ISTANBUL TECHNICAL UNIVERSITY  GRADUATE SCHOOL OF SCIENCE

ENGINEERING AND TECHNOLOGY

EVALUATION OF CATIONIC POLYMER VECTORS FOR NON-VIRAL

GENE DELIVERY

M.Sc. THESIS

Hatice İmran GÜNGÖRDÜ

Department of Chemistry

Chemistry Programme

Anabilim Dalı : Herhangi Mühendislik, Bilim

Programı : Herhangi Program

JUNE 2012
ISTANBUL TECHNICAL UNIVERSITY  GRADUATE SCHOOL OF SCIENCE
ENGINEERING AND TECHNOLOGY

EVALUATION OF CATIONIC POLYMER VECTORS FOR NON-VIRAL

GENE DELIVERY

M.Sc. THESIS

Hatice İmran GÜNGÖRDÜ

(509101061)

Department of Chemistry

Chemistry Programme

Thesis Advisor: Prof. Dr. Gülaçtı TOPÇU

Anabilim Dalı : Herhangi Mühendislik, Bilim
Programı : Herhangi Program

JUNE 2012
İSTANBUL TEKNİK ÜNİVERSİTESİ  FEN BİLİMLERİ ENSTİTÜSÜ

KATYONİK POLİMERLERİN GEN TAŞIYICI SİSTEMLERİ OLARAK

DEĞERLENDİRİLMESİ

YÜKSEK LİSANS TEZİ

Hatice İmran GÜNGÖRDÜ

(509101061)

Kimya Anabilim Dalı

Kimya Programı

Tez Danışmanı: Prof. Dr. Gülaçtı TOPÇU

Anabilim Dalı : Herhangi Mühendislik, Bilim
Programı : Herhangi Program

HAZİRAN 2012
Hatice İmran GÜNGÖRDÜ, a M.Sc. student of ITU Graduate School of Science
Engineering and Technology student ID 509101061, successfully defended the
thesis entitled “EVALUATION OF CATIONIC POLYMER VECTORS FOR
NON-VIRAL GENE DELIVERY”, which she prepared after fulfilling the
requirements specified in the associated legislations, before the jury whose signatures
are below.

Thesis Advisor : Prof. Dr. Gülaçtı TOPÇU

İstanbul Technical University

Jury Members : Prof. Dr. Bahire Filiz ŞENKAL

İstanbul Technical University

Prof. Dr. Jülide AKBUGA

Marmara University

Date of Submission : 04 May 2012

Date of Defense : 08 June 2012

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 Dedicated to my beloved family and
everyone in pursuit of his curiosity,

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viii
FOREWORD

First of all, I am greatly indebted to my thesis advisor in Istanbul Technical

University, Prof. Dr. Gülaçtı Topçu who has inspired me with her hard-working and
enthusiastic personality as a scientist. She has always supported me about my area of
interest and rendered the possibility for an international research experience for me.

I feel honored to express my gratitude to my thesis advisor in Ghent University, Prof.

Dr. Kevin Braeckmans as well as his colleagues Prof. Dr. Stefaan De Smedt and
Prof. Dr. Jo Demeester for giving me the opportunity to pursue my thesis study as a
member of their interdisciplinary and extraordinary group.

My very sincere thanks to my supervisor Thomas Martens for his guidance,

encouragements and teaching me all the skills that I needed to work on this project
independently. As a chemist, gene therapy was a brand new research area for me.
During my thesis studies, he has shared all his knowledge and experience with me
and expedited my adaptation to this research.

Despite my short stay in Ghent, I had the chance to collaborate with two different
groups. I would like to mention my thanks to Prof. Dr. Johan Engbersen and Dr.
Marc Ankone from University of Twente and Prof. Dr. Peter Dubruel and Dr.
Kehinde Adesanya from Ghent University for their kind collaborations and helps for
polymer synthesis.

I am also thankful to everyone in “Laboratory of Biochemistry and Physical

Pharmacy”, especially Dr. Joanna Rejman, Dr. Katrien Remaut and Dr. Bart Lucas,
for their warmest welcome and sharing their experience whenever I needed.

I cannot forget to thank the dear people, both my lab-mates and home-mates, that
beautiful city of Ghent brought to me during those unforgettable days of my Erasmus
experience. I would also like to say my warmest thanks to my lab-mates and
colleagues in İTÜ, Dr. Fatemeh Bahadori, Demet, Burcu, Tuba, Seda, Anıl, Tayyibe,
and Mervenur for their moral support during my studies.

Finally, I owe the greatest gratitude to my beloved family who stands by me

unconditionally with full support and advice all the time.

June 2012 Hatice İmran GÜNGÖRDÜ

(Chemist)

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TABLE OF CONTENTS

Page

FOREWORD ............................................................................................................. ix
TABLE OF CONTENTS .......................................................................................... xi
ABBREVIATIONS .................................................................................................. xv
LIST OF TABLES ................................................................................................. xvii
LIST OF FIGURES ................................................................................................ xix
SUMMARY ........................................................................................................... xxiii
ÖZET....................................................................................................................... xxv
1. INTRODUCTION .................................................................................................. 1
1.1 Purpose of The Thesis ........................................................................................ 1
1.2 Outline of The Thesis ......................................................................................... 2
2. BASICS OF CELL BIOLOGY ............................................................................. 3
2.1 The Cell .............................................................................................................. 3
2.2 Cell (Plasma) Membrane .................................................................................... 3
2.3 Cellular Entry of Extracellular Material ............................................................. 5
2.3.1 Endocytosis and endosomes........................................................................ 5
2.3.2 Lysosome .................................................................................................... 7
2.4 Intracellular Environment .................................................................................. 8
2.4.1 Cytoplasm and cytosol ................................................................................ 8
2.4.2 Cytoskeleton................................................................................................ 8
2.5 Cell Nucleus ....................................................................................................... 9
2.5.1 Function ...................................................................................................... 9
2.5.2 Nuclear envelope and pores ........................................................................ 9
2.6 Gene Expression ............................................................................................... 10
2.6.1 Transcription ............................................................................................. 10
2.6.2 Translation ................................................................................................ 11
3. GENE THERAPY ................................................................................................ 13
3.1 A Brief History of Gene Therapy ..................................................................... 13
3.2 Barriers for Gene Delivery ............................................................................... 15
3.2.1 Stability in the extracellular environment ................................................. 15
3.2.2 Cellular attachment and adhesion ............................................................. 16
3.2.3 Cellular uptake .......................................................................................... 17
3.2.4 Endosomal escape ..................................................................................... 18
3.2.5 Cellular translocation to the nucleus ......................................................... 19
3.2.6 Nuclear import ......................................................................................... 20
3.3 Methods for Gene Delivery .............................................................................. 21
3.3.1 Physical methods....................................................................................... 21
3.3.1.1 Gene gun ............................................................................................ 21
3.3.1.2 Electroporation ................................................................................... 21
3.3.1.3 Gene transfer by ultrasound radiation .............................................. 22
3.3.2 Viral vectors .............................................................................................. 23

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 3.3.2.1 Adeno-associated virus ...................................................................... 23
3.3.3 Non-viral vectors ....................................................................................... 23
3.3.3.1 Lipid-based gene delivery systems .................................................... 24
3.3.3.2 Polymer-based gene delivery systems................................................ 25
4. MATERIALS AND METHODS......................................................................... 29
4.1 Polymer Synthesis ............................................................................................ 29
4.1.1 Synthesis of pDMAEMA by radicalic polymerization ............................. 29
4.1.2 Synthesis of SS-PAAs by Michael-type polyaddition .............................. 34
4.2 Cell Culture ...................................................................................................... 37
4.2.1 Materials .................................................................................................... 37
4.2.2 Methods used in cell culture...................................................................... 37
4.3 Nucleic acids used in this study........................................................................ 38
4.3.1 pGL4.13 plasmid ....................................................................................... 38
4.3.2 gWiz™-GFP plasmid ................................................................................ 39
4.3.3 YOYO®-1 iodide labelled pGL4.13 plasmid ............................................ 40
4.4 Preparation and Physicochemical Characterization of Polyplexes .................. 40
4.4.1 Dynamic light scattering ........................................................................... 40
4.4.1.1 Theory and working principles .......................................................... 40
4.4.1.2 Particle size and surface charge measurements.................................. 43
4.4.2 Preparation of polyplexes .......................................................................... 43
4.4.2.1 pDMAEMA polyplexes ..................................................................... 44
4.4.2.2 SS-PAA polyplexes ............................................................................ 46
4.4.3 Agarose gel electrophoresis ...................................................................... 47
4.4.3.1 Principles ............................................................................................ 47
4.4.3.2 Experimental part ............................................................................... 48
4.5 In vitro Biological Evaluation of Polyplexes ................................................... 49
4.5.1 Flow cytometry ......................................................................................... 49
4.5.1.1 Principles ............................................................................................ 49
4.5.2 Uptake and transfection efficiency studies................................................ 50
4.5.3 Cell viability .............................................................................................. 51
4.5.3.1 MTT assay .......................................................................................... 51
4.5.3.2 Experimental ...................................................................................... 52
5. RESULTS AND DISCUSSION........................................................................... 53
5.1 Polymer Synthesis ............................................................................................ 53
5.1.1 Characterization of polymers .................................................................... 53
5.1.1.1 pDMAEMA ........................................................................................ 53
5.1.1.2 SS-PAAs............................................................................................. 53
5.2 pDMAEMA polyplexes.................................................................................... 58
5.2.1 Physicochemical characterization ............................................................. 58
5.2.1.1 Particle size and surface charge measurements.................................. 58
5.2.1.2 pDNA complexation efficiency ......................................................... 59
5.2.2 In vitro biological evaluation .................................................................... 61
5.2.2.1 Uptake efficiency ............................................................................... 61
5.2.2.2 Transfection efficiency....................................................................... 62
5.2.2.3 Cell viability ....................................................................................... 62
5.2.3 Discussion ................................................................................................. 63
5.3 SS-PAA Polyplexes .......................................................................................... 66
5.3.1 p(CBA-ABOL) polyplexes ....................................................................... 67
5.3.1.1 Physicochemical characterization ...................................................... 67
5.3.1.2 In vitro biological evaluation ............................................................ 70

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 5.3.1.3 Discussion .......................................................................................... 72
5.3.2 p(CBA-ABOL/DMEDA/PEG) polyplexes ............................................... 74
5.3.2.1 Physicochemical characterization ...................................................... 75
5.3.2.2 In vitro biological evaluation ............................................................ 76
5.3.2.3 Discussion .......................................................................................... 79
5.3.3 HA-coated p(CBA-ABOL) polyplexes ..................................................... 80
5.3.3.1 Physicochemical characterization ...................................................... 80
5.3.3.2 In vitro biological evaluation ............................................................ 83
5.3.3.3 Discussion .......................................................................................... 86
5.3.4 p(CBA-HIS) polyplexes ............................................................................ 87
5.3.4.1 Physicochemical characterization ...................................................... 87
5.3.4.2 In vitro biological evaluation ............................................................ 90
5.3.4.3 Discussion .......................................................................................... 93
6. CONCLUSION..................................................................................................... 95
REFERENCES ......................................................................................................... 97
APPENDICES ........................................................................................................ 109
APPENDIX A. ..................................................................................................... 111
CURRICULUM VITAE ........................................................................................ 113

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ABBREVIATIONS

ABOL : 4-aminobutan-1-ol
AIDS : Acquired immunodeficiency syndrome
App : Appendix
APS : Ammonium persulfate
CBA : N,N′-cystaminebisacrylamide
CF : Cystic Fibrosis
DLS : Dynamic Light Scattering
DMAEMA : Dimethylaminoethyl methacrylate
DMEDA : N, N’-dimethyl ethyl-1,2-diamine
DMEM : Dulbecco's modified Eagle's medium
DNA : Deoxyribonucleic acid
DTT : Dithiothreitol
ECM : Extracellular matrix
FC : Flow Cytometry
GAG : Glycosaminoglycan
GFP : Green Fluorescent Protein
GPC : Gel Permeation Chromatography
HA : Hyaluronic acid (Hyaluronan)
HEPES : 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIS : Histamine
HS : Heparan Sulphate
HSPG : Heparan sulphate proteoglycan
IF : Integrated filament
kDa : Kilodalton
LF : Lipofectamine™ 2000 Reagent
MF : Microfilament
mRNA : Messenger Ribonucleic Acid
MT : Microtubule
MTT : 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Mw : Molecular weight
MWCO : Molecular weight cut-off
NLS : Nuclear localization signal
NMR : Nuclear Magnetic Resonance
NPC : Nuclear pore complex
OTC : Ornithine transcarbamylase
P/S : Penicillin-Streptomycin
PAA : Poly(amidoamine)
PAMAM : Poly(amidoamine)
PBS : Phosphate buffer saline
pDI : Polydispersity index
pDNA : Plasmid DNA
PEG : Poly(ethylene glycol)

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PEI : Poly(ethylene imine)
PG : Proteoglycan
pHPMA : Poly(hydroxypropyl methacrylate)
PLL : Poly(L-lysine)
Pplx : Polyplex
PS : Polstyrene
RGD : Arginine-glycine-aspartate
RNA : Ribonucleic acid
RPE : Retinal Pigment Epithelium
RT : Room Temperature
SCID : Severe combined immunodeficiency
SS-PAA : Linear, disulfide-containing poly(amidoamine)
TBE : Tris-base boric acid EDTA
TMS : Tetramethylsilane
US : Ultrasound

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LIST OF TABLES

Page

Table 5.1 : The amounts of pDMAEMA polymer and pDNA which was used to
obtained desired polymer/pDNA (w/w) ratios ........................................ 59
Table 5.2 : Illustration of average size, pDI and charge values for pDMAEMA
polyplexes................................................................................................ 59
Table 5.3 : The amounts of p(CBA-ABOL) polymer and pDNA which was used to
obtain desired polymer/pDNA (w/w) ratios. The polyplexes were
prepared with increasing amount of polymer while the amount of plasmid
was the same for each.............................................................................. 67
Table 5.4 : Illustration of average size, pDI and charge values for p(CBA-ABOL)
pplxes with different polymer/pDNA mass ratios .................................. 68
Table 5.5 : Illustration of average size, pDI and charge values for PEGylated
p(CBA-ABOL) polyplexes with different polymer/pDNA mass ratios . 75
Table 5.6 : Illustration of the amount of HA and pDNA used to apply the coating in
appropriate HA/pDNA molar ratio ......................................................... 81
Table 5.7 : The amounts of p(CBA-HIS) polymer and pDNA which was used to
obtain desired polymer/pDNA (w/w) ratios. The polyplexes were
prepared with increasing amount of polymer while the amount of plasmid
was constant. ........................................................................................... 88
Table 5.8 : Illustration of average size, pDI and charge values for p(CBA-HIS)
polyplexes with different polymer/pDNA mass ratios ............................ 88

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LIST OF FIGURES

Page

Figure 2.1 : Comparison of sizes of precellular organisms with an average cell in

human body. ............................................................................................. 3
Figure 2.2 : A cross section from cell membrane (Bolsover, 2004) .......................... 4
Figure 2.3 : A general molecular formula of HSPGs where R= SO3- (Gallagher et al,
1986).......................................................................................................... 5
Figure 2.4 : a. Categorisation of different endocytotic pathways, adapted from
(Mayor and Pagano, 2007). b. Multiple routes of endocytosis into
mamallian cells (Conner and Schmid, 2003). ........................................... 6
Figure 2.5 : The mechanism of endocytosis. ............................................................. 7
Figure 2.6 : Cytosolic transport and nuclear import of the cargo, adapted from
(Wong et al, 2007). .................................................................................... 8
Figure 2.7 : The structure of cell nucleus [Url-1]. ...................................................... 9
Figure 2.8 : Illustration of gene expression process [Url-2] ..................................... 10
Figure 3.1 : a. Number of approved gene therapy clinical trials worldwide 1989-
2011, b. Clinical phases of approved gene therapy trials, adapted from
[Url-3]...................................................................................................... 14
Figure 3.2 : Intracellular barriers for gene delivery, adapted from (De Smedt et al,
2005)........................................................................................................ 15
Figure 3.3 : Schematic illustration of proton sponge hypothesis .............................. 19
Figure 3.4 : Chemical structure of cationic lipid (A) DOTMA, (B) DOTAP and (C)
an illustration of liposome, adapted from (Remaut et al, 2007). ............. 24
Figure 3.5 : Chemical structures of selected polymers , adapted from (Wong et al,
2007)........................................................................................................ 26
Figure 4.1 : Chemical structures of a. DMAEMA monomer, b. pDMAEMA
polymer.................................................................................................... 30
Figure 4.2 : Illustration of radicalic polymerization reaction mechanism ................ 31
Figure 4.3 : Illustration of Michael type polyaddition of a primary amine to bisacryl
amide monomer, adapted from (Lin et al, 2007a) ................................... 35
Figure 4.4 : Illustration of Michael type addition polymerization of a. p(CBA-
ABOL), b. p(CBA-HIS), adapted from(Coue and Engbersen, 2011) ..... 35
Figure 4.5 : Illustration of Michael type addition polymerization of a p(CBA-
ABOL/DMEDA/PEG) copolymer, adapted from (Vader et al, 2012) .... 36
Figure 4.6 : Schematic representation of pGL4.13 plasmid. The plasmid encodes for
luc2 gene under the control of SV40 promoter. Ampr: ampicilline
resistance marker [Url-4] ........................................................................ 39
Figure 4.7 : Schematic representation of gWiz™-GFP plasmid. kanr: kanamycin
resistance [Url-5] ..................................................................................... 40
Figure 4.8 : Schematic illustration of zeta potential [Url-6] ..................................... 42
Figure 4.9 : Main components and optical configurations of a Nano Ζeta-sizer
instrument for DLS measurements [Url-7] ............................................. 44

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Figure 4.10 : Formation of polymer/DNA nanocomplex (polyplex) by self-assembly,
adapted from (Coue and Engbersen, 2011) ............................................. 45
Figure 4.11 : The scattering of laser in flow cytometer [Url-8] ................................ 50
Figure 4.12 : Reduction of MTT reagent to formazan .............................................. 52
Figure 5.1 : Illustration of 1H-NMR spectrum of DMAEMA monomer .................. 54
Figure 5.2 : The 1H-NMR spectrum relative to TMS (CDCl3, 300 MHz and
expressed in ppm) of pDMAEMA .......................................................... 55
Figure 5.3 : 1H-NMR spectrum relative to D2O (D2O, 300 MHz and expressed in
ppm) of p(CBA-ABOL) .......................................................................... 56
Figure 5.4 : The 1H-NMR spectrum relative to D2O (D2O, 300 MHz and expressed
in ppm) of p(CBA-HIS) .......................................................................... 56
Figure 5.5 : Illustration of the Mw distribution of p(CBA-ABOL) obtained by gel
permeation chromatography .................................................................... 57
Figure 5.6 : Illustration of the Mw distribution of p(CBA-HIS) obtained by gel
permeation chromatography .................................................................. 57
Figure 5.7 : Illustration of a. particle size and polydispersity, b. Ζ-potential of
polyplexes formed by pDMAEMA with different Mw and mass ratios . 60
Figure 5.8 : Agarose gel electrophoresis of pDMAEMA polyplexes Mw 50 kDa and
600 kDa with different mass ratios. The lanes with the polyplexes contain
20 µL of sample (corresponding to 200 ng of pDNA per well). As a
negative control, 20 µL of heparin (7 μg/μL) was added to last 4 samples
to simulate pDNA displacement.............................................................. 61
Figure 5.9 : Comparison of cellular internalization of p(DMAEMA) polyplexes with
different Mw and mass ratios. ................................................................. 62
Figure 5.10 : Comparison of transfection efficiency of pDMAEMA polyplexes with
different Mw and mass ratios and LF as a positive control ..................... 63
Figure 5.11 : Cell viability results of pDMAEMA polyplexes with different Mw and
polymer ratios .......................................................................................... 64
Figure 5.12 : Illustration of a. particle size and polydispersity, b. ζ-potential of
p(CBA-ABOL) polyplexes with different mass ratios. ........................... 68
Figure 5.13 : Agarose gel electrophoresis of p(CBA-ABOL) pplxes with different
mass ratios. Each sample was colored with 5 µL loading buffer. The
polyplex samples contain 0.2 µg of pDNA. As a negative control, 10 µL
of Heparin (7 μg/μL) was added to last sample ...................................... 69
Figure 5.14 : Comparison of cellular internalization of p(CBA-ABOL) polyplexes
with different mass ratios ........................................................................ 70
Figure 5.15 : Comparison of transfection efficiencies of p(CBA-ABOL) polyplexes
with various mass ratios compared to LF. ............................................... 71
Figure 5.16 : Cell viability results of p(CBA-ABOL)/pDNA polyplexes with
different mass ratios ................................................................................ 72
Figure 5.17 : Illustration of the effect of PEGylation on a. particle size and
polydispersity, b. ζ-potential ................................................................... 75
Figure 5.18 : Agarose gel electrophoresis for p(CBA-ABOL/DMEDA/PEG)
complexes. The lanes with the polyplexes contain 20 µL of sample
(corresponding to 200 ng of pDNA per well). As a negative control, 5 µL
of Heparin (7 μg/μL) was added to last 3 samples to simulate pDNA
displacement. ........................................................................................... 77
Figure 5.19 : The effect of PEGylation on cellular internalization of p(CBA-ABOL)
polyplexes with different mass ratios in ARPE-19 cells. ........................ 78

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Figure 5.20 : Comparison of the effect of PEGylation on transfection efficiency of
p(CBA-ABOL) polyplexes with various mass ratios in ARPE-19 cells.78
Figure 5.21 : Illustration of the effect of HA-coating on a. particle size and
polydispersity, b. ζ-potential of p(CBA-ABOL) polyplexes with different
HA/pDNA molar ratios ........................................................................... 81
Figure 5.22 : Agarose gel electrophoresis of HA-coated p(CBA-ABOL) polyplexes
with different HA/pDNA molar ratios. The lanes with the polyplexes
contain 20 µL of sample (corresponding to 200 ng of pDNA per well).
As a negative control, 5 µL of Heparin (7 μg/μL) was added to last 4
samples to simulate pDNA displacement................................................ 83
Figure 5.23 : The effect of HA-coating on cellular internalization of p(CBA-ABOL)
polyplexes with different HA/pDNA molar ratios in ARPE-19 cells ..... 84
Figure 5.24 : Comparison of transfection efficiencies of different gene carriers HA-
coated p(CBA-ABOL) with different HA/pDNA molar ratios, non-
coated p(CBA-ABOL) and LF complexes. ............................................. 84
Figure 5.25 : Effect of HA-coating on the viability of ARPE-19 cells ..................... 85
Figure 5.26 : Illustration of a. particle size and polydispersity, b. ζ-potential of
pDMAEMA polyplexes with different Mw and mass ratios .................. 89
Figure 5.27 : Agarose gel electrophoresis of p(CBA-HIS) polyplexes with different
mass ratios. The lanes with polyplexes contain 20 µL of sample
(corresponding to 200 ng of pDNA per well). As a negative control, 3 µL
of Heparin (7 μg/μL) was added to last 5 samples to simulate pDNA
displacement. 48/1 p(CBA-ABOL) polyplexes were applied as a
comparative reference. ............................................................................ 90
Figure 5.28 : Comparison of internalization of different gene carriers p(CBA-HIS)
with different mass ratios, p(CBA-ABOL) and LF in ARPE-19 cells.... 91
Figure 5.29 : Comparison of transfection efficiencies of different gene carriers
p(CBA-HIS) with different mass ratios, p(CBA-ABOL) and LF in
ARPE-19 cells........................................................................................ 91
Figure 5.30 : Cell viability results of p(CBA-HIS)/pDNA pplexes with different
mass ratios ............................................................................................... 92
Figure A.1 : Preliminary study for HA-coating ..................................................... 111

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 EVALUATION OF CATIONIC POLYMER VECTORS FOR NON-VIRAL
GENE DELIVERY

SUMMARY

Gene therapy has been a promising way to treat human genetic disorders. The major
goal of gene therapy is to repair the genetic cause underlying the disorder by
transferring the therapeutic nucleic acid with a safe and efficient carrier. For this
purpose, two main classes of vectors are explained: viral and non-viral vectors. Even
though, viral vectors are the most efficient carriers known, they have restrictions
about their safety and production process. Cationic polymers have been the mostly
investigated non-viral vector systems as a safe and easy-to-produce alternative to
viral carriers. Moreover, numerous research have been done to increase their
transfection efficiency.
In this thesis study, two different types of cationic polymers were synthesized and
investigated for their non-viral gene carrier potentials. One of them is a cationic, non-
biodegradable poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA). The
second type of carriers is cationic and biodegradable linear, disulfide linkage
containing poly(amidoamine)s (SS-PAAs) that include four different polymers with
different side groups and coating strategies. The SS-PAA family consists of p(CBA-
ABOL), p(CBA-ABOL/DMEDA/PEG) with PEGylation strategy, p(CBA-ABOL)
with HA-coating and lastly, p(CBA-HIS).
The main objectives of this study were first of all, physicochemical characterization
of the polymer/pDNA complexes with different polymer Mw and polymer/pDNA
ratio in terms of size, polydispersity and surface charge which were measured by
DLS. Secondly, the pDNA complexation ability of polyplexes was investigated by
performing agarose gel electrophoresis. Thirdly, in vitro biological evaluation of
polymers on ARPE-19 cells were performed. For this purpose, uptake and
transfection efficiencies of the polymers and the effect of different polymer Mw’s
and different polymer/pDNA ratios on uptake and transfection efficiency of
polyplexes were evaluated by Flow Cytometry. Finally, the effect of polyplexes on
the viability of ARPE-19 cells was analyzed with MTT Assay.
While evaluating the pDMAEMA complexes, they showed the ability to form stable
complexes in HEPES buffer and were able to efficiently complex pDNA.
Nevertheless, the cationic nanoparticles formed with these polymers only exhibited
signs of transfection in concentrations already toxic for the cells. This is why a new
family of biodegradable polymers- the disulfide-containing PAAs, were investigated.
p(CBA-ABOL) particles were also able to form stable polyplexes with efficiently
complexed pDNA and also showed very high transfection efficiency, without
significant effect on cell viability. Unfortunately, these nanoparticles were not stable
in mimicked physiological conditions, which is why the same polymer was
investigated with a PEG-chain as a universal coating strategy. Even though these
particles were stable under physiological conditions, their uptake was drastically
xxiii
hindered. A different coating strategy with hyaluronic acid showed promising
preliminary results, but the same results could not be observed with the research
grade purity low Mw HA. Subsequently, another member of SS-PAA family with a
different side chain and buffering capacity was evaluated. p(CBA-HIS) polymer was
able to condense the pDNA in HEPES buffer and transfected the cells with almost no
cytotoxic effect. However, the colloidal stability of p(CBA-HIS) polyplexes in
physiological conditions should be further increased.

xxiv
 KATYONİK POLİMERLERİN GEN TAŞIYICI SİSTEMLERİ OLARAK
DEĞERLENDİRİLMESİ

ÖZET

Gen tedavisi, genetik kaynaklı birçok hastalığa çözüm sunma potansiyeli açısından
son yıllarda oldukça rağbet gören bir çalışma alanı olmuştur. Gen tedavisinin ana
amacı hastalıkların ortaya çıkardığı semptomları iyileştirmeye çalışmak yerine,
hastalığa sebep olan genetik bozuklukları ortadan kaldırmaktır. Bu tedavide, tedavi
edici özelliği olan genler, en güvenli ve verimli bir taşıyıcı ile hedef dokuya ya da
hücreye transfer edilmektedir. Bu amaçla kullanılan taşıyıcılar viral ve viral olmayan
taşıyıcılar olmak üzere ikiye ayrılır. Viral taşıyıcılar, hedefe ulaşma verimliliği
açısından bilinen en verimli taşıyıcılar olmakla birlikte güvenilirlik ve üretim
aşamaları açısından uygulamaları çok kısıtlıdır. Bu sebeple, güvenli ve üretimi kolay
alternatif bir metod olarak katyonik polimerler araştırılmaktadır. Literatürde,
katyonik polimerlerin transfeksiyon verimliliklerini artırmak için yapılmış pek çok
çalışmaya ulaşılabilir.
Bu tez çalışmasında, gen tedavisi potensiyelleri açısından iki farklı polimer türü
sentezlendi ve çeşitli özellikleri bakımından incelendi. Bu polimerlerin ilki, katyonik
fakat biyobozunur olmayan poli(2-(dimetilamino)etil metakrilat) (pDMAEMA)
polimeridir. İkinci tür taşıyıcı sistem ise katyonik ve biyobozunur disülfür bağı
içeren, lineer poli(amidoamin) (SS-PAA) polimer türevleridir. SS-PAA ailesine ait
farklı yan grup ve kaplama stratejileri ile sentezlenen 4 farklı polimer bu çalışmada
incelendi. Bunlar poli-(N,N’-sistamin bisakrilamit-4-amino bütanol) p(CBA-ABOL),
PEG’leme stratejisi ile geliştirilen p(CBA-ABOL/DMEDA/PEG), hiyaluronik asit
(HA) kaplanmış p(CBA-ABOL) ve son olarak da poli-(N,N’-sistamin bisakrilamit-
histamin) (p(CBA-HIS))’dir. Bu çalışmanın ilk aşaması olarak farklı molekül
ağırlıklı ve farklı polimer oranlarına sahip polimer/pDNA (plazmit DNA)
kompleksleri (polipleks) fizikokimyasal özellikleri açısından incelendi.
Bu amaçla boyut, polidispersite ve yüzey yükü ölçümleri DLS cihazı ile yapıldı.
İkinci olarak poliplekslerin pDNA’yı kaplayarak kararlı kompleksler oluşturma
etkinlikleri agaroz jel elektroforez ile belirlendi. Bu karakterizasyonları takiben,
polimer/pDNA komplekslerinin ARPE-19 hücreleri ile in vitro biyolojik
değerlendirmeleri amaçlandı. Farklı molekül ağırlığına (MA) ve farklı polimer
oranlarına sahip polipleksler hazırlanarak hücrelere transfer edildi ve hücre içine
alınma ve genetik materyali hedefe ulaştırma verimlilikleri Hücre Sitometresi (Flow
Cytometry) cihazı ile ölçüldü. Son olarak, poliplekslerin hücreler üzerindeki
sitotoksik etkisi MTT Testi ile analiz edildi.
pDMAEMA polimeri ile oluşturulan 2 farklı MA ve farklı polimer oranına sahip
poliplekslerin HEPES tamponu ortamında yapılan boyut ve yük tayini sonuçlarına
göre bu ortamda pDNA ile kararlı komplekler yapabildiği görüldü. Yapılan
ölçümlerin fizyolojik ortama uygunluğunun denenmesi için aynı seri ölçümler bir de
serum içermeyen transfeksiyon ortamı olan OptiMEM ortamında gerçekleştirildi.

xxv
OptiMEM ortamında kompleks kararlılığının azaldığı ve agregasyon oluşumu
gözlendi. Bu ölçümleri takiben pDMAEMA polimerinin pDNA’yı kondanse
edebilme özelliğinin analizi için pDMAEMA polipleksleri agaroz jel elektroforeze
uygulandı. Her iki MA ve polimer oranındaki kompleksler pDNA kaplamaya
muktedirdi. Polimer MA ve oranının polipleks kararlılığına bariz bir etkisinin
olmadığı gözlendi fakat yüksek polimer oranına sahip kompleksler daha küçük
parçacık boyutu gösterdiler. pDMAEMA poliplekslerinin in vitro biyolojik
değerlendirilmesi sonuçlarına göre pDMAEMA/pDNA komplekslerinin hücre içine
alınma verimlilikleri oldukça tatmin ediciydi. Fakat hücre içine alınan poliplekslerin
çekirdeğe ulaşma potansiyelleri oldukça düşük ve ancak hücreler için toksik olan
polimer miktarlarında ilgi çekici sonuçlar verdi.
SS-PAA polimerleri içerdikleri tekrarlanan sülfür köprüleri sayesinde biyobozunur
polimerler olduklarından gen salımı konusunda iyi bir potansiyele sahiptirler. Bu
ailenin ilk üyesi olarak p(CBA-ABOL) polimeri gen taşıyıcısı olarak değerlendirildi.
pDMAEMA polipleksleri için yapılan tüm işlemler p(CBA-ABOL)/pDNA
komplekleri için de denendi. Farklı polimer oranlarıyla oluşturulan p(CBA-ABOL)
komplekslerinin yüzey yükü ve boyut tayini HEPES ve OptiMEM ortamlarında
gerçekleştirildi. HEPES ortamında artan polimer miktarının daha kararlı kompleksler
oluşturmasına karşılık, aynı komplekslerde OptiMEM ortamında agregasyon
gözlendi. Agaroz jel elektroforez sonuçlarına göre yüksek polimer oranları 48/1 ve
96/1 ile oluşturulan kompleksler pDNA’yı kaplama açısından daha elverişli idiler.
p(CBA-ABOL) poliplekslerinin in vitro biyolojik değerlendirilmelerinin sonucunda
ise p(CBA-ABOL) poliplekslerinin genetik materyalin hücre içine alınması ve
ardından çekirdeğe ulaştırılması açısından pDMAEMA komplekslerine göre daha
verimli olduğu ayrıca ARPE-19 hücreleri için yalnızca ihmal edilebilir düzeyde bir
toksisiteye sebep olduğu görüldü.
p(CBA-ABOL) komplekslerinin fizyolojik ortamdaki agregasyonunu engellemek
amacıyla evrensel bir strateji olan PEG’leme stratejisi kullanıldı. PEG’lenen p(CBA-
ABOL) polimeri ile oluşturulan kompleksler de daha önceki ölçümlere tabi tutuldu.
PEG’leme sonucunda komplekslerin OptiMEM ortamındaki kolloidal kararlılığında
artış gözlenirken in vitro biyolojik değerlendirme açısından PEG’lenen p(CBA-
ABOL) polimeri ile yapılan ölçümler istenilen verimi göstermedi. Hücre içine alınma
verimliliği açısından PEG’lenmiş komplekslerin yok denecek kadar az bir
potansiyellerinin olduğu gözlendi. Kompleksler hücre içine alınmadığından
sitotoksisite analizine gerek duyulmadı.
p(CBA-ABOL) komplekslerinin hücre içine alınma kapasitelerinin artırılması için
kompleksler bir hücre zarı proteini olan HA ile elektrostatik olarak farklı HA/pDNA
oranlarında kaplanarak önceki polimerler ile aynı şekilde fizikokimyasal ve biyolojik
değerlendirmelere tabi tutuldu. Komplekslerin dış kabuğu anyonik HA ile
kaplandığından yüzey yükünün pozitiften negatife döndüğü gözlendi. Parçacık
boyutu ve kolloidal stabilite açısından ise kaplanmamış p(CBA-ABOL)
komplekslerine göre OptiMEM ortamında daha kararlı kompleksler elde edildi.
Biyolojik değerlendirme sonuçlarında, poliplekslerin hücre içine alımında HA
kaplama ile elde edilmek istenen artış gözlenmedi.
En son taşıyıcı sistem olarak SS-PAA polimerlerinin bir türevi olan p(CBA-HIS)
polimeri ile oluşturulan kompleksler gen taşıma kapasiteleri açısından
değerlendirildi.

xxvi
HIS yan grubu, ABOL yan grubuna göre daha asidik olduğundan komplekslerin
hücre içine endozomlar vasıtasıyla alınmasını takiben endozomlardan çıkışı
artıracağı ve buna bağlı olarak çekirdeğe ulaşan kompleks miktarının artacağı
amaçlandı. p(CBA-HIS) kompleksleri kolloidal karalılık ve pDNA kondenzasyonu
açısından p(CBA-ABOL) kompleksleri ile benzer özellikler gösterdiler. Aynı şekilde
in vitro ölçümler sonucuda p(CBA-HIS) komplekslerinin hücre içine alınma
kapasitelerinin daha yüksek olmasına rağmen genetik materyalin çekirdeğe ulaşma
verimliliği açısından amaçlanan artış gözlenmedi.
Sonuç olarak pDMAEMA polimer/pDNA komplekslerinin artan molekül ağırlıkları
ve polimer miktarları hücreler üzerinde toksik etki yaparken, SS-PAA polimerlerinin
transfeksiyon açısından iyi bir potansiyellerinin olduğu tespit edildi. Bu gruptan son
olarak incelenen p(CBA-HIS) poliplekslerinin HEPES tamponu ortamında pDNA’yı
kondanse etmesi ve transfekte ettiği hücreler üzerinde sitotoksik aktivite
göstermemesine rağmen agregasyon oluşumu tamamen engellenemedi. Bu nedenle
bu poliplekslerin fizyolojik şartlardaki davranışlarının daha ileri araştırmalarla
incelenmesi gerekmektedir.

xxvii
xxviii
1. INTRODUCTION

Gene therapy has been a promising way to treat human genetic disorders during the
last two decades. The major goal of gene therapy is to repair the genetic cause
underlying the disorder by transferring therapeutic nucleic acids with a safe and
efficient carrier to the target cells. For this purpose, two main classes of vectors are
explained: viral and non-viral vectors. Even though viral vectors are the most
efficient carriers known, they have restrictions about their safety and production
process and they are reported to have immune response after repeated
administrations (Thomas et al, 2003). Synthetic non-viral vectors such as cationic
polymers have been the mostly investigated gene delivery systems as a safer and
easy-to-produce alternative to viral carriers (De Smedt et al, 2000).

The most recently studied examples of cationic polymer vectors include

polyethylenimine (PEI), poly(L-lysine) (PLL), poly(amidoamine) (PAA) and poly(2-
(dimethylamino)ethyl methacrylate) (pDMAEMA). In this project pDMAEMA, and
a new class of PAAs with repetitive disulfide linkages in the polymer backbone (SS-
PAAs) were chosen to be synthesized and evaluated as potential gene delivery
systems.

pDMAEMA has attracted the attentions by being readily synthesized via radicalic
polymerization and having relatively high transfection efficiency (van de Wetering et
al, 1997). Moreover, PAAs are widely investigated since they are highly
biocompatible and biodegradable (Ferruti et al, 2002). SS-PAAs have been
previously described as a new bioreducible class of PAAs. They can be synthesized
by Michael-type addition polymerization of amine compounds to disulfide
containing bisacrylamides (Lin et al, 2007a).

1.1 Purpose of The Thesis

The main purpose of the study described in this thesis is first of all, synthesis of
cationic polymer vectors for gene delivery applications. Secondly, physicochemical
characterization of the polymer/pDNA complexes with different polymer Mw and

1
polymer/pDNA ratio was done in terms of size, polydispersity and surface charge.
Thirdly, the ability of the polymers to condense and retain the plasmid DNA in an
electrostatic complex was investigated. Subsequently, in vitro biological evaluations
of polymers were aimed. For this purpose, the effect of different polymer Mw’s and
different polymer/pDNA ratios on uptake and transfection efficiencies was
evaluated. Finally, the influence of polyplexes on the viability of cells was analyzed.

1.2 Outline of The Thesis

In the first following chapter, in order to give a background about general cell
biology, the main components of an eukaryotic cell that are related to the journey of
a gene carrier system are briefly explained.

In the “Gene Therapy” chapter, because the understanding of the efficiency of the
polymers to transfer the therapeutic genetic material to its target depends on
understanding the various gene delivery barriers that a carrier system has to
overcome, the major barriers for gene delivery are explained in relation to the
biological chapter. Subsequently, the most leading gene delivery methods including
physical, viral and synthetic methods to overcome these barriers are introduced
according to literature.

The other two chapters describe the materials and methods used in this study and the
results obtained, respectively. Finally, the concluding remarks about the results of
this project can be found in conclusion.

2
2. BASICS OF CELL BIOLOGY

2.1 The Cell

The cell is the smallest living unit of an organism. The term “cell” was defined for
the first time by Robert Hooke (1635-1702), an English physicist (Mazzarello, 1999).
Following the developments and technical improvements in microscopy science in
late 18th century, scientists became interested in visualizing the living organisms.
Then it was stated by a German physician and physiologist Theodore Schwann
(1810-1882) that animal tissues are comprised of cells and each cell has a nucleus
(Hajdu, 2002). Three centuries after the first discovery of the cell, today it is
estimated that there are ±10 trillion cells in a human body. Still, not all the cells have
the same function in the body and are specialized to carry out different functions,
such as epithelium, muscle and neuron cells. In general, cells have a size ranging
from 10 µm to 50 µm, yet depending on its function this can vary (See Figure 2.1).

Figure 2.1 : Comparison of sizes of precellular organisms with an average cell

in human body.

2.2 Cell (Plasma) Membrane

The cell membrane (or plasma membrane) is the protective boundary of a cell with a
thickness of 3 nm (Chen and Moy, 2000). The semi-permeability of the cell
membrane regulates what is being taken into cell and what is being given out of the

3
cell. Besides, this selectively permeable membrane controls the electrical and
chemical signals between cells. Lipids form half of the mass of cell membrane, while
the other half is made up of proteins, cholesterol and sugars (carbohydrates), (See
Figure 2.2).

The lipid bilayer of the membrane is built up through self assembly of phospholipid
molecules. These lipids contain a hydrophilic phosphate head, which is soluble in
water, and a hydrophobic fatty acid tail, which is soluble in lipid. Because
hydrophobic tails are repelled by the water molecules of intracellular and
extracellular fluids, they are located in the inner part of the bilayer membrane while
the hydrophilic heads are in contact with intracellular and extracellular fluids
(Bolsover, 2004).

Figure 2.2 : A cross section from cell membrane (Bolsover, 2004).

The proteins in the cell membrane maintain the chemistry of the cell and the transfer
of the chemical messages into the cell. The highly hydrophobic environment of the
lipid bilayer makes it easier for the fat soluble molecules such as oxygen, carbon
dioxide and alcohols to enter the cell. On the other hand, specific proteins form pores
on the membrane and this way, also water soluble molecules such as ions, glucose,
amino acids and urea can diffuse into the cell (Edidin, 2003), (Poo and Cone, 1974).

There are sugar chains which are called glycosaminoglycans (GAGs) attached via
protein moeties to the outer surface of the membrane. These protein-carbohydrate
complexes, called proteoglycans (PGs) play a crucial role in determining the charge
characteristic of the cell membrane. The most abundant PG on the cell surface is the

4
polyanionic heparan sulphate proteoglycans (HSPGs), which makes the cell
membrane negatively charged (Perrimon and Bernfield, 2000), (See Figure 2.3).
Thus, any compound with negative electrical charge will be electrostaticaly repelled
by the cell membrane, whereas positively charged particles will readily adhere to the
cell surface.

Figure 2.3 : A general molecular formula of HSPGs where R= SO3- (Gallagher et al,
1986).

2.3 Cellular Entry of Extracellular Material

2.3.1 Endocytosis and endosomes

A cell internalizes the nutrients which are necessary to maintain its functions from
the extracellular environment. Diffusion and active transport are the most common
ways that small substances such as amino acids, sugars and certain ions use to pass
through the cell membrane (Conner and Schmid, 2003). Diffusion is the simple and
passive movement of a particle (atom, ion or molecule) according to concentration
gradient. Particles are transported from a region in which they have higher
concentration to a region of lower concentration. Active transport is an energy-
demanding process that can occur when a particle needs to be passed against the
concentration gradient, i.e., from lower concentration to higher concentration. It is
assisted by special protein carriers.

However, very large molecules cannot enter through cell membrane neither via
diffusion nor active transport. They use a specialized function of the cell membrane
which is called endocytosis to be taken up by the cell. There are several routes
described for endocytosis, but the main mechanism remains the same. In general,
endocytotic pathways can be divided into two major categories: phagocytosis, which
is the uptake of large particles, and pinocytosis, which entails the uptake of fluid and

5
solutes (See Figure 2.4). Phagocytosis can be seen only in specialized mammalian
cells while pinocytosis occurs in all cell types (Conner and Schmid, 2003).
Moreover, pinocytosis has 4 subcategories: macropinocytosis (Apodaca, 2001),
clathrin-dependent endocytosis, caveolae-mediated endocytosis and clathrin- and
caveolae- independent endocytosis (Gong et al, 2008) (Nichols and Lippincott-
Schwartz, 2001). These routes differ from each other with regard to the size and
formation mechanism of the vesicle and the nature of the cargo that is being taken up
(Conner and Schmid, 2003).

Figure 2.4 : a. Categorization of different endocytotic pathways, adapted from

(Mayor and Pagano, 2007). b. Multiple routes of endocytosis into
mammalian cells (Conner and Schmid, 2003).

During endocytosis, the plasma membrane invaginates and engulfs some of the
extracellular fluid containing the foreign material (cargo). When two portions of the
plasma membrane reencounter each other, a part of the membrane pinches off and
forms an internal membrane-bounded vesicle named endosome (See Figure 2.5).

6
 Figure 2.5 : The mechanism of endocytosis.

The pH of the endosomal compartment is around 6 when the vesicle is near the cell
membrane. As the endosomal vesicles mature and get closer to a lysosomal state and
then to the nucleus it becomes more acidified (pH ~5) by the proton pumps (H+-
ATPase) of the endosome (Wong et al, 2007), (Luby-Phelps, 2000). The endocytotic
transport pathway is followed by lysosomal digestion of the cargo material.

2.3.2 Lysosome

Lysosomes are the organelles containing hydrolitic enzymes which are specialized in
the digestion of intra- and extracellular compounds (Ciftci and Levy, 2001). The
enzymes are optimally active at pH 5 to digest damaged cellular structures,
exogeneous materials -the compounds that are not wanted inside the cell- and the
food particles that have been taken into the cell (Rybak and Murphy, 1998).

Immediately after the cargo is taken up by endosomes, one or more lysosomes attach
to the endosome and the vesicle becomes acidified by proton pumps. The vesicular
hydrolytic enzymes of the lysosomes start to digest proteins, carbohydrates and lipids
to their building blocks amino acids, glucose and phosphates respectively. These
small digestion products can be delivered out of the vesicle to the cytosol. The
indigestible substances (residual body) are excreted through the cell membrane by a
process called exocytosis –the opposite mechanism of endocytosis.

7
2.4 Intracellular Environment

2.4.1 Cytoplasm and cytosol

Cytoplasm - the intracellular matrix - contains high volume of water while holding
the organelles inside. The watery-liquid part of the cytoplasm without any organelles
is defined as cytosol. It is a mixture of substances dissolved in water such as salts,
nutrients and some macromolecules. Additionally, cytosol contains nucleolytic
enzymes (nucleases), which are ready to degrade nucleic acids, distributed over the
cytoskeleton (Lechardeur and Lukacs, 2006) and intracellular reduction agents that
can cleave bioreducible foreign materials (Saito et al, 2003). The pH of intracellular
fluid ranges between 6.8 - 7.4 depending on the cell type and biological mechanism
and viscosity of the cytosol is nearly the same as pure water (Bright et al, 1987). The
major function of the cytosol is to help the transport of the metabolites and
compounds, through cytoskeleton, from their place of production to the place that
they will be used.

2.4.2 Cytoskeleton

The cytoskeleton is a dynamic structure, maintaining the cell shape, mechanical

resistance of the cell and cytoplasmic transport of organelles and large molecules
inside the cell (Luby-Phelps, 2000). It has a network of proteins which form the
microtubules (MTs), actin filaments (microfilaments) (MFs) and integrated filaments
(IFs), (See Figure 2.6).

Figure 2.6 : Cytosolic transport and nuclear import of the cargo, adapted from
(Wong et al, 2007).

8
The cytoplasmic MTs are responsible for intracellular trafficking of vesicles, proteins
and organelles (Fuchs and Yang, 1999). The actin MFs gives the cell polarity and
contractility for migratory processes (Apodaca, 2001). Finally, the IFs provides the
need for mechanical integrators of cytoplasm (Fuchs and Yang, 1999).

2.5 Cell Nucleus

2.5.1 Function

The cell nucleus is the control center of the eukaryotic cells. It is the largest organelle
in animals cells and can fill up to 10 % of the cell space with an average diameter of
6 μm (Lodish, 2004). Nuclear envelope, nuclear pores, nucleolus and genetic
material (chromosomes and chromatins) form the main structure of a cell nucleus
(See Figure 2.7). The nucleus coordinates essential cellular activities including
growth, protein synthesis and cell division. The genetic material in the nucleus is
very well protected by the nuclear envelope - the bilayer membrane of the nucleus.

Figure 2.7 : The structure of cell nucleus [Url-1].

2.5.2 Nuclear envelope and pores

The nuclear envelope encloses the nucleus separating the nucleoplasm and genetic
material from the cytoplasm by a double membrane (Gorlich and Mattaj, 1996). The
inner membrane which is called nuclear lamina is made from proteins and gives extra
rigidity to nucleus. The inner and outer membranes are penetrated with thousands of
nuclear pores.

The transport of macromolecules, such as proteins, from cytoplasm to the interiors of

nucleus, is controlled by highly selective membrane proteins through the formation

9
of an aqueous channel (Laskey et al, 1996). These proteins are termed as nuclear
pore complexes (NPCs). The NPCs recognize the macromolecules which are
forwarded to the nucleus and provide the energy for their translocation into nucleus.
The recognition process is guided by nuclear localization signals (NLSs) (Pouton,
1998). NPCs allow passive diffusion of metal ions, small metabolites and molecules
less than 40kDa in mass with diameters of 5 nm (Wente and Rout, 2010).
Nevertheless, there are particles known with a diameter up to 26 nm that can pass the
nuclear pores if a suitable NLS guides the particle (Dworetzky and Feldherr, 1988).
Moreover, breakdown of the nuclear envelope during the cell division, gives a
possibility for nuclear entry of some macromolecules like exogenous DNA (Wilke et
al, 1996).

2.6 Gene Expression

Gene expression is the process of synthesis of a functional gene product by using the
information from another gene (See Figure 2.8).

Figure 2.8 : Illustration of gene expression process [Url-2].

2.6.1 Transcription

Transcription is the first step of gene expression that takes place in the nucleus by
means of enzymatic complexes that are produced by specific genes. In this step
ribonucleic acid (RNA) copies of deoxyribonucleic acid (DNA) are produced. The
three major classes of RNA are ribosomal RNA (rRNA), transfer RNA (tRNA), and
messenger RNA (mRNA). All play crucial roles in protein synthesis. During

10
transcription, the double helix of DNA is opened, and one strand of DNA is used as a
pattern for the production of an RNA molecule (Bolsover, 2004).

2.6.2 Translation

Genes encoding mRNAs are known as protein-coding genes. Translation follows the
transcription process. In this step a single strand mRNA, which was transcribed from
double stranded DNA in the nucleus, produces proteins (polypeptides) (Lodish,
2004).

11
12
3. GENE THERAPY

Gene therapy is the treatment of human diseases with an underlying genetic cause by
delivering therapeutic genetic material (transgene) into the diseased cells of the
patient. It has been a promising way to treat genetic diseases over the last decades
because it offers a solution to treat the causes of diseases rather than curing the
symptoms (Mountain, 2000). When a gene has a mutation, it becomes unable to
express the proteins that it encodes for or the mutation can cause the production of
inactive or toxic proteins. In both cases, the vital cell functions are affected and
eventually induce symptoms. Consequently, gene therapy can be used to treat
diseases caused by a defect in a particular gene such as cystic fibrosis (CF) (Russ and
Wagner, 2007), or to treat acquired redoubtable diseases such as cancer and acquired
immunodeficiency syndrome (AIDS) (Ledley, 1995). The efficiency of gene therapy
depends on the delivery of DNA to target cells and long-term expression of the gene
(Naik et al, 2009).

3.1 A Brief History of Gene Therapy

The first approved clinical trials of gene therapy took place in United States in 1990
(Scollay, 2001). One of those two ex vivo trials was employed to cure an enzyme
deficiency that causes severe combined immunodeficiency (SCID) and the other as
an immunotherapy for melanoma (Blaese, 1995). Both of them relied on viral vectors
for the transfection of the therapeutics into cells, but neither of them were successful
applications. Then scientists had an encouraging result in 1995, with the treatment of
a four year old girl – an SCID patient. Although the disease was not treated, it was
under a better control after the treatment (Blaese, 1995). Nearly a decade after the
initial thoughts occurred about gene therapy, more than 175 clinical trials were done
and 2000 patients were already treated (G. Ross et al, 1996). However, the first
decade ended tragically with the death of a 18-year old patient who suffered from a
hepatic metabolic disorder - ornithine transcarbamylase (OTC) deficiency (Edelstein
et al, 2004). He was being treated with a viral vector but during the clinical safety

13
trial, he had an adverse patient reaction which affected his immune system and
caused fatality in 1999 (Thomas et al, 2003).

Then the millennium brought the success together for gene therapy. In 2000, three
children had been treated for SCID and the first actual healing with gene therapy was
reported (Cavazzana-Calvo et al, 2000). Unfortunately the success lasted shortly,
because three years after, two of these patients were diagnosed with leukemia
(Thomas et al, 2003).

After all the trials with failure, there were some lessons to be learned. Scollay (2001)
suggested that scientists had to base their studies more on the development of safer
and feasible technologies rather than unrealistic expectations.

According to the survey of Journal of Gene Medicine Clinical Trial Database (See
Figure 3.1), there have been 1786 approved clinical trials evidenced in worldwide
since the year 1989. Although the idea of treating the causes of diseases is only three
decades old and most of the clinical trials are still on phase I, with the acceleration
and success gained, it is a candidate to revolutionize the treatment of genetic
disorders.

Figure 3.1 : a. Number of approved gene therapy clinical trials worldwide

1989- 2011, b. Clinical phases of approved gene therapy trials,
adapted from [Url-3].

14
3.2 Barriers for Gene Delivery

In general, gene therapy involves the delivery of the transgene which is encoded to
produce a specific protein, into the patient’s somatic cells. In order for the
therapeutic nucleic acid to be effective, it should overcome both extracellular and
intracellular barriers. Delivering the pDNA has additional hurdles than delivering
mRNA, since the pDNA has to be tranfered into nucleus for mRNA transcription,
whereas the translation of mRNA occurs in cytoplasm.

These barriers for pDNA delivery can be summarized into following steps as in
Figure 3.2:

I. stability in the extracellular environment,

II. cellular attachment and adhesion,
III. cellular uptake,
IV. endosomal escape,
V. cellular translocation and
VI. nuclear import.

Figure 3.2 : Intracellular barriers for gene delivery, adapted from (De Smedt et al,
2005).

3.2.1 Stability in the extracellular environment

Due to the phosphate groups present in the DNA backbone, it is negatively charged.
Also, DNA is not a small molecule that can easily be delivered. It has a bulky
structure which has to be condensed in order to ensure cellular internalization.
Moreover, the plasmid should be protected against the existence of degradation

15
enzymes (nucleases) both in the extracellular and intracellular environment
(Abdelhady et al, 2003) (Lechardeur et al, 1999). The stability of the therapeutics in
the extracellular environment such as intravascular spaces is highly related to the
chemical stability of DNA. The exogenous negatively charged materials such as
GAGs and serum albumin cause instability of non-viral cationic carriers. The
potential carrier which will answer the requirements mentioned above has to provide
colloidal stability for carrier/DNA complexes. It is observed for the systems, which
require high concentration of cationic lipid or polymer particles to deliver the
therapeutic DNA molecule, that in the presence of excess positive charge complexes
readily aggregate (Davis, 2002).

There are different strategies which have been tried to avoid aggregation of the
complex particles in heterogeneous extracellular milieu. One of them is the covalent
attachment of biocompatible hydrophilic polymers such as polyethylene glycol
(PEG) or polyhydroxypropyl methacrylate (pHPMA) to the cationic carrier, to
provide a steric barrier against aggregation. Since PEG is the most favored polymer
for this purpose, it has given the name to the strategy as PEGylation. Covalent
coupling of PEG can be performed either before the polyplex formation
(prePEGylation) (Kursa et al, 2003) or after the polyplex formation (postPEGylation)
(Ogris et al, 1999). The effectiveness of PEGylation to form stable complexes both
for cationic lipid and polymers has been proved (Hong et al, 1997) (Y. H. Choi et al,
1998). In addition to increased solubility (Ogris et al, 2003) and stability, PEGylation
also provides lower toxicity and extended circulation time in blood, which can be
perceived as a heterogeneous extracellular compartment with high ionic strength
(Wagner and Kloeckner, 2006).

3.2.2 Cellular attachment and adhesion

When the stability of the complexes is ensured, the first obstacle is the attachment to
the cell membrane. There are two main factors affecting the cellular association of
cationic lipid or polymer carriers. First of all, the colloidal stability of the complexes
has a major impact on the cellular attachment of the complexes. It has been shown
that particles which form aggregates can better manifest the cellular attachment in
vitro (P. C. Ross and Hui, 1999). Nevertheless, from a pharmaceutical point of view
these aggregation events are dangerous and undesirable. Secondly, the cellular

16
attachment and adhesion of cationic particles is thought to be mediated by the
interactions with anionic cell surface HSPGs (Wiethoff and Middaugh, 2003). These
PGs are involved in various cellular processes as well as cellular attachment and
adhesion (Bernfield et al, 1999). The interruption of HSPG expression on the cell
surface results in a decrease in the amount of cellular associated-DNA and posterior
transgene expression in vitro (Mislick and Baldeschwieler, 1996). Furthermore, for
cationic lipid or polymer based gene delivery systems it is clearly shown that, the
presence of HSPGs increases the efficiency of gene delivery (Belting and Petersson,
1999).

3.2.3 Cellular uptake

After successful attachment to the cell membrane, the second challenge for a gene
delivery system is being taken up by the cell. Due to size restrictions, passive
diffusion through the cell membrane is not possible for these systems. In general,
cationic carrier systems enter the cell via endocytosis with non-specific electrostatic
interaction between positively-charged lipoplexes or polyplexes and negatively-
charged cell membrane GAGs. As it is described in the section 2.3.1, endocytosis
occurs mainly in 5 different pathways: phagocytosis, pinocytosis, clathrin-dependent,
caveolae-mediated and clathrin- and caveolae independent endocytosis. Most of the
lipid carrier systems are explained to be taken up via clathrin-dependent endocytosis
(Pichon et al, 2010). The uptake mechanism of polyplexes is more diverse depending
on the cell size and type, the charge and side group characteristics of the polymer
(Midoux et al, 2008).

In addition to the non-specific endocytotic entry to the cell, targeting is a strategy to

obtain cell specific gene delivery and increase the uptake specificity for the
internalization of the carrier system. Targeting the gene delivery system to a specific
cell can be employed by using target specific receptors or ligands. This strategy gives
the chance to design the delivery system containing the target-ligand according to the
aimed cell type. Mainly, electrostatic, conjugative or covalent coupling are being
used for ligand binding purposes. The most favored and successful targeting
molecules are transferring (Schatzlein, 2003), galactose (Zanta et al, 1997) and the
RGD (arginine-glycine-aspartate) peptide (Kunath et al, 2003). Additionally, cell-
penetrating peptides are being investigated as a strategy to enhance the cellular

17
uptake (Heitz et al, 2009). These are positively charged peptides which are derived
from viral proteins and have a length of 5-40 amino acids. They can be conjugated to
the cargo material either covalently or non-covalently and their uptake mechanism is
clearly explained by El-Andaloussi et al. (2005).

Moreover, as cellular internalization and uptake of polyplexes are mediated by cell

surface GAGs, it has been shown that the use of high concentrations of exogenous
GAGs for targeting purposes increased the uptake and transgene expression
efficiency of complexes (Mounkes et al, 1998). One example of the GAGs is
hyaluronic acid (HA) which is used as a shielding and coating material to increase
specific cellular uptake. The uptake mechanism of HA-coated particles has been
explained to be receptor-mediated endocytosis that is facilitated by CD-44 receptors
(Knudson et al, 2002). HA and CD44-mediated uptake and targeting is specifically
used in tumor targeting (K. Y. Choi et al, 2010).

3.2.4 Endosomal escape

Once the complexes are internalized by endosomal vesicles, the endosomal

compartment becomes acidified by proton pumps (H+-ATPase) and the luminal pH
drops. The therapeutic polymer-gene complexes undergo a pH drop from 7.4 on the
cellular surface to 5.5 inside the endosomal lumen. As the vesicle matures from
endosome to lysosome the pH gets even more acidic around pH 4.5-5.0, and the
therapeutic DNA is faced with different types of nucleases in the lysosome. Hence,
to avoid degradation of the therapeutic nucleic acids in the lysosomes and ensure
high transfection in the diseased cell, it is essential for any gene delivery vector to be
able to escape from the endosomes in time (Wiethoff and Middaugh, 2003).

For cationic lipid based gene delivery systems, their escape from endosomes is
thought to be mediated by lipid mixing (flip flop mechanism) between endosomal
and cationic lipid membranes and subsequently destabilization of the membrane and
release of DNA into cytoplasm (Xu and Szoka, 1996).

In case of the cationic polymer vectors, there are two possible hypotheses involved in
their endosomal escape mechanism. One of them supports that the direct electrostatic
interaction between negatively charged endosomal membrane and cationic polymer
causes membrane disruption. Such a mechanism has been suggested for poly(amido
amine) (PAMAM) dendrimers and poly(lysine) vectors (Zhang and Smith, 2000).

18
The second and most common explanation for the endosomal escape mechanism of
cationic polymers with ionizable amine groups is the proton sponge hypothesis
(Boussif et al, 1995), even if it is debatable (Funhoff et al, 2004). This hypothesis
came up with the poly(ethyleneimine) (PEI) polyplexes, since PEI has a high
cationic-charge-density potential, due to the presence of multiple protonable amino
groups. Besides, PEI is only 20% protonated at physiological pH and can have a
buffering effect nearly at any pH (Boussif et al, 1995). During the maturation of the
endosomal vesicle, as it pumps protons and becomes more acidic, PEI-like polymers
can act like a proton sponge, accept the protons and effectively buffer the decreasing
pH. On the other hand, these accumulated protons disturb the endosomal pH and
endosomal ATPases tend to keep the pH constant in the endosomes (See Figure 3.3).
They balance the H+ influx by the influx of counter Cl- anions to maintain the
electroneutrality. This huge increase of ionic concentration inside the endosomes
causes osmotic swelling by the influx of water and results in the bursting of
endosomal membrane, eventually releasing its content.

Figure 3.3 : Schematic illustration of proton sponge hypothesis.

3.2.5 Cellular translocation to the nucleus

Polyplexes that enter to cytosol following the endolysosomal release, are

immediately faced with a contentious environment. The nucleolytic degradation
enzymes are ready to degrade unprotected DNA, and the actin and tubulin filaments
from the cytoskeleton form a dense network which can act as a diffusional barrier.
The diffusion coefficient of the naked DNA in the cytoplasm is found to be highly

19
dependent on the plasmid size. However, the cytoskeleton does not allow the
diffusion of naked DNA greater than 85 nm, thus polyplexes (150-200 nm) can reach
to the nucleus only through active transport, provided by molecular motor proteins
which are attached to the MTs or MFs (Luby-Phelps, 2000), (Dauty and Verkman,
2005).

3.2.6 Nuclear import

The translation of mRNA occurs in cytosol while pDNA transcription occurs in the
nucleus. For this reason, pDNA therapy has to deal with one additional barrier during
the entry to nucleus.

There are three possible routes for a molecule to overcome the nuclear envelope and
enter the nucleus. One of them suggests that as the nuclear membrane breaks down
during cell division, the DNA molecule can diffuse into the nuclear environment of
the dividing cell (Gorlich and Mattaj, 1996). Most of the gene delivery systems are
thought to transport their genetic material by this way. Second way is the passive
diffusion of the molecules less than 5 nm through nuclear membrane (Wente and
Rout, 2010). However, this way is not applicable due to size restrictions of the DNA
molecule. The third route depends on the ability of a macromolecule to interact with
the nuclear transport system. Highly selective membrane proteins -the nuclear pore
complexes (NPCs) - present on this transport system, recognize a macromolecule by
the guidance of nuclear localization signals (NLSs) (Pouton, 1998). Particles less
than 26 nm in diameter can be actively transported through NPCs. DNA molecules
can be condensed by NLSs via electrostatic interactions or NLSs can be bound to the
polymer vector for nuclear targeting. Zanta et al. have shown that a 10- to 1000-fold
enhanced transfection can be achieved with NLS conjugated DNA compared to DNA
without NLS (1999).

Another important barrier to obtain an effective gene transfer for a non-viral vector
system is the unpacking of the vector (Schaffer et al, 2000). A successful
transcription and enhanced gene expression require safe and effective dissociation of
the vector/DNA complex. As polymeric vectors are the subject of this thesis, gene
unpackaging strategies for polymers will be discussed in the next section.

20
Overall, the successful transfer of nucleic acids to the nucleus and dissociation of
DNA is followed by the transcription of the genes to mRNA and subsequently
translation to the therapeutic protein in the cytosol.

3.3 Methods for Gene Delivery

The previously mentioned challenges for gene delivery, make the delivery of naked
DNA inefficient and enforces to use a physical method or combine it with a vector.
Many types of physical methods and vector systems have been developed for this
purpose.

3.3.1 Physical methods

3.3.1.1 Gene gun

This is a method based on applying physical force for the gene transfer. For the
delivery process, DNA-coated gold nanoparticles are accelerated by a high-pressure
helium steam (Mhashilkar et al, 2001). This method is also called “needle-free
injection” because the method allows gene delivery injection without the use of
needles (Mountain, 2000). The created force can deliver the DNA directly into the
cytoplasm and can be used as a promising alternative to the intramascular injection
of naked DNA for genetic vaccination (Condon et al, 1996). Because of the easier
physical accessibility the gene gun method has been widely applied for gene delivery
to the skin and encouraging immune responses are reported in initial clinical studies
(Tacket et al, 1999).

Large amounts of DNA can be delivered by this method, however, the introduction
of foreign materials such as gold particles, the relatively low transfection efficiency
and possible physical damage to the cells remain disadvantages of the gene gun
(Bleiziffer et al, 2007).

3.3.1.2 Electroporation

Electroporation is a method based on the administration of short (nanosecond range)

high-voltage pulses to temporarily disrupt the cell membrane barrier (Gehl, 2003).
The effect of the electric field disrupts the lipid and lipid-protein interactions and the
membrane becomes porous and permeable, allowing reversible transitions (Neumann

21
et al, 1999). In short, cells and DNA are put into a cuvette in vitro and then placed
between electrodes. After the electrical field is applied for a short incubation time to
allow the DNA pass the cell membrane, cells are restored to normal culture
conditions (Makrides, 2003). Cell size, temperature, post-pulse manipulation and
composition of electrodes and pulsing medium are important parameters to be
considered while performing electroporation (Gehl, 2003). Improved transfections
for in vivo electroporation applications have been reported as electrochemotherapy,
for the delivery of drugs and genes to malignant tumors, in brain (Nishi et al, 1997),
skin (Dujardin et al, 2001) and muscle (Mir et al, 1999) tissues.

Nevertheless, the need for a surgical procedure to place the electrodes into the organs
(Gao et al, 2007) and the applied high voltage which results in inevitable tissue
damage because of thermal heating (Durieux et al, 2004), limit the applicability of
electroporation facilitated gene delivery methods

3.3.1.3 Gene transfer by ultrasound radiation

The first statement that ultrasound (US) may improve the transdermal penetration of
drugs was established in 1954 (ter Haar, 2007). Following the developments in
therapeutic applications of US, it was discovered that US waves can be used for gene
transfer purposes both on the cellular (Kim et al, 1996) and tissue (Liang et al, 2004)
levels. The term sonoporation is also used for this method, which is based on
creating temporary plasma membrane defects by using US to generate acoustic
cavitation (Mehier-Humbert and Guy, 2005). During each ultrasonic cycle, the tissue
absorbs the energy of the propagating wave which results in thermal heating that
affects the cell membrane structure (Al-Dosari and Gao, 2009). This way, the DNA
can be transferred into the cytoplasm via passive diffusion through created
membrane pores (Kim et al, 1996). The transfection efficiency of the system is
controlled by the frequency, the output strength of the US wave, the duration of the
treatment and the amount of the plasmid DNA used (Gao et al, 2007).

The major improvement was observed in US mediated gene transfer when the
technique was combined with contrast agents or microbubbles (Endoh et al, 2002).
Microbubbles are compressible gas-field vesicles which are stabilized by surface
active molecules such as cholesterol (Al-Dosari and Gao, 2009). Their ability to
expand and shrink immediately under US application enhances the transfection

22
efficiency (Bao et al, 1997). Furthermore, two successful in vivo examples can be
stated for US-mediated gene delivery combined with a contrast agent: one of them
with the aim of intravenous injection (Unger et al, 2001) and the second for
disrupting the blood-brain barrier (BBB) (Sheikov et al, 2008).

3.3.2 Viral vectors

The use of viral vectors for gene delivery purposes has been the most efficient way
(Loser et al, 2002). Viral particles contain a nucleic acid genome surrounded by a
capsid of proteins. The introduction of a gene of interest into a host cell by a virus is
defined viral transduction. They can easily gain entry to host cells where the genetic
information is replicated by the host’s machinery. In the ideal case, these viral
vectors utilize the viral infection pathway but avoid the subsequent expression of
viral genes as this may lead to self-replication and potential toxic effects. Thus, the
gene must be combined into a virus that is replication-deficient (Jang et al, 2004).
There are large number of viral vector systems (i.e. retrovirus, adenovirus and adeno-
associated virus) used for gene therapy applications but the most frequently used
adeno-associated virus will be explained in this section.

3.3.2.1 Adeno-associated virus

Adeno-associated virus (AAV) is frequently chosen because it is able to transduce a

variety of cells, including non-dividing cells, provide long-term gene expression, and
offer minimalised immune response (Jang et al, 2004). However, applications are
limited with AAV vectors due to a small packaging capacity, inefficient transgene
expression and the need for duplication of the single-stranded DNA genome
(Thomas et al, 2003).

In addition to these issues, the reported failures of clinical trials with viral vectors
accelerated the developments of safer, less pathogenic and immunogenic gene
delivery alternatives such as non-viral vectors including lipid and polymer based
delivery systems.

3.3.3 Non-viral vectors

Non-viral vectors have been developed as alternative gene delivery systems to viral
vectors because they offer a number of potential advantages. These advantages can
be listed as non-immunogenicity, ability for repeated administration, ease of

23
production and lower costs (Brown et al, 2001). The introduction of DNA by means
of non-viral delivery system is defined as transfection process. This process includes
a successful gene transfer and expression (Mountain, 2000). Most commonly used
synthetic non-viral delivery systems will be described in the following.

3.3.3.1 Lipid-based gene delivery systems

Lipid-based systems have a variety of applications for drug and gene delivery in
pharmaceutical industry. Liposomes are spherical bilayered phospholipid structures
which are formed by the self assembly of a hydrophilic polar head group and
hydrophobic hydrocarbon tail. In an aqueous media, this bilayered structure consists
an internal aqueous phase and a water phase between the alternating bilayers
(Remaut et al, 2007). Due to the earlier mentioned physicochemical characteristics of
DNA molecule, cationic liposomes are the most frequently investigated carriers to
encapsulate the anionic surface charge of DNA by electrostatic interactions. The
cationic liposome and nucleic acid complexes are called as lipoplexes.

Commonly used cationic lipids include N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-

trimethyl-ammoniumchloride (DOTMA) and 1,2-dioleoyl-3-
trimethylammoniumpropane (DOTAP) as illustrated in Figure 3.4.

(A)

(C)

(B)

Figure 3.4 : Chemical structure of cationic lipid (A) DOTMA, (B) DOTAP and (C)
an illustration of liposome, adapted from (Remaut et al, 2007).

The use of lipids as gene delivery systems offers some advantages, such as their lack
of immunogenicity which makes repeated delivery in vivo possible. Another
advantage is the potential to deliver large amounts of DNA and the protection of
DNA against physical forces and enzymatic degradation. However, there are some

24
major limitations of these liposomes such as the duration and level of transgene
expression (Brown et al, 2001).

3.3.3.2 Polymer-based gene delivery systems

As it was explained in the previous section that the understanding of the barriers that
a vector system should overcome provides the development of safe and efficient
vectors for the delivery of the plasmids. With this in mind, an ideal polymeric vector
should meet the requirements of being able to neutralize the negative charge of the
DNA to avoid charge repulsion against the anionic cell surface, to condense the
DNA molecule to an appropriate length scale, and to protect the DNA from
degradation by extracellular nucleases. Since cationic polymers are capable of
condensing the negative charge of DNA, tremendous research has been done to
improve their potential as non-viral gene carriers. They offer enhanced bio-safety
and biocompatibility, a high flexibility regarding the size of the delivered nucleic
acid, repeated administration and can be synthesized at low cost in large quantities
(Wagner and Kloeckner, 2005).

The nano-sized self assemblies of positively charged polymers and negatively

charged DNA are defined as polyplexes (pplexes). The packaging of therapeutic
genetic material by a cationic polymer can be based on three different ways including
electrostatic interactions, encapsulation within the spherical polymer structure and
adsorption onto biodegradable nanosphere (Wong et al, 2007).

There have been many novel polymer structures developed for gene therapy
applications but the most frequently investigated polymer systems include poly(L-
lysine) (PLL) (Pack et al, 2005), poly(ethyleneimine) (PEI) (Akinc et al, 2005),
methacrylate polymers (Dubruel et al, 2003) and poly(amidoamine) (PAA) (Liu et al,
2010), (See Figure 3.5).

The ratio of amine groups present in the cationic polymer to phosphates on the
plasmid affects the effective diameter and determines the surface charge of the
polyplex.

Poly(L-lysine) (PLL) was one of the first polycations used for polyplex formation. At
physiological pH the amino groups of PLL are positively charged and interact
ionically with the negatively charged phosphate groups of the DNA.

25
 Figure 3.5 : Chemical structures of selected polymers, adapted from (Wong et al,
2007).

PLL/DNA polyplexes have a highly positive zeta potential, interact electrostatically

with a negatively charged cell surface, and are taken up by absorptive endocytosis
(Wagner and Kloeckner, 2005). The efficiency of uptake can be enhanced by
covalently coupling PLL with ligands that can specifically target cells and promote
receptor-mediated uptake (Zauner et al, 1998).

Poly(ethylenimine) (PEI) is an organic macromolecule with the highest cationic

charge-density potential, allowing for a tight compaction of nucleic acids (Boussif et
al, 1995). Linear PEI is solid at room temperature and contains only secondary
amines, while branched PEI (bPEI) is liquid at all molecular weights and contains
primary, secondary and tertiary amine groups (Ogris, 2004). Usually the branched
form of PEI is used for gene delivery purposes. Because every third amino nitrogen
atom is protonated, PEI has a buffer capacity at virtually any pH value (Boussif et al,
1995).

Methacrylate based polymers including poly(2-(dimethylamino)ethyl methacrylate))

(pDMAEMA) can be easily synthesized by radicalic polymerization. pDMAEMA
apparently has advantageous properties to escape the endosome due to its average

26
pKa value of 7.5. This polymer is partially protonated at physiological pH and might
behave as a proton sponge that can cause a disruption of the endosome, which results
in the release of both the polyplexes and cytotoxic endolysosomal enzymes into the
cytosol (van de Wetering et al, 1998). Moreover, pDMAEMA was shown to be an
efficient vector for gene delivery to OVCAR-3 cells (van de Wetering et al, 1999).

Poly(amidoamine)s (PAAs) are synthetic tert-amino polymers obtained by stepwise

polyaddition of primary or secondary aliphatic amines to bisacrylamides. Linear
polymers are produced during the polymerization of both primary and secondary
amines (Ferruti, et al, 2002). A novel bioreducible, disulfide containing family of
PAAs were introduced by Lin et. al. (2007a). These polymers showed relative
stability in physiological conditions but were rapidly degraded in reductive
environment. SS-PAAs possessed higher buffer capacities than PEI in endosomal pH
range, which may contribute to the endosomal escape of the polyplexes. Some
members of SS-PAA family also showed higher transfection efficiency in COS-7
cells than PEI with low cytotoxicity (Lin et. al, 2007a). Dendrimer form of
polyamidoamines (PAMAM) also shows high transfection potential in the absence of
any additional membrane-active agent. Due to the low pKa of its terminal and
internal amines this polymer can act as an endosomal buffering agent by this way
preventing DNA degradation within the endolysosomes (Wagner and Kloeckner,
2005).

A variety of polymer systems have been developed to maintain an efficient

DNA/vector dissociation. These strategies include thermoresponsive polymers,
bioreducible disulfide bonds and ester bonds (Wong et al, 2007).

The polymeric gene delivery vectors have relatively low transfection efficiency when
it is compared with viral and lipid based systems. Mainly, this delivery inefficiency
of the polymeric vectors relies on their inability to overcome numerous barriers
encountered between the site of administration and the localization of nucleus
(Wiethoff and Middaugh, 2003). Thus, numerous strategies were applied to improve
the efficiency of polymer vector systems.

27
28
4. MATERIALS AND METHODS

The aim of the research described in this work is to evaluate cationic polymers in
terms of their physicochemical and biological suitability for gene therapy
applications. For this purpose, non-biodegradable poly((2-dimethylamino)ethyl
methacrylate) (pDMAEMA) polymer and bioreducible disulfide containing
poly(amidoamine)s (SS-PAAs) were synthesized and evaluated.

4.1 Polymer Synthesis

4.1.1 Synthesis of pDMAEMA by radicalic polymerization

As a leading compound, poly((2-dimethylamino)ethyl methacrylate) (pDMAEMA),

is chosen as a candidate for gene delivery studies since it is relatively easy to obtain
by radical polymerization from dimethylaminoethyl methacrylate (DMAEMA) and
its molecular weight (Mw) can be controlled by controlling the amount of initiator
and monomer.

(4.1)

DP = Degree of Polymerization (units of monomers in a polymer chain).

[M] = concentration of monomer.
[I] = concentration of initiator.

Moreover, DMAEMA has amine groups which are protonated at physiological pH.
Due to a neighboring effect present in the polymer, some groups remain
unprotonated and therefore enable the polymer to express buffering capacities. In
gene therapy, the buffering capacity of a polymer is extremely important as it enables
the polymer to escape from endosomes.

The low Mw pDMAEMA (50 kDa) and high Mw pDMAEMA (600 kDa) which are
evaluated in this thesis were obtained from Ghent University Polymeric Biomaterials
Group (Ghent, Belgium).

29
 CH3
a. H2C
b.
O

N
H3C CH3

Figure 4.1 : Chemical structures of a. DMAEMA monomer, b. pDMAEMA

polymer.

The synthesis of pDMAEMA (250 kDa) was performed by thermally initiated

radicalic polymerization reaction, as a model reaction for this study.

It should be noted that even though the general set up and mechanisms are the same
for low and high Mw pDMAEMA synthesis, it is not possible to synthesize high Mw
pDMAEMA with the same initiator. As it was previously stated by van de Wetering
et. al., high Mw (>300 kDa) pDMAEMA polymers were obtained when ammonium
persulfate (APS) radicalic initiator was used in aqueous solution conditions (1998).

As it is illustrated in Figure 4.2, the AIBN initiator splits into two radical molecules
in the initiation step. Afterwards, the propagation occurs while the radical attacks to
the DMAEMA monomer. Then the monomer radical attacks to the other DMAEMA
monomer and the polymerization continues by the formation of polymer radicals.
The termination of the polymerization can be either via combination of two radicalic
polymers with each other or disproportionation.

The DMAEMA monomer is commercially available from Fluka Analytical

(Belgium). The pDMAEMA polymer was synthesized according to a previously
published protocol (Dubruel et al, 2000). In short, first the monomer is stabilized
with inhibitors and it is necessary to carry out a vacuum distillation prior to
polymerization. Therefore, the monomer was vacuum distilled for 2 hours. Although
it contained polymerization inhibitor, 10% of the monomer was polymerized and
sticked to the flask. At the end of the distillation, the distilled monomer liquid was
decanted gently from the flask.

30
 AIBN

DMAEMA

Figure 4.2 : Illustration of radicalic polymerization reaction mechanism.

31
 Figure 4.2 (continued) : Illustration of radicalic polymerization reaction
mechanism.

Meanwhile, the solvent of the reaction was distilled nearly for 3 hours in order to get
rid of any possible impurities. The solvent purification was performed in the
presence of Ca(OH)2 to dry out existing water or moisture in the solvent. The choice
of the solvent and initiator depends on the solubility of the monomer as well as the
polymer which will be synthesized inside and the desired Mw of the polymer. As an
appropriate solvent, both for monomer and polymer, toluen was used and azo-bis-
isobutyronitrile (AIBN) was chosen as an initiator for the polymerization reaction.

Prior to polymerization, a cooler, a valve and a two neck, round-bottomed flask were
put in the drying oven, to remove any possible moisture. Firstly, the flask was placed
in an oil bath which was placed on a magnetic stirrer, to allow monitoring of both the
temperature and the stirring speed. Secondly, the reflux cooler was fixed to the flask
and above this cooler the valve was placed with a connection to a balloon filled with

32
N2. The other neck of the flask was covered with a septum to avoid air-contact.
Finally, after addition of monomer and solvent, all contacts and seals were checked.

Before and after the addition of the appropriate amount of initiator, the reaction
mixture is degassed and purged with N2, by means of a vacuum pump and a balloon
filled with N2. This procedure was performed in order to remove the O2 present in the
set up as it may disturb the polymerization due to trapping of initiator radicals
(Osinglet – Otriplet transition) which may lead to the absence of the initiator-propagation
transition (Cherng et al, 1996).

When all of the reagents were purified and ready for the synthesis, the reaction was
started by adding the calculated amounts of reagents according to the desired Mw of
polymer. . A Mw of 250 kDa (250000 g/mol) was desired, 7.74 mg of AIBN was
added to 10 % DMAEMA (25.3 mL) in toluene as shown by the following
calculation. The density and Mw of DMAEMA are 0.93 g/ml and 157.21 g/mol
respectively. The Mw of AIBN is 164.21 g/mol. Based on these values, the following
calculations were made :

Aimed DP = Mw polymer / Mw monomer

= (250000g/mol) / (157.21g/mol) = 1590.23 units

Amount of monomer : If we start with 0.15 moles of monomer [M],

;
0.15 mol = m monomer / 157.21g/mol
m monomer = 23.58 g
;
0.93 g/ml = 23.58 g / Vmonomer
Vmonomer = 25.3 mL
Amount of initiator : [I] = [M]/ DP
[I] = 0.15 mol / 1590.23 = 9,43 x 10-5 mol
minitiator = [I] x Mw
minitiator = 9,43 x 10-5 mol x 164,21 g/mol = 0,01548 g

At the initiation step of the reaction, as every 1 mole of initiator gives rise to 2 moles
of radicals as illustrated in figure 4.2, the calculated amount of the initiator should be
divided by 2. This results in, 0.01548 g / 2 = 0.00774 g = 7.74 mg of AIBN.

33
After the addition of AIBN the polymerization reaction is started by heating the oil
bath to approximately 75⁰C, in order to obtain a temperature of 65 - 70⁰C in the
flask, for 24 hours. 24 hours later, the polymerization reaction was stopped and the
obtained reaction mixture is brought to room temperature. The mixture in toluene
was precipitated drop wise in ice-cooled distilled pentane (5x volume of the reaction
mixture). As a non-polar solvent pentane can remove the solvent of the reaction
(toluen), the residue of the monomer (DMAEMA) and the initiator (AIBN). The
synthesized polymer was separated from the pentane by decantation. After the
precipitation procedure, a sticky and gummy polymer was obtained. Then the
polymer was left to be dried and re-precipitated in ice-cooled ultra pure acetone (5x
volume of the reaction mixture) and again it was left to be dried. Finally, the polymer
was dissolved in ultrapure water. To ensure a good dissolution of the polymer in
water, a few drops of 0,5 M HCl are added. This solution was purified by dialysis
through a membrane with a Mw cut-off of approximately 1/5th of the Mw of the
polymer. The dialysis procedure was carried out for 48 hours against ultrapure water,
0,1 M NaCl solution and 0,1 M HCl solution respectively. Subsequently, the
dialyzed polymer solution was freeze-dried, then the final product was characterized
by NMR (300 MHz, CDCl3, relative to TMS) and stored at -20⁰C for further
experiments.

p(DMAEMA): 1H-NMR (CDCl3) δ (ppm) = 1.83 (CH3C(CH2)CH2); 1.92

((CH3)CCH2C(CH3)); 2.29 (CH2N(CH3)CH3); 2.57 (CH2N(CH3)CH3); 4.06
(O=COCH2CH2).

4.1.2 Synthesis of SS-PAAs by Michael-type polyaddition

Poly(amidoamine)s (PAAs) have attracted significant interest due to their

biocompatibility and biodegradability as potential gene delivery systems. A new
series of PAAs containing repetitive disulfide linkages (SS-PAAs) in the polymer
backbone was previously introduced by Lin et. al. (2007a). Moreover, Zintchenko
et.al. have reported an improved synthesis strategy for SS-PAAs by means of Ca2+
catalysis in order to increase polymerization times of conventional synthesis (2011).

In this study, three types of SS-PAAs were synthesized by Ca2+ catalyzed Michael
type polyaddition of a primary amine to N,N’-cyctaminebisacrylamide (CBA)

34
monomer according to published protocol (Zintchenko et al, 2011). The
polymerization reactions are illustrated in following figures (Figure 4.3, 4.4, 4.5) :

CBA a primary amine Poly(amidoamine)

Figure 4.3 : Illustration of Michael type polyaddition of a primary amine to

bisacryl amide monomer, adapted from (Lin et al, 2007a).

a.
CBA ABOL

b.
CBA HIS

Figure 4.4 : Illustration of Michael type addition polymerization of a. p(CBA-

ABOL), b. p(CBA-HIS), adapted from(Coue and Engbersen, 2011).

All monomers, 4-amino-1-butanol (ABOL, Aldrich), histamine (Fluka), N,N’-

cystaminebisacrylamide (CBA, polysciences, USA), were purchased in the highest
purity and no further purification was used. All reagents and solvents were of reagent
grade and were used without further purification.

Solutions of CaCl2 in methanol and in water (0.4 M) were prepared separately and
mixed together at the ratio of 3/1 (v/v) just before the synthesis. The polymerizations
were carried out with 1:1 molar ratio between the reactants.

35
 CBA
ABOL
PEG-NH2

DMEDA p(CBA-ABOL/DMEDA/PEG)

Figure 4.5 : Illustration of Michael type addition polymerization of a p(CBA-

ABOL/DMEDA/PEG) copolymer, adapted from (Vader et al,
2012).

Solutions of CaCl2 in methanol and in water (0.4 M) were prepared separately and
mixed together at the ratio of 3/1 (v/v) just before the synthesis. The polymerizations
were carried out with 1:1 molar ratio between the reactants. As a typical reaction
ABOL (0.34 g, 3.85 mmol ) was added to CBA (1.0 g, 3.85 mmol), 2 ml of solvent
was added, and the reaction flask was sealed. The polymerization was performed at
50⁰C and the reaction was allowed to proceed for 30 h, yielding a viscous solution.
Subsequently, the polymerization was quenched with 10 mol% excess of ABOL
(0.034g, 0.385 mmol) in order to consume any unreacted acrylamide groups and
stirring was continued for 1 h at 50⁰C. The resulting solution was diluted with water
to about 20 ml, acidified with 6 M HCl to pH ~ 4, and then purified using a
ultrafiltration membrane (MWCO 1000 g/mol). After freeze-drying, the p(CBA-
ABOL) polymer was collected as its HCl–salt form. The same procedures were
applied for the synthesis of p(CBA-HIS) polymer. The p(CBA-
ABOL/DMEDA/PEG) polymer was obtained from Engbersen Lab. (University of
Twente, The Netherlands). The polymerization of PEGylated p(CBA-ABOL) was
also performed with the same procedure. The Michael type addition of ABOL and
PEG-NH2 to CBA was followed by a second addition of DMEDA monomer as
illustrated in figure 4.5.

NMR spectra of final products were recorded on Varian Unity 300 (1H NMR 300
MHz) using tetramethylsilane (TMS) as the internal standard.

36
 1
p(CBA-ABOL) : H NMR (D2O) δ (ppm) = 1.73 (CH2CH2OH, 2H); 1.94
(CH2CH2CH2OH, 2H); 2.91(2 × NHCOCH2, 4H); 3.00 (2 × SSCH2CH2, 4H); 3.36
(NCH2CH2CH2CH2OH, 2H); 3.59 (CH2CH2NCH2CH2, 4H); 3.66 (2 × SSCH2CH2,
4H); 3.75 (CH2OH, 2H), 4.90 (2 x NH, 2H) .

p(CBA-HIS) : 1H NMR (D2O) δ (ppm) = 2.77 (2 × NHCOCH2, 4H); 2.95 (2

× SSCH2CH2, 4H); 3.17 (NCH2CH2, 2H); 3.38 (CH2N(CH2)CH2, 6H); 3.61 (2 ×
SSCH2CH2, 4H); 7.26 (C=CH–N, 1H); 8.38 (C–N=CH–N, 1H).

The molecular weight and polydispersity (Mw/Mn) of the synthesized SS-PAAs

were determined by gel permeation chromatography (GPC) relative to PEO
standards (Polymer Labs) as suggested by Jiang et.al. (2006). A Viscotek GPCMax
pump, autoinjector and two thermostated (30°C) PL aquagel-OH 30 columns (8 μm,
300×7.5 mm, Polymer Labs, with a low-molar-mass separation range 200 – 40,000)
were used during the analysis. Data were collected using a TDA302 Tripledetector
with RI, Visc and LS (7 and 90°). 0.3 M NaAc aqueous solution, pH 4.4, with 30%
methanol was used as eluent at a flow rate of 0.7 ml/min).

4.2 Cell Culture

In this project, in vitro studies were done with ARPE-19 cells (retinal pigment
epithelial cell line; ATCC number CRL-2302), which are described by Dunn et al. as
an adherent, human retinal pigment epithelium (RPE) cell line (1996).

4.2.1 Materials

Dulbecco’s modified Eagle’s medium (DMEM), serum-free transfection medium

OptiMEM™, L-glutamine, fetal bovine serum (FBS), penicillin-streptomycin
solution (5000 IU/mL penicillin and 5000 μg/mL streptomycin) (P/S), and
phosphate-buffered saline (PBS) were provided from GibcoBRL (Merelbeke,
Belgium). Trypsin was purchased from Sigma Aldrich (Bornem, Belgium) and used
as 0.05% trypsin/ 0.02% ethylenediaminetetraacetate (EDTA) solution.

4.2.2 Methods used in cell culture

The cells are grown in polystyrene (PS) culture bottles in RPE cell culture medium
(CCM), (DMEM:F12 supplemented with 10% fetal bovine serum (FBS), 2 mM

37
l-glutamine, and 1% penicillin/streptomycin (P/S)), and incubated at 37⁰C and in the
presence of 5% CO2 (Nu-5510 NuAire incubator).

All cell work is done under sterile laminar airflow conditions. When the monolayer
of ARPE-19 cells has become 70-80 % confluent in culture bottle, the cell line is
spliced to a new cell culture bottle. Prior to cell splicing procedure, the cells are
always checked under the microscope for possible contamination. First of all, the old
CCM is removed to waste. Then the cells are washed once with PBS solution without
Ca2+ and Mg2+, to get rid of the dead cells floating inside the bottle and any
remaining serum, which might inhibit trypsin activity. Afterwards, the flask is
shaken gently and the PBS is aspirated from the bottle. Secondly, the cells are
trypsinized with pre-warmed trypsin/EDTA solution and incubated at 37⁰C for 5
minutes. Trypsin provides the cell detachment from the bottle surface and EDTA
chelates the remaining divalent cations, necessary for Ca2+-dependent cellular
adhesion molecules. When cell detachment is verified by light microscopy, trypsin is
neutralized and inhibited by means of pre-warmed fresh CCM since the serum in the
CCM contains trypsin inhibitors. Fourthly, a certain amount of cell suspension is
transferred to a new culture bottle. Then the cells are re-incubated at 37⁰C and in the
presence of 5% CO2 until the next passage. If the cells are needed the next day for a
transfection experiment, then the cells are counted, seeded and plated following the
trysinization step.

4.3 Nucleic Acids Used in This Study

4.3.1 pGL4.13 plasmid

Bacterial plasmids are described as closed circular molecules of double-stranded

DNA. Their size can range between 1-200 kb. There are genes which code for
enzymes in plasmids. pGL4.13 is a plasmid of the series of pGL4-luciferase reporter
vectors of Promega (Leiden, The Netherlands). It encodes for luciferase reporter
gene (luc2) and is designed for high expression and reduced anomalous transcription.
Besides, the genes of pGL4.13 plasmid encodes for ampicilline (Ampr) resistance
(See Figure 4.6).

38
During this thesis study, pGL4.13 plasmid is isolated from Eschericia Coli bacteria
by using Qiafilter Plasmid Purification Kit (Qiagen, Venlo, The Netherlands)
according to the instructions manual.

Figure 4.6 : Schematic representation of pGL4.13 plasmid. The plasmid encodes for
luc2 gene under the control of SV40 promoter. Ampr: ampicilline
resistance marker [Url-4].
This assay is based on growing the bacteria containing the plasmid in appropriate
medium and then isolation of the plasmid from the grown bacteria culture. The
isolation includes filtration of alkaline lysate of the bacteria, centrifugation of lysate,
anion-exchange chromatography for selective isolation of pDNA from lysate and
finally purification of plasmid by alcohol precipitation (QIAGEN, 2005). The
concentration and purity of isolated pDNA were determined by measuring the UV
absorption at 260 and 280 nm with Nano Drop 2000c UV-Vis Spectrophotometer
(Thermo Scientific, USA). Subsequently, pDNA solution was diluted to 1 mg/ mL
and stored at -20⁰C.

4.3.2 gWiz™-GFP plasmid

The gWiz™-GFP plasmid which encodes for green fluorescent protein is an

expression vector from the gWiz™ series of Aldevron (Freiburg, Germany). As a
bacterial selection marker, kanamycin (kanr) resistance was used in this plasmid. The
gWiz™-GFP plasmid is designed for the highest level of gene expression in various
types of mammalian cells and tissues (See Figure 4.7). In this study, transfection
efficiency studies were analyzed in terms of GFP expression by the use of gWiz™-
GFP plasmid.

39
 Figure 4.7 : Schematic representation of gWiz™-GFP plasmid. kanr: kanamycin
resistance [Url-5].

4.3.3 YOYO®-1 iodide labeled pGL4.13 plasmid

For uptake efficiency purposes, pGL4.13 plasmid was fluorescently labeled with
YOYO®-1 iodide dye (λex = 491 nm, λem = 509 nm). This is a green fluorescent dye
from a series of dymeric cyanines obtained from Molecular Probes (Merelbeke,
Belgium). The plasmid and YOYO-1 (1 mM in DMSO) iodide was mixed at a ratio
of 0.15/1 (v/w), to provide a theoretical labeling density of 1 YOYO-dye molecule
per 10 bp. Then the mixture was incubated at room temperature for 4 h in the dark.
Free YOYO-1 dye and DMSO were removed by precipitating the labeled plasmid.
For this purpose 2.5 volumes of ice-cold ethanol and 0.1 volume of 5 M NaCl were
added to the plasmid/YOYO-1 solution. After incubation for 30 min at -80°C, the
centrifugation (17000 g, 30 min) and washing with clean 70 % ethanol, fluorescently
labelled plasmid was finally resuspended in 25 mM HEPES, pH 7.2. The
oncentration of the plasmid was again determined by UV absorption at 260 and 280
nm with Nano Drop 2000c UV-Vis Spectrophotometer (Vercauteren et al, 2011).

4.4 Preparation and Physicochemical Characterization of Polyplexes

4.4.1 Dynamic light scattering

4.4.1.1 Theory and working principle

Dynamic Light Scattering (DLS) is a technique which is used to measure the size-
related Brownian motion of particles. Particles suspended within a liquid will move
randomly under influence of the surrounding solvent molecules. The larger the
particle, the slower the movement will be. The translational diffusion coefficient D is

40
a measure for the diffusional motion, from which the hydrodynamic diameter is
calculated by using the Stokes-Einstein equation (4.2) :

(4.2)

where:

d(H) = hydrodynamic diameter (m)

D = translational diffusion coefficient (m².s-1)
K = Boltzmann’s constant (1.38 x 10-23 m².kg.s-2.K-1)
T = absolute temperature (K)
η = viscosity (kg.m-1.s-1)
The diameter that is obtained by DLS is the diameter of a sphere that has the same
diffusion coefficient as the measured particle. This coefficient will depend not only
on the size of the particle core, but also on any surface structure, as well as the
concentration and type of ions in the medium.

The Brownian motion of particles is measured by determining the rate at which the
intensity of light, scattered from the particles, fluctuates when detected with a
suitable optical arrangement. The signal intensities at different time points are
compared and a correlogram is constructed by a digital auto correlator in the DLS
machine. This device will also determine the correlation function of the scattered
intensity, from which the size of the particles can be calculated by using a suitable
algorithm. The size distribution obtained from the DLS measurements is a plot of the
relative intensity of light scattered by particles of various sizes and is therefore an
intensity-dependent size distribution.

The other important parameter that determines the stability of nanoparticles is zeta
potential which is defined as the measure of the magnitude of the electrostatic or
charge repulsion or attraction between particles. Its measurement gives detailed
information about the causes of dispersion, aggregation or flocculation, and can be
used to improve the formulation of dispersions, emulsions and suspensions. When
charged surfaces are put in aqueous solution an electric double layer forms at the
particle-liquid interface. The electric double layer consists of two parts: an immobile
layer of adversely charged ions where in this Stern layer the ions are bound tightly to
the surface. Secondly, there is an outer layer, which is a diffuse layer where a

41
balance of electrostatic forces and random thermal motion determines the ion
distribution. The potential in this region, therefore, decays with increasing distance
from the surface until, at sufficient distance, it reaches the bulk solution value,
conventionally agreed to be zero as illustrated in figure 4.6. The magnitude of the
zeta potential gives an idea about colloidal stability of the suspension. If all the
particles have a large value of positive or negative surface charge, then they will tend
to repel each other and there will be no tendency for the particles to come together
and form aggregates. In reverse case, if they have a low zeta potential then there will
be no force to prevent the particles floculating. The main factors that affect the zeta
potential of a particle can be stated as pH of the solution, conductivity and
concentration of a formulation component (See Figure 4.8).

Figure 4.8 : Schematic illustration of zeta potential [Url-6].

The measurement of the zeta potential was performed on a Zetasizer. This apparatus
calculates the zeta potential by determining the electrophoretic mobility (μ) of the
polyplex. The latter is obtained by measuring the velocity of particles using Laser
Doppler Velocimetry (LDV). The velocity and electrophoretic mobility are related
by the following equation (4.3) :

(4.3)

42
By applying the Henry equation the zeta potential can be calculated from the
following formula (4.4) :

(4.4)

where;
μ = the electrophoretic mobility in m /Vs
2

ζ = the zeta potential in V

= the dielectric constant in F/m
= the viscosity of the medium in cP
f(ka) = Henry’s function

In this project, a Nano-ZS (Malvern Instruments, Hoeilaart, Belgium) is used for

DLS measurements. This instrument consists of the following main components (See
Figure 4.9) : a laser beam (1) illuminates the sample contained in a cell (2). To
prevent that the detector becomes saturated or that an insufficient amount of light
reaches the detector, a variable attenuator (4) is placed between the laser and the
sample, allowing to reduce or increase the intensity of the laser source and hence the
intensity of scattering. The laser beam is scattered by the particle within the sample
at all angles and a detector (3) measures the scattered light. In the Nano-ZS model,
the detector is positioned at 173°. The signal from the detector is then passed to a
correlator (5) that compares the scattering intensity at successive time intervals to
derive the rate at which the intensity is varying. The information obtained by this
digital processing board is then passed to a computer, equipped with Nano software
to analyze the data and derive size information.

4.4.1.2 Particle size and surface charge measurements

All DLS measurements were performed in triplicate on a Nano-ZS (Malvern

Instruments, Hoeilaart, Belgium), equipped with an Argon laser beam (488 nm) at a
scattering angle of 173° and a temperature of 25°C. The preparation of polyplexes
occurs as described in the following section.

4.4.2 Preparation of polyplexes

Polymer/DNA complexes were obtained by mixing the cationic polymer solution and
anionic pDNA as illustrated in Figure 4.10.

43
 Figure 4.9 : Main components and optical configurations of a Nano Ζeta-sizer
instrument for DLS measurements [Url-7].

The formation of polyplexes are spontaneous and depends on electrostatical

interaction between polymer and pDNA. All the polyplexes in this study were
prepared in a filtered (0.2 μm mesh size), 25 mM, pH 7.2 HEPES buffer solution
under dust-free laminar air flow conditions.

4.4.2.1 pDMAEMA polyplexes

pDMAEMA polyplexes were prepared with two different polymer Mw 50 kDa and
600 kDa, and in two different polymer/pDNA mass ratios of 1.5/1 and 12/1. As
plasmid DNA, pGL4.13 plasmid is used for all DLS measurements in this thesis. The
DLS samples with different polymer/pDNA mass ratios were prepared with changing
amounts of polymer but the same amount of plasmid (10μL). The polymer stock
solution (5 mg/mL) was diluted in HEPES buffer solution to have a concentration of
0.6 mg/mL. The pDNA stock solution (1mg/mL) was also diluted until a
concentration of 0.05 mg/mL. Then the formation of polyplexes were achieved

44
Figure 4.10 : Formation of polymer/DNA nanocomplex (polyplex) by self-assembly,
adapted from (Coue and Engbersen, 2011).

by mixing the appropriate amount of polymer with pDNA solution which

corresponds to 10 μg of plasmid. The amount of polymer for each ratio was
calculated as follows :

(1.5/1) Polyplex preparation :

M1.V1 = M2.V2

8.33x dilution of polymer stock (100 μL polymer + 733 μL HEPES)

-> 5 mg/mL x 100 μL = M2x 833 μL
M2 = 0,6 mg/mL

20x dilution of pDNA stock (10 μL pDNA + 190 μL HEPES)

-> 1 mg/mL x 10 μL = M3 x 200 μL
M3= 0,05 mg/mL

Add 25 μL of 0,6 mg/mL polymer to 200 μL of 0,05 mg/mL pDNA then dilute to
1ml with 775 μL HEPES.

1.5/1 polyplex contains:

[(5μg/μLx 100 μL) x 25/833] = 15 μg of polymer
(1μg/μL x 10 μL) = 10 μg of pDNA

(w/w) ratio -> 15 μg / 10 μg = 1.5/1

When the polyplexes were formed after 20 minutes, first of all the particle size and
polydispersity index (pDI) of the samples were measured in low volume polystyrene
(PS) cuvettes to avoid any electrical effect that may disturb the polyplex formation.
Subsequently, ζ-potential measurements were performed in clear disposable zeta
cells.

Additionally, prior to the in vitro evaluation studies which are performed in

OptiMEM™ GibcoBRL (Merelbeke, Belgium) serum free transfection medium, the

45
particle size and pDI measurements were also applied for the polyplexes in
OptiMEM, representative for the stability in physiological media. For this purpose,
polyplexes were prepared in the previous way but after the polyplex formation in
HEPES, polyplexes were diluted 2 times in OptiMEM. Since OptiMEM media is rich
in different types of proteins and dissolved ions, charge measurements were not
carried out in OptiMEM media.

4.4.2.2 SS-PAA polyplexes

The polyplexes with p(CBA-ABOL), p(CBA-ABOL/DMEDA/PEG) and p(CBA-

HIS) polymers were prepared in the same way as the pDMAEMA polyplexes were
done. The amounts of polymers and pDNA that are calculated for polyplex formation
are shown as a model for 48/1 ratio below :

48/1 (polymer/pDNA) polyplex preparation :

M1.V1 = M2.V2

8.33x dilution of 5 mg/mL polymer stock (100 μL polymer + 733 μL HEPES)

-> 5 mg/mL x 100 μL = M2x 833 μL
M2 = 0.6 mg/mL

20x dilution of 1 mg/mL pDNA stock (10 μL pDNA + 190 μL HEPES)

-> 1 mg/mL x 10 μL = M3 x 200 μL
M3= 0.05 mg/mL

Add 800 μL of 0,6 mg/mL polymer to 200 μL of 0,05 mg/mL pDNA.

48/1 polyplex contains:

[(5μg/μL x 100 μL) x 800/833] = 480 μg of polymer
(1μg/μL x 10 μL) = 10 μg of pDNA

(w/w) ratio -> 480 μg / 10 μg = 48/1

For HA-coated p(CBA-ABOL) polyplexes, research grade HA with a known Mw

range of 20-40 kDa was purchased from Lifecore Biomedical (MN, U.S.A). The
chosen optimum polymer/pDNA ratio 48/1 was applied for the preparation of
polyplexes. Polyplexes with 48/1 ratio were prepared as previously explained. When
the polyplexes were formed, HA-coating was performed on 200 μL pplx
(corresponding to 2 μg pDNA), with varying amounts of HA for the same amount of
pDNA. Afterwards, the polyplex-HA mixture was vortexed for 10 seconds and
allowed to stabilize for 15 minutes. Particle size and polydispersity of HA-coated

46
polyplexes were measured both in HEPES and OptiMEM™ media. As is mentioned
before, the ζ- potential is only measured for the complexes in HEPES.

For 25.5/1 (HA/pDNA) ratio :

12,4 μL of 5 mg/mL stock HA solution is diluted to 800 μL with HEPES

5 mg/mL x 12.4 μL = 62 μg HA

Mw HA = 402 g/mol
Mw pDNA = 330 μg/μmol

Molar ratio (HA/pDNA) -> (62 µg / 402 µg/µmol) / (2 µg / 330 µg/µmol) = 25.5/1

4.4.3 Agarose gel electrophoresis

4.4.3.1 Principles

Gel electrophoresis is a technique in which a gel matrix and an electric field are used
to separate molecules based on their size and surface charge. DNA molecules will
migrate to the positively charged electrode when forced over a gel by an electric
current due to the negative charge on their phosphate backbone. Larger molecules
will migrate more slowly because they move less easily through the gel network.
After the separation is completed, the DNA fragments can be visualized with
ethidium bromide, a fluorescent dye that intercalates between the heterocyclic bases
in DNA helixes. This intercalation strongly intensifies the signal obtained when the
gel is exposed to UV light. Thus, different DNA fragments in the sample can be
distinguished and their size can be determined by adding a DNA ladder (a sample
containing different DNA molecules of known size) on the gel in parallel with the
unknown sample.

In this study, agarose gel electrophoresis was used as a tool in determining the pDNA
condensation ability of polymers and optimal polymer/pDNA ratio for polyplex
formation. It allows to detect if there is still uncomplexed DNA present in the
samples after complexation. If so, a migration band located toward the positive
electrode will clearly be visible after electrophoresis. Usually, free pDNA is added to
the gel to confirm the band in the polyplex well indeed corresponds to free pDNA.
As this is unwanted, this indicates that a higher polymer ratio is needed to form
stable polyplexes.

47
4.4.3.2 Experimental part

To visualize the pDNA complexation ability of polymer, polyplexes were applied on

agarose gel electrophoresis.

For the preparation of TBE Electrophoresis buffer; 10.8 g/L Tris-base (Sigma,
Bornem, Belgium), 2.5 g/L Boric acid (Merck, Brussels, Belgium) and 0.58 g/L
EDTA (Titriplex®; Merck, Brussels, Belgium) were dissolved in double distilled
water.

A 1% agarose gel was prepared using electrophoresis grade agarose powder

(Invitrogen, Merelbeke, Belgium) and 10x TBE electrophoresis buffer (10 mL buffer
in 100 mL water). The powder was dissolved by heating the suspension to 90°C in
microwave. When the agarose was completely dissolved, the solution was cooled
down till approximately 50°C, then solution was poured into a gel casting tray
containing a sample comb and was allowed to solidify at room temperature. The
solution is left to solidify gently at room temperature, to prevent clumping inside the
agarose network.

In the meantime, the complexes were prepared with the appropriate ratios as
explained in section 4.2.2. Then 20 μL samples (including 0.2 μg pDNA) were taken
from each polyplex solution. The samples were mixed with 5μL loading buffer (50%
sucrose solution in TBE) containing bromophenol blue as colorant. The loading
buffer makes the samples more dense to remain at the bottom of the gel lane and
bromophenol blue present in the loading buffer helps to visualize the migration of the
complexes.

When the gel was formed, the polyplex samples were loaded into gel, and subjected
to electric field at 100 V for 40 minutes in 1x TBE buffer. 1 kbp DNA ladder
(BIORON) was added as a Mw marker and pure pGL4.13 plasmid as a reference
were applied into gel. Heparin, as a molecule with the highest known anionic charge
density was used as a control for DNA displacement. After that, the gel was stained
by incubation on ethidium bromide (EtBr) bath for ±1 hour. Subsequently, the gel
was observed by UV-illumination. At last, the photograph of the gel was recorded by
a camera and processed by the software (Kodak Digital Sicence).

48
4.5 In vitro Biological Evaluation of Polyplexes

The major goal of gene therapy is the introduction of a vector carrying the gene of
interest into cells and to allow these cells to express that specific gene. Besides,
during this transfer process, the carrier should not harm the exposed cells. In this
study, after the polymer/pDNA complexes were investigated for their stability and
DNA condensation ability, it is aimed to evaluate the synthesized polymers by means
of their cellular uptake and gene expression efficiencies as well as the viability
response of the ARPE-19 cells to the introduction of cationic vectors synthesized in
this thesis.

4.5.1 Flow Cytometry

4.5.1.1 Principles

Flow cytometry is a technique that is capable of performing simultaneous

measurements such as counting, examining and analyzing cells in a flowing
suspension depending on the certain chemical and physical properties of cells. With a
flow cytometer, a cell suspension is analyzed cell per cell using a capillary tube
through which the cell suspension passes along a laser beam that is perpendicular to
the capillary tube (See Figure 4.11). The instrument can measure up to 3000 cells
passing in the capillary tube per second. The laser light is scattered in the forward
(forward scatter, FS) and sideward scatter (SS) direction, and by using a combination
of dichroic mirrors and filters, fluorescence emission light is sent to individual
detectors and in different spectral channels. The detectors convert the optical signal
to an electrical signal. The scatter signal is used to derive information on cell size
and granularity, while fluorescence is used to obtain additional information, such as
uptake and transfection efficiency of the non-viral particles which are being
evaluated.

The equipment contains 3 subsystems: fluidics, optics and detectors. In the fluidics-
subsystem, a stream of cells is introduced into the flow and prepared for single cell
measurement. This is done by hydrodynamic focusing: pressure causes the sheath
buffer to be driven through a flow cell. The sample stream, entering the system
between two sheath buffer streams, is compressed by these surrounding streams,

49
which causes the arrangement of the cells in the sample like pearls on a string. This
way, the cells are passed through the flow cell one at a time.

Figure 4.11 : The scattering of laser in flow cytometer [Url-8].

4.5.2 Uptake and transfection efficiency studies

For uptake and transfection efficiency studies cells were seeded as follows: after the
trypsinization of the ARPE-19 cells, the cells were counted with Burker Counting
Chamber and diluted to a concentration of 10x104 cells per mL. Then cells were
seeded into 12-well plates with a confluence of 10x104 cells per well (2,5x104 cells
per cm2, 1 mL of diluted cell suspension). Next, they were allowed to attach on the
plate overnight. The day after, CCM was removed from the wells and freshly
prepared polymer/pDNA complexes in OptiMEM were added to the cells, with a
pDNA concentration of 2 μg per well (for polyplex preparation see section 4.4).
During the uptake experiments, YOYO-1 labelled pGL4.13 plasmids were used for
the preparation of the polyplexes. One set of the cells with the same polyplexes were
put at 4⁰C as it is known that 4°C almost completely inhibits cellular uptake. After 2
h incubation, the extracellular fluorescence of the cells, caused by the fluorescent
polyplexes attached to the cell surface but not taken up, was quenched with trypan
blue (0,20%) for 5 minutes at RT after which the cells were washed with PBS.
Subsequently, they were detached by trypsinization. When they were loosened
enough, cell pellets were collected by centrifugation for 7 min at 300g. Prior to the

50
measurements, the cells were resuspended in flow buffer (PBS with 0,1% azide, 1%
BSA).

For transfection experiments, the gWiz™-GFP plasmid was used which expresses
the green fluorescent protein (GFP) and enables the quantification of a green
fluorescent signal in ARPE-19 cells. Non-treated cells and the cells which were
transfected with non-fluorescent pGL4.13 plasmid were negative controls for
transfection experiments. 2 hours after the addition of polyplexes, the complexes
were removed and cells were incubated 22 h more in full CCM. After 22 h, the cells
were washed, trypsinized and centrifuged. Appropriate gating was applied by means
of the scatterplot of untreated cells to select for intact cells. Then the cells were
resuspended in flow buffer and the average GFP expression of the total gated
population of cells and the amount of GFP-positive cells in the same gate were
subsequently measured by FACS Calibur flow cytometer (Beckton Dickinson,
Erembodegem, Belgium). The analyses were done by using Cellquest software
(Beckton Dickinson, Erembodegem, Belgium).

4.5.3 Cell viability

In order to analyze the cytotoxicity related to the application of different polymers

with different Mw’s and mass ratios, viability measurements were performed. Cell
viability measurements determine healthy cells in a sample. This can be
accomplished either by directly counting the number of living cells or by measuring
an indicator related to the amount of living cells in cell populations. An increase in
cell viability shows cell proliferation, while a decrease in viability may be the result
of either toxic effects of the particular polymer or inappropriate culture conditions
(Roche, 2003).take and transfection efficiency studies.

4.5.3.1 MTT assay

Most viability assays are based on one of two characteristic parameters, namely
metabolic activity or cell membrane integrity of healthy cells. In this study, the
metabolic activity was measured in cell populations by incubating the cells with a
product, called MTT reagent. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) reagent is a yellow colored tetrazolium salt, that is
reduced into a purple colored product, which is formazan, by metabolically active
cells.

51
 Figure 4.12 : Reduction of MTT reagent to formazan.

This colorimetric test also gives the possibility to see the color change and viability
of cells visually. More dense purple color indicates more MTT reagents that is
reduced to formazan which means more living cells. Finally, the analyses were
quantified by measurement of the absorbance by Micro Plate Reader.

4.5.3.2 Experimental

The cells were seeded into 96-well plates with a confluence of 1,0 x 104 cells/well
(0,25x104 cells per cm2) and allowed to adhere overnight. The next day, freshly
prepared polyplexes, including 0,2 µg of plasmid/well, were added to the cells. The
concentration of pDNA was proportional to the amount of cells, thus the same
concentration was used as uptake and transfection experiments. Then, the same
procedure as transfection studies was applied. 24 h after the addition of polyplexes,
the old CCM was removed with fresh CCM and 10 µL MTT labeling reagent was
added per well. After 4 h incubation, 100 µL (x10 of first reagent) of solubilizing
reagent was added incubated overnight. As a negative control, some wells were only
treated with MTT reagents. Next, the cell viability was analyzed by measuring the
absorbance at 590 nm with a reference at 690 nm by a Micro Plate Reader.

52
5. RESULTS AND DISCUSSIONS

5.1 Polymer Synthesis

5.1.1 Characterization of polymers

5.1.1.1 pDMAEMA

Characterization of pDMAEMA polymer was performed at the end of purification

and lyophilization steps. The 1H-NMR (300 MHz) spectrum of pDMAEMA was
recorded in CDCl3 solvent after the radical polymerization with AIBN initiator. The
measurement was applied relative to tetramethylsilane (TMS) at 25⁰C.

The absence of sharp monomer peaks at 5.5 ppm and 6 ppm (See Figure 5.1) which
represent the double bonds verifies the purity of the polymer. As these peaks are not
observed in the spectrum of synthesized pDMAEMA (See Figure 5.2), it can be
concluded that complete conversion of DMAEMA monomer to pDMAEMA
polymer took place or that unreacted monomers were removed during the
purification process.

5.1.1.2 SS-PAAs

Three different types of linear, disulfide linkage containing PAAs were synthesized
via Michael type polyadditon of corresponding primary amine monomers to N, N’-
cystaminebisacrylamide (CBA). The addition polymerization occurs in a stepwise
process thus in order to obtain a polymer with highest theoretical Mw equal
monomer ratios were used for the synthesis. Any possible toxic acrylamide end
group residues that were not polymerized during the reaction were consumed by
adding excess amine monomer to the reaction media in the final stage. The obtained
polymers are easily soluble in water, alcohols and dimethyl sulfoxide, but not in
chloroform or ether (Lin and Engbersen, 2009). The 1H-NMR spectrum of the HCl
salts of the synthesized SS-PAAs in D2O were recorded on Varian Inova
spectrometer operating at 300 MHz.

53
 a bb b
a

b a

Figure 5.1 : Illustration of 1H-NMR spectrum of DMAEMA monomer.

54
Figure 5.2 : The 1H-NMR spectrum relative to TMS (CDCl3, 300 MHz and expressed in ppm) of pDMAEMA. The spectrum was recorded after
radicalic polymerization of DMAEMA monomer with AIBN initiator. The corresponding H-atoms are shown on the polymer
molecule structure.

55
As the double bond proton signals of acrylamide monomer at 5 and 7 ppm were not
observed in 1H-NMR spectra of final products, the purity of synthesized polymers
was verified (See Figure 5.3 and 5.4).

Figure 5.3 : The 1H-NMR spectrum relative to D2O (D2O, 300 MHz and expressed
in ppm) of p(CBA-ABOL).

Figure 5.4: The 1H-NMR spectrum relative to D2O (D2O, 300 MHz and expressed in
ppm) of p(CBA-HIS).

Gel Permeation Chromatography (GPC) measurements were performed using a

Waters 2695 LC system and two thermostated (30°C) PL aquagel-OH 30 columns (8

56
μm, 300×7.5mm, with a low-molar-mass separation range (200 ~ 40,000)) as
previously described by Lin et. al. (2007a). Data were collected using a differential
refractometer (Model 2414). 0.3 M NaAc aqueous solution (pH 4.4) plus methanol
(70/30, v/v) was used as eluent at a flow rate of 0.5 mL/min. The GPC
chromatograms (GPC) showed that the weight-average molecular weight (Mw) of
p(CBA-ABOL) was 5.24 kDa and p(CBA-HIS) 7.48 kDa, with polydispersity
indexes (pDI) 1.29 and 1.66, respectively (See Figure 5.5 and 5.6).

p(CBA-ABOL)

Figure 5.5: Illustration of the Mw distribution of p(CBA-ABOL) obtained by gel

permeation chromatography.

p(CBA-HIS)

Figure 5.6: Illustration of the Mw distribution of p(CBA-HIS) obtained by gel

permeation chromatography.

Additionally the buffering capacity and polymer degradation studies were previously
performed by Lin et. al. (2007a). The buffer capacity of the SS-PAA polymers,
defined as the percentage of amine groups that becomes protonated when pH
decreases from 7.4 to 5.1 (i.e., the pH change from the extracellular environment to

57
the lower pH of the endosomes), was measured by acid-base titration. A known
amount of polymer which was isolated as its HCl-salt form was dissolved and titrated
by 0.1 M NaCl. p(CBA-ABOL) that is a polymer lacking a proton-acceptor side
group shows good buffer capacity of 72% whereas, p(CBA-HIS) with imidazole side
chain has a buffer capacity of 58%. These values are reported to be significantly
higher than that of PEI (24%). The hydrolytic and reductive degradation profile of
SS-PAAs was analyzed by GPC in the absence and presence of reduction agent
dithiothreitol (DTT, 2.5 mM), mimicking the physiological and intracellular
environment, respectively. The analyses indicated that SS-PAAs were reduced very
slowly in the absence of DTT however, in the reductive environment with DTT a
rapid degradation of polymers within 5 min. was reported.

5.2 pDMAEMA Polyplexes

Since naked DNA is not capable of passing the cellular membrane efficiently, a
carrier system is needed for gene delivery purposes (Behr, 1993). Cationic polymers
were widely investigated as safe carrier systems. As a promising candidate of
cationic polymer vectors, pDMAEMA was evaluated in terms of its physicochemical
characteristics and biological efficiency on ARPE-19 cells.

5.2.1 Physicochemical characterization

Stability in the extracellular environment is the first necessary condition for an

efficient vector system. The size and surface charge of pDMAEMA/pDNA
complexes were characterized by DLS. The influence of Mw and different
polymer/pDNA (w/w) ratios on the polyplex stability was investigated this way.
Moreover, agarose gel electrophoresis was applied to analyze the pDNA
condensation ability of pDMAEMA.

5.2.1.1 Particle size and surface charge measurements

Prior to the in vitro evaluation of the pDMAEMA polymer, the complex stability
characteristics such as particle size, pDI and surface charge of pDMAEMA/pDNA
complexes were analyzed. The amounts of polymer and plasmid that are used to
prepare the polyplexes are given in Table 5.1.

58
It can be seen from Table 5.2 that the average particle size of all polyplexes are in the
range 100-150 nm. The increase in the Mw does not have an indicative effect on
charge and pDI of the polyplexes (See Figure 5.7). However, as the polymer/pDNA
ratio increases particles get smaller. It is observed that in OptiMEM, polyplexes with
higher polymer/pDNA ratio are more densely condensed than the polyplexes with a
lower amount of polymer.

Table 5.1 : The amounts of pDMAEMA polymer and pDNA which was used to
obtained desired polymer/pDNA (w/w) ratios.

Polymer Mw mass ratio mpolymer (μg) mpDNA (μg)

pDMAEMA

1.5/1 15 10
50 kDa
12/1 120 10
1.5/1 15 10
600 kDa
12/1 120 10

Table 5.2 : Illustration of average size, pDI and charge values for pDMAEMA
polyplexes.
pDMAEMA pplx HEPES OptiMEM
Ζ-potential
Mw mass ratio size (nm) pDI (mV) size (nm) pDI
1.5/1 140,9 0,35 30,4 323,9 0,21
50 kDa
12/1 103,3 0,32 31,8 104,1 0,2
1.5/1 146,4 0,16 24,9 432,5 0,19
600 kDa
12/1 110,8 0,37 31,2 120,8 0,22

5.2.1.2 pDNA complexation efficiency

The influence of both Mw and mass ratio of pDMAEMA on pDNA condensation

and polyplex formation ability was analyzed by agarose gel electrophoresis, the
results of which are presented in Figure 5.8. Lane number 3,4,5 and 6 indicate the
different polyplexes prepared in HEPES buffer (25 mM, pH 7.2). The following four
lanes, numbers 7,8,9 and 10, are loaded with the same polyplexes which are diluted
in OptiMEM after polyplex formation. Finally, the last four lanes show the
polyplexes prepared in HEPES buffer, to which 20 μL heparin solution (7 μg/μL)
was added. The Et-Br signals are localized in the wells, indicating that all the
complexes condense the pDNA efficiently. Surprisingly, pDNA displacement by
heparin did not yield a similar signal as observed in lane 2, yet it was still different
from the normal complexed pDNA in the former 8 lanes.

59
 a. 600
Size and pDI of pDMAEMA pplxes 1,0
0,9
500
0,8
Z-average diameter (nm)

0,7
400
0,6

pDI
300 0,5
0,4
200
0,3
0,2
100
0,1
0 0,0
1.5/1 12/1 1.5/1 12/1 1.5/1 12/1 1.5/1 12/1 size HEPES
size OptiMEM
50 kDa 600 kDa 50 kDa 600 kDa
pDI HEPES
pDI OptiMEM

b. Zeta potential in HEPES

40
35
zeta potential (mV)

30
25
20
15
zeta
10
5
0
1.5/1 12/1 1.5/1 12/1

50 kDa 600 kDa

The charts give the output of DLS analyses. The results are average of 3
measurements and shown with standard deviations. For size and pDI measurements
complexes were prepared both in HEPES buffer solution (25 mM, pH 7.2) and
OptiMEM serum free transfection medium. Since OptiMEM media is rich in different
types of proteins and dissolved ions, charge measurements were only carried out with
the polyplexes that are prepared in HEPES. In Figure a., the primary y-axis shows the
Z-averaged size in nm and secondary y-axis shows the corresponding pDI. The x-axis
indicates the polymer type, the polymer/pDNa (w/w) ratio and Mw of the polymer
used. It can be seen in figure a. only influence of polymer ratio is that a lower ratio
gives rise to large particles in OptiMEM, possibly aggregates. Figure b. shows the zeta
potential measurements in HEPES media, which appear more or less constant around
+30 mV for all ratios and MW’s.

Figure 5.7 : Illustration of a. particle size and polydispersity, b. ζ-potential of

pDMAEMA polyplexes with different Mw and mass ratios.

60
 1. Ladder (1kb)
2. Pure pGL4.13 plasmid
3. 50 kDa 1.5/1 in HEPES
4. 50 kDa 12/1 in HEPES
1 2 3 4 5 6 7 8 9 10 11 12 13 14 5. 600 kDa 1.5/1 in HEPES
6. 600 kDa 12/1 in HEPES
7. 50 kDa 1.5/1 in OptiMEM
8. 50 kDa 12 in OptiMEM
9. 600 kDa 1.5 in OptiMEM
10. 600 kDa 12 in OptiMEM
11. 50 kDa 1.5 + 20 µL Heparin
12. 50 kDa 12 + 20 µL Heparin
13. 600 kDa 1.5 + 20 µL Heparin
. 14. 600 kDa 12 + 20 µL Heparin

Figure 5.8 : Agarose gel electrophoresis of pDMAEMA polyplexes Mw 50 kDa and

600 kDa with different mass ratios. The lanes with the polyplexes
contain 20 µL of sample (corresponding to 200 ng of pDNA per well).
As a negative control, 20 µL of heparin (7 μg/μL) was added to last 4
samples to simulate pDNA displacement.

5.2.2 In vitro biological evaluation

The ability of the polymer to form stable complexes and condense the pDNA
efficiently is the first key for an efficient cellular uptake and transfection. Following
the complex characterization measurements, in vitro biological evaluations of the
polyplexes both with different Mw and polymer mass ratios, in terms of uptake and
transfection efficiency and cell viability were investigated.

5.2.2.1 Uptake efficiency

The uptake efficiency of stable pDMAEMA/pDNA complexes was analyzed by flow

cytometry. The negative control samples of both untreated cells and the cells at 4⁰C
did not show any cellular uptake more than the autofluoresence of the cells, as
expected. Because as it is known, at 4⁰C the metabolic activity of cells slows down
as well as the uptake. Figure 5.9 shows the uptake efficiency results of pDMAEMA
polyplexes based on two different Mw and mass ratios with their negative controls. It
can be seen from figure that the percentage of positive cells is higher for the cells
treated with 12/1 polyplexes, indicating a more efficient uptake. Moreover, the effect
of Mw is clear for the complexes with 1.5/1 mass ratio. The increment in the Mw for
the same polymer/pDNA ratio shows 3 times increase for the uptake efficiency.

61
 Uptake efficiency of ARPE-19 cells
100 80
90
% Uptake positive cells

Mean cellular uptake

70
80
60
70
60 50
50 40
40 30
30
20
20
10 10
0 0
cells 1.5/1 12/1 1.5/1 12/1 1.5/1 12/1 1.5/1 12/1

50 kDa 600 kDa 50 kDa 600 kDa % pos

Mean

p(DMAEMA) polyplexes with fluorescently labeled pGL4.13 plasmid (2 μg pDNA per

well) were added to the cells the next day of the seeding. After 2 hours, extracellular
fluorescence, which is emitted by the polyplexes that are attached to the cell, but not
taken up, was quenched with 0.2 % trypan blue solution. As a negative control for uptake,
one set of the same polyplexes were put at 4⁰C. Flow cytometry was used to analyze the
uptake efficiency of ARPE-19 cells by the means of the mean YOYO-1 fluorescence
(green bars) and blue bars represent the % uptake positive cells. The data are shown with
standard deviation of three independent samples.

Figure 5.9 : Comparison of cellular internalization of p(DMAEMA) polyplexes

with different Mw and mass ratios.

5.2.2.2 Transfection efficiency

The influence of pDMAEMA Mw and polymer mass ratio on transfection efficiency

of ARPE-19 cells was determined by flow cytometry. The samples were analyzed 24
h after the addition of polyplexes. % positive GFP expression of polyplex samples
and their controls is shown in Figure 5.10. Similar to uptake negative control, no
transfection was observed for cells and the negative control for transfection. The
positive control, LF transfected nearly 50% of the cells. The pDMAEMA complexes
with lower polymer amount almost did not transfect the cells. On the other hand, the
polyplexes with 12/1 ratio showed very low transfection efficiency.

5.2.2.3 Cell viability

The cytotoxic effect of pDMAEMA polyplexes on ARPE-19 cell viability was

analyzed with MTT assay by means of absorbance measurement of formazan dye
that comes from the reduced MTT by metabolically active cells.

62
 Transfection efficiency of ARPE-19 cells
100 3000

Mean GFP expression

90
2500
% GFP positive cells
80
70 2000
60
50 1500
40
30 1000
20 500
10
0 0
cells LF 1.5/1 12/1 1.5/1 12/1 LF 1.5/1 12/1 1.5/1 12/1
% pos
pDMAEMA pDMAEMA pDMAEMA pDMAEMA Mean
50 kDa 600 kDa 50 kDa 600 kDa

Data shown here represents average values of 3 independent measurements, where the
errors bars denote the standard deviation between these measurements. The next day of
the seeding, cells were transfected with polyplexes that are prepared with gWiz™-GFP
plasmid which encodes a gene that translates into green fluorescence protein (GFP)
(containing 2 μg pDNA per well). Hence, this protein emits green fluorescence under the
right excitation light. The gating, which is the selection of the appropriate cells based on
their size and granularity, was applied by adjusting the FSC and SSC. After 24 h the
amount of GFP-positive cells (purple bars) and the mean average GFP expression of
the total gated population of cells (pink bars) were measured by flow cytometry. Non-
treated cells and the cells which were transfected with non-fluorescent pGL4.13 plasmid
were negative controls for transfection experiments.

Figure 5.10 : Comparison of transfection efficiency of pDMAEMA polyplexes with

different Mw and mass ratios and LF as a positive control.

In Figure 5.11, it can be seen that adding more polymer to the cells resulted in a
significant decrease of metabolic cell activity, which can indicate higher cytotoxicity.
The effect of polymer Mw on toxicity of polyplexes is less clear in case of 1.5/1 ratio
as both Mws show relatively moderate viability of cells.

5.2.3 Discussion

In this part of the study, pDMAEMA homopolymers were synthesized and evaluated
as a candidate to be a gene delivery vector. Since the stability in the extracellular
environment is the primary condition for an efficient carrier, the size and surface
charge of pDMAEMA/pDNA complexes were characterized by DLS in both HEPES
buffer and OptiMEM, the latter being representative for physiological conditions.

63
 The results are shown with standard deviations of 5 independent samples. The cells
were seeded on transparent 96-well plates at 1x104 cells per well. The wells were
treated in the same way with transfection studies but with a pDNA concentration of 0,2
µg per well (see section 4.5). As a negative control, the cells only with MTT reagents
were applied. LF/pDNA complexes were prepared as positive control. After 3 days, the
absorbance was measured by Microplate Reader. The influence of higher polymer
ratios on cell viability can be significantly observed. (°) normalized against cells which
were not incubated with polyplexes. (*) indicates significant differences with p< 0.05
in comparison to LF (ANOVA).

Figure 5.11 : Cell viability results of pDMAEMA polyplexes with different Mw

and polymer ratios.

The difference in Mw for the same polymer/pDNA (w/w) ratios did not show a
significant effect on the polyplex stability (See Figure 5.7). This was not surprising
because the charge density per weight would be the same both for 50 kDa and 600
kDa polymer. Hence the amount of positive charges of polymer that will bind the
negative charge of pDNA and form complexes does not change in both cases.
However, the increase in polymer/pDNA ratio resulted in smaller particles that could
be because more positive charges lead to stronger complexation. For the complexes
with lower polymer mass ratio in OptiMEM, the interactions between proteins and
polyplexes may cause an increase in particle size, resulting in more stable complexes
than the ones in HEPES buffer. Secondly, agarose gel electrophoresis was applied to
analyze the pDNA condensation ability of polyplexes as shown in Figure 5.8. It was
reported by Arscott et al. that DNA condensation occurs when 90% of the anionic
charges of pDNA is condensed by the positive charges of the polymer (1990). All the
samples showed a great ability to form stable complexes and condense the pDNA

64
efficiently, even the 1.5/1 polyplexes with lowest polymer/pDNA mass ratio.
Heparin was added to the complexes as a negative control to simulate the pDNA
displacement. The effect of heparin did not displace the pDNA completely as
expected, which could be due to the plasmid still being bound to smaller fractions of
polymer. Nevertheless, even if the complete pDNA displacement effect of heparin
cannot observed, it should be emphasized that when it is compared with the polyplex
samples that were not treated with heparin, no pDNA displacement was seen for
them.

The physicochemical characteristics of pDMAEMA polyplexes were verified with

the satisfying results of stable complexes and efficiency for pDNA complexation.
Afterwards, the cellular processing of the pDMAEMA/pDNA complexes on ARPE-
19 cells was performed. In terms of biological evaluation of pDMAEMA polymers
the uptake and transfection efficiency of polyplexes were investigated by flow
cytometry and then cell viability was analyzed by an MTT assay. The MTT assay
measures metabolic activity by the increase of absorbance by formazan crystals that
are reduced. pDMAEMA/pDNA complexes with higher Mw and higher
polymer/pDNA ratio showed promising results for cellular uptake (See Figure 5.9).
They were taken up by the cells with a maximum of 70%. The influence of polymer
amount on pDMAEMA uptake efficiency was previously explained by van de
Wetering et al. that the amount of free polymer would increase as the polymer ratio
in the polyplex increases which results in higher membrane destabilization thereby
facilitating the cellular uptake of polyplexes (1997). Moreover, the increment in the
Mw for the same polymer/pDNA ratio showed 3 times increase for the uptake
efficiency. It was also stated that higher Mw polymers form smaller particles because
they can condense the pDNA more strongly. Thus polyplexes can permeate through
the membrane easily and this results relatively increased uptake and number of
transfected cells (van de Wetering et al, 1997). However, the fact that this effect did
not observed for cellular uptake of the polyplexes with 12/1 mass ratio with 600 kDa
polymer could be explained by the maximization of uptake around 80% for
pDMAEMA polyplexes.

Following the cellular entry by means of endosomal vesicles, another key is the
escape from endosomes prior to the transport to the nucleus. The pDMAEMA
homopolymer has tertiary amine groups which are partially protonated at

65
physiological pH and therefore can be further protonated at endosomal pH. As it was
indicated by van de Wetering et.al, pDMAEMA shows an average pKa value of 7.5
where 50% of the amino groups in the polymer chain are protonated (1999). This
answers the requirement for endosomal escape for methacrylate based polymers
according to the proton sponge hypothesis (Dubruel et al, 2003). However, the
transfection efficiency of pDMAEMA was very limited. The polyplexes both with
the ratio of 1.5/1 and 12/1 showed almost no transfection for ARPE-19 cells. This
fact was previously explained by Jones et. al. that although, pDMAEMA disrupts the
endosomes when internalized by the endosomal vesicles, it does not induce the
release of therapeutic material into the cytosol (2004). This could be because of the
strong DNA binding ability of the polymer that does not allow the plasmid to be
released once it reaches the cytosol or there may be another factor that limits the
disassembly of the complexes to be released in the cytosol.

Subsequently, when the cell viability was evaluated by MTT assay, it was also
observed that higher polymer ratio gave rise to higher cytotoxic effect on ARPE-19
cells in vitro. Since many cationic polymers, including pDMAEMA, were reported to
have higher cytotoxicity (Layman et al, 2009), the investigation of an alternative
delivery system with higher transfection efficiency and less cytotoxic effects was
necessary.

5.3 SS-PAA Polyplexes

Cationic polymers have been attractive research materials as non-viral gene delivery
vectors due to their lack of specific immune response, large scale DNA loading
capacity and ease of production (Pack et al, 2005). Besides numerous types of
cationic polymers which are investigated as gene carriers, poly(amidoamine)s
(PAAs) are a unique family of cationic polymer therapeutics. They are reported to
have a good solubility in water, be hydrolytically more stable and biodegradable
(Ferruti et al, 2002). Recently, a new series of linear PAAs containing repetitive
disulfide linkages in their main chain has been developed by the Engbersen Lab. (Lin
et. al, 2007a). The disulfide linkage present in SS-PAAs, which are relatively stable
in extracellular environment, provides bioreducibility in the intracellular media.

66
In this project, physicochemical and biological properties of SS-PAAs with different
polymer/pDNA (w/w) ratios, variable coating strategies and buffering capacity were
evaluated.

5.3.1 p(CBA-ABOL) polyplexes

p(CBA-ABOL) is one of the examples of SS-PAAs as a potential gene delivery

vector. It was obtained by Michael type polyaddition of 4-amino-1-butanol (ABOL)
to N,N’-cystaminebisacrylamide (CBA). The ABOL-side group provides the positive
charge and buffering capacity, while the repetitive disulfide linkage present in its
main chain makes the polymer bioreducible and a good candidate for gene delivery
applications.

5.3.1.1 Physicochemical characterization

Particle size and surface charge measurements

The particle size and surface charge measurements of p(CBA-ABOL) polyplexes

with various polymer/pDNA mass ratios were determined by DLS, in order to have
an idea about the stability range of the p(CBA-ABOL) polyplexes. The amount of
polymer and pDNA which was used while preparing the complexes is illustrated on
Table 5.3 and the measurement values can be found on Table 5.4. As it is shown in
Figure 5.12 a, the complexes in HEPES indicates a narrow size range between 120-
150 nm with a stable pDI value. Polyplexes with the ratio of 96/1 has the most
positive surface charge with +45.53 mV. However, in OptiMEM media the
increasing amount of polymer resulted in very large aggregates.

Table 5.3: The amounts of p(CBA-ABOL) polymer and pDNA which was used to
obtain desired polymer/pDNA (w/w) ratios. The polyplexes were
prepared with increasing amount of polymer while the amount of plasmid
was the same for each.

mass ratio m polymer (μg) m pDNA (μg)

12/1 120 10
24/1 240 10
48/1 480 10
96/1 960 10

67
 Table 5.4 : Illustration of size, pDI and charge values for p(CBA-ABOL) pplxes
with different polymer/pDNA mass ratios.

p(CBA-ABOL) HEPES OptiMEM

mass ratio Z-potential
size (nm) pDI (mV) size (nm) pDI
12/1 147,93 0,1 27,57 449,70 0,14
24/1 134,27 0,11 29,83 511,00 0,20
48/1 122,47 0,11 43,1 744,00 0,37
96/1 132,17 0,07 45,53 1335,00 0,62

pDNA complexation efficiency

The relation between the amount polymer and DNA condensation ability while
forming stable complexes were analyzed by agarose gel electrophoresis. Polyplexes
which were prepared in HEPES buffer with increasing mass ratio of p(CBA-ABOL)
were loaded into lanes number 3,4,5 and 6, respectively (See Figure 5.13). It can be
seen from the figure that the free DNA bands become less clear above a
polymer/pDNA mass ratio of 12/1. This indicates that increasing polymer ratios form
complexes with increased pDNA complexation efficiency. The following four lanes,
number 7,8,9 and 10, were loaded with the same polyplexes which were diluted in
OptiMEM after polyplex formation in HEPES buffer. The bands of the pDNA that
migrated from the polyplexes in OptiMEM become more visible which corresponds
to more pDNA release.

Figure 5.12 : Illustration of a. particle size and polydispersity, b. ζ-potential of

p(CBA-ABOL) polyplexes with different mass ratios.

68
Figure 5.12 (continued) : Illustration of a. particle size and polydispersity, b. ζ-
potential of p(CBA-ABOL) polyplexes with different mass
ratios.

When it is compared with lane numbers 7,8,9 and 10, it can be concluded that the
release in OptiMEM is not negligible but there is still some plasmid left in the wells.
The pDNA binding ability of the polyplexes with 48/1 ratio supports the stable
results which were obtained from DLS, as well.

1. Ladder (1 kb)
1 2 3 4 5 6 7 8 9 10 11
2. Pure pGL4.13 plasmid
3. Pplx 12/1 in HEPES
4. Pplx 24/1 in HEPES
5. Pplx 48/1 in HEPES
6. Pplx 96/1 in HEPES
7. Pplx 12/1 in OptiMEM
8. Pplx 24/1 in OptiMEM
9. Pplx 48/1 in OptiMEM
10. Pplx 96/1 in OptiMEM
11. Pplx 48/1 + 10 µl Heparin

Figure 5.13 : Agarose gel electrophoresis of p(CBA-ABOL) pplxes with different

mass ratios. Each sample was colored with 5 µL loading buffer. The
polyplex samples contain 0.2 µg of pDNA. As a negative control, 10
µL of Heparin (7 μg/μL) was added to last sample.

69
5.3.1.2 In vitro biological evaluation

After the physicochemical characterization of p(CBA-ABOL) polyplexes, the effect

of polymer/pDNA ratio on biological evaluation of the polyplexes in terms of uptake
and transfection efficiency and cell viability was analyzed.

Uptake efficiency

In order to determine effect of mass ratio on the uptake efficiency of p(CBA-ABOL)

polyplexes, ARPE-19 cells were incubated with polyplexes and analyzed by flow
cytometry (Figure 5.14). It can be seen from the figure that for the samples at 37⁰C,
an increasing amount of polymer results in a higher uptake efficiency. The uptake
efficiency of 96/1 ratio reaches to 90% whereas it results in 10% for 12/1.

Uptake efficiency of ARPE-19 cells

100 250
90

Mean cellular uptake

% Uptake positive cells

80 200
70
60 150
50
40 100
30
20 50
10
0 0
cells 12/1 24/1 48/1 96/1 12/1 24/1 48/1 96/1
%pos
CBA-ABOL CBA-ABOL Mean

The data are shown with standard deviation of three independent samples. p(CBA-
ABOL) polyplexes with fluorescently labeled pGL4.13 plasmid (2 μg pDNA per well)
were added to the cells the next day of the seeding. After 2 hours, extracellular
fluorescence, which is emitted by the polyplexes that are attached to the cell, but not
taken up, was quenched with 0.2 % trypan blue solution.As a negative control for
uptake, one set of the same polyplexes were put at 4⁰C. Flow cytometry was used to
analyze the uptake efficiency of ARPE-19 cells by the means of the mean YOYO-1
fluorescence (green bars) and blue bars represent the % uptake positive cells.

Figure 5.14 : Comparison of cellular internalization of p(CBA-ABOL) polyplexes

with different mass ratios.

70
Transfection efficiency

p(CBA-ABOL) polyplexes with different polymer/pDNA (w/w) ratio were evaluated

in terms of GFP expression on ARPE-19 cells. Figure 5.15 shows the transfection
efficiencies of polyplex samples and their negative controls analyzed by flow
cytometry. The negative control samples which were prepared with non-fluorescent
pGL4.13 plasmid and cells showed no transfection, not more than auto-fluorescence.
The polyplexes with 12/1 ratio indicate very low transfection efficiency while the
complexes with higher polymer ratios show about 30% transfection efficiency. The
GFP expression of 96/1 ratio reaches to 40%. Whereas, the transfection efficiency of
positive control samples with LF that is commercially available and known to have
good transfection, reaches to 30 %.

Transfection efficiency of ARPE-19 cells

100 3000
90

Mean GFP expression

80 2500
% GFP positive cells

70 2000
60
50 1500
40
30 1000
20 500
10
0 0
cells 12/1 24/1 48/1 96/1 12/1 24/1 48/1 96/1 % pos

Mean
LF CBA-ABOL LF CBA-ABOL

The experiments were carried out in triplicate and data are shown with standard
deviation. The day after the seeding, the cells were transfected with p(CBA-ABOL)
polyplexes prepared with gWiz™-GFP plasmid (2 µg pDNA concentration per well).
Then the mean average GFP expression of the total gated population of cells (pink
bars) and the amount of GFP-positive cells (purple bars) were measured by flow
cytometry. Non-treated cells and the cells which were transfected with non-fluorescent
pGL4.13 plasmid were negative controls for transfection experiments.

Figure 5.15 : Comparison of transfection efficiencies of p(CBA-ABOL) polyplexes

with various mass ratios compared to LF.

71
Cell viability

The cytotoxic effects of p(CBA-ABOL) complexes with different polymer mass

ratios (w/w) were analyzed by means of the MTT assay. The cells were incubated
with the same polymer/pDNA ratios as was used for the transfection experiments.
However, the amount of sample which was added to the cells was proportional to the
cell confluence. The cell viability percentage of ARPE-19 cells which were treated
with p(CBA-ABOL) complexes is shown in Figure 5.16. The 48/1 ratio was seen to
have the lowest cytotoxic effect on the cells, equal to that of LF. The increase in the
amount of polymer for 96/1 ratio resulted in more toxicity and less viability for
ARPE-19 cells.

The cells were treated in the same way with transfection studies but with a pDNA
concentration of 0,2 µg per well. As a negative control, only MTT reagents were added
to the cells. LF/pDNA complexes were prepared as positive control. The third day, the
absorbance was measured by Microplate Reader at 590 nm. The results are shown with
standard deviations of 5 independent samples. (°) normalized against cells which were
not incubated with polyplexes. (*) indicates significant differences with p< 0.05 in
comparison to LF (ANOVA).

Figure 5.16 : Cell viability results of p(CBA-ABOL)/pDNA polyplexes with

different mass ratios.

5.3.1.3 Discussion

In the first part of this study pDMAEMA polyplexes were evaluated as potential
gene delivery vectors. Even though pDMAEMA formed stable complexes with

72
pDNA, it could only transfect the cells somewhat at polymer/pDNA ratios which
were toxic to the cells. As SS-PAAs were reported to be efficient p(CBA-ABOL)
was chosen to be evaluated as a promising member of this family. The repetitive
disulfide linkages present in the CBA monomer chain makes the polymer
bioreducible, whereas hydroxybutyl groups in the side chain originating from the
ABOL unit enrich the efficiency in DNA transfection by increasing the
endosomolytic properties of polyplexes (Lin et. al, 2007a).

In this section, prior to biological evaluation of p(CBA-ABOL), the polymer/pDNA

complexes were physicochemically characterized. The ability of the polymer to form
stable complexes with DNA and the influence of polymer/pDNA (w/w) ratios on
colloidal stability of complexes were analyzed by DLS. The size, polydispersity and
surface charge measurements of p(CBA-ABOL) polyplexes showed that the
increase in the amount of polymer resulted in more stable and more positive
complexes in HEPES buffer.. Hence, all polyplexes with 4 different mass ratios
(12/1, 24/1, 48/1 and 96/1) were capable to form nanosized polyelectrolyte
complexes by the means of self assembly in HEPES buffer. The same measurements
were also performed in OptiMEM to simulate physiological conditions. However,
due to the the interactions of proteins and various ions that are present in OptiMEM,
polyplex particles caused very large aggregates as the polymer amount increases.
According to agarose gel electrophoresis results, all p(CBA-ABOL) polyplexes were
able to condense the pDNA but the ones with 48/1 and 96/1 polymer/pDNA ratio
were more efficient in terms of DNA binding (See Figure 5.13). As the amount of
polymer increases corresponding to the same amount of pDNA, when the pDNA is
condensed with the cationic polymer at some point, this could be that free polymer
causes the increment in surface charge and pDNA binding ability.

To further evaluate the effect of polymer/pDNA (w/w) ratio on biological efficiency

of p(CBA-ABOL) polyplexes the same 4 ratios were used for uptake and transfection
efficiency experiments as well as for MTT assay. The uptake efficiency results,
which were determined by flow cytometry, clearly indicated that increasing amount
of polymer resulted in higher percentages of cellular uptake (See Figure 5.14). When
it is compared with pDMAEMA, p(CBA-ABOL) polyplexes have higher uptake that
is probably due to more positive charges present in the polyplexes with higher
amount of polymer. This effect could be facilitated the interactions between anionic

73
cell membrane HSPGs. Especially the ratios 48/1 and 96/1 were observed to be more
efficient in terms of uptake. After the cellular entry via endocytosis, the escape from
endosomes and the release of the pDNA from polymer are the requirements for gene
expression. The p(CBA-ABOL) polyplexes showed highly efficient transfection
relative to LF, which was used as a positive control, by increasing amount of
polymer. If it is compared with previous carrier system pDMAEMA, we assume the
effect of hydrophobic side chain such as ABOL and rapid release of pDNA following
the endosomal escape by the means of disulfide cleavage in the intracellular
reductive environment give rise to higher pDNA transfection and increased level of
gene expression (Lin and Engbersen, 2009). Cell viability of p(CBA-ABOL)
polyplexes with different mass ratios which was analyzed by MTT assay showed that
p(CBA-ABOL)/pDNA complexes possess very low cytotoxicity on ARPE-19 cells
relative to LF especially compared to pDMAEMA. Nevertheless, 96/1 ratio with the
maximum transfection efficiency showed significantly lower viability on ARPE-19
cells.

In conclusion, p(CBA-ABOL) was determined as a good non-viral carrier. As the

pDMAEMA polymer, it was able to form stable polyplexes, yet it also showed high
cellular uptake and transfection efficiency results with very low cytotoxicity. As 96/1
ratio was stated to be relatively cytotoxic, 48/1 ratio polyplexes with similar
physicochemical properties and biological efficiency but higher cell viability were
chosen as a comparative reference for further experiments. Nevertheless, the
tendency of the polyplexes to form large aggregates in physiological conditions
dictate further optimizations for p(CBA-ABOL) to be a better vector.

5.3.2 p(CBA-ABOL/DMEDA/PEG) polyplexes

In order to increase the stability of gene delivery vectors in the extracellular

environment, PEGylation is a universal strategy that is widely applied. This method
is performed by grafting the non-ionic and hydrophilic polyethylene glycol (PEG) to
the gene delivery vector. Since p(CBA-ABOL) polyplexes was promising non-viral
carriers with good uptake and transfection efficiency and favorable cell viability,
PEGylation was applied to increase their colloidal stability. In the following section,
PEGylated particles were physicochemically and biologically evaluated.

74
5.3.2.1 Physicochemical characterization

Particle size and surface charge measurements

p(CBA-ABOL/DMEDA/PEG) complexes were characterized in terms of their

particle size, surface charge and polydispersity by DLS. Complexes were prepared
with the same amounts of polymer and pDNA of p(CBA-ABOL) polyplexes for the
corresponding polymer/pDNA (w/w) ratio. It can be seen from Table 5.5 that
polyplex particles in HEPES get smaller as the amount of polymer increases. On the
other hand, complexes have very slight positive charge even with the highest amount
of polymer for 96/1 ratio. For the measurements in OptiMEM, it is clearly observed
from Figure 5.17 that polyplexes show a great stability with a size range between
120-160 nm.

Table 5.5 : Illustration of average size, pDI and charge values for PEGylated
p(CBA-ABOL) polyplexes with different polymer/pDNA mass ratios.

HEPES OptiMEM
ABOL/DMEDA/PEG)

mass ratio Z-potential

size (nm) pDI (mV) size (nm) pDI
p(CBA-

12/1 134,63 0,41 5,29 160,87 0,52

48/1 114,6 0,34 8,35 121,33 0,40

96/1 108.9 0,32 9,40 152,70 0,49

Figure 5.17 : Illustration of the effect of PEGylation on a. particle size and

polydispersity, b. ζ-potential.

75
Figure 5.17 (continued) : Illustration of the effect of PEGylation on a. particle size
and polydispersity, b. ζ-potential.

pDNA complexation efficiency

Agarose gel electrophoresis was applied to investigate the DNA binding capability of
PEGylated polymers. Lane numbers 3,4 and 5 indicate the polyplexes prepared in
HEPES and number 6,7 and 8 show the complexes diluted in OptiMEM (See Figure
5.18). It can be observed from the band intensities that PEGylated polymers have a
more strong DNA binding ability both in HEPES and OptiMEM. Moreover, the
polymer mass ratio above 48/1 show more stable complexes for PEGylated
polymers.

5.3.2.2 In vitro biological evaluation

Uptake efficiency

The uptake efficiency of PEGylated p(CBA-ABOL) polymer complexes with

different polymer/pDNA (w/w) ratios on ARPE-19 cells was evaluated by flow
cytometry. According to the results of the stability and DNA condensation ability of

76
 1. Ladder (1kb)
1 2 3 4 5 6 7 8 9 10 11
2. Pure pGL4.13 plasmid
3. PEG-pplx 48/1 in HEPES
4. PEG-pplx 72/1 in HEPES
5. PEG-pplx 96/1 in HEPES
6. PEG-pplx 48/1 in OptiMEM
7. PEG-pplx 72/1 in OptiMEM
8. PEG-pplx 96/1 in OptiMEM
9. PEG-pplx 48/1+ 5 µl Heparin
10. PEG-pplx 72/1+ 5 µl Heparin
11. PEG-pplx 96/1+ 5 µl Heparin
Figure 5.18 : Agarose gel electrophoresis for p(CBA-ABOL/DMEDA/PEG)
complexes. The lanes with the polyplexes contain 20 µL of sample
(corresponding to 200 ng of pDNA per well). As a negative control, 5
µL of Heparin (7 μg/μL) was added to last 3 samples to simulate pDNA
displacement.
the polymer, the most capable two ratios, 48/1 and 96/1, were chosen and applied for
the uptake experiment.The non-PEGylated 48/1 p(CBA-ABOL) polyplexes were
also applied as a comparison. Figure 5.19 shows the efficiencies of polyplex samples
and their negative controls. The negative control samples which were put at 4⁰C,
showed no uptake, as expected. As expected, the cells showed positive uptake for
p(CBA-ABOL)/pDNA complexes with 80%. However, it can be seen from Figure
5.19 that PEGylated p(CBA-ABOL) polyplexes were almost not taken up by ARPE-
19 cells.

Transfection efficiency

In order to determine transfection efficiency of PEGylated p(CBA-ABOL)

complexes ARPE-19 cells were treated with polyplexes having different mass ratios
and the transfection efficiency was analyzed by flow cytometry. LF/pDNA
complexes and non-PEGylated p(CBA-ABOL) polyplexes were applied as positive
controls. As it is illustrated in Figure 5.20 that the positive control samples showed a
GFP expression around 25% on the other hand, PEGylated p(CBA-ABOL)
complexes did not transfect.

77
Figure 5.19 : The effect of PEGylation on cellular internalization of p(CBA-ABOL)
polyplexes with different mass ratios in ARPE-19 cells.

Figure 5.20 : Comparison of the effect of PEGylation on transfection efficiency of

p(CBA-ABOL) polyplexes with various mass ratios in ARPE-19 cells.

78
5.3.2.3 Discussion

p(CBA-ABOL) polyplexes were evaluated as the first member of SS-PAA family in

the previous section. p(CBA-ABOL) polymer was able to efficiently transfect the
ARPE-19 cells, however, the complexes formed large aggregates in the extracellular
environment. PEGylation was applied as a strategy to increase the colloidal stability
of p(CBA-ABOL)/pDNA complexes in the extracellular environment. Synthesis of
p(CBA-ABOL/DMEDA/PEG) was performed by Michael addition polymerization.
p(CBA-ABOL/DMEDA) was first synthesized by the same reaction and then
PEGylation was performed by PEG-NH2 quenching. In addition to the bioreducibility
and DNA transfection ability of p(CBA-ABOL), N,N’-dimethylethane-1,2-diamine
(DMEDA) group increases the positive charges present in the polymer chain which
improves complexation and electrostatic interactions between polymer and pDNA
(Vader et al, 2011). The effect of PEGylation on polyplex particle size and surface
charge was characterized by DLS. It was clearly seen that PEGylated polyplexes
form more stable complexes than unPEGylated p(CBA-ABOL) polyplexes in
OptiMEM with a hydrodynamic diameter of 152 nm in 96/1 ratio. The surface
charge measurements indicated less positive complexes with p(CBA-
ABOL/DMEDA/PEG) even with the highest polymer ratio of 96/1 which is because
of PEG molecule that has a neutral surface charge in buffer conditions (Brumbach et
al, 2010). To determine the ability of PEGylated polymer to condense the pDNA,
agarose gel electrophoresis was applied. As it was shown in Figure 5.18, p(CBA-
ABOL/DMEDA/PEG) polyplexes were able to condense DNA in HEPES for all
applied ratios. Additionally, the samples in OptiMEM showed less clear bands that
represent the pDNA than p(CBA-ABOL) polyplexes which means PEGylated
polymers can condense the pDNA more densely with the facilitating effect of
increased polymer/pDNA mass ratio.

In vitro biological evaluation of p(CBA-ABOL/DMEDA/PEG) polyplexes was

analyzed by flow cytometry by the means of uptake and transfection efficiency
which showed that these complexes showed almost no cellular uptake (See Figure
5.19). This fact was explained by Coi (1998) and Oupicky et al. (2002) that
PEGylation reduces the cellular association and uptake of polyplexes due to the
steric shielding of polyplex particles. When the PEGylated particles were
investigated for transfection efficiency on ARPE-19 cells, they did not show any

79
GFP expression where the unPEGylated p(CBA-ABOL) has transfected similar to
positive control lipoplexes. As Lin and Engbersen stated that PEGylated poylplexes
indicated very low transfection efficiency because their neutral surface appeases
efficient cellular association and internalization (2009). Furthermore, the endosomal
escape of polyplexes from acidic endosomes is also diminished by PEGylation
(Knorr et al, 2007). Since the results of biological evaluation of PEGylated particles
address the “PEG dilemma”, PEGylated polyplexes was not investigated in terms of
cellular viability, because particles which are not taken up by the cell will have
minimal influence on cell viability.

To conclude, PEGylation of polyplexes results in reduced interaction with OptiMEM

components giving rise to more stable particles. Nevertheless, this extracellular
stability comes at the cost of an inefficiency for uptake and transfection observations,
possibly due to the shielding of the surface charge, which emphasizes the need for
alternative strategies to increase colloidal stability of p(CBA-ABOL) particles.

5.3.3 HA-coated p(CBA-ABOL) polyplexes

Hyaluronic acid (HA) is a cell surface GAG which is highly abundant in extracellular
matrix (ECM). It is popularly investigated for drug delivery purposes because of
having good biocompatibility and biodegradability (Han et al, 2009). Moreover, it
has been reported to be responsible of cellular internalization and uptake of gene
therapy vectors. In this section, HA-coated p(CBA-ABOL) polyplexes were
investigated in terms of increase in the stability of complexes and in the uptake
efficiency.

5.3.3.1 Physicochemical characterization

Particle size and surface charge measurements

Particle size and surface charge measurements were carried out for a series of HA-
coated p(CBA-ABOL)/pDNA complexes with different HA/pDNA molar ratios. The
characterization was performed by DLS. The coating was applied on p(CBA-ABOL)
polyplex with 48/1 ratio with different amount of research grade HA that is
purchased from Life Core Scientific. As it is shown in Figure 5.21, increasing
amount of HA results in smaller, more negative and stable complexes in HEPES

80
buffer. The effect of increasing amount of HA is less clear for size measurements in
OptiMEM because all the complexes have an average size of 500 nm.

Table 5.6 : Illustration of the amount of HA and pDNA used to apply the coating in
appropriate HA/pDNA molar ratio.

molar ratio V HA m HA m pDNA

(HA/pDNA) (μL) (μg) (μg)
p(CBA-ABOL) 25.5 /1 12.4 62 2
HA-coated

63/1 30.6 153 2

125/1 61.1 305.5 2
190/1 92 460 2
250/1 122.2 611 2
313/1 153 765 2
377/1 184 920 2
505.5/1 244.4 1222 2

Figure 5.21 : Illustration of the effect of HA-coating on a. particle size and

polydispersity, b. ζ-potential of p(CBA-ABOL) polyplexes with
different HA/pDNA molar ratios.

81
Figure 5.21 (continued) : Illustration of the effect of HA-coating on a. particle size
and polydispersity, b. ζ-potential of p(CBA-ABOL)
polyplexes with different HA/pDNA molar ratios.

pDNA complexation efficiency

The influence of HA-coating on the DNA condensation behavior of p(CBA-ABOL)

complexes was observed by agarose gel electrophoresis. Based on DLS results, the
most stable complexes were chosen and applied into gel. As a reference, non-coated
p(CBA-ABOL) complexes with 48/1 ratio was also loaded in gel. Polyplexes which
were prepared in HEPES buffer with increasing HA/pDNA ratio were loaded into
lanes numbers 4,5 and 6 (See Figure 5.22). In terms of pDNA band intensity the
complexes in HEPES, looks more or less have the same stability with non-coated
polyplexes. Nevertheless, the slight bands of the lane numbers 8,9 and 10 that are
loaded with HA-coated complexes in OptiMEM, clearify the effect of HA-coating on
the stability of complexes.

82
 1. Ladder (1kb)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 2. Pure pGL4.13 plasmid
3. Pplx CBA-ABOL 48/1 in HEPES
4. HA-coated 190/1 in HEPES
5. HA-coated 313/1 in HEPES
6. HA-coated 501,5/1 in HEPES
7. Pplx CBA-ABOL 48/1 in OptiMEM
8. HA-coated 190/1 in OptiMEM
9. HA-coated 313/ in OptiMEM
10. HA-coated 501,5/1 in OptiMEM
11. Pplx CBA-ABOL 48/1 + 5 µL Heparin
12. HA-coated 190/1 + 5 µL Heparin
13. HA-coated 313/1 + 5 µL Heparin
14. HA-coated 501,5/1 + 5 µL Heparin

Figure 5.22 : Agarose gel electrophoresis of HA-coated p(CBA-ABOL) polyplexes

with different HA/pDNA mass ratios. The lanes with the polyplexes
contain 20 µL of sample (corresponding to 200 ng of pDNA per well).
As a negative control, 5 µL of Heparin (7 μg/μL) was added to last 4
samples to simulate pDNA displacement.

5.3.3.2 In vitro biological evaluation

Uptake efficiency

The influence of HA-coating on the uptake efficiency of stable p(CBA-ABOL)

complexes was analyzed by flow cytometry. A series of HA/pDNA ratios were
applied for physicochemical characterization of HA-coated p(CBA-ABOL)
polyplexes but only the more stable ones were chosen for the uptake experiment.
Non-coated p(CBA-ABOL) complexes were also applied in order to compare the
effect of HA-coating. Figure 5.23 shows the uptake efficiency results of HA-coated
p(CBA-ABOL) polyplexes based on three different HA/pDNA molar ratios with
their controls. The positive control samples with non-coated p(CBA-ABOL)
polyplexes show 80% cellular uptake. The desired effect of HA-coating and
increasing HA/pDNA ratio was not observed in terms of uptake efficiency because
all the ratios show similar efficiency around 10% of uptake positive cells.

Transfection efficiency

The transfection efficiency of HA-coated p(CBA-ABOL) polyplexes with different

HA/pDNA molar ratios was determined by flow cytometry. Polyplexes with non-
coated p(CBA-ABOL) and LF/pDNA complexes were prepared as positive controls.
The samples were analyzed 24h after the addition of the complexes. % positive GFP
expression of HA-coated p(CBA-ABOL) polyplex samples and their controls is

83
shown in Figure 5.24. The positive control LF transfected nearly 30% of the plasmid
while p(CBA-ABOL) had 15% GFP expression. The HA-coated polyplexes showed
very low transfection, even with the highest HA/pDNA ratio for 400/1.

Figure 5.23 : The effect of HA-coating on cellular internalization of p(CBA-ABOL)

polyplexes with different HA/pDNA molar ratios in ARPE-19 cells.

Figure 5.24 : Comparison of transfection efficiencies of different gene carriers HA-

coated p(CBA-ABOL) with different HA/pDNA molar ratios, non-
coated p(CBA-ABOL) and LF complexes.

84
Figure 5.24 (continued) : Comparison of transfection efficiencies of different gene
carriers HA-coated p(CBA-ABOL) with different
HA/pDNA molar ratios, non-coated p(CBA-ABOL) and
LF complexes.

Cell viability

The effect of HA-coating for p(CBA-ABOL) polyplexes on ARPE-19 cell viability

was analyzed with MTT Assay and the absorbance was measured by Microplate
Reader. LF/pDNA and non-coated p(CBA-ABOL)/pDNA complexes were applied
as positive controls. It is clearly seen on Figure 5.25 that HA-coated complexes
indicate higher cell viability when than the positive controls. This may because of
they are taken up with a lower efficiency with comparison to the control samples.

The HA-coated p(CBA-ABOL) polyplexes with different HA/pDNA molar ratios were
added to the cells the day after the cell seeding. The cells were treated in the same way with
transfection studies but with a pDNA concentration of 0,2 µg per well. As a negative
control, the cells only with MTT reagents were applied. LF/pDNA complexes were applied
as positive control and p(CBA-ABOL) polyplexes as comparative control. The third day,
the absorbance of cells was measured by Microplate Reader at 590 nm. No significant
difference was observed according to statistical tests. The results are shown with standard
deviations of 5 independent samples. (°) normalized against cells which were not incubated
with polyplexes. (*) indicates significant differences with p< 0.05 in comparison to LF
(ANOVA).

Figure 5.25 : Effect of HA-coating on the viability of ARPE-19 cells.

85
5.3.3.3 Discussion

In the previous sections, pDMAEMA polyplexes were reported to be stable but had a
very low transfection efficiency. Then as an alternative vector system p(CBA-
ABOL) polymer was chosen to be evaluated for its potential to be a gene delivery
vector. p(CBA-ABOL) complexes were stable in HEPES and had promising results
for transfection. However, they were not stable in the extracellular environment.
Thus PEGylation was applied to increase the stability of p(CBA-ABOL) polyplexes
in the extracellular environment. PEGylated particles formed stable complexes in the
extracellular media, but were not taken up and did not transfect efficiently. p(CBA-
ABOL) polyplexes should be improved in terms of stability in the extracellular
environment and transfection efficiency. HA-coating was applied by means of
electrostatic binding of negatively charged low molecular weight HA polymer to
positively cored polyplexes. The choice of hyaluronan Mw was determined by the
previous study of Hornof et. al. (2008) and the ratio of complexes based on the
molar ratio between HA and pDNA. By adding the HA to the polyplexes as the outer
core of the polyplexes was coated with anionic HA, the ζ-potential of complexes
turned negative from positive (See Figure 5.21) and resulting in a lowest value of -35
mV. According to the Z-average diameter measurements in OptiMEM, particles with
all ratios show a clear stability around 500 nm in average. When it is compared to
uncoated polyplexes this drastic change in the size of particles could be because the
polyplexes are completely restructured, perhaps with more plasmids per polyplex,
and more polymer as well. The pDNA condensation efficiency of the polyplexes in
OptiMEM can be seen from agarose gel electrophoresis (See Figure 5.22). The HA-
coated particles with the lowest ratio showed a slight pDNA release and the other
two ratios were able to completely condense the pDNA which means HA-shielding
has a positive effect on pDNA binding ability of polymer.

For biological evaluation of HA-coated particles, uptake and transfection efficiency

analyses were performed. In contrast to generally obtained results, the desired
increasing effect of HA-coating could not be observed for p(CBA-ABOL) uptake
efficiency studies. Previously, Wang et al. have reported a facilitating effect for
cellular uptake of HA-shielded PEI/pDNA complexes (2011).

In a preliminary study, we obtained low Mw HA with enzymatic digestion of

hyaluronan (See App. A). The transfection efficiency results showed higher gene

86
expression than the results obtained with research grade purity HA. This could be
due to the purity and decreased effect of HA Mw. Although we aimed low Mw by
the enzymatic digestion, there is a possibility that the high Mw HA particles could
not be eliminated completely by ultrafiltration. Then the mixture of low and high
Mw HA may have resulted in increased transfection efficiency in the preliminary
study. Moreover, the MTT assay results indicated that the cell viability is higher for
the cells treated with HA-coated particles than uncoated particles. This could be
because they are less toxic for the cells, but it could also be because they are not
taken up as much as uncoated particles. Hence, the application of HA-coating should
be further investigated.

5.3.4 p(CBA-HIS) polyplexes

p(CBA-HIS) was chosen to be used as a gene delivery vector due to the imidazole
groups present in the side chain. These groups have a good buffering capacity in
physiological conditions that is expected to be more efficient in terms of delivery of
the plasmid to the cell nucleus.

5.3.4.1 Physicochemical characterization

Particle size and surface charge measurements

The particle size and surface charge measurements of p(CBA-HIS) polyplexes with
various polymer/pDNA mass ratio were determined by DLS. The p(CBA-
HIS)/pDNA complexes were prepared with 4 different mass ratios which were the
same ratios that applied for p(CBA-ABOL) polyplexes. By this way, it was possible
to compare the polyplex formation characteristics of two types of polymers. The
amount of polymer and pDNA which was used while preparing the complexes is
illustrated on Table 5.7 and the measurement values are given on Table 5.8. As it is
shown in Figure 5.26, increasing amount of polymer results in smaller, more positive
and stable complexes in HEPES buffer. However, measurements in OptiMEM
media indicates that the increase in the polymer/pDNA ratio causes very large
particles, even >1 μm for 96/1 ratio.

87
 Table 5.7: The amounts of p(CBA-HIS) polymer and pDNA which was used to
obtain desired polymer/pDNA (w/w) ratios. The polyplexes were
prepared with increasing amount of polymer while the amount of
plasmid was constant.

mass ratio m polymer (μg) m pDNA (μg)

12/1 120 10
24/1 240 10
48/1 480 10
96/1 960 10

Table 5.8 : Illustration of average size, pDI and charge values for p(CBA-HIS)
polyplexes with different polymer/pDNA mass ratios.
HEPES OptiMEM
mass ratio Z-potential
p(CBA-HIS)

size (nm) pDI (mV) size (nm) pDI

12/1 157,07 0,08 26,87 509,70 0,05
24/1 117,70 0,09 31,67 598,10 0,13
48/1 101,80 0,10 43,70 778,97 0,22
96/1 96,78 0,15 46,77 1036,33 0,34

pDNA complexation efficiency

Agarose gel electrophoresis was performed to analyze the pDNA condensation

ability of homopolymer p(CBA-HIS)/pDNA complexes. As it is shown in the Figure
5.27, the polyplexes were loaded into gel starting from lane number 3 and
polymer/pDNA ratio of 12/1. 48/1 p(CBA-ABOL)/pDNA complex was also applied
as a reference. It can be seen from the figure that above the ratio of 24/1, the bands
become almost invisible and polymer strongly binds the pDNA. If the polyplexes
with the same amount but different type of polymer are compared (lane number 5
and 7), it can be concluded that p(CBA-HIS) has strong ability to condense the
pDNA molecule. For the samples in OptiMEM, the release of pDNA is observed
however, the bands are less bright than the ones with heparin where the pDNA was
almost totally displaced.

88
 a.1200 Size and pDI of p(CBA-HIS) pplxes
1,0
Z-average diameter (nm) 0,9
1000 0,8
800 0,7
0,6

pDI
600 0,5
0,4
400 0,3
200 0,2
0,1
0 0,0
12/1 24/1 48/1 96/1 12/1 24/1 48/1 96/1 size HEPES
size OptiMEM
CBA-HIS CBA-HIS pDI HEPES
pDI OptiMEM

b. Zeta potential in HEPES

50
zeta potential (mV)

40

30

20

10
zeta
0
12/1 24/1 48/1 96/1

The charts give the output of DLS analyses. The results are average of 3 measurements
and shown with standard deviations. For size and pDI measurements complexes were
prepared both in HEPES buffer solution (25 mM, pH 7.2) and OptiMEM serum free
transfection medium. Since OptiMEM media is rich in different types of proteins and
dissolved ions, charge measurements were only carried out with the polyplexes that are
prepared in HEPES. In Figure a., the primary y-axis shows the Z-averaged size in nm
and secondary y-axis shows the corresponding pDI. The x-axis indicates the polymer
type and the polymer/pDNa (w/w) ratio. It can be seen in figure a. that the influence of
increasing polymer ratio gives rise to large particles in OptiMEM, possibly aggregates.
Figure b. shows the zeta potential measurements in HEPES media, which appear to be
maximum around +50 mV for 96/1 ratio.

Figure 5.26 : Illustration of a. particle size and polydispersity, b. ζ-potential of

pDMAEMA polyplexes with different Mw and mass ratios.

89
 1. Ladder (1 kbp)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 2. Pure pGL4.13 plasmid
3. 12/1 in HEPES
4. 24/1 in HEPES
5. 48/1 in HEPES
6. 96/1 in HEPES
7. Pplx CBA-ABOL 48/1 in HEPES
8. 12/1 in OptiMEM
9. 24/1 in OptiMEM
10. 48/1 in OptiMEM
11. 96/1 in OptiMEM
12. 12/1 + 3 µl Heparin
13. 24/1 + 3 µl Heparin
14. 48/1 + 3 µl Heparin
15. 96/1 + 3 µl Heparin
16. Pplx CBA-ABOL 48/1 + 3 µl Heparin

Figure 5.27: Agarose gel electrophoresis of p(CBA-HIS) polyplexes with different

mass ratios. The lanes with polyplexes contain 20 µL of sample
(corresponding to 200 ng of pDNA per well). As a negative control, 3
µL of Heparin (7 μg/μL) was added to last 5 samples to simulate
pDNA displacement. 48/1 p(CBA-ABOL) polyplexes were applied as
a comparative reference.

5.3.4.2 In vitro biological evaluation

Uptake efficiency

The influence of increased buffering capacity on the uptake efficiency of ARPE-19

cells was determined by flow cytometry (See Figure 5.28). Two mass ratios of
p(CBA-HIS), 24/1 and 48/1, with the best physicochemical characteristics were
chosen and applied for uptake experiment. p(CBA-ABOL) 48/1 ratio was used to
prepare the positive control samples. Untreated cells and one set of the polyplexes
were put at 4⁰C as negative controls. p(CBA-HIS) with 48/1 mass ratio polyplexes
showed nearly 95% uptake which is higher than the p(CBA-ABOL) polyplexes with
the same polymer/pDNA (w/w) ratio.

Transfection efficiency

Following the uptake efficiency experiment, the p(CBA-HIS) polyplexes were

evaluated in terms of their transfection efficiency. ARPE-19 cells were treated with
polyplexes having different mass ratios and the transfection efficiency was analyzed
by flow cytometry. LF/pDNA complexes and p(CBA-ABOL) polyplexes were
applied as positive controls. For positive control samples, nearly 30% GFP
expression was observed while p(CBA-ABOL) showed a transfection efficiency

90
around 10% (See Figure 5.29). On the other hand, p(CBA-HIS) complexes showed
very low transfection for both selected polymer/pDNA ratios.

Figure 5.28 : Comparison of internalization of different gene carriers p(CBA-HIS)

with different mass ratios, p(CBA-ABOL) and LF in ARPE-19 cells.

Figure 5.29 : Comparison of transfection efficiencies of different gene carriers

p(CBA-HIS) with different mass ratios, p(CBA-ABOL) and LF in
ARPE-19 cells.

91
Figure 5.29 (continued) : Comparison of transfection efficiencies of different gene
carriers p(CBA-HIS) with different mass ratios, p(CBA-
ABOL) and LF in ARPE-19 cells.

Cell viability

The cell viability for p(CBA-HIS) polyplexes on ARPE-19 cells was analyzed with
MTT Assay by means of absorbance measurements on Microplate Reader. LF/pDNA
and p(CBA-ABOL)/pDNA complexes were applied as controls. It can be seen on the
Figure 5.30 that, p(CBA-HIS)/pDNA complexes have significantly favorable cell
viability both for 24/1 and 48/1 mass ratios.

The day after the seeding, cells were treated in the same way with transfection
studies but with a pDNA concentration of 0,2 µg per well. As a negative control,
the cells only with MTT reagents were applied. LF/pDNA complexes were
prepared as positive control and p(CBA-ABOL) complexes as comparative control.
The third day, the viability of cells was determined with MTT assay. The results are
shown with standard deviations of 5 independent samples. (°) normalized against
cells which were not incubated with polyplexes. (*) indicates significant differences
with p< 0.05 in comparison to LF (ANOVA).

Figure 5.30 : Cell viability results of p(CBA-HIS)/pDNA pplexes with different

mass ratios.

92
5.3.4.3 Discussion

p(CBA-HIS) is a homopolymer with imidazole side groups that was synthesized by

Michael type polyaddition of CBA monomer with repetitive disulfide linkages to
histamine (HIS) molecule. p(CBA-HIS) homopolymer has a buffering capacity of
58% due to the imidazole groups (pKa ~6.5) present in the side chain (Lin et al,
2007b). The same 4 mass ratios as p(CBA-ABOL) polyplexes were applied for both
physicochemical and biological evaluation of p(CBA-HIS) by this way, it was
possible to compare the effect of buffer capacity of the polymer. First of all, the
particle size and surface charge of the polyplexes was analyzed by DLS. According
to the data obtained by DLS for the same mass ratios, p(CBA-HIS) polyplexes
formed smaller complexes in HEPES buffer than p(CBA-ABOL) polyplexes which
may be because of the imidazole groups in p(CBA-HIS) giving rise to stronger
interactions with DNA by the means of increased cationic charge (Coue and
Engbersen, 2011). Nevertheless, the same aggregation problem in OptiMEM
conditions was also observed for p(CBA-ABOL) polyplexes (See Figure 5.26).
Secondly, agarose gel electrophoresis was employed to investigate the pDNA
binding ability of p(CBA-HIS) polyplexes with various mass ratios. As it is shown in
Figure 5.27, the DNA release bands become less clear starting from 24/1 ratio,
indicating a stronger condensation. Moreover, p(CBA-HIS) polyplexes were able to
condense the pDNA more efficiently in comparison to the bands of p(CBA-ABOL)
polyplexes with the same mass ratio of 48/1.

The uptake and transfection efficiency of p(CBA-HIS) polyplexes on ARPE-19 cells

was studied with 2 stable mass ratios of 24/1 and 48/1. Furthermore, cell viability of
ARPE-19 cells that are incubated with p(CBA-HIS) polyplexes was analyzed by
MTT assay. The uptake efficiency results clearly showed that, p(CBA-HIS)
polyplexes are taken up with a higher percentage. Especially at 48/1 polymer/pDNA
mass ratios where pDNA is condensed into smaller and more positively-charged
nanoparticles, the cellular uptake is much increased with comparison to p(CBA-
ABOL) polyplexes. The transfection efficiency results of p(CBA-HIS) polyplexes
were quite unexpected. The pKa of imidazole groups present in histamine side chains
is in the range of endosomal acidification (pH 7.4–5.1) (Coue and Engbersen, 2011).
By this way, these polyplexes were supposed have an advantage of extra pH-
responsiveness during endosomal escape which would result in higher transfection

93
efficiency. However, as it is illustrated in Figure 5.29 that p(CBA-HIS) polyplexes
have very low GFP expression where the LF control and p(CBA-ABOL) complexes
show an acceptable efficiency. This may be explained by the stronger pDNA binding
ability of p(CBA-HIS). Finally, the cell viability experiment indicated that p(CBA-
HIS) polyplexes have a negligible cytotoxicity relative to LF complexes and as well
as p(CBA-ABOL).

All in all, p(CBA-HIS) is stated to form stable complexes in HEPES but not in the
extracellular environment, have high uptake efficiency and good cell viability. The
same strategies as for p(CBA-ABOL) polyplexes can be used to increase the stability
of p(CBA-HIS) complexes in OptiMEM media and facilitate the transfection
efficiency.

94
6. CONCLUSION

Gene therapy is the treatment of human diseases with an underlying genetic cause by
delivering therapeutic genetic material (transgene) into the diseased cells of the
patient. It has been a promising way to treat genetic diseases over the last decades
because it offers a solution to treat the causes of diseases rather than curing the
symptoms.

In this project, pDMAEMA polymer/pDNA complexes were evaluated as an

example of non-biodegradable polymers. These polyplexes were able to form stable
complexes with pDNA and transfect the ARPE-19 cells with a moderate efficiency.
Apart from lower gene expression, pDMAEMA polymer had a cytotoxic effect on
ARPE-19 cells.

Secondly, SS-PAAs were evaluated as an example of biodegradable and bioreducible

polymer systems. Additionally different coating strategies and side chains were
applied to improve the efficiency of these systems. All types of SS-PAA polymer
were able to form stable complexes with pDNA. p(CBA-ABOL) was reported to a
good transfection agent with relatively high cell viability however, its stability in the
physiological mimicking media should be increased. PEGylation strategy was
expected to increase the stability of p(CBA-ABOL) complexes and the positive
effect was observed. Nevertheless, PEGylated polymers were not able to taken up by
the ARPE-19 cells and transfect. HA-coating was an alternative way to increase the
stability of particles and facilitate their uptake efficiency. The experiments resulted
in expected way however, the effect of HA Mw on the transfection efficiency should
be further investigated.

Finally, p(CBA-HIS) was chosen to be evaluated in terms of cellular uptake and

transfection. This polymer was supposed to have an increased transfection efficiency
due to the buffering capacity and higher pKa of imidazole groups. The expected
efficiency for transfection did not observed with p(CBA-HIS) polyplexes which their
DNA release profile in the intracellular environment should be further analyzed.

95
96
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APPENDICES

APPENDIX A: Preliminary study with HA

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APPENDIX A

Preliminary Study for HA-coated p(CBA-ABOL) Polyplexes

Prior to the studies that were performed by research grade HA obtained from Life
Core, the enzymatically degraded HA was evaluated. For this purpose the HA
powder with unknown Mw was dissolved in water. Then the hyaluronan degradation
enzyme hyaluronidase was dissolved in gelatin hydrolyzed and this enzyme solution
was used to digest the HA by stirring overnight. Afterwards, the digested HA
solution was filtered by ultrafilters with 30 kDa and 50 kDa molecular weight cut-off
(MWCO) and centrifuged. Finally obtained HA was assumed to have a Mw of 30-50
kDa.

Transfection efficiency of ARPE-19 cells

100 2500
90
80 2000
70
60 1500
% pos

Mean
50
40 1000
30
20 500
10
0 0
48/1 400/1 48/1 400/1
% pos

cells LF CBA- HA- LF CBA- HA- Mean

ABOL coated ABOL coated

The next day of the seeding, freshly prepared HA-coated polymer/pDNA complexes
were added to cells with a 2 µg concentration per well. For transfection experiments,
gWiz-GFP plasmid was used. Then the average GFP expression of the total gated
population of cells (pink bars) and the amount of GFP-positive cells (purple bars)
were measured by flow cytometry. Non-treated cells and the cells which were
transfected with non-fluorescent pGL4.13 plasmid were negative controls for
transfection experiments. As a positive control, three wells were treated with
lipoplexes formed by LF. p(CBA-ABOL) polyplexes were used as comparative
reference. The measurements were carried out in triplicate and data are shown with
standard deviation of three independent samples.

Figure A.1 : Comparison of transfection efficiency of enzymatically degraded HA-

coated p(CBA-ABOL) polyplexes on ARPE-19 cells with LF and non-
coated p(CBA-ABOL) complexes.

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CURRICULUM VITAE

Name Surname: Hatice İmran Güngördü

Place and Date of Birth: Istanbul, 1989

E-Mail: [email protected]

B.Sc. : Istanbul Technical University, Chemistry (2010)

M.Sc. : Istanbul Technical University, Chemistry (2012)

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