examples of computational studies on each aspect of

interest for each nanoparticulate DDS type in separate sections, and then comment on challenges in this

field. We will focus on atomistic, CG MD simulations, and DPD simulations of nanoparticulate DDSs;

these fall mainly in categories defined as dendrimers/dendrons, polymer-, protein/peptide-, nucleic acid-,

lipid-, carbon-based DDS, as well as gold nanoparticles (AuNPs). We emphasize studies published since

2010. There are computational studies on other DDSs that are outside the scope of this review, e.g.

graphene oxides and reduced graphene oxides [19, 162], silicon NPs [19, 163], nanodiamond [164, 165],

layered drug delivery carriers [166, 167], zeolites [168-170], metal-organic frameworks [171, 172], and

other porous materials. These are excluded because they are not NPs, there are limited computational

studies available, or because their focus is different from the studies described below.

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3.5.1 Dendrimers and dendrons.

Dendrimers are a class of branched globular materials with broad application in nanotechnology and

nanomedicine, including for drug/gene delivery [9-11]. Their monodispersity, modifiable surface, shell

and core characteristics, and their high loading capacity make them suitable carriers for therapeutic

agents, with promising applications in gene delivery through complex formation with nucleic acids, so-

called dendriplexes [173-177]. Drugs can be covalently or noncovalently incorporated in dendrimers to

overcome solubility problems and to improve targeting and cellular uptake. Dendrimers have been studied

in many computational papers to characterize their structure in solution, their interactions with drugs and

biomolecules [62, 175], and their response to external stimuli [178]. All parts of dendrimers can be

modified chemically; core, shell and surface. The surface can be optimized for different biological

activities by functionalizing the terminal groups. Thus, dendrimers are a flexible class of materials with

the potential for almost infinite variation.

Different dendrimers have been modeled including PPI, triazines and PAMAM. Simulations can

be used to investigate their interactions with other molecules, the effect of solvents and counter-ions, pH,

salt concentration, different generation number, the nature of terminal groups and chemical modifications.

Computer simulations have also been used to study deformation and conformational changes of

dendrimers upon interaction with nanopores [179], lipid membranes [180], and nucleic acids [181] as a

function of generation number, pH, functionalization, and ionic strength. For example, Nandy et al. [181]

simulated a 38 base pair double stranded DNA and three different generations of PAMAM dendrimer

(G3, G4, G5). They found that DNA caused deformations in lower-generation dendrimers (G3) while

higher generation PAMAM (G5) bent DNA [Figure 3.4]. Generation number, pH, and architecture have

been shown to affect dendron’s flexibility and rigidity, and as a result influence their binding efficiency to

84
nucleic acids [182, 183]. Several studies have investigated the effect of chemical modification on self-

assembly, conformation, and drug/gene binding [184-186].

Figure 3.4: PAMAM dendrimer-DNA interaction and deformation as a function of generation

number.

a) For the G3 dendrimer, very little deformation in DNA was observed, while the dendrimer was bent

considerably by the DNA. b) For G4, both DNA and dendrimer were deformed. c) For G5, DNA was

deformed, but the dendrimer behaved almost as a rigid body. Adapted with permission from B. Nandy,

P.K. Maiti, A. Bunker, Force Biased Molecular Dynamics Simulation Study of Effect of Dendrimer

Generation on Interaction with DNA, (2013). Copyright 2013 American Chemical Society.

Self-assembly of Janus dendrimers, comprised of hydrophobic and hydrophilic parts, has been

studied both experimentally and computationally. They form onion-like dendrimers and other complex

architectures [187, 188].

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 Computer simulation has been used to study how chemical modification and surface

functionalization (e.g. PEGylation, acetylation, folic acid groups, peptides, and targeting ligands of the

surface) influences the structure [189], and the interactions with cargos [190, 191]. Computer modeling

can also be used to determine the optimal grafting densities and patterns based on structural and drug-

loading properties, and interactions with target membranes [154, 192-195]. Effect of PEGylation size and

grafting density on the structure of dendrimers, and also the structure of the PEG layer itself have been

widely studied [154, 196-198]. PEGylation expands the core of dendrimers, effectively reduces their

surface charge density and increases the overall size of dendrimers [196]. The effect of PEGylation on

structure and drug loading capacity of PAMAM-G4 dendrimers was investigated by an all-atom MD

simulation [154]. Different PEG densities (25%, 50%, 75% and 100%), were tested for complexation and

loading capacity for 5-fluorouracil (5-FU), as a model anticancer drug. The simulations suggested that

25% PEG is the most suitable PEGylation density for drug delivery purposes. This grafting density can

retain the internal drug complexation capability, and also assist in retaining drugs. For higher PEGylation

degrees than 25%, 5-FU molecules to a high extent interact with external PEG chains than dendrimer

branches. Pearson et al. [193] used MD simulation to understand why PEGylated dendron-based

copolymers functionalized with different terminal groups, -NH2, -COOH, and –Ac, do not exhibit a

charge-dependent cellular interaction. Simulations suggest that interactions between positively charged

terminal groups with PEG molecules sequester the charges and cause this low level of cellular interaction

for NH2 terminated micelles.

Interactions between dendrimers with lipid membranes, permeation of dendrimers as a function

of generation number (size), protonation state, functionalization, and dendrimer concentration, as well as

the effect of bilayer asymmetry, bilayer tension, lipid tail length, and lipid phase (temperature) all have

been studied by simulations [124, 180, 195, 199-204]. Tian and Ma [124] studied interactions of G4-

PAMAM dendrimers with both symmetric and asymmetric negatively charged bilayers, with and without

86
tension, at both neutral and low pH. It was shown that both membrane tension and electrostatic

interactions between charged dendrimers and tensed membrane play important role in dendrimer

penetration through membrane. Based on their simulation results authors proposed a mechanism of

endosomal escape for pH-responsive gene delivery vectors [Figure 3.5]. Xie et al. [204] found that low

generations leave gel-phase membranes intact while higher generations fluidize the membrane and cause

a gel-fluid phase transition around the dendrimer binding site. Higher generation cationic dendrimers are

capable of membrane disruption in both low and high temperatures [204]. Functionalization can influence

these interactions since it affects the physicochemical properties of dendrimers [195].

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Figure 3.5: Proposed mechanism of endosomal escape for pH-responsive dendrimers.

In endosomes (pH ~ 5), dendrimers are protonated, leading to increasing osmotic pressure and dendrimer

swelling, putting the membrane under tension. Meanwhile, electrostatic interactions between charged

dendrimers and asymmetric negatively charged endosomal membrane cause a drop in the critical

membrane tension required for membrane disruption, allowing NP escape from endosomes. Bottom row:

snapshots of a MARTINI simulation of interactions between a G4 dendrimer (orange beads) and a

membrane (with lipid tails represented with cyan beads, and lipid headgroups represented with green and

blue beads) at different pH levels. Reproduced from [124] with permission from The Royal Society of

Chemistry.

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 Dendrimers can condense DNA and siRNA, and stabilize small molecules in their core, shell and

interface. Several studies have considered the loading, release, interactions and distribution of small

molecules in dendrimers [174, 205-213]. For example, Jain et al. [206] used all atom MD simulations and

molecular mechanics Poisson-Boltzmann surface area analysis to study the solubility and drug release

profile of the hydrophobic drugs famotidine and indomethacin in G5 ethylenediamine cored PPI

dendrimers under different pH conditions. They found fast drug release in low pH, intermediate values in

neutral pH and a slow release in high pH. These results and further simulations on the effect of dendrimer

chemistry and topology on drug loading and release suggest that both the pKa of the drug and pH-

dependent changes in the dendrimer influence these dendrimer-drug interactions and complex stability.

Loading and release of the antibiotic rifampicin, from G4-PAMAM dendrimer has been studied by Bellini

et al. [205] in a combined experimental and simulation approach. Experimentally, approximately 20 drug

molecules were found as the maximum potential loading capacity of this dendrimer at neutral pH. In

simulation, the model of the dendrimer with 20 drug molecules in neutral pH was more stable than in low

pH, where drug molecules got expelled rapidly and simultaneously to the solvent. This suggests a pH

dependent drug release desirable for drug delivery applications. Simulations can also be used to study the

effect of PEG lipids on loading capacity of dendrimers, and on surface ligand’s targeting capability [154,

211, 214].

Based on a comparison of several generations of PAMAM dendrimers, G4 PAMAM might be

optimal as vector for gene delivery, based on PAMAM interactions with bilayers and siRNA

complexation [203, 215]. For gene delivery applications, generation number (size), flexibility, hydrogen

bonding capability, pH, ion concentration and salt type have been considered in simulations [183, 216-

219]. Electrostatic interactions and the entropy gain from counterion release upon binding have been

89
proposed as important driving forces determining the interactions between dendrimers and nucleic acids

[181, 212, 213].

As one final example, we would like to design drug specific nanocarriers by including optimal

drug-binding molecules in the structure. Shi et al. [220] used de novo design of dendrimers via

optimizing building blocks and including optimal drug-binding molecules with a final goal the design of a

drug-specific nanocarrier. They combined virtual screening with molecular docking and MD simulations

on DOX-dendrimer complexes. Using Rhein, a molecule with strong DOX binding, they achieved Rhein-

containing nanocarriers with a suitable DOX profile.

3.5.2 Polymer-based delivery systems.

Polymeric NPs are of interest in drug delivery applications and more generally because of their stability

and easily modifiable surface [21, 31]. Block-copolymers [92] composed of hydrophobic and hydrophilic

parts can form a variety of self-assembled structures [221] of interest in drug delivery applications. These

structures, e.g. polymeric micelles [222], are capable of accumulating drug molecules in their core,

interface and shell [121]. There are several reviews of computational studies on different aspects or

specific type of polymers [23, 221, 223-225]. Here, we will discuss some examples.

Amphiphilic polymers form a variety of structures in solvents, e.g. core-shell, Janus particles,

micelles, and rod-like micelles [102, 226, 227]. Several factors affecting aggregate structures, stability

and phase transitions between different structures have been studied: for example, solvent polarity [130],

polymer architecture, concentration and physicochemical properties [226, 228, 229], mixture components

and mixing ratios of comprising polymers [230, 231], pH, temperature [226, 232], and the addition of

components, e.g. lipids, cholesterol and ligands [102, 233, 234]. Huynh et al. [122] performed all atom

MD simulations on star copolymers (SCPs) to understand why SCP micelles are unstable and form

multimolecular micelles in solution, and to assist in the rational design of stable SCP unimolecular

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micelles in solution. Each SCP has a central connecting part and six attached arms, with each arm

comprised of a hydrophilic part (PEG) and a hydrophobic one (PCL). These SCPs were different in their

PCL and PEG length and the resulting molecular weight. They form core-shell structures, in which PCL

blocks form a dense hydrophobic core while PEG blocks form a shell around the PCL core. The

simulations suggested that partial water exposure of the PCL core drives aggregation into multimolecular

micelles. Increasing PEG length protects the PCL core from exposure to the water but it also increases the

size of micelles. Therefore, since smaller micelles are preferred in DDS applications, predicting the

minimum number of PEG units for a specific number of PCL blocks required to fully protect the PCL

core is important. The authors found a quantitative relation between hydration of PCL core and both

number and molecular weight of PEG and PCL blocks [122].

The internal structure of the thermoresponsive polymer blend NPs, and the phase transition

between core-shell and Janus structures were investigated by DPD simulations by Guo et al. [226]. Chen

and Ruckenstein [230] looked at the structure and mechanism of formation and the degradation process of

multicomponent multicore micelles via DPD simulations. Taresco et al. [231] used a combined

computational/experimental approach to study the self-assembly and structure of copolymer micelles

formed by two different types of monomers. Wang et al. [233] studied the structural characterization of

cholesterol-functionalized CD micelles by all-atom MD simulation. Tan et al. [102] by both experiment

and DPD simulations showed that introducing a second hydrophilic phosphatidylcholine group into the

polymer chains causes a phase transition from sphere to rod-like in multiblock polyurethane micelles.

Hybrid systems composed of polymers and other NPs, e.g. AuNPs and QDs, also show

interesting structural and phase transition behaviour [227, 235]. Using both experiments and DPD

simulations, Cai et al. [227] studied the effect of adding AuNPs on block copolymer self-assembly.

Adding AuNPs caused a transformation from long cylindrical micelles to short cylindrical micelles and

finally to spherical micelles.

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 Interactions with and translocation through cell membranes for polymer-based DDSs have been

studied both by experiment and simulations [236-238]. Li et al. [236] studied the interactions between a

novel amphiphilic polymer, PMAL, and siRNAs, combining experiment and CG MD simulations.

Potential of mean force calculations showed that siRNA by itself experiences a large free energy barrier

for translocation while the siRNA-PMAL complex entered the membrane spontaneously, consistent with

experiment. The PMAL polymers induced pore formation to facilitate siRNA translocation. Srinivas et al.

[237] used CG MD simulation and showed that these patchy polymeric micelles are capable of

accommodating and transporting hydrophilic contents across a lipid membrane into a lipid vesicle.

Ding and Ma [238], using DPD simulations, investigated receptor-mediated endocytosis for a

new type of pH-responsive DDS composed of NP (radius of 4 nm) and some pH-sensitive polymers (of

twelve beads length). Beads on the NP surface were treated as ligands and assigned +e charges. The

polymer had N randomly distributed negatively charged (-e) beads, where N depends on the pKa of the

polymer and the system’s pH. Half of the model lipids in the membrane were treated as receptors by

replacing charged beads in the lipid’s head group with neutral beads. They introduced a modified

Lennard-Jones potential to account for receptor-ligand interactions. Eighteen-microsecond simulations for

three different pH conditions combined with potential of mean force calculations showed a triple-pH-

response. The NP can only be engulfed by cell membrane when the pH is higher or lower than the

polymer’s pKa, whereas endocytosis is blocked when the pH equals the polymer’s pKa. It was also found

that the properties of NPs, pH-sensitive polymer and cell membranes, and the external environment, all

affect NP engulfment by the membrane, at least in this simplified DPD model [Figure 3.6].

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Figure 3.6: Time evolution of nanoparticle-polymer complex endocytosis as a function of pH.

The endocytosis process of a nanoparticle-polymer complex (nanoparticle with pH-sensitive polymers

absorbed on its surface) depends on the pH. For pH values lower (a) and higher (c) than the polymer’s

pKa, the nanoparticle can be fully engulfed by the membrane. However, for pH = pKa (b), endocytosis is

blocked. Membrane lipid head groups are shown in green, purple, and blue correspond to +e, -e, and

neutral beads, respectively. Lipid tail are in orange, receptor heads are in red, and nanoparticle beads are

in yellow. Polymer beads are in cyan (-e) and pink. Reprinted by permission from Macmillan Publishers

Ltd: Scientific reports H. Ding, Y. Ma, Controlling Cellular Uptake of Nanoparticles with pH-Sensitive

Polymers., Sci. Rep. 3 (2013) 2804., copyright 2013.

The effect of functionalization on interactions with the membrane, uptake mechanism, as well as

effect on the structure and conformation of drugs/polymer complexes have been studied by simulation

[118, 239-243]. Liao et al. [240] used both experiment and all atom MD simulations to study the uptake

93
of chitosan/DNA complexes coated with anionic poly(gamma-glutamic acid) (gamma-PGA). This coating

enhanced the cellular uptake of chitosan/DNA complexes via a specific protein-mediated endocytosis.

Both the structure of this ternary complex (CS/DNA/PGA), as well as the interaction between gamma-

PGA and glutamyl transpeptidase proteins were studied by MD simulations. Sun et al. [239, 244] studied

the effect of polyethylenimine polymer (PEI) modification by lipids on nucleic acid compaction and

aggregation. They found that lipid association as an additional mechanism of aggregation resulted in more

compact and stable structures.

Several studies have focused on interactions between polymeric DDS and small molecule cargos,

investigating driving forces for partitioning [60, 245-249], complex stability [250], drug loading capacity

and rate, and drug distribution and release [121, 247, 251-255] as a function of temperature [256], pH

[228, 232, 257, 258], concentration of drugs [232], physicochemical and structural properties of polymers

and cargos [259, 260]. CD is a well-known family of cyclic oligosaccharides with great promise as

solubilizing agent for poorly soluble drugs [246, 261]. He et al. [246] performed MD simulations to

investigate the binding mode and affinities of the antifungal drug amphotericin B drug with γ- and β-CD.

Consistent with experiments, simulations showed a significantly higher binding affinity for γ-CD than β-

CD. The binding mechanism of the drugs bexarotene and human vasoactive intestinal peptide (VIP) to

highly PEGylated sterically stabilized micelles (SSMs) has been investigated by Vukovic et al. [121] both

experimentally and computationally. Using free energy profiles, they predicted the drug distribution in

SSMs. Single bexarotene, as a poorly water-soluble drug, resides in the ionic interface with its polar end

exposed to solvent. With multiple bexarotene molecules, the preferred distribution is as clusters in the

alkane core of SSM [Figure 3.7]. Dominated by electrostatic interactions, VIP molecules, with two

regions with positively charged residues, interact with negatively charged -PO4 groups in the ionic

interface. These simulations suggest the importance of the balance between hydrophobic and Coulomb

interactions between drugs and phospholipid polymers in stabilization of drugs in SSMs.

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Figure 3.7: Solubilization of bexarotene in sterically stabilized micelles (SSM).

Left) Free energy profiles for systems composed of 1, 3, and 5 bexarotenes in SSMs. SSMs are either

composed of 10 (small) or 90 (large) monomers, in water and 0.16 M NaCl, respectively. Single drugs

prefer the ionic interface, represented with vertical arrows, in both SSMs. For multiple drugs accumulated

in the SSM core (shown as a gold surface), a deeper minimum develops in the core. This is because

several bexarotene (e.g. 5) cluster together (Right) via a hydrogen bond network between their –COOH

groups. Reprinted (adapted) with permission from L. Vukovic, A. Madriaga, A. Kuzmis, A. Banerjee, A.

Tang, K. Tao, et al., Solubilization of Therapeutic Agents in Micellar Nanomedicines, (2013). Copyright

2013 American Chemical Society.

The interaction and complexation of nucleic acids, including DNA and siRNA with cationic

polymers has been studied extensively [223]. PEI, as an example of cationic polymers, is one of the most

promising polymers which has been widely utilized in studies for gene delivery applications. Both

experimental and computational studies have provided a better understanding of PEIs’ binding modes

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with DNA/siRNA [223]. Molecular modeling has had a significant impact on our understanding of

critical steps in gene delivery, including complexation of carriers with nucleic acids, as well as nucleic

acid condensation and aggregation by gene carriers [177]. Simulations can be used to investigate the

factors determining interactions between cationic polymers and nucleic acids, including complexation and

decomplexation. Such factors, similar to those in other DDSs, include chemical surface composition as

amine-to-phosphate ratio [51, 262], charge density, protonation state, polymer molecular weight, polymer

chemical structure and charge distribution [262-267], the effect of endogenous molecules [136], and

multivalent ions, e.g. Fe(III) [137, 268]. miRNA as endogenous molecule might cause decomplexation.

To test this hypothesis, Meneksedag-Erol et al. [136] used both experiment and all atom MD to study the

interactions between miRNA and pre-formed PEI-siRNA complexes. PEI was found to bind more

strongly to miRNA than siRNA. However, miRNA could not disrupt the integrity of PEI-siRNA but

formed a layer on the complex, leading to a siRNA-PEI-miRNA complex.

Interactions between polymeric DDS and nucleic acids for gene delivery purposes can be studied

indirectly using polymers. Polycations and polyanions can be considered as models for DDS and nucleic

acids, respectively [269-271]. Zhao et al. [271] utilized polycations and polyanions and studied the effect

of charge distribution along the chain on complexation behavior and the structure of the resulting

complexes. They found that charge distribution has a significant influence on aggregation and the

complex structure.

3.5.3 Peptide- and nucleic acid-based delivery systems.

There are several peptide-based DDSs, e.g. protein- [272] and cyclic peptide-based [273, 274], and cell

penetrating peptides (CPPs) [275]. There are also DDSs based on nucleic acids. RNA and DNA have

been used as either building blocks or surface ligands to design a variety of three-dimensional

nanostructures, including rings, tubes, cubes, cages, and spheres [276-279]. As with other DDSs, the

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structural properties of these systems, and their interactions with endogenous biological molecules, as a

function of environmental conditions are of interest [280-282]. Badu et al. [281] performed atomistic MD

simulations on RNA nanotubes and investigated their structural properties. Juul et al. [282] studied DNA

cages loaded with active enzymes both experimentally and by all-atom MD simulation and found a

temperature-dependent conformational change from an ‘open’ to a ‘closed’ state, in principle enabling

controllable encapsulation and release [Figure 3.8].

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Figure 3.8: DNA nanocage with temperature-controlled conformational transition capability.

The cage is closed at 4 C (a), and in a more open conformation at 37 C (b). Heating the nanocage to

37 C in presence of horseradish peroxidase (HRP), followed by cooling to 4 C, allows HRP enzymes to

be encapsulated in nanocages (c). Reheating to 37 C causes enzyme (orange) release (not shown).

Reprinted with permission from S. Juul, F. Iacovelli, M. Falconi, S.L. Kragh, B. Christensen, R. Frøhlich,

et al., Encapsulation and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA

Nanocage, (2013) 9724–9734. Copyright 2013 American Chemical Society.

Protein-based NPs are promising because of their unique characteristics, including

biocompatibility, biodegradability, low cytotoxicity, and non-antigenicity [283, 284]. Some proteins, e.g.

albumin, can be used for targeted delivery to tumor cells and inflamed tissues since they are preferentially

taken up by these types of cells [284]. Luo et al. [272] using both theory and experimentations

investigated the binding mode, affinity and interactions between bovine serum albumin (BSA) and

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interferon alpha-1b (IFN) as delivery system and protein drug, respectively. Simulations predicted domain

III of BSA as the most probable binding sites, as well as hydrogen bonds and salt bridges as the main

contributors in binding between BSA and IFN. Cyclic peptide-based nanotubes (CPNTs) can be used as a

transporter for ions and small molecules [273, 274]. Vijayaraj et al. [273] investigated the transport

mechanism of 5-FU through a variety of CPNTs and determined the driving forces and free energy

barriers affecting this transport. They found that transport was driven by direct or water-mediated

hydrogen bonding and hydrophobic interactions between CPNT and 5-FU.

CPPs are of interest because of their intrinsic ability to enter cells, their low cytotoxicity, and

their versatility. They can facilitate the transport of small molecules (e.g. drugs, imaging agents),

macromolecules (e.g. DNA, siRNA, proteins, peptides) and NPs/DDS. Therefore, CPPs can be used as

drug/gene delivery systems, as well as carriers for imaging agents, proteins and peptides [275]. Recently,

it has been of great interest to combine the benefits of nanomaterials and CPPs. CPPs can be

functionalized with other delivery systems, like QDs, liposomes, and dendrimers, potentially improving

CPPs intracellular drug release ability and decreasing their toxicity. DDSs such as polymeric micelles,

dendrimers, liposomes, QDs, and inorganic nanocarriers (e.g. gold-, silver-, and iron-based NPs) can be

functionalized with CPPs to enable their cellular uptake [275]. CPPs' unique physicochemical properties,

their uptake mechanisms, classifications, and applications in medicine and biotechnology have recently

been reviewed by Durzynska et al. [275].

Key elements involved in CPPs' roles in drug delivery have been studied computationally [285-

288]. Atomistic MD simulations revealed the importance of arginine, lysine and tryptophan in the

interaction between two CPPs, penetratin [285] and transportan [286], and

dipalmitoylphosphatidylcholine (DPPC) membranes, as well as their translocation mechanism. CPP

conjugation can help translocate hydrophobic and hydrophilic drugs across lipid membranes [289, 290].

Teixeira et al. [290], via both MD simulations and experiments, found that lysine-based surfactants

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enhanced the transdermal permeation of two topically administered hydrophilic drugs, tetracaine and

ropivacaine hydrochloride. As mentioned earlier, NPs can be functionalized with CPPs. The

concentration and distribution of CPPs on the NP's surface influence the conformation of the peptide

layer, which in turn affects the CPP-functionalized NPs’ activity and the efficiency of cellular

internalization. So, designing a DDS of desired activity requires knowledge of the structure and dynamics

of CPPs on NPs in solution prior to interacting with membranes. Todorova et al. [291] using both

experimental and atomistic MD simulations investigated the effect of the grafting density and surface

distribution of HIV-derived trans-activator of transcription (TAT) peptide on NPs cell internalization.

They found a correlation between the surface properties, e.g. positive charge distribution, of these TAT

conjugated NPs in solution, and their membrane permeation.

New DDS design strategies combine the benefits of several systems such as block copolymers

and CPPs [292], or mimic natural systems such as intrinsically disordered proteins (IDPs) [293]. Sanchez-

Sanchez et al. [293], using both experiments and MD simulations, designed and characterized single

chain NPs called artificial IDP mimetics. These IDP mimetics are capable of simultaneous release of two

dermal bioactive molecules, folic acid and hinokitiol, into aqueous solution in a pH-dependent manner.

Computer simulations can provide insight into drug loading, distribution/complex structure and

release from peptide self-assemblies [294, 295], as well as the influence of pH and temperature [282, 294,

296] on these processes. Guo et al., using both experiments and DPD simulations investigated the effect

of pH on microstructure of peptidic micelles [296]. Micelles were self-assemblies of HR-20 (Histidine10-

Arginine10) peptides conjugated with cholesterol, either loaded or not with DOX. Micelles had more

compact structure at pH > 6 [Figure 3.9]. For pH < 6, channels in swollen micelles might cause drugs to

diffuse out of micelles. This was verified experimentally as the DOX release rate was influenced by pH

values, consistent with simulation results.

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Figure 3.9: Effect of pH on microstructure of self-assembled micelles.

Starting from a homogeneous state (a), cholesterol conjugated peptides form compact core/shell micelles

at pH > 6.0 (b). Decreasing the pH loosens these micelles (c) and the swelling facilitates DOX release.

Arginine, histidine, and cholesterol, are shown in green, brawn, and black, respectively. Water has not

been shown for clarity. Reprinted with permission from X.D. Guo, L.J. Zhang, Z.M. Wu, Y. Qian,

Dissipative Particle Dynamics Studies on Microstructure of pH-Sensitive Micelles for Sustained Drug

Delivery, Macromolecules. 43 (2010) 7839–7844. Copyright 2010 American Chemical Society.

Finally, physicochemical properties of peptides and solvent conditions can govern self-assembly

and structural properties of peptide-based carriers. Computer simulation can be used to design peptides

which can self-assemble, and to study the mechanism of self-assembly into nanostructures.

Computational approaches can also be used to study how factors such as solvent and temperature

influence both self-assembly and transitions between different morphologies [297-301].

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3.5.4 Carbon-based delivery systems.

Several different types of carbon-based nanoparticles (CNPs) have the potential to act as DDS [12, 302].

Here we focus on DDS based on CNTs and fullerenes but DDS using graphene, graphene oxide, and

nanodiamond [19, 162, 165, 168] are also of interest.

Computational research has played a major role in exploring drug loading of [164, 303] and

release [132, 133, 304, 305] from CNPs, as well as interactions of cargos with carbon nanocarriers [306]

as a function of internal and external stimulus, e.g. pH, temperature, torsion, and external magnetic field.

CNTs are capable of loading therapeutic agents on their surface or inside their cylindrical hollow [19,

307]. Saikia et al. [305] performed MD simulation to study the C60 fullerene-mediated release of

multiple pyrazinamide molecules from the interior of a single walled CNT as a function of temperature.

Chaban et al. [304] investigated the effect of temperature and concentration on drug release from CNTs

using MD simulations. The effect of external magnetic fields has been studied on hybrid systems

composed of CNTs with magnetic NP caps [132].

Understanding the interactions of CNTs with biological macromolecules is also of great interest

[308-313]. Wu et al. [313] studied DNA ejection from CNTs as a function of temperature, torsion loading

and CNT size by MD simulation. Santosh et al. [309] studied the binding of siRNA and DNA to the

surface of single walled CNTs via MD simulations. Chen et al. [311] studied the dynamic mechanism of

encapsulation of HIV replication inhibitor peptide into CNTs using MD simulation. It is also important to

understand the interactions of carbon-based DDS with human serum albumin (HSA), the most abundant

protein in blood. Interaction of DDS with HSA is crucial in DDS absorption, distribution and metabolism

[314].

The membrane associations of carbon-based DDS have been studied by computer simulation

[315-317]. Several factors affect these associations: DDS structural properties [318-321]; clustering [320-

322]; functionalization [319, 323, 324]; concentration [319] and lipid membrane properties [321, 325].

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The effects of size and clustering of fullerene NPs, and fullerene concentration on a DPPC monolayer as a

model for pulmonary surfactant was investigated by CG MD simulation by Chiu et al. [321]. Free energy

calculations suggest that all fullerene systems in this study (C60, C180, C540, and cluster of five C60

fullerenes) spontaneously diffuse into the hydrophobic region of both monolayer and bilayer membranes.

Large fullerene molecules were found to prefer partitioning into bilayers rather than monolayers,

however, which can influence the monolayer-to-bilayer transition in the respiratory cycle. This may

suggest a possible mechanism of internalization of CNPs through lung inhalation.

The effects of using polymers, peptides, and lipids as surface functionalization agents in carbon-

based DDS were studied by computational methods. Chehel Amirani and Tang [308] comprehensively

reviewed studies, mainly at the quantum mechanical level, of graphene and CNT functionalization, and

their binding energies with nucleobases. Lai and Barnard [131] have reviewed recent studies on

functionalized nanodiamonds and their importance for biomedical applications. Functionalization can

prevent NPs from aggregating [323, 326], increase their stability [327], and influence their interactions

with lipid membranes [323, 326, 328]. Surface functionalization can also promote drug delivery through

improving translocation across lipid bilayers [328]. Sridhar et al. [328] used CG MD simulations to look

at effects on fullerene (C60) translocation through the membrane due to polar and nonpolar

functionalization, temperature, and fullerene concentration. Although none of their models showed

complete translocation, they found that Janus particles having half the surface modified by polar groups

were the most promising form of functionalized fullerenes in terms of bilayer translocation.

A NP's behavior depends on the conformation of the functionalization layer on its surface. The

coating mechanism used to functionalize the NP, e.g. covalent vs. non-covalent attachment, as well as the

grafting density are two factors which can affect this conformation [329, 330]. For example, Lee [330]

found, using CG MD simulation, that the PEGylation method can influence PEG distribution on CNTs.

They also found that PEG size and grafting density affected the PEG layer's conformation [Figure 3.10].

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Designing functionalized NPs with desired properties will require a detailed understanding of these

factors.

Figure 3.10: Effect of PEGylation method, and PEG chain size and grafting density on the

conformation of PEG layer on carbon nanotube.

Non-covalently modified single walled CNT (SWNT) (Left) is more exposed to water than covalently

modified SWNT (Right). PEG (red) size and grafting density also influence the conformation of PEG

layer on SWNT (grey cylinder). RF is the mushroom radius and L is the thickness of brush state. SWNT is

shown as grey cylinders. Reprinted with permission from H. Lee, Molecular Dynamics Studies of

PEGylated Single-Walled Carbon Nanotubes: The Effect of PEG Size and Grafting Density, (2013).

Copyright 2013 American Chemical Society.

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3.5.5 Lipid-based delivery systems.

Recently, lipid-based DDS such as liposomes, micelles and LNPs have attracted much attention [7, 8,

331, 332] due to their ability to encapsulate and transport drugs as well as biomolecules, the versatility of

their structures and compositions, and their inherent selectivity to tumor cells and inflammation sites.

There are currently more than ten approved lipid-based drug formulations and many more in clinical trials

[7, 8]. Specifically, sterically stabilized liposomes, i.e. PEGylated liposomes, are widely used as DDS.

PEGylation achieves long circulation times for liposomes in vivo due to the formation of a protective PEG

layer over the liposomal surface. This layer sterically prevents the coating of liposomes by opsonins, thus

reducing drug uptake by cells of the immune system [333]. However, the mechanism through which

PEGylated lipids interact with different components in the liposomes, its function in drug distribution,

localization and retention as well as the influence of external environmental factors such as salt

concentration are not completely understood.

Since atomistic simulations of full liposomes are computationally expensive, a common approach

is to infer results on liposomes from lipid bilayer simulations. Dzieciuch et al., [334] for instance,

performed atomistic MD simulation to study the effect of liposome PEGylation on their drug-loading

efficiency. They compared the effect of zwitterionic and PEGylated membranes on the location and

orientation of a model hydrophobic compound, 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin (p-

THPP). p-THPP enters both types of lipid bilayers, which agreed with experimental results, but in

PEGylated liposomes p-THPP also localizes to the outer PEG corona where porphyrins are wrapped by

PEG chains. Thus, in PEGylated liposomes p-THPP showed a greater exposure to the water than in

zwitterionic liposomes. These results highlight that PEGylation enhances the drug-loading efficiency of

membranes and support fluorescence experiments carried out in this study [334]. Simulations also

predicted strong interaction between PEG lipids and porphyrin [334]. This interaction is particularly

important in drug delivery studies as it may be an extra barrier to drug release. This finding supports

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previous computational studies that showed that PEG interacts with hydrophobic molecules [127, 335,

336]. Similarly, MD simulations combined with free energy calculations were used to elucidate binding

mechanisms and divulge the exact location and organization of two small drugs, bexarotene and human

VIP, within PEGylated micellar nanocarriers [121].

PEGs strongly interact with salts. This property has been utilized in lithium ion batteries with

PEGs as polymer electrolytes [337, 338]. PEGs are soluble in a wide variety of both polar and nonpolar

solvents [339]. Although both of these general properties have been shown previously by computational

studies [337, 338, 340] and experiments [341, 342], for DDS applications it is important to understand

how the PEG layer on DDS, e.g. PEGylated liposomes, interacts with salts in physiological conditions

and how these interactions affect the structure of the PEG layer [118, 343, 344]. Stepniewski et al. [344]

modeled the effect of NaCl on the surface structure of PEGylated liposomes. They varied the salt

concentration in Langmuir monolayer film experiments and added ions at the physiological level in

atomistic MD simulation studies. The results showed that the PEG surface layer should not be treated as

generic hydrophilic molecules completely outside the bilayer, nor should it be considered as totally

neutral as was previously accepted. PEG molecules are able to penetrate into the liquid-crystalline lipid

bilayer, which may affect the permeability and structure of the membrane. It was also noted that Na + ions

bind to PEG chains, changing the surface charge of the liposome and enhance opsonization, whereas Ca 2+

did not interact with PEG [343]. These results are not surprising, however. In fact, when PEGs are

attached on DDS surface, e.g. liposomes, they seem to show similar properties as previously reported for

other applications [337, 338, 340-342]. As another example, Pannuzo et al. [345] performed CG

simulations to study the effect of Ca2+ and PEG molecules on membrane fusion. They showed that for a

rapid fusion between negatively charged apposed membranes both Ca2+ and PEG were required.

An important property governing drug release from liposomal DDS is bilayer permeability [346,

347]. Magarkar et al. [346] performed atomistic simulations using the OPLS-AA force field to investigate

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the high permeability observed for "inverse-phosphatidylcholine" (CPe) liposomes in experimental

studies [348]. CPe is a synthetic analog of phosphatidylcholine (PC) with a reversed zwitterionic

headgroup and has been proposed for use in liposomal DDS formulations. The larger surface area

observed for the CPe bilayer compared with the PC bilayer may cause CPe's higher permeability. Adding

cholesterol to liposomal formulations reduces leakage from liposomes due to its well-known role in lipid

packing and stability, but the mechanism for this is not understood. Magarkar et al. [336], by performing

MD simulation using OPLS force field, proposed a model to explain the interaction between PEG

molecules and cholesterol. PEG molecules tend to interact with the ß side of cholesterol, disrupting the

membrane structure. Contrary to the expectation that cholesterol should make the membranes more stable

and compact, adding cholesterol in the presence of PEG caused membrane destabilization. This result

may explain possible effects of cholesterol on the permeability and compressibility of PEGylated

liposomes.

Self-assembly is another feature of special interest in nanotechnology and nanomedicine as its

understanding provides a framework for developing new nanoscale materials with desirable properties

[116]. CG and DPD simulations have been performed for the investigation of lipid-based aggregates

relevant for drug delivery [115, 349-352]. For example, Lee and Pastor [349] used the Martini CG force

field to study mixtures of lipids and PEGylated lipids in water at different sizes and concentrations of

PEGylated lipids. They found that the mixtures self-assembled to liposomes, bicelles and micelles. The

analyses and simulations indicated that the average aggregate sizes decreased when the concentration of

PEGylated lipid increased, in agreement with experimental results. Janke et al. [352] used CG MD

simulations to study the phase behavior of oleic acid aggregation at various concentration and protonation

states. They observed a range of structures including micelles, vesicles and oil phases depending on the

protonation state of the oleic acid head group.

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 Simulations can be used to calculate drug release rates from lipid-based DDS [353, 354]. For

instance, the release of encapsulated materials from systems including emulsions (100% liquid lipid

phase) and nanostructured lipid carriers, which have a combination of solid and liquid lipid domains, was

investigated by Dan using Monte Carlo simulations [353]. The results obtained suggest that the size of

lipid domains does not significantly affect the rate of release, but the location and distribution of the solid

domains have a notable impact. When solid domains are concentrated near the solution interface,

transport is inhibited due to a reduction in the accessible surface area. However, when the solid domain is

located in the center of the LNP the release rate is increased and may even be higher than in the

corresponding emulsion particle. In another study by Dan [354], Monte Carlo simulations were performed

to study drug release in response to electric field-induced liposome pores.

There are also other interesting studies, three of which are mentioned briefly here. Computer

simulations were combined with experiments to investigate the effect of penetration and membrane

behavior of three terpenes, which are effective chemical permeation enhancers in nanostructured lipid

carriers [355]. A detailed model of the distribution of drug molecules inside a liposome was obtained

using the Martini force field [356]. Jämbeck et al. [356] performed large scale simulations of hypericin, a

photosensitizing drug, in a DPPC liposome [Figure 3.11] and calculated the distribution and orientation

of the drugs in the lipid bilayer. Hoof et al. [357] studied the encapsulation of proteins in spontaneously

forming vesicles using MD simulation with a CG force field. They showed that the interactions of

proteins with membranes govern the encapsulation efficiency, but the size of the encapsulated proteins

and the speed of the vesicle formation did not seem to have a significant effect.

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Figure 3.11: Distribution and orientation of hypericin within liposome.

a) Snapshots from MD simulations taken after 10 μs. Phosphorus groups (orange), choline groups (blue)

and hydrophobic tails (green) of the lipids are represented, with different numbers of drug molecules

(yellow). b) Liposomes with some lipids removed to show the binding of hypericin to DPPC; the

hydrophilic parts of hypericin are shown in red and the hydrophobic parts in yellow. Reprinted with

permission from J.P.M. Jämbeck, E.S.E. Eriksson, A. Laaksonen, A.P. Lyubartsev, L.A. Eriksson,

Molecular Dynamics Studies of Liposomes as Carriers for Photosensitizing Drugs: Development,

Validation and Simulations with a Coarse-Grained Model, (2013). Copyright 2013 American Chemical

Society.

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 A promising gene therapy approach is based on siRNA. Sophisticated delivery systems are

required to protect and deliver siRNA effectively to the target tissues [358, 359]. CG molecular

simulations together with a variety of experimental techniques were able to give a more detailed view of

the structure of a LNP containing siRNA [Figure 3.12] [79, 360]. Simulations of small systems

containing 8 duplex 12 base pair DNA complexes, DLinKC2-DMA (an ionizable cationic lipid), DSPC,

cholesterol, a PEG-lipid, water and ion molecules were performed. These molecules self-assembled and

organized in an ordered structure where, for instance, the PEG-lipids were distributed in the outer layer of

the LNP. This was the first study to show that LNP siRNA systems likely have a nanostructured core,

with the encapsulated siRNA located in internalized inverted micelles complexed to cationic lipids. The

in silico finding agreed with 31P NMR, FRET, cryo-TEM, and RNase digestion data and it was possible to

understand the mechanism whereby LNP siRNA systems are formed. This insight is being applied to the

design and construction of new LNP systems. In addition, both CG and atomistic computer simulation

have been used to study the DNA adsorption onto anionic lipid membranes induced by multivalent

cations [361].

110
Figure 3.12: Lipid nanoparticle.

External and a cross-sectional view of the LNP (top). Components are represented by their molecular

densities: protective PEG-lipid (magenta), therapeutic siRNA (red), cholesterol (green), DSPC (yellow),

ionizable cationic lipid (DLin-KC2-DMA) (blue) and water (cyan). Adapted from D. Rozmanov, S.

Baoukina, D.P. Tieleman, Density Based Visualization for Molecular Simulation., Faraday Discuss. 169

(2014) 225–43. doi:10.1039/c3fd00124e. under a Creative Commons Attribution 3.0 Unported License.

3.5.6 Gold-nanoparticles.

AuNPs are of particular importance in drug delivery research due to their unique properties including

their stability, ease of synthesis, and the ability to manufacture a range of sizes [15, 362]. They can be

loaded with various therapeutics including small molecules, peptides, proteins, and nucleic acids [19],

either conjugated to AuNPs covalently or noncovalently. There are several computational studies on the

properties of AuNPs and their interactions with other molecules [135, 227, 363-370].

111
 The effect of AuNP conjugation on peptide conformational flexibility and structure was studied

by Lee and Ytreberg [366]. They examined the structure and dynamics of six peptides that were either

free or conjugated to AuNPs in water. Conjugation affected both structure and dynamics in an amino acid

sequence dependent way. Peptides with little or no secondary structure in solution were adsorbed on the

AuNP surface. This causes peptides to lose their specific interactions with cell components. For drug

delivery purposes, this suggests that peptides with significant secondary structures in solution are suitable

candidates for peptide-NP conjugation. Interactions between ubiquitin and AuNPs have also been studied

by Brancolini et al. [135] using computer simulation.

Van Lehn and co-workers performed a series of computational studies on AuNPs coated by

lipids. Among other properties, they investigated the structure of the monolayer coating the AuNP under

different conditions [369], lipid composition and AuNP size [368], the interactions of a monolayer-coated

AuNP with model membranes relevant for uptake [367], suggesting similarities in uptake process with

bilayer-bilayer fusion [371], and interactions between AuNPs [370].

3.6 Conclusions and Future Perspectives

We have reviewed common biophysical and computational approaches to studying DDSs, focusing in

particular on computational studies of several major classes of NPs used in drug delivery research. The

strength of computer simulations in general is their ability to give very detailed insight into the structure

of NPs and their interactions with drugs and model membranes under a range of conditions. Clearly, the

efficiency of DDSs depends on a large number of other parameters, including stability in the bloodstream,

targeting to the desired tissues, uptake by endocytosis or other mechanisms, and final release of the drugs.

Although experimental biophysical and computational studies can characterize important aspects of

DDSs, a major challenge for the near future is to go beyond individual drug carriers and to investigate at a

mechanistic level the larger-scale processes that determine the success of DDSs.

112
 At the computational level, currently we are limited by some of the standard limitations of

biomolecular simulation. In general, these involve limited sampling time, limited length scale, and

expensive or difficult access to important thermodynamic variables. Simulations can access time scales of

the order of microseconds or for coarser models tens or hundreds of microseconds, at length scales of ca.

10 × 10 nm for atomistic and up to 150 × 150 nm for CG simulations. Thus, it is now possible to simulate

entire nanocarriers, which will open up new areas of study for simulations. However, the process of NP

formation by e.g. microfluidics remains outside reach of direct detailed simulations. Questions regarding

drug loading, the stability of complexes, and the interactions of NPs with membranes essentially involve

free energies of binding, partitioning, and membrane pore formation. These can all be studied by free

energy methods, but the current state of the literature remains somewhat qualitative. This is an obvious

area for future work, which is already feasible with current methods and computational resources. Some

of the most interesting simulations currently available use CG MD or DPD. This is an exciting

development, but also comes with its own limitations; in particular, CG models need to be carefully tested

for their ability to reproduce the effect of important factors in DDSs such as salt concentration, the

distribution of chemical functional groups over the surface of a DDS, and the effect of pH.

DDSs are an interesting example of multi-scale computational problems, with a growing amount

of data from biophysical characterization. They have an obvious biomedical and biotechnological

importance, and bridge chemistry, nanoscience, materials, basic biology and medical applications in a

unique way. Although there already is a large body of literature as reviewed in this paper, in our

assessment the impact of computational work in this important area is likely to grow significantly in the

near future as model systems become more realistic and more tightly coupled to experiment.

113
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156
 Chapter Four: Ionizable Amino Lipid Interactions with POPC: Implications for Lipid

Nanoparticle Function

Contributions:

I ran and analyzed all the simulations and wrote the major part of this chapter. I had major contributions

in methodology, data interpretation, hypothesizing, model development and validation, and visualization.

The experimental data and methods sections were provided by Dr. Jenifer L. Thewalt, Dr. Sherry S.W.

Leung, Dr. Miranda L. Schmidt, and Iulia Bodnariuc at the Simon Fraser University, and by Dr. Pieter R.

Cullis and Dr. Jayesh A. Kulkarni at the University of British Colombia.

Abbreviations:

LNP, lipid nanoparticle; ICL, ionizable cationic lipid; 2H NMR, deuterium nuclear magnetic resonance;

MD, molecular dynamics; 𝑆𝐶𝐷 , deuterium order parameter for C-D bond; POPC, 1-palmitoyl-2-oleoyl-sn-

glycero-3-phosphocholine; SAXS, small angle X-ray scattering; KC2, DLin-KC2-DMA ionizable

cationic lipid; KC2H, KC2-protonated; KC2(H), KC2 or KC2H; Cryo-TEM, cryogenic transmission

electron microscopy; siRNA, small interfering ribonucleic acid.

4.1 Abstract

Lipid nanoparticles (LNPs) based on ionizable cationic lipids are currently the leading systems for siRNA

delivery in liver disease, with a major limitation of low release efficacy in the cytoplasm. Ionizable

cationic lipids are known to be of critical importance in LNP structure and stability, siRNA loading, and

siRNA release stage. However, their distribution inside the LNPs and their exact role in delivery efficacy

are not known. A recent study [Kulkarni et al, ACS nano, 2018] on siRNA-LNPs containing cationic lipid

157
DLin-KC2-DMA (also known as KC2 with an apparent pKa of ca. 6.7) suggested that neutral KC2

segregates from other components and form an amorphous oil droplet in the core of LNPs. However, no

direct evidence for the presence of KC2 in the core was provided.

We further studied KC2 segregation in the presence of POPC using molecular dynamics

simulation, deuterium NMR, SAXS, and cryo-TEM experiments. Our findings strongly suggest that

neutral KC2 has a high tendency to separate from POPC dispersions, further supporting the model

proposed in [Kulkarni et al, ACS nano, 2018]. KC2 confinement, upon raising the pH during the

formulation process, could rearrange the internal structure of LNPs and consequently affect the LNP

transfection efficacy. As interactions between cationic KC2 and anionic endosomal lipids are thought to

be a key factor in cargo release, KC2 confinement inside the LNP may lower the efficacy of release.

4.2 Introduction

Lipid nanoparticles (LNPs) containing ionizable cationic lipids (ICLs) are among the leading drug

delivery systems with promising applications for siRNA and mRNA delivery [1-4]. One current

limitation of these systems is the low release efficacy of siRNA, estimated to be 1-2% [5]. The size,

surface composition, and lipid distribution inside the LNP, as well as the internal structure of these

systems are known to affect their transfection efficacy to a considerable extent [2, 6-8]. The ionizable

cationic lipid (ICL) component of these siRNA-LNPs, with an apparent pKa of less than 7.0 [9], plays a

critical role in siRNA loading, internal arrangement of biomolecules in the LNPs, and endosomal escape

of the therapeutics.

DLin-KC2-DMA, also known as KC2, is one of the recently developed and optimized ICLs with

a reported apparent pKa of ca. 6.7 [9, 10]. This pKa ensures a neutral or low cationic surface charge for

the LNP in physiological pH and a high positive surface charge in endosomal pH. Electrostatic

interactions between cationic lipids and naturally occurring anionic lipids in endosomal membranes have

158
been proposed as the underlying mechanism of drug release for RNAi-LNPs containing (ionizable)

cationic lipids [2, 9, 11]. Thus, the presence of protonated KC2 on the LNP surface is assumed to be

required for destabilizing the endosomal membrane and releasing siRNA. However, there are still

discrepancies regarding the detailed internal structure and lipid distribution across this class of LNPs [2,

12, 13]. Previous work, involving cryo-TEM, has shown that LNP-siRNA systems containing KC2 form

structures with electron dense cores [6, 12-15]. This electron-dense core was hypothesized to be the result

of inverted micellar structures generated through rapid-mixing procedures [12, 14]. More recently, it was

determined that LNPs containing KC2 generated liposomal structures when the KC2 is protonated at pH

4, while forming electron-dense structures at pH 7.4 [13]. This suggests that KC2 might adopt an oil-

phase when neutralized. However, no direct evidence for localization of neutral KC2 in the LNP’s core

was found.

Understanding the structural properties of LNPs is difficult due to the inherently complex nature

of LNP manufacturing. To what extent are the fundamental interactions between ionizable cationic lipids

such as KC2 and phospholipids responsible for the sequestration of neutral KC2 away from other typical

LNP constituents such as phosphatidylcholine? In this study we used molecular dynamics (MD)

simulation, solid state deuterium NMR (2H NMR) and small-angle X-ray scattering (SAXS) experiments

to investigate the interactions between KC2 and POPC – lipid bilayers in simulations and multilamellar

dispersions in experiments - as a function of pH, temperature and mixing ratio. To further investigate

KC2 localization in LNPs we performed cryo-TEM experiments on LNPs composed of POPC, KC2 and

cholesterol.

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4.3 Methods

4.3.1 Approach.

We used 2H NMR to measure the deuterium order parameter (𝑆𝐶𝐷 ) for the POPC palmitoyl chain in

POPC/KC2 and POPC/KC2H bilayers, to study the effect of KC2 and its protonated state (KC2H) on the

POPC bilayer structure. POPC/KC2 systems of four different mixing ratios (100/00, 90/10, 80/20, and

70/30) were studied at two pH levels of 4.4 and 8.1, and at several temperatures in a range of 288 to 313

K (section 4.3.3).

The 𝑆𝐶𝐷 profiles at 313 K were further used to develop force field parameters for KC2. Both KC2

and KC2H were parameterized using the CHARMM36 force field (C36 FF) [16]. Bilayers composed of

POPC and KC2 or KC2H were simulated, and the 𝑆𝐶𝐷 profiles for the POPC palmitoyl acyl chain

obtained from simulations were compared with the corresponding profiles extracted from 2H NMR

experiments (section B.1).

Simulations using the validated models were used to study the POPC/KC2 binary mixtures as a

function of pH and mixing ratio [Table 4-1] (section 4.3.2). Herein again, systems with all the above-

mentioned mixing ratios were simulated at acidic, neutral and basic pH levels. Considering the pKa of ~

6.7 for KC2, the POPC/KC2 systems, where all the KC2 were taken as neutral, correspond to pH levels of

7.4 to 8.1. Also, the POPC/KC2H bilayers, with all the KC2 taken as protonated (KC2H), correspond to

experimental systems at pH of ~ 4.

Based on the results from MD simulations, additional SAXS and cryo-TEM experiments were

designed and conducted. SAXS experiments were used to measure the bilayer repeat spacing (d-spacing)

in the POPC/KC2 lamellar phase for several mixing ratios at basic pH values, whereas cryo-TEM

experiments were conducted to detect possible KC2 segregation from POPC. In the cryo-TEM

experiments, cholesterol was added to the POPC/KC2 and POPC/KC2H systems (section 4.3.3).

160
4.3.2 Computational method.

4.3.2.1 System construction.

All the systems (binary systems and pure POPC bilayers) were constructed using an in-house developed

script which is based on GROMACS package [17] tools as described in section 2.5.1 of Chapter 2.

Briefly, for the binary mixtures, two monolayers each composed of only one lipid type were built first.

Each monolayer was composed of 100 lipids distributed on a two-dimensional grid (10 by 10). Two

GROMACS modules, gmx editconf and gmx genconf, were used to translate and rotate the lipids on each

grid point. Next, a set of random numbers from 1 to 100 was generated to satisfy the desired mixing ratio.

For instance, if POPC/KC2H (80/20) was of interest, 20 random numbers were generated. These 20

numbers were used to both keep the KC2H in 20 grid points, and to delete the POPC in the same grid

points. Finally, the coordinate files from two modified monolayers were merged resulted in one mixed

monolayer with a desired mixing ratio; which was taken as the upper leaflet. Assuming this monolayer is

in the XY-plane, its rotation (using gmx editconf module) around either the X or Y axis gives the lower

leaflet. Adjusting the center of both leaflets to (0,0,0) and subsequently merging the coordinate files for

these two leaflets resulted in the POPC/KC2H (80/20) bilayer. Waters were subsequently added on the

top and bottom of this system. Finally, counter ions (Cl-) were added to neutralize the systems composed

of POPC and KC2H. For the neutral POPC/KC2 systems no ions were added. When excess ion

concentration was of interest, structures at 200 ns from pure POPC simulations and 500 ns from

POPC/KC2H simulations were extracted, and Na+ and Cl- ions were added to these structures to reach the

150 mM salt concentration.

161
4.3.2.2 Molecular dynamics simulations.

All the simulations were conducted using the GROMACS package (v. 2016.3) (see Table 4-1 for a full

list of systems simulated in this study). The CHARMM36 FF [16] was used to model the molecules in the

system, and about 70 TIP3P water molecules [18, 19] were added to each system to fully hydrate the

bilayer. Periodic boundary conditions were applied in all directions. A time step of 2 fs was used, and the

coordinates were saved every 50000 steps. A semi-isotropic pressure coupling was applied to the system,

with Z direction (the normal direction to the bilayer surface) treated independently from the XY-plane.

The Parrinello-Rahman pressure coupling [20, 21] with a time constant of 5.0 ps was used to keep

the pressure at 1 bar. The temperature was maintained at either 288 K or 313 K using Nose-Hoover

extended ensemble [22, 23]. The temperature for two groups of lipids and water plus ions were coupled

separately using a time constant of 1 ps, and the center of mass translation of these groups were removed

every 100 steps independently.

The nonbonded interactions were treated using Verlet cut-off scheme. The van der Waals and

Coulomb interactions were switched at 0.8 nm and 0.0 nm, respectively, whereas both were cut-off at 1.2

nm. No dispersion correction was applied, and PME [24, 25] was used to treat the long-range electrostatic

interactions after the cut-off. The force-switch and potential-shift functions were used as van der Waals

and the coulomb modifier functions, respectively. All bonds involving hydrogen atoms were constrained

using the LINCS algorithm [26].

The other simulation parameters were mainly the ones recommended by CHARMM-GUI [27] for

using C36 FF in GROMACS package for simulation of lipid bilayers, except that the van der Waals

interactions were switched at 0.8 instead of 1.0 nm [28]. The rest of the parameters were kept as default

within GROMACS (v. 2016.3).

Pure POPC, POPC/KC2, and POPC/KC2H systems were simulated for 200 ns, 700 ns, and 700

ns, respectively. The systems with 150 mM salt concentration were simulated for 300 ns [Table 4-1].

162
Table 4-1: Summary of simulations.

System Mixing ratio NaCl concentration Simulation time

(mM) (ns)

POPC ------ 0 200

POPC ------ 150 300

POPC/KC2 90/10 0 700

80/20

70/30

POPC/KC2H 90/10 0 700

80/20

70/30

POPC/KC2H 90/10 150 300

80/20

70/30

4.3.2.3 Simulation Analysis.

All analyses were performed using GROMACS (v. 2016.2) modules, except otherwise mentioned. The

last 100 ns of each simulation was used for the analysis. VMD [29] and Xmgrace were used for

generating the figures and graphs, respectively.

163
Deuterium order parameters: The order parameter is defined as:

< 3𝑐𝑜𝑠 2 𝜃 − 1 > Equation 4-1

|𝑆𝐶𝐷 | = | |
2

where θ is the angle between the C-D bond in the methylene group and the vector normal to the bilayer,

and the angular brackets represent the ensemble average. The SCD parameters for the POPC palmitoyl

chain in each system were calculated from the simulations. The script to calculate the SCD from

simulations was provided by Dr. Thomas Piggot [30]. Given the all-atom nature of the C36 FF, the actual

angle between the C-H bond in each methylene group in the POPC palmitoyl chain and the Z axis (the

normal direction to the membrane surface) was used for the SCD calculation.

Electron and number densities: All the electron/number densities were calculated using the gmx density

module of the GROMACS package. The box in the Z direction was divided into 100 slices and the

density was symmetrized along the Z axis.

POPC P→N vector angle distribution: The distribution of the angle between the POPC P→N vector

and the vector normal to each bilayer leaflet pointing in the direction of the water phase was calculated

using the gmx gangle module of the GROMACS package. The bin width was 0.5 degrees, and graphs

show a running average over 20 data points (10 degrees) to highlight the observed trend in tilt angle

change due to KC2H concentration.

164
4.3.3 Experimental methods.

4.3.3.1 Materials and sample preparation.

Lipids:

1-Palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (POPC-d31) was purchased in powder from Avanti

Polar Lipids Inc. (Alabaster, AL). The POPC-d31 was dissolved in 80/20 benzene/methanol (v/v) and then

the solvent was removed under vacuum to obtain a completely dry powder. The ionizable cationic lipid

2,2dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (KC2) [9] was donated by AlCana Technologies

Inc., (Vancouver BC). KC2 came in an oil form and was not lyophilized prior to use. All lipids were

stored at -20°C.

Buffers:

HEPES buffer was prepared by dissolving the salts in deuterium depleted water (reagents from Bioshop

Canada Inc., (Burlington, ON) and Sigma-Aldrich Canada (Oakville, ON) respectively). The pH was

adjusted to the desired value by adding NaOH (EM Science, affiliates of Merck KGaA (Darmstadt,

Germany)).

Multilamellar vesicle sample preparation:

Appropriate amounts of each lipid were weighed out and the lipids were co-dissolved in 1-2 mL of a

mixture of 80/20 (v/v) benzene/methanol. The solvent was removed in two steps: first N2 gas was used to

evaporate the bulk of the solvent until only a thin lipid film was left, then any residual solvent was

eliminated by lyophilization of the sample using a strong vacuum pump for several hours or overnight

(until the sample weight was stable).

165
 Samples contained approximately 50 – 60 mg total lipids and were hydrated in an excess of 10

mM HEPES buffer (~625-650 μL) at the desired pH. A series of five freeze (in liquid N2) – thaw (room

temperature) – vortex cycles were performed to ensure sample homogeneity. The sample was then

transferred into the NMR tube and/or a quartz capillary tube (Charles Supper Company Inc. (Natick,

MA)) for X-ray analysis. Samples in NMR tubes were stored at -20° C. However, since the quartz

capillary tube were sealed with wax, samples for X-ray analysis were put into the capillary tube either

immediately before the experiment or left in the fridge overnight.

After the experiments under basic pH conditions were completed, the pH of the sample was

changed inside the sample tube by adding an appropriate amount of glacial acidic acid (Anachemia

Science Canada Inc. (Vancouver, BC)), and then vortexing the sample to ensure homogeneous mixing.

The final sample pH was verified using pH paper.

Preparation of LNPs containing KC2 for cryo-TEM of LNPs:

LNPs were prepared as previously described [13]. Briefly, lipid components (POPC, cholesterol and

KC2) dissolved in ethanol were mixed at the appropriate ratios to a final lipid concentration of 20 mM.

The lipids in ethanol were mixed with 25 mM Na Acetate pH 4 buffer using a T-junction mixer [31-33].

The resulting suspension was dialysed against 1000-fold volumes of pH 4 buffer or phosphate buffered

saline (PBS) overnight. The LNPs were then concentrated using 10 kDa NWCO Amicon centrifugal units

to a final lipid concentration of ~20 mg/mL and analyzed by Cryo-TEM.

4.3.3.2 Deuterium NMR (2H NMR).

2
H NMR experiments were performed using an Oxford 300 MHz magnet with a 2H frequency of 46.8

MHz and a TecMag Scout (TecMag, Inc. (Houston, TX)) spectrometer. The standard quadrupolar echo

pulse sequence was used. The two out of phase 90° pulses were 3.95 μs long, the interpulse delay was 40

166
μs and the recycle delay was 300 ms. Data was collected using 8-cycle CYCLOPS phase cycling. All

temperatures investigated were well above the gel-fluid transition of POPC, and an equilibration time of

45 minutes was used for each 5 degree temperature increment. 50 000 scans were collected to ensure an

adequate signal-to-noise ratio in the spectra that were de-Paked using the iterative method described by

Sternin et al. [34].

The first moment of a deuterium NMR spectrum is proportional to the average quadrupolar

splitting and is determined by

𝑥
1 Equation 4-2
𝑀1 = ∑ |𝜔|𝑓(𝜔)
𝐴
𝜔=−𝑥

where ω is the frequency, f(ω) describes the spectrum centered at 0, A is the spectral area ( A =

∑xω=−x f(ω)), and ±x is chosen such that the entire spectral area is contained within the limits [35]. The

orientational order parameter of a C-D bond in the hydrocarbon chain is

< 3𝑐𝑜𝑠 2 𝜃 − 1 > Equation 4-3

|𝑆𝐶𝐷 | = | |
2

where θ is the angle between the C-D bond vector and the bilayer normal, and can be obtained from the

spectrum via the relationship

3 Equation 4-4
𝛥𝜈𝑄 = (167𝑘𝐻𝑧)|𝑆𝐶𝐷 |
4

167
where ΔνQ is the quadrupolar splitting and 167 kHz is the quadrupolar coupling constant. The smoothed

order parameter profiles were obtained experimentally by measuring the quadrupolar splittings of the de-

Paked 2H spectra [36].

4.3.3.3 Small angle X-ray scattering (SAXS).

Small-angle X-ray scattering (SAXS) curves were obtained using a SAXSLAB Ganesha 300XL

(Skovlunde, Denmark) setup with a Linkam variable temperature sample stage. The X-ray source was Cu-

Kα (λ= 1.54 Å). The Linkam stage sample to detector distance was calibrated using AgBeh and the

temperature was calibrated using the pre-transition and chain melting transition of both DPPC and DMPC

bilayers. All temperatures reported are the corrected temperatures. Disposable thin-walled quartz capillary

tubes (80 mm long, 1.5 mm (outer diameter), 0.01 mm thickness) were used to hold the samples. To

ensure that the sample was equilibrated and stable, two sets of data were typically collected at each

temperature: one after 30 minutes, and the second 20 minutes after the completion of the first run (an hour

after the setpoint temperature was reached). Each data collection run was 600 s. Initial and final data were

collected at the same temperature to check for any changes in the sample over the course of the

temperature run.

The bilayer repeat spacing (d-spacing) was measured from the SAXS scattering curves using the

following relationship

2𝜋𝑛 Equation 4-5

𝑑=
𝑞𝑝𝑒𝑎𝑘

where qpeak is the position of the peak and n is its order number. The peak order number depends on the

symmetry of the system and in the case of stacked bilayers, n = 1, 2, 3, 4, … [37].

168
4.3.3.4 Cryo-TEM of LNPs.

Cryo-TEM was performed as previously described [13]. Briefly, 3-5 L of concentrated LNP suspensions

were added to glow-discharged copper grids. A FEI Mark IV Vitrobot (FEI, Hillsboro, OR) was used to

plunge-freeze blotted grids into liquid ethane. Vitrified samples were stored under liquid nitrogen. Frozen

grids were imaged using a FEI Tecnai G2 (FEI, Hillsboro, OR) operating at 200 kV in low-dose mode

and images were obtained using an FEI Eagle 4K CCD camera (FEI, Hillsboro, OR). All sample

preparation and imaging were performed at the UBC BioImaging Facility (Vancouver, BC).

Using the present technique, we have shown that POPC liposomes produced by rapid-mixing are

extremely small and poorly resolved by cryo-TEM. However, systems containing cholesterol are larger

and thus can be imaged easily [38, 39]. Additionally, it was shown that replacement of PC-lipid with

ICLs resulted in decreased particle sizes [13]. Thus, in order to study the effect of KC2(H) on

nanoparticles (at sizes that are readily resolved by the present technique) cholesterol was included in the

formulation.

4.4 Results and Discussion

In simulations, starting from a binary lipid mixture composed of POPC and neutral KC2 in a bilayer form,

almost all the neutral KC2 segregated from POPC and incorporated into the hydrophobic interior between

two POPC monolayers [Figure 4.1-a, pH 7.4]. In contrast, KC2H, with a net charge of +e, stays in the

lipid-water interface and interacts with water molecules, ions, and POPC headgroups [Figure 4.1-a, pH

4], at both temperatures and all mixing ratios studied [Table 4-1]. These observations in simulations were

further investigated using cryo-TEM, 2H NMR, and SAXS experiments.

169
 The cryo-TEM micrographs of POPC/KC2/cholesterol dispersions are shown in Figure 4.1-b.

These micrographs suggest that all particles (regardless of KC2 content) displayed bilayer structures at

pH 4.0, but not at pH 7.4. For systems with no KC2, raising the pH from 4.0 to 7.4 changed the particle

structures from unilamellar vesicles to bi-/oligo-lamellar structures. When 30 mol % KC2 was added,

unilamellar vesicles but with smaller average sizes compared to the POPC/cholesterol dispersions were

formed at low pH. However, at pH 7.4 larger multi-lamellar vesicles with electron-dense cores were

formed. The appearance of this electron dense core upon increasing pH level supports the KC2 separation

from POPC and cholesterol, and formation of the oily cores. Furthermore, systems with 30 mol% KC2 at

pH 7.4 were larger than the same systems at pH 4.0, implying that particle fusion has occurred [13].

Taken together, the separation of POPC and KC2 is supported by the cryo-TEM of

POPC/KC2/cholesterol mixtures [Figure 4.1-b]. However, systems studied in cryo-TEM contained

cholesterol which makes them different from POPC/KC2(H) dispersions studied in simulations. As

mentioned in the Methods sections, addition of cholesterol to POPC/KC2(H) was required to form

nanoparticles which could be imaged easily. On the other hand, these systems are more directly related to

actual LNPs because cholesterol is a major component of LNPs [3].

170
Figure 4.1: KC2 segregation from POPC as a function of pH.

a) Bilayers are composed of POPC and KC2 or KC2H. Snapshots are taken from systems with 70 mol%

POPC. Green and red spheres represent the nitrogen atoms of KC2 and phosphorus atoms of POPC,

whereas the blue and orange lines are representative of KC2 and POPC acyl chains, respectively.

171
Hydrogen atoms, ions, and water molecules are not shown for clarity. The simulation box is shown as a

blue rectangle. The vertical line represents the phosphate-phosphate bilayer thickness defined as the

ensemble averaged distance between the red spheres (phosphorus atoms of POPC) in two POPC leaflets.

b) Cryo-TEM micrographs of POPC/cholesterol (55/45) and POPC/KC2(H)/cholesterol (25/30/45)

mixtures at pH 4.0 and 7.4.

Order parameter ( 𝑆𝐶𝐷 ) profiles from 2H NMR can also be used for characterizing lipid

organization and interactions in lipid mixtures. 2H NMR 𝑆𝐶𝐷 profiles of POPC-d31/KC2(H) systems are

shown in Figure B.2 and Figure B.3. At 288 K, adding KC2H to POPC-d31 increases palmitoyl chain

order [Figure B.2], implying that KC2H intercalates between POPC-d31 molecules as predicted by the

MD simulations. In contrast, adding even 30 mol% KC2 to POPC-d31 has little to no effect on the

conformational freedom of the palmitoyl chain. This suggests that KC2 does not interact with POPC-d31

chains to a significant extent, since KC2H does affect POPC-d31 palmitoyl chain order. Note, though,

that it is possible for amphiphiles to intercalate among phospholipid chains without affecting chain order.

For example, 25 mol% 1-decanol does not affect DPPC palmitoyl chain order parameters [40, 41]. But

given that KC2H significantly orders POPC-d31, despite its charged dimethylamine, it is likely that

KC2’s lack of effect on POPC-d31 chain order stems from a lack of interaction. The observed differences

between POPC/KC2(H) order parameter profiles at low and high pH become less significant with heating

and are insignificant at 313 K [Figure B.3], likely because thermal energy enhances chain fluctuations

and loosens lateral packing.

Our SAXS results can also be interpreted to suggest that KC2 and POPC are segregated [Figure

B.4]. The broadening of scattering peaks upon the incorporation of KC2 implies that the lamellae are

significantly less correlated than in pure POPC, as would be the case if they were interrupted by pools of

172
KC2. KC2 is silent in the SAXS patterns, and is likely separating into oily droplets associated with the

POPC lamellae and thereby disrupting their stacking regularity. As well, the increases in lamellar repeat

spacing observed for POPC/KC2 multilamellar dispersions at pH 8.1 or 8.5 (section B.3) are consistent

with the interbilayer repulsion expected for the small amount of KC2H at these pH values [42].

Both simulations and experiments suggest that KC2 separates from POPC, but the distribution of

KC2 in the system is not the same between two approaches. In simulations, the segregated KC2 lipids are

confined within the bilayer center. This confinement results in a systematic increase in the POPC inter-

leaflet distance [Figure 4.2]; from 3.72 nm to 4.18 nm, 4.44 nm, and 5.32 nm upon raising the KC2 molar

fraction from 0 % to 10 %, 20 %, and 30 %, respectively. The KC2 incorporation in the bilayer center was

not, however, supported by the SAXS experiments. In the cryo-TEM experiments, furthermore, the

neutral KC2 goes to the nano-formulation core [Figure 4.1-b]. This difference in KC2 distribution

between simulations and experiments might be a direct effect of periodic boundary condition in the

simulation. The complete separation of KC2 to the aqueous phase is not energetically favorable (or

allowed) due to the applied boundary condition. Indeed, in the simulations confinement into the bilayer

interior is the only possible scenario, unless a massive system is simulated that allows (energetically

speaking) the KC2 expulsion from the bilayer.

173
Figure 4.2: Systematic increase in POPC inter-leaflet distance upon rising the KC2 concentration at

basic pH level.

Symmetrized electron densities for the phosphorus atoms of POPC were calculated from the simulated

systems with different mixing ratios at neutral/basic pH and T = 313 K. The X axis shows the relative

position from the bilayer center in the direction normal to the bilayer surface. Each double-headed arrow

represents the peak-to-peak distance for a mixing ratio, corresponding to the phosphate-to-phosphate (P-

P) inter-leaflet distance. For the pure POPC at 313 K, the P-P distance is about 3.72 nm.

174
 The KC2 incorporation into the POPC bilayer center is not, however, impossible, and might be

the case for systems with small content of KC2. There are experimental reports on the alkanes segregating

into the middle of bilayers [43], or into the voids in the inverted hexagonal structures [44]. Furthermore,

there are reports on the confinement of water-soluble polymers into the center of bilayers composed of

non-ionic surfactants [45]. There are also computational studies where partitioning of hydrophobic

alkanes and polymers in the bilayer interior has been reported [46]. Although there are numerous

examples on partitioning of both small and large drug molecules into the lipid membranes, there is no

simple explanation for KC2 separation from PC lipids.

Simulation also suggests that the level of KC2 confinement was a function of KC2 concentration

[Figure 4.3]. That being said the KC2 percentage confined in the hydrophobic section is higher for the

system with a higher concentration of KC2. For the 10 mol% KC2, a few percent of the lipids could stay

in the lipid-water interface but as the concentration of KC2 increased to 20 % and 30 %, more and more

KC2 were confined in the membrane center [Figure 4.3].

175
Figure 4.3: KC2 confinement as a function of mixing ratio.

The symmetrized number densities for the KC2 nitrogen atoms and POPC phosphorus atoms are shown at

neutral/basic pH and T =313 K for two mixing ratios: Left) POPC/KC2 (90/10), and Right) POPC/KC2

(70/30). Assuming the bilayers are centered at zero, the densities for the positive positions are only

shown. The X axis shows the direction normal to the bilayer surface. The insets are snapshots taken from

the corresponding systems. The red and green spheres represent the POPC phosphorus atoms and KC2

nitrogen atoms, respectively. The blue box represents the simulation box. Water, ions, and other atoms in

POPC and KC2 are not shown for clarity. System with 30 mol% KC2 seems to have less KC2 headgroups

in the lipid-water interface (shown as the POPC phosphorus atom densities) compared to the system with

10 mol% KC2.

These results suggest that an increase in pH from 4.4 to 7.4/8.1 causes the KC2 to separate from

POPC. Results from POPC/KC2(H) and POPC/KC2(H)/cholesterol systems can be further related to the

LNPs because phosphatidylcholine, KC2(H) and cholesterol are three major components of LNPs.

Therefore, interactions between these molecules play critical roles on the internal structure and

176
consequently the drug delivery efficacy of LNPs. However, LNPs are more complex and contain other

molecules in their formulations. For instance, LNPs contain distearoylphosphatidylcholine (DSPC) which

is different from POPC structurally, and PEG-lipids which assist in stabilizing the LNPs [3]. These

differences could result in different molecular behavior than what was observed in the simple model

systems in this study.

Considering these limitations, our findings suggest that KC2 is the major component forming the

oil droplets observed in the core of LNPs [12, 13]. As proposed elsewhere [13], these oil droplets are

expected to be stabilized by a lipid monolayer on the surface. The material for this covering layer would

be drawn from the rest of the LNP particle, which consequently rearranges the structural components of

the LNP membranes. This rearrangement of the molecules in the LNP could affect the internal structure

of LNP and destabilizes it. Given a certain number of phospholipids present in the formulation, these

destabilized LNPs are fused together and form larger LNPs which are more stable. The underlying reason

for this stability is that, assuming a spherical shape for the LNP, the surface to volume ratio for the

spherical nanoparticles decreases as the radius increases. Thus, larger LNPs will be more stable because

their phospholipid content is now sufficient to cover the surface [13].

The confinement of KC2 inside the LNP might also cause a delay in the detection of endosomal

acidification, and a consequent delay in KC2 protonation and their transfer to the LNP surface. Lack of

sufficient KC2H on the LNP’s surface causes a delay in the LNP’s attachment to and effective

interactions with negatively charged endosomal membrane. Assuming that LNPs attached to the internal

side of the endosomal membrane, the destabilizing step of endosomal membrane will cause another level

of delay to the drug release process.

Despite the neutral KC2 which were segregated from POPC, KC2H stays in the bilayer along

with POPC and interact with other molecules in the lipid-water interface. Both 2H NMR experiments and

simulations suggest that including KC2H in the system slightly induces order in the POPC palmitoyl

177
chain in a concentration-dependent way [Figure B.2 and Figure B.3]. In the simulations, KC2H were

shown to leave the P-P distance almost unaffected [Figure B.5]. Moreover, adding KC2H to the systems

was shown to attract Cl- ions to the membrane surface, and push the Na+ ions far from the lipid-water

interface, as expected [Figure B.6]. It also affects the POPC choline group distribution which

consequently affects the POPC P→N vector orientation [Figure B.7]. These observations are consistent

with the previous reports on other zwitterionic-cationic lipid mixtures [47].

4.5 Conclusions

Our results suggest neutral KC2 is segregated from POPC, while charged KC2H mixes with POPC in the

lipid bilayer. KC2H affects the POPC head group orientation but had no effect on the bilayer thickness.

The segregation of KC2 supports the recently proposed model for LNPs containing KC2 [13]. The

sequestration of KC2 from POPC might also explain why this class of LNPs suffers from a low efficacy

of drug release from endosomes.

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lipid bilayers: Molecular dynamics study. Biophysical Journal, 86(6), 3461-3472.

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 Chapter Five: Structural Properties of Inverted Hexagonal Phase: A Hybrid

Computational and Experimental Approach

Contributions:

I ran and analyzed all the simulations and wrote the major part of this chapter. I had major contributions

in methodology, data interpretation, hypothesizing, model development and validation, and visualization.

The experimental data and methods sections were provided by Dr. Jenifer L. Thewalt, Dr. Miranda L.

Schmidt, and Bashe Y. M. Bashe at the Simon Fraser University, and by Dr. Paul E. Harper, Jason R.

Pruim, and Matthew L. Link at the Calvin College.

Abbreviations:

HII, reverse/inverted hexagonal phase; APL, area per lipid; MD, molecular dynamics; DOPE, 1,2-

dioleoyl-sn-glycero-3-phosphoethanolamine; PE, phosphatidylethanolamine; POPE, 1-palmitoyl-2-

oleoyl-sn-glycero-3-phosphoethanolamine; SCD, deuterium order parameter for C-D bond; SAXS, small-

angle X-ray scattering; dhex, lattice distance; Rw, water core radius; nw, water molecules per lipid inside

the lipid cylinder (or equivalently hydration level); nc, number of lipids per slice along the lipid cylinder;
2
H NMR, deuterium nuclear magnetic resonance

5.1 Abstract

Inverted/reverse hexagonal (HII) phases are of special interest in several fields of research, including

nanomedicine. Study of the structural properties of HII phase at low hydration levels, as well as

determination of the internal dimensions of HII structure in presence of excess water could be challenging

experimentally.

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 We used molecular dynamics (MD) simulation to study HII systems composed of DOPE and

POPE at several hydration levels and temperatures. The effect of hydration level on several HII structural

parameters, including deuterium order parameters, was investigated. We further used MD simulations to

estimate the maximum hydrations of DOPE and POPE HII lattices at several given temperatures. Finally,

the effect of acyl chains on HII structure was studied via comparing the DOPE with POPE HII systems.

In addition to MD simulations, we used deuterium NMR (2H NMR) and small-angle X-ray scattering

(SAXS) experiments to measure the DOPE acyl chain order parameters, lattice spacings, and the water

core radius in the DOPE HII systems at several temperatures in the presence of excess water.

Structural parameters calculated from MD simulations are in excellent agreement with the

experimental data. In addition, the water core radius calculated directly from MD simulations agrees with

the estimated value based on electron density profiles in SAXS. Dehydration is shown to increase the

lipid cylinder length and decrease the radius of water core. An increase in hydration level slightly

increased the deuterium order parameter of lipids acyl chains, whereas an increase in temperature

decreased it. Lipid cylinders were shown to be undulated along the cylinder axis as a function of

hydration level. Provided that a single experimental measurement for the lattice distance be available in

the presence of excess water, the maximum hydration of PE HII phases at the corresponding temperature

were successfully predicted by MD simulations. An increase in temperature were shown to decrease the

maximum hydration, and consequently the radius of water core and lattice distances. Finally, DOPE was

shown to form HII structures with a higher curvature compared to POPE, as expected. We propose a

general protocol for constructing computational HII systems corresponding to the experimental systems.

This protocol could be used to study HII systems composed of other molecules than PEs, for which

experimental data are not available.

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5.2 Introduction

Inverted/reverse hexagonal (HII) phase is a non-lamellar mesophase formed by self-assembly of

amphiphiles (e.g. lipids, peptides, co-polymers) dispersed in polar solvents, e.g. water. Lipid tubules are

filled with water and ions, and are arranged in a hexagonal geometry [1] [Figure 5.1]. Given its special

geometry, HII phase has been of special interest for its (potential) use in several research areas [1-7],

including drug/gene delivery systems [8-10] and drug release mechanism [11-14].

Structural properties of HII has been the subject of many experimental studies. Lattice distance

(dhex), radius of water core (Rw), area per lipid (APL), deuterium order parameter (SCD), and shape of the

water core are a few examples of the parameters studied using experimental techniques [15-20]. Several

factors have been shown to affect the HII structure significantly, including hydration level, temperature,

lipid type and mixing ratio.

Experimental studies on structural characterization of HII are usually conducted in the excess

water regime [Figure 5.1], where two phases coexist. The first phase is the HII lattice formed from lipid

cylinders filled with a certain number of water molecules (referred as the maximum hydration) allowed.

The excess water forms the second phase; a bulk of free water molecules outside of the HII lattice.

Estimation of volume fraction of water in each phase is not readily doable [15]. However, volume fraction

of water is required to determine internal dimensions of the HII phase. There are several experimental

approaches and methodologies developed to assist in determination of maximum hydration and internal

dimensions of HII phase. One traditional approach, developed by Tate and Gruner [15], is to conduct a

series of SAXS experiments in low hydration levels and measure the dhex at each hydration level. The

hydration level which dhex saturates is considered as the maximum hydration taken up by the HII lattice.

An alternative approach is based on the diffraction pattern in the SAXS scattering curves [16, 17, 21].

Using the methods developed by Turner and Gruner [16] and Harper et al. [17, 21], the peak intensities in

a scattering curve can be used to reconstruct the electron density profile for the HII structure, from which

187
the Rw can be estimated. Given these values, the internal dimensions of the HII, including the volume

fraction of water in the HII lattice, can be calculated using geometric considerations and the symmetry of

HII phase. In this approach, a single sample prepared in presence of excess water is enough to extract the

HII structural dimensions. This method was shown to result in structural parameters in close agreement

with the traditional method by Tate and Gruner [15], although in a relatively easier way.

Experimental approaches are usually limited in terms of resolution. Regarding the HII phase,

conducting experiments at low hydration levels are usually challenging. It is also challenging to study the

effect of several factors independently. For instance, an increase in temperature results in a decrease in

maximum hydration of HII phase, and therefore the lattice distance [15, 17]. Therefore, to what extent the

structure of HII system and its comprising molecules are affected by hydration level or temperature is

difficult to assess experimentally.

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Figure 5.1: HII phase in experiments and simulation.

Right) A schematic view for a test tube containing lipid/water dispersions in experiments. HII phase

containing lipids and water molecules form at the bottom of the tube. The excess waters are located on the

top of lipid dispersions, outside of the HII lattice. Left) A schematic view of the part of lipid dispersion

which is modeled and used for the simulations. The blue box represents the simulation box containing one

single lipid tubule and its corresponding water channel. HII phase is a group of these lipid tubules

arranged in hexagonal geometry. The tan colored spheres represent the phosphorus atom of phospholipids

whereas the red spheres are the carbon atoms of CH3 groups at the end of each acyl chain. Water

molecules inside the central lipid tubule is represented as the cyan dots. The rest of the atoms, as well as

the water molecules in the neighbor cells are not shown for clarity. The pink, yellow, and white lines are

the radius of water core (𝑅𝑤 ), lattice distance (𝑑ℎ𝑒𝑥 ), and unit cell spacing (𝑎), respectively. The white

and orange arrows represent the interstitial and interaxial directions, respectively. VMD [22] was used to

make the figure.

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 Computer simulation is a complementary approach to experimental techniques, which can

provide additional details in a molecular level and be used to study each effect independently [23].

Molecular dynamics simulations have been previously used to study the structural and dynamical

properties of HII phase [24-26] and lamellar-to-HII phase transition process as a function of hydration,

temperature, and mixing ratio [27, 28]. There are also computational studies of HII phase used to

calculate the bending free energies, spontaneous curvatures of lipid monolayers, and bending modulus

[29, 30]. It has also been used to determine the pivotal plane - the plane at which the area per molecule at

the given curvature is the same with the flat bilayer - for lipid molecules [31].

Ideally and in most cases, comparing the results from simulations with experiments requires

equivalent molecular systems in both approaches. However, construction of such equivalent systems in

HII phase are not straightforward, especially for systems for which experimental data are not available.

Herein again, one of the key challenges is that the maximum hydration at a certain temperature is not

always known for an arbitrary molecule/mixing ratio. Assuming the hydration level is known, it is still

challenging to make reasonable initial guesses for the water core radius and other HII internal dimensions

to construct the HII structure with. Assuming that both hydration level and system dimensions can be

determined, construction of stable HII systems with the desired mixing ratio is another challenge to

overcome. There are several computational tools designed to assist in overcoming the last challenge [32-

35]. CHARMM-GUI is a popular online toolkit for construction of molecular systems to the pre-

production run step for a variety of systems. However, there are shortcomings when simulation of HII

phase is of interest. For instance, not all the lipids or molecules (e.g. copolymers, detergents, synthetic

lipids, and peptides) are available in the CHARMM-GUI library. In addition, CHARMM-GUI requires

alkanes to be added to the corners of the hexagonal box, does not provide enough control of the number

of waters inside the HII channels, and does not support adding other solvents than water to the HII phase.

190
The output files given by CHARMM-GUI are only for all-atom CHARMM36 force field (C36 FF) but

not other force fields (FFs). These output files are for use in the NAMD package and can not be used in

the GROMACS package directly. Finally, the hexagonal box shape given by CHARMM-GUI is not yet

supported in GROMACS package.

Therefore, the first objective of this study was to develop a general protocol for construction and

simulation of HII systems composed of amphiphilic molecules using the minimum amount of

experimental data. The second goal was to evaluate the level of agreement between the structural

parameters, including the maximum hydration at a given temperature, predicted by this protocol and the

known experimental values. Finally, it was interesting to see whether this protocol could reproduce the

expected structural differences between structurally similar molecules in HII phase (i.e. sensitivity of the

protocol and simulations in reproducing the structural properties in HII phase).

In this study, we used MD simulations to investigate the structural properties of DOPE and POPE

HII systems as a function of hydration and temperature. Phosphatidylethanolamines (PEs) such as DOPE

and POPE are known to form HII phases at high temperatures and low hydration levels [36]. Inverted

hexagonal systems composed of DOPE and POPE have been extensively studied experimentally, which

could be used for both validation of our protocol and comparing the structural parameters with

simulations. Small angle X-ray scattering experiment was used to measure the lattice distance and water

core radius in DOPE HII phase at several temperatures. Also, deuterium NMR ( 2H NMR) was used to

measure the deuterium order parameter (SCD) for the last carbon atoms in DOPE acyl chains. Several

structural parameters of DOPE and POPE HII systems, including water core radius and SCD, were

calculated from MD simulations and compared with experimental data. The results from DOPE and

POPE simulations at 323 K were further compared to investigate the effect of different acyl chains and to

evaluate the sensitivity of our protocol. Molecular dynamics simulations were also used to estimate the

maximum hydration for both DOPE and POPE HII systems at several temperatures, and to investigate the

191
effect of temperature on both lattice distance and maximum hydration. Finally, a protocol for constructing

HII molecular systems equivalent to the experimental systems is presented.

5.3 Methods

Although experimental and computational data on both DOPE and POPE HII systems are available, none

of these data were used in construction of the HII molecular systems. This way, we could validate our

protocol and see whether MD simulations could blindly reproduce the experimental data.

For each desired water per lipid ratio inside the cylinder (nw), an initial lipid cylinder filled with

required amount of water molecules was constructed (see below for details). Next, each system was

relaxed during the simulation to adjust its radius and length. Based on temperature, lipid type, and n w,

lipid cylinders were either stretched or shrunk along the cylinder axis during the equilibration step, as a

direct result of simulations. The structural parameters were then calculated from these equilibrated

systems and compared with available experimental data.

As mentioned in the caption of Figure 5.1, the excess water in the experimental setups is not

modeled in simulations. Therefore, any water exchange between the excess water and waters inside the

HII lattice is prohibited in simulations. This water exchange, however, is possible in the experiments, for

instance upon a change in the temperature [15].

5.3.1 Computational method.

5.3.1.1 System construction.

A single tube composed of either DOPE or POPE lipids was constructed and oriented in the Z direction

using GROMACS [37] modules, as described in section 2.5.2 in Chapter 2 [38]. Next, CHARMM-GUI

Solvator module [39] was used to build a water column with the same dimensions and orientation. The

192
atomic coordinates from these two systems were subsequently merged to give a water channel covered by

lipids. Water molecules were gradually deleted to give the system with the desired n w. A triclinic box was

employed for the simulations and periodic boundary conditions (PBC) were applied in all the three

directions. A few lipid and water molecules were deleted from several DOPE HII systems due to bad

contacts. This resulted in slightly higher hydration for DOPE HII systems than POPE (see Table 5-1 for

details). Each system was subsequently energy minimized using steepest descent algorithm and used for

the next steps, i.e. equilibration and production runs.

Preliminary simulation suggested that system with 4 nw tends to deform from its cylindrical

geometry and might not be stable in HII phase. For this system, therefore, a larger system composed of

four lipid cylinders in a rectangular box [40] was simulated instead to allow any possible phase transition

to happen if energetically favorable.

Table 5-1: Simulated HII systems.

Lipid Pressure Coupling Temperature Water per lipid Simulation time

(K) (nw) (ns)

DOPE Anisotropic 283, 303, 323 10.26, 14.35, 16.41, 20.26, 500

22.55, 24.61, 26.65, 30.77

DOPE Semi-isotropic 283, 303, 323 10.26, 20.26, 30.77 300

DOPE Anisotropic 4.10 300

, 283, 303, 323 Note: Systems were composed

Semi-isotropic of four cylinders with 1404

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 lipids (351 lipids per cylinder)

POPE Anisotropic 323, 348, 358 10, 14, 16, 20, 22, 24, 26, 30 500

5.3.1.2 Molecular dynamics simulation.

GROMACS version 2016.3 [37] was used for all the simulations. The force field parameters for the

molecules in the system were taken from CHARMM36 FF version Nov.2016 [41], and the standard

TIP3P water model [42, 43] was used for these simulations.

Each energy minimized system was first simulated under NPT ensemble. An anisotropic pressure

coupling was applied, to control the pressure in each direction independently, using the Berendsen [44]

weak coupling algorithm. Next, the output structures were used for the longer equilibration and

production runs of either 300 ns or 500 ns time lengths [Table 5-1].

For the production runs, a time-step of 2 fs was employed, and atom coordinates were saved

every 100 ps. The van der Waals interactions were switched off and cut-off at 0.8 and 1.2 nm,

respectively, and force-switch function was used to smoothly switch off the interactions. The short-range

electrostatic interactions were cut-off at 1.2 nm and PME [45, 46] was used for treatment of the long-

range electrostatic interactions. Lipid cylinders and solvent were coupled to the heat bath separately.

Temperature was controlled using Nose-Hoover coupling [47, 48] with a time-constant of 1.0 ps.

Parrinello-Rahman extended-ensemble pressure coupling [49, 50] with a time-constant of 5.0 ps was used

to control the pressure. For most of the simulations in this study, an anisotropic pressure coupling was

used, which requires 6 values for each compressibility and reference pressure. A compressibility of 4.5e-5

bar -1 and a reference pressure of 1.0 bar were used for all the diagonal components (i.e. XX, YY, and ZZ)

of compressibility and reference pressure matrices, respectively, whereas all the off-diagonal components

(i.e. XY/YX, XZ/ZX, and YZ/ZY) in these matrices were set to zero. This combination of parameters

194
maintained the box shape through the simulation course while still allowing the box sizes to scale in each

direction independently. In cases, where the effect of pressure coupling was of interest, several systems

were also simulated using the semi-isotropic coupling [Table 5-1]. Assuming the lipid cylinders are along

the Z axis, a semi-isotropic coupling allows the Z direction and XY-plane to be scaled independently.

Herein again, all the off-diagonal components in both compressibility and pressure matrices were set to
-1
zero, whereas a compressibility of 4.5e-5 bar and a reference pressure of 1.0 bar were used for the

diagonal components. LINCS [51] was employed to constrain all the bonds involving hydrogen atoms.

Visual Molecular Dynamics (VMD) [22] and Xmgrace were used to generate the figures and graphs,

respectively, while Python 3.6.0 and Excel was used for some of the analysis.

5.3.1.3 Simulation analysis.

Several structural parameters of HII systems were calculated from the last 100 ns of each simulation. The

data corresponding to every 100 ps in both structure and energy files were used for the analysis, resulting

in a total of 1000 data points for calculation of averages and standard deviation of means. Each of the

following structural parameters was first calculated for each frame. Then the average and standard

deviations of mean were calculated from these data and shown as the error bars on the corresponding

graphs. The following sections explain the process (in order) by which these parameters were calculated.

First, the PBC effects in simulations were fixed using gmx trjconv module of GROMACS

package; all the broken molecules were made as whole (using the -pbc whole option), and all the atoms in

the system were put at the closest distance from the simulation box center (using the -ur compact option).

Next, the system was centralized in the simulation box (using -center option).

195
Deuterium order parameter:

Given the PBC effects being fixed, the SCD parameters were calculated according to the formula in

Equation 5-1.

< 3𝑐𝑜𝑠 2 𝜃 − 1 > Equation 5-1

|𝑆𝐶𝐷 | = | |
2

Where θ is the angle between the C-D bond in the methylene group and the Z axis (i.e. the cylinder axis

which is the lipid’s symmetry axis in HII phase). The angular brackets represent the ensemble average. A

script provided by Dr. Thomas Piggot [52] was used to calculate the 𝑆𝐶𝐷 from simulations. This script is

based on the actual angle between the C-H bond and the Z axis. Since the cylinder axis is aligned with the

Z axis, this angle will be the angle between the C-D bond and the cylinder axis (see next paragraph).

Experimentally, the SCD parameters are calculated from the quadrupolar splittings in the de-Paked
2
H NMR spectra [53, 54] (see section 5.3.2.4 and Equation 5-9). These de-Paked spectra correspond to

the sample orientations which lipid’s symmetry axis is parallel to the magnetic field direction. For

lamellar phase, this means that the bilayer normal is aligned with the magnetic filed, therefore the θ is the

angle between the C-D bond vector and the bilayer normal. For the HII phase, the lipid’s symmetry axis is

the cylinder axis which is oriented parallel to the magnetic field direction after de-Pake-ing. Therefore, if

the same formulae in Equation 5-1 and Equation 5-9 are going to be used for HII phase, the angle θ will

represent the angle between the C-D bond vector and the cylinder axis [53, 54].

Structural dimensions of HII systems:

Assuming a cylindrical geometry for the lipid tubules in the HII phase, Figure 5.2 illustrates several

structural parameters calculated from MD simulations in this study. As it is often assumed in HII phase

196
studies, lipids are assumed to be distributed radially around the cylinder axis, and their long axis is on a

plane approximately perpendicular to the cylinder surface [18].

This cylinder is covered by Nlipids of lipid molecules, filled with Nwater water molecules. This

molecular composition result in a HII system with Nwater/Nlipids of water per lipid inside the cylinder

(referred as nw in this study), a radius of Rw, and a length of Z along the cylinder axis. Each lipid, with

headgroup pointing towards the water core is taking an average area per lipid of APL at the lipid-water

interface [Figure 5.2].

197
Figure 5.2: Definitions for several structural parameters calculated from MD simulations.

A single lipid tubule in the HII phase. Rw, APL, and Z represent the radius of water core, area per lipid at

the lipid-water interface, and the instantaneous length of lipid cylinder along the cylinder axis,

respectively. The solid blue cylinder represents the water core, whereas the dotted green cylinder

represents the pivotal plane for this lipid at this curvature. The pivotal area per lipid for DOPE (at 275 K)

and POPE (at 348 K) is estimated to be ca. 0.65 nm2 [19, 55], from which its square root (~ 0.806 nm) can

be taken as the approximate average length for a PE lipid along the lipid cylinder [18]. In a study by Rand

and Fuller [18], the average number of DOPE were counted for a length of L = 0.806 nm slice along the

cylinder axis (slice colored in green). The same value (referred to as nc in this study) was calculated from

simulations to compare.

198
 The gmx energy module of GROMACS was used to extract the length of simulation box in X, Y,

and Z directions from the energy file (.edr). The average length of the simulation box in Z direction was

calculated and taken as the average length of lipid cylinder <Z> [Figure 5.2].

The number of lipids per slice of length L = 0.806 nm along the lipid cylinder (nc) were obtained

using Equation 5-2:

(𝑁𝑙𝑖𝑝𝑖𝑑𝑠 ) (0.806) Equation 5-2

𝑛𝑐 =
𝑍

Where Nlipids is the number of lipids forming the lipid cylinder, and Z is the instantaneous length of lipid

cylinder measured in nm.

The lattice distance (dhex) for each system was calculated using simulation box sizes in X and Y

directions [Equation 5-3].

Equation 5-3
𝑑ℎ𝑒𝑥 = ((√3⁄2) 𝑋 + 𝑌) /2

This was done for each time frame and the average was reported in this study. The standard deviations of

means were calculated from this data and shown as the error bars in the corresponding figures.

For the rest of structural parameters, the volume fraction of lipids (𝜑𝑙 = 𝑉𝑙𝑖𝑝𝑖𝑑𝑠 / 𝑉𝑡𝑜𝑡𝑎𝑙 ) and

waters (𝜑𝑤 = 1 − 𝜑𝑙 ) were required. In this equation, 𝑉𝑡𝑜𝑡𝑎𝑙 is the total volume of the simulation box,

and 𝑉𝑙𝑖𝑝𝑖𝑑𝑠 is the volume taken by the lipids in the system. To calculate 𝜑𝑙 and 𝜑𝑤 , the following

approach was taken. First, a system composed of 15000 water molecules was simulated at four

temperatures (288 K, 303 K, 313 K, 348 K) and the volume for a single water molecule at each

199
temperature was calculated. A linear fit to these four volumes was made using Xmgrace, from which the

volume for a single water molecule at all the temperatures of interest was extracted. Given the total

number of water molecules inside the lipid cylinder, the volume taken by the water molecules inside a

cylinder was calculated. Next, the total volume of the simulation box (𝑉𝑡𝑜𝑡𝑎𝑙 ) for each HII system was

calculated using gmx energy module of GROMACS. The difference between the total volume and volume

taken by the water molecules was taken as 𝑉𝑙𝑖𝑝𝑖𝑑𝑠 . Given all this data, the 𝜑𝑙 and 𝜑𝑤 was calculated for

each system and were used for calculation of water core radius (Rw) and area per lipid (APL) at the lipid-

water interface as follows.

Assuming the water cores are cylindrical in shapes, the radius of water core was calculated

according to Equation 5-4 [15].

Equation 5-4
𝑅𝑤 = 𝑎 √𝜑𝑤 ( √3 / 2 𝜋)

Where 𝑎 is the unit cell spacing (spacing between adjacent cylinders) [Figure 5.1] and is equal to

2
( 3) dhex.
√

Finally, the area per lipid (APL) at the lipid-water interface (corresponding to Rw) was calculated

using Equation 5-5 [18].

𝐴𝑃𝐿 = (√3 𝜋 𝑅𝑤 𝑉𝑙 )⁄(𝑑ℎ𝑒𝑥 2 𝜑𝑙 ) Equation 5-5

Where 𝑉𝑙 is the volume taken by a lipid molecule in the system.

In both simulations and experiments [Figure 5.3], Rw and APL parameters were calculated based

on geometrical assumption that the lipid tubules are cylindrical in shape and have an approximate uniform

200
circular cross section along the Z direction [18, 56]. In this study, the SAXS scattering curves were

further analyzed to reconstruct the electron density profiles for the HII systems (see Section 5.3.2.3) [17].

From these electron densities, the water core radius at the water-lipid interface was estimated. The peaks

in these electron density profiles correspond to the DOPE phosphate groups [Figure 5.4-left]. The water

core radius for one of the simulated systems was also calculated from simulations as described below and

compared with the experimental data. We chose DOPE HII system at 303 K for comparison because the

maximum hydration for DOPE at 303 K is about 16 nw [15] and simulation for this specific system was

conducted in this study. Herein again, this analysis was done on the last 100 ns of the simulation with

frames saved every 100 ps. Considering that the lipid tubules in simulations are not perfectly cylindrical

and straight along the Z direction, the following approach was taken to calculate the water core radius

based on the probability distribution of DOPE phosphorus (P) atoms.

First, the coordinates of DOPE P atoms for each frame was saved separately resulted in 1001

structure files; each structure was centered in a cubic box. For this specific system, the average length of

lipid cylinder was about 14 nm. Thus, each structure was divided into 14 slices/rings; each ring had an

approximate length of 1 nm, resulted in 14014 rings in total. Each ring was subsequently centered in the

box so that the P atoms were radially distributed around the Z axis in the XY-plane. The radial distance

for each P atom from the Z axis was then calculated based on its coordinates:

𝑅𝑎𝑑𝑖𝑎𝑙_𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 = √(𝑋 2 + 𝑌 2 ) Equation 5-6

Next, the average radial distance of P atoms from the center was calculated for each ring. Finally, a

histogram was made from these 14014 average values as shown in [Figure 5.4-right].

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5.3.2 Experimental methods.

5.3.2.1 Sample preparation.

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) for SAXS was obtained in powder form from

Avanti Polar Lipids Inc. (Alabaster, AL). The DOPE was transferred from the sealed glass ampule into a

scintillation vial using an 80/20 (v/v) benzene/methanol mixture. The lipid/solvent mixture was frozen in

liquid nitrogen for approximately ten minutes and then the solvent was removed by lyophilization over a

period of about 12 hours (until the weight had stabilized). The result was a condensed, powdered lipid

that could be weighed for sample preparation. When not in use, lipids were stored at -20 °C. In order to

prepare the sample for SAXS analysis, 100 μL of deuterium depleted water (ddw) (Sigma-Aldrich

Canada (Oakville, ON)) was added to 10 mg of dry, powdered DOPE. A series of five freeze (in liquid

N2) – thaw (room temperature) – vortex cycles were performed to ensure full hydration of the hexagonal

phase. The sample was carefully transferred into to a disposable thin-walled quartz capillary tube (80 mm

long, 1.5 mm (outer diameter), 0.01 mm thickness) which was then sealed with wax and analyzed

immediately.

For the 2H NMR experiments, partially deuterated DOPE-d18 was purchased in chloroform from

Avanti Polar Lipids Inc. (Alabaster, AL). In DOPE-d18, the protons on the last four carbons of each chain

(carbons 15-18) were replaced with deuterons. The DOPE-d18/chloroform mixture was transferred to a

pre-weighed scintillation vial and the bulk of the solvent was evaporated using a gentle flow of nitrogen

gas. Any residual solvent was then removed under vacuum over the course of 6 hours (until the weight

had stabilized). The resulting lipid film was used in its entirety to prepare the 2H NMR sample. The

approximately 41 mg of DOPE-d18 lipid were mixed with 700 μL ddw and a series of five freeze (in

liquid N2) – thaw (room temperature) – vortex cycles were performed. The hydrated lipid mixture was

transferred into an NMR tube for analysis.

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5.3.2.2 Small angle X-ray scattering (SAXS).

A SAXSLAB Ganesha 300XL (Skovlunde, Denmark) setup with a Linkam variable temperature sample

stage was used to obtain the SAXS scattering curves. The Ganesha 300XL system has a Cu-Kα X-ray

source with a wavelength λ= 1.54 Å. The sample-to-detector distance for the Linkam stage setup was

calibrated using AgBeh and the sample temperature was calibrated using the pre-transition and chain

melting transition of both DPPC and DMPC bilayers. At each temperature, two sets of data were

collected: one after 30 minutes of equilibration, and a second 20 minutes after the completion of the first

acquisition (about an hour after the setpoint temperature was reached). Each data collection run was 600

s. Both the initial and final runs were collected at the same temperature to monitor sample stability across

the experiment.

The lattice distance (dhex) can be measured directly from the SAXS scattering curves via

2𝜋𝑛 Equation 5-7

𝑑ℎ𝑒𝑥 =
𝑞𝑝𝑒𝑎𝑘

where qpeak is the position of the peak in the scattering curve and n is its order number which is dictated

by the symmetry of the system. The Miller indices to define the hexagonal phase peaks can be written in

terms of h, and k, as (h,k). The corresponding order number for each peak is 𝑛 = √ℎ2 + ℎ𝑘 + 𝑘 2 . For the

hexagonal phase the order numbers for the peaks follow the sequence 𝑛 = 1, √3, 2, √7, 3, √12, … [57].

5.3.2.3 Electron density reconstruction and water core radius estimation.

Diffraction data was used to build electron density reconstructions by established techniques, as

detailed in Harper et al. [17, 21]. Briefly, X-ray diffraction patterns were radially integrated, and

203
the peaks fitted to Gaussians. After applying the Lorentz and multiplicity corrections to the peak

intensities, the peak amplitudes were extracted. In order to complete the electron density

reconstructions, the proper phasing or sign for each of the peak amplitudes must be obtained. For

DOPE in excess water, it is well established that the proper phasing is “+,-,-,+,+,+,+” [16, 17].

The electron density can then be succinctly written as

ℎ,𝑘 𝑚𝑎𝑥

𝜌(𝒓) = ∑ 𝛼ℎ𝑘 𝐹ℎ𝑘 cos(𝒃ℎ𝑘 ∙ 𝒓)

(ℎ,𝑘)≠(0,0) Equation 5-8

where ρ(r) is the electron density relative to the mean electron density as a function of position r,

h and k are the Miller indices, αhk and Fhk are, respectively, the phase and amplitude for each set

of Miller indices and bhk are the reciprocal lattice vectors.

The electron density profiles along the (1,0) and (1,1) directions, as well as the 3-D

electron density profile in the unit cell were then reconstructed. It has been shown that the peak

maxima fortuitously correspond to the location of the Luzatti interface [16], which is the

boundary between lipid and water when they are modeled as separate, non-overlapping

components. Hence, the average radius of the peak maxima of the electron density reconstruction

was used as the water core radius.

5.3.2.4 Deuterium NMR (2H NMR).

2
H NMR experiments on DOPE-d18 were carried out using an Oxford 300 MHz magnet with a 2H

frequency of 46.8 MHz and a TecMag Scout (TecMag, Inc. (Houston, TX)) spectrometer. The standard

204
quadrupolar echo pulse sequence with two out of phase 90° pulses was used. The 90° pulses were 3.95 μs

long with an interpulse delay of 40 μs and a recycle delay of 2 s. To eliminate artefacts, data was

collected using 8-cycle CYCLOPS phase cycling. These 2H NMR experiments were performed at a series

of temperature ranging from 20 °C to 40 °C where DOPE is in the hexagonal phase. Between each 5

degrees temperature increment the sample was allowed to equilibrate for 45 minutes before the 20,000

scan experiment was run. Again, the first and last experiments were run at the same temperature to ensure

that there was no sample degradation. 20,000 scans provided a sufficient signal-to-noise ratio in the

resulting de-Paked spectra for the order parameters to be determined. De-Pake-ing was performed using

the iterative method as described by Sternin et al. [53]. Smoothed order parameter profiles for the C-D

bonds were obtained using the method outlined by Lafleur et al. [54]. The order parameter, SCD can be

calculated using the relationship

4 ℎ Equation 5-9
|𝑆𝐶𝐷 | = 𝛥𝑣𝑄
3 𝑒 2 𝑞𝑄

𝑒 2 𝑞𝑄
where ℎ
≈ 167 kHz and Δ𝑣𝑄 is the quadrupolar splitting of the C-D bond. The quadrupolar splittings

for carbons 15-17 on the sn-1 and sn-2 chains were calculated using a fitting function for the 12

deuterons. The left- and right-side data were averaged at each temperature. The twelve ΔνQ values

obtained, which were organized from largest to smallest, were assigned pairwise to the carbons from the

sn-1 and sn-2 chains. The first two were assigned to the carbon 17 sn-2- deuterons, the next two were

assigned to the same carbon on the sn-1 chain, this pattern was continued alternating between the sn-2 and

sn-1 chains and decreasing carbon number until the peaks for the carbon 15 sn-1 chain were assigned.

205
5.4 Results

The results presented in the following sections are calculated from simulations conducted using

anisotropic pressure coupling. Effects of using semi-isotropic pressure coupling on HII structures and

their comparison with anisotropic simulations are discussed in Appendix C. Briefly, for hydration levels

less than 20 nw, the dhex values and therefore other structural properties calculated from dhex agreed

between anisotropic and semi-isotropic simulations.

5.4.1 HII structural parameters as a function of hydration.

One of the main objectives in this study was to see whether the constructed HII systems were correct and

MD simulations could reproduce the experimentally known structural parameters of HII system. To do so,

several structural parameters were calculated from DOPE HII simulations and are shown as a function of

hydration level (nw) in Figure 5.3. Experimental data from Rand and Fuller [18] are also shown for

comparison.

Structural parameters calculated from simulations are in excellent agreement with experimental

data [Figure 5.3]. In both simulations and experiments, there is a linear increase in dhex, Rw, and nc

parameters with hydration level [Figure 5.3, a-c]. For the APL, a logarithmic fit described the observed

trend in Figure 5.3-d better according to the chi-squared values for the fitted lines. APL increases as the

hydration increase and reaches a plateau at approximately 20 nw, which the APL is estimated to be about

50 A2 at. Among these four parameters, the Rw was the most affected by hydration level. An increase of

18 nw in the hydration level caused about 400 %, 75 %, 100 % and 100 % increase in R w, dhex, nc, and

APL, respectively.

206
Figure 5.3: Structural parameters for DOPE HII system as a function of hydration.

Data represent the average a) number of PE lipids per 0.806 nm slice along the cylinder axis (see Figure

5.2 for definitions), b) area per lipid, c) radius of water core, and d) lattice distance. Numbers on the X

axis are the number of water molecules inside the lipid cylinder per lipid. The maximum hydration for

207
DOPE at 303 K is about 16 nw [15]. Black circles and green diamonds represent data calculated from MD

simulations (T = 303 K) and the experimental data (T = 295 K) from Rand and Fuller [18], respectively.

Lines are linear (a, c, d) or logarithmic (b) fits to each data sets. Error bars are the standard deviation of

means for simulation data, and their size is comparable with the symbol sizes. See Figure 5.5 for POPE

and DOPE HII systems at 323 K.

The Rw for DOPE HII systems were also calculated from the electron density profiles

reconstructed from SAXS scattering curves. The three- and two-dimensional electron density profiles for

DOPE system at 303 K are shown in Figure 5.4-left. The peaks in these profiles correspond to the

phosphate groups of DOPE. The radial distance of these peaks from the center of water core are 19.91 and

20.22 angstroms in (1,0) and (1,1) directions, respectively. The average, 20.07 angstroms, was taken as

the average Rw for this system at this temperature.

The Rw for the corresponding system was also calculated from MD simulations via calculating

the probability distribution of DOPE P atoms with respect to the lipid cylinder axis [Figure 5.4-right].

System with 16 nw at 303 K was chosen for this analysis [15]. The distribution reaches its maximum

around 20.01 angstroms, in an excellent agreement with the experimentally determined value. Both the

Rw from electron density profiles and the one calculated directly from MD simulations are also in good

agreement with Rw calculated from geometrical assumptions (~ 20 angstroms) shown in Figure 5.3-c.

208
Figure 5.4: Radius of water core from reconstructed electron density profiles and MD simulation.

Left) The two-dimensional electron density profile for DOPE HII system at 303 K is shown in (1,0) and

(1,1) directions. The inset shows the three-dimensional profile for the same system. Right) The

probability distribution of DOPE P atoms calculated from MD simulations from DOPE HII systems with

16 nw at 303 K.

5.4.2 Structural differences between DOPE and POPE HII systems.

DOPE has a lower bilayer to HII phase transition temperature than POPE [36, 58], which is due to

structural differences between the two lipid types. While DOPE has two oleoyl chains, POPE has one

oleoyl and one palmitoyl chain. Therefore, at a given temperature (e.g. T = 323 K), DOPE is expected to

form HII structures with higher curvature and smaller Rw compared to POPE. To see whether these

structural differences and expectations in HII phase can be reproduced by MD simulations and the

protocol used, simulations for DOPE and POPE HII systems were compared at 323 K [Figure 5.5].

The overall trends for all the structural properties are similar between DOPE and POPE. POPE

HII systems have a slightly higher nc, Rw, and dhex but lower APL than DOPE at each hydration level.

209
POPE HII systems contain approximately one more lipid per slice than DOPE for low hydration levels.

As the hydration level increases, this difference also increases from one to two molecules.

Figure 5.5: Structural parameters of DOPE and POPE HII systems as a function of hydration.

210
Data represent the average a) number of PE lipids per 0.806 nm slice along the cylinder axis (see Figure

5.2 for definitions), b) area per lipid, c) radius of water core, and d) lattice distance. Numbers on the X

axis are the number of water molecules inside the lipid cylinder per lipid. Black circles and red squares

represent data for DOPE and POPE, respectively. Lines are linear (a, c, d) or logarithmic (b) fits to each

data sets. Error bars are the standard deviation of means for simulation data, and their size is comparable

with the symbol sizes. Data corresponds to the temperature of 323 K for both DOPE and POPE HII

systems.

A change in the nc as a function of dehydration is expected to be associated with a change in the

length of lipid cylinders [18]. To study the effect of dehydration on lipid cylinder elongation, we further

calculated the average length of lipid cylinders for DOPE and POPE HII systems from simulations for

several hydration levels and compared [Figure 5.6]. For both lipid types, dehydration caused an increase

in the average length of lipid tubules along the cylinder axis. For instance, removing water molecules

from 20 nw to 10 nw resulted in ~ 61 % increase in the DOPE cylinder length. Moreover, POPE cylinders

were slightly shorter in length than DOPE cylinders.

211
Figure 5.6: Cylinder length in DOPE and POPE HII systems as a function of hydration.

Average size of simulated lipid cylinder in Z direction (see Figure 5.2 for definitions) is calculated from

simulations and plotted versus hydration level (nw). Lines are the inverse (1/(aX+b)) curves fitted to the

data, where a and b are two fitting parameters. Error bars represent the standard deviation of means. Data

corresponds to the temperature of 323 K for both DOPE and POPE HII systems.

5.4.3 Effect of hydration level on shape of lipid cylinders.

The water core of HII systems are usually assumed to be cylindrical in geometry for systems with a dhex

of ~ 65 angstrom or less [16]. There are also experimental studies that suggest the lipid tubules in HII

phase are not perfectly straight [59]. This further motivated us to look at the HII structures in more

details.

212
 Figure 5.7 shows snapshots from DOPE HII systems with ca. 4 nw (a, b), 10 nw (c, d), and 16 nw

(e, f) simulated at 303 K. Among the low hydration systems (i.e. up to 16 n w) studied, systems with 4 nw

were the most deviated systems from a cylindrical geometry and were highly curved/undulated along the

cylinder axis. In addition, the water cores in these systems had a strip-like shape instead of a cylindrical

geometry. Nevertheless, the water channels maintained their hexagonal packing in the system [Figure

5.7-a]. As the hydration level increased, both the deviations from a cylindrical geometry and the

undulations along the cylinder axis decreased.

213
Figure 5.7: Effect of hydration level on lipid tubules shape in DOPE HII systems.

The snapshots corresponding to the last frame of each simulation for DOPE HII systems with different

hydration levels at 303 K are shown in both XY-plane (Left) and along the Z direction (Right). Systems

with ca. 4 nw (a, b), 10 nw (c, d), and 16 nw (e, f) are shown. Yellow and red spheres represent the DOPE

214
phosphorus atoms and the methyl group’s carbon atoms in acyl chains, respectively. Orange lines

represent the lipid acyl chains, and water is shown as the cyan surface. The blue box is the simulation box

containing four lipid cylinders in a rectangular box for system with 4 n w, and one lipid cylinder in a

triclinic box for systems with 10 nw and 16 nw. The red spheres are not shown in the right column for

clarity. Structures correspond to the simulations at 303K.

5.4.4 SCD parameters as a function of hydration and temperature.

Structural changes in a molecular system are associated with the changes in structure, orientation and

organization of its molecular components. Deuterium order parameter (SCD), a parameter which can be

both measured by deuterium NMR (2H NMR) technique and be calculated directly from simulations, can

provide insights into such structural changes in the comprising molecules.

Deuterium order parameter (SCD) for the last carbon atoms (carbon atom numbers of 15-17) of

DOPE in sn-1 and sn-2 chains were measured in DOPE systems at 303 K using 2H NMR experiments [

Figure 5.8-a]. The same parameters were calculated from MD simulations of DOPE system with 16 nw at

the same temperature to compare. Systems with 16 nw were used for comparison because the maximum

hydration for DOPE lipids at 303 K (and for POPE at 348 K) has been experimentally estimated to be ca.

16 nw [15, 19]. Simulations and experiments agree on the general trend observed in the SCD profiles. sn-1

is shown to be more disordered than sn-2, and SCD decreases along the acyl chains from carbon number

15 to 17. However, SCD parameters from simulations are higher than experimental data by ~ 0.01.

Deuterium order parameters for sn-1 of POPE HII system with 16 nw at 348 K were also

calculated from simulations, sorted descending, and compared with experimental data from Lafleur et al.

[60] [Figure 5.8-b]. In both simulations and experiments, the SCD for POPE palmitoyl chain decreases

along the acyl chain towards the methyl terminus. The computational SCD are higher than experimental

215
values, and the difference between the two data set is highest for the middle segment of sn-1. Overall, the

computational SCD for both DOPE and POPE are in good agreement with the corresponding experimental

values.

We further investigated and compared the effect of hydration on SCD parameters in sn-2 chain of

DOPE and POPE HII systems at 323 K [Figure 5.8, c and d]. In DOPE systems, a change of nw from 4 to

10 increased the SCD significantly [Figure 5.8-c], and the segment containing the unsaturated bond was

the least affected segment upon hydration. For nw higher than 10, hydration level had little to no effect on

SCD of DOPE and POPE [Figure 5.8, c and d], although dehydration tends to decrease the SCD. For

example, in both DOPE and POPE systems, systems with 20 nw has the highest SCD values.

Effect of temperature on SCD parameters in sn-2 chain of DOPE and POPE were also studied from

simulations [Figure 5.8, e and f]. The results for three temperatures for each lipid type (283, 303, and 323

K for DOPE, and 323, 348, and 358 K for POPE) are shown to compare. Increasing temperature induced

disorder in both DOPE and POPE systems. Finally, the SCD for sn-2 is similar between DOPE (shown in

green) and POPE (shown in black) at 323 K, although the terminal segment (i.e. carbon numbers 12 to

17) of POPE is more ordered than DOPE. This might be due to an increase in van der Waals interactions

of sn-2 chain with fully saturated and more ordered palmitoyl chain in POPE than unsaturated oleoyl

chain in DOPE [61].

216
Figure 5.8: Deuterium order parameter (SCD) for DOPE and POPE in HII phase.

SCD parameters calculated from simulations of DOPE (Left) and POPE (Right) HII systems. a) SCD

parameters for the last carbon atoms in both DOPE sn-1 and sn-2 acyl chains calculated from system with

16 nw at 303 K, and compared with data measured using 2H NMR technique at 303 K. b) SCD parameters

for POPE sn-1 chain calculated from system with 16 nw at 348 K, sorted descending, and compared with

217
the experimental data from Lafleur et al. [60] conducted at T = 348 K. c, d) SCD parameters for DOPE and

POPE sn-2 acyl chains calculated from simulations with different levels of hydrations at 323 K. e, f) SCD

for DOPE and POPE sn-2 acyl chains calculated from simulations with 16 nw at three different

temperatures.

5.4.5 Estimation of the maximum hydration in HII phase.

The method used in Tate and Gruner [15] was used to estimate the maximum hydrations inside the lipid

cylinders (see Figure 5.9 caption). However, in our hybrid approach, MD simulations were used to obtain

the dhex values corresponding to the low hydration levels.

For DOPE systems, the experimental dhex for each temperature were taken from Tate and Gruner

[15]. These dhex values correspond to experiments conducted in the presence of excess water and were

served as horizontal lines in Figure 5.9-left. The estimated maximum hydrations using this hybrid model

are compared with the experimentally measured values [15] in Table 5-2. Assuming a 0.5 uncertainty in

experimental dhex measurements [17], the uncertainty in maximum hydration calculated using the current

hybrid approach is ca. 0.3 water molecules per lipid. The predicted values for T = 303 K and 323 K are in

good agreement with corresponding experimental data. For the lower temperature of 283 K, the hybrid

model gave higher hydration than experiment. Although no simulation was conducted at 295 K,

interpolation of simulation data in Table 5-2 suggest that the maximum hydration of DOPE HII systems

at 295 K should be between 19.7 (for 283 K) and 16.6 (303 K). This agrees with the reported values of

18, 18±1, and 19 for DOPE at 295 K [18, 62, 63]. Finally, in both simulations and experiments, an

increase in the temperature resulted in lower maximum hydration [Table 5-2] as expected [15].

For POPE, the experimental dhex at the excess water regime are taken from Rappolt et al. [19]

[Figure 5.9-right]. Herein again, the maximum hydrations estimated computationally are in a good

218
agreement with experimental values. In experiments, increasing temperature from 348 K to 358 K

increased the maximum hydration by 1 nw. However, in simulations, the same increase in temperature

caused a slight decrease in maximum nw from 16.8 to 16.2, which is more in line with experimental

expectations upon rising the temperature [15].

Figure 5.9: Estimation of maximum hydration in HII systems at several temperatures.

Left) DOPE, Right) POPE HII systems. Simulations were used to calculate the dhex for HII systems at

each hydration level and temperature. These data are plotted for each nw and the linear fit to each data set

is shown. The dhex values corresponding to the horizontal lines are taken from experiments conducted at

excess water regime by Tate and Gruner [15] (for DOPE), and Rappolt et al. [19] (for POPE). Each

horizontal line corresponds to one temperature. The nw where these horizontal and diagonal lines cross is

taken as the maximum hydration for that system at that specific temperature [15].

Table 5-2 can also be used to compare the maximum hydration of DOPE and POPE HII systems

at 348 K. At this temperature, POPE systems were estimated to have ~ 16 nw (experiments) or 16.8 nw

219
(simulation) [Table 5-2]. The maximum hydration for DOPE HII systems at 348 K is not known, but it

can be extrapolated from the data. According to simulation data and the trend observed in Table 5-2, the

maximum hydration for DOPE should be less than 15.1 nw. This is, again, in agreement with the

expectations that at a given temperature, DOPE form HII structures with higher curvature and therefore,

with a smaller Rw and number of trapped water molecules in their core than POPE.

Table 5-2: Maximum hydrations for DOPE and POPE HII systems at several temperatures.

DOPE POPE

283 303 323 348 358

T (K)

Experiment 18.0 16.2 14.9 16 17

Hybrid 19.7 16.6 15.1 16.8 16.2

5.4.6 Temperature effect on DOPE HII lattice distance and maximum hydration.

The effect of hydration level and temperature are coupled in HII systems [15, 17]. An increase in

temperature results in a decrease in the maximum number of water molecules taken up by the HII lattice.

The change in dhex has been explained to affect the effective shape of the comprising lipids, and

consequently the hydration level and Rw. We further studied this experimental observation as follows.

Lattice distances for DOPE HII systems with different hydration levels (nw = 10, 14, 16 and 20)

were calculated for three temperatures (T = 283 K, 303 K, and 323 K) from simulations and are shown in

220
Figure 5.10. The experimental data measured in the present study, and the data from Harper et al. [17] are

also shown for comparison (Our experimentally measured dhex values are in excellent agreement with data

in Harper et al. [17]).

Experimentally, 42 degrees increase in temperature from 281 K to 323 K resulted in ~ 8

angstroms decrease in dhex [Figure 5.10]. In simulations, the lines connecting the values for each nw are

approximately horizontal meaning that for a given hydration level, increasing temperature has little to no

effect on dhex. However, a systematic increase in dhex was observed upon hydration of HII system at each

temperature. Adding enough water inside the cylinders to double the nw from 10 to 20 resulted in an

increase of ca. 17 angstroms in the dhex at 303 K [Figure 5.10].

221
Figure 5.10: Lattice distance as a function of temperature for DOPE HII systems.

Lattice distances for DOPE HII systems were calculated from MD simulations and are shown for each n w

and temperature. Data are the means and error bars represent standard deviations of means. Experimental

dhex measured in this study are shown as squares colored in magnet, whereas the experimental data from

Harper et al. (2001) [17] are shown as orange circles. Experimental uncertainties in dhex measurements are

ca. 0.5 angstrom [17].

The number of water molecules inside the lipid cylinders at each temperature could also be

estimated from Figure 5.10. The diagonal line (colored in orange) corresponding to the experimental data

crosses the green line (nw = 16) at 303 K, suggesting the experimental systems, most likely, have had

222
about 16 nw inside their lipid cylinders. This agrees with 16.2 nw reported by Tate and Gruner (1989) [15]

[Table 5-2]. By extrapolations of each line in Figure 5.10, the orange line seems to have crossed the red

line (nw =14) at 333 K. Simulation data for other hydration levels are not available. However, according

to the observed trend, the lines corresponding to 15 nw are expected (by interpolation) to be located

somewhere between the red (nw =14) and green (nw =16) lines. This further suggests that the orange line

would have, most likely, crossed the line corresponding to 15 nw at 323 K. Therefore, simulations suggest

that experimental DOPE HII systems studied by Harper et al. (2001) [17] have had about 16, 15, and 14

nw at 303 K, 323 K, and 333 K, respectively.

5.5 Discussion

5.5.1 MD predicts the HII structural parameters as a function of hydration.

In a study by Rand and Fuller [18], X-ray diffraction technique was used to study the structural

dimensions of DOPE dispersions in HII phase as a function of hydration. Dehydration was shown to

reduce the dhex, Rw and APL, and to cause formation of longer lipid cylinders with less lipid molecules per

nm slice along the cylinder axis [18]. The same parameters calculated from MD simulations using both

anisotropic and semi-isotropic pressure coupling were in excellent agreement with experimental data

[Figure 5.3, Figure 5.4, Figure 5.6, Figure C.1].

The structural parameters of this study were calculated using the formulae described in Rand and

Fuller [18], which are based on the geometrical assumption that the cross-section area of water core is

cylindrical. This was done to illustrate how MD simulation could reproduce the experimental data using

the same assumptions used in experiments. However, structural parameters could also be calculated

directly from MD simulations, without any assumption on cylindrical geometry. The Rw calculated from

223
the positions of DOPE P atoms for a test system was in excellent agreement with the R w measured from

reconstructed electron density profile [Figure 5.4].

Deuterium order parameter (SCD) calculated from MD simulations were also in good level of

agreement with experimental data measured by 2H NMR experiments [Figure 5.8, a and b]. This further

validates the methods used to simulate HII systems and to calculate the SCD parameters from simulations

in this phase (see Methods). SCD parameters in simulations were, however, higher than experimental data,

especially for the last carbon atoms of DOPE acyl chains [Figure 5.8-a]. The most likely reason for such

a difference could be that the hydration level and actual temperature in the experimental setup might be

slightly different than the computational systems. It might also be partially because of using

CHARMM36 FF in GROMACS package [64, 65]. Moreover, the force field parameters for DOPE are

not perfect and could affect the structural properties in HII phase. However, DOPE parameters in

CHARMM36 FF have been shown to be transferable to the simulation of HII systems [29]. Considering

the algorithm used to calculate the SCD (see Methods), lipid cylinder undulations along the cylinder axis

and deviation of the water core from a cylindrical geometry in computational systems might also cause

those differences in SCD. However, systems with 16 nw were almost cylindrical and straight in shape [

Figure 5.7, e and f].

There are other computational studies on HII systems using both atomistic and coarse-grained

models [24-30]. In a study by Marrink and Mark [24], the dhex values for DOPE HII systems predicted by

simulations were larger than experimental values by ca. 10 percent. This increase was originated from

using coarser models and consequently an increase in the length of DOPE acyl chains. Therefore, the all-

atom nature of the simulations in the present study could be one of the reasons for a better agreement of

dhex data with experiments.

224
5.5.2 HII water channels are not straight and cylindrical at low hydrations.

The assumption of circular cross-section for the water core might not be necessarily true for all hydration

levels. Systems with high hydration levels corresponding to the dhex of ~ 65 angstroms and higher are

known to deviate from circularity significantly [16, 24], whereas for systems with lower hydration levels

the water core was circular within 5 % of Rw [16]. The systems studied in [16] were all in presence of

excess water and changes in the hydration were due to the changes in temperature. Therefore, these

experimental findings might not be applicable to very low hydration levels such as 4 nw simulated at a

given temperature and in absence of excess water.

In our simulations, the water core in systems with 4 nw deviated from circularity and had a strip-

like shape [Figure 5.7]. However, coarse-grained simulations using MARTINI model suggested that this

system had a circular water core [24]. This discrepancy with the coarse-grained simulation might be due

to the coarser models for DOPE and water molecules, or different number of water channels simulated in

two studies. It might also be because of using the fully anisotropic pressure coupling in [24], which

allowed the simulation box to deform. In our simulations, although an anisotropic pressure coupling was

used, the box shape was fixed during the simulation course while the box sizes were allowed to vary. The

HII formation process as well as equilibration process and different time scales between two studies could

be among other reasons for such a difference in water core shapes. Disregard of water core shape, the dhex

and other structural properties of system with 4 nw followed the overall trend observed for higher

hydrations [Figure 5.3].

HII structures were also shown to be curved/undulated along the cylinder axis [Figure 5.7],

which is consistent with the experimental finding that HII cylinders are not perfectly straight [59, 62]. The

level of curvature/undulation was further a function of hydration level. Systems with lower hydration

experienced higher levels of undulations [Figure 5.7]. This might be because of the change in mechanical

properties and bending modulus of lipid tubules as a function of hydration level. Alternatively, it could be

225
because of the larger size of the equilibrated systems with lower hydrations along the cylinder axis.

Undulations have been previously reported for lipid bilayers [66]. These undulations could be supressed

by using small simulation boxes [67].

5.5.3 MD reproduces the differences between DOPE and POPE in HII.

MD simulation could successfully reproduce the structural differences between DOPE and POPE in HII

as expected [4]. At a given temperature, DOPE was shown to form HII systems with higher curvature

than POPE [Figure 5.5 and Figure 5.6]. Due to the two oleoyl acyl chains in DOPE, the lipid tails splay

apart more than they do in POPE. This makes DOPE less ordered and more cone-shaped which support

HII structures with higher curvatures than POPE [4].

5.5.4 Both dehydration and raising temperature reduces SCD in HII.

Increasing temperature is known to decrease the acyl chain order in both lipid bilayers [68, 69] and HII

phases [60, 70]. In HII experiments, increasing temperature is associated with a decrease in the number of

water molecules inside the lipid cylinders [15] [Table 5-2]. Thus, the observed decrease in SCD profiles

[60, 70] in HII phase is indeed a sum of two effects: temperature and dehydration. Each effect was

investigated by simulations independently. Increasing temperature had similar effects to dehydration on

the SCD profiles in HII phase; both resulted in smaller SCD parameters [Figure 5.8]. The reduction of SCD

by an increase in temperature was expected due to the entropic effects [71], but decrease of SCD with

dehydration was not.

Experimentally, it has been observed that the HII dehydration decreases the average length of

DOPE lipid hydrocarbons in both interstitial and interaxial directions [Figure 5.1] [18]. This shorter

hydrocarbon lengths might mean that the lipid tails are more splayed in lower hydration levels or are

226
tilted more towards the regions formed by three neighboring lipid cylinders [18, 72]. This observed

shortening in lipid hydrocarbon chains can explain the observed decrease in SCD parameters upon

dehydration, or vice versa. In bilayers, also, there is a correlation between the bilayer hydrophobic

thickness and SCD profiles [68]. Lipid tails with smaller SCD parameters are less ordered and are more

splayed. This results in a shorter length for the projection of acyl chains along the bilayer normal, and

consequently shorter hydrophobic thickness.

5.5.5 Temperature affects the maximum hydration and lattice distances in HII.

The maximum hydrations estimated from our hybrid model were in an excellent agreement with the

estimated values from experiments [Table 5-2]. Increasing temperature caused a decrease in the

maximum hydration in HII phase [Figure 5.9]. This was further shown to be the main factor for the

experimentally observed temperature-dependent reductions in dhex [15, 17] [Figure 5.10]. As explained

elsewhere, an increase in temperature affects the molecular shapes, and consequently decreases the lipid

monolayer spontaneous radius of curvatures (Ro) [15]. In the presence of excess water, the Rw is near Ro

so that the system is in its minimum free energy state. Therefore, HII systems with smaller R w and less

amount of water molecules inside the lipid cylinder are more energetically favorable systems at higher

temperatures. Radius of water core and dhex are also directly correlated [Figure 5.3], meaning that a

decrease in Rw decreases dhex as well. Furthermore, temperature is known to have little effect (~ 1

angstrom for 80 degrees increase in temperature) on the hydrophobic length of DOPE tails in HII phase

[15]. Therefore, most of the changes in dhex as a function of temperature is due to change in maximum

hydration.

227
5.5.6 Protocol for construction of equivalent HII systems to experiments.

As demonstrated in Figure 5.9, MD simulations for low hydration levels can be used in combination with

a single SAXS measurement for dhex (in presence of excess water) to estimate the maximum hydration for

the HII systems at a certain temperature. This maximum hydration can be further used for construction of

molecular systems which are equivalent to experimental systems.

This protocol should be applicable to other amphiphilic molecules, e.g. polymers, in HII phase.

However, like other protocols it has a few limitations which should be considered. First, at least one d hex

value measured in presence of excess water at the temperature of interest is required. Second, at least two

hydration levels need to be simulated to obtain the diagonal line. Simulation of systems with higher

hydration levels (i.e. more than 10 nw) is recommended because systems are straighter and more

cylindrical in geometry. Third, equilibration of lipid tubules in simulations might be in the order of 200 ns

for systems which the initial dimensions are far from equilibrium state. However, considering the recent

advances in computing resources, this would not be a problem. Fourth, although coarse-grained models

could also be used to measure the dhex and estimate the maximum hydrations, such coarser models might

result in larger dhex values for some molecules [24]. This will result in non-accurate estimations for the

maximum hydrations. Finally, the force field parameters are critical to result in correct structural

parameters such as dhex. Non-accurate parameters could result in inaccurate APL and consequently dhex

and maximum hydrations.

As an application example, this protocol can be used for parameterizing new molecules, e.g.

synthetic lipids, using the target data in HII phase [Figure 5.11]. Force field development and validation

usually requires comparing data between equivalent systems in simulations and experiments. Most of the

structural parameters of HII systems were shown to be highly affected by the hydration level. Therefore, a

reasonable estimation of maximum hydration is a necessity for such applications. Assuming one single

measurement for dhex in excess water regime at the desired temperature (red dotted horizontal line in

228
Figure 5.11-left), and at least one target data for the corresponding systems are available, the

parameterization process can be done as follows. In this example, SCD parameters are used as the target

data (black line in Figure 5.11-right).

First, reasonable initial parameters (parameter set 1) are assigned to each atom in the molecule

according to the parent force field, e.g. CHARMM36 force field. Using parameter set 1, two hydration

levels (e.g. with nw of 10 and 22) are constructed and simulated using semi-isotropic pressure coupling to

obtain the first diagonal line (the solid pink line in Figure 5.11-left). The nw where this line crosses with

the red dotted line is extracted (i.e. 20 nw in this example). Next, the HII system with 20 nw is simulated

and SCD parameters are compared with the experimental SCD data. According to the differences between

computational and experimental SCD profiles, force field parameters are further adjusted resulting in

parameter set 2. Parameter set 2 are used to simulate HII systems with 10 and 22 n w again. This results in

a new diagonal line (the solid blue line in Figure 5.11-left) and estimation for the maximum hydration

(i.e. 18 nw in this example). Next, HII system with 18 nw is simulated and SCD are compared between

simulation and experiment. As we iterate through this process, the simulation data are expected to

approach the experimental data. This method might require several iterations till estimated n w or the SCD

converge.

229
Figure 5.11: A proposed protocol for molecule parameterization in HII phase.

Left) Hypothetical dhex values as a function of hydration calculated using parameter sets 1 and 2. The

parameter set 1 is the initial set of parameters assigned to the molecule, whereas the parameter set 2 refers

to the modified parameters after one iteration. The black line represents the experimental dhex data for low

hydration levels (which is not known!). The dotted line represents the experimentally measured dhex in

presence of excess water at the desired temperature. The dashed vertical lines point to the n w where the

horizontal and diagonal lines cross at. Right) Typical SCD profiles obtained by parameter sets 1 and 2

from systems constructed with 20 nw and 18 nw, respectively. These nw were estimated from the left panel.

The experimental SCD are shown in solid black line corresponding to system with 16 nw (This is known

and is the target data used in this example).

5.6 Conclusions

We show that MD simulations of DOPE and POPE HII phase successfully reproduce d hex, Rw, APL, SCD,

nc, and length of the lipid cylinders in the HII phase at different hydration levels, including low hydration

230
levels where conducting experiments is usually challenging. Dehydration was shown to result in longer

lipid cylinders with smaller Rw, APL, nc, and SCD parameters. HII structures were shown to be undulated

along the cylinder axis and deviate from a perfect cylindrical geometry at 4 nw. Hydration was further

shown to reduce the level of undulations. We obtained reasonable estimates of the maximum hydration

for PE HII structures at several studied temperatures, using only a single experimental d hex measured from

a single sample in the excess water regime. In agreement with experimental data, increasing the

temperature decreased the maximum hydration in HII systems, accounting for the experimentally

observed temperature-dependent reductions in dhex. The POPE HII systems studied followed the same

trends as DOPE, although as expected DOPE formed HII structures with a higher curvature than POPE.

We proposed a general protocol for constructing HII phases and molecular parametrization using the

target data in HII phase using MD simulations, 2H NMR and SAXS experiments.

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239
 Chapter Six: An Auto-Inhibitory Helix in CTP:Phosphocholine Cytidylyltransferase

Hijacks the Catalytic Residue and Constrains a Pliable, Domain-Bridging Helix Pair

Copyrights:

This research was originally published in the Journal of Biological Chemistry. Ramezanpour, M.*, Lee,

J.*, Taneva, S.G., Tieleman, D.P., & Cornell R.B (2018). An auto-inhibitory helix in

CTP:phosphocholine cytidylyltransferase hijacks the catalytic residue and constrains a pliable, domain-

bridging helix pair. Journal of Biological Chemistry, 293(18), 7070 –7084. (* contributed to work

equally). Copyright © the American Society for Biochemistry and Molecular Biology.

Contributions:

I ran all the simulations and analyzed all the simulation data. I had contributions in methodology,

conceptualization, data interpretation, hypothesizing, validation, and experimental design. I wrote the

computational methods section and had contribution in editing the other sections in the manuscript. The

experimental data and methods section were provided by Dr. Rosemary B. Cornell, Dr. Jaeyong Lee, and

Dr. Svetla G. Taneva at the Simon Fraser University.

Abbreviations:

CCT, CTP:phosphocholine cytidylyltransferase; AI, auto-inhibitory; PC, egg phosphatidylcholine; PG,

egg phosphatidylglycerol; PE, phosphatidylethanolamine; PDB, Protein Data Bank; MD, Molecular

Dynamics; RMSF, Root Mean Square Fluctuation; NOE, Nuclear Overhauser Enhancement; PR,

Positional Restraint; DTT, dithiothreitol; TCEP, tris(2-carboxyethyl)phosphine.

240
6.1 Abstract

The activity of CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme in phosphatidylcholine

synthesis, is regulated by reversible interactions of a lipid-inducible amphipathic helix (domain M) with

membrane phospholipids. When dissociated from membranes, a portion of the M domain functions as an

auto-inhibitory (AI) element to suppress catalysis. The AI helix from each subunit binds to a pair of 

helices (E) that extend from the base of the catalytic dimer to create a four-helix bundle. The bound AI

helices make intimate contact with loop L2, housing a key catalytic residue, Lys-122. The impacts of the

AI helix on active-site dynamics and positioning of Lys-122 are unknown.

Extensive MD simulations with and without the AI helix revealed that backbone carbonyl

oxygens at the point of contact between the AI helix and loop L2 can entrap the Lys-122 side chain,

effectively competing with the substrate, CTP. In silico, removal of the AI helices dramatically increased

E helix dynamics at a predicted break in the middle of these helices, enabling them to splay apart and

forge new contacts with loop L2. In vitro cross-linking confirmed the reorganization of the E element

upon membrane binding of the AI helix. Moreover, when E bending was prevented by disulfide

engineering, CCT activation by membrane binding was thwarted. These findings suggest a novel two-part

auto-inhibitory mechanism for CCT involving capture of Lys-122 and restraint of the pliable E helices.

We propose that membrane binding enables bending of the E helices, bringing the active site closer to

the membrane surface.

6.2 Introduction

Many regulatory enzymes are silenced by inter-domain interactions that are broken by activating ligands.

The inhibitory interactions can involve a direct steric block of the active site by occupation of a

pseudosubstrate or ligand-binding domain in its apo form [1, 2] or by binding of the unoccupied ligand-

241
binding domain to an allosteric site that shifts the equilibrium toward unproductive configurations of an

element in the active site [3-5]. An X-ray structure of the catalytically silenced form of

CTP:phosphocholine cytidylyltransferase (CCT) as well as molecular dynamics simulations hinted at a

novel allosteric silencing mechanism involving electrostatic re-direction of a key catalytic residue [6]. We

have probed the silencing mechanism in depth in this publication.

The enzyme CCT catalyzes a rate-limiting step in phosphatidylcholine (PC) synthesis and

controls PC homeostasis by being active only when the membrane PC content is low. Its conserved

catalytic domain is linked to a weakly conserved regulatory domain via a highly conserved short linker

segment [Figure 6.1-A]. CCT’s regulatory domain (domain M) is an inducible 60-70-residue membrane-

binding amphipathic helix [7-10], and the membrane is its “ligand”. The enzyme can thus interconvert

between a soluble, inactive form and a membrane-bound, active form by transformation of domain M

from mostly disordered into a long  helix [Figure 6.1-B]. CCT membrane binding and hence its

catalytic power are regulated by physical properties of the membrane [11]. PC-rich membranes have low

net surface charge and tight packing between neighboring lipid molecules of the bilayer. PC-deficient

membranes have a higher surface negative charge, and increased packing stress [12]. CCT’s domain M

responds to the latter properties, binds and inserts partway into the membrane as an amphipathic helix,

and this binding event is communicated to the active site to enhance catalysis more than two orders of

magnitude by effects on both kcat and Km for CTP [13, 14]. This process accelerates PC synthesis to

restore PC compositional homeostasis. CCT mutations are linked causatively to three human diseases

that impair development of bone, cartilage, and/or retinal tissue [15-17] or lipid metabolism [18].

The catalytic domain of CCT derives from an ancient domain designed to catalyze nucleotide

transfers to a variety of metabolites, such as the transfer of AMP from ATP to amino acids and CMP from

CTP to phosphoalcohols, such as phospho-glycerol, ethanolamine or choline [19, 20]. In the

cytidylyltransferases (glycerol phosphate (GCT), choline-phosphate (CCT), and ethanolamine-phosphate

242
(ECT)) catalysis is accomplished by charge-stabilization of the highly charged CTP with basic residues

residing in loops L1, L2, and L6, and in the junction of 4/L5. Residues at the N-terminus of helix E

make contacts with CTP to promote a contorted U-shape [21]. This enables an in-line attack of the

nucleophilic phosphate at the -phosphate of CTP to displace diphosphate [20]. The crystal structure of

the catalytically silenced form of CCT [6] as well as fluorescence anisotropy analyses [22] showed that

when not engaging membranes, the M domain is composed of a disordered leash segment of ~40 residues

followed by a ~22-residue auto-inhibitory (AI) helix that docks onto two elements of the catalytic

domain: the E helices, and loop L2 [Figure 6.1-C]. These two elements are special in that their

sequence differs between lipid-regulated CCTs and non-regulated cytidylyltransferases, such as GCT and

ECT. The E helices are much longer in the CCTs (~22 residues versus 10-12 residues in GCT or ECT)

and are predicted to be interrupted by a short disordered segment in the middle [Figure 6.1-A] [23]. Thus,

the long uninterrupted E helices observed in the structure of the silenced form may be stabilized by the

AI helices, which, together with the two E helices, form a four-helix bundle and may be destabilized

once the AI helices are dissociated by membrane binding. The L2 sequence in all cytidylyltransferases

contains one or more lysines that participate in catalysis based on mutagenesis [24, 25] and solved

structures with substrate and product [6, 20, 21, 25]. In rat CCT, a substitution with arginine at Lys-122 in

L2 results in a ~5-order of magnitude decrease in catalytic efficiency [24], the strongest mutagenic effect

for any single residue tested in CCT. Any impact on Lys-122 dynamics or alignment could have severe

consequences on enzyme activity.

We proposed, based on the structure of the silenced enzyme [6], that CCT’s mode of auto-

inhibition involves a partial occlusion of the active site entrance by the AI helix and clamping of the

active site in a non-productive configuration by the AI helix. Limited molecular dynamics simulations

suggested that the docking of the AI helix could restrict the dynamics of the E and L2 loops. Most

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intriguingly, where the end of the AI helix contacts loop L2 (the AI turn), the backbone carbonyls of the

turn steered the key catalytic residue, Lys-122, away from its orientation into the active site. This

electrostatic steering involved a carbonyl on loop L2 at Phe-124 and a carbonyl at Phe-293 on the AI turn,

and we refer to this interaction as the “backbone trap” [6]. These single-replicate 200-ns simulations were

carried out in the absence of substrate.

A key unanswered question arising from the previous work was whether the backbone trap can

operate when the active site is occupied with substrate. Can the backbone carbonyls compete with

substrate for the key catalytic residue? In the present work, we determined the impact of the AI helix on

active site dynamics and positioning of Lys-122 in the presence and absence of the substrate CTP. The

new data, involving a total of 40 separate atomistic 1-s simulations, suggest that the backbone trap can

effectively compete with CTP. We also tested loop L2 flexibility in vitro and its role in catalysis by

mutation of a conserved glycine, Gly-123. Furthermore, simulations in the absence of the AI helices

revealed a remarkable plasticity for the E helices once the AI helices are displaced, generating bends in

the E helices at the predicted helix break [Figure 6.1-A]. Cross-linking measurements in vitro

confirmed a reorganization of the E helices upon membrane binding. Straightjacketing the helices with a

disulfide prevented lipid activation, suggesting that the conformational changes observed upon removal of

the AI helices may reflect the active membrane-bound configuration of the E. We discuss various

hypotheses for how the bent E helices might accelerate the CCT-catalyzed reaction.

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Figure 6.1: Structure of mammalian CCT and its active site.

A) domain map. The N region is non-conserved and disordered, and the catalytic (C) domain is a

conserved dinucleotide fold encompassing ∼150 residues. The locations of the two elements in domain C

where the AI helices dock (L2 and αE) are indicated. In lipid-regulated CTs, the αE helix is predicted to

be two helices (αEN and αEC) with a helical break at residues ∼212–214. When not membrane-bound, the

M domain consists of a disordered leash followed by the AI helix. The phosphorylation (P) region is

poorly conserved and disordered. B) schematic of the CCT fold for silenced and active forms. Left,

residues 40–223 of the catalytic dimer (cyan and green chains) with the AI helices docked onto the αE

helix pair to create a four-helix bundle. Elements in the crystallized protein that are not visible in the

solved structure (PDB code 4MVC) are represented as dotted lines. The box encompasses one active site,

enlarged in (C). Right, a proposed membrane-bound active form using the catalytic dimer from the

solved structure of CCT236 (PDB code 3HL4) and the M domain modeled as a simple unbroken helix

(not to scale). The αEC and linker are of unknown configuration in the active form and are represented as

dotted lines. CDP-choline occupies both active sites. C) close-up of the CCT active site showing

conserved residues that influence catalysis (stick representation of Lys-122, Tyr-173, Arg-196, and Thr-

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202), the backbone-to-backbone contact between the AI turn and loop L2 (amide N at Phe-124 and

carbonyl oxygens at Met-292 and Phe-293; spheres with H-bonds shown as dashed lines), and the

hydrophobic pocket (side-chain spheres) that is created by the confluence of the AI helix, αEN, and loop

L2. This image is from the silenced form (PDB code 4MVC), with CDP-choline removed.

6.3 Results

6.3.1 Optimal catalysis requires a flexible Loop L2 at Lys-122, enabled by Gly-123.

Loop L2 has been identified as a key loop in catalysis based on studies with CCT and a related bacterial

cytidylyltransferase, GCT [24, 25], which is involved in cell-wall biosynthesis and is not regulated by

lipids. The loop L2 sequence of GCT differs from that of the lipid-regulated eukaryotic CCTs. Whereas

the latter contain a single lysine (Lys-122) followed by an invariant glycine, L2 in GCT has two

conserved lysines and no glycine [Figure 6.2-A]. Mutagenesis of B. subtilis GCT showed that both Lys-

44 and Lys-46 are required for catalysis [25]. In the crystal structure of B. subtilis GCT in complex with

CTP (PDB code 1COZ), Lys-46 contacts the -phosphate of CTP. In the structure of the same enzyme in

complex with the product CDP-glycerol, Lys-46 contacts the - and -phosphates of CDP-glycerol and

Lys-44 contacts the -phosphate (PDB code 1N1D) [Figure 6.2-B]. Thus, it appears that two lysines

participate in multiple alternative interactions with substrate and product during a catalytic cycle. The

backbone configurations of L2 at these lysines are similar in both crystal structures, and movement of the

lysines to engage the two phosphates of the product are rigid body translocations of  3 Å (21).

In CCT, however, loop L2 (residues 122-127) is 3 residues shorter and likely to be less mobile,

and Lys-122 is the sole lysine. In the crystal structures of CCT in complex with CDP-choline (PDB codes

3HL4 and 4MVC), Lys-122 occupies a position between that of Lys-44 and Lys-46 of GCT, and contacts

246
both the - and -phosphate of CDP-choline [Figure 6.2-B]. The backbone at Lys-122 in CCT occupies a

configuration in the -helix portion of a Ramachandran plot, whereas the , angles at the lysines in the

crystal structures of GCT occupy a configurational space within or near the β portion. We hypothesized

that the single lysine in L2 of CCT must be agile during a reaction cycle so that its -amino group can

partner with either the -, β- or -phosphate oxygens of CTP, or with phosphocholine, and that this

requires a very flexible backbone, enabled by Gly-123. This hypothesis was explored by mutagenesis to

alanine or proline, which would restrict the , angles and present a larger barrier opposing backbone

conformational sampling at Lys-122. The data in Figure 6.2-C show that neither Ala nor Pro can

effectively substitute for the glycine at residue 123. The Pro substitution was completely inactivating in

the context of CCT236, which lacks the membrane-binding domain. In a construct with domain M

(CCT312), the effect of the proline substitution on the lipid-activated enzyme was curiously less severe,

suggesting that when membrane-bound, additional factors may mitigate the detrimental loss of flexibility.

Further experiments will be needed to establish the basis for the less stringent L2 flexibility requirement

of membrane-bound CCT. These data do support the hypothesis that configurational lability of loop L2

promotes catalysis. In support of Lys-122 as a primary target of regulation in lipid-dependent CCTs, the

positions of four other functional active-site residues, Arg-196, Thr-202, His-89, and His-92, super-

impose within <1.5 Å with the analogous residues in the GCT structures with substrate or product.

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Figure 6.2: CCT relies on a single lysine followed by glycine in loop L2 for catalysis.

A) sequence comparison of the L2 loop. B) overlay of GCT–CTP (1COZ), GCT–CDP-glycerol (1N1D),

and CCT–CDP-choline (3HL4) showing H-bonds between the lysines in loop L2 and CTP, CDP-glycerol,

or CDP-choline. C) impact of mutations at Gly-123 in L2 on the activity of CCT. Purified CCTs were

assayed for activity under conditions optimal for the CCT312 or CCT236 constructs in the absence (gray

bars) or presence (green bars) of 0.2 mM PC/PG (1:1) vesicles. Data are means ± average deviation (error

bars) of 4–6 independent determinations.

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6.3.2 The AI helices selectively repress the dynamics of helix αE.

To explore the impact of the AI helix on the dynamics of loop L2 and other active site loops and catalytic

residues, we performed 1-µs MD simulations of the CCT dimer, residues 40–223, comprising the

complete catalytic domain, with and without the AI helices. A 3.0 Å structure of this segment with bound

AI helices [6] provided the starting coordinates for the simulations. The effect of the substrate CTP was

also probed in each of these sets. The final 8 residues of the E helices were restrained by either of two

methods (referred to as PR or NOE) to prevent their unwinding and migration deep into the active site,

which was observed in a preliminary 200-ns simulation. Five replicates of each of the four conditions

(protein alone; +AI; +CTP; +CTP and +AI) were simulated for each restraining method, for a total of 40

independent simulations (40 µs total). We discovered early on that the mobility and interactions of key

loops and residues were frequently dissimilar for the two active sites. For example, when Lys-122

interacted with CTP in chain A, it might be contacting a carbonyl of the backbone trap in chain B. Thus,

effectively, the simulations span 80 µs of CCT monomer dynamics.

In the 40 chains containing CTP in the active site, CTP was maintained in the pocket except for

the last 150 ns of just one replicate, where it diffused away from most of its partners in the starting

structure (e.g. Arg-196, Thr-202). More common was a somewhat fixed position of the cytosine and

ribose while a rotation about the ribose C1 oxygen -  phosphorus bond redirected the - and -

phosphates away from the starting contact with Thr-202 in E and toward Lys-122 and loop L2. This

transformed the CTP from its U-shaped starting conformation to a more stretched conformation.

The loops contributing to the active site are L1, L2, L5 and L6 [Figure 6.1]. The N terminus of

E (EN) also contributes Thr-202 and Ser-203 to the active site. The docking site for each AI helix is

formed from the E helices of both chains and loop L2 of one chain [Figure 6.1]. This prompted an

249
investigation of the impact of the AI helices on L2 and E dynamics by computing the RMSF for

backbone atoms and key catalytic residues housed in these loops. Loops L5 and L6, which are longer and

more dynamic, were also investigated. From a visual inspection of the many simulations, it was obvious

that L1 is not mobile, and neither CTP nor the AI reduced its mobility further. Figure 6.3 shows that the

dynamics of L2 was reduced a small and variable degree by the AI and by CTP in isolation, but the

combination of CTP and the AI helix reduced dynamics significantly by 20%. The same effect was

observed on the dynamics of Lys-122 in L2, where the combination of CTP and the AI resulted in a

significantly depressed RMSF (24%), but neither CTP nor the AI alone showed a significant effect on

mobility. The immobilization of EN and its resident active site residue, Thr-202, reflected the combined

impact of active site residues bonding with the ligand and the AI. The AI helix dampened the dynamics of

the EN backbone and Thr-202 side-chain by ~ 30%. The dynamics at Thr-202 was reduced  40% by

CTP, in keeping with a direct H-bonding contact. Together, the AI and CTP reduced Thr-202 RMSF by

>2-fold [Figure 6.3-A and Figure D.1].

The dynamics of loop L5 and its resident active site residue Tyr-173 were high and were not

suppressed by either CTP or the AI helix [Figure 6.3]. The dynamics of loop L6 and its active site

residue, Arg-196, were not impacted by the AI helix, but were reduced 40-50% by CTP, with which it

formed direct H-bonds. The dynamics of the loop/hinge region linking helix EN and helix EC was

profoundly reduced by the AI helix in the NOE-restrained simulations [Figure D.5-B]. The high mobility

of this region when the AI is not present is discussed below. In summary, the docking of the AI helices to

form the four-helix bundle has a strong immobilizing effect on the E helices and only weakly constrains

loop L2, which is ordered even with no AI present.

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Figure 6.3: Effect of the AI helix and CTP on the dynamics of active-site loops and side chains.

A) RMSF of heavy backbone atoms of the indicated loops/structural elements and the heavy side-chain

atoms of the indicated residues were computed as described under “Materials and Methods.” The

residues of the loops/elements are as follows: L2 (Lys-122–Met-127), L5 (Asp-170–Tyr-182), L6 (Gln-

195–Ser-201), and αEN (Thr-202–Val-210). The data are means ± average deviation (error bars) of five

replicates for each condition, indicated in the matrix at the top. n = 10 for each condition, as there are two

active sites for each CCT dimer. p values are provided with reference to the first set (white bar). *, p <

0.05; **, p < 0.005. These analyses used simulations with NOE restraints applied on helix αE. The same

analysis on 20 simulations using positional restraints is available in Figure D.1. B) dynamics of the

active-site loops and select side chains are shown, visualized by the overlay of protein coordinates from

simulations at 100-ns intervals at 0 (gray), 100 (red), 200 (orange), 300 (yellow), 400 (green), 500 (cyan),

251
600 (blue), 700 (magenta), 800 (wheat), 900 (pale green), and 1000 ns (black). The side chains of the

active site residues Lys-122, Tyr-173, Arg-196, and Thr-202 are shown as sticks. Each set is from a

representative simulation, and the presence/absence of the AI and CTP is indicated on the figure. For

clarity, neither CTP nor AI is displayed.

To investigate L2 dynamics in vitro we examined the effect of a docked AI helix on the mobility

of a tryptophan substituted for Phe-124 in loop L2. The F124W substitution did not affect the activity in

the presence or absence of lipid. We also monitored an engineered F121W in the B helix adjacent to

loop L2, and a native Trp (Trp-151) located in the active site [20]. The location of these Trp residues is

shown in Figure D.2-A. The fluorescence anisotropy of Trp-124 was compared for this CCT construct in

its soluble form and when bound to a lipid micelle that induces dissociation of the AI helices from the

catalytic domain [22] and fully activates the enzyme [10]. The anisotropy measurements are sensitive to

motions in the 1-10 ns regime. The anisotropy values for each of the three Trp residues were 0.15 – 0.17,

typical values for a Trp that is constrained in a folded element of a protein [26]. There was only a

marginal decrease in the anisotropy of Trp-124 upon binding lipids (12 ± 3% decrease), and a slightly

stronger effect on Trp-121 (20  8 % decrease; Figure D.2-B). In keeping with these decreases in

dynamics, the RMSF value for Phe-124 computed from a set of 10 NOE simulations was reduced 18% by

the AI (P = 0.01). There was no change induced by lipids when Trp-124 was evaluated in the context of a

CCT lacking an M domain (CCT236; Figure D.2-B), confirming that the effect on dynamics is due to

lipid binding. Thus, in keeping with the simulation results, docking of the AI onto loop L2 has only a

small restraining effect on the dynamics of a hydrophobic side chain at residue 124 in L2. Phe-124

rotations are sustained within a hydrophobic pocket that is created from side chains of L2 (Val-126) and

the AI helix (Met-292, Phe-293, and Phe-289) [Figure D.2-A].

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6.3.3 The AI helix supplies H-bonding partners for Lys-122 that compete with CTP.

Previous published simulations without substrate suggested that the AI interactions could steer the Lys-

122 N into an H-bonding trap with backbone carbonyls contributed from the AI and the L2 loop [6].

When the active site is occupied by ligand, can the AI compete with the phosphates of the ligand for

contact with Lys-122 to inhibit catalysis? To further explore the likelihood of the backbone trap for Lys-

122, we mined the full set of 40 simulations with and without CTP and the AI helix for the frequency of

Lys-122 H-bonding with one or more oxygen atoms from 5 residues that were frequently found in shared

H-bonds with Lys-122. These are the Phe-124 carbonyl in loop L2, Asp-86 carboxyl and backbone

carbonyl in loop L1, and, in the presence of the AI helix, three backbone carbonyls at the C-terminal turn

of the AI at Phe-293, Gly-294, and Pro-295. Figure 6.4-A shows a snapshot of a common 3-way

interaction with Lys-122. In some of the simulations in the presence of the AI, contact between the AI C-

terminus and loop L2 was lost for prolonged time intervals during the simulations. The contact between

AI and L2 was monitored by the formation of an H-bonding interaction between Met-292 carbonyl and

Phe-124 backbone NH, a contact that was also observed in the crystal structure of the silenced CCT (PDB

code 4MVC). For analysis of the impact of the AI helix on Lys-122 H-bonding partners, we deleted time

intervals where the Met-292 – Phe-124 contact was disengaged for more than 50 ns. This step reduced

total simulation time associated with the production runs from 38.4 to 32.4 µs.

In both the PR and NOE-restrained simulations, there was a significant effect of the AI helix on

Lys-122 contact with the trapping atoms [Figure 6.4-B]. The frequency of contact with these atoms due

to the AI alone increased >2-fold, from 32% to 83% (PR simulations) and from 31 to 72% (NOE

simulations). When CTP occupied the active site, the AI helix increased the frequency of Lys-122 contact

with the backbone trap 3 – 4-fold, from 14 to 38% (PR) and from 8 to 36% (NOE). At the same time, the

H-bonding frequency with CTP decreased from 67.5 to 43% (PR) and from 67 to 39% (NOE). This

253
analysis strongly supports the hypothesis that the interaction of the AI helix with the L2 loop steers Lys-

122 away from CTP by forging direct contacts with backbone atoms. Figure 6.4-C shows an image of the

electrostatic surface surrounding the active site, with and without the docked AI helix. The C-terminus of

the AI helix bends at its juncture with loop L2, and that bend is highly electronegative, thus exerting an

electrostatic pull on the Lys-122 amino group. With no side-chain, Gly-123 enables close docking of the

AI-turn on loop L2, enhancing this pull. The AI helix did not affect the H-bonding of two other near-by

active site residues that are significant partners with CTP: Arg-196 in loop L6 and Thr-202 in the EN

(data not shown). These residues have no or little interaction with the atoms of the backbone trap, and

their stable interactions with CTP were not disturbed by the AI helix. Thus, it appears that the backbone

trap device is selective for Lys-122.

Figure 6.4-B also shows that CTP and/or the AI helix reduce the frequency of Lys-122 partnering

with other atoms and the time spent searching for partners. The other atoms that served as prominent

partners for Lys-122 in the absence of CTP or the AI include Asp-82 carboxyl and Gly-83 carbonyl in 1,

Glu-198 in loop L6, and Thr-202 in helix E. Partnering with side chain or backbone atoms of loop L5,

such as Tyr-173, Ser-174, and Ser-175, was also seen. Many of these contacts are not compatible with an

active site occupied by CTP, phosphocholine or CDP-choline, as the position of the side chains would

thwart substrate entry and/or productive binding.

We noticed that the backbone of L2 at Lys-122 can flip between - and - dihedral

configurations, facilitated by Gly-123, and that the  backbone configuration predominated over the 

configuration when Lys-122 was contacting the trapping atoms. This motivated us to analyze whether the

backbone configuration could deter contact of Lys-122 with the substrate, CTP. The analyses of the

simulations (see supplemental information in Appendix D and Figure D.3) showed that CTP contact was

254
compatible with either the  or  configuration, and that even when the AI was present and L2 was in the

 configuration Lys-122 could readily contact CTP.

Figure 6.4: Backbone trap for Lys-122.

A) left, active site of chain A with CTP in its productive U-shape, interacting with Ser-203 in αE, and

Lys-122 H-bonding with CTP. Right, 10 ns later in the same simulation, Lys-122 is in a split H-bond with

Asp-86 in loop L1, Phe-124 in loop L2, and Phe-293 in the AI. These interactions compete with CTP for

Lys-122 for the duration of the 1-μs simulation. B) effects of AI and CTP on Lys-122 H-bonding

partners. The simulation conditions are indicated in the matrix at the top. Lys-122 H-bonding frequency

with the indicated residues or CTP was analysed using the GROMACS tool, g_hbond. A contact

involving 1–3 H-bonds to a partner was scored as one H-bond. Lys-122 H-bonded with the α-, β-, or γ-

255
phosphate oxygens of CTP. The group of five included the carboxyl of Asp-86 and the carbonyl of

residues 124, 293, 294, and/or 295. None, frequency of H-bonds with solvent; other, any other protein

atom. Data are the means ± average deviation (error bars) of 10 replicates for each condition (5

simulations × 2 chains), except 9 replicates for the condition PR-restrained, +CTP, +AI. p values for the

effect of the AI are indicated as follows: *, p < 0.05; **, p ≤ 0.005. C) electrostatic surface of active site

with and without the AI helix. PDB images shown are of 4MVC with the AI removed (left) and AI

present (right). The electrostatic surface was calculated using default settings in PyMOL.

6.3.4 Removal of the constraining AI helices allows bending of the E helices.

In 17 of 20 simulations carried out in the presence of the AI helices the 4-helix bundle remained intact for

the large majority of the simulation time, with occasional unwinding of the N-terminal portion of one AI

helix at the base of the 4-helix bundle. The contacts between the 4 helices are mostly hydrophobic, and

the AI-helix-turn/loop L2 interaction involves an aromatic cluster [Figure D.4]. In these simulations, the

E segments remained unbroken -helices with minimal backbone fluctuation, even in the hinge region.

The E - E contact was maintained by the hydrophobic side chains of Ile-206, Ile-209, and Val-210 in

the N-terminal segment of the E [Figure D.4]. This hydrophobic interaction is also seen in the crystal

structure [6]. The two turns at the C-terminus of the E helices have fewer interactions but were fixed by

restraints. In 3 of the 20 simulations, one of the AI helices disengaged for hundreds of ns at the N

terminus [Figure D.4, image on right] and rotated on its axis to become nearly perpendicular to the E

helices. Despite this rotation, hydrophobic contacts between the middle of the AI (Phe-285, Ile286, Phe-

289) and the E helices (Val-210, Tyr-213, Tyr-216) were maintained. The two E helices in all

simulations remained stable in their conformation and interactions with each other [Figure D.4].

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 One of the most striking observations, which occurred in all 10 NOE-restrained simulations

without the AI helices, was the bending of helix E at the hinge (residues 211-215). We refer to this

helical break as a hinge following the definition of Wolfson and colleagues as a region about which

protein domains rotate [27]. In the equilibrated starting structures, the two E helices were unbroken

[Figure 6.5-A], and their interaction was mediated by the hydrophobic cluster described above. The two

helices cross-over at the Ile-209 –Val-210 contact site. In all 10 simulations, the helices drifted together

toward one active site for 10 – 200 ns, and then the segment between residues Arg-211- Val-215

unwound and/or bent in both helices, enabling contact of at least one EC with loop L2. In 6 out of 10

dimer simulations, the bend at this loop accompanied the complete dissociation of the EC portions from

each other (EC splay; configurations 4, 5, and 6; Figure 6.5). The EC in the splayed conformation

formed contacts with loop L2, principally via an aromatic cluster composed of Phe-124 in L2 and Tyr-

213 and Tyr-216 in the EC of the opposite chain [Figure 6.5, C-E]. In 15 of 20 simulated chains, this

aromatic contact (with a minimum distance of ~3Å) persisted for more than 550 ns. The EN retained its

tight inter-chain hydrophobic interaction for the duration of the simulations. There were at least 5

common configurations of the CCT dimer with bent E helices [Figure 6.5], and these were observed in

the absence or presence of CTP, but never in the presence of the AI helices. These bent configurations

could interconvert (i.e. 2  3 and 4  5  6) but did not return to the starting configuration. Once the

two EC helices had engaged separate L2 loops, they did not break away to re-forge contacts with each

other. In 6 replicates, the splayed configurations were maintained for more than 500 ns, for a cumulative

total of 4 µs of a total of 10 µs. In the splayed configurations 4-6, the frequency of H-bonding of Tyr-213

or Tyr-216 hydroxyl with Phe-124 carbonyl in L2 increased, especially when assuming a configuration

with the EC helix at an approximate right angle beneath L2 [Figure 6.5-E].

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 The simulations with CTP present revealed a frequent H-bond between Arg-223 at the C-

terminus of EC and the -phosphate of CTP [Figure D.5-A]. This occurred in 5 of 10 chains for a total

of ~2.7 µs of 5.0 µs. This contact was specifically associated with splayed EC configurations that

featured close packing of EC under and perpendicular to loop L2 [Figure 6.5, D and E]. The CTP

contact with Arg-223 did not preclude an interaction of Lys-122 with CTP [Figure D.5-A]. The impact of

the AI on the flexibility of the E hinge is shown in Figure D.5-B. The RMSF of residues 211-215 was

reduced by >2-fold, both in the presence and absence of CTP. For the most part, the AI helices constrain

the helices of the 4-helix bundle to diffuse as a unit.

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Figure 6.5: αE conformational change triggered by removal of the AI helices.

The central panel shows a time plot of the interchain distance between Tyr-213 and Phe-124 (top), and

the interchain distance between the two Tyr-213 hydroxyls of the dimer (bottom) for one replicate of the

NOE simulations. The abrupt change after 180 ns correlates with the loss of αE–αE contact. A–E,

configuration 1 is the equilibrated starting structure. Configuration 2 occurs after a short period of drift.

The αE helices remain in contact with each other, and one αE forges contact with L2 via the aromatic

cluster composed of Tyr-213 and Tyr-216 in the αE and Phe-124 in L2 (gray arrow). Configuration 3 is

shown in Figure D.6. Configuration 4 has both αE helices splayed apart and interacting with L2 via the

aromatic cluster. In configuration 5, a sharp bending at the hinge in one of the αE helices positions the

αEC at right angles to loop L2; this orientation is stabilized by aromatic cluster and Arg-223–CTP

259
contacts. Configuration 6 features both αE helices adopting a close orientation under L2, with αE helices

anti-parallel to each other.

6.3.5 Membrane binding reorganizes the E helices, a pre-requisite for activation.

Membrane binding dissociates the AI helices, which incorporate into the long M-helices. If the E helices

are malleable and splay apart upon dissociation of the AI helices, as suggested by the MD simulations,

then their average inter-chain distances should increase upon membrane binding [Figure 6.6-A]. We

substituted cysteine in place of a nonconserved, native alanine at residue 217 in the EC helix in a

cysteine-free CCT-312 background where five native cysteines were changed to serines [22, 28]. The

activity was lowered ~2-fold by the substitution from Ala to Cys at position 217, relative to the Cys-free

enzyme. We probed inter-chain cross-linking efficiency using a set of sulfhydryl-reacting bis-maleimides.

Inter-E helix cross-bridging will generate a covalent CCT dimer which can be detected on gels [Figure

6.6-B]. Because cross-bridging is in competition with reactions of each cysteine individually with

separate bis-maleimides, monomer-to-dimer conversion will necessarily be incomplete. All three bis-

maleimides were modestly effective at cross-linking cysteines at residue 217, which is buried within the

four-helix bundle in the silenced CCT form. Displacement of the AI helices by lipid vesicle binding

should increase their accessibility, but we detected much less covalent dimer in the presence of a large

molar excess of lipid [Figure 6.6-B and Figure D.7]. These results support an increase in average

distance between sites in the EC upon membrane binding and dissociation of the AI from the E helices.

260
Figure 6.6: Lipid vesicles reduce interchain cross-linking efficiency between the two αEc helices.

A) increased interchain distance between Ala-217 in the αEC accompanies αE hinge bending. Images

from simulations in the presence (left) or absence (right) of the AI helices are shown. This site was

substituted with cysteine in a Cys-free background for conjugation with the indicated cross-linkers. B)

lipid vesicles reduce covalent dimer formation. Sulfhydryl-directed cross-linking reactions with three bis-

261
maleimide reagents with the indicated optimal cross-linking distances for each [29]. Coomassie-stained

11% reducing gels show the cross-linking reaction as a conversion of CCT312 (A217C) monomers to

dimers in the absence (−) or presence (+) of 100-fold molar excess lipid vesicles (PC/PG (1:1)). Bottom

panels, quantification of band density using ImageQuant version 5.2: gray symbols, no lipid; green

symbols, with lipid. Data are means of two images ± range (error bars).

To assess whether restructuring of the E helices is required for enzyme activation upon binding

to membrane vesicles, we purified CCT312-A217C and its cysteine-free analog under mild oxidizing

conditions. In contrast to the cysteine-free control enzyme, the A217C variant was partially oxidized to

produce a covalent dimer at Cys-217, and 1 mM DTT was sufficient for its complete reduction [Figure

6.7-A]. Figure 6.7-B shows the impact of oxidation and reduction of this sulfhydryl on CCT activity in

the presence of excess lipid vesicles. DTT had a small (~ 20%) positive impact on the activity of the Cys-

free control, suggesting a minor oxidation effect unrelated to disulfide bond formation. However, the

activity of oxidized CCT-A217C was only 12  5 % of the activity of the reduced A217C, assayed in the

presence of 10 mM DTT, an ~8-fold effect of reduction. To ensure that the low activity of the disulfide-

bridged CCT was not due to an impairment of the membrane binding of domain M, we compared

oxidized and reduced CCT in an assay that monitors fluorescence resonance energy transfer (FRET)

between Trp-278 in the M domain and dansyl-phosphatidylethanolamin in lipid vesicles. The FRET

signal was the same for both oxidized and reduced CCT-A217C and was the same as FRET observed

with a Cys-free CCT resistant to disulfide formation [Figure D.8]. These data suggest that preventing

restructuring of the E by di-sulfide bonds (or alternatively by formation of a stable 4-helix bundle with

the AI helices) blocks lipid activation.

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Figure 6.7: A disulfide bond linking the αE helices at Cys-217 inhibits CCT activity.

CCT312 (Cys-free) and CCT312 (A217C) with a single cysteine in the αE were purified under oxidizing

conditions. A) DTT reduction of oxidized CCT. Oxidized CCT preparations were incubated for 10 min at

37 °C in the presence of the indicated DTT concentration before analysis by nonreducing SDS-PAGE of

monomeric (36-kDa) and dimeric (72-kDa) species. B) reduction of the disulfide between αE helices is

263
required for lipid activation. In a separate experiment, enzyme activities were determined for A217C (▲),

and Cys-free (♦) using standard conditions, 0.2 mM egg PC/egg PG (1:1) sonicated vesicles, and the

indicated concentration of DTT. The data are means of two independent experiments with ranges (error

bars) and are expressed as units/mg of CCT normalized to peak activity at 10 mM DTT (10378 ± 1654

units/mg for the Cys-free CCT and 4460 ± 780 for CCT-A217C).

6.4 Discussion

In the MD simulations the docked AI helices did not cause any major rearrangements of the active site.

The AI helices reduced access to the active site from the underside of the catalytic domain, although the

lateral opening remained sufficiently wide for entry and escape of CTP and CDP-choline (~16 Å × 10 Å,

larger than the length of CDP-choline at 13.5 Å; Figure 6.4-C). The sequence of the AI helix-turn is

acidic and glycine-rich across species that show lipid regulation. The placement of the electronegative AI

helix-turn at the mouth of the active site might also deter an approaching CTP or phosphocholine

molecule from entering the active site. In addition to the restricted access of substrates and release of

products, we have considered three possible mechanisms for auto-inhibition by the AI helices: (i)

restricting the dynamics of active site loops and participating residues; (ii) hijacking the catalytic lysine;

and (iii) preventing a conformational transition in the E helices that enables an EC interaction with the

active site. The AI helices dock onto the E helices and loop L2. Thus, we focused on these elements, but

in doing so we are not excluding other indirect impacts of the AI.

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6.4.1 AI restriction of L2 dynamics.

Our previous single 200-ns simulation showed that the AI helix reduced the root mean square deviation

from the starting structure for all backbone atoms in loop L2 (residues 121-128) by ~ 1 Å and the RMSFs

by ~ 60% [6]. The more extensive analysis here suggests that the AI has specific effects on the dynamics

of L2 (modest) and the E helices (strong). The RMSF analysis we performed reports on the nanoscale

fluctuations of active site loops and residue side-chains. A 20 - 30% reduction in fluctuations, which we

observed over 20 × 1 s, could influence catalysis by limiting the range of conformational sampling, and

this may reduce the frequency of productive orientations for groups participating in catalysis [30].

However, given a kcat of 50 ms for fully active CCT, the dampening of nanosecond fluctuations may be

irrelevant.

On the other hand, the conversion of the L2 backbone at Lys-122 between  and  configurations

does occur on the nano-second timescale. The  and  Lys-122 configurations favour inward (into the

active site) and outward orientation, respectively. The loss of activity when the conserved Gly in loop L2

next door to Lys-122 was substituted with ala or pro supports a requirement for L2 backbone flexibility,

spurring the question as to whether the docking of the AI helix could impede transitions between the 

and  configurations of the backbone and in this way impede contact with substrates in the active site.

However, our analysis showed that although the  configuration was highly correlated with the backbone

trap, Lys-122 contact with CTP was compatible with either configuration [Figure D.3]. Notably, the 

configuration did not preclude an interaction of Lys-122 with CTP when the AI was present. The side

chain of Lys-122 can effectively contort to make contact with CTP. We cannot exclude a requirement for

the  configuration (which is enabled by glycine at residue 123) for Lys-122 interaction with

phosphocholine or the transition state. Our simulations did not test this. Thus, the data do not provide

support for a lock-in of a particular L2 backbone conformation as a means for AI suppression of catalytic

265
function; however, a role for backbone flexibility of loop L2 to facilitate Lys-122 sequential access to

different partners during a catalytic cycle remains a viable hypothesis.

6.4.2 Novel backbone competition for the catalytic lysine.

Another more obvious mechanism to reduce the frequency of productive conformations is to capture

alternative non-productive conformations. In CCT, the AI helix-turn forged frequent contacts with the

Lys-122 -amino group. Even with the active site occupied by CTP, the backbone carbonyls of the AI-

turn were effective at steering the catalytic lysine away from the substrate (the backbone trap increased

from 8-14% frequency to ~40% frequency). The reaction rate will scale inversely with the cumulative

time spent in non-productive alignments of a key catalytic residue. There are other enzymes that use

electrostatic steering to forge inhibitory contacts with an active site residue, but utilizing side-chain rather

than backbone carbonyls as the interloper. For example, Src kinases employ one or more basic residues in

the regulatory A loop to trap the glutamate in helix C that is a component of the KDE triad involved in

ATP binding. Activation by phosphorylation of certain tyrosines steers the arginines away from the

glutamate [5]. By analogy, in CCT, the catalytic residue Lys-122 is competitively pulled away from an

optimal orientation for substrate contact by the auto-inhibitory helix-turn, which presents a strongly

electronegative pull. Another example of electrostatic steering is found in PGE2 synthase, where an

oxidation step requiring Arg-126 is blockaded by an electrostatic interaction with Asp-49 [31]. In PGE2

synthase the cofactor glutathione (GSH) can overcome this inhibitory interaction, whereas in CCT,

effective desilencing requires larger domain movements (i.e. the dissociation of the AI helix-turn away

from loop L2 and Lys-122).

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6.4.3 Silencing by constraining the E helices.

In addition to influencing the positioning of Lys-122, we suggest that the AI helices inhibit the CCT

reaction by preventing conformational change in the E helices. The simulations in the absence of the AI

helices with NOE-type restraints on helices EC show that the E helices are highly dynamic in a hinge

segment between residues ~210 and 215. The EN region is much less so. The hinge undergoes many

different contortions, enabling splaying apart of E helices to contact loop L2 at the base of the active

site. The contact with L2 can be maintained for hundreds of nanoseconds, and in one replicate, the

splayed conformation was acquired during the equilibration and persisted in both chains for the entire

production run (960 ns). The principle stabilizing force for this interaction is an aromatic cluster between

Phe-124 in L2 and the two tyrosines of the E hinge. This aromatic cluster was observed in 17 of 20

chains in the NOE simulations. In the other three chains the EC of that chain did not migrate toward L2,

but stayed linked to its partner EC. In addition, a hydrogen bond between the Tyr-213 hydroxyl in the

E hinge and the Phe-124 backbone carbonyl in L2 was observed for hundreds of ns in splayed

conformations. We note that Phe-124 of the aromatic cluster is not universally conserved. However, the

residues that can substitute for it are leucine (hydrophobic), arginine, and glutamine. The latter two may

replace a purely aromatic interaction with a π – cation/amine interaction [32]. It is intriguing that the

aromatic clusters that mediate the loop L2 – AI helix interaction may be replaced with an alternative

aromatic cluster between L2 and the E hinge upon dissociation of the AI helices.

The entire C-terminus of E may be intrinsically malleable in the absence of the AI helices.

There was no electron density for these residues in the crystal structure of CCT236, which contains all of

catalytic domain, the E, and the linker to domain M (but no AI helix). However, we were forced to put

some constraints on the C-terminus of the E helices, because without them the tail end entered into the

unoccupied active site.

267
In vitro cross-linking also provided evidence that lipid-induced dissociation of the AI resulted in

increased distance between the two EC helices, consistent with the splaying observed in silico with the

AI helices removed. An alternative hypothesis, that lipid vesicles reduce the accessibility and/or reactivity

of the cross-linkers, is unlikely for two reasons: (i) the disassembly of the four-helix bundle should

increase the exposure of the reactive cysteines, leading to elevated cross-linking; and (ii) in separate

experiments, we found that the EC does not bury in the membrane in the CCT mem form. 2 Most

importantly, straightjacketing the E helices by disulfide engineering prevented activation by lipid

vesicles. The disulfide was located just C-terminal to the E hinge segment. This suggests that

reorganization and/or dissociation of the EC helices is required for activation of the membrane-bound

CCT, and that the four-helix bundle silences CCT by virtue of the constraining action on the two E

helices.

What is the potential impact of the E plasticity on catalysis? We know that membrane binding

displaces the AI helices, which would allow a large increase in conformational sampling of the E

helices. But perhaps only a few of those conformers are productive for speeding up catalysis. We

hypothesize that membrane binding of the M domain (and perhaps the short amphipathic linker segment

intervening between the E and domain M) promotes that productive conformation. As illustrated in the

captured images [Figure 6.5] and in the model shown in Figure 6.8, the bent helix E configurations

could enable close approach of the catalytic domain to the membrane surface. Whether this is important

for catalysis or for efficiency of PC synthesis remains to be probed in future studies. Delivery of product

(CDP-choline) at the membrane would facilitate its utilization in the last step of the CDP-choline

pathway, which is catalyzed by an integral membrane protein, choline phosphotransferase. Pioneering

2
D.G. Knowles and R.B. Cornell, unpublished results

268
studies by George et al. [33, 34] showed that [3H]choline flux into PC in cultured glioma cells was not

reduced by excess CDP-choline delivered by cell permeabilization, providing evidence for metabolite

channeling within the CDP-choline pathway. Although an intriguing idea, CCT and the

phosphotransferase often localize to different membrane systems in cells [35]. We propose three

hypotheses for how the bent E with its close contacts with loop L2 could facilitate catalysis. (i) The bent

E could eliminate the backbone trap. In simulations without the AI helices, bent E helices enabled H-

bonding between the hydroxyl of Tyr-213 with the Phe-124 carbonyl, removing this carbonyl oxygen

from a trapping interaction with Lys-122. A hydrogen bond between these two atoms was observed in one

chain of the partially active CCT236 crystal structure [20]. Loss of H-bonding potential at Tyr-213 by

mutation to phenylalanine partially inhibited lipid activation, 3 implying a function for the Tyr-213

hydroxyl. (ii) Residues in the bent E helices may participate in one or more steps in the catalytic cycle.

Arg-223 at the C-terminus of the E made frequent interactions with the -phosphate of CTP that lasted

for up to 700 ns. Thus, upon membrane binding and acquisition of a bent E, Arg-223 may assist in the

binding of the substrate CTP. Alternatively, Arg-223, which bonds only to the -phosphate, may provoke

Lys-122 to interact with the -phosphate of CTP, phosphocholine, or the transition state. A third

possibility is that Arg-223 may assist in displacement of the product, diphosphate. Interestingly CCT-

R223S is one of several alleles in humans that suffer from an inherited disorder, SMD-CRD [15]. CCT-

R223S has a 16-fold lower kcat/Km for both substrates than the WT enzyme. 4 The contact frequency

between Ser-223 and CTP phosphates would be much lower than that for Arg-223. (iii) Residues from

the bent E may seal off the opening of the active site bounded by Loop L2 and the EN of both chains

3
J. Lee, S.G. Taneva, and R.B. Cornell, unpublished results
4
R.B. Cornell, unpublished results

269
[Figure 6.4-C]. This could assist catalysis by trapping the reaction intermediates and perhaps excluding

water. It is tempting to speculate that the bent helix conformation might be dominant in one step of the

catalytic cycle (e.g. in the lead-up to the chemical step) and that subsequently its reorganization to a more

open conformation allows product release.

Signaling helix pairs are emerging as agents of communication between domains of regulatory

proteins, such as receptor tyrosine kinases [36], two-component signaling systems [37], and cytokine

receptors [38-40]. The helix deformations that transduce inter-domain signals can occur within

microseconds after a trigger is applied in vitro [37], lending validity to our in silico results. We propose

that the conformational malleability of the E helix pair that bridges the membrane binding and catalytic

domains of CCT makes it an ideal element adapted by evolution for transducing signals from membrane

to active site.

270
Figure 6.8: New model for CCT activation.

In its soluble form, the two AI helices silence CCT by (i) capturing the catalytic lysine (Lys-122) with

backbone carbonyls and (ii) restraining helix αE dynamics. In response to increased anionic charge on the

membrane surface, the positively charged leash segment is attracted to the membrane, and its binding

facilitates dissociation of the AI helices. The αE helices gain conformational freedom, but the binding of

the linker segment to the membrane creates a platform to stabilize αE conformations that are productive

271
for catalysis. We propose that the bend at the αE hinge is featured in productive conformations. The linker

is represented as a simple trapezoid because its structure is unknown. The linker-membrane interaction

may be dynamic, enabling sampling of different αE (and linker) conformations during a catalytic cycle.

The bent αE helices and the folding of the linker brings the catalytic domain closer to the membrane

surface. Images are from NOE-restrained simulations. The disordered N and P regions are not displayed.

6.5 Materials and Methods

6.5.1 Molecular dynamics simulations.

The crystal structure of the rat CCT dimer, CCT1-312(238-269), was used as the starting structure for

simulation (PDB code 4MVC) [6]. The N termini of each chain were acetylated, and the C termini were

amidated to eliminate charged ends. The crystal structure was solved in complex with CDP-choline. To

create a CTP-bound model, the crystal structure of GCT (PDB code 1COZ) [21] was first aligned to

4MVC using the superimpose module of Coot software [41]. Next, the CTP coordinates from the aligned

GCT were combined with the coordinates of the CCT protein from 4MVC. Hydrogens were added to the

CTP molecule using the program Avogadro [42]. The CTP acidic oxygens were deprotonated, giving it a

charge of -4 e. Hydrogens were added to the protein using the pdb2gmx module of the GROMACS

package. Each structure was solvated with TIP3P water molecules [43, 44] in a truncated dodecahedron-

shaped simulation box with a minimum distance of 1.2 nm around the protein. Na+ and Cl- ions were

added to neutralize the protein/ligand charge and then to obtain a 0.15 M ionic concentration.

MD simulations were run with the GROMACS version 4.6.7 package [45, 46]. The protein was

described using the all atom AMBER99SB-ILDN force field [47]. The parameters for CTP were taken

272
from Demir and Amaro [48] and were adapted for compatibility with GROMACS using the python script

ACPYPE [49].

The simulations fall into four different groups distinguished by the presence or absence of CTP,

and the presence or absence of the AI helix. In addition, we applied two methods for restraining the EC

helix, for a total of eight conditions, each with five replicates. In preliminary simulations without external

restraints, the C-terminal ends of the E helices were prone to unwinding and migration into the active

site pockets. To prevent this, we applied two different restraining methods [Figure D.9]. In the first, the

heavy backbone atoms of EC residues (216-223 inclusive) were restrained using position restraints with

a force constant of 1000 kJ mol-1 nm-2. This ensures these helices are locked in their crystal conformation

and pinned in space. In the second method, we applied NMR NOE-type distance restraints between all

pairs of heavy backbone atoms in the EC segment, using force constants of 1000 kJ mol-1 nm-2. This

maintained helicity, while allowing the helices freedom to move relative to each other. The side chain

atoms were unrestricted in both restraining conditions.

All systems were energy minimized using the steepest descent algorithm, first with position

restraints applied on the non-hydrogen atoms of protein and CTP molecules, and then without. The

resulting structures were equilibrated by simulation for 40 ns at a temperature of 310 K using the v-

rescale thermostat [50] with a time constant of 1.0 ps and a pressure of 1 bar using the Berendsen pressure

coupling [51] with a p of 5.5 ps. This step was followed by 960 ns of production simulation at 310 K, in

which the pressure was maintained at 1 bar using the isotropic Parrinello-Rahman barostat [52, 53] and a

p of 5.5 ps. The van der Waals and the electrostatic interactions were both cut-off at 1.0 nm, whereas the

long-range electrostatic interactions were treated with the PME algorithm [54, 55]. The potential-shift-

Verlet modifier was used to shift both the van der Waals and coulomb potentials to zero at the cut-off. All

273
bonds were constrained using the LINCS algorithm [56] and a 2 fs time-step was used for the simulations.

Atomic coordinates were saved every 100 ps during the 40 ns equilibration and 960 ns production runs.

The coordinates of every 100 ps of each trajectory was fitted (rotated and translated) to the Cα

atoms of five stable beta strands on each monomer (residues 77-83, 106-113, 144-149, 165-169, and 191-

194), and the fitted trajectories were used for the analyses. Distances between select atoms, H-bonding

analysis, dihedrals at Lys-122, and RMSF analysis of select residues were carried out with GROMACS

tools. For the H-bond analysis, a value of 3 Å was used as the “donor-acceptor” distance cut-off. The

RMSF analysis used the coordinates at the end of the 40 ns equilibration as the reference for the change in

position of (i) heavy atoms of a specified residue side chain or (ii) The N, Cα, and carbonyl backbone

atoms of specified loops using the GROMACS module g_rmsf (with options -res and -nofit). Outputs of

the analyses were imported into Excel for averaging and statistical analyses. Unpaired 2-tailed t-tests

were used to compute the probability of sameness between sample sets.

6.5.2 Construction of CCT mutants.

All CCT constructs were derived from Rattus norvegicus pcyt1a (α isoform; UniProtKB/Swiss-Prot

accession number P19836). All primer designs and codon substitutions were performed using

QuikChange site directed mutagenesis (Agilent). Schematics of all constructs are shown in Figure D.10.

6.5.3 pET14b-CCT constructs.

The preparation of pET14b-CCT236 and pET14b-CCT312 was described in Lee et al. [6]. These were

used as the templates for engineering an Ala or Pro substitution at Gly-123 by site directed mutagenesis.

pET14b-CCT236 was also used for engineering a Trp replacement at Phe-124. All pET14b CCT

274
constructs contained a plasmid-derived His tag and a thrombin cleavage sequence at the N terminus

(MGSSH6SSGLVPRGSH).

6.5.4 pET24a-CCT312 constructs.

C-terminally truncated proteolytic fragments were sometimes found as contaminants in the His-CCT312

preparations. To facilitate the removal of these fragments during purification, we prepared pET24a-

CCT312 with the His tag at the C-terminus (LEHHHHHHH). Fragments lacking the C-terminus would

not be retained on nickel-agarose. To prepare pET24a-CCT312-His, we amplified the open reading frame

(codons 1-312) and engineered NdeI and XhoI restriction sites at 5’ and 3’ ends, respectively, by standard

PCR methods using the template pAX142-His-CCT312, whose construction was described in Dennis et

al. [57]. The amplicon was inserted into NdeI/XhoI-cut pET24a. This construct was subsequently used to

create CCT312-His with two native tryptophans at Trp-151 and Trp-278 mutated to phenylalanine

(CCT312 (W151F/ W278F)). We then used the Trp-free construct to engineer CCT312-His F121W or

F124W.

The construction of CCT312 with five Cys to Ser mutations (pET24a-CCT312 (Cys-free))

utilized the template pAX142-HisCCT367-T207C, described in Huang et al. [22], which contained a

single cysteine at T207C in the context of a full-length CCT. The Cys-207 mutation was first converted

back to Thr by site-directed mutagenesis, and an internal KpnI/SacI fragment, which encompassed all five

Cys to Ser mutations was transferred into pET24a-CCT312. This construct was subsequently modified by

removal of the nuclear localization sequence (12RKRRK16) by site directed deletion. The NLS

modification was to eliminate CCT dimer-mediated vesicle cross-bridging and aggregation [58, 59]. The

resulting pET24a-CCT312 (Cys-free) NLS plasmid was then used to engineer a single Cys mutation at

Ala-217, and this construct was used for the cross-linking studies.

275
6.5.5 Expression and purification of CCT constructs.

All proteins were expressed in Escherichia coli (Rosetta-DE3) cells as His6 fusion proteins and were

purified using nickel-agarose affinity chromatography as previously described [6, 28, 58] with

modifications described below. The His tag was at the N terminus of all CCT236 constructs as well as

CCT312 (WT), CCT312 (G123A), and CCT312 (G123P). The tags were not cleaved after protein

purification. The His tag was at the C terminus of CCT312 (F121W), CCT312 (F124W), and CCT312

with a single Cys at 217.

His-CCT236 constructs were purified from the 20,000 × g × 15 min supernatant fraction of the

cell lysates. They were eluted with 350 mM imidazole, 300 mM NaCl, 10 mM Tris, pH 8.0, and 2 mM

DTT and subjected to Q-Sepharose ion-exchange chromatography to remove the imidazole. The 312His

F121W and 312His F124W proteins were mostly in the 10,000 × g pellet and were purified after

solubilization of this pellet with 6 M guanidine hydrochloride at room temperature with occasional

vortexing over 30-60 min. The denatured proteins were refolded while bound on the nickel-agarose resin

using a 6 M to 0 M urea gradient in 50 mM NaH2PO4 (pH 8.0), 0.5 M NaCl, 25 mM imidazole and 1 mM

DTT at a flow rate of ~ 2 mL/min over  1 h. The renatured proteins were eluted and dialyzed against 200

mM NaCl, 10 mM Tris, pH 7.4, and 2 mM DTT to remove the imidazole.

The single Cys CCT312His (NLS) A217C protein used in cross-linking experiments was

purified from the 10,000 × g pellet as described above; however, the elution and dialysis buffers

contained TCEP as a reducing agent instead of DTT. TCEP is compatible with the bis-maleimide cross-

linkers (Thermo Fisher Scientific); DTT is not.

276
6.5.6 Preparation of lipid vesicles or micelles.

Lyso PC (18:1)/egg PG (4:1) micelles used for fluorescence spectroscopy were prepared as described in

Taneva et al. [10]. Small unilamellar vesicles, egg PC/egg PG (1/1), were prepared by sonication as

described [60], and were used for enzyme activity assays and cross-linking reactions.

6.5.7 Tryptophan fluorescence anisotropy.

Steady-state fluorescence anisotropy measurements were made using a Horiba Jobin Yvon FluoroLog 3

spectrometer equipped with polarizers. Intensities with excitation polarization either Vertical (V) or

Horizontal (H) and emission polarization either V or H (IVV, IVH, IHH, and IHV) were measured and the

anisotropy (r) was calculated by the software of the instrument. The temperature was controlled at 20 oC

with a thermo-regulated cell holder. The integration time was 1 s. The excitation wavelength was 295 nm,

the emission wavelength was 345 nm, and the excitation and emission bandwidths were 4 and 12 nm,

respectively. Anisotropy values are the average of 4-6 replicates for each sample. The protein

concentration was 5 µM and the lipid/protein molar ratio was 150.

6.5.8 CCT activity assay.

CCT activity was performed as described previously [61]. The standard reaction mixture contained 50 nM

CCT, 20 mM Tris, pH 7.4, 10 mM DTT, 88 mM NaCl, 12 mM MgCl2, 2 mM [14C]phosphocholine (1

mCi/mmol), and either 10 mM CTP (CCT312 constructs) or 20 mM CTP (CCT236 mutants). Incubations

were for 10 min at 37 ˚C with or without 0.2 mM PC/PG (1/1) SUVs.

277
6.5.9 Bis-maleimide cross-linking.

Thiol-specific homo bifunctional cross-linkers were obtained from Sigma (N,N’-o-phenylene-

dimaleimide) and TCI America (1,2-(Bis-maleimido)ethane and 1,4-(bis-maleimido) butane).

Immediately before use, the cross-linkers were dissolved in DMSO at a 25-50 mM concentration.

The single-Cys CCT312His (NLS; A217C) was purified and stored in 1 mM TCEP to prevent

oxidation of Cys residues. Chemical cross-linking was carried out at 37 ˚C for 10 min in a volume of 50

µl, containing ~2 µM CCT, 0.5 mM TCEP, and variable concentrations of cross-linkers and phospholipid

vesicles (egg PC/egg PG (1/1)). The reactions were initiated with the cross-linking agent and were

terminated with 5.5 mM DTT. Control reactions in parallel were pre-quenched with 5.5 mM DTT at time

zero. Aliquots were mixed with reducing SDS sample buffer, heated to 85 ˚C and evaluated using 11%

SDS-PAGE. Gels were stained with Coomassie Blue or proteins were electro blotted onto

poly(vinylidene difluoride) membranes (Immun-Blot PVDF, Bio-Rad) and probed with antibody directed

against residues 164-176 of domain C [28].

6.5.10 Disulfide bond formation by air oxidation.

CCT312His (NLS; A217C) and Cys-free CCT312His (NLS) were purified from the 10,000 × g pellet

under denaturing conditions as described above. Low concentrations of the reducing agent DTT were

used throughout the protocol to prevent misfolding of the denatured proteins. The concentration of DTT

in the refolding, elution and dialysis buffer were 100, 50 and 10 µM, respectively. Gentle oxidation of the

single Cys CCT312His (NLS; A217C) was achieved by exposure to the atmospheric air during the

purification. We avoided copper phenanthroline-catalyzed disulfide induction because copper inhibits

CCT activity.

278
6.6 Bibliography

[1]. Huse, M., & Kuriyan, J. (2002). The conformational plasticity of protein kinases. Cell, 109(3),

275-282.

[2]. Kazanietz, M. G., & Lemmon, M. A. (2011). Protein kinase C regulation: C1 meets C-tail.

Structure, 19(2), 144-146.

[3]. Filippakopoulos, P., Müller, S., & Knapp, S. (2009). SH2 domains: Modulators of nonreceptor

tyrosine kinase activity. Current Opinion in Structural Biology, 19(6), 643-649.

[4]. Wu, W. I., Voegtli, W. C., Sturgis, H. L., Dizon, F. P., Vigers, G. P., & Brandhuber, B. J. (2010).

Crystal structure of human AKT1 with an allosteric inhibitor reveals a new mode of kinase

inhibition. PLoS ONE, 5(9), e12913.

[5]. Xu, W., Doshi, A., Lei, M., Eck, M. J., & Harrison, S. C. (1999). Crystal structures of c-Src

reveal features of its autoinhibitory mechanism. Molecular Cell, 3(5), 629-638.

[6]. Lee, J., Taneva, S. G., Holland, B. W., Tieleman, D. P., & Cornell, R. B. (2014). Structural basis

for autoinhibition of CTP:phosphocholine cytidylyltransferase (CCT), the regulatory enzyme in

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 Chapter Seven: Conclusions and Future Directions

7.1 Conclusions

Computer simulation can assist in a better understanding of the biomolecules, including lipid aggregates,

proteins, and nanoparticles [1]. A comprehensive review on how molecular simulation techniques,

including MD simulation, have enhanced our current understanding of nanoparticle-based drug/gene

delivery systems was provided in Chapter 3 [2]. In this thesis, three more examples for the successful

application of MD simulation were presented.

In Chapter 4, binary systems composed of POPC and KC2 were studied as a function of pH and

mixing ratio. The results strongly suggest that the interactions between neutral KC2 and POPC are highly

dependent on the pH level. Neutral KC2 were shown to segregated from POPC at high pH level.

However, the KC2H, corresponding to the low pH levels, stayed in the lipid-water interface and interact

with the POPC and solvent. This segregation was further found to be a function of mixing ratio. Systems

with less content of KC2 had larger proportion of KC2 stayed in the lipid-water interface. The effect of

KC2H on POPC was further shown to be similar to the effect of other cationic lipids on PC lipids

reported in literature [3]. Adding KC2H were shown to affect the ion distribution in vicinity of bilayer

surface. The POPC headgroup tilt angle, i.e. the angle between POPC P→N vector and the vector normal

to the bilayer surface were also shown to decrease upon adding KC2H to the system. Segregation of

neutral KC2 from POPC was proposed to be the underlying reason for the observed electron dense cores

in LNPs [4, 5], and limited drug release for LNPs containing KC2 [6].

In Chapter 5, DOPE and POPE HII systems were studied using MD simulations. Simulations,

using both semi-isotropic and anisotropic pressure couplings, could successfully predict the structural

parameters of HII systems as a function of hydration level and temperature. HII structure was shown to be

287
highly affected by the hydration level. The temperature and lipid type were two other factors affected the

HII structure. Dehydration resulted in the formation of longer lipid cylinders, with smaller R w, APL, and

SCD parameters. Increasing temperature was shown to decrease both the maximum hydration of HII lattice

and SCD parameters. Provided that a single measurement of dhex in the presence of excess water be

available, the maximum hydration of HII lattice could also be estimated using MD simulations.

Accordingly, a protocol for construction of computational molecular systems equivalent to the

experiments, and parameterization of new molecules using target data in HII phase was proposed.

In Chapter 6, MD simulation was used to study the structure and dynamics of CCT enzyme in

both presence and absence of two autoinhibitory (AI) helices [7]. The interactions between CCT and its

substrate (CTP) were also studied for each of these two conditions. The results suggested that AI helices

have a dual role in inhibiting CCT activity. AI helices were shown to affect the dynamics of loop L2 in

the active site. Both the dynamics of loop L2, and its interaction with CTP are believed to be important

for the catalytic activity of CCT. AI helices were also shown to repress the dynamics of two helices

(referred as αE helices). Upon the removal of AI helices, these αE helices were shown to bend towards

the active sites and interact with the substrates. This bending made the contacts between residues, e.g.

Arg-223, at the C-terminus of αE helices with CTP possible. Finally, a new model for the CCT activation

was proposed. In this model, the CCT structure with the bent αE helices was proposed as the

conformation corresponding to the membrane-mediated active state of CCT enzyme.

In addition to these specific scientific findings from each chapter, a more general conclusion can

also be made. Both MD simulation and experimental techniques have their own limitations which must be

considered when the data are interpreted and compared between two approaches. This will prevent any

overinterpretation of the data, or making any misleading conclusions from them.

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 There are several factors which determines the reliability of MD simulations [8]. The reliability

of an MD simulation and any computational technique is highly dependent on the assigned parameters to

the comprising molecules in the system. Parameters developed for any new molecule need to be well

tested and validated before being used. This validation should be done, preferably, by direct comparison

with available experimental data [9]. However, to make such a direct comparison, the systems in both

simulation and experiments should be equivalent as much as possible. If not, comparing the data between

two sets could be misleading.

Simulation time is another important factor to be considered [8, 10]. The appropriate time scale is

dependent on the questions asked and the properties of interest in the system. Short simulation times

could result in lack of enough sampling and, consequently, in inaccurate data and conclusions.

Regarding the experimental techniques, the data obtained from one technique might not be

enough to provide a correct/complete description for the molecular system under study. Thus,

characterizing a molecular system usually requires conducting several complementary experiments. The

study conducted on POPC/KC2 mixtures was such an example, where MD simulation, 2H NMR, SAXS,

and cryo-TEM were used to investigate the KC2 localization in binary mixtures. Investigation of CCT

enzyme activation and inhibition mechanism was another example, where both computational and

experimental techniques were combined in a complementary way to detect, test, and validate the

observations.

7.2 Future Directions

The studies and findings presented as well as the methods developed in this thesis could be used for

future studies as follows.

• KC2 is one of the most efficacious ICLs for gene silencing in liver. DLin-MC3-DMA (also known as

MC3) [Figure 7.1] is another ICL, which is known to be 10 times more potent than KC2 in silencing

289
 a gene in hepatocytes in mice [11-13]. However, the underlying molecular interactions causing this

difference are not known. Simulations of MC3 and comparing the results with KC2 simulations might

assist in determining the structural factors responsible for this observed difference in efficacy, and

potentially assist in rational design of new ICLs with a higher efficacy. For instance, it would be

interesting to study interactions between MC3 and POPC to see if the observed segregation is

happening for MC3 or not. The force field parameters for MC3 and its protonated state are not

available and need to be developed. The protocol developed for parameterizing KC2 and KC2H could

be used for parameterization of MC3 in C36 FF.

Figure 7.1: Two-dimensional structure of KC2 and MC3.

Hydrogen atoms, except the protonation one, are not shown for clarity. Avogadro [14] and Chimera [15]

were used to generate the figure.

290
• LNPs containing ICLs also contain DSPC and cholesterol [Figure 1.4] [12]. Looking at interactions

between KC2 and KC2H with these molecules could be directly related to the nano-formulations. It

would be interesting to see if the KC2 segregation is also happening in presence of DSPC and

cholesterols, and if yes, how a change in DSPC/KC2/cholesterol mixing ratio will affect the KC2

solubility.

• This class of LNPs are the leading delivery systems for siRNA molecules [16, 17]. The siRNA

molecules are known to interact with KC2H inside the LNPs. Interactions between siRNAs and

KC2H in presence of water and ions could be useful to look at. As an example, effect of KC2H

concentration, siRNA sequence and length, and ion concentration and types are several factors to

study. Looking at these interactions could provide insight into the stability of the LNPs core structure

under different conditions.

• Given the large size of LNPs (~ 100 nm in diameter), simulation of these systems in a full atomistic

resolution is not currently feasible. Therefore, coarse-grained simulations in which several atoms are

usually treated as a single interacting bead, should be used instead. MARTINI [18] is a popular

coarse-grained model which has been successfully used in many studies on biomolecules, including

siRNA-LNPs [4]. The validated all-atom parameters for KC2 and KC2H, and the atomistic

simulations conducted in this study can be further used for developing, validating, and improving

such coarse-grained models for KC2 and KC2H in MARTINI.

• The current siRNA-LNPs containing ICLs have reached the encapsulation efficacies of 100 % [19].

However, currently, only ca. 1-2 % of the internalized siRNAs are released to cytoplasm while others

are transferred to the lysosome and degraded [6]. Therefore, further optimization should be done on

drug release from endosomes. To do so, an understanding of the underlying mechanism and

interactions involved in the drug escape from endosome for this class of LNPs is required. According

to the proposed mechanism of drug release from endosome for these systems, endosomal membrane

291
 destabilization is due to cationic-anionic lipids interactions between cationic lipids of siRNA-LNPs

and negatively charged lipids of endosomal membrane [20]. Forming ion-pairs with an effective

cone-shaped morphology was hypothesized to cause such a phase transition from lamellar to HII

phase, and consequently destabilizes the endosomal membrane required for drug release.

Therefore, study of KC2H interactions with negatively charged lipids in HII phase would be

interesting. Both early endosomes and late endosomes are known to contain negatively charged lipids,

e.g. phosphatidylserine (PS) and lysobisphosphatidic acid (LBPA), in their lipid composition [21].

The C36 FF parameters for LBPA lipids have been recently developed in our group [22]. Given the

validated parameters for KC2 and KC2H (Chapter 2 and 4), validated HII system setups (Chapter

5), and available parameters for LBPA, this study is possible now.

• Cholesterol is of special interest in lipid research, and is known to affect the structural properties of

lipids in both bilayers and HII phases [23]. It would be interesting to study the effect of cholesterol

concentration on structure of DSPS/KC2H or LBPA/KC2H systems in HII phase [Figure 7.2], and

see how cholesterol is distributed in HII phase. These simulations could potentially provide insight

into the role of cholesterol in HII formation and consequently in drug release.

292
Figure 7.2: HII phase corresponding to DSPS/KC2H/cholesterol (35/35/30).

Left) A single lipid tube composed of DSPS, KC2H and cholesterol, filled with water molecules. Red,

dark blue, and green spheres represent the nitrogen atom of DSPS, nitrogen atom of KC2H, and oxygen

atom in cholesterol molecules, respectively. Orange and light blue lines represent the DSPS and KC2H

tails, whereas the cholesterol body are colored in tan. Water represented as the cyan surface. Hydrogens

are not shown for clarity. Right) a small part of HII phase composed of such single water channel. Lipid

cylinders are arranged in a hexagonal symmetry. Blue box represents the triclinic simulation box. VMD

[24] was used to make the figures.

• Finally, the study on CCT enzyme, determined a new role for AI helices (inhibition of the αE

bending) and supported previously reported role (the backbone trap) [25]. These simulations also

resulted in new structures due to αE helices bending, as a result of in silico removal of AI helices.

These new structures were hypothesized to be corresponding to the active state of CCT upon its

binding and interaction with the PC-deficient lipid membranes [7]. These structures could be used as

the starting structure for future simulations in presence of lipid membranes. It would be interesting to

293
 study how CCT enzyme and its residues interact with a PC-poor and PC-rich membranes. To do this

study, however, the structure for the linker region connecting the αE helices to the M domains should

be determined first.

• CCT enzyme with and without CDP-choline in its active sites has been previously studied using both

MD simulations and experimental techniques [25]. The focus of that study (200 ns simulations) was

on the effect of AI helices on loop L2 dynamics in presence and absence of CDP-choline. It would be

interesting to study this system again using the technique used in Chapter 6, i.e. using NOE

restraints, and compare the results with CCT-CTP system. This might assist in clarifying the role of

bent αEs in different stages of catalysis process. There were more hypothesis and mechanisms

regarding αE bending discussed in Chapter 6 which could be a subject for future experimental and

computational studies.

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375
 Appendix A: Parameters for the Parameterized Segment in the KC2 and KC2H

The final atom types, partial charges, and the bonded parameters for the parameterized segment

[Figure A.1] in KC2 and KC2H lipids are provided here as a reference. The index numbers in

these tables are the same with the atom index numbers in the .itp files for KC2 and KC2H lipids.

These numbers are shown in Figure A.1 for simplicity.

376
Figure A.1: Atom index numbers for the parameterized segment of KC2(H).

Atom index numbers are according to the KC2(H) .itp files. Atom 126 is the proton attached to the

nitrogen atom. This atom is therefore present in KC2H but not in KC2. All the hydrogens are shown with

grey lines representing the bonds, except the protonation hydrogen which is shown with a thick red line.

377
Table A-1: Parameters for the ring segment in KC2 and KC2H.

Index 1, 7 2, 8 3, 9 15, 26 16 17, 18 19 20

Atom CG321 CTL2 CTL2 CG321 CG3C50 OG3C51 CG3C52 CG3C51

Type

Partial -0.2655 -0.180 -0.180 -0.233 0.972 -0.582 0.048 0.151

Charge

Table A-2: Parameters for the amino segments in KC2 and KC2H.

Atom Protonation 21 22 23 24, 25 126

Index

KC2 CG321 CG321 NG301 CG331 --

Atom

Type KC2H CG321 CG324 NG3P1 CG334 HGP2

KC2 -0.116 -0.057 -0.507 -0.110 --

Partial

Charge KC2H -0.068 0.146 -0.446 0.061 0.346

378
Table A-3: Parameters for the hydrogen atoms in KC2 and KC2H.

Atom 49, 50, 51, 52, 47, 48, 59, 60, 74, 75, 76, 78 83, 84, 85,

Index 61, 62, 63, 64 77, 79, 80, 81, 82, 89, 90 86, 87, 88

Atom HAL2 HGA2 HGA1 HGA3

Type

Partial 0.090 0.090 0.090 0.090

Charge

Table A-4: Parameters for several bonds defined in KC2 and KC2H.

These parameters were added to the C36FF ffbonded.itp file, in the bonds section.

* b0 and kb represents the equilibrium bond length and the corresponding force constant in the harmonic

term in the potential function, respectively.

Atom Atom Function

Type Type Type *b0 *kb

𝒏𝒎 𝒌𝑱 𝒎𝒐𝒍−𝟏 𝒏𝒎−𝟐

CG321 CTL2 1 0.153 186188

CG321 CG3C50 1 0.1528 186188

CG3C50 OG3C51 1 0.1425 292880

CG321 NG301 1 0.1474 220078.4

379
Table A-5: Parameters for several angles defined in KC2 and KC2H.

These parameters were added to the C36FF ffbonded.itp file, in the angles section.

* theta0 and ktheta are the equilibrium angle and the force constant in the harmonic term in the potential

function, respectively.

** ub0 and kub mean the equilibrium distance between i-k atoms in an i-j-k angle formed by three

connected atoms, and the force constant in the Urey-Bradley term in the potential function, respectively.

Atom Atom Atom Function

Type Type Type Type *theta0 *ktheta **ub0 **kub

𝒅𝒆𝒈𝒓𝒆𝒆 𝒌𝑱 𝒎𝒐𝒍−𝟏 𝒓𝒂𝒅−𝟐 𝒏𝒎 𝒌𝑱 𝒎𝒐𝒍−𝟏 𝒏𝒎−𝟐

CG321 CG321 CTL2 5 113.6 488.2728 0.2561 9338.69

CG321 CTL2 CTL2 5 113.6 488.2728 0.2561 9338.69

HGA2 CG321 CTL2 5 110.1 221.752 0.2179 18853.1

HAL2 CTL2 CG321 5 110.1 221.752 0.2179 18853.1

CG321 CG321 CG3C50 5 111 446.4328 0.2561 6694.4

CG321 CG321 CG3C51 5 111 446.4328 0.2561 6694.4

CG321 CG321 NG301 5 112.2 365.6816 0 0

CG3C50 CG321 HGA2 5 110.1 289.5328 0.2179 18853.1

NG301 CG321 HGA2 5 109.5 271.1232 0.213 41840

NG301 CG331 HGA3 5 109.7 255.224 0.214 41840

CG321 CG3C50 CG321 5 113.5 488.2728 0.2561 9338.69

380
CG321 CG3C50 OG3C51 5 111.5 376.56 0 0

OG3C51 CG3C50 OG3C51 5 108.1 711.28 0 0

CG3C50 OG3C51 CG3C51 5 111 794.96 0 0

CG3C50 OG3C51 CG3C52 5 111 794.96 0 0

CG324 CG321 CG3C51 5 110.5 488.2728 0.2561 9338.69

CG334 NG3P1 CG334 5 109.5 376.56 0 0

CG321 NG301 CG331 5 110.9 443.504 0 0

Table A-6: Parameters for several dihedrals defined in KC2 and KC2H.

These parameters were added to the C36FF ffbonded.itp file, in the proper dihedrals section.

* phi0 and kphi are the phase shift and the force constant in the sinusoidal expansion term in the potential

function corresponding to the proper dihedral energy, respectively.

Atom Atom Atom Atom Function

Type Type Type Type Type *phi0 *kphi multiplicity

𝒅𝒆𝒈𝒓𝒆𝒆 𝒌𝑱 𝒎𝒐𝒍−𝟏

CG321 CG321 CTL2 CTL2 9 0 0.422584 2

CG321 CG321 CTL2 CTL2 9 180 0.594128 3

CG321 CG321 CTL2 CTL2 9 0 0.309616 4

CG321 CG321 CTL2 CTL2 9 0 0.405848 5

CG321 CTL2 CTL2 CTL2 9 0 0.422584 2

381
CG321 CTL2 CTL2 CTL2 9 180 0.594128 3

CG321 CTL2 CTL2 CTL2 9 0 0.309616 4

CG321 CTL2 CTL2 CTL2 9 0 0.405848 5

HGA2 CG321 CG321 CTL2 9 0 0.81588 3

HAL2 CTL2 CG321 G321 9 0 0.81588 3

HAL2 CTL2 CG321 HGA2 9 0 0.92048 3

HGA2 CG321 CTL2 CTL2 9 0 0.81588 3

CG3C50 CG321 CG321 HGA2 9 0 0.81588 3

CG3C51 CG321 CG321 HGA2 9 0 0.81588 3

NG301 CG321 CG321 HGA2 9 0 0.66944 3

CG321 CG321 CG3C50 CG321 9 0 0.8368 3

HGA2 CG321 CG3C50 CG321 9 0 0.79496 3

HGA2 CG321 CG3C50 OG3C51 9 0 0.66944 3

CG321 CG321 CG3C51 CG3C52 9 180 2.092 4

CG321 CG321 CG3C51 HGA1 9 0 0.81588 3

HGA2 CG321 NG301 CG331 9 180 0 3

HGA3 CG331 NG301 CG321 9 180 0 3

CG321 CG3C50 OG3C51 CG3C51 9 0 1.2552 3

CG321 CG3C50 OG3C51 CG3C52 9 0 1.2552 3

OG3C51 CG3C50 OG3C51 CG3C51 9 180 6.9036 3

OG3C51 CG3C50 OG3C51 CG3C52 9 180 6.9036 3

CG321 CG3C51 CG3C52 OG3C51 9 0 0 3

382
OG3C51 CG3C51 CG3C52 OG3C51 9 0 1.08784 3

CG321 CG3C51 OG3C51 CG3C50 9 0 1.2552 3

CG3C52 CG3C51 OG3C51 CG3C50 9 0 2.092 3

HGA1 CG3C51 OG3C51 CG3C50 9 0 1.2552 3

CG3C51 CG3C52 OG3C51 CG3C50 9 0 2.092 3

HGA2 CG3C52 OG3C51 CG3C50 9 0 1.2552 3

CG3C51 CG321 CG324 HGA2 9 0 0.81588 3

CG324 CG321 CG3C51 CG3C52 9 180 2.092 4

CG324 CG321 CG3C51 OG3C51 9 180 14.2256 1

CG324 CG321 CG3C51 HGA1 9 0 0.81588 3

HGA3 CG334 NG3P1 CG334 9 0 0.4184 3

Table A-7: Dihedral parameters specific to KC2.

These parameters were included in KC2.itp file:

* phi0 and kphi are the phase shift and the force constant in the sinusoidal expansion term in the potential

function corresponding to the proper dihedral energy, respectively.

Atom Atom Atom Atom Function

Index Index Index Index Type *phi0 *kphi multiplicity

𝒅𝒆𝒈𝒓𝒆𝒆 𝒌𝑱 𝒎𝒐𝒍−𝟏

1 26 16 17 9 180 8.61063 1

1 26 16 17 9 180 0.98874 2

383
1 26 16 17 9 0 2.05603 3

1 26 16 17 9 180 0.36095 4

1 26 16 17 9 180 0.04944 6

1 26 16 18 9 180 8.61063 1

1 26 16 18 9 180 0.98874 2

1 26 16 18 9 0 2.05603 3

1 26 16 18 9 180 0.36095 4

1 26 16 18 9 180 0.04944 6

2 1 26 16 9 0 2.96923 1

2 1 26 16 9 0 0.52548 2

2 1 26 16 9 180 1.41779 3

2 1 26 16 9 0 0.71499 4

2 1 26 16 9 180 0.27977 6

7 15 16 17 9 180 8.61063 1

7 15 16 17 9 180 0.98874 2

7 15 16 17 9 0 2.05603 3

7 15 16 17 9 180 0.36095 4

7 15 16 17 9 180 0.04944 6

7 15 16 18 9 180 8.61063 1

384
7 15 16 18 9 180 0.98874 2

7 15 16 18 9 0 2.05603 3

7 15 16 18 9 180 0.36095 4

7 15 16 18 9 180 0.04944 6

8 7 15 16 9 0 2.96923 1

8 7 15 16 9 0 0.52548 2

8 7 15 16 9 180 1.41779 3

8 7 15 16 9 0 0.71499 4

8 7 15 16 9 180 0.27977 6

17 20 21 22 9 180 1.81701 1

17 20 21 22 9 0 0.95898 2

17 20 21 22 9 0 2.26375 3

17 20 21 22 9 180 0.63793 4

17 20 21 22 9 180 0.03559 6

20 21 22 23 9 180 0.90678 1

20 21 22 23 9 0 0.45645 2

20 21 22 23 9 0 0.23682 3

20 21 22 23 9 0 0.34473 4

20 21 22 23 9 180 0.27423 6

385
21 22 23 24 9 180 6.44817 1

21 22 23 24 9 0 3.26084 2

21 22 23 24 9 0 5.26565 3

21 22 23 24 9 180 0.40517 4

21 22 23 24 9 0 0.36597 6

21 22 23 25 9 180 6.44817 1

21 22 23 25 9 0 3.26084 2

21 22 23 25 9 0 5.26565 3

21 22 23 25 9 180 0.40517 4

21 22 23 25 9 0 0.36597 6

Table A-8: Dihedral parameters specific to KC2H.

These parameters were included in KC2H.itp file:

* phi0 and kphi are the phase shift and the force constant in the sinusoidal expansion term in the potential

function corresponding to the proper dihedral energy, respectively.

Atom Atom Atom Atom Function

Index Index Index Index Type *phi0 *kphi multiplicity

𝒅𝒆𝒈𝒓𝒆𝒆 𝒌𝑱 𝒎𝒐𝒍−𝟏

1 26 16 17 9 180 8.61063 1

1 26 16 17 9 180 0.98874 2

386
1 26 16 17 9 0 2.05603 3

1 26 16 17 9 180 0.36095 4

1 26 16 17 9 180 0.04944 6

1 26 16 18 9 180 8.61063 1

1 26 16 18 9 180 0.98874 2

1 26 16 18 9 0 2.05603 3

1 26 16 18 9 180 0.36095 4

1 26 16 18 9 180 0.04944 6

2 1 26 16 9 0 2.96923 1

2 1 26 16 9 0 0.52548 2

2 1 26 16 9 180 1.41779 3

2 1 26 16 9 0 0.71499 4

2 1 26 16 9 180 0.27977 6

7 15 16 17 9 180 8.61063 1

7 15 16 17 9 180 0.98874 2

7 15 16 17 9 0 2.05603 3

7 15 16 17 9 180 0.36095 4

7 15 16 17 9 180 0.04944 6

7 15 16 18 9 180 8.61063 1

387
7 15 16 18 9 180 0.98874 2

7 15 16 18 9 0 2.05603 3

7 15 16 18 9 180 0.36095 4

7 15 16 18 9 180 0.04944 6

8 7 15 16 9 0 2.96923 1

8 7 15 16 9 0 0.52548 2

8 7 15 16 9 180 1.41779 3

8 7 15 16 9 0 0.71499 4

8 7 15 16 9 180 0.27977 6

17 20 21 22 9 180 1.81701 1

17 20 21 22 9 0 0.95898 2

17 20 21 22 9 0 2.26375 3

17 20 21 22 9 180 0.63793 4

17 20 21 22 9 180 0.03559 6

20 21 22 23 9 180 1.90831 1

20 21 22 23 9 180 0.50746 2

20 21 22 23 9 180 0.26074 3

20 21 22 23 9 0 0.50625 4

20 21 22 23 9 180 0.6132 6

388
21 22 23 24 9 180 0.18613 1

21 22 23 24 9 0 0.18274 2

21 22 23 24 9 0 2.52813 3

21 22 23 24 9 0 0.189 4

21 22 23 24 9 0 0.41145 6

21 22 23 25 9 180 0.18613 1

21 22 23 25 9 0 0.18274 2

21 22 23 25 9 0 2.52813 3

21 22 23 25 9 0 0.189 4

21 22 23 25 9 0 0.41145 6

389
 Appendix B: Supplementary Information for Chapter Four

B.1 KC2 and KC2H Parameterization in the CHARMM36 Force Field

Although the parameters for POPC are available, optimized and well-tested in C36 FF [1], parameters for

KC2 and KC2H are not present and need to be developed. The step-by-step protocol for parameterization

of a novel molecule in C36 FF is documented in detail in several publications [2, 3]. In this study, several

computational tools, including CHARMM-GUI [4], GAAMP [5], and ParamChem [6, 7], were used to

develop reasonable models for KC2 and KC2H compatible with C36 FF.

The KC2(H) molecules were first divided into two main segments: lipid tails and headgroup. The

parameters for the tails were simply taken from DUPC downloaded from CHARMM-GUI [4], with atom

types taken from C36 FF. Next, the headgroup was further divided into two smaller segments (the ring

and the amino segments) and each segment was parameterized using the GAAMP server [5], with atom

types taken from CHARMM General force field (CGenFF) [3]. The parameters developed for the

segments were subsequently combined to give the parameter set for the whole lipid molecules. Where

needed, parameters were further modified in an iterative procedure until the desired level of agreement

between computational and experimental data was achieved (see below). A more detailed description of

the parameterization process, and the final parameter set are provided in section 2.4.1 of Chapter 2, and

Appendix A, respectively.

B.1.1 KC2 and KC2H model validation.

The amount of target data to validate parameters against are limited for the case of KC2 and KC2H. In

this study, the effect of adding KC2(H) on POPC palmitoyl chain order parameters in POPC/KC2(H)

mixtures at 313 K was used for KC2(H) model validation.

390
 The SCD profiles for the POPC palmitoyl chain extracted from 2H NMR spectra and calculated

from simulations of POPC/KC2(H) binary mixtures are shown in Figure B.1. In simulations, systems

with KC2 and KC2H correspond to the 2H NMR experiments conducted at pH 8.1 and 4.4, respectively.

The SCD parameters from simulations are in good agreement with the experimental data. The

overall trend in SCD profiles is similar between simulations and experiments. The acyl chain order

gradually decreases along the acyl chain towards the terminal methyl group. Also, in both simulations and

experiments, the pH seems to have no or little effect on acyl chain order parameter at 313 K. The only

difference was observed between pure POPC bilayers at two pH values. We note that the experimental

POPC palmitoyl chain order parameters are smaller at pH 4.4 than at pH 8.1. This is likely due to the

higher interfacial surface tension observed in POPC membranes at pH 4.4 [8]. In simulations, however,

the SCD parameters for palmitoyl chain are the same between two pH levels. That is mainly because the

effect of buffer and the higher surface tension were not considered in our simulations.

Finally, the SCD parameters calculated from simulations are slightly higher than the corresponding

experimental values. This is also true for POPC which the parameters are optimized and well tested in

C36 FF.

391
Figure B.1: Validation of developed models for KC2 and KC2H.

Smoothed deuterium order parameter (SCD) for the palmitoyl chain of POPC in POPC/KC2(H) binary

mixtures were measured using 2H NMR experiments (Left column). The same parameter was calculated

from simulations and smoothed (Right column). This comparison was done for two pH levels of 4.4 (a

and b) and 8.1 (c and d) at temperature of 313 K.

392
B.2 KC2(H) Effects on POPC as a Function of Temperature and Mixing Ratio

The POPC-d31 order parameter (SCD) profiles for POPC/KC2(H) systems at different temperatures and

mixing ratios were measured using 2H NMR technique. The effect of KC2 and KC2H on POPC sn-1 acyl

chain SCD was shown to be a function of both temperature and mixing ratio [Figure B.2 and Figure B.3].

For both pH 4.4 and 8.1, and all the mixing ratios studied, higher temperature resulted in lower SCD

values as expected. For the pure POPC system, the POPC palmitoyl chain order parameters are

significantly smaller at pH 4.4 than pH 8.1. At higher temperatures (303 K and 313 K), pH has little to no

effect on chain order in POPC/KC2(H) systems. At 288 K and 293 K, KC2 has no effect on POPC chain

order, while KC2H increases it.

393
Figure B.2: SCD parameters for the palmitoyl chain (sn-1) of POPC at 288 K and 293 K.

Experimental SCD parameters (smoothed) measured using 2H NMR technique for binary POPC/KC2(H)

mixtures at (a) pH = 4.4 and T = 288 K, (b) pH = 8.1 and T = 288 K, (c) pH = 4.4 and T = 293 K, and (d)

pH = 8.1 and T = 293 K.

Such an induced order in PC lipids due to adding cationic lipids to the system has been previously

reported [9]. Including cationic lipids (DMTAP) in a zwitterionic (DMPC) membrane was reported to

induce acyl chain order in DMPC in a concentration-dependent way, up to a 0.50 mole fraction of

DMTAP [9]. Increasing the DMTAP mole fraction above 0.50, however, started to decrease the induced

394
order. To explain this observation, the authors proposed a scenario as follows. Given that both DMTAP

and DMPC have the same acyl chains, a small amount of DMTAP tends to reorient the nearby DMPC

headgroups tilt angle to smaller angles. This will result in a more packed structures with a reduced area

per lipid and increased acyl chain order. Upon increase in the DMTAP concentration beyond a limit, the

electrostatic repulsions between the positively charged lipids dominate which result in the membrane

surface expansion and a decrease in the acyl chain order.

395
Figure B.3: SCD parameters for the palmitoyl chain (sn-1) of POPC at 303 K and 313 K.

Experimental SCD parameters (smoothed) measured using 2H NMR technique for binary POPC/KC2(H)

mixtures at (a) pH = 4.4 and T = 303 K, (b) pH = 8.1 and T = 303 K, (c) pH = 4.4 and T = 313 K, and (d)

pH = 8.1 and T = 313 K.

The same scenario might also be valid here and could explain the induced chain order at pH 4.4

[Figure B.2]. However, since the acyl chains in KC2H and POPC are not the same, the behavior of

POPC/KC2H systems are rather more complex to explain. This complex pattern (i.e. temperature-

concentration dependency) of induced order might be a collective effect of several factors, including the

396
interfacial surface tension [8], which is likely relaxed due to KC2H, the observed decrease in POPC P→N

vector tilt angle (see section B.6) [9], lipid-ion interactions (see section B.5) [10], and lipid-lipid

interactions [9, 11]. Other factors might also contribute to such a complex behavior. The so-called free

volume in the center of the bilayer [12] could be one such factor. In a study by Kupianien et al. [13], it

was shown that a reduction in the free volume of membrane result in a tighter molecular packing and

enhanced ordering of the acyl chains.

B.3 KC2(H) Effect on POPC Lamellar Repeat Spacing

If neutral KC2 are segregated and confined in the POPC bilayer center, systems with a higher

concentration of KC2 are expected to result in systems with larger repeat spacings (d-spacing) at higher

pH values. According to this expectation and the observations from simulations, small angle X-ray

scattering experiments were carried out on POPC multilamellar vesicles containing 0, 20 or 30 mol %

KC2 at pH 8.1, 8.5 or 10 [Figure B.4]. The major finding is that added KC2 causes a dramatic reduction

in structure correlations in the sample, as exemplified by the broad scattering peak [Figure B.4]. A

second result is that the bilayer repeat spacing increases from 65 angstroms to 68 angstroms upon the

addition of 20 mol % KC2 to POPC at pH 8.1. Increasing the pH to 8.5 results in a reduction in lamellar

repeat spacing to 67.5 angstroms, and increasing it to 10 brings the lamellar repeat spacing back to 65

angstroms. These changes in lamellar repeat spacing can be explained by the presence of a small fraction

of positively charged KC2H at pH 8.1 and 8.5 and are consistent with the observations of B. Pozo Navas

et al. [14] on POPC/POPG mixtures. KC2 is silent in the SAXS patterns, and is likely separating into oily

droplets associated with the POPC lamellae and thereby disrupting their stacking regularity.

397
Figure B.4: SAXS curves for POPC/KC2 multilamellar vesicles at several basic pH values.

SAXS curves for POPC multilamellar vesicles containing 0, 20 or 30 mol % KC2 at the indicated pH.

B.4 KC2H has Little to no Effect on the POPC Bilayer Thickness

Disregard of the KC2H content in the mixtures, simulations suggest that all KC2H stay in the lipid-water

interface [Figure 4.1]. Figure B.5 shows the symmetrized electron densities for the POPC phosphorus

atoms in POPC/KC2H systems at different mixing ratios, pH = 4.4 and temperature 313 K. For the pure

398
POPC, the inter-leaflet distance is approximately 3.72 nm. Adding KC2H had little to no effect on POPC

P-P distance.

Figure B.5: Effect of adding KC2H on POPC inter-leaflet distance.

The electron density of the phosphorus atoms of POPC were calculated from simulations of binary

POPC/KC2H mixtures at pH = 4.4 and T = 313 K. The X axis shows the relative distance from the

bilayer center. Each double-headed arrow represents the peak-to-peak distance for a mixing ratio,

corresponding to the P-P inter-leaflet distance. The P-P distance for the pure POPC system at 313 K is

about 3.72 nm.

399
B.5 KC2H Repels Na Ions from and Attracts Cl Ions to the Lipid-Water Interface

Given the positive charge of amino group in KC2H, the electrostatic interactions with both negatively and

positively charged groups (e.g. ions and POPC headgroups) in the system are expected. To study how

KC2H affect the structural properties of the system, we further looked at the ion distribution and POPC

headgroup tilt angle as a function of KC2H concentration in the system at 313 K.

Electron densities for the Cl and Na ions in POPC/KC2H systems simulated at acidic pH and

temperature 313 K are shown in Figure B.6. Adding KC2H to the system redistributes the ions. For the

pure POPC system, the Cl ions density is zero till ca. 1.8 nm from the bilayer center [Figure B.6-a]. It

then increases smoothly and reaches its maximum at ca. 2.9 nm. Finally, it decreases slightly and reaches

a plateau corresponding to a uniform distribution of ions in the aqueous phase. Adding KC2H to the

POPC system populates the hydration layer with Cl ions. The Cl ion density in the hydration layer was

directly correlated with the KC2H concentration in the bilayer. Furthermore, in the presence of KC2H, the

peak corresponding to the maximum Cl ion density were shifted towards the bilayer center, compared to

the pure POPC system.

400
Figure B.6: Effect of KC2H on ion distribution.

Symmetrized electron densities of a) Cl and b) Na ions for systems with different mixing ratios simulated

at pH = 4.4 and T = 313 K. Assuming the bilayers are centered at zero, only the densities for the positive

positions are shown. The X axis shows the direction normal to the bilayer surface.

Na ions [Figure B.6-b] are distributed deeper in the hydration layer, compared to the Cl ions. The

Na density peaks around 1.8 nm. The Na density near the bilayer surface is slightly less than Na density

in aqueous phase. Adding KC2H to POPC repels the Na ions from the bilayer.

The Cl and Na ions electron densities in pure POPC system [Figure B.6] agree with previous

studies on ion distributions in lipid bilayers [15, 16]. Furthermore, Na ions were found to be deeper in the

hydration layer compared to Cl ions, as expected. Figure B.6 suggests that adding KC2H affects the ion

distribution along the bilayer normal. Upon adding KC2H to the system, the Cl ions are attracted towards

the lipid-water interface, while the Na ions are repelled from the interface. This behavior is also consistent

with previous reports on membranes containing cationic lipids [9, 15]. Presence of positively charged

KC2H in the membrane increases the effective positive charge of the membrane surface. This

401
consequently attracts the negatively charged Cl ions and deplete the lipid-water interface from positively

charged Na ions.

B.6 KC2H Affects the POPC Headgroup Distribution

The tilt angle for the POPC headgroup (i.e. the angle between the POPC P→N vector and outward normal

to the leaflet) were calculated for each system at 313 K and are shown in Figure B.7. For the pure POPC

simulations, this angle is approximately 70 degrees [Figure B.7], in a close agreement with previous

studies [17-19]. Adding KC2H to the POPC bilayers is shown to affect the distribution of POPC

headgroups. More KC2H in the system resulted in smaller tilt angles so that the POPC choline group

moves towards the aqueous phase. For instance, 30 mol% KC2H reduced this angle from ~ 70 to ~ 43

degrees. KC2, on the other hand, did not have any effect on the average POPC headgroup angle

distribution.

402
Figure B.7: Effect of KC2 and KC2H on POPC P→N vector angle distribution.

POPC headgroup angle distribution for each simulated system at T = 313 K are shown. The X axis shows

the angle between the POPC P→N vector with the outward direction normal to the leaflet. Adding KC2H

to the system reduces the POPC headgroup tilt angle at the acidic conditions but leaves it intact at the

basic condition (systems with KC2).

Similar to the change in the ion distributions, the POPC headgroup will also interact with cationic

lipids in the system. These electrostatic interactions will straighten the POPC headgroup and move the

choline group to the aqueous phase [9, 20]. Consistent with what was observed in a study by Gurtovenko

403
et al. [9] on zwitterionic-cationic lipid mixtures, including positively charged lipids in the PC bilayer

shifts the PC headgroup tilt angle to the smaller angles, i.e. make it more perpendicular to the bilayer

surface. The observed changes in the POPC headgroup tilt angle in this study might partially explain why

adding KC2H to the POPC dispersions results in smaller vesicles [21].

B.7 Bibliography

[1]. Klauda, J.B., Venable, R.M., Freites, J.A., O’Connor, J.W., Tobias, D.J., Mondragon-Ramirez,

C., Vorobyov, I., MacKerell Jr, A.D. & Pastor, R. W. (2010). Update of the CHARMM all-atom

additive force field for lipids: Validation on six lipid types. The Journal of Physical Chemistry B,

114(23), 7830-7843.

[2]. Foloppe, N., & MacKerell, Jr, A. D. (2000). All‐atom empirical force field for nucleic acids: I.

Parameter optimization based on small molecule and condensed phase macromolecular target

data. Journal of Computational Chemistry, 21(2), 86-104.

[3]. Vanommeslaeghe, K., Hatcher, E., Acharya, C., Kundu, S., Zhong, S., Shim, J., Darian, E.,

Guvench, O., Lopes, P., Vorobyov, I. & Mackerell Jr, A. D. (2010). CHARMM general force

field: A force field for drug‐like molecules compatible with the CHARMM all‐atom additive

biological force fields. Journal of Computational Chemistry, 31(4), 671-690.

[4]. Jo, S., Kim, T., Iyer, V. G., & Im, W. (2008). CHARMM‐GUI: A web‐based graphical user

interface for CHARMM. Journal of Computational Chemistry, 29(11), 1859-1865.

[5]. Huang, L., & Roux, B. (2013). Automated force field parameterization for nonpolarizable and

polarizable atomic models based on ab initio target data. Journal of Chemical Theory and

Computation, 9(8), 3543-3556.

404
[6]. Vanommeslaeghe, K., & MacKerell Jr, A. D. (2012). Automation of the CHARMM General

Force Field (CGenFF) I: Bond perception and atom typing. Journal of Chemical Information and

Modeling, 52(12), 3144-3154.

[7]. Vanommeslaeghe, K., Raman, E. P., & MacKerell Jr, A. D. (2012). Automation of the

CHARMM General Force Field (CGenFF) II: Assignment of bonded parameters and partial

atomic charges. Journal of Chemical Information and Modeling, 52(12), 3155-3168.

[8]. Petelska, A. D., & Figaszewski, Z. A. (2000). Effect of pH on the interfacial tension of lipid

bilayer membrane. Biophysical Journal, 78(2), 812-817.

[9]. Gurtovenko, A. A., Patra, M., Karttunen, M., & Vattulainen, I. (2004). Cationic DMPC/DMTAP

lipid bilayers: Molecular dynamics study. Biophysical Journal, 86(6), 3461-3472.

[10]. Pandit, S. A., Bostick, D., & Berkowitz, M. L. (2003). Molecular dynamics simulation of a

dipalmitoylphosphatidylcholine bilayer with NaCl. Biophysical Journal, 84(6), 3743-3750.

[11]. Pandit, S. A., Bostick, D., & Berkowitz, M. L. (2003). Mixed bilayer containing

dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine: Lipid complexation, ion

binding, and electrostatics. Biophysical Journal, 85(5), 3120-3131.

[12]. Marrink, S. J., Sok, R. M., & Berendsen, H. J. C. (1996). Free volume properties of a simulated

lipid membrane. The Journal of Chemical Physics, 104(22), 9090-9099.

[13]. Kupiainen, M., Falck, E., Ollila, S., Niemelä, P., Gurtovenko, A.A., Hyvönen, M.T., Patra, M.,

Karttunen, M. & Vattulainen, I. (2005). Free volume properties of sphingomyelin, DMPC, DPPC,

and PLPC bilayers. Journal of Computational and Theoretical Nanoscience, 2(3), 401-413.

[14]. Navas, B.P., Lohner, K., Deutsch, G., Sevcsik, E., Riske, K.A., Dimova, R., Garidel, P. & Pabst,

G. (2005). Composition dependence of vesicle morphology and mixing properties in a bacterial

model membrane system. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1716(1), 40-48.

405
[15]. Gurtovenko, A. A., Miettinen, M., Karttunen, M., & Vattulainen, I. (2005). Effect of monovalent

salt on cationic lipid membranes as revealed by molecular dynamics simulations. The Journal of

Physical Chemistry B, 109(44), 21126-21134.

[16]. Böckmann, R. A., Hac, A., Heimburg, T., & Grubmüller, H. (2003). Effect of sodium chloride on

a lipid bilayer. Biophysical Journal, 85(3), 1647-1655.

[17]. Pluhackova, K., Kirsch, S. A., Han, J., Sun, L., Jiang, Z., Unruh, T., & Böckmann, R. A. (2016).

A critical comparison of biomembrane force fields: Structure and dynamics of model DMPC,

POPC, and POPE bilayers. The Journal of Physical Chemistry B, 120(16), 3888-3903.

[18]. Doux, J. P., Hall, B. A., & Killian, J. A. (2012). How lipid headgroups sense the membrane

environment: An application of 14N NMR. Biophysical Journal, 103(6), 1245-1253.

[19]. Semchyschyn, D. J., & Macdonald, P. M. (2004). Conformational response of the

phosphatidylcholine headgroup to bilayer surface charge: Torsion angle constraints from dipolar

and quadrupolar couplings in bicelles. Magnetic Resonance in Chemistry, 42(2), 89-104.

[20]. Bechinger, B., & Seelig, J. (1991). Interaction of electric dipoles with phospholipid head groups.

A deuterium and phosphorus-31 NMR study of phloretin and phloretin analogs in

phosphatidylcholine membranes. Biochemistry, 30(16), 3923-3929.

[21]. Kulkarni, J.A., Darjuan, M.M., Mercer, J.E., Chen, S., van der Meel, R., Thewalt, J.L., Tam,

Y.Y.C. & Cullis, P. R. (2018). On the formation and morphology of lipid nanoparticles

containing ionizable cationic lipids and siRNA. ACS nano, 12(5), 4787-4795.

406
 Appendix C: Supplementary Information for Chapter Five

C.1 Semi-isotropic versus Anisotropic Pressure Coupling

Anisotropic pressure coupling has been used in many simulations on HII phase [1-3]. In those studies, the

pressure in each direction was treated independently, and both the simulation box size and shape were

allowed to change. This way, system can undergo deformations and form new phases, if energetically

favorable. Assuming the lipid cylinders in HII phase are along the Z axis, HII systems are symmetric in

the XY-plane. Therefore, simulations of HII phase could also be conducted using the semi-isotropic

pressure coupling, where the pressure in Z direction and in XY-plane are treated independently. From a

computational point of view, it is important to evaluate the extent which each pressure coupling could

affect the structural properties of HII systems. All the results presented in Chapter 5 were based on the

simulations conducted with an applied anisotropic pressure coupling, although the shape of simulation

box kept fixed. A few systems in this study were also simulated using semi-isotropic coupling [Table

5-1], and the results were compared with anisotropic simulations.

Figure C.1 shows the dhex for DOPE HII systems calculated from simulations at 303 K using two

different pressure coupling algorithms. The dhex values calculated from two approaches agree with each

other. One exception was the HII system with 26 nw, which the predicted dhex from anisotropic simulation

is higher than the interpolated value (according to the fitted line) from semi-isotropic simulations.

The molecular systems were further inspected visually. For the hydration levels less or near the

maximum hydration (i.e. up to 20 nw), systems simulated using both coupling methods were similar in

their water core shapes at each hydration level. However, using semi-isotropic and anisotropic couplings

caused differences in HII structures at higher hydration levels. For instance, the systems with 26 nw and

407
30 nw simulated using anisotropic coupling were stretched in the Y direction, whereas the system with

even 30 nw simulated using semi-isotropic coupling maintained its hexagonal shape.

To conclude, both semi-isotropic and anisotropic could be used for hydration levels less or near

the maximum hydration. For the higher hydration levels, choosing the appropriate coupling method

depends on the question of interest.

408
Figure C.1: Lattice distance as a function of hydration for DOPE HII systems (semi-isotropic

versus anisotropic pressure coupling).

Lattice distances for DOPE HII systems were calculated from simulations conducted at T = 303 K using

either semi-isotropic or anisotropic pressure couplings and results are compared. The red line is the linear

fit to the data from semi-isotropic simulations. The orange box points to several systems simulated using

anisotropic pressure coupling (black circles) which, based on visualization inspection, the water core

shape of these systems tended to deviate or deviated from their cylindrical/hexagonal shape. Error bars

represent the standard deviation of means, and their size are comparable to the symbols.

409
C.2 Bibliography

[1]. Marrink, S. J., & Mark, A. E. (2004). Molecular view of hexagonal phase formation in

phospholipid membranes. Biophysical Journal, 87(6), 3894-3900.

[2]. Marrink, S. J., De Vries, A. H., & Mark, A. E. (2004). Coarse grained model for semiquantitative

lipid simulations. The Journal of Physical Chemistry B, 108(2), 750-760.

[3]. Knecht, V., Mark, A. E., & Marrink, S. J. (2006). Phase behavior of a phospholipid/fatty

acid/water mixture studied in atomic detail. Journal of the American Chemical Society, 128(6),

2030-2034.

410
 Appendix D: Supplementary Information for Chapter Six

Figure D.1: Dynamics of active site loops and side chains based on the 20 simulations with

positional restraints (PR) on residues 216-223 of the EC helices.

411
RMSF of heavy atoms of the indicated residues, excluding backbone, were computed as described in the

Methods. The data are means  average deviation of 10 replicates (5 CCT dimer simulations × 2 chains

each) for each condition indicated in the matrix at the top of the figure. The asterisks above each bar

indicate that the value is significantly different (*, P < 0.05; **, P < 0.0005) with reference to the first set

(white bar). The results resemble those for the analyses on 20 simulations using NOE restraints [Figure

6.3] with two exceptions. In the NOE-restrained analysis there was no significant effect of the AI on

R196 or K122 RMSF. Whereas in the PR analyses R196 was significantly restrained by the AI (RMSF

decreased 27%); and K122 showed a ~25% higher RMSF in the absence of AI and CTP. An explanation

for the lower RMSF for K122 in the NOE restrained simulations (Figure 6.3; RMSF = 2.3 0.6 Å) is that

the Ec helices bend to make contact with L2 and this restrains the L2 backbone and K122. This bending

is not possible with PR restraints.

412
Figure D.2: Dynamics of Loop L2 in vitro.

A) Location of F-121 and F-124, sites in L2 for tryptophan substitution. Image is from a simulation with

AI helices, and PR applied to αEC. The residues highlighted in ball and stick form a hydrophobic pocket

for F124. B) Effect of lipid vesicles on the fluorescence anisotropy of tryptophans in loop L2. Purified

CCTs were incubated with or without lipid (Lyso PC/PG (4/1) micelles) for 5 min prior to acquisition of

steady state fluorescence anisotropy for the indicated Trp residue. The data are averages ± S.D. of 5

replicates. ** P< 0.005.

413
Supplemental text related to Figure D.3.

Gly123-dependent flexibility of the L2 backbone allows alternate backbone configurations at K122.

Do these configurations correlate with CTP vs backbone trap contacts?

The data showing that non-glycine substitutions at G123 in L2 inhibited CCT activity spurred an

investigation of the impact of the L2 backbone configuration on contact preferences for the catalytic

residue, K122. We examined the L2 backbone configurations of all 40 simulations (20 with positional

restraints on the EC helix and 20 with NOE restraints) and found facile switching between the  and 

backbone configurations, which strictly correlated with a switch in the , angles at G123 from glycine-

restricted values to -helical values. We mined the simulations to generate correlation maps between the

dihedrals at K122 and H-bonding partners for K122. We found that the AI helix promoted the 

configuration at K122, but only when CTP was absent. With CTP present there was an even distribution

of  and  configurations [Figure D.3-A].

The correlation analysis revealed that the interaction of K122 with CTP was compatible with

either the  or  configuration at K122 in the absence of the AI; but the AI helix hampered bonds to CTP

when K122 was in the  configuration [Figure D.3-B]. The interaction with the backbone trapping atoms

was strongly correlated with the  configuration [Figure D.3, C and D]. In PR simulations with the AI

present and where the backbone trap interaction was dominant, extending for >750 ns, the  configuration

was highly preferred and was also maintained for >750 ns. This occurred in 8 of 10 chains. In the NOE

simulations with AI present 6 of 10 chains showed continuous backbone traps for >500 ns, and the L2

configuration was β in five and  in one of the six. In simulations lacking the AI helix we also found that

the backbone trap correlated strongly with the  configuration at K122 [Figure D.3-C].

These data suggest that the  configuration at K122 does not preclude an interaction with CTP,

but that the  configuration is strongly preferred for trapping by the backbone atoms. This is a reasonable

414
outcome because in the  configuration the lysine is directed away from the interior of the active site

compared to in the  configuration.

We examined individual time plots to further probe whether the  configuration prevents K122

interaction with CTP when the AI is present. When CTP and AI were both present, interaction of K122

with the AI was strongly correlated with the  configuration, while contact with CTP was only weakly

correlated with the  configuration [Figure D.3, B-D]. In 7 of 19 chains with the AI present there were

instances of prolonged contacts with CTP (> 100 ns) while the L2 backbone configuration was ; 12

while the L2 backbone was . Figure D.3-E shows some representative time-plots comparing H-bonding

to CTP and backbone configuration. Even when the AI was present and L2 was in the  configuration,

K122 could H-bond with CTP.

These findings suggest that the conformational switching of the L2 backbone between  and 

structure does not dictate strict silencing and activating positions for K122; the presence of CTP in the

active site can over-ride the influence of the AI on the L2 backbone configuration. In other words, the

mechanism whereby the AI helix silences catalysis is not primarily by freezing the L2 loop at K122 into a

conformation that prevents it from engaging substrate. An alternative hypothesis, that remains untested, is

that this backbone flexibility simply facilitates K122 sequential access to different partners during a

catalytic cycle, including phosphocholine, which was not present in any of the simulations. The 

configuration may be required for K122 to extend all the way to the phosphocholine phosphate, which is

deep within the active site.

415
Figure D.3: Correlations between K122 backbone dihedrals and H-bonding partners.

A–D) The data from 5 NOE and 5 PR-restrained simulations for each condition described in the matrix at

the top of this figure was mined for an α or βconfiguration at the K122 backbone and corresponding H-

bonding partner for K122. Alpha was defined as Ψ angle < 60o, and β was defined as Ψ angle > 60o. An

H-bond to CTP, F124, or F293 was defined by a distance < 4 Å to any oxygen. A) Frequency of α versus

β configurations for K122. B-D) Frequency of K122 H-bonds to CTP, F124, or F293 while sampling

either the α or β configuration. The data for the NOE set and PR set were averaged to generate the error

bars. E) Time correlations between H-bonds and backbone dihedrals. Upper panels: The Ψ angle for

K122 in the presence of both the AI and CTP are plotted versus simulation time. Representative results

from four separate chains are shown. Lower panels: Corresponding distance between K122 ε-amino and

any CTP phosphate oxygen. The red dashed lines indicate H-bonding distance (2.5 -3.5 Å).

416
Figure D.4: Stability of 4-helix bundle during 1000 ns simulation time.

Snapshot of one replicated simulation at 40 ns (left) and 1000 ns (middle), where both AI helices remain

part of the 4-helix bundle for the duration. Right image is a snapshot of the endpoint of a separate

simulation where the N-terminal portion of AI-2 (yellow) unravels, yet the αE helices remain associated.

The hydrophobic cluster that stabilizes packing of the αE helices at the cross-over is highlighted in ball

and stick (I206, I209, V210). The side chains of the aromatic/hydrophobic residues involved in AI – L2

interactions are highlighted in stick representation (F124, F289, M292, F293). Color coding of chains is

as in previous images.

417
Figure D.5: Alpha-E hinge dynamics enables contacts with the active site.

A) R223 in helix αEC contacts CTP. R223 of chain A αE contacts the -phosphate of CTP in the active

site of chain B, without eliminating the K122 -  phosphate contact. The aromatic cluster is captured in

the opposite chain, composed of Y213 and Y216 in the αE and F124 in L2. B) RMSF of the αE hinge.

The residues of the hinge are 211-215, inclusive. Data are means ± Ave Dev of 5 NOE simulations for

each condition (10 chains). P values for the effect of the AI are indicated with ** for P < 0.005, and # for

P ≤ 0.001.

418
Figure D.6: Alpha-E configuration 3.

The αEC helices have dissociated and one is in close contact with L2, the other is not. With few

exceptions this is usually not long-lived and transitions to configuration 2, 4 or 5 within 20 ns.

419
Figure D.7: Lipid vesicles reduce inter-chain cross-linking between αEc.

A separate reaction of CCT312 (A217C) with BMOE was imaged by immunoblot using an antibody

against the catalytic domain, and the signal was quantified using Image Quant 5.2. The data are means 

range of 2 determinations.

420
Figure D.8: CCT(A217C) binding to lipid vesicles via domain M is not impaired by oxidative

disulfide formation.

Oxidized CCT-312(A217C) or its Cys-free parent was incubated with (■) or without (◊) 10 mM DTT for

10 min at 37 oC prior to addition of the indicated concentration to 50 µM sonicated lipid vesicles

containing egg PC/egg PG/Dansyl-PE (47.5/50/2.5). The increase in fluorescence of Dansyl-PE, due to

FRET from Trp-278 in domain M, was measured with a Varian Eclipse spectrofluorimeter. As a negative

control, CCT-236 with no M domain and a single Trp (W151) in the catalytic domain was tested in the

presence of 10 mM DTT (○, right panel). The excitation wavelength was 280 nm and the emission was

scanned between 294 and 550 nm. FRET denotes the fluorescence increase at 520 nm, calculated as the

fractional increase relative to the Dansyl-PE-containing vesicles in the absence of CCT, where

fluorescence of vesicles alone = 0. Data are means  range of 2 determinations; lines drawn to guide the

eye.

421
Figure D.9: PR vs NOE restraints.

The heavy backbone atoms in the αEC of both monomers (residues 216 to 223 inclusive, colored blue and

red) were treated either with PR or NOE restraints. PR restraints pin these atoms in space (with respect to

the coordinate system origin), which maintains the helicity of the segments and fixes the inter-helical

distances for the duration of the simulation. The rest of the enzyme can fluctuate freely. The NOE

restraints can be imagined as a group of virtual bonds between the heavy backbone atoms in each αE C

segment that maintain the helicity of each independently from the other αEC segment. Consequently, the

two helices can drift apart.

422
Figure D.10: Detailed description of CCT mutant constructs.

The length of the CCT mutant constructs and the locations of the His-tag are displayed. The green box

(residues 40-223) indicates the region corresponding to the resolved segment in the X-ray structure

(PDB:4MVC). The positions of the mutated residues are indicated by red bars. For the single tryptophan

and single cysteine mutants the sites of the native Trps and Cys that were mutated to Phe or Ser are also

indicated. The deleted NLS sequence (residues 12-16) served to prevent vesicle aggregation.

423
 Appendix E: Copyright Permissions

Permission from the Journal of Biological Chemistry:

As the first author of this manuscript, no permission was required for reusing it in this thesis.

424
Permission from the journal of Biochimica et Biophysica Acta (BBA) - Biomembranes:

Permission for Figure 1.4-right:

Kulkarni, J. A., Darjuan, M. M., Mercer, J. E., Chen, S., van der Meel, R., Thewalt, J. L., Tam Y. Y. C. &

Cullis, P. R. (2018). On the Formation and Morphology of Lipid Nanoparticles Containing Ionizable

Cationic Lipids and siRNA. ACS nano, 12(5), 4787-4795.

425
426
Permission for Figure 1.4-left:

Tam, Y. Y. C., Chen, S., & Cullis, P. R. (2013). Advances in lipid nanoparticles for siRNA

delivery. Pharmaceutics, 5.3, 498-507.

Copyright permissions are owned by the authors and this figure was reproduced (adapted) with

permission from Dr. Yuen Yi C. Tam, Dr. Sam Chen, and Dr. Pieter R. Cullis.

427
Permission from co-authors/collaborators to include our study as the Chapter 3 of this thesis:

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Permission from co-authors/collaborators to include our study as the Chapter 4 of this thesis:

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Permission from co-authors/collaborators to include our study as the Chapter 5 of this thesis:

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Permission from co-authors/collaborators to include our study as the Chapter 6 of this thesis:

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