Advanced Drug Delivery Reviews 156 (2020) 80–118

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

journal homepage: www.elsevier.com/locate/addr

Polymeric micelles for the delivery of poorly soluble drugs:

From nanoformulation to clinical approval
Duhyeong Hwang a,1, Jacob D. Ramsey a,1, Alexander V. Kabanov a,b,⁎
a
Center for Nanotechnology in Drug Delivery and Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC
27599, USA
b
Laboratory of Chemical Design of Bionanomaterials, Faculty of Chemistry, M. V. Lomonosov Moscow State University, Moscow 119992, Russia

a r t i c l e i n f o a b s t r a c t

Article history: Over the last three decades, polymeric micelles have emerged as a highly promising drug delivery platform for
Received 10 May 2020 therapeutic compounds. Particularly, poorly soluble small molecules with high potency and significant toxicity
Received in revised form 18 September 2020 were encapsulated in polymeric micelles. Polymeric micelles have shown improved pharmacokinetic profiles
Accepted 21 September 2020
in preclinical animal models and enhanced efficacy with a superior safety profile for therapeutic drugs. Several
Available online 24 September 2020
polymeric micelle formulations have reached the clinical stage and are either in clinical trials or are approved
Keywords:
for human use. This furthers interest in this field and underscores the need for additional learning of how to
Active pharmaceutical ingredient best design and apply these micellar carriers to improve the clinical outcomes of many drugs. In this review,
Block copolymer we provide detailed information on polymeric micelles for the solubilization of poorly soluble small molecules
Clinical trials in topics such as the design of block copolymers, experimental and theoretical analysis of drug encapsulation
Drug in polymeric micelles, pharmacokinetics of drugs in polymeric micelles, regulatory approval pathways of
Drug delivery nanomedicines, and current outcomes from micelle formulations in clinical trials. We aim to describe the latest
Polymeric micelle information on advanced analytical approaches for elucidating molecular interactions within the core of poly-
Solubilization
meric micelles for effective solubilization as well as for analyzing nanomedicine's pharmacokinetic profiles.
Taking into account the considerations described within, academic and industrial researchers can continue to
elucidate novel interactions in polymeric micelles and capitalize on their potential as drug delivery vehicles to
help improve therapeutic outcomes in systemic delivery.
© 2020 Elsevier B.V. All rights reserved.

Abbreviations: %ID, percent injected dose; 17-AAG, 17-allylamino-17-demethoxygeldanamycin; ABC, accelerated blood clearance; ANVISA, Brazilian Health Surveillance Agency; API, ac-
tive pharmaceutical ingredient; AUC, area under the curve; CDDP, cisplatin, cis-dichlorodiaminepatinum(II); CED, cohesive energy density; CI, combination index; CL, clearance; CMC, critical
micelle concentration; CQA, critical quality attribute; DACHPt, dichloro(1,2-diaminocyclohexane)platinum(II); DPD, dissipative particle dynamics; ELP, elastin-like polypeptide; EMA,
European Medicines Agency; GCM, group contribution method; GMP, good manufacturing process; GRAS, generally regarded as safe; HPMA, poly[N-(2-hydroxypropyl)methacrylamide];
ICH, International Conference on Harmonisation; LC, loading capacity; LCRP, living cationic ring-opening polymerization; LCST, lower critical solution temperature; LE, loading efficiency;
MAA, marketing authorization application; MBC, metastatic breast cancer; MD, molecular dynamics; MDR, multidrug resistant; MHLW, the Ministry of Health, Labor, and Welfare; MHRA,
Medicines and Healthcare Products Regulatory Agency; mPEG, methoxy-PEG; mRNA, messenger RNA; MTD, maximum tolerated dose; MW, molecular weight; NCL, Nanotechnology
Characterization Laboratory; NDA, New Drug Application; NMPA, the National Medical Products Administration; NMR, nuclear magnetic resonance; NSCLC, non-small cell lung cancer;
OH-PEG, hydroxy-PEG; Oxaliplatin, cis-oxalato-(trans-l)-1,2-diaminocyclohexane‑platinum(II); P(Asp), poly(aspartic acid); P(Glu), poly(glutamic acid); PAMAM, polyamidoamine; PBAE,
poly(β-amino ester); PBLA, poly(β-benzyl-L-aspartate); PBLG, poly(γ-benzyl-α, l-glutamate); PBuOx, poly(2-n-butyl-2-oxazoline); PBuOzi, poly(2-n-butyl-2-oxazine); PCL, poly(Ɛ-
caprolactone); PDI, polydispersity index; PDLLA, poly(D,L-lactide); PDMA, Pharmaceutical and Medical Devices Agency; (PDMA), poly(N,N-dimethylacrylamide); pDNA, plasmid DNA;
PEG, polyethyleneglycol; PEO, poly(ethylene oxide); PEtOx, poly(2-ethyl-2-oxazoline); PFS, progression free survival; PiPrOx, poly(2-isopropyl-2-oxazoline); PK, pharmacokinetic; PLGA,
poly(D,L-lactide-co-glycolide); PLLA, poly(L-lactide); PMeOx, poly(2-methyl-2-oxazoline); PMMA, poly(methacrylic acid); (PMMA), poly(methyl methacrylate); POx, poly(2-oxazoline);
POzi, poly(2-oxazine); PPO, poly(propylene oxide); PPrOx, poly(2-n-propyl-2-oxazoline); PPrOzi, poly(2-n-propyl-oxazine); PTX, paclitaxel; PVP, poly(vinylpyrrolidone); QSPR, quantitative
structure property relationship; RES, reticuloendothelial system; RLD, reference listed drug; ROP, ring-opening polymerization; ROS, reactive oxygen species; SANS, small angle neutron scat-
tering; siRNA, small interfering RNA; SITUA, stable isotope tracer ultrafiltration assay; SP, solubility parameter; ssNMR, solid-state NMR; TEM, transmission electron microscopy; TGA,
Therapeutic Goods Administration; UCST, upper critical solution temperature; USFDA, US Food and Drug Administration; Vd, volume of distribution.
⁎ Corresponding author at: Center for Nanotechnology in Drug Delivery, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 125 Mason Farm Road, Marsico Hall,
Office #2012, Campus Box 7362, Chapel Hill, NC 27599-7362, USA.
E-mail address: [email protected] (A.V. Kabanov).
1
Authors contributed equally on this review.

https://doi.org/10.1016/j.addr.2020.09.009
0169-409X/© 2020 Elsevier B.V. All rights reserved.
D. Hwang, J.D. Ramsey and A.V. Kabanov Advanced Drug Delivery Reviews 156 (2020) 80–118

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
2. Functionalities of polymeric micelles as a delivery platform for poorly soluble small molecules . . . . . . . . . . . . . . . . . . . . . . . . . 83
2.1. Hydrophilic blocks and anti-fouling polymers in block copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
2.1.1. Polyethylene glycol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
2.1.2. Hydrophilic poly(2-oxazoline)s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
2.1.3. Other reported anti-fouling polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
2.2. Hydrophobic polymers in block copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
2.2.1. Polyethers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
2.2.2. Polyesters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
2.2.3. Hydrophobic poly(amino acid)s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
2.2.4. Polyoxazolines and polyoxazines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
2.3. Stimuli-responsive block copolymers in polymeric micelle formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
2.4. Drug conjugates and complexes with block copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
2.5. Unimolecular micelles and cross-linked micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
3. Drug-polymer interactions within polymeric micelles: Theory, modeling and experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
3.1. Theoretical and computational approaches. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
3.1.1. Hildebrand and Hansen solubility parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
3.1.2. Flory-Huggins solution theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
3.1.3. Molecular dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
3.1.4. Quantitative structure property relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
3.2. Experimental approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
3.2.1. Interactions with core and shell-forming blocks in drug partitioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
3.2.2. Drug-polymer interactions in polymeric micelles by NMR spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
3.2.3. Microstructure of polymeric micelles by small angle neutron scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
3.2.4. Host-guest interactions by fluorescence analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
3.2.5. In vitro drug release analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
4. Drug loading, pharmacokinetics and distribution of polymeric micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
4.1. Drug loading and excipient derived toxicity in polymeric micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
4.2. Pharmacokinetic analysis of polymeric micelle drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.3. Hydrodynamic size and morphology of polymeric micelles and drug distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
4.4. Polymeric micelle combination therapies and effects on drug retention and pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . 102
5. Polymeric micelles in clinical trials and regulatory approval for human use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
5.1. Polymeric micelle manufacturing considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
5.2. Regulatory approval of nanomedicines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
5.3. Clinical status of polymeric micelle formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.3.1. Genexol® PM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.3.2. Nanoxel® M . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
5.3.3. NK105 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
5.3.4. NC-6004 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
5.3.5. SP1049C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
6. Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Declaration of Competing Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

1. Introduction molecular interactions which result in the protection of the cargo from
the external environment and improvements of the pharmacokinetic
Biocompatible polymers have been extensively employed in phar- (PK) profile [14]. Ideal polymeric micelle formulations are expected to
maceutical science as excipients for traditional pharmaceutical formula- improve therapeutic outcomes of the encapsulated drug due to the
tions and more recently in nanomedicines for enhancing therapeutic functionalities of the formulation conferred by the polymer. The PK pro-
outcomes of potent drugs [1,2]. About three decades ago, micelles file of the therapeutic compounds encapsulated in polymeric micelles
formed by amphiphilic block copolymers in aqueous solution were con- differ from native compounds, because micelles are capable of releasing
ceived as carriers for poorly soluble therapeutic compounds that were the cargo in a controlled manner from the core during systemic circula-
either covalently attached to polymer chains [3] or non-covalently tion. Structural features of polymeric micelles, such as the hydrophilic
incorporated in the micelles [4]. Since then, the applications of amphi- shell, help to avoid both unexpected drug loss from serum components
philic block copolymers in the design of polymeric micelles as therapeu- and prevent opsonization by the complement system which typically
tics have been extensively studied [5–8]. A variety of novel block result in the rapid clearance of drugs from systemic circulation [15,16].
copolymers have been proposed to develop micelle-based delivery sys- Based on these functionalities derived from the polymer formulations,
tems as potential nanomedicines for humans [9–12]. Many significant the overall PK profile of the therapeutic compounds such as maximum
advances in polymeric micelles have been made to optimize the deliv- systemic concentration, area under the curve (AUC), clearance (CL), vol-
ery of therapeutic molecules. Such advances have driven an increasing ume of distribution (Vd), and biodistribution can be improved [13]. Fur-
number of polymeric micelle drug formulations to enter clinical trials thermore, ideal polymeric micelles are expected to reduce the toxicity
for regulatory approval [12,13]. of the therapeutic compounds. The safety profile of compounds within
The design of block copolymers is intended to effectively encapsu- polymeric micelles could improve therapeutic outcomes by expanding
late therapeutic compounds into polymeric micelles by various the therapeutic window. Side effects could be largely mitigated in

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D. Hwang, J.D. Ramsey and A.V. Kabanov Advanced Drug Delivery Reviews 156 (2020) 80–118

both preclinical and clinical studies by using polymeric micelle formula- the micelle core is the most feasible path to clinical translation for poly-
tions. This could also greatly affect the quality of life of the patients meric micelles, so this is where we will focus our efforts.
[11,17]. In the subsequent sections, we consider the current state of the poly-
Polymeric micelle systems exploit block copolymers for the delivery meric micelle field from four different principal angles. In Section 2, we
of therapeutic compounds such as small molecule drugs, proteins, and focus on the polymeric materials used for the manufacturing of poly-
nucleic acids [2]. The desired physicochemical properties of block meric micelles with a particular emphasis on the materials which
copolymers will vary based on the physicochemical properties of each have been or could be developed for clinical use. The various possible
therapeutic compound. Generally speaking, hydrophobic small mole- blocks in block copolymers such as hydrophilic shell forming blocks
cules could be encapsulated in amphiphilic block copolymers which with anti-fouling properties, hydrophobic blocks, and blocks with alter-
have both hydrophilic and hydrophobic blocks [4,18,19]. The native interaction mechanisms are described along with their roles in
amphiphilic block copolymers can spontaneously self-assemble into a the solubilization of poorly soluble small molecules.
core-shell polymeric micelle even in the absence of the therapeutic In Section 3, we consider the pivotal problem of drug loading in
molecules. The drug molecules can be physically entrapped (“solubi- polymeric micelles with the aim to maximize the payload of the drug
lized”) in the hydrophobic core of such micelles. Small, water insoluble and decrease relative amounts of polymeric excipient used. To this
drugs can also be chemically conjugated to the core-forming block of a end, we analyze the drug-polymer interactions within polymeric mi-
block copolymer and the resulting amphiphilic block copolymer conju- celles focusing on the theory, modeling and experiment. Here, multidis-
gates then self-assemble into a polymeric micelle containing the conju- ciplinary approaches for investigating detailed molecular interactions
gated drug in the core. Meanwhile, biopolymers usually require charged between drugs and block copolymers are introduced to improve the un-
blocks to be encapsulated in polymeric micelles by electrostatic interac- derstanding of the solubilization processes and to aid in the informed
tions [20,21]. development of polymeric micelles for effective drug delivery. We also
In this review we focus on the applications of the polymeric mi- discuss in this section key innovations in the analysis of high loaded mi-
celle technology for the delivery of poorly soluble small molecules. celles, such as drug partitioning measurements, nuclear magnetic reso-
Among these applications, the exploitation of amphiphilic block co- nance (NMR) spectroscopy, small angle neutron scattering (SANS), and
polymers as carriers for poorly soluble small molecules in polymeric fluorescence analysis of host-guest interactions.
micelles has shown the potential to improve therapeutic outcomes, In Section 4, we focus on the highly complex problems of polymeric
and several polymeric micelle drugs have reached the clinical stage micelle drug PK and biodistribution that remains a constant subject
of evaluation and regulatory approval for cancer treatment. For for active research. Here we focus on key metrics for PK studies of
example, paclitaxel has been physically encapsulated in polymeric nanoformulations, in particular those which relate to the tumor distri-
micelles to improve the systemic PK profile and alleviate drug- bution of the polymeric micelle drugs. We discuss advanced experimen-
induced side effects, such as neurotoxicity in both preclinical studies tal methods of PK analysis of polymeric micelle drugs as well as
and clinical trials [6,7,9,22]. One such formulation, Genexol® PM, has theoretical and modeling approaches. The role of the drug release
received regulatory approval in South Korea and other countries as a characteristics along with the hydrodynamic size and morphology of
cancer therapeutic. polymeric micelles in the drug distribution to the disease site is thor-
Many comprehensive reviews and collections on polymeric micelles oughly discussed. We also discuss the potential for the combination
have been published over last two decades which describe the general therapy with multiple drugs co-loaded in polymeric micelles from the
aspects of formulation and applications for the delivery of small drugs standpoint of both improved PK and delivery as well as improved ther-
and biopolymers [2,23], or that highlight specific delivery strategies, apeutic efficacy. Last but not the least, this section focuses on the rela-
such as oral drug delivery [24], biological response modifying effects tionship between the drug loading and excipient derived toxicity in
of block copolymers [25] or field-responsive micelles [26,27]. We refer polymeric micelles as a foundation for successful clinical translation of
the readers to these and other publications for additional insights and the polymeric micelle drugs.
historical perspectives. In this review, we focus on the basic principles Finally, in Section 5 we focus on the preclinical and clinical transla-
and current advances in polymeric micelle systems for the delivery of tion of polymeric micelle products. Here we discuss various methods
poorly soluble small molecules with a particular emphasis on the sys- for polymeric micelle preparation from the standpoint of their scalabil-
temic drug delivery (Fig. 1). The current state of the literature reflects ity and translational potential. The discussion of the regulatory approval
that the physical entrapment of water insoluble small molecules into of nanomedicines, including polymeric micelles, is provided with the

Fig. 1. Schematic illustration of polymeric micelles for delivery of poorly soluble drugs.

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D. Hwang, J.D. Ramsey and A.V. Kabanov Advanced Drug Delivery Reviews 156 (2020) 80–118

objective to assist academic and industrial scientists in considering reg- interactions other than purely hydrophobic have recently been devel-
ulatory approval challenges and opportunities throughout the formula- oped for the encapsulation of poorly soluble compounds [28]. These
tion discovery and development process. Lastly, we discuss several key block copolymers exhibit very complex and interdependent inter-
examples of polymeric micelles which have been translated successfully actions. For example, the work of Kozlov et al. showed that both hyd-
to the clinic. The concluding Section 6 focuses on future directions. rophobic and hydrophilic blocks participate in formation of the
We would like to emphasize that polymeric micelle systems are spe- microenvironment for poorly soluble compounds in the micelles [29].
cial and present unique advantages over many other nanosized drug The Luxenhofer group has shown that hydrophilic blocks can play a sub-
carrier systems. One issue with many nanoparticle carriers is that if stantial role in drug polymer interaction, especially in highly drug
they penetrate into tumors, or other sites of action, the drug release is loaded polymeric micelles [30]. Not only does the structural composi-
slow, uncontrolled, or inefficient. Polymeric micelles are dynamic sys- tion of each block play a role, but the length of each block, and thus poly-
tems. Because of this, they release the drug to their target much easier mer molecular weight, are important as well. Additionally, the amount
than many more rigid, “solid” nanoparticle systems. On the other of drug loaded into the micelles can affect stability, morphology, and
hand, due to their dynamic character, the micelles can lose drug on the size of the micelles in aqueous solution. The complex interdepen-
the way to the target. In light of this, the pharmaceutical development dency of block structure and block lengths make for highly tunable
of these formulations must balance both drug loading and release to im- properties with unique capacities for drug solubilization. However, it
prove drug therapeutic indices by polymeric micelle delivery. These also makes understanding these interactions and the intelligent design
properties are governed by drug-polymer interactions as well as struc- of block copolymers more challenging.
tural parameters of the block copolymers, which could be finely When considering biological interactions, the hydrophilic shell plays
optimized. Therefore, this review has a particular emphasis on such pa- a critical role in the polymeric micelles. Utilizing hydrophilic blocks
rameters which have maximal influence over these properties as well as which have “anti-fouling” properties reduces the binding of serum com-
recent advances in the analysis of the complex interactions between ponents (serum proteins and complement system) and protects the en-
drugs and block copolymers in these uniquely dynamic drug delivery capsulated drug, thus avoiding the unexpected loss of the cargo during
systems. systemic circulation. To this end, polymeric micelles should be designed
to minimize these interactions. Otherwise, polymeric micelles could be
2. Functionalities of polymeric micelles as a delivery platform for readily cleared from the body by plasma protein adsorption and/or
poorly soluble small molecules complement activation leading to the removal of the entire micelle
along with the drug within its core by the reticuloendothelial system
Amphiphilic block copolymers self-assemble in aqueous media to (RES) [31,32]. The RES removes immune complexes in healthy people
form micelles that have hydrophilic shells and hydrophobic cores. The and consists of phagocytic cells in circulation and tissues. To avoid this
shell prevents aggregation and precipitation of the micelles while also system, several hydrophilic blocks have been introduced into the struc-
protecting the therapeutic cargo. The core holds the micelle together ture of block copolymers to endow anti-fouling properties to the
and solubilizes poorly soluble small molecules. In general, diblock co- polymeric micelles (Table 1) [33,34]. The functionalities of the
polymers (A–B) or triblock copolymers (A–B–A) of hydrophilic hydrophilic shells were extensively studied and according to those
(A) and hydrophobic (B) blocks are most often employed for the prep- studies, physicochemical properties of hydrophilic polymers such as
aration of polymeric micelle formulations. However, B-blocks exhibiting molecular weight and surface density were closely related to the

Table 1
Hydrophilic polymers commonly used for constructing amphiphilic block copolymers.

Polymer Chemical structure Synthesis Properties and comments Ref

PEG1 Living anionic Most often used hydrophilic polymer with stealth [40–43]
ring-opening property. Used in clinically approved
polymerization (ROP) of nanoformulations including polymer micelle
ethylene oxide (Genexol® PM). Potential immunogenicity and
accelerated blood clearance (ABC) phenomenon. The
only shell-forming polymer that is used in clinically
approved products as of today.
Poly(2-oxazoline)s Living cationic Both polymers are evaluated as PEG replacement [22,44–46]
ring-opening PMeOx is more hydrophilic than PEG.
polymerization (LCRP) of
2-oxazoline monomers

Poly(sarcosine) Living polymerization of Evaluated as PEG replacement. Biodegradable. [47–49]

α-amino acid-N
carboxyanhydrides

Polysaccharides Enzymatic synthesis Used as a component in block and graft copolymers. [50–53]
Highly variable molecular weight. Dextran has been
used as excipient in clinically approved injectable
products (FERAHEME®). Biodegradable.

Miscellaneous Atom transfer radical Potential immunogenicity noted (PVP). [57–64]

polymerization; Nonbiodegradable.
reversible addition
fragmentation chain
transfer

1
Polyethyleneglycol, 2methoxy-PEG, 3hydroxy-PEG, 4poly(2-methyl-2-oxazoline), 5poly(2-ethyl-2-oxazoline), 6poly(vinylpyrrolidone), 7poly(N,N-dimethylacrylamide), 8poly[N-
(2-hydroxypropyl) methacrylamide], 9poly(methyl methacrylate).
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stability, systemic circulation time, and biodistribution of polymeric which may cause the clearance of the cargo and polymeric micelles by
micelles in vivo [16,35]. the RES [35]. PEG with molecular weights ranging from 1 to 6 kDa are
The hydrophobic block of block copolymers is intended to solubi- an ideal molecular weight for endowing nanoparticles with efficient
lize poorly soluble drugs in the core and control the release of the anti-fouling properties and are frequently being employed to prepare
drug from the polymeric micelles [36–38]. Hydrophobic interactions block copolymers for drug delivery [34,69,70]. The anti-fouling mecha-
between drugs and hydrophobic blocks of amphiphilic block copoly- nism of PEG has been comprehensively investigated in many studies.
mers are very well recognized as one principal factor in solubilizing They reveal that primarily steric repulsion by PEG minimizes the ad-
the drugs in polymeric micelles. Such interactions help to retain the sorption of plasma components on polymeric micelles and the physical
drug in the core and may retard the release rate of the drug to the ex- properties of PEG, such as sufficient flexibility and aqueous solubility,
ternal solution. Additional molecular interactions existing in the play a significant role in the anti-fouling properties as well [38]. Both
core, such as hydrogen bonding and pi-pi interactions, are no less the surface density and the MW of PEG are critical parameters when
significant as they can strengthen the molecular interactions be- forming the shell. These both influence the conformation of PEG on
tween the polymer and the drug in the core [28,39]. Many hydropho- the surface of the polymeric micelle where a “brush-like” conformation
bic polymers have been synthesized and evaluated as core-forming is preferred to sterically repel complement and plasma proteins [35,71].
blocks in polymeric micelles and show the capacity to solubilize It was also reported that PEG conformation ultimately affects the circu-
poorly soluble drugs (Table 2). lation time and clearance of polymeric micelles in vivo [35]. That is, mi-
In this section, frequently employed blocks of block copolymers will celles with a higher PEG density and a brush-like conformation had
be identified and their functionality in delivery platforms will be increased AUC in vivo which is essential for improved efficacy of
discussed. polymeric micelle formulations. PEG polymers have also been used in
delivery involving mucosal barriers, because PEG can confer mucus pen-
2.1. Hydrophilic blocks and anti-fouling polymers in block copolymers etrating properties. These mucus penetrating properties are conferred
by the polarity of the molecule and overall net neutral charge which
2.1.1. Polyethylene glycol has been shown to enhance the penetration of nanomedicines through
Polyethyleneglycol (PEG) (also known as poly(ethylene oxide) mucosal barriers [72,73].
(PEO)) has been the most frequently employed hydrophilic, shell- Synthesis of PEG is usually done by anionic ring opening polymeriza-
forming block in polymeric micelles thus far due to its safety profile in tion (ROP) of ethylene oxide and this synthetic process generates well-
humans and classification as “Generally Regarded as Safe” (GRAS) by defined PEG with a narrow molecular weight distribution [43,74,75].
the US Food and Drug Administration (USFDA). Low molecular weight The modification on the end group of PEG by appropriate chemical re-
PEG and PEG-conjugates of 20 kDa or less, have a low incidence of tox- agents (end-capping moiety) can expand the structural versatility of
icity [66–68]. PEG has been the gold standard for anti-fouling polymers PEG [76]. The chemical versatility of the end group of PEG includes ad-
throughout nanomedicine. When PEG forms the hydrophilic shell of the ditional reactive moieties for ligand labeling which enables further con-
polymeric micelles, its hydrophilicity and flexibility help the micelle jugation with other species of polymers to prepare target-specific block
avoid the adsorption of plasma proteins and opsonization processes copolymers.

Table 2
Hydrophobic polymers commonly used for constructing amphiphilic block copolymers.

Polymer Chemical structure Synthesis Ref

Polyethers Anionic ROP of PPO as a component of poloxamers – PEO-PPO-PEO [43,54,55]

respective alkylene triblock copolymers that are widely used in
oxides pharmaceutical formulations including SP1049C
polymeric micelles in clinical trials. Commercially
available (poloxamers).
Polyesters ROP of cyclic PLA is used clinically approved polymeric micelles drugs [40,56–58]
monomers (Genexol® PM, Nanoxel® M). PLGA has been used as
biodegradable surgical suture in clinic (Vicryl®).
Biodegradable.
Poly(amino acid)s Living Biodegradable. Increased hydrophobicity by benzyl [59–61]
polymerization of pendant group.
α-amino acid-N
carboxyanhydrides

Poly(2-oxazoline)s LCRP of 2-oxazoline Versatile library of polymer structures, ultra-high loading [22,45,62]
monomers capacity for several poorly-soluble drugs (ex. paclitaxel,
etoposide)

Poly(2-oxazine)s LCRP of 2-oxazine Ultra-high loading capacity several poorly-soluble drugs [30,46,63–65]
monomers (ex. curcumin)

1
Poly(propylene oxide), 2poly(D,L-lactide), 3poly(D,L-lactide-co-glycolide), 4poly(ε-caprolactone), 5poly(β-benzyl-L-aspartate), 6poly(γ-benzyl-α, l-glutamate), 7poly(2-isopropyl-2-
oxazoline), 8poly(2-n-propyl-2-oxazoline),9 poly(2-n-butyl-2-oxazoline), 10poly(2-n-propyl-oxazine), 11poly(2-n-butyl-2-oxazine).

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Recently, the phenomenon of accelerated blood clearance (ABC) of As for the anti-fouling properties of POx, Zhang et al. reported that
PEG has gained a lot of attention due to its detrimental effects on the both PMeOx and PEtOx had extremely low protein adsorption and cell
nanoparticle therapeutics which utilize PEG shielding [77]. It is well adhesion that is comparable to that of PEG-coating [87]. Interestingly,
studied that systemic exposure to PEG may cause ABC in humans [78]. the modification on the end group of those polymers had minimal effect
This phenomenon mainly arises from development of anti-PEG antibod- on the protein adsorption, unlike with PEG. However, the length of the
ies. ABC primarily occurs in human patients treated with PEGylated pro- polymer was significantly related to the anti-fouling properties with
teins as well as liposomal formulations coated with PEG [79]. In a Phase I longer block lengths exhibiting better anti-fouling up until a certain
study of refractory gout, patients were treated with a PEG-uricase ther- point where the effect of additional block length was negligible. Another
apy. About one third of the treatment group, who had previously been study done by Pidhatika et al. clarified long-term anti-fouling properties
treated with pegylated therapies at some point, had previously devel- of PMeOx coatings [88]. They found that PMeOx had excellent anti-
oped anti-PEG antibodies in the body. This resulted in lower AUC's fouling properties comparable to PEG for short term protein exposures.
and poor efficacy of the PEG-uricase treatment [80]. Another study by However, for a long-term exposure to media, it was found that only
Sherman et al. reported that the end-group of PEG contributed to the PMeOx, but not PEG, could maintain the anti-fouling properties. This su-
immune response to PEG-protein conjugates [81]. They compared the periority of PMeOx was due to the lack of degradation of PMeOx in bio-
immunogenicity of mPEG-protein conjugates and HO-PEG-protein con- logical fluids. In the case of PEG, though it had anti-fouling properties at
jugates using enzyme-linked immunosorbent assays. It was found that the early time points, it gradually degraded in biological fluids resulting
the methoxy group of the mPEG-protein contributed to a significantly in the loss of anti-fouling properties. This study, along with others, indi-
higher immune response than that of hydroxy group of HO-PEG. This cate that PMeOx may actually have superior anti-fouling properties to
study indicates that the end-group of PEG may affect the ABC phenom- that of PEG [89]. Currently, only PEtOx is approved as food additives
enon due to differential affinity to anti-PEG-antibodies. by the USFDA [90] and the safety profile of POx in humans, such as
The clinically approved doxorubicin liposome formulation, DOXIL®, the biodegradation of POx, needs to be investigated for the further clin-
has also shown the ability to induce the ABC phenomenon in human pa- ical development of POx-based micelle formulations. POx hydrophilic
tients mainly due to the development of anti-PEG antibodies in the body blocks have also demonstrated improved mucus penetrating properties
after the initial treatment of DOXIL® [82]. The effect of hydrophilic which could be useful in the oral delivery of polymer micelles [91]. They
chains of liposomal formulation on the genesis of ABC phenomenon showed that PMeOx had superior muco-penetrating properties, as mea-
was extensively studied by Dr. Szoka's group [83]. They found that sured by the diffusion coefficient in gastric mucus, compared to silica
both PEG hydrophilic shells and PMeOx hydrophilic shells on liposomes nanoparticles. PEtOx also showed some muco-penetrating enhance-
induced the ABC phenomenon in rats after the initial dose of the same ment, but less so than PMeOx. Overall, hydrophilic POx polymers, and
liposomes. Other hydrophilic polymers such as poly[N-(2-hydroxypro- especially PMeOx, have emerged as highly attractive anti-fouling stealth
pyl) methacrylamide] (HPMA), poly(vinylpyrrolidone) (PVP), poly(N, polymers which have the potential to replace PEG in these applications.
N-dimethylacrylamide) (PDMA) and poly(N-acryloyl morpholine) did
not induce ABC, indicating these polymers may have superior anti- 2.1.3. Other reported anti-fouling polymers
fouling properties. However, these polymers did not have as long of cir- Several other hydrophilic polymers have been identified which
culation times during the initial dose. In our view, the conclusion that show anti-fouling properties in preclinical models, suggesting their
these polymers may have superior anti-fouling properties requires potential to be applied as shielding agents in polymeric micelles. Hydro-
more extensive verification. philic poly(amino acid)s were employed in amphiphilic block copoly-
Interestingly, in contrast to ABC phenomenon induced by lipo- mers as anti-fouling agents to form the outer shell of the polymeric
somes with PEG shielding and PEGylated proteins, previous studies micelles. The biodegradability of poly(amino acid)s by endogenous pro-
revealed that polymeric micelle formulations with PEG shielding teases in vivo potentially confers the safety of these materials in the
did not induce significant ABC phenomenon in preclinical animal body [92]. However, this could mean that anti-fouling properties are
models as determined by the reduced anti-PEG antibody production. not sustained for long durations like those seen with POx systems. The
According to Shiraishi et al., anti-PEG antibodies did not affect the PK synthesis of hydrophilic poly(amino acid)s can be done via anionic
of PEG-b-poly(b-benzyl L-aspartate) (PEG-b-PBLA) polymeric mi- ROP using the N-carboxyanhydride of amino acids to generate poly
celles, while the PK profile of PEG-liposomes was marked by signifi- (aspartic acid) (P(Asp)), poly(glutamic acid) (P(Glu)), and poly(sarco-
cantly decreased circulation times after repeated dosing [84]. sine) [93]. Among hydrophilic poly(amino acid)s, poly(sarcosine) has
Another study revealed that the hydrophobic block of PEG- shown effective anti-fouling properties in recent studies [49,94,95].
conjugates was closely related to the binding of anti-PEG antibodies Polysaccharides such as dextran, heparin, chitosan, hyaluronic acid,
[85]. That is, proximal hydrophobic blocks are another key factor for and chondroitin sulfate have also shown anti-fouling properties and
the binding of PEG-specific anti-PEG antibodies to PEG moieties. inhibited protein adsorption on the particle surface in biological fluids.
Thus, polymeric micelle formulations with optimal PEG length and Interestingly, some studies revealed that dextran as a shielding agent
density on the surface may be less of a concern in promoting the for nanoparticles displayed anti-fouling effects and prolonged circula-
ABC phenomenon than their liposomal counterparts. Nevertheless, tion in animal models [96,97]. A comprehensive and concise review
the ABC phenomenon remains a concern for the use of PEGs and on polysaccharides as anti-fouling agents was reported by Doh et al.
the field is actively searching for suitable replacements. and this review may provide useful information for researchers in
selecting suitable polysaccharides with anti-fouling properties [50].
2.1.2. Hydrophilic poly(2-oxazoline)s Several studies have investigated the anti-fouling properties of PVP.
Poly(2-oxazoline) (POx)-based block copolymers recently gained a PVP can be synthesized via radical polymerization, and it has tradition-
lot of interest as novel biomaterials due to their biocompatibility and ally been used as an excipient in formulation design [98]. Allegedly, both
chemical versatility [44,45]. Hydrophilic POx such as poly(2-methyl-2- the pyrrolidone moiety and amide groups in the side chain are closely
oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx) have related to the anti-fouling properties of PVP, but comprehensive mech-
shown the anti-fouling properties to avoid rapid clearance by the RES anisms of these properties are still unknown [99].
in vivo. These studies demonstrated the potential of these hydrophilic Several other hydrophilic polymers such as PDMA, HPMA, and other
POx as stealth polymers [86]. POx can be readily synthesized via living zwitterionic polymers have been reported as anti-fouling macromole-
cationic ring opening polymerization (LCRP) and recently block copoly- cules [100–102]. Those polymers are expected to be suitable for the de-
mers composed of POx have demonstrated scalable synthesis and velopment as block copolymers for the efficient delivery of poorly
chemical versatility [45]. soluble small molecules in polymeric micelles formulations.

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2.2. Hydrophobic polymers in block copolymers and hydrophilic block, such as PEG, were often utilized to formulate
micelle systems. For example, micelle formulations prepared using
Hydrophobic segments of block copolymers play an essential role in PCL-b-PEG-b-PCL showed high loading up to 28% of paclitaxel [109].
solubilizing and encapsulating poorly soluble drugs in the core of poly- The clinically approved polymeric micelle formulation of paclitaxel
meric micelles. The core of the polymeric micelles features a hydropho- Genexol® PM formulation exploits mPEG-b-PDLLA to solubilize pacli-
bic environment which allows for the entrapment of poorly soluble taxel and is discussed in greater detail in Section 5.3 [110].
drugs via hydrophobic and potentially other types of interactions. This
allows encapsulated drug to stably reside in the core during systemic 2.2.3. Hydrophobic poly(amino acid)s
circulation and gradually be released to the external environment. Poly(amino acid) have often been used as hydrophobic core-
Hydrophobic segments of block copolymers can vary widely in their forming blocks in amphiphilic block copolymers for solubilizing
structure in order to effectively encapsulate poorly soluble drugs poorly-soluble drugs. Synthesis of poly-amino acids is usually done via
(Table 2). Commonly employed hydrophobic polymers are polyethers living polymerization of α-amino acid N-carboxyanhydrides [61]. Com-
and polyesters. More recently, a variety of POx and poly(2-oxazine) monly used hydrophobic poly(amino acid)s are poly(β-benzyl-L-aspar-
(POzi) based polymers such as poly(2-n-butyl-2-oxazoline) (PBuOx) tate) (PBLA) and poly(γ-benzyl-α, l-glutamate) (PBLG). According to
and poly(2-n-butyl-2-oxazine) (PBuOzi) have gained much attention Thambi et al., PEG-b-PBLG bearing the disulfide bond (PEG-SS-PBLG)
due to their high loading for physically encapsulating drugs [45,63,65]. could solubilize poorly soluble camptothecin and form micelles in solu-
tion [59]. The micelles displayed 20–125 nm size and the drug loading
2.2.1. Polyethers capacity was up to 12%. PEG-b-PBLA block copolymer was employed
Polyethers have been used as the core-forming segment for encap- to form polymeric micelles for the physical encapsulation of doxorubi-
sulating hydrophobic drugs. Generally, polyethers are synthesized via cin [2]. The micelle formulation exhibited 15–20% of doxorubicin load-
ring-opening anionic polymerization of alkenes to produce well- ing and a 57–70 nm of size distribution.
defined polymers with low polydispersity index (PDI) and molecular
weight (MW) distributions [43,54]. PPO and poly(butylene oxide) 2.2.4. Polyoxazolines and polyoxazines
have shown hydrophobic properties and, when incorporated in block POx and POzi block copolymers were recently introduced for drug
copolymers, have the capacity to solubilize hydrophobic drugs [4,103]. delivery applications and have shown high potential as materials for
PEO-PPO-PEO copolymers, which are called poloxamers (also known polymeric micelle drug carriers [45,63,111]. The synthesis of POx and
under the trademark of BASF formerly as Pluronic® and currently POzi can be achieved via LCRP process which results in strictly linear
Kolliphor® P grade), are often exploited as block copolymers for solubi- polymers of low molar mass distribution (PDI = Mw/Mn from 1.01 to
lizing hydrophobic drugs and preparing polymeric micelle formulations 1.3) and defined degrees of polymerization [45,86]. POx and POzi
[23]. In fact these block copolymers were the first used for the delivery represent a versatile library of polymer structures. Depending on the
of non-covalently incorporated drug in polymeric micelles. This concept 2-substitution of the 2-oxazoline or 2-oxazine monomers, the water-
was introduced by our group in the late 1980s and was initially termed solubility of the resulting polymers range from highly hydrophilic
“micellar microcontainer”, but is now widely known as a “micellar MeOx or EtOx described above to highly hydrophobic, e.g. 2-nonyl-2-
nanocontainer” [4]. In that study, Pluronic® block copolymer micelles oxazoline (NOx) [112]. Such structural variability makes easily accessi-
were used to solubilize a neuroleptic drug, haloperidol, and the micelles ble an expanded library of POx- and POzi-based block copolymers that
were conjugated with insulin or antibody to neurospecific antigens to can be used to produce polymeric micelle formulations of structurally
deliver this neuroleptic to the brain. Subsequently, Pluronic® block co- diverse, poorly soluble drugs [64].
polymers were extensively studied by our group and many others as Triblock A-B-A copolymers of POx consisting of hydrophobic PBuOx
materials for the design of polymeric micelles for drug delivery. A nota- block with two flanking hydrophilic PMeOx blocks, PMeOx-b-PBuOx-b-
ble property of select Pluronic® block copolymers to act as biological re- PMeOx, have shown unprecedentedly high loading for many poorly
sponse modifiers to sensitize multidrug resistant (MDR) and cancer soluble drugs [22,45,113,114]. Our group, and others, have reported
stem cells with respect to anticancer chemotherapeutics was widely re- several polymeric micelle systems composed of POx-based block copol-
ported and reviewed from mechanistic and translational points of view ymers [22,28,63,113–115]. We have screened potential hydrophobic
[25,104]. Pluronic® block copolymers were also used in the first poly- drug candidates and found that many hydrophobic drugs can be
meric micelle drug formulation for cancer chemotherapy that entered efficiently solubilized in these POx systems with extremely high drug
clinical evaluation in the early 2000s. Particularly, Pluronic® block co- loading (sometimes approaching or even exceeding 50% by weight
polymers were employed to manufacture the SP1049C formulation drug loading) [116]. For example, paclitaxel was extremely well-
which is composed of an anticancer drug, doxorubicin, solubilized in solubilized in POx up to a paclitaxel concentration of 40 mg/mL in aque-
the mixture of Pluronic® F127 and Pluronic® L61 [23]. This polymeric ous solution to form well-defined spherical micelles with a size of less
micelle drug is discussed in further detail in Section 5.3. Extensive re- than 50 nm [22]. The maximum loading of paclitaxel in POx was up to
view of the properties of poloxamers as micellar carriers for small mol- 50% which potentially minimizes the amounts of excipients in formula-
ecule drugs as well as biological response modifiers can be found tion design (Fig. 2). Stability studies confirmed that the POx-paclitaxel
elsewhere [23,25]. polymeric micelles were stable in aqueous media for a month without
any loss of paclitaxel. A number of other hydrophobic drugs such as
2.2.2. Polyesters etoposide, 3rd generation of taxanes, and vismodegib as well as multi-
Polyesters are other exemplary hydrophobic polymer candidates ple drug combinations were shown to be solubilized in the POx micelle
which are frequently used in the formulation design of polymeric mi- system with high loading [113,117,118]. Due to its high drug loading ca-
celles. Synthesis of polyesters is commonly done by ring-opening poly- pacity and safety profile, the POx system has drawn a lot of interest for
merization of cyclic esters and this synthetic strategy is known to use as a polymer carrier for drug delivery.
produce high molecular weight polyesters with narrow polydispersity Interestingly, it was recently reported that such high loading capac-
[105]. One major advantage of using polyesters is their biodegradability ity of poorly soluble drugs in POx micelle was due to the structure of
[60]. The in vivo degradation process of the polyester backbone pre- both the hydrophilic and hydrophobic blocks in POx triblock copoly-
vents the undesired accumulation of the polymer in the body, thus re- mers [119]. For example, PMeOx (which is more hydrophilic than
ducing the risk of chronic toxicity [60]. Examples of polyesters for PEG) was well-hydrated in the shell of highly drug-loaded POx micelles
solubilizing hydrophobic drugs are PCL, PDLLA and PLGA [106–108]. and had less interaction with loaded drug compared to PEtOx (which is
The block copolymers composed of the hydrophobic polyester block similarly hydrophilic to PEG). With highly hydrophilic properties,

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2.3. Stimuli-responsive block copolymers in polymeric micelle formulations

Polymers with stimuli-responsive properties are of interest as ma-

terials for polymeric micelle design due to their ability to modulate
drug delivery by internal chemical or external physical stimuli. Upon
exposure to either chemical stimulus (pH, hypoxia, redox, enzyme ac-
tivity) or physical stimulus (light, temperature), the physicochemical
properties of polymers, e.g. solubility, within the micelles can be al-
tered. This can be tailored to modulate the delivery functionality of
polymeric micelles, such as their accumulation at disease sites or re-
lease rate of the drug cargo. This section introduces some stimuli-
responsive polymers as components of amphiphilic block copolymers
for the delivery of poorly soluble drugs and discusses their principal
functions in drug delivery.
Temperature-responsive hydrophilic polymers are often incorpo-
Fig. 2. Comparison of various paclitaxel (PTX) formulations that are either clinically
rated in block copolymers used for drug delivery. Such polymers are
approved (Abraxane and Taxol by USFDA, Genexol-PM by South Korea's Ministry of
Food and Drug Safety) or undergone clinical trials (NK105) with the POx/PTX polymeric
characterized by lower critical solution temperature (LCST) and/or
micelle formulation. Taxol contains only about 1% wt. of active ingredient (m(PTX)max/m upper critical solution temperature (UCST) which define the mode of
(total)), while Genexol-PM and NK105 have much higher drug loadings. The maximal modulation of the polymer solubility by temperature. Thus far, poly-
paclitaxel concentration in solution (PTXmax) achieved with all four formulations is mers with LCST have been preferably employed in temperature-
below 10 g/L, while POx/PTX can reach almost 50 g/L. Compared to Abraxane and
responsive polymeric carriers for drug delivery. At temperatures
POx-PTX, NK105 and Genexol-PM formulations are significantly diluted down for injec-
tion, so that final PTX concentrations ([PTX]inj) are well below 1 g/L [6,7]. Of all compared below LCST such polymers are hydrophilic and water-soluble, but
PTX formulations, the novel POx/PTX polymeric micelle formulation exhibits the highest above LCST they dehydrate and transform to hydrophobic polymers
maximum tolerated dose (MTD) in mice. Reprinted with permission from [22] Copyright that phase-separate from a solution. If such a polymer comprises the
2016, Elsevier.
shell of a polymeric micelle that is stable in dispersion below the LCST,
once the temperature exceeds the LCST (such as upon injection or traf-
ficking to a hyperthermic site) the micelle precipitates and the drug
PMeOx in the shell could support super high drug loading micelle could be rapidly released to the environment [120]. Due to this
formulation and stabilize the micelle structure in solution. Also, a vari- temperature-responsive property, polymeric micelles with shell-
ety of hydrophobic POx blocks have been used to produce micelles forming constituents exhibiting LCST behavior could be employed as
with different cores which show differential solubilization profiles carriers for therapeutic drugs in conjunction with local hyperthermia
with respect to various poorly soluble drugs [63]. For example, one [121]. Commonly used polymers with LCST in the physiologically
study varied the side chain structure in the core forming blocks of POx meaningful range include poly(N-isopropylacrylamide) [122], poly(N,
and POzi A-B-A triblock polymers to determine the effect on drug load- N-dimethylacrylamide) [123] and their copolymers. The LCST values
ing [63]. The triblock copolymers composed of PMeOx-b-PBuOx-b- of such polymers are well-defined and can be fine-tuned by changing
PMeOx and PMeOx-b-PBuOzi-b-PMeOx were exploited to prepare the copolymer composition to enable temperature response of
these polymeric micelle formulations. In particular, the solubility of the resulting polymeric micelles within a desired temperature
the drug curcumin was compared between the two triblock polymers. range [124,125].
The POzi system has an extra carbon in the hydrophobic block backbone For example, Sun et al. reported that hydrophilic poly
compared to the PMeOx-b-PBuOx-b-PMeOx system, and this confers (N-isopropylacrylamide) in amphiphilic block copolymers exhibited
differential solubilizing capacity – the same drug, curcumin, is much temperature-sensitive behavior that facilitated the release of a loaded
less soluble in the POx only system than the POzi polymer. Meanwhile, drug (doxorubicin) from the polymeric micelles [126]. An in vitro
paclitaxel was highly soluble in PMeOx-b-PBuOx-b-PMeOx but is less cytotoxicity assay confirmed the temperature-sensitive micelle formu-
soluble its POzi analog [63]. lation showed enhanced cytotoxicity in MCF-7 cells above LCST
Also, we have recently reported a POx based diblock copolymer (37 °C) compared to that below LCST (20 °C). Wang et al. reported on
which has a hydrophobic block consisting of a triazine ring and exhibits a thermo-sensitive amphiphilic diblock copolymer, poly(N,
both hydrophobic interaction and likely pi-pi stacking capabilities [28]. N-isopropylacrylamide-co-N-hydroxymethylacrylamide)-b-PCL with
In this work, first PMeOx-b-poly(2-methoxycarboxyethyl-2-oxazoline) an LCST of ~38 °C [127]. The polymeric micelles of this copolymer loaded
is synthesized followed by the conversion of the methyl ester group to a with doxorubicin released the drug in vitro in a temperature-dependent
triazine ring structure via the condensation of N,N-dimethylbiguanide. fashion. At 14 °C only 20% of doxorubicin was released during 200 h,
Prior to adding the ring structure, the polymer was unable to form mi- while at 43 °C over 80% of the drug was released during the same time.
celles in aqueous solution. This polymer structure conferred some Additional useful temperature-responsive polymers with LCST be-
unique solubilization characteristics likely due increased modalities of havior are poly(2-isopropyl-2-oxazoline) [128], elastin-like polypep-
drug-polymer interaction, such as hydrogen bonding and pi-pi stacking. tides (ELPs) [129], and some substituted polysaccharides [130]. ELPs
These examples illustrate the breadth of chemistry and functional- are polypeptides composed of amino acids which exhibit elastin like
ization that can readily be performed with POx and POzi based systems properties that potentially induce LCST phase transition behavior. The
for the rational design of polymeric micelle systems. LCST behavior of ELPs is dependent on the additional amino acid moiety
POx-based polymers have shown little degradation in biological among amino acid sequence as repeating unit. Frequently employed
fluids in the short-term, but it was reported that POx can be de- ELPs has polypeptide structure with valine (Val), proline (Pro), glycine
graded in the long-term via oxidative degradation [45]. Although (Gly), and additional amino acid which may determine the physico-
an extensive body of work in preclinical models is available about chemical properties of ELPs such as LCST and their assembly properties
the safety profile of PMeOx-b-PBuOx-b-PMeOx (see for example in solution.
[22]), the safety profile analysis of other amphiphilic block copoly- Another important class of stimuli-responsive polymers are pH-
mers containing various hydrophobic POx or POzi blocks in sensitive polymers that are employed for targeted drug delivery to
humans has not been conducted. This evaluation is needed before acidic compartments in the body. Physicochemical properties of such
proceeding to clinical use in the future. pH-sensitive polymers can be modulated by changes in the

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environmental pH enabling the release of the cargos at target sites. conjugated drug was not easily released from the micelle core, resulting
Endosomes and the tumor microenvironment where the local pH is in negligible drug activity; albeit, the toxicity to the body was also
slightly acidic are the targeted spaces for such pH-sensitive polymers. decreased and circulation time greatly increased [149]. Therefore, in
Several pH-sensitive polymers have been employed in polymeric mi- the subsequent development, free doxorubicin was added in the
celle formulations including polycations such as poly(histidine) [131], formulation and solubilized in the hydrophobic core formed by PEG-b-
poly(4-vinylpyridine) [132], poly(N,N-dimethylaminoethylmetha- poly(Asp)-doxorubicin conjugate utilizing the “like-dissolves-like”
crylate) [133], poly(β-amino ester) (PBAE) [134], and polyanions, principle [150].
such as poly(acrylic acid) [135], poly(methacrylic acid) (PMAA) [136], In subsequent studies, a different type of “conjugate” was developed
and poly(sulfonamides) [137]. using PEG-b-P(Asp) and PEG-b-P(Glu) – which can form a coordination
For example, the carboxylic groups in PMAA are ionized and charged complex with drugs containing transition metal complexes such as cis-
at the physiologic pH 7.4 which renders the polymer soluble, while at dichlorodiammineplatinum(II) (cisplatin, CDDP), dichloro(1,2-
acidic pH these groups protonate and the polymer becomes insoluble diaminocyclohexane)platinum(II) (DACHPt) and cis-oxalato-(trans-l)-
due to the presence of methyl groups in the backbone of the main 1,2-diaminocyclohexane-platinum(II) (oxaliplatin) [151]. In this case
chain [136]. Therefore, PMAA can be used as pH-sensitive shell- the micelle formation of the block copolymer is driven by complexation
forming block in polymeric micelles that can enable precipitation of among the carboxylic acid groups on the polyacid block and the metal of
the micelle and release of the drug in the acidic environment. On the the drug molecule. One example is CDDP-containing micelles formed by
other hand, the imidazole groups in poly(histidine) are uncharged at reacting PEG-b-poly(Asp) and CDDP [152]. The complex of PEG-b-P
the physiologic pH 7.4 and become protonated at acidic pH. Therefore, (Asp) and CDDP spontaneously self-assembled into polymeric micelles
poly(histidine) can be used as a core-forming block in the polymeric mi- with a very narrow size distribution. The drug was released from the
celles that is hydrophobic at the extracellular pH but becomes positively micelles via ligand exchange with chloride ions in biological milieu. A
charged and soluble upon acidification, which can result in the local re- similar polymeric micelle formulation where CDDP was coordinated
lease of an incorporated drug [131]. with PEG-b-P(Glu) [153] evolved into a clinically evaluated polymeric
Another class of pH-responsive polymers are degradable polymers micelle drug, NC-6004 which is described below in Section 5.3.
such as PBAEs synthesized by Michael step-growth polymerization Kataoka's group also reported on PEG-b-P(Glu) polymeric micelles con-
using diacrylates and amines [138]. Due to the tertiary amine groups taining another platinum drug DACHPt [154]. Similarly to CDDP,
in the polymer structure, PBAEs exhibit pH-sensitive behavior with DACHPt was attached via coordination bonding of the platinum to the
the polymer being insoluble at neutral pH but degrading to soluble frag- carboxylic groups of P(Glu) block and the active drug was released via
ments in acidic and alkali environments. Such properties allow the use ligand exchange of DACHPt with chloride ions in the environment.
of PBAE as a core-forming segment for solubilizing poorly soluble The polymeric micelle drug NC-4016 based on the PEG-b-P(Glu) and
drugs in PEG-b-PBAE polymeric micelles at physiological pH 7.4 [139]. DACHPt complex is undergoing clinical trials and is described in
In weakly acidic environments the PBAE protonates, the micelle core Section 5.3.
swells, and the drug is rapidly released. Due to the presence of the cat-
ionic charge, PBAE-based copolymers were also employed as carriers for 2.5. Unimolecular micelles and cross-linked micelles
nucleic acids [140]. The molecular diversity of diacrylates and amines
greatly expands the library of PBAE-based copolymers available for de- The performance of polymeric micelles is intimately related to mi-
livery of drugs and nucleic acids [140–142]. celle stability, drug loading, release kinetics, circulation time, and
biodistribution. The formation of polymeric micelles from amphiphilic
2.4. Drug conjugates and complexes with block copolymers block copolymers in solution is thermodynamically favorable when
above the critical micelle concentration (CMC) of the amphiphilic mac-
The drug-polymer conjugates were introduced in the early work of romolecules. Below the CMC, micelles in solution tend to dissociate and
Helmut Ringsdorf in the 1970s to improve drug solubility, toxicity, the loaded drug may be unexpectedly dispersed in the solution. Since
and body biodistribution, which was followed by extensive studies on polymeric micelles are significantly diluted upon administration into
the development of drug-polymer conjugates [143,144]. One problem the body, classical amphiphilic block copolymers may disassemble
often encountered with hydrophobic drugs conjugated to water- after injection, which results in the loss of micelle functionalities. For
soluble polymers is that as the amount of drug conjugated increases these reasons, structurally or chemically modified polymeric carrier sys-
the hydrophobicity of the conjugate also increased, resulting in its ag- tems have been introduced aiming for the optimal drug release from the
gregation. This was the rationale for the proposal by Helmut Ringsdorf micelles. Such approaches are unimolecular micelles, core-crosslinked
to use block copolymers in which one block is used as solubility en- micelles, and shell-crosslinked micelles.
hancer – e.g. hydrophilic PEG and another as drug attachment scaffold Unimolecular micelles are topologically similar to self-assembled
that can be highly modified with drug [145]. In the early 1980s, micelles, but consist of single polymer molecules with covalently linked
Ringsdorf used PEG-polypeptide block copolymers with hydrophobic amphiphile block copolymer chains [155]. Dendrimers are commonly
and cyclophosphamide-containing side groups, where upon conjuga- used as building blocks to prepare unimolecular micelles, because of
tion of the drug, the drug conjugate block becomes hydrophobic and their well-defined globular architecture, high-branching, and controlled
the entire block copolymer becomes amphiphilic resulting in drug- surface functionality [156,157]. To increase the loading of poorly soluble
conjugate self-assembly into the polymeric micelles [143]. In these mi- drugs the dendrimer core can be modified with a hydrophobic block,
celles, the core was formed by the conjugated block and shell from the followed by the attachment of the hydrophilic chains. For example,
PEG block. According to their approach, a drug is chemically conjugated Wang et al. reported on an amphiphilic 16-arm star block copolymer
to the core-forming block of the copolymer via a carefully designed pH- consisting of inner lipophilic PCL and outer PEG blocks [158]. The core
or enzyme-sensitive linker, that can be cleaved to release a drug in its of the polymer was a polyamidoamine (PAMAM) dendrimer of genera-
active form within a cell. The appropriate choice of conjugating bond tion 2 with 16 terminal OH groups. These OH groups were used to initi-
depends on specific applications. ate polymerization of ε-caprolactone to form PCL blocks and then the
This same concept was used by Kataoka and colleagues in a series of free ends of PCL were coupled with PEG chains. The micelle formulation
works leading to development of polymeric micelle NK911, which was from the resulting 16-arm star-block copolymer, stPCL-PEG16 exhibited
evaluated clinically [146,147]. The original approach developed by this high loading of a hydrophobic drug, etoposide, up to 22% w/w and did
group used doxorubicin conjugated to the poly(Asp) chain of PEG-b-P not show toxicity on porcine kidney epithelial cells. However, it was
(Asp) block copolymer through an amide bond [3,148]. However, the pointed out that despite the star-block architecture the drug loaded

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micelles still represented aggregates of several unimolecular micelles 3. Drug-polymer interactions within polymeric micelles: Theory,
assembled together due to relatively loose 16 PEG chain outer shell modeling and experiment
[158]. To increase the density of the PEG chains in the shell a similar de-
sign was employed to manufacture a 32-arm star-block copolymer The self-assembly of amphiphilic block copolymers composed of im-
using PAMAM dendrimer of generation 3 coupled with either PCL or miscible blocks elicits the formation of core-shell micelle architecture in
poly(L-lactide) (PLLA) and then with PEG [159]. In this case, however, aqueous media when the block copolymer concentration is above the
the loading with respect to the same drug, etoposide was much lower CMC [182]. Several factors may affect the size and morphology of mi-
- 7.8 w/w for stPCL-PEG32 and 4.3 w/w % for stPLLA-PEG32. Generally celles in solution. These factors include 1) structural parameters of the
speaking, loading of unimolecular star-block copolymer micelles with block copolymer, such as chemical structure of the repeating units in
hydrophobic drugs is more challenging than that of linear amphiphilic each block, molecular mass of the blocks, and their mass ratio,
block copolymers, probably, due to lack of conformational flexibility of 2) block copolymer concentration, as well as 3) the environmental pa-
polymeric chains covalently attached to the same structural node. rameters, such as the temperature, ionic strength and/or pH for blocks
Chemical crosslinking approaches were also employed to im- containing ionizable groups (e.g. polyelectrolytes) [182]. The assembled
prove the stability and circulation time of self-assembled polymeric micelles feature highly ordered macromolecular structure having segre-
micelles. Either the shell or core could be crosslinked, and the drug gated hydrophobic compartment in the core surrounded by a hydro-
release could be modulated by cleavage of the crosslinks [160–163]. philic shell on the outer surface of the micelle which confines the
The resulting cross-linked micelles are, in essence, single molecules overall micelle architecture. From a thermodynamic perspective, the
of nanoscale size that are stabile upon dilution, shear forces and en- self-assembly process is driven by the minimization of the interfacial
vironmental variations (e.g. changes in pH, ionic strength, solvents free energy [14]. As a first approximation, 1) the hydrophobic segment
etc.) [155]. Various shell-crosslinked polymeric micelles were re- collapses and aggregates, which decreases the contact area of this seg-
ported by several groups, showing the effect of shell-crosslinking ment with the aqueous environment, and 2) the hydrophilic segment
on the drug release profile from the micelle formulations becomes hydrated and forms a shell, which further masks the core sur-
[164–167]. For example, Chang et al. reported a shell-crosslinkable face and reduces the interaction between the hydrophobic segment and
poly(methyl methacrylate)-b-poly(N-isopropylacrylamide-co-N- water. In addition to the interfacial free energy, the micelle thermody-
acryloxysuccinimide) block copolymer. Shell-crosslinking of the namics and the resulting micelle shapes are critically dependent on
polymeric micelle was done by the addition of ethylenediamine in the steric repulsion of the hydrophilic chains in the shell and the
the micelle solution. An in vitro release study showed a stretching of the hydrophobic chains in the core [183].
more sustained release of prednisolone acetate from the shell- Hydrophobic small molecules can be physically encapsulated (solu-
crosslinked micelle compared to uncrosslinked micelles [168]. bilized) in the core of the micelle during the self-assembly of amphi-
Bronich et al. reported polymeric micelles with cross-linked ionic philic block copolymers, which brings about the formation of
cores prepared by using complexes of PEG-b-PMAA copolymer and di- polymeric micelles in aqueous solution. A traditional view is that the
valent metal cations [169]. These complexes self-assemble into small solubilization process is primarily driven by hydrophobic interactions
spherical micelles with PEG shell and PMAA -cation complex core. between the incorporated molecules and the hydrophobic domains of
Crosslinking of the core followed by removal of the divalent metal cat- the micelles formed by segregated copolymer blocks in the micelle
ions by dialysis in the presence of a chelating agent results in formation core. The molecular interactions between encapsulated drugs and hy-
of a nanogel-like structure with covalently linked PMAA core and outer drophobic blocks not only assist the formation of the micelle, but also
PEG shell. The core can be further loaded with various drugs that can in- further stabilize micelle structure in solution. Additional cohesive forces
teract with the carboxylic groups, such as CDDP or doxorubicin. As an such as van der Waals forces, driven by the proximity of hydrophobic
example in a study by Kim et al. the cross-linking was done by using drug and hydrophobic segment of the polymer in the core, could
1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride and lower the CMC of the micelle, resulting in further stabilization of the mi-
1,2-ethylenediamine [170]. After removal of the divalent cations doxo- celle structure upon dilution [184,185]. Recent studies on the experi-
rubicin was incorporated into the core of such micelles via electrostatic mental analysis of polymeric micelles have shown that the drug-
and hydrophobic interactions, and the micelle exhibited high loading of polymer compatibility was achieved via more complex molecular
the drug up to 50% wt/wt. In acidic solution, the protonation of carbox- mechanisms of interaction than simple hydrophobic interactions
ylic acid moieties on PMAA accelerated the release of doxorubicin. This [63,65,115]. Other molecular interactions such as hydrogen bonding or
design was used extensively in subsequent studies with a number of pi-pi interactions are known to affect CMC values of drug-loaded poly-
cross-linked core micelles prepared using either PEG-b-PMAA or PEG- meric micelles and facilitate enhanced stability of the micelles [186].
b-poly(Glu) or other similar copolymers which in some cases were ad- In some cases, these interactions can involve not only the core-
ditionally modified with hydrophobic groups in the core-forming blocks forming hydrophobic blocks but also the shell-forming hydrophilic
to facilitate their self-assembly before cross-linking [171–175]. Various blocks and select drugs can be at least partially incorporated in the
crosslinkers including biodegradable ones were used to facilitate load- shell of the micelle [46,65]. In other cases, the hydrophilic blocks were
ing and release of the drugs in such micelles. Notable examples of reported to interpenetrate into the core of the micelle and affect the mi-
these studies include loading of the core of the core-crosslinked micelles celle CMC as well as the partitioning of the drug between the micelle
with CDDP, DACHPt and other anti-cancer drugs and multiple drug and the external milieu [29]. For the drug containing micelles, the mo-
combinations that then were used to treat cancer in animal models lecular interactions between the solubilized drug and the micelle are
[176–178]. Increased blood circulation time and tumor distribution of also important parameters that can affect not only the stability of the
drugs incorporated in such core-crosslinked micelles along with de- micelle but also its size and morphology, thereby strongly influencing
creased toxicity and improved anti-tumor effects of the micellar drugs the biological performance of the polymeric micelles as drug delivery
were reported. Successful targeted delivery of these drug-loaded mi- vehicles [114,115].
celles to the tumors using tumor-specific ligands attached to the micelle The CMC and the partitioning of the drug between the micelle and
outer PEG chains were also described [179–181]. However, the clinical the external milieu has long been considered a thermodynamic mea-
translation of the cross-linked micelle designs has not occurred yet, sure of the stability of the micelle as a drug carrier in equilibrium condi-
with one of the greatest challenges in our view being consistency of tions [29,187]. The extent of the partitioning is defined by the value of
the cross-linking chemistry and characterization of the chemical com- the partitioning coefficient, which depends on the drug solubility in
position including the number and spatial distribution of the crosslinks the aqueous solution and drug-polymer interactions in the micelle
in the resulting crosslinked micelles. core. Upon micellar drug dilution in water, the drug partitions itself in

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the external solution. Once the block copolymer concentration drops interaction energies and is expressed in MPa1/2. This parameter is deter-
below the CMC the micelle disintegrates into single block copolymer mined by Eq. (3):
molecules (“unimers”) releasing the remaining drug. In more complex rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
biological environments, the various biological molecules present in pffiffiffiffiffiffiffiffiffi H coh −RT
δHIL ¼ CED ¼ ð3Þ
these environments, such as serum proteins in blood, can bind the V
drug, thereby shifting the equilibrium towards drug release. Kinetic sta-
where δHIL is the Hildebrand SP, Hcoh is the cohesive enthalpy needed to
bility of the assembled micelles reveals the dynamic character of the mi-
infinitely separate a unit volume of molecules from each other, R is the
celles in aqueous media and its stability in solution over time [23,182].
universal gas constant, and V is the molar volume [213].
Upon dilution or external environmental changes, the dynamics
The entropy term −TΔSmix in this equation is negative and therefore
among individual micelles, such as exchange of polymer chains and
the substances are miscible when the enthalpy term ΔHmix is negligible.
the merging/disruption of the micelle structure, determine the stability
Based on this the two components with similar SP values are predicted
of the micelle structure over time [23,182]. The dynamic distribution of
to be miscible. According to the Hildebrand's SPs, two components are
the drugs between the polymeric micelles and various body compart-
predicted to be miscible when the difference in SPs is less than
ments after administration of drug-containing micelles in the organism
2 MPa1/2 [214,215].
plays a pivotal role in the PK of the drug [188].
In order to predict the solubility of polymers in solvents and account
A theoretical understanding of the solubilization processes of poorly
for a broader range of molecular interactions, such as dissimilar patterns
soluble drugs by amphiphilic block copolymers can be helpful to inform
of polar and hydrogen-bonding interactions, Hansen has proposed to
the design of novel drug delivery carriers. Computational approaches
use a multi-dimensional SP expressed as the square root of a sum of dis-
can facilitate formulation discovery and design by sparing loss of time
persion, polar, and hydrogen-bonding components as shown in Eq. (4):
and cost for experiments based on trial-and-error learning for drug sol-
ubilization in polymeric micelles. Various computational approaches rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ffi
have been proposed to predict the compatibility between the drug δT ¼ δ2d þ δ2p þ δ2h ð4Þ
and polymer during the encapsulation process, such as using the solu-
bility parameters (SPs) [189], Flory-Huggins interaction parameters where δT, is the Hansen's SP, δd, δp, and δh are the partial dispersion,
[190], Molecular Dynamics (MD) [191], and quantitative structure dipole-dipole and hydrogen-bonding components, each corresponding
property relationship (QSPR) [116] (Table 3). Experimental validation to their respective partial energies of cohesion [211].
of the prediction data from the aforementioned approaches has been In the case of Hansen's SP, two components are predicted to be mis-
performed. In-depth physicochemical analysis of drug-loaded poly- cible when the difference in SPs is less than (or equal) to the interaction
meric micelles by experimental approaches have revealed detailed mo- sphere radius defined by Eq. (5):
lecular interactions between drug and polymer systems which form
 2
micelles. These interactions play important roles both during the self- 4ðδd1 −δd2 Þ2 þ δp1 −δp2 þ ðδh1 −δh2 Þ2 ≤ R2o ð5Þ
assembly process and in the dynamic behavior of polymeric micelles
in solution, such as drug release to the external milieu. Investigation of where δd, δp, and δh are the partial SP of the components 1 and 2, and Ro
the molecular interactions provides explanations for the role of struc- is radius of interaction sphere in Hansen space.
tural factors of both components in polymeric micelles. The interpreta- The Hansen's SPs of various substances can be determined experi-
tion from those investigations gives us comprehensive insight into the mentally or estimated using the group contribution method (GCM),
plausible molecular interactions during micelle formation and guidance which estimates CED as the sum of partial contributions from all func-
in the development and intelligent design of polymeric micelle-based tional and structural groups of a molecule (see [213] for further review).
delivery systems. In this section, we will describe computational and ex- Several studies on drug and macromolecule compatibilities revealed
perimental approaches which explain the drug solubilization process by that the enthalpies of mixing derived from the calculation of Hansen's
polymeric micelles as well as showcase the recent progress in the char- SP could predict the solubilization of hydrophobic drugs by amphiphilic
acterization of polymeric micelles for the efficient design of polymeric block copolymers. Although, in some cases the predictions were not sat-
micelle-based delivery systems. isfactory (Table 3).
An exemplary study from the Allen group showcased the application
3.1. Theoretical and computational approaches of SPs as an indicator of polymer-drug compatibility in order to formu-
late the anticancer drug, ellipticine [192]. The physicochemical analysis
3.1.1. Hildebrand and Hansen solubility parameters of polymer–drug pairs was performed and the difference in total and
Two substances are mutually soluble when the free energy of their partial SPs, as well as enthalpies of mixing, were compared using a
mixing (ΔGmix) is negative. The free energy is defined by the following range of biodegradable polymers. The partial and total SPs of polymer
Eq. (1): candidates and ellipticine were calculated using the GCM. Interestingly,
the efficiency of drug loading in micelle formulations for PEG-b-PCL and
ΔGmix ¼ ΔH mix −TΔSmix ð1Þ PEG-b-PDLLA block copolymers was in good agreement with the predic-
tion of drug compatibility with the core-forming polymer blocks using
SPs. Also, along with the compatibility prediction, the release profile of
where ΔHmix is the enthalpy of mixing, ΔSmix is the entropy of mixing,
ellipticine from each formulation was closely related to the SPs. Specif-
and T is the absolute temperature.
ically, a compatible polymer, such as PCL, sustained the release of
According to the modern interpretation of the Hildebrand approach
ellipticine for over 6 days, while a less compatible polymer, such as
(for historical reference, please, see [211,212]), the mixing enthalpy can
PDLLA, showed a faster release of ellipticine which was complete within
be estimated from Eq. (2):
3 days. These results indicate that Hansen's SPs could predict polymer–
drug compatibility in the context of both drug solubilization and drug
ΔHmix ¼ ϕ1 ϕ2 ðδ1 −δ2 Þ2 ð2Þ release in polymeric micelles.
A subsequent study by the Kissel group, using similar PEG-b-PCL,
where ϕ1 and ϕ2 are the volume fractions of the drug and the polymer PEG-b-PDLLA and PEG-b-PLLA block copolymers and an anti-cancer
and δ1 and δ2 are the SPs of the drug and the polymer, respectively. drug, sagopilone, concluded to the contrary-that Hansen's SPs were
Hildebrand's SPs are determined by the square root of the cohesive not in good agreement with the experimental data such as solubiliza-
energy density (CED) that corresponds to the molecular self- tion capacity and micelle stability [193]. In this case the drug loading

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Table 3
Theoretical approaches for solubilization of poorly soluble drugs in polymeric micelles.

Approach Advantage Limitations Examples and result

Hansen's SP
Prediction of miscibility of a drug Allows for simple and rapid Does not account for excluded Ellipticine with PEG-b-PCL and PEG-b-PDLLA. Hansen's SPs
and core-forming block based estimate of drug-polymer volume, concentration of predictions correlate with experimental drug loading and release
on similarity of their Hansen's miscibility solutes, configuration and [192]
SPs estimated using GCMs conformation of molecules and Sagopilone with PEG-b-PCL, PEG-b-PDLLA and PEG-b-PLLA. Hansen's
drug interactions with shell SPs are not predictive. Drug solubilization accompanied by
supersaturation [193]
Five drugs with eighteen POx and POzi-based triblock copolymers.
Hansen SPs predicted solubilizing trends for a given drug among
different copolymers. The prediction would not allow comparing
different drugs with each other. [64]
Flory-Huggins interaction parameter
Uses a classic Flory-Huggins Prediction miscibility of binary Does not account for Eleven drugs with PEG-b-PCL. χFH ranks drug solubilization
solution theory for a binary mixtures with accurate polymer-solvent interactions, consistent with experimental data for a large drug set. [194]
mixture. Predicts miscibility of estimation of enthalpy changes. excluded volume effects, and Five drugs with mPEG-b-PCL. χFH ranks drug solubilization for any
a drug and a block copolymer In many cases allows correct geometry of molecules. Cannot one copolymer; not the dependence on PCL length [195]
based on the Flory-Huggins ranking of solubilization of distinguish between isomers Eight drugs in PEG-b-poly(ε-caprolactone-co-trimethylene
interaction parameter χFH different drugs in one polymer, or that have identical chemical carbonate) micelles. χFH predicts trend in drug solubilization and
which is estimated using compares solubilization of one structures but different effect of core forming block composition. Uses interaction
Hansen's SPs. drug in different polymers constitution and configuration. parameters for core and shell blocks. [196]
Underestimates polar and Doxorubicin with di-block copolymers of mPEG and modified PCL.
Coulomb interactions. χFH successfully predicts experimental drug solubilization
depending on core block composition. [197]
Cucurbitacin I and di-block copolymers of PEG and modified PCL. χFH
successfully predicts experimental drug solubilization depending on
core block composition. [198]
Bicalutamide and di-block copolymers of mPEG and PLLA-based
blocks. χFH successfully predicts experimental drug solubilization
depending on core block composition. [199]
Indomethacin with mPEG-b-poly(ε-decalactone) and mPEG-b-PCL.
χFH gives opposite results for drug solubilization in poly
(e-decalactone) vs PCL core, presumably, due to difference in core
crystallinity. [200]
Five drugs in eighteen POx and POzi-based triblock copolymer. χFH
determined by different methods are not predictive of drugs
solubilization. [64]
Molecular dynamics
Computer simulation method for Allows computing Hansen's SPs, Limitation on time span and the Nimodipine, fenofibrate Cucurbitacin B and Cucurbitacin I in
analyzing the physical Flory-Huggins interaction system size PEG-b-PCL diblock copolymer and branched multi-block copolymer.
movements of atoms and parameters and free energy of χFH values are consistent with experimental drug solubilization. MD
molecules. mixing. Can successfully predict simulations account for drug interaction with both PEG and PCL
the solubility of the drugs in the blocks and correctly predict binding of drugs with linear and
micelles, localization of the drug multi-block copolymers. [201–203]
in the micelle, size and Pyrene, nile red, and indomethacin with mPEG-b-PDLLA. χFH and
morphology of the drug-loaded free energy of mixing from MD simulations correctly predict the
micelles, drug localization within trend in the solubilization of drugs in the micelles. MD simulations
the micelle. correctly account for effects of block length and ratio. [204]
Itraconazole with PEG-b-PLGA. MD simulation reveals that the drug
localizes primarily at the interface, while the core of the micelle
remains empty; explains relatively low loading of this drug. [205]
Curcumin, paclitaxel and vitamin D3 with PEG-b-oligo
(desaminotyrosyl-tyrosine octyl ester suberate)-b-PEG. χFH and free
energy of mixing from MD simulations correctly predict a trend in
drug solubilization. [206]
Doxorubicin with poly
{γ-2-[2-(2-methoxyethoxy)-ethoxy]-ethoxy-ε-caprolactone}-b-poly
(γ-alkoxy-ε-caprolactone). MD simulations predict drug solubility
for polymers differing in side chains structures. [207]
Camptothecin with mPEG-b-PBAE. Predicts drug solubility and
provide information about the shape, size and morphological
transitions in drug loaded micelles. Provides insight on in the drug
release mechanism. [208]
QSPR
Powerful model with sufficient Requires large dataset for model Doxorubicin with 15 star polymers of different architecture
amount of dataset for statistics development containing PCL, poly[(2-diethylamino)ethyl methacrylate] and poly
can predict property such as drug (poly(ethyleneglycol)methacrylate) blocks. The QSPR approach was
loading in the micelle able to establish A quantitative relationship between the polymer
architecture and drug loading established. [209,210]
Many drugs in several POx-based amphiphilic copolymers. Based on
large data set, predicted loading of eight drugs in the micelles with
75% accuracy. [116]

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was high and the micelles were supersaturated with the drug, which attempt to lower χFH and thereby improve the solubilization in the mi-
could have contributed to the lack of correlation with the SPs. Interest- celle. This approach was shown to be successful as the prodrugs with
ingly, there was a drastic difference in the drug solubilization in the mi- the lower interaction parameters indeed demonstrated enhanced solu-
celles with PDLLA and PLLA core forming blocks. This was not reflected bilization. Similar observations were reported by Helen Burt's group
in the SPs which do not distinguish between stereoisomers. The PDLLA who examined five poorly soluble drugs (etoposide, paclitaxel,
and PLLA also differed in their aggregation behavior and degree of plumbagin, curcumin, and indomethacin) in polymeric micelles of
crystallinity which most likely affected the placement of the drug in mPEG-b-PCL [195]. They also described inverse correlations between
the core. the drug solubilization and χFH for any single copolymer. However,
Recently Luxenhofer's group published a comprehensive study in- this study also revealed a limitation of the classic Flory-Huggins ap-
volving five different drugs and eighteen A-B-A type amphiphilic proach for the polymeric micelles. That is, for each single drug the ex-
block copolymers with hydrophilic PMeOx block (A) and hydrophobic perimentally measured partitioning between the micelle and external
POx- or POzi-based blocks (B) [64]. The Hansen's SPs were calculated solution depended on the PCL block length, which was not reflected in
using GCMs as well as estimated experimentally by determining solu- the estimated χFH.
bility of polymers in solvents with different polarity. The experimental Nevertheless, several other studies compared the solubilization of
approach was proven to be more complex and difficult to interpret be- model drugs in polymeric micelles formed by various PCL- and PLA-
cause of the possibility of self-assembly of the block copolymers. The ex- based copolymers using experimental and theoretical approaches.
perimentally derived Hansen's SPs poorly correlated with the solubility Latere Dwan'Isa et al. reported on eight poorly soluble drugs (carbamaz-
data. However, the SPs obtained using Hoftyzer−van Krevelen method, epine, cimetidine, furosemide, hydrocortisone, indomethacin,
and to a lesser extent the computer-aided Yamamoto Molecule Break ketoconazole, ketoprofen, risperidone) in PEG-b-poly(ε-caprolactone-
method, were able to classify the drug-polymer compatibility fairly co-trimethylene carbonate) micelles [196]. An interesting aspect of
well. However, the computational methods in some cases were mis- this work is that to predict the drug-polymer compatibility they took
leading, especially in the case of non-solubilizable drugs. They also into account interaction parameters for both hydrophilic and hydropho-
could not account for small changes in the chemical structure of the bic blocks, de facto suggesting that the drug could interact not only with
side chains of the polymers that had tremendous impact on polymer the core but also with the shell. The resulting rankings agreed reason-
−drug compatibility. No differentiation between two different drugs ably well with the experimentally measured drug solubilities in the mi-
would have been possible using these SPs only. Overall, the study sug- celles. For the block copolymers with varying composition of the
gested limited applicability of the Hansen's SP approach for predicting core-forming block the solubility of a single drug (furosemide) posi-
drug solubilization in POx- and POzi-based triblock copolymer systems. tively correlated with the content of the trimethylene carbonate
co-monomer in this block. This also was consistent with the drug-
3.1.2. Flory-Huggins solution theory polymer miscibility prediction based on χFH. Yan et al. estimated the
The classic Flory-Huggins solution theory is based on the lattice drug core compatibility for doxorubicin in the polymeric micelles of
model to describe the thermodynamic behavior of polymer solutions. mPEG-b-PCL and mPEG-b-poly[(ε-caprolactone-co-γ-(carbamic acid
According to this theory, the free energy of mixing of a solvent and a benzylester)-ε-caprolactone] [197]. They reported that the Flory-
polymer at a constant temperature and pressure is expressed by Eq. (6): Huggins parameters could predict differences in drug solubilization
and controlled release between these micelle systems. Mahmud et al.
ΔGmix ¼ RT ½n1 ln ϕ1 þ n2 ln ϕ2 þ n1 ln ϕ2 χ FH  ð6Þ examined solubilization and release of cucurbitacin I in PEG-b-PCL,
PEG-b-poly(R-benzylcarboxylate-ε-caprolactone) and PEG-b-poly(R-
where ϕ1 and ϕ2 are the volume fractions of the solvent and the poly-
cholesteryl carboxylate-ε-caprolactone) micelles [198]. They also
mer, n1 and n2 are the number of moles of the solvent and the polymer,
found that the interaction parameter correctly predicted the trend of
and χFH is the Flory-Huggins interaction parameter.
the drug loading. However, in this case the release profiles of the drug
In the classic Flory-Huggins theory the interaction parameter χFH is a
from the micelle were not predictable. Rather, the release of the drug
scalar quantity that accounts for the enthalpy of mixing of a solvent and
depended on the core viscosity which likely controls the drug diffusion
a polymer and is expressed by Eq. (7):
from the micelle. Danquah et al. compared the solubilization of
V1 bicalutamide in polymeric micelles formed by PLLA-based copolymers
χ FH ¼ ðδ1 −δ2 Þ2 ð7Þ [199]. The drug solubilization in mPEG-b-poly(carbonate-co-lactide),
RT
containing additional carbonate groups in the core-forming block,
where δ1 and δ2 are the SPs of the solvent and the polymer, and V1 is the exceeded that in mPEG-b-PLLA, which was again consistent with the
molar volume of the solvent [216,217]. prediction based on the Flory-Huggins interaction parameter. However,
According to the Flory-Huggins theory, two components are pre- both Mahmud et al. [198] and Danquah et al. [199] noted that the nu-
dicted to be miscible if χFH is less than 0.5, or phase separated merical values of estimated χFH substantially exceeded 0.5 even for
if χFH > 0.5. The χFH values can be determined by estimating SP the drug-polymer pairs that displayed good solubilization. The lack of
values for each component as described above in Section 3.1.1. consistency with the classic Flory-Huggins theory which uses,
Several studies were reported that related experimental results and χFH < 0.5, for polymer-solute miscibility was accounted for by non-
theoretical estimates using classic Flory-Huggins theory for drug solubi- random chain distribution within the micelle and specific molecular in-
lization in polymeric micelles (Table 3). These publications suggest that teractions formed or destroyed upon incorporation of the drug in the
the interaction parameters in some cases were able to correctly predict polymeric micelle [199]. Likewise, it was pointed out that the use of
the trends in solubilization of different drugs in a block copolymer mi- the 0.5 as a miscibility cutoff is not suitable in the cases when Coulombic
celle or rank the solubility of a drug in different micelles. In most of or hydrogen bonding interactions are involved [198]. As a result, in most
these studies the χFH values were estimated using the partial Hansen's cases the classic theory χFH can only be used for polymer-drug interac-
SP calculated by GCM. tions ranking to reveal possible trends in drug solubilization and in
One of the earliest works in this area by Glen Kwon's group reported some cases it can be false.
on eleven drugs in polymeric micelles of PEG-b-PCL [194]. They esti- Indeed, several authors suggested limitations of the Flory-Huggins
mated the Flory-Huggins parameter for drug-PCL interactions and con- approach for predicting the solubility of the drugs in polymeric micelles.
cluded that as the value of this parameter decreased the solubility of the For example, Kakde et al. pointed out the discrepancy of the prediction
drug in the micelles increased. Then they prepared a series of prodrug using χFH estimates and experimental data for indomethacin in mPEG-
derivatives of geldanamycin with varying alkyl chain lengths in an b-poly(ε-decalactone) and mPEG-b-PCL micelles [200]. The χFH values

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D. Hwang, J.D. Ramsey and A.V. Kabanov Advanced Drug Delivery Reviews 156 (2020) 80–118

estimated in this work predicted that the drug solubility in poly(ε- A (fA) and B (fB) and the binary interaction parameters χAD, χBD and
decalactone) core was less than that in PCL core, which contradicted χAB related by Eq. (9):
the experimental data. The erroneous prediction was attributed to the
decreased crystallinity of the poly(ε-decalactone) core compared to χ eff ¼ f A χ AD þ f B χ BD − f A f B χ AB ð9Þ
PCL that was not accounted for in the classic Flory-Huggins approach.
An extensive study using five drugs and eighteen various POx- and Patel et al. successfully performed a series of MD modeling studies to
POzi-based block copolymers by Lubtow et al. reported that the Flory elucidate the essential factors in the solubilization of hydrophobic drugs
−Huggins interaction parameters estimated using various GCMs did (fenofibrate, nimodipine, cucurbitacin B and cucurbitacin I) in mPEG-b-
not correctly estimate the experimental drug solubilization [64]. As PCL micelles [201–203]. They found that Flory-Huggins interaction pa-
discussed in the previous section, they also concluded that the Hansen rameters computed by MD simulations could successfully predict the
SP values could better predict some trends in identifying good and experimental solubility of the drugs in the micelles, whereas those cal-
poor solubilizers among the block copolymers. culated by the GCM deviate from the experimental observations. Their
Overall, further advancement in the application of the Flory-Huggins MD simulations accounted for the drug interactions with both PEG
theory in polymeric micelle field is needed to correctly predict the solubi- and PCL blocks. They predicted that the increase in the PCL block length
lization of diverse drugs in a wider variety of block copolymers. The clas- should enhance solubilization of the drugs due to additional polar inter-
sic method fails to take into consideration the geometry of the molecules actions and hydrogen bond formation between a drug and PCL. More-
involved, the excluded volume interactions that are especially prevalent over, the simulations suggested differential localization of drugs
for long-chain copolymers, and cannot distinguish between isomers within a micelle depending on the drug and block copolymer ratio.
that have identical chemical structures but different constitution and For example, nimodipine at high drug loading has higher solubility in
configuration [201]. It also tends to underestimate polar and coulomb in- PCL and localizes deeper within the core, whereas fenofibrate clusters
teractions, and does not provide a straightforward approach for account- around both PCL and PEG. The MD simulations also predicted the effects
ing for the drug interactions with both core- and shell-forming block. of matching molecular architecture of the drug molecule and polymer
chain. A branched multi-block copolymer with three PCL blocks at-
tached to the same PEG chain was predicted to be a better solubilizer
3.1.3. Molecular dynamics
for cucurbitacin [203]. Due to an even distribution of the hydrogen
Computational approaches, such as MD, have been extensively uti-
bond donors and acceptors, the molecules could form a greater number
lized to investigate the molecular interactions in polymeric micelles
of hydrogen bonds with branching PCL chains compared to a linear PEG-
and obtain microscopic insights into the solubilization of small drug
b-PCL. An opposite trend in solubilization was predicted for nimodipine
molecules in the polymeric micelles [218]. MD is based on application
and fenofibrate that have the hydrogen bond acceptors only and could
of Newton's second law for the computation of the interactions among
not bind effectively with the branched multiblock copolymer, although
molecules to trace the successive molecular motions or conformational
no experimental evidence was provided to validate these predictions in
changes of the components in the solution [219]. In the case of poly-
this study.
meric micelles, MD simulations of the interactions between hydropho-
More recently Erlebach et al. applied MD simulation to elucidate the
bic drug and block copolymers in a given model are computed to
polymer–drug compatibility in mPEG-b-PDLLA micelles [204]. To facili-
generate the next successive conformations. This can simulate the pro-
tate the computation, they obtained Flory−Huggins interaction param-
cess of self-assembly during drug loading in the micelle core [218]. The
eters from the MD simulations that avoided explicit consideration of the
time scale required for these simulations is usually on the order of mi-
actual copolymer chains. The predictions from those Flory−Huggins pa-
croseconds or longer, which often makes fully atomistic simulations im-
rameters correctly accounted for the effects of the block length and the
possible, or at the very least extremely expensive and impractical with
PEG:PDLLA ratio and were in reasonable agreement with the experi-
the currently available computational power. Therefore, coarse-
mental data for the encapsulation of several hydrophobic molecules
grained MD simulations are more frequently exploited, in which the
(pyrene, Nile red, and indomethacin). MD simulation was also used to
number of the degrees of freedom is reduced in order to simplify and
understand a relatively low loading of itraconazole in the PEG-b-PLGA
expedite the simulations [218]. For example, in the dissipative particle
polymeric micelles [205]. The results demonstrated that the drug load-
dynamics (DPD), a coarse-grained methodology commonly used for
ing primarily occurred at the water-polymer interfaces, while the core
simulating the dynamic and rheological properties of fluids, several
of the micelle tends to remain empty. MD simulation could also rank
atoms or repeat units are grouped together and presented by a single
order drugs by their loading in polymeric micelles in a good agreement
bead [208]. Similar to MD simulation, time evolution in DPD is governed
with experimental data. For example, Costache et al. used a combination
by Newton's equation of motion.
of MD and docking calculations which predict preferred orientation of a
Several studies have investigated the drug loading and molecular in-
drug to a polymer for ranking three drugs (curcumin, paclitaxel and
teractions within polymeric micelles using the MD modeling approach
vitamin D3) by the energies of binding with PEG-b-oligo(desamin-
(Table 3). In contrast to the SP approach, the MD simulations are able to ac-
otyrosyl-tyrosine octyl ester suberate)-b-PEG micelles [206]. Further-
count for specific factors, such as hydrogen bonding or spatial distribution
more, MD simulations can predict drug solubility for polymers
of hydrophobic drugs in the micelle core [203]. Therefore, the combination
differing in side chain structure. Hao et al. successfully used MD simula-
of Flory-Huggins theory and MD could produce more reliable predictions
tions to predict particle size and drug (doxorubicin) loading in the mi-
of the drug-polymer compatibility than the interaction parameter calcu-
celles of various substituted poly{γ-2-[2-(2-methoxyethoxy)ethoxy]
lated from SP alone. Moreover, the MD simulations can also account for
ethoxy-ε-caprolactone}-b-poly(γ-alkoxy-ε-caprolactone) [207].
drug interactions with both shell and core forming blocks as well for the in-
In addition to the drug loading characteristics the MD simulations
teractions of the blocks with each other. For a mixture of a drug D contain-
could provide information about the shape, size and morphological
ing volume fractions of a drug D (ϕD) and a diblock copolymer A-B (ϕAB=
transitions in drug loaded polymeric micelles. For example, the MD
ϕA + ϕB) the free energy of mixing can be determined by Eq. (8):
and DPD simulations were integrated to investigate the micellization
 
ϕ of the pH-sensitive amphiphilic block copolymer, mPEG-b-PBAE [208].
ΔGmix ¼ RT AB ln ϕAB þ ϕD ln ϕD þ ϕAB ϕD χ eff ð8Þ
r AB This multi-scale simulation suggests that the PBAE block upon proton-
ation undergoes the transition from hydrophobic to hydrophilic. This
where rAB is the sum of the ratios of the molar volumes of each A and B is accompanied by a transition of the shape of the polymeric aggregates
to D, and χeff is the effective interaction parameter [204]. This effective from spherical to disk-like micelles and finally to vesicles, as dictated by
interaction parameter is defined using the volume fractions of blocks the counterbalance of free energies for the formation of shell, interface,

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and core. The loading of a drug (camptothecin) in such aggregates pre- which produced over four hundred data points reporting various drug
dicted by the Flory-Huggins parameter and MD simulation was in good solubilities. The study demonstrated that the computational QSPR
agreement with the experimental values. The drug was loaded into both modeling could predict the solubility of water-insoluble drugs with a
the hydrophobic core and the core's interface with the hydrophilic shell. high accuracy of up to 75%. These models were employed for virtual
Similar to literature reports, high loading could induce a morphology screening of drug libraries, and eight drugs predicted to have either
transition from micelles to vesicles. The simulations also provided in- good or poor solubilization in these polymeric micelles were selected.
sight in the drug release and suggested that camptothecin was released Three putative positives, as well as three putative negative hits, were
from the micelles and/or vesicles upon protonation of the PBAE block. confirmed experimentally. The success of this computer-aided strategy
Overall, these studies elucidated the molecular interactions occur- suggests its broad utility for predicting drug compatibility in a given mi-
ring in the core of the drug-containing micelles and suggested that celle system. Further work in this QSPR modeling involving adding
MD simulation can be useful for analyzing some important trends of more polymers to the database could allow for the informed selection
drug solubilization in polymeric micelle systems. of a given polymer to solubilize a desired drug molecule.

3.1.4. Quantitative structure property relationship 3.2. Experimental approaches

MD approaches are often inappropriate for large data sets commonly
encountered in polymeric micelle drug formulation research where the Many experimental approaches have been employed for the physi-
MD simulations could require enormous time, cost, and computational cochemical characterization of block copolymer micellization, structure,
power [218]. Instead, statistical approaches such as QSPR can be and morphology [221]. Generally, the polymeric micelle stability, size
exploited for the prediction of polymer–drug compatibility. Several and size distribution, along with the drug loading and drug release pro-
promising studies were published recently that conducted QSPR model- files are considered key properties. These properties of the polymeric
ing for the prediction of drug solubilization in polymeric micelles. QSPR micelle-based drug delivery systems affect their performance in vivo.
modeling is based on the statistical analysis of the data sets and has The combination of several physicochemical techniques is normally
been frequently used in the field of medicinal chemistry and chemical used to determine the relevant parameters of drug-loaded polymeric
toxicology for the prediction of efficacy and/or toxicity of small mole- micelles. Typically, (a) the zeta potential, z-average size, and polydis-
cules [220]. Wu et al. reported the development of a series of QSPR persity of polymeric micelle particle over time is determined by dy-
models to assess the loading of doxorubicin in polymeric micelles namic light scattering; (b) the particle number-average size, size
using the genetic function approximation algorithm [209,210]. In this distribution, and concentration by nanoparticles tracking analysis; and
study the polymeric micelles were formed by either four-arm or six- (c) the particle morphology by transmission electron microscopy
arm star polymers consisting of PCL, poly[(2-diethylamino)ethyl meth- (TEM), cryo-TEM, and/or atomic force microscopy. Size exclusion chro-
acrylate], and poly(poly(ethyleneglycol)methacrylate) blocks. The matography and gel permeation chromatography are also often used to
QSPR approach was able to establish a quantitative relationship be- determine MW and PDI of the polymers. NMR spectroscopy can be used
tween the polymer structure and drug loading. to confirm polymer structure as well as monitor reaction progress and
To maximize the quality of prediction by QSPR, a large data set is re- determine block lengths of block copolymers. The drug-polymer inter-
quired. Recently, Alves et al. have shown possibility of generalizing the actions and partitioning are sometimes characterized by fluorescence-
use of QSPR for cheminformatics-driven discovery of polymeric micelle or ultraviolet spectroscopy [29,30]. The drug concentration present in
formulations across multiple drugs to predict their solubilization in polymeric micelle dispersion is determined by high performance liquid
PMeOx-b-PBuOx-b-PMeOx tri-block copolymer micelles (Fig. 3) [116]. chromatography, matrix-assisted laser desorption/ionization time-of-
A total of 41 hydrophobic compounds were tested at various drug flight mass spectrometry, or similar analytical techniques. The
concentrations either individually or in combination with each other, polymeric micelle stability after preparation and storage in aqueous

Fig. 3. Study design of cheminformatics-driven discovery of polymeric micelle formulations for poorly soluble drugs. From [116]. Reprinted with permission from AAAS.

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D. Hwang, J.D. Ramsey and A.V. Kabanov Advanced Drug Delivery Reviews 156 (2020) 80–118

dispersions are commonly assessed for over two weeks by measuring The P value is a general thermodynamic characteristic depending on
the particle size and polydispersity index, drug concentration, and par- all types of interactions existing between the drug and polymer in the
ticle concentration [22]. Also, the drug release profiles in vitro are stud- micelles and drug and solvent in the external solution. It represents a
ied by dialysis under “sink conditions”, i.e. against external solutions measure of the free energy of transfer from the solution to the micelle,
containing excess of drug-binding molecules, such as serum albumins. ΔGmw, as presented by Eq. (11):
The retention of the drug in polymeric micelles in the presence of the
serum proteins in vitro is characterized by the adsorption column chro- log ðP Þ ¼ −ΔGmw =RT ð11Þ
matography [22].
In this section, we would like to focus mainly on those approaches The partitioning coefficients have been determined for many drugs
that are applied to drug-polymer interactions in polymeric micelle for- in polymeric micelles (for example, poloxamers) [29]. By comparing
mulations. We will not cover the more traditional physicochemical the partitioning coefficients between different polymeric micelles for a
characterization mentioned in the previous paragraph which is com- given drug, one can probe differences in the thermodynamic contribu-
monplace in the field. A more-fine, molecular-level characterization of tions of drug interactions with these micelles. The work by Kozlov
the drug and polymer interactions, as well as the short- and long- et al. determined the incremental contributions of the free energy of
range order of the drug and polymer chain distribution within the mi- the transfer of methylene group from an aqueous environment into
celle are also needed to understand the size, morphology, and stability the micellar hydrophobic core for a significant number of poloxamers
of drug-containing polymeric micelles in aqueous dispersions. Such (triblock copolymers consisting of PEO-b-PPO-b-PEO) that differ in the
studies can update the simplistic representation of polymeric micelles PEO and PPO block lengths (Fig. 4). This was accomplished by measur-
as a simple core-shell structure with strictly segregated polymer chains ing the partitioning coefficients for a homologous series of alkylated
and drug being exclusively localized in the micelle core. It is necessary fluorescein probes with different lengths of alkyl substituents and de-
to explore the microstructures within the drug-containing polymeric termining the partial contribution of the free energy per one methylene
micelles that potentially govern the solubilization capacity as well as group. Typically, the free energy of transfer from aqueous to a water-
the drug distribution in vivo. Several such studies describing these intri- immiscible organic solvent of a methylene group is −3.1 kJ/mol.
cacies are described below. Surprisingly, for the poloxamer polymeric micelles this value was less
suggesting that the core environment was less hydrophobic than that
3.2.1. Interactions with core and shell-forming blocks in drug partitioning of organic solvent and the free energy of transfer strongly depended
Studying of drug partitioning between the polymeric micelles and on both hydrophobic PPO and hydrophilic PEO length. This implies
external solution has been one approach to probe drug-polymer inter- that both types of blocks contributed to the microenvironment of the
actions and determine the thermodynamic characteristics of such inter- methylene group in the polymeric micelle cores. In other words, the
actions. Generally, the partitioning of the drug between the micelle and probe molecule in the micelle was coming in contact with both hydro-
external solution is determined by the partition coefficient, P philic and hydrophobic blocks in the core. The more hydrophobic copol-
determined by Eq. (10): ymers, P103 and P123, having long PPO and short PEO blocks had values
Cm similar to that of an immiscible organic solvent. As the length of PEO
P¼ ð10Þ chain increased the apparent penetration of PEO chain in the core also
Cw
increased and the core environment became less hydrophobic. The au-
where Cm and Cw are the concentrations of the drug in the micelle and in thors detailed that an increase in the PEO length allowed increased dis-
the water, respectively. tribution of ethylene oxide units and water into the micellar core,
decreasing its hydrophobicity and lowering the partitioning coefficient
[29]. In contrast, as the length of the PPO chain increased the core was

Fig. 4. (A) Partitioning coefficients of CH3−(CH2)N−Flu probes vs the number of methylene groups in the alkyl radical obtained for P103, P105, and F108. (B) Schematic presentation of
Pluronic micelle using spherical lattice model described in [222–225]. Hydrophobic PO units (black filled circles) localize in the central part of the micelle, while hydrophilic EO units (black
empty circles) and water molecules (empty cells) fill external layers. Parts a–c show incorporation of a solute (probe) having alkyl radicals of varying length (gray filled circles) and a polar
fluorescent group (gray empty circle). Note unfavorable contacts between hydrophobic groups of the solute and water molecules or EO units in the case of the probes with shorter radicals.
Reprinted with permission from [29] Copyright 2009, American Chemical Society.

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D. Hwang, J.D. Ramsey and A.V. Kabanov Advanced Drug Delivery Reviews 156 (2020) 80–118

becoming more hydrophobic due to a greater share of propylene oxide revealed that the degree of curcumin loading in polymeric micelle af-
units in the core. Using alkylated fluorescein probes with varying fected the localization of the drug in the micelle. At a low loading, the
lengths of alkyl substituents the “size” of the core was also probed as drug primarily accumulated in the core of the micelle where it
the break point in the incremental dependence of the partitioning coef- interacted with the hydrophobic PPrOzi block via hydrogen bonding be-
ficient (−ΔGmw) on the alkyl substituent length [29]. tween phenolic OH group of curcumin and the amide group in the poly-
The study also pointed out that the log(P) of the drug in a given poly- mer backbone. At higher loading, there was an increased interaction of
mer was strongly correlated to log(CMC) suggesting that similar types the drug with the carbonyl-carbon of the hydrophilic PMeOx blocks.
of interactions are guiding the partitioning of the hydrophobic drug Haider et al. followed up on this previous work and examined drug-
and self-assembly of poloxamer block copolymers into a micelle. The polymer interactions in polymeric micelles formed by several A-B-A tri-
limitation of this approach was that the partition coefficient probe mea- block copolymers, that had the same core forming block but different
surement was done at very low drug loading when the molecules incor- hydrophilic blocks - PMeOx-b-PPrOzi-b-PMeOx, PMeOx-b-PPrOzi-b-
porated in the micelles did not perturb strongly the polymer chains PEtOx and PEtOx-b-PPrOzi-b-PEtOx [46]. The drug (curcumin) loading
arrangement within the micelle structure. For the highly drug-loaded was extremely high when more hydrophilic PMeOx was used as the
polymeric micelles, different techniques are needed to characterize shell forming block in the polymeric micelles. Such micelles were also
the drug-polymer interactions. highly stable in solution. On the contrary, PEtOx was much less efficient
as a hydrophilic shell and the respective micelles did not solubilize as
3.2.2. Drug-polymer interactions in polymeric micelles by NMR much curcumin and were less stable at higher loading. The micelles
spectroscopy with the mixed PEtOx and PMeOx shell displayed an intermediate re-
Solid-state NMR (ssNMR) spectroscopy can be a valuable experi- sult. The authors further used several physicochemical methods to dem-
mental approach to study drug-containing polymeric micelles since onstrate the interactions of the drug with the polymers. They confirmed
the chemical shifts and the respective peak widths depend on the their previous observation that at low drug loading the drug is predom-
local environment and interactions between the drug and polymer mol- inantly localized within the hydrophobic core. As the drug loading in the
ecules. Advanced NMR techniques have been employed for the charac- polymeric micelle increased the interactions of the hydrophilic blocks
terization of drug and polymer interactions within the polymeric with the drug became evident. Notably, they observed drastic differ-
micelles. Callari et al. showcased the utility of 1D and 2D ssNMR to in- ences in drug interaction with PMeOx and PEtOx. 1H NMR spectroscopy
vestigate the correlation between drug loading and size of polymeric revealed that the latter displayed much greater propensity for
micelles [226]. They used a diblock copolymer composed of polymer- interacting with the drug at the lower loadings, which is possibly reduc-
ized fructose methacrylate as a hydrophilic shell-forming block and ing the colloidal stability of the drug loaded micelles by shrinking the
PMAA as a core-forming block which was conjugated with a platinum shell. This study suggests a significant role for drug interactions with
drug (dichloroplatinum;1,10-phenanthroline) via coordination bonds. the hydrophilic block in achieving extremely-high drug loadings. Over-
They compared micelles with varied drug loading and from ssNMR all, 1D ssNMR is able to probe the polymer-drug interactions of super
analysis concluded that as the drug loading increased the chain mobility high loaded polymeric micelles.
and swelling in the core and shell of the micelle decreased. They further However, not all intermolecular interactions with these amorphous
related these observations to the cytotoxicity of the higher and lower polymer-drug micelles can be easily probed using 1D ssNMR. The inclu-
loaded micelles in the cells [226]. The lower loaded micelle had greater sion of highly complex molecules, such as paclitaxel, which has 51 indi-
uptake and in vitro cytotoxicity, presumably due to the softer micellar vidual protons, can significantly complicate the analysis. To this end,
structure facilitating interactions between the micelles and the cells. Grune et al. have recently utilized 14N\\1H Heteronuclear Multiple
Another study using ssNMR by Pöppler et al. investigated polymeric Quantum Coherence (HMQC) ssNMR to show the interactions of the
micelles with various drug loading by physical entrapment (Fig. 5) [65]. PMeOx-b-PBuOx-b-PMeOx triblock copolymer and paclitaxel [227]. Fol-
They used amphiphilic A-B-A triblock copolymer PMeOx-b-poly(2-n- lowing the work of Callari et al. and Pöppler et al., they found additional
propyl-2-oxazine)-b-PMeOx (PMeOx-b-PPrOzi-b-PMeOx) and curcu- ways in which the loading of drugs in the polymeric micelle affect the
min as a model hydrophobic drug. Interestingly, ssNMR analysis of the intermolecular interactions. In the HMQC ssNMR they can visualize
changes in the chemical shifts and cross-peaks in 2D correlation the cross-peak interactions between nitrogens and hydrogens [227].

Fig. 5. Schematic model of the structural changes of the polymeric micelles upon loading with curcumin based on the solid-state NMR data and complementary insights. For each loading
stage, the additionally occurring interaction site is depicted.
Reproduced with permission from [65].

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At a low 10:2 (weight polymer:weight paclitaxel) loading, all cross which may be due to hydrogen bonding between the keto and
peaks occur in the aliphatic region of the 1H NMR spectrum indicating enol groups of curcumin and the carbonyl groups on the polymer,
that primarily interactions between aliphatic hydrogens on the polymer which indicates that hydrogen bonding plays a key role in curcumin sol-
and the nitrogens on the polymer backbone are occurring. Due to the ubilization. Additionally, a shift in the absorbance maximum from
micellar structure, these interactions are most likely intermolecular in 432 nm to 414 nm was observed as the concentration of curcumin in
nature (i.e. not within the same unimer). At a higher loading of 10:4, ad- the POzi-based polymeric micelle increased indicating drug localization
ditional peaks on the HMQC can be seen which are in the 4.5–8.0 ppm in a less polar microenvironment and the further exclusion of water
range on the 1H NMR spectrum. These peaks indicate that as paclitaxel from the core with increased curcumin loading.
loading increases, there is an increased interaction between polymer ni- Interestingly, as the curcumin loading increased, the micelle size as
trogens and hydrogens involved in the aromatic ring systems and hy- measured by dynamic light scattering first decreased and then in-
drogen bonding of paclitaxel [227]. The signal at the negative ppm creased [30]. Possibly, at low loading both curcumin and water are pres-
shift on the nitrogen NMR spectrum continued to increase with in- ent in the micelle core. As curcumin accumulates the size decreases as
creased paclitaxel loading indicating increased interactions with the water is excluded from the core, and then the size proceeds to increase
polymer amides. Therefore, 2D HMQC ssNMR provides additional struc- due to the continued accumulation of curcumin in the core.
tural insights, and in combination with 1D NMR, SANS, MD, and other Additionally, the quantum yield of curcumin was able to elucidate
advanced characterization techniques, yields a better understanding of additional properties of the polymer-drug interactions [30]. At the
drug-polymer interactions. same level of curcumin absorbance (same curcumin concentration), a
higher fluorescence intensity was observed in the POx polymer com-
3.2.3. Microstructure of polymeric micelles by small angle neutron pared to the POzi polymer. This must mean that the fluorescence quan-
scattering tum yield is higher in the POx polymer due to a decrease in non-radiative
SANS is an experimental method to probe materials structure at the decay pathways. They hypothesize that this is due to the decreased mo-
nanometer to micrometer scale using elastic neutron scattering at small bility of the POx polymer when compared to the POzi. The POzi has an
angle of scattering. This method has been previously used to reveal the additional methylene unit in the hydrophobic block background which
microstructure of the core and shell of drug-free polymeric micelles confers an added degree of flexibility and enables alternative non-
[228] and more recently extended to the thorough characterization of radiative decay pathways for excited state curcumin. This indicates
drug-loaded micelles. Schultz et al. reported on the morphological that the added degree of flexibility may play a critical role in the differ-
change of micelle structure upon the incorporation of paclitaxel in ential solubilization capacity of POzi with regard to curcumin.
PMeOx-b-PBuOx-b-PMeOx polymeric micelles [115]. In the absence of Additionally, they used fluorescence to probe the dynamic behavior
the drug, this particular block copolymer formed worm-like micelles of these micelles [30]. The addition of powder polymer to the polymeric
as shown in atomic force microscopy and cryo-TEM with an effective micelle formulations altered the Polymer/Drug ratio without changing
hydrodynamic diameter of about 200 nm. As the drug was loaded in the concentration of the curcumin. After this addition, in both the POx
the micelles at some critical point (>8% paclitaxel loading w/w), the mi- and POzi polymers, the curcumin fluorescence increased indicating
celles underwent a structural transition from worm-like micelles to the mobility and dynamic exchange/behavior of these polymer systems.
small and uniform spherical micelles of about 45–50 nm. Further phys- The work of the Luxenhofer lab in this regard shows the powerful na-
icochemical analysis of these paclitaxel loaded polymeric micelles by ture of using curcumin and other microenvironment sensitive
SANS revealed that the micelle core represents a raspberry-like sphere fluorophores for probing host-guest interactions in polymeric micelles.
morphology with a diameter of 4 to 6 nm. This was interpreted as the
presence in the core of multiple paclitaxel-rich domains (raspberry 3.2.5. In vitro drug release analysis
nodules) which increased in size from about 2.4 to 3.4 nm as the drug One essential step in the physicochemical characterization of poly-
loading increased. This was the first study to show the microstructures meric micelle systems is the in vitro drug release studies. Utilizing
in polymeric micelles, which could possibly assist ultra-high drug- these studies, one can visualize general release kinetics from a particular
loading of POx-based polymeric micelles. formulation or gain insight into the role of different drug-polymer inter-
The Luxenhofer group recently followed with a similar analysis action mechanisms occurring within the polymeric micelle formulation.
using the SANS technique which was extended to polymeric micelles These experiments are often performed using dialysis under sink condi-
containing a different drug, curcumin [46]. In this case a broader set of tions. Sink conditions ensure that complete dissolution of the drug from
POx- and POzi-based triblock copolymers was used. Consistent with the polymeric micelle system is feasible. Literature varies on the defini-
the NMR study discussed in the previous section, this drug revealed dif- tion of sink conditions, but in general the value is around 5 times the
ferential localization depending on its loading amount in the micelles. volume necessary for saturation of the drug in the system [229]. That
As the drug loading amount increased, the SANS data suggests that is, the drug must be released in a volume of liquid which is 5 times
curcumin initially located in the micelle core shifted to the periphery greater than that which is needed to solubilize the total amount of
forming an inner “shell” within the micelles. These data clearly show drug in the system. Performing these experiments under sink condi-
that a better understanding of the inner morphology of the drug- tions is essential for reliable data.
loaded micelles can be achieved using the SANS technique. There are two issues with drug dissolution and sink conditions only
addresses one of them. Sink conditions address the potential for satura-
3.2.4. Host-guest interactions by fluorescence analysis tion of the drug in the system, but not for the potential of saturation of
Fluorescence spectroscopy analysis can yield valuable insights into drug in the dosage form. Water must penetrate into the polymeric mi-
drug-polymer interactions in polymeric micelles in the case of drugs celle system and solubilize the drug for it to be released into the sur-
with fluorophore groups which are sensitive to the microenvironment. rounding media. Thus, the drug dissolution into the water solubilized
For example, Luxenhofer and colleagues have utilized curcumin as a phase in the micelle can be a rate limiting step for the overall release.
means for investigating host-guest interactions in polymeric micelles. However, this same kind of limitation exists during in vivo circulation,
Curcumin is a very hydrophobic drug with fluorescent properties, so the use of sink conditions for in vitro release is still a good initial as-
such as peak positions and quantum yield, which are highly dependent sessment of drug release behavior [229].
on the local microenvironment [30]. Major changes in curcumin peak Additionally, these in vitro release experiments can be performed in
absorption position were observed when this drug was encapsulated either buffers, such as PBS, or in a serum solution which better mimics
into POx- and POzi-based micelle systems. The absorption band at the in vivo environment. Studies performed in serum will often show
345 nm completely disappeared when encapsulated in both polymers quicker release profiles as hydrophobic drugs can directly transfer

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from the micelle to the serum protein molecules and the latter can serve excipient use and improving overall drug PK and therapeutic indices.
as carrier for the drug to the external solution. Experiments in both con- This has led to many formulations going into clinical trial. There are
ditions are important and give a more complete picture of polymeric many intertwined factors which influence polymeric micelle behavior
micelle drug release behavior. To establish sink conditions for very in vivo and the pharmacodynamic response. This complex interplay be-
poorly soluble drugs in the dialysis experiment serum albumin is tween polymeric micelle physicochemical properties and PK behavior is
added sometimes to the external solution to avoid the use of excessively not well understood. As a whole, in nanomedicine PK behavior is com-
large volumes. plex, and it is even more complex for polymeric micelles due to their
A study by He et al. demonstrated how one could probe the distri- unique, dynamic nature. Many factors influence this PK behavior such
bution of hydrophobic drugs between the micellar fraction and as drug loading, particle size, morphology, and the presence of multiple
serum bound fraction [22]. They incubated POx block copolymers therapeutic agents. Each of these can uniquely affect polymeric micelle
with serum solutions for 1 and 4 h to mimic what the polymeric mi- PK and add additional layers and considerations to PK modeling. In this
celles might experience in vivo. This was followed by the separation section, we will not consider polymeric micelle-cell interactions which
of the samples on a reversed phase solid phase extraction column. effect internalization. Rather, we will cover fundamental considerations
The column could effectively bind the serum albumin (and associ- for approaching and understanding the PK of polymeric micelles. With
ated drug) as well what little free, unbound drug was in the system. this information in-hand, we hope the reader can better design and ex-
Micelle bound drug was quickly eluted from the column. An acid/ ecute PK experiments to expedite the progress of promising formula-
methanol wash could then release the rest of the drug from the col- tions in the pre-clinical stage.
umn. Using this technique, they were able to show that about 83% of
paclitaxel was in the micellar fraction of the solution. The commer- 4.1. Drug loading and excipient derived toxicity in polymeric micelles
cial Taxol formulation only showed 20% of paclitaxel being eluted
in this fraction. This technique can be used to effectively probe The physicochemical properties of polymeric micelle products are
how micelles alter drug distribution in the body. closely related to the success of novel polymeric micelles in clinical tri-
As discussed above (Section 3.1.1), the Allen group used Hansen's SPs als. These properties must be well analyzed to validate the characteris-
to predict whether or not a given drug, ellipticine, was compatible with tics of the polymeric micelle products in the form of a certificate of the
varying block copolymer systems including PEG-b-PCL and PEG-b- analysis to identify unexpected inconsistencies during the manufactur-
PDLLA [192]. They then performed in vitro drug release studies. The ing process [221,235].
polymer with the lowest enthalpy of mixing (most negative and thermo- One critical parameter is the loading efficiency (LE) which repre-
dynamically favorable), PEG-b-PCL showed the strongest retention of the sents the portion of the drug entrapped in the micelles during the
drug when compared to the PEG-b-PDLLA system. The release rate was process:
also dependent on the drug: polymer loading ratio. The higher loading
ratios of 1:4 showed slower release rates than the 1:10 ratio. This was at- mdrug
LE ð%Þ ¼  100 ð12Þ
tributed to the “like dissolves like” principle as more drug present in the mdrug added
micelle core creates a more suitable and stable environment for the drug
to exist in. In another study, Hwang et al. synthesized an A-B diblock co- It is important for formulation to maintain a high LE to ensure that
polymer with a triazine ring based hydrophobic B block and PMeOx A the formulation process is economical and the drug loss is minimized.
block [28]. They compared the drug release of paclitaxel and bruceantin Another parameter is the loading capacity (LC) of the drug within
from the micelles of this copolymer to their release from a PMeOx-b- the polymeric micelle which is defined as:
PBuOx-b-PMeOx triblock copolymer micelles. While the rate of release
of bruceantin from both types of the micelles was approximately the mdrug
LC ð%Þ ¼  100 ð13Þ
same, the rate of release of paclitaxel was drastically different. Their mdrug þ mpolymer
work showed that over 24 h paclitaxel was not completely released
from the PBuOx polymer while it was completely released from the tri- The ratio of drug to excipient can be one of the significant factors for
azine ring-based polymer. While limited in scope, this study shows successful polymeric micelle products as therapeutics. High LC of poly-
that in vitro release can give some insights into the differential affinity meric micelles is desirable as it reduces the amount of excipient being
between drugs and various polymeric micelle core structures. used in the formulation, thus minimizing undesired toxicity derived
A study by the Kataoka group showed the development of from the excipients.
pH sensitive drug release from PEG-b-P(aspartate hydrazone Excipient derived toxicity has often occurred in patients and was a
adriamycin) [147]. This kind of technology can improve drug targeting dose limiting factor in clinical treatments. For example, the Taxol® for-
abilities by only allowing drug release from the micelles under certain mulation of paclitaxel consists of Cremophor EL and anhydrous ethanol
external stimuli. Other groups have explored this concept as well [236]. Though Taxol® has shown anti-cancer efficacy in human patients
[230,231]. These studies highlight the need to explore the in vitro and is still approved for humans, these excipients cause toxicity and
drug release of formulations in different pH conditions. For example, limit the ability to dose Taxol® [237]. Patients must often be pre-
the tumor microenvironment is relatively acidic compared to that of cir- treated with a corticosteroid before being administered Taxol® to pre-
culating blood. Analysis of in vitro drug release in lower pH's can aid in vent these hypersensitivity reactions. Due to the biocompatible nature
the optimization of pH-sensitive formulations and limit the number of of some block copolymers, polymeric micelles that encapsulate pacli-
costly in vivo studies by ruling out certain formulations prior to taxel have shown reduced excipient-derived toxicity. For instance, it
in vivo analysis. Other common stimuli which are used to trigger drug was shown that the Cremophor EL-free Genexol® PM formulation of
release are temperature and local redox environment [27,231]. We paclitaxel (approved in South Korea and several other countries) was
refer the readers to the following reviews for details on the design and much safer than Taxol® in cancer patients (390 and 200 mg/m2 doses
implementation of stimuli sensitive polymeric micelles [232–234]. approved, respectively) and there was much less concern for hypersen-
sitivity reactions compared to those that can be observed during Taxol®
4. Drug loading, pharmacokinetics and distribution of polymeric treatment [11]. Some other paclitaxel polymeric micelle formulations,
micelles such NK105, were also reported to have lower toxicity in clinical trials
compared to Taxol® [238].
Polymeric micelles have shown the unique potential to deliver crit- Our group has reported on the ultra-high LC of paclitaxel in POx mi-
ically important drugs with high drug loadings thereby minimizing celles which resulted in superior safety of the drug in preclinical animal

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models. POx formulation could encapsulate paclitaxel up to 4:5 weight 4.2. Pharmacokinetic analysis of polymeric micelle drugs
ratio of drug and excipient, resulting in LC of ~45%. Maximum tolerated
dose (MTD) of paclitaxel in mouse was about 7.5-fold higher in POx mi- Knowing the PK profile for all medicines, traditional and nano-
celle (150 mg/kg) compared to that of Taxol (20 mg/kg) [22]. Also, ac- based, is extremely important. The primary goals of clinical PK is to en-
cording to Alves et al., POx micelles could solubilize many poorly hance the efficacy and decrease the toxicity of a patient's drug therapy.
soluble small molecules with high LC (Table 4) [116]. Another paper Researchers look for strong correlations between a drug's concentration
out of our lab showed decreased liver and kidney toxicity in animals in various compartments (plasma, tumor, liver, kidney, etc.) and phar-
treated at the MTD dose of POx-Paclitaxel versus the clinically approved macologic responses. Insights into these relationships and mechanistic
Abraxane and Taxol formulations. The POx formulation showed no indi- PK differences between formulations can elucidate mechanisms of
cations for organ toxicity. Additionally, the activation of the comple- pharmacodynamic activity and improve patient outcomes. However,
ment system, which could lead to hypersensitivity reactions and the application of basic PK principles to nano-based pharmaceuticals
premature clearance of the formulation by macrophages, was evalu- is not trivial and requires some additional considerations. The advent
ated. The Taxol formulation showed a significantly higher activation of of nanomedicines introduces additional complexities to traditional PK
the complement pathway when compared to the POx formulation. studies, modeling, and analysis. This is reflected in the increasing num-
This is partly due to the significantly decreased amount of excipient nec- ber of studies focusing on the delivery of the nanoparticles to tumors,
essary to deliver the given PTX dose. This could also be due to an in- which is the most extensively studied facet of nanoparticle drug PK.
creased biocompatibility and/or decreased immunoactivation in Despite a significant body of literature on the use of various nano-
response to POx polymer [118]. This study indicates that both the bio- particles for cancer drug delivery, the complex interactions between
compatibility of excipients and high LC are required to minimize solid tumor physiology and nano-sized drugs are not fully understood.
excipient-derived toxicity. A high LC also increases the amount of drug One notable example is an analysis by Wilhelm et al. that has
per micelle, which can increase the amount of drug internalized into questioned the utility of nanoparticles for the treatment of solid tumors
cells [114]. This is especially true if the internalization processes become due to presumed low tumor delivery efficiency and extent of tumor
saturated so only a given number of micelles can be taken up per unit penetration [241]. However, they used a non-standard metric
time. It has also been noted that as polymer concentrations increase, for tumor delivery defined as percent injected dose (%ID) in tumor =
this can inhibit the caveolae mediated endocytosis at least in the case (AUCtumor/tend)*tumor mass. This metric reduces the time-
of certain poloxamers with sufficiently long PPO blocks [239]. For concentration data to a single average value, neglects overall exposure
these reasons, it is critical to maximize LC in polymeric micelle time and does not relate the tumor and systemic exposure [242,243].
formulations. A recent re-analysis by Price et al., based on the same dataset, has cast
High LC is also important for combination polymeric micelle formu- major doubt on the validity of Wilhelm et al. conclusions [244]. The
lations that contain two or more drugs in a single micelle for combina- studies included in the Wilhelm et al. analysis which reported matched
tion therapy. For the effective and safe delivery of drug combinations, tumor and blood concentration versus time data were re-evaluated by
the combination drugs should be well-solubilized in the polymeric mi- Pierce et al. using classical PK endpoints. These classical PK endpoints
celle so that the amount of the excipient and injection volume can be were compared to the unestablished %ID in tumor metric used in the
minimized for parenteral injection [114,188,240]. The effects of the co- Wilhelm et al. study. The conclusion was that the %ID in tumor was
loading of drug combinations in the same polymeric micelle carriers poorly correlated with the standard PK metrics which describe nano-
are further considered below. particle tumor delivery (AUCtumor/AUCblood ratio). The relative tumor
delivery of nanoparticles was ~100-fold greater as assessed by the stan-
Table 4
dard AUCtumor/AUCblood ratio than by %ID in tumor. Therefore, the flaw
Examples of active pharmaceutical ingredients with improved solubility in PMeOx-b- of the Wilhelm et al. analysis is that %ID in tumor does not relate
PBuOx-b-PMeOx micelles ranked by LE and LC. (Modified from Ref. [116]. Reprinted with tumor exposure to systemic exposure, as AUC ratio does, and is there-
permission from AAAS). fore not a true measure of the tumor delivery efficiency. Moreover, a rig-
Compound Aqueous log Pa Solubility in Fold LE % LC % orous nanoparticle PK analysis of tumor drug delivery should have
solubility POx micelle increased (mean) (mean) accounted for Cmax in the tumor, nanoparticle drug release, tumor free
(mg/mL)a (10 mg/mL POx) drug exposure and overall exposure time.
ABT-263 0.000212 7.77 8.00 37,736 100.0 44.4 Another important issue is that most studies have only measured
Podophyllotoxin 0.114 1.5 7.62 67 95.2 43.2 total drug (i.e., encapsulated plus released), and not the released drug
Etoposide 0.1b 0.60c 7.34 73 91.8 42.3
which is pharmacodynamically active. In many cases, the nanoparticle
Simvastatin 0.0122 4.68c 6.98 572 87.2 41.1
Efavirenz 0.00855 4.6c 6.90 807 86.2 40.8
encapsulated drug dominates the total drug profile for nanoparticle for-
Cisplatin Insoluble – 6.78 – 84.8 40.4 mulations. Thus, the nanoparticle-encapsulated drug uptake into the
prodrug (C6) tumor can often be inferred from the total drug profile. However, in
VE-822 0.0401 3.1 6.42 160 80.2 26.7 many cases, a considerable portion of the drug can be released by nano-
Paclitaxel 0.00556 3c 5.05 908 63.1 30.4a
particles before they reach their final destination. This is definitely the
AZD5363 Insoluble 1.31 4.98 – 62.3 33.3
Cisplatin Insoluble – 4.68 – 58.5 31.9 case of such dynamic systems as polymeric micelles that can partition
prodrug (C4) the drug between the micelle and its external environment. Therefore,
Teniposide 0.0598 1.24c 4.58 77 57.2 31.4 it is always best to measure these distinct fractions individually as it is
Cisplatin Insoluble – 4.30 – 53.7 23.9 the released drug fraction that correlates with toxicity and efficacy
prodrug (C10)
AZD8055 0.241 2.87 4.06 17 50.8 28.9
[245]. The PK analysis of traditional, excipient-based formulations usu-
Docetaxel 0.0127 2.4 3.71 292 46.4 19.0 ally treats the drug in two forms: that which is protein bound and that
Rutin 0.125 0.15 3.61 29 45.1 26.5 which is free and unbound. The unbound drug is the fraction which is
a
Data obtained from drugbank (https://www.drugbank.ca/) and predicted by either considered the pharmacodynamically active form. The protein bound
ALOGPS (http://www.vcclab.org/lab/alogps/) or ChemAxon (https://chemaxon.com/) drug is not pharmacologically active, and not available for metabolism.
when available, in other cases we refer to compounds “insoluble” if their solubility is less However, a lot of PK analysis of nanoparticle formulated agents mea-
than 0.1 mg/mL.
b
sures the total drug in the blood or plasma, and does not consider the
US patent (US4772589A).
c
Experimental data obtained from drugbank (https://www.drugbank.ca/), The exper-
difference between protein bound and free drug. The introduction of
imental value is from [22] and is different from that listed in [116], which was a mean of nanocarriers adds an additional layer of complexity to PK analysis, as
several values obtained for different conditions of micelle preparation. there is another drug fraction to consider that is a nanoparticle

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encapsulated drug. The existence of three drug fractions, protein bound, this novel methodology showcased advanced analysis of drug subpopu-
nanoparticle bound/encapsulated, and free unbound drug makes it dif- lations of nanoformulations, which are closely related to therapeutic
ficult to interpret PK profiles. Both unbound and nanoparticle bound outcomes, as well as a pragmatic analytical approach to determine the
drug can enter tumor microenvironments, but only the unbound drug bioequivalence of follow-on formulations and reference products, in
has pharmacological activity, thus the nanoparticles must release their order to facilitate 505(b)(2) regulatory review. Therefore, SITUA
encapsulated drug to the tumor microenvironment after local uptake. method fills a major gap in nano-based pharmaceutical research-that
Additionally, the protein bound drug fraction in target tissues can be dif- is, the analysis of distinct drug fractions in PK. It is of paramount impor-
ferent than when in the blood. Thus, one must measure all three frac- tance that we can measure all three fractions over time, and SITUA is
tions in all tissues in order to obtain the full PK picture. However, to one of the first steps towards this goal to elucidate how PK of various
the best of our knowledge there are no methods at this time to measure fractions influences pharmacodynamic activity.
encapsulated/protein bound/unbound drug fractions in tissue and this While this SITUA assay is an invaluable tool for nanomedicine PK
is an important area of research. analysis, its application to polymeric micelles which encapsulate hydro-
The Nanotechnology Characterization Laboratory (NCL) has recently phobic drug by physical entrapment may have additional levels of com-
developed the Stable Isotope Tracer Ultrafiltration Assay (SITUA) to plexity. It is important to consider that, in contrast to other common
probe these complex nanomedicine PK profiles [246]. The SITUA con- nanoformulations, polymeric micelles formulations exist as dynamic
cept is based on an assumption that a tracer amount of isotopically la- structures in which drugs are loaded via non-covalent interaction
beled drug in plasma will behave identically to drug that is released resulting in gradual drug release to the external environment and can
from the nanomedicine with regard to protein binding. The isotopically be reabsorbed by the micelles [246]. The SITUA assay assumes the isoto-
labeled tracer added to nanomedicine-containing plasma becomes a pically labeled drug, D*, only equilibrates with the plasma protein
measure of the free drug fraction in the system, which can then be bound drug and not with the nanomedicine bound drug (polymeric mi-
used to calculate nanomedicine encapsulated, unencapsulated protein celle encapsulated in our case) during the timescale of this assay. Thus,
bound, and unbound drug fractions simultaneously. The system is the assay assumes that the polymeric micelles are not behaving dynam-
spiked with the isotopically labeled tracer which rapidly achieves bind- ically under the timespan of the assay: that is, there is no exchange be-
ing equilibrium with plasma proteins. The plasma sample is then trans- tween free D* and drug which is encapsulated in the polymeric micelles.
ferred to an ultrafiltration device and the sample is separated by If exchange between unbound drug and micelle-bound drug is rapid,
centrifugation. One first determines the % bound isotopically labeled similar to protein binding, then the SITUA identifies the micelle binding
drug (D*) using Eq. (14) below. A known amount of D* is spiked into as an increase in protein binding. This was the case for the SITUA when
the plasma sample, and measurement of the D* in the reservoir and fil- it was applied to the Taxol formulation in which Cremophor EL micelles
trate are then used to determine % Bound D*, which the NCL claims be- bind in rapid equilibrium with unbound drug; this Cremophor EL mi-
haves similarly to the non-isotopically labeled drug, D. From there, celle binding was observed as an increase in protein binding that corre-
Eq. (15) can be used to determine the unencapsulated drug concentra- lated with formulation concentration, and influenced paclitaxel PK by
tion. With these two values determined, the nanomedicine encapsu- decreasing both clearance and volume of distribution [247]. While mi-
lated drug can be determined using Eq. (16). These equations yield celle drug exchange may occur at a slower rate than protein drug ex-
values for nanomedicine encapsulated drug, unencapsulated protein change, this was not the case for Taxol, and there is a significant need
bound drug, and unencapsulated free drug. for the evaluation of this phenomena. It is possible that this dynamic ex-
change of D* into the micelles occurs on a longer timescale than the
%Bound D∗ ¼ ð½Total D∗ −½Ultrafilterable D∗ Þ ∗ 100=½Total D∗  ð14Þ assay takes to run (~10 min, dependent upon the time for the tracer to
reach equilibrium with protein). If so, then the assay should be straight-
½Unencapsulated D ¼ ½Ultrafilterable D=ð1−ð%Bound D∗ =100ÞÞ ð15Þ forward to identify the polymeric micelle encapsulated drug fractions.
However, this could be different for all polymeric micelle systems and
½Encapsulated D ¼ ½Total D−½Unencapsulated D ð16Þ should be individually evaluated. Until then, the SITUA assay should
be cautiously applied to polymeric micelle drug systems and additional
This approach enables the analysis of the aforementioned subpopu- considerations such as dependence of the bound D* fraction on the for-
lations of drugs derived from nanomedicines in the systemic circulation mulation concentration should be taken into account. This is contrary to
in PK studies and can also be used clinically [247]. In a recent study, the polymeric micelle formulations prepared via stable covalent conjuga-
authors applied the SITUA method to analyze the PK profiles of several tion which may only react upon certain external stimuli and release
clinically approved nanomedicines and their generic or nanosimilar for- the cargo via the cleavage of the covalent bond.
mulations, some of which lack bioequivalence testing. This study show- Overall, polymeric micelle formulations that physically encapsulate
cases the utility of pragmatic methodology for the measurement of drug hydrophobic drugs are gradually releasing drug via both diffusion of
subpopulations in plasma [247]. They reported extensive bioequiva- the drug from the core to the external solution and drug binding to
lence studies on several nanomedicines, to compare PK profiles of plasma proteins. Thus, polymeric micelle formulations may exist in sys-
follow-on formulations to reference products (Janssen's DOXIL® vs. temic circulation as a dynamic system, comprised of the multiple forms
Sun Pharma's doxorubicin hydrochloride liposome formulation and of hydrophobic drug as discussed above. This dynamic nature must be
Celgene's Abraxane® vs. Samyang's Genexol® PM) [247]. SITUA was accounted for in the development of PK models. A recent study by our
employed in the analysis of plasma samples from animal PK studies in lab explored the complex, dynamic nature of POx polymeric micelles
order to quantify the subpopulation of each product. They demon- and the PK activity of these formulations [188]. This has also been stud-
strated that both doxorubicin (DOXIL® and Sun Pharma's formulation) ied earlier by Bulitta et al., who came to develop similar models to rep-
and paclitaxel (Abraxane® and Samyang's Genexol® PM) nanoformul- resent the regular micelle and emulsions systems [248]. They compared
ations had comparable encapsulated/unencapsulated/unbound PK the PK in cancer patients of Taxol and a Cremophor-free D-α-Tocoph-
parameters. Bioequivalence analysis by statistical analysis (two one- erol PEG succinate (TPGS)-based nanoemulsion formulation of pacli-
sided t-test) revealed that the Abraxane® and Genexol® PM formula- taxel. They sought to develop a mechanistic model in humans to
tions were bioequivalent in total drug PK parameters. However, the model both the free and total paclitaxel in the system. Although the
generic Taxol formulation did show some marked differences in key Bulitta et al. model was more comprehensive, there were some key sim-
PK parameters when compared to Abraxane® and Genexol® PM. This ilarities between these two studies and we will consider the Wan et al.
study could have been improved by using a higher animal number model [188] as an example. Rather than treating the drug as being
and a crossover study design to increase data variability. Nonetheless, injected into a single central compartment, the system must be modeled

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D. Hwang, J.D. Ramsey and A.V. Kabanov Advanced Drug Delivery Reviews 156 (2020) 80–118

as “polymeric micelle” compartment and a “plasma bound” compart-

ment. In the Wan et al. study, this modeling approach was applied
using a POx-paclitaxel polymeric micelle formulation. They also used a
tumor “effector” compartment as seen in Fig. 6. There was no measure-
ment of the free, unbound drug which is the shortcoming of this model
as the unbound drug is pharmacologically active and contributes to the
transfer of drug between compartments. However, the unbound drug
fraction is minimal in systemic circulation and neglecting its measure-
ment in a model is inconsequential as this fraction is generally negligi-
ble, and thus can be represented by the total unencapsulated drug
profile. The work showed that the critical parameter was the micelle en-
capsulated drug permeability into the tumor effector compartment.
When the micellar drug permeability into the tumor is much less than
the plasma bound drug permeability (Scenario A), then changes in the
micelle drug retention have little effect on the tumor AUC. However,
when the micelle permeability is similar to or better than that of plasma
bound drug (Scenario B), then the micelle retention has a major effect of Fig. 7. The drug amount-time profiles for the tumor obtained using a three-
the tumor AUC. That is, stronger micelle retention leads to significantly compartmental model. Simulations for basic scenarios A (solid lines) and B (dashed
increased tumor AUC (Fig. 7). This is because for both scenarios the lines) are shown. The three-compartmental model is presented in Fig. 6 and the values
of the PK parameters used in simulation were varied. In the A scenario, the penetration
clearance of plasma bound drug is assumed, based on experimental ev-
of micellar bound drug into the tumor is much less than that of plasma bound drug. In
idence, to be higher than that of micelle bound drug. Scenario B, in our scenario B, the micellar bound drug penetration is comparable to that of plasma bound
opinion, is the more realistic and biologically relevant scenario. Thus, drug. As you move through A0 and B0 up to A5 and B5, this shows the effect of
it is important for formulation designers to maximize the drug retention increased drug retention in the micelle. All units are arbitrary. Reprinted with
in the micelles to improve overall tumor drug exposure profiles. permission from [188].

The Wan et al. study did not discriminate between the micelle bound
and released drug within the tumor assuming that all drug will be phar-
macologically active if delivered to the tumor, which may be a fair as- would involve dependence of the PK on the micelle concentration and
sumption for the micelles used in the study. However, as a general would need to be considered separately. An additional layer of complex-
case, both drug subpopulations should be accounted for as the Cmax of ity for the modeling of the PK of polymeric micelle drugs is that in sys-
the unbound form achieved in the tumor is strongly correlated to phar- temic circulation, just as in vitro, the CMC of polymeric micelle materials
macodynamic activity. Measuring drug concentrations in tumors can be can play a central role in the PK. Upon dilution of the polymeric micelles,
a bit more complex than measuring concentrations in plasma, as there is should the polymer concentration decrease below the CMC, the micelles
some in the tumor interstitial area as well as intracellularly. The lysis of will disassemble over time altering the drug populations in circulation.
cells and digestion of tumors is often necessary for these measurements. In this case, there will eventually be no “nanoparticle encapsulated”
Microdialysis can help separate the two subpopulations and in some fraction thus increasing the protein bound and free, unbound concen-
cases noninvasive imaging techniques such as NMR, quantitative auto- trations in circulation. The CMC of polymeric micelles should be low
radiography, positron emission tomography can be used to visualize enough to endure dilution upon infusion, thus the micelle structure
drug concentrations and localization in the tumors [249]. would be intact during the systemic circulation and avoid unexpected
It should be pointed out that both Wan et al. [188] and Bulitta et al. drug loss [23]. While concerns over CMC are valid, it is also important
[248] models relied on stability of the micelles and did not account for to note that this is not an instantaneous process, rather it could be kinet-
a situation where formulation could influence the unbound drug con- ically dominated. While the micelle disassembly upon dilution below
centration, like in the case of equilibrium micelle binding. Such a case the CMC is thermodynamically favored, the disassembly process can

Fig. 6. A three-compartmental model describing the PM drug delivery to a tumor. The drug is administered as bolus in the form of PM (1) and is subsequently distributed between the
plasma (2) and tumor (3) compartments. The PK constants correspond to: k12 - rate of drug transfer from PM to plasma; k21 – rate of drug re-capture from plasma to PM; k13 - rate
of transfer (permeability) of the micellar drug to tumor; k23 - rate of transfer of the plasma bound drug to tumor; k31 and k32 – rates of drug reabsorption from tumor to PM and
plasma, respectively; k10 and k20 - micellar and plasma bound drug clearances, respectively. The model assumes that the drug solubility in blood is very low and the free drug form in
the blood is therefore neglected. Reprinted with permission from [188].

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take hours, especially in micelles with “glassy cores” [250,251]. It is also Not only hydrodynamic size, but also micelle morphology can play a
important to note that the CMC of a micelle system could depend on the role in PK and distribution. For example, the Discher group was to our
drug loading in the micelle. A higher drug loading could improve micelle knowledge the first to report on drastic PK differences between spheri-
stability in circulation. A previous study by Batrakova et al. showed that cal and worm-like polymeric micelles made from the same block copol-
the clearance of the polymer (Pluronic P85) was determined by the re- ymer [256]. The Discher Lab used PEO-b-PCL block copolymers and have
lease of the single polymer chains (unimers) and was unaffected by de- reported on the increased systemic circulation times of worm-like poly-
livery of a dose with the polymer concentration being above or below meric micelles compared to spherical micelles. The Discher lab did not
the CMC [252]. This indicates that unimers were cleared by the kidneys use drug loaded micelles for their study so only monitored the polymer
while micelles bypassed glomerular filtration. However, this does not distribution. In contrast, Wan et al. have recently reported on the highly
apply to drug which would be contained in the micelles. When polymer loaded worm-like POx-based micelles with therapeutically relevant
concentration drops below the CMC, the redistribution of drug into the concentrations of the drugs [114]. This study suggested that the
unbound fractions can then increase the observed clearance (but not worm-shaped POx micelles co-loaded with two drugs (etoposide and
necessarily depending on rate limiting metabolic processes). Only un- hydrophobic cisplatin analog) have increased accumulation of the
bound drug can be metabolized, so as polymer concentration is de- drugs into tumor compared to spherical micelles loaded with just one
creased below the CMC, the equilibrium shifts as drug is slowly drug. They reported that the overall AUC of the drugs in the tumor
removed from the “micellar compartment” and the unbound concentra- from worm-like micelles were higher than from spherical micelles.
tion can increase making more drug available for metabolism. However, other factors, in particular better retention of co-loaded
While nanomedicine PK is highly complex, it is clear that the dy- drugs compared to single-drug micelles, rather than the shape alone
namic nature of polymeric micelles presents many additional consider- could have also contributed to this phenomenon as discussed below.
ations and opportunities in analytical processing of samples and the PK In addition, although worm-like micelles circulate longer and could
modeling of drug systems. This is still an area where there is an im- eventually accumulate more in the tumor, they can release the drug be-
mense need for further work and characterization of these fundamental fore they reach the tumor. Therefore, as drug is released from the mi-
PK processes. Additional analytical techniques may need to be devel- celles over time this does not necessarily mean that drug exposure in
oped to probe polymeric micelle systems which can account for their the tumors is higher from worm-like micelles. The further comparison
dynamic nature. For now, we must be sure to utilize proper PK metrics, of highly loaded spherical and worm-like polymeric micelles is needed
such as the tumor/plasma AUC ratio, and validate novel metrics against to consider morphology of micelles and how this may affect circulation,
traditional analyses. A proper understanding of this complex PK and de- biodistribution, accumulation, and targeting.
sign of polymeric micelle PK analysis will ensure that literature moving Other factors must be considered as well which may affect the hy-
forward is reliable and truly represents the field. drodynamic size and shape of polymeric micelles in systemic circula-
tion. One such factor is the effect of dilution of polymeric micelles by
plasma. This dilution as discussed above could possibly result in a con-
4.3. Hydrodynamic size and morphology of polymeric micelles and drug centration below the CMC causing the disassembly of the polymeric mi-
distribution celles altogether but could also lead to changes in size and morphology
or morphology rather than complete disassembly. Additionally, surface
Hydrodynamic size of polymeric micelles is another important factor charge of polymeric micelles could affect their biodistribution. Lastly,
to consider for clinical applications. It is well-established that the size the release profile of drug from polymeric micelles can subsequently af-
distribution of nano-sized particles may affect their biodistribution fect the hydrodynamic size of polymeric micelles over time. Some stud-
when administered, resulting in either extended systemic circulation ies have shown that the release of drug, and lower Drug/Polymer ratios
or faster clearance from the body. Particles with size ranging over allow the incorporation of solvent (water) into the micelle core which
200 nm could be caught by the liver, while smaller nanoparticles less increases the hydrodynamic size [30]. If this size increase is large
than 10 nm would be easily cleared by the kidney [239]. In some previ- enough, this could affect key PK parameters such as clearance and vol-
ous studies, it was shown that sizes from 50 nm–100 nm are effective in ume of distribution as well as penetration of micelles in the tumors.
preclinical models, suggesting ideal particle size distribution is neces-
sary for efficacy of polymeric micelles [253,254]. The size of polymeric 4.4. Polymeric micelle combination therapies and effects on drug retention
micelles currently in clinical trials are less than 100 nm [6,7]. Thus, and pharmacokinetics
one may speculate that size range from 20 nm to 100 nm can be effica-
cious as polymeric micelle formulations. In fact, the study by Kataoka's Combination therapy has been largely exploited for the treatment of
group has shown that tumor accumulation of the polymeric micelles de- various types of cancer based on related pathways of oncogenesis or
pends both on the particle size and tumor desmoplasticity with smaller utilizing a combination of agents which affect the tumor microe-
polymeric micelles (size) displaying better accumulation in more nvironment [257,258]. Frequently employed combinations are 1) a
desmoplastic tumors [255]. To increase penetration into such tumors combination of several chemotherapies for killing cancer cells, 2) a com-
the particle size of the drug carriers must be minimized. Kataoka and bination of chemotherapy with additional agents, such as tumor micro-
colleagues have shown that when the drug is encapsulated in small environment modifiers, or more recently 3) a combination of
polymeric micelles (~30 nm), both the penetration of the micelles into immunotherapy with anticancer agents [259]. One recent study
the desmoplastic tumors and anti-tumor effect of the drug can be im- highlighted that even the best combinations of the chemotherapeutic
proved [255]. An alternative approach discussed in the next section, is agents determined in vitro as having the most potent and highly syner-
the co-delivery of two drugs where one drug is used to modify the gistic effect against non-small cell lung cancer (NSCLC) cells may not be
tumor microenvironment and reduce desmoplasticity while the other effective against the in vivo tumor even at the MTD of those combina-
drug acts as a traditional chemotherapy. Recently, a study from our tions [259]. In contrast, resiquimod, an imidazoquinoline TLR 7/8 ago-
lab showed the improved PK profile of the drug vismodegib when en- nist, solubilized in POx block copolymer (PMeOx-b-PBuOx-b-PMeOx)
capsulated in a POx micelle for treatment of a pediatric brain tumor – micelles had a superior tumor inhibitory effect in a metastatic model
medulloblastoma [113]. The POx-vismodegib formulation showed a of lung adenocarcinoma, relative to anti-PD1 immune checkpoint block-
1.6-times increase in tumor AUC and overall improved delivery to the ade therapy as well as platinum-based chemotherapy, which is the
brain. The micelle formulation also resulted in a lower Vd which could mainstay of treatment for NSCLC. Investigation of the in vivo immune
lead to fewer off-target affects than the conventional formulation's status following resiquimod in polymeric micelles treatment showed
less than ideal distribution to peripheral tissues [113]. that resiquimod-based stimulation of antigen-presenting cells in the

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tumor microenvironment resulted in the mobilization of anti-tumor

CD8+ immune response [259]. This study demonstrates the promise
of optimally delivered and nanoformulated immunomodulating thera-
peutic agents and possibly their combinations with chemotherapy for
treatments of metastatic NSCLC. Ideal combination therapy requires
precise drug exposure to induce synergistic therapeutic effects. Thus,
the administration and subsequent disposition of combinations to tar-
get tissue with the desired ratio of the delivered agents is highly
warranted.
Hydrophobic small molecule combination therapy is hindered due
to poor PK profiles of the molecules, similar to that of single drug
therapies. However, these combination therapies are complicated
even further by the inclusion of additional active pharmaceutical in-
gredient (API). One of the main factors that determine PK profiles of
small molecule drugs is the aqueous solubility which hinders adminis-
tration of such molecules via the parenteral route. For this reason, the
effective solubilization of hydrophobic small molecules and co-
encapsulation of drug combinations in a single micelle is important
Fig. 8. Multiple chemotherapeutic agents in high capacity poly(2-oxazoline) micelles.
for successful combination therapy. The ability to solubilize multiple (Drug designations: BTZ – bortezomib, DTX – docetaxel, ETO – etoposide, PTX –
drugs in the same micelle can yield more predictable PK profiles and paclitaxel).
allow for the solubility of new molecules and additional combination Reprinted with permission from [117] Copyright 2012, American Chemical Society.
options. Although, as of today no polymeric micelle formulation for
combination therapy is USFDA approved, but the design of pharmaco-
logically effective drug combinations in polymeric micelles is highly micelles co-loaded with three drugs (paclitaxel, 17-AAG, and
warranted. etoposide) displayed improved formulation stability in aqueous
Kwon and colleagues conducted a series of studies on a drug com- media. Presumably, multiple drug components in the core of poly-
bination therapy for cancer therapy using polymeric micelles meric micelle could further stabilize the polymeric micelle systems.
[240,260,261]. In 2009, they reported that paclitaxel, etoposide, doce- Alternatively, drugs which form highly stable single drug systems
taxel, and 17-AAG were solubilized in PEG-b-PDLLA block copolymers (like paclitaxel), could increase the stability of those drugs which do
as either single drug or combination therapy. Polymeric micelles of 2- not form stable single drug micelles. This stabilization can allow for
and 3-drug combinations such as paclitaxel/17-AAG, etoposide/17- unique therapeutic combinations which are not available in single
AAG, docetaxel/17-AAG and paclitaxel/etoposide/17-AAG were small drug micelle systems. An in vitro cytotoxicity assay confirmed syner-
(~30–40 nm) and displayed enhanced drug solubility up to gistic cytotoxicity of micellar etoposide/17-AAG and micellar
3–4 mg/mL in aqueous media [260]. In a follow-up study, 3-in-1 bortezomib/17-AAG in cancer cells.
PEG-b–PDLLA micelles were prepared for poorly soluble multidrug Wan et al. reported POx-based combination therapy of etoposide
systems such as 17-AAG, paclitaxel, and rapamycin [240]. The 3-in-1 and an alkylated cisplatin prodrug for the treatment of lung cancers
micelle featured 40 nm hydrodynamic size and improved drug solu- [114]. The POx block copolymer (PMeOx-b-PBuOx-b-PMeOx) was
bility (approximately up to 3 mg/mL in aqueous solution). In vitro cy- employed to solubilize drug combinations in a single high-capacity ve-
totoxicity studies and combination index (CI) analysis displayed a hicle (over 50% wt. drug in dispersed phase). The combination poly-
synergistic effect of drug components in 3-in-1 micelles for MCF-7 meric micelle featured nanosized and worm-like morphology which
and 4 T1 breast cancer cell lines. This group also reported that 3-in- was rarely reported previously for drug-loaded polymeric micelles. In-
1 PEG-b-PDLLA micelle delivering high doses of paclitaxel, 17-AAG, terestingly, drug release from combination polymeric micelle was
and rapamycin significantly increased the exposures of paclitaxel slower, presumably due to the formation of stable micelle formulations
and rapamycin in mice compared to single drug micelles [261]. In con- with the co-loading of drugs. PK analysis of combination polymeric mi-
trast, at a lower dose of 3-in-1 micelle, PK differences of individual celles exhibited enhanced drug exposure to the target site compared to
drugs were marginal. These data indicate that the PK profile of drug single micelle, mixture of single micelle, and free drugs. Along with the
combinations in polymeric micelle formulations can be improved by strong synergistic effect of the combination, a superior anti-tumor activ-
the functionalities of polymeric micelles such as the solubilization ity of combination polymeric micelles was observed in preclinical lung
capacity and increased dose tolerability. cancer models.
Often times, the inclusion of a drug which is highly soluble in a mi- Wan et al. also reported the co-delivery of paclitaxel and an
celle system with a drug which is not typically highly soluble in the alkylated cisplatin prodrug as polymeric micelles for the treatment
system, can synergistically increase the solubility of the non-soluble of ovarian and breast cancer [188]. The drug combination was effec-
drug in the system. In POx micelles, the inclusion of poorly compatible tively solubilized in a POx-based block copolymer with high LC (over
drugs with well-compatible, such as paclitaxel, allows for their encap- 50%) and stable polymeric micelles for two-drug combinations were
sulation and increased aqueous solubility. This alters the PK of both formed. Drug-coloaded micelles had slower release of the drug com-
paclitaxel and the other drug. Han et al. reported the synergistic solu- ponents to plasma and improved drug exposure of both drugs to the
bilization of multiple drugs in a single micelle system with high LC tumor site (Table 5). Superior anti-tumor activity of combination ther-
resulting in formation of ultra-high loaded polymeric micelle drug for- apy was confirmed in ovarian and breast cancer preclinical models.
mulations. A variety of poorly-soluble drugs (paclitaxel, docetaxel, Interestingly, PK simulations of the polymeric micelles in a three-
etoposide, 17-allylamino-17-demethoxygeldanamycin (17-AAG), compartment model as previously discussed revealed that a decreased
bortezomib) were solubilized in POx-based block copolymer release rate of drug components from the micelle could be related to
(PMeOx-b-PBuOx-b-PMeOx) to prepare binary and ternary drug com- the improved tumor delivery of the drug combination (Fig. 7). This
binations (Fig. 8) [117]. POx-based polymeric micelles were able to study indicates to us that co-loading of drugs in a single micelle
solubilize multi-drug combinations with an extremely high LC of up would be beneficial for the delivery of the combination therapy.
to 50% (48.7% LC for 1:1:1 ratio of paclitaxel:17-AAG:etoposide and Also, an in-depth analysis of the PK profile/simulation and its
48.4% LC for 1:1:1 ratio of paclitaxel:17-AAG:bortezomib). Also, POx

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Table 5
PK parameters of C6CP and paclitaxel in plasma and tumor after administering polymeric micelle in A2780/CisR tumor bearing mice.
Reprinted with permission from [188] Copyright 2018, Elsevier.

Parametersa C6CPb, 20 mg/kg Paclitaxel, 20 mg/kg

Paclitaxel/C6CP C6CP Parameter Paclitaxel/C6CP Paclitaxel Parameter

polymeric polymeric ratioc polymeric polymeric ratiod
micelle micelle micelle micelle

Plasma t1/2, α (h) 7.67 6.20 1.24 5.12 3.67 1.40

Cmax (μg/mL) 9.40 4.71 1.99 6.51 4.62 1.41
AUClast 34.80 30.00 1.16 18.41 9.78 1.88
(h*μg/mL)
Clobs (mL/h/kg) 527.40 541.00 0.97 984.06 1434.66 0.69
Vdobs (mL/kg) 5077.11 7963.56 0.64 10,112.89 7689.6 1.32
Tumor AUClast (h*μg/g) 110.92 38.68 2.87 111.14 87.28 1.27
Cmax (μg/g) 9.86 4.34 2.27 8.34 5.38 1.55
Tmax (h) 1 1 1 1
a
t1/2, α, half-life at the biodistribution phase; Cmax, maximum plasma concentration; AUClast, area under the curve from time 0–15 h; Clobs, observed total body clearance; Vdobs, total
volume of distribution observed; Tmax, time of maximum concentration.
b
Hydrophobic derivatives of cisplatin with aliphatic chains of 6 carbon atoms at the axial positions.
c
paclitaxel/C6CP polymeric micelle: C6CP polymeric micelle.
d
paclitaxel/C6CP polymeric micelle: paclitaxel polymeric micelle.

correlation with physicochemical properties of polymeric micelles solution and sonicated to form micelles [193]. In that study, when com-
would be helpful for designing ideal polymeric micelle formulations. paring the sonication method and thin film hydration technique, they
determined that the process which forms more stable micelles is poly-
mer dependent. Some key factors are the block copolymer chemical
5. Polymeric micelles in clinical trials and regulatory approval for composition, block lengths, and the polymer/drug ratio. However,
human use in our experience, the sonication process alone yields poor results,
because the drug tends to precipitate faster than it solubilizes into the
5.1. Polymeric micelle manufacturing considerations micelle core.
One way to control this process is by using flash precipitation, which
The manufacturing process of polymeric micelles should be consid- was in part pioneered by the Prud'Homme lab. In flash precipitation, an
ered throughout formulation development in order to produce micelle aqueous and organic phase are mixed rapidly with engineered geome-
formulations with consistent physicochemical properties and achieve tries in a special “jet mixer” instrument which allows for the synthesis
the desired scale of production. The selection of applicable of uniform nanoparticles [266,267]. This process has been brought to
manufacturing processes may impact characteristics of the final poly- scale by BASF for the generation of β-carotene nanoparticles [268]. It
meric micelle formulation such as drug loading, size distribution, and can be optimized by increasing mixing times to obtain drug-
stability in aqueous media, which are critical factors for translation containing polymeric micelles and block copolymer coated drug nano-
[262]. Several processes are available to produce polymeric micelles particles with desired size, coating, and other characteristics [269].
with uniform size distributions and stability. However, not all pro- The mixing performance can be maximized by using microfluidic
cesses are created equal. That is, what works for one polymer-drug mixer systems, leading to the highest mixing efficiency and homoge-
system may not be feasible in another, and not all processes are con- neous reaction environment of the mixed solutions under continuous
ducive to a manufacturing scale. flow condition. However, the process is not universal and has challenges
One of the most common drug-loaded polymeric micelle prepara- especially for drugs with relatively low logP values that tend to dissolve
tion techniques is the thin film hydration method, which is fairly condu- faster than they incorporate into nanoparticles. Moreover, the organic
cive to scale-up in a batch-wise manner. In this method, polymers and solvents must still be removed by dialysis or freeze drying, which is
drugs are solubilized in a common organic solvent and the solution is discussed below. Overall the use of rapid mixing for preparation of
evaporated under air flow or at a reduced pressure to form a polymer- drug containing micelles is in our view an area for further research
drug thin film. This film is then hydrated with an aqueous solution and development, potentially at industrial scale.
and the polymer and drug are dispersed into the solution as drug loaded One technique that is commonly used in laboratory micelle prepara-
micelles [22,263–265]. In this technique, it is often critical that a dry thin tion which is not conducive to industrial scale up is dialysis. Pure or-
film is achieved with no residual solvent. Often times after thin film for- ganic solutions of micelle and drug, with appropriate dialysis cut-off
mation, the film will be placed under vacuum for several hours to en- membranes, are dialyzed against pure water for a few days while the
sure solvent removal is complete. Additionally, solvent selection is a water is changed out frequently. As organic solvent in the dialysis bag
key parameter which is explored in several studies [116,193,264]. First is replaced with aqueous media, drug loaded polymeric micelles begin
of all, both the drug and polymer must be soluble in the selected organic to form [270–273]. With dialysis, the processing times are long and
solvent. However, this does not necessarily mean that all good solvents the complete removal of organic solvent and free, unloaded drug from
for a polymer-drug system will produce stable, uniform polymeric mi- the solution is difficult. This is often paired with freeze drying for a
celles. It is important to explore several solvent systems when possible. more complete solvent removal, but scale up of the dialysis method re-
There must be intimate mixing to avoid crystallization of the drug dur- mains a challenge. In one study, the use of just dialysis did not produce a
ing the thin film formation, and this cannot be achieved in every solvent. uniform particle population or nanosized polymeric micelles [274].
Alternatively, if melting temperature allows, the co-melting of the poly- They compared this pure dialysis approach with a hybrid approach. In
mer and drug can be performed without a solvent in order to prepare the hybrid approach, polymer (PEO-b-PCL) and drug are dissolved in
the micelles. an organic phase which is added dropwise to an excess of aqueous
One study compared the use of thin film hydration and sonication media. As the organic phase enters aqueous solution, polymeric micelles
alone. In the sonication process an anticancer drug, sagopilone, and spontaneously form which encapsulate the drug. These solutions can
PEG-b-PLLA, PEG-b-PDLLA or PEG-b-PCL were added to an aqueous

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then be dialyzed against aqueous media to remove solvent and free excipient (de-facto LE), sterilization process for endotoxin-free formu-
drug. This approach yielded smaller (20–50 nm) and more uniform mi- lation, and final formulation design (solution or lyophilized powder
celle sizes. In both methods, the particle size was stable after dilution. for reconstitution). Minimizing the steps in the manufacturing process
The selection of organic solvent used also had an effect on polydispersity to increase LE and mitigate the detrimental side effects of manufactur-
once again highlighting the need to evaluate multiple solvent systems in ing processes (e.g. formulation instability) is highly desirable. For this
order to optimize formulation stability. purpose, the thin film hydration method could be the most plausible op-
An alternative to dialysis is using cosolvent evaporation. Drug and tion since 1) there are the fewest steps 2) organic solvent removal is
polymer are dissolved in organic solvent and are added dropwise, or easy during film formation 3) it avoids dialysis and potential contami-
sometimes rapidly, to an aqueous phase. The solution can then be nation from water. For the final formulation, the lyophilized powder
heated and/or placed under vacuum to remove the volatile organic sol- form is the better option since it may be helpful to avoid contamination
vents and produce stable aqueous micellar solutions [275,276]. If there or drug release/degradation of micelle formulations in aqueous media.
is any residual drug precipitated, then a brief centrifugation step can re- At a larger scale of production, the manufacturing of polymeric
move drug precipitates (this is applicable to any of the discussed micelle products with reproducible physicochemical properties is of
methods). It is also important to note that any combination of the afore- importance. For this purpose, proper establishment of the good
mentioned methods could potentially be used to prepare stable mi- manufacturing process (GMP) is necessary to follow industrial stan-
celles. In these studies, organic solutions were added to an aqueous dards and ensure the quality of the final nanomedicine products. Also,
media, dialyzed, sonicated, and then freeze dried to produce stable for- adapting a systemic management system based on the principle of
mulations [273,277]. Quality by Design for the manufacturing is useful in order to develop ef-
Another technique, oil in water emulsions, is common in pharma- ficient quality control systems and define key parameters for mass pro-
ceutical development and can be applied to polymer micelle prepara- duction of polymeric micelle formulation. The key parameters such as
tion. Polymer and drug are dissolved in a water immiscible organic amount of API and excipients, purity of solvents, presence of impurities
solvent which is then added to an aqueous solution to prepare an emul- and processing variables (time, temperature and pressure) can be useful
sion [278,279]. The mixture is then stirred and/or heated to remove the factors to define the cause of unexpected quality issues or non-
organic solvent. Oil in water emulsions are commonly produced at reproducibility of the products during the production. International
manufacturing scales, but the complete removal of organic solvent Conference on Harmonisation (ICH) guidelines, Q8 Pharmaceutical De-
from them can be difficult. Thus, the use of common emulsion solvents velopment of the European Medicines Agency (EMA) and Q11 Develop-
such as tetrahydrofuran, chloroform, and acetone could be problematic ment and Manufacture of Drug Substances (Chemical Entities and
for the development of injectable polymeric micelle formulations. How- Biotechnological/Biological Entities) of the USFDA provide such guide-
ever, the addition of a freeze-drying step could help completely remove lines to provides knowledge on the application of scientific approaches
undesired excipients from the formulation. and quality risk management for the development of a product and its
Freeze drying, when done under particular conditions, can be used manufacturing process [286,287].
to remove small amounts of residual organic solvents. Moreover, freeze
drying alone has been used to prepare stable micelle formulations. In 5.2. Regulatory approval of nanomedicines
one study, a tert-butanol solution of drug was added to an aqueous so-
lution of a polymer (PVP-b-PDLLA), and then the mixture was freeze No new medical product is without risks, and these must be assessed
dried to form stable micelles upon resuspension in aqueous media throughout the drug development pipeline, and will ultimately be eval-
[280,281]. The tert-butanol is highly compatible with the one-step uated to either approve or reject the use in human or veterinary patients
freeze drying process. However, the use of tert-butanol in this process by regulatory agencies worldwide, based on evaluation of quality, safety
was unique to the system of PVP-b-PDLLA block copolymer and could and efficacy, as well as a final risk-benefit assessment. Examples of such
not be applied, for example, to PEG-b-PDLLA since PEG is practically in- Agencies include the USFDA, the UK Medicines and Healthcare Products
soluble in tert-butanol. Regulatory Agency (MHRA), the EMA, Brazilian Health Surveillance
The freeze drying process is highly conducive to scale up. Freeze dry- Agency (abbreviated in the home country as ANVISA), Health Canada,
ing after preparation of micelles can also be used to stabilize the formu- Australian Therapeutic Goods Administration (TGA), Japanese Pharma-
lation for longer periods of time [264]. Sometimes, the aqueous stability ceuticals and Medical Devices Agency (PMDA) and the Ministry of
of polymeric micelles can be limited, so long term storage of powdered Health, Labor, and Welfare (MHLW), the National Medical Products Ad-
polymeric micelle solutions could be more feasible. To ensure the rapid ministration (NMPA) of China, and the Ministry of Health of Russian
dissolution of the powdered polymeric micelles, their mixing with ex- Federation to name a few. The purpose of these agencies is to protect
cipients such as lactose makes this composition instantly dissolvable public health through the evaluation and review of the quality, efficacy,
in water allowing the product to be dried to a solid. This approach was and safety for innovative medical products at the moment of marketing
used, for example, for preparation of the dry from of SP1049C polymeric approval and to provide a framework for the continued evaluation of
micelle formulation of doxorubicin [282]. technologies and therapeutic options. Below, we will focus on the ap-
Lastly, a more recent and complex method which has been devel- proval process used in the US and Europe. The move towards nano-
oped involves the use of supercritical fluids [283,284]. A full review on based pharmaceutical platforms are expected to improve one or several
the theory of using supercritical fluids in drug delivery systems can be aspects related to drug dissolution, bioavailability, metabolism, clear-
found here [285]. Using supercritical fluids in polymeric micelle prepa- ance, and distribution profiles, improving the therapeutic index, often
ration allows for faster processing times, easy solvent removal, and no by reduction of safety risks in clinical use [288–290]. While improving
long freeze drying or dialysis steps. Common solvents for this are many facets of drug delivery, the use of nanocarriers changes the
trifluoromethane and carbon dioxide. A solution is brought up to pres- frame of traditional regulatory procedures. Encapsulation in nanoparti-
sure to dissolve the polymer and drug as a supercritical fluid and then cles changes the size, surface properties, and other characteristics which
pressure is quickly removed allowing for micelles to form and the sol- affect in vivo drug behavior when compared to traditional formulations
vent to evaporate. It can then be washed with water to form stable composed of low molecular weight API and excipients. Regulatory
aqueous solutions. We refer the readers to this more comprehensive re- appraisal has to deal with its impact on safety and efficacy profiles.
view for additional information on supercritical fluid use [285]. This calls for increased scrutiny and rigor in the review process
As we have explained, several factors in these processes should be [288,291,292].
considered critical such as the selection of organic solvent, number of In the US, all new pharmaceuticals are reviewed and approved based
overall steps, yield of the polymeric micelle from the pure drug and on the well-established regulatory framework which is based on a

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weight-of-evidence approach. Such an approach considers each new synthetic in origin and do not fall under the ‘biologics’ class of drugs.
drug on a case-by-case basis. Since each new formulation has unique However, there are some biologics which are nanosized and exhibit
properties, the USFDA recommends sponsors engaging the Agency unique properties due to these dimensions. This creates a highly com-
early in the development and pre-submission process so as to plex classification of nanoscale drugs. This high degree of complexity
obtain timely and adequate guidance for studies critical for the given can make complete physicochemical characterization a significant chal-
drug product. The available guidance for industry documents lenge, especially for follow-on or generic formulations, which has also
containing specific recommendations for different categories of drug been addressed at different levels both by the NCL (USA) and
products are accessible on the USFDA website (https://www.fda.gov/ European Pharmacopoeia [293,299].
drugs/guidance-compliance-regulatory-information/guidances-drugs). Similar to the USFDA's 351(k) route for biosimilars, the EU regula-
Among them are specific recommendations for nano-based products tory bodies have developed a ‘nanosimilar’ route for nano-based phar-
(https://www.fda.gov/science-research/nanotechnology-programs- maceuticals. For the approval of nanosimilars, the EMA follows a
fda/nanotechnology-guidance-documents, https://www.fda.gov/ stepwise approach [293,299] which includes in vitro quality assess-
science-research/nanotechnology-programs-fda/nanotechnology- ments, pre-clinical biodistribution, clinical PK, and then therapeutic
guidance-documents) [291]. equivalence. The nanosimilar formulation must show comparable char-
The traditional application process in the US for New Drug Applica- acter at each step of the process and cannot rely on in vitro or preclinical
tion (NDA) of novel human pharmaceuticals, including those containing animal model data alone, making a case for having some level of clinical
nanomaterials, occurs through the 505(b)(1) regulatory pathway. In evaluation to be necessary for nanosimilars. On the other hand, the
addition, the 505(b)(2) pathway exists and applies to products closely USFDA does not rely on pre-clinical animal data and instead focuses
related to the innovators. If an API has been reformulated into a new on extensive physicochemical characterization followed by clinical PK
‘nanocarrier’, then it may proceed along either the 505(b)(1) with data [291,293]. These data sets provide necessary information to predict
non-similar PK profile to the innovator or the 505(b)(2) route if the “sameness” of two nanoformulations. However, there is still a high attri-
PK profile is similar. Utilizing the 505(b)(2) pathway then allows for tion rate for new drugs, and new nano-based pharmaceuticals are sub-
the use of clinical data from the Reference Listed Drug (RLD). However, ject to this as well. The development of new clinical models which
by following the 505(b)(2) pathway, one must still demonstrate some better replicate patient pathophysiology are highly warranted, and
kind of improved outcome whether therapeutic, improved shelf-life/ this is a key place where physician-scientists can help to evaluate
stability, increased convenience, reduced metabolic burden, or some these formulations and push the field forward by lowering the attrition
other quality which distinguishes it from the innovator product. The rate of new medicines. That is, the responsibility does not fall solely on
505(j) pathway for the approval of generics requires the demonstration the USFDA, but also on researchers (both in Academia and Industry) to
of “sameness” with only small changes to formulation allowed. This develop better pre-clinical models that can more accurately assess the
pathway can be more challenging for nano-based pharmaceuticals potential for clinical efficacy similarity.
than traditional small molecule formulations due to the added complex- The nanosimilarity approach should not be confused with the fact
ity inherent when working with nanomaterials. These pathways are that a nano-formulation of an already existing API, is to be considered
authorized by the Food, Drugs, and Cosmetics Act and apply to non- by default as a new medicinal product, that needs to go through an al-
biologic drugs. Biologics, on the other hand, are licensed through the most full dossier for marketing authorization application (MAA). That
Public Health Service Act. Innovators follow a 351(a) pathway whereas was the case for Caelyx (DOXIL® in USA) in 1996 (https://www.ema.
‘biosimilars’ are approved through the 351(k) pathway [293]. Nano- europa.eu/en/medicines/human/EPAR/caelyx-pegylated-liposomal) or
based pharmaceuticals and biologics represent the category of complex later on for Abraxane® in 2005 (https://www.ema.europa.eu/en/
drug formulations which are often heterogeneous in nature. This het- documents/assessment-report/abraxane-epar-public-assessment-
erogeneous nature makes it impossible to produce identical follow-on report_en.pdf), or more recently in 2018 to Vyxeos® (https://www.ema.
drugs and demands a more adequate route to regulatory approval. europa.eu/en/documents/assessment-report/vyxeos-epar-public-
That is, there is not a distinct or definitive set of critical quality attributes assessment-report_en.pdf) just to give three distinct, well-known
(CQAs), which can accurately predict the in vivo similarity of two dis- examples.
tinct nano-based pharmaceuticals or biologic products. Thus, a wide va- The EMA has developed since 1995 a Scientific Advise procedure
riety of evidence is needed to show equivalence which includes long that is very useful for companies to use when assessing the relevance
term clinical monitoring and extensive PK data [288,293–295]. For ex- of the evidence gathered at the moment of designing their clinical stud-
ample, the SITUA method (see Section 4.2) developed by the NCL has ies. This procedure is well supported for small enterprises and larger
shown the potential to probe the complex PK behaviors of nano-based companies dealing with orphan drugs, which makes EMA an attractive
pharmaceuticals [247]. This kind of analysis can provide strong evidence pole for translational research in highly entrepreneurial ecosystems
for the similarity of two nanomedicines. around academia involving new company incubators and translational
A robust and consistent manufacturing process for nano-based phar- accelerators that foster the launch of active and innovative Start-Ups.
maceuticals is of paramount importance to regulatory approval. While The system is expected to have a high impact in the future of medicines
drug nanocrystal, liposomal, iron nanoparticle, and a few others have innovation in Europe (https://www.ema.europa.eu/en/human-
been approved by the USFDA, there is not yet a clinically approved poly- regulatory/overview/supporting-smes).
meric micelle formulation in the United States [296,297]. In fact, the The application of the complexity issue to block copolymers is ap-
USFDA has released a specific guidance for Liposomal formulations as parent. Block copolymers and polymeric micelles do not consist of one
they have become more common in the marketplace [298]. Two poly- single molecular weight or sized species. Rather, they consist as a distri-
meric micelle formulations, Genexol® PM and Nanoxel® M, have been bution range and thus should be described by their averaged molecular
approved by regulatory agencies as discussed in Section 5.3. The rela- masses and mass distribution for block copolymers and mean size and
tionship between nano-based pharmaceutical physicochemical proper- PDI for micelles as well. Both of these must be under tight control for op-
ties and in vivo behavior is an active area of investigation. Unlike in timal consideration in the regulatory process. Micelle morphology and
traditional drug formulations, similar physicochemical properties of zeta potential could also play critical roles in the interactions of poly-
nano-based pharmaceuticals are not often a predictor of similar meric micelles with cells and proteins. Performing in vitro drug release
in vivo behavior [288,293,295] Once thoroughly assessed, our under- studies, in the most biologically relevant matrix possible, is essential to
standing of these relationships can help to improve how we design predicting in vivo stability. The ideal matrix for these studies would be
and evaluate nano-based pharmaceuticals and, in particular, block co- blood plasma, but a buffer containing albumin can be helpful as well,
polymer micelle systems. Nano-based pharmaceuticals are usually when blood serum is unavailable [221,235].

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The in vivo stability of polymeric micelles in particular can be diffi- slowly released from the micelle with 65% released at 24 h and 95% re-
cult to assess. The dynamic nature of polymeric micelles means the leased at 48 h [110].
polymeric micelle dissociates over time often leading to changing mea- In a phase I clinical trial in South Korea, twenty-one patients entered
surements of free, unbound drug and that which is physically encapsu- into a dose-escalation study and were treated with Genexol® PM rang-
lated in the micelle. It is important to measure all “species” of drug ing from 135 mg/m2 to 390 mg/m2 without premedication of hydrocor-
present in the blood and determine which of these is most critical to tisone and histamine blocker [11]. No acute hypersensitivity reactions
therapeutic efficacy [291]. Otherwise, extensive characterization using were observed, while neuropathy, myalgia, and neutropenia were ob-
the methods discussed in Section 3 above are useful to support ap- served which limited the highest dose. The MTD was determined to
proval. In general, for the nano-based pharmaceuticals the physico- be 390 mg/m2 for Genexol® PM, which is higher than that of the com-
chemical sameness is more difficult to relate to the same therapeutic mercially available paclitaxel formulations Taxol® (200 mg/m2) and
performance because the physiochemical characteristics that may affect Abraxane® (300 mg/m2) [11].
performance of a delivered drug are more complex and could affect Based on the established toxicity profile, a multi-centered phase II
therapeutic performance in different ways. However, as the under- study of Genexol® PM with cisplatin was performed on patients with
standing of this relationship improves one should expect that a more ro- advanced NSCLC in South Korea [301]. In this study, the patients were
bust and well-defined regulatory process develops. In the meantime, administered Genexol® PM 230 mg/m2 and cisplatin 60 mg/m2. The
the researchers in the field of translational nanomedicine could utilize overall response rate was 37.7% and the median survival period was
resources available from the government sponsored programs 21.7 months, which indicated significant improvements compared to
(e.g., the U.S. National Cancer Institute Sponsored NCL, https://ncl. previous clinical trials conducted with Taxol® 175–200 mg/m2 and cis-
cancer.gov/; the USFDA Learning Portal, https://www.fda.gov/training- platin 75–80 mg/m2 [301]. Another phase II study in South Korea re-
and-continuing-education/fda-learning-portal-students-academia- ported the clinical benefit of Genexol® PM in patients with metastatic
and-industry; European Union Nanomedicine Characterization Lab, breast cancer [9]. Forty-one patients were enrolled in the study and ad-
http://www.euncl.eu/; EMA, https://www.ema.europa.eu/en/partners- ministered with a Genexol® PM infusion (300 mg/m2) every 3 weeks.
networks/international-activities/training-opportunities-non-eu- The overall response rate was 58.5%, which is superior to that of
regulators). Some of these resources including the USFDA and EMA, Abraxane® (47.6%) and Taxol® (21–54%). With improved therapeutic
contain trainings and workshops for students and pharmaceutical sci- outcomes in clinical trials conducted in South Korea, Genexol® PM re-
entists to help better understand the drug approval process. Moreover, ceived regulatory approval in South Korea and was marketed there
since 2012 the USFDA implemented Generic Drug User Fee Amendment since 2007 for the treatment of MBC, NSCLC, and ovarian cancers. In ad-
(GDUFA) that provides additional resources for developers of generic dition to South Korea it received marketing approval in several other
medicines (https://www.fda.gov/industry/fda-user-fee-programs/ countries including India, Serbia, Philippines, and Vietnam. A completed
generic-drug-user-fee-amendments). Other available resources, such phase III study in South Korea further evaluated the efficacy and safety
as the NCL provide a standardized assay cascade protocols (https://ncl. of Genexol® PM (260 mg/m2) compared to paclitaxel (175–200 mg/
cancer.gov/resources/assay-cascade-protocols) and assist nanome- m2) in recurrent or MBC (ClinicalTrials.gov: NCT00876486) [302], but
dicine community with conducting IND-enabling preclinical studies the results of this trial have not been reported yet. Other clinical trials
(https://ncl.cancer.gov/working-ncl/ncl-assay-cascade-application- of Genexol® PM were conducted for patients with advanced biliary
process). Consolidated efforts among researchers in regulatory agen- tract cancer (Genexol® PM 100 mg/m2 with gemcitabine 1000 mg/
cies, government, industry and academia, and other nanomedicine m2) [303], with unresectable thymic epithelial tumors (Genexol® PM
stakeholders (e.g., educators, patients, physicians) are required to ad- 230 mg/m2 with cisplatin 70 mg/m2) [304], with ovarian cancer (as
vance the science and make regulatory approval for nanomedicines first-line treatment) (Genexol® PM 260 mg/m2) [305], which posted
more straight forward [235]. positive results on therapeutic outcomes from treatment with Genexol®
PM. In conclusion, Genexol® PM could improve the dosing of paclitaxel
5.3. Clinical status of polymeric micelle formulations and the safety of the drug in patients. In clinical trials Genexol® PM
demonstrated improved therapeutic outcomes as a formulation which
A number of polymeric micelle formulations utilizing the physical limited hypersensitivity reactions in patients through the elimination
entrapment of poorly soluble small molecules have been reported in lit- of toxic excipients. Although Genexol® PM has shown some hypersensi-
erature. Since then, many polymeric micelle formulations have reached tivity reaction in the clinic, probably due to the PEG, this is not nearly as
clinical trials in several countries [9,10,300]. Examples of polymeric mi- much of a concern as for Taxol® which contains Cremophor EL [301].
celle drugs which have obtained regulatory approval or clinical evalua- The tolerable dose of paclitaxel was increased by virtue of polymeric mi-
tion are presented in Table 6. These drugs have all been for cancer celle formulation, resulting in an increased MTD. From the results of a
indications. The first polymeric micelle which was clinically approved bioequivalence study, it is expected that it could gain regulatory ap-
(in South Korea) was Genexol® PM. Of the listed drugs, two have been proval in the United States via the 505(b)(2) pathway.
approved (Genexol® PM and Nanoxel® M in South Korea) and two The clinical development of Genexol® PM in the USA (under the
have completed phase 3 (NK105 and NC-6004). Some selected exam- trade name of Cynviloq™) was initiated by Sorrento Therapeutics
ples are considered below in greater detail. after the exclusive distribution rights to Genexol® PM were acquired
by Sorrento Therapeutics in 2013. In 2014, bioequivalence studies of
5.3.1. Genexol® PM Cynviloq™ versus Abraxane® were conducted in patients with metasta-
Genexol® PM is a Cremophor EL-free, polymeric micelle formulation tic or locally recurrent breast cancer and patients with NSCLC
incorporating paclitaxel as the API. Originally developed by Samyang (ClinicalTrials.gov: NCT02064829). Preliminary positive data in eight
Biopharmaceuticals Corp., this is the first polymeric micelle formulation patients was reported in 2014 which potentially supported the bio-
approved for human use in South Korea and several other countries for equivalence of the two products (https://sorrentotherapeutics.com/
patients with metastatic breast cancer (MBC), NSCLC, and ovarian can- sorrento-announces-first-patient-dosed-in-registration-trial-to-
cer. Genexol® PM consists of paclitaxel in polymeric micelles of mPEG- evaluate-bioequivalence-between-cynviloq-and-abraxane/). The bio-
b-PDLLA (mPEG: 2000 g/mol, PDLLA: 1750 g/mol, PDI: 1.0–1.2). The mi- equivalence of Cynviloq™ to Abraxane® could grant 505(b)(2) pathway
celles are manufactured by the solid dispersion method using the thin approval by the USFDA and expedite the regulatory process by avoiding
film hydration approach and are nano-sized particles with well- extensive clinical trials to validate efficacy versus the standard of care
defined spherical structure in aqueous media (20–50 nm in diameter [13]. No updates are available on the bioequivalence of these
and 16.7% LC of paclitaxel) [6]. Under sink conditions, paclitaxel was

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Table 6
Examples of polymeric micelle-based drug products in clinical trials or approved.

Product API Polymer Physicochemical Development stage Comments Trial code

code name properties and/or Ref.

Genexol® Paclitaxel mPEG-b-PDLLA Size: 20–50 nm, Approved in South Enabled higher doses of paclitaxel with decreased toxicity in [9,11]
PM1 LC: 16.7% Korea, Philippines, phase I study, Improved overall response rate (58.5%) compared NCT00876486
India, and Vietnam to that of Taxol® (21–54%) in patients with metastatic breast NCT02064829
cancer in phase II study.
Nanoxel® Docetaxel mPEG-b-PDLLA Size: 25.4 nm Approved in South Comparable efficacy and superior safety profile compared to that NCT01336582
M1 Korea of conventional docetaxel formulated in polysorbate 80 NCT02639858
(Taxotere) [13]
NK1052 Paclitaxel mPEG-b-modified P Size: 85 nm Phase 3 (completed) Improved plasma AUC of paclitaxel and reduced hypersensitivity NCT01644890
(Asp) LC: 23% reaction compared to conventional paclitaxel [238]. Failed to
improve efficacy of paclitaxel in phase III study [307].
NC-60043 Cisplatin PEG-b-P(Glu) Size: 28 nm Phase 3 (completed) Improved safety profile and patient's quality of life, while NCT02043288
coordination LC: 39% showed modest efficacy in phase II study [309].
complex
SP1049C4 Doxorubicin Pluronic® L61 and Size: < 30 nm Phase 2 (completed) Shown efficacy of doxorubicin as a single agent in phase II study [10,17]
F127 LC: 8.2%
NK0125 SN-38 PEG-b-P(Glu) Size: 20 nm Phase 2 (completed) Showed efficacy in patients with sensitive relapsed small cell NCT00951054
covalent LC: 20% lung cancer and toxicity was manageable. NCT00951613
drug-copolymer
conjugate
6
CPC634 Docetaxel PEG-b-P Size: 66 nm Phase 2 (recruiting) Possible improved safety profile, skin toxicity was seen at high NCT02442531
(CriPec®) (HPMAm-Lacn) LC: 12% dose in phase I study NCT03742713
covalent
drug-copolymer
conjugate
NK9117 Doxorubicin PEG-b-P(Asp) Size: 40 nm Phase 1 (completed) Comparable toxicity profile of doxorubicin to free doxorucin, No [146]
covalent infusion-related reaction in phase I study
drug-copolymer
conjugate
NC-40168 Oxaliplatin PEG-b-P(Glu) Size: 40 nm Phase 1 (completed) No results available yet NCT03168035
coordination LC: 32%
complex
1
Developed by Samyang Biopharmaceuticals Corp.
2
Originally developed by NanoCarrier Co., Ltd. and licensed to Nippon Kayaku Co., Ltd.
3
Developed by NanoCarrier Co., Ltd. in collaboration with Orient Europharma Co., Ltd. (drug is bound to the P(Glu) block of the block copolymer via coordination bonds).
4
Originally developed by Supratek Pharma Inc. and acquired by SoftKemo Pharma Corp. (now termed SKC1049).
5
Developed jointly by NanoCarrier Co., Ltd. and Nippon Kayaku Co., Ltd. (SN-38 is chemically conjugated to the P(Glu) block of the copolymer).
6
Developed by Cristal Therapeutics (cdocetaxel is covalently conjugated m-PEG-b-poly[N-(2-hydroxypropyl)methacrylamide lactate] (mPEG-b-p(HPMAm-Lacn) copolymer).
7
Developed by Nippon Kayaku Co., Ltd. (doxorubicin is chemically conjugated to the P(Asp) block of the copolymer, and the free drug is solubilized in the micelles of the resulting
conjugate).
8
Developed by NanoCarrier Co., Ltd. (drug is bound to the P(Glu) block of the block copolymer via coordination bonds).

formulations since Cynviloq™ was acquired in 2015 by NantWorks, squamous cell carcinoma (Nanoxel® M 75 mg/m2, ClinicalTrials.gov:
which was founded by Dr. Patrick Soon-Shiong who developed NCT02639858), Nanoxel® M and oxaliplatin for patients with metasta-
Abraxane®. tic esophageal squamous cell carcinoma (Nanoxel® M 75 mg/m2 and
oxaliplatin 120 mg/m2, ClinicalTrials.gov: NCT03585673), and the
5.3.2. Nanoxel® M safety of Nanoxel® M in patients with other various types of cancers
Nanoxel® M is a docetaxel-loaded polymeric micelle formulation (ClinicalTrials.gov: NCT04066335).
which was developed by Samyang Biopharmaceuticals Corp. and re-
ceived regulatory approval in South Korea in 2012 (according to an- 5.3.3. NK105
nouncement from Samyang Biopharm (https://samyangbiopharm. NK105 is a paclitaxel-loaded polymeric micelle formulation which
com/eng/ProductIntroduce/injection) and Korea Pharmaceutical Infor- was originally developed by Kataoka's group and NanoCarrier Co., Ltd.
mation Center (http://www.health.kr/searchDrug/result_drug.asp? in the early 1990s and advanced to clinical trials (phase III completed,
drug_cd=2012122700008). Also, Nanoxel® M is currently under clin- ClinicalTrials.gov: NCT01644890). NK105 is composed of paclitaxel
ical evaluation in the USA. The formulation is composed of docetaxel and modified mPEG-b-P(Asp) block copolymer (mPEG = 12,000 g/
in mPEG-b-PDLLA (mPEG: 2000 g/mol and PDLLA: 1765 g/mol) poly- mol and P(Asp) = 8000 g/mol) where half of the carboxylic groups P
meric micelle [306]. It is manufactured by the thin-film hydration (Asp) block is modified with hydrophobic 4-phenyl-1-butanol to in-
method, which produces uniform micelles with a hydrodynamic size crease its hydrophobicity and improve drug incorporation [7]. The mod-
of 25.4 nm [306]. However, Nanoxel® M has limited micelle stability ified P(Asp) block, which forms the hydrophobic core of the micelle,
in solution after reconstitution (up to 6 h in saline) [306]. A phase I enhances the drug loading in the micelle core via physical entrapment
clinical trial was conducted in South Korea (NCT01336582) and it [7]. NK105 exhibited 23% LC of drug loading with a hydrodynamic size
was reported that Nanoxel® M (70 mg/m2) exhibited an improved of approx. 90 nm [7].
drug safety profile compared to the conventional docetaxel formula- In a phase I trial of NK105, nineteen patients with various type of
tion in patients with advanced solid tumors [13]. This is believed to cancers (pancreatic, bile duct, gastric, and colonic) were recruited to ex-
be due to the removal of toxicity of Polysorbate 80 that is contained amine the safety and PK of NK105. NK105 doses ranging from 10 mg/m2
in a conventional docetaxel formulation, Taxotere. Currently, addi- to 180 mg/m2 were administered to patients without premedication.
tional clinical trials are recruiting participants to evaluate the efficacy The MTD of NK105 was determined to be 180 mg/m2 due to dose limit-
and safety of Nanoxel® M in recurrent or metastatic head and neck ing hematological toxicity (neutropenia). The PK profile of NK105

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showed that the plasma AUC of paclitaxel from NK 105 (at 150 mg/m2) adverse events were leukopenia and thrombocytopenia. Tumor shrink-
was approximately 15-fold higher than that of paclitaxel from Taxol® age, partial responses, and stable disease were observed in 55%, 15%,
(210 mg/m2) [238]. Also, NK105 was well tolerated exhibiting reduced and 70% of total patients, respectively [309]. A Phase III clinical trial of
hypersensitivity reactions in patients. NC-6004 with gemcitabine in patients with locally advanced or meta-
A phase II trial of NK105 recruited 57 patients with advanced gastric static pancreatic cancer has been completed, but no results reported
cancer after the failure of first-line chemotherapy [300]. The patients yet (ClinicalTrials.gov: NCT02043288).
were administered NK105 (at 150 mg/m2 paclitaxel) without anti- Overall, NC-6004 formed a stable polymeric micelle with a sustained
allergic premedication. The results of the phase II study revealed that release of cisplatin. Clinical trials of NC-6004 demonstrated long circula-
NK105 showed modest activity and tolerability for paclitaxel. The over- tion and sustained drug release of NC-6004 in patients. Also, NC-6004
all response rate was 25% with median progress free survival of could improve the drug toxicity profile, such as reducing nephrotoxicity.
3.0 months and median overall survival of 14.4 months [300]. In this Additional clinical trials are ongoing on NC-6004 in patients with head
study, conventional drug was not administered to patients since there and neck cancer and pancreatic cancers.
was no standard of care for advanced gastric cancer. Thus, the interpre-
tation of the results from the phase II study of NK105 is difficult. In July
2016, an open-label phase III non-inferiority trial of NK105 in patients 5.3.5. SP1049C
with metastatic or recurrent breast cancer was completed SP1049C is a doxorubicin-loaded Pluronic®-based formulation
(ClinicalTrials.gov: NCT01644890) [307]. Four hundred thirty-six pa- which was developed by Supratek Pharma Inc. and was the first poly-
tients were enrolled in the study and administered either NK105 meric micelle drug formulation to enter clinical trials in 1999 [17]. It
(65 mg/m2) or conventional paclitaxel (80 mg/m2). The results of the was then evaluated in Phase II clinical trials and has shown positive re-
phase III trial revealed that the primary endpoint (statistical non- sults [10]. The formulation was prepared by the blending of two
inferiority of progression-free survival) was not met (the median Pluronic® block copolymers (L61 and F127) which consist of PEO and
progression free survival (PFS) of 8.4 and 8.5 months for NK105 and PPO at a 1:8 weight ratio of L61:F127 [23] and compounding this mix-
paclitaxel, respectively, and the median overall survival of 31.2 and ture with doxorubicin. Initially the micellar formulation was prepared
36.2 months, and overall response rates of 31.6% and 39.0%, respec- before injection by dissolving doxorubicin in the sterile aqueous solu-
tively) [307]. However, the incidence of peripheral sensory neuropathy tion of the block copolymer mixture. Subsequently the dry form of the
among patients treated with NK105 was decreased compared to that of SP1049C was developed that is reconstituted by adding saline. Physico-
paclitaxel, indicating an improved toxicity profile of the drug in NK105 chemical analysis of SP1049C revealed that the micelles had well-
[307]. Overall, NK105 successfully improved the PK and safety profiles defined spherical morphology with a size of less than 30 nm, and the
of paclitaxel by incorporating the drug into a polymeric micelle formu- doxorubicin LC was 8.2%. According to Batrakova et al., Pluronic® L61
lation. However, NK105 failed to improve the efficacy of paclitaxel in exhibited sensitization of multidrug-resistant cancer cells, thereby en-
clinical trials. hancing the cytotoxicity of doxorubicin while F127 showed stabilization
of the doxorubicin-loaded micelle formulation in aqueous solution [23].
5.3.4. NC-6004 In drug-sensitive tumors, SP1049C has the same efficacy as doxorubicin
Originally developed by Kataoka's group and NanoCarrier Co., Ltd., but, at the same time, is highly active against multidrug-resistant
NC-6004 is a cisplatin-containing polymeric micelle formed via coordi- tumors and cancer stem cells [310,311].
nation of platinum drug with the P(Glu) segment of the PEG-b-P(Glu) In the phase I clinical trial, SP1049C exhibited a similar PK profile of
block copolymer (PEG: 12,000 g/mol and P(Glu): 6000 g/mol) [153]. doxorubicin to the conventional doxorubicin formulation and a similar
The complex was formed between the platinum metal and the carbox- MTD of 70 mg/m2 in the patients with metastatic or recurrent solid tu-
ylic acid group in the P(Glu) segment of the block copolymer resulting mors [17]. Interestingly, doxorubicin-related toxicity, such as hand–foot
in a cisplatin-loaded core in NC-6004. NC-6004 exhibited stable micelles syndrome was less prevalent in the SP1049C treated patients compared
in solution and slowly released cisplatin from the micelle for over 150 h to those treated with conventional doxorubicin. Later, a Phase II trial
in physiological saline. NC-6004 exhibited a narrow size distribution in demonstrated that SP1049C has a notable single-agent activity as well
solution with a size of 28 nm and loading of cisplatin in NC-6004 was up as an acceptable safety profile in patients with advanced carcinoma of
to 39% LC [153]. the esophagus and gastroesophageal junction [10]. Patients treated
In a phase I clinical trial, a total of 17 patients with various type of ad- with SP1049C at a dose 75 mg/m2 (doxorubicin equivalents) had an ob-
vanced solid tumors were recruited to evaluate the efficacy and safety of jective response rate of 47% in the evaluable patient population, and 43%
NC-6004 [308]. NC-6004 was administered in doses ranging from in the intent-to-treat population along with the median overall survival
10 mg/m2 to 120 mg/m2. Drug toxicities were observed at the dose of of 10 months and PFS of 6.6 months. The principal toxicity concern, neu-
90 mg/m2 of NC-6004, and 120 mg/m2 of NC-6004 was determined as tropenia, was manageable and reversible, and in line with that expected
the MTD due to renal impairment and hypersensitivity reactions in pa- from doxorubicin 75 mg/m2 in the standard formulation. Notably, doxo-
tients [308]. Plasma samples were processed via gel-filtration and ultra- rubicin (API of SP1049C) is considered to be inactive (response rates
filtration methods to analyze the subpopulations of cisplatin in plasma less than 20%) in advanced adenocarcinoma of the esophagus and is
such as total platinum, platinum in NC-6004, and extra-micellar plati- not used in this indication. In 2008, the SP1049C obtained a special pro-
num [308]. In the PK profile, the amounts of low-molecular mass plati- tocol assessment on a single approvable Phase 3 trial in refractory upper
num, including cisplatin released from NC-6004, were marginal gastrointestinal adenocarcinoma and has obtained an orphan drug des-
compared to gel-filterable platinum (micelle encapsulated) and total ignation in adenocarcinoma of the esophagus from USFDA. In addition,
platinum, indicating sustained release of cisplatin from NC-6004 in sys- SP1049C has obtained two USFDA orphan drug designations for the car-
temic circulation. Also, NC-6004 exhibited extended half-life and in- cinoma of the esophagus and gastric cancer. However, no clinical data
creased AUC compared to cisplatin infused as an aqueous solution due has been reported since then. The development of SP1049C was
to the prolonged blood circulation of NC-6004. suspended as a result of the economic crisis of 2008. In late 2016 patent
A Phase Ib/II trial of NC-6004 with gemcitabine was conducted in pa- rights to SP1049C were acquired by SoftKemo Pharma Corp. to complete
tients with advanced solid tumors to evaluate the safety and tolerability the final development of the novel anticancer therapeutic now code
(ClinicalTrials.gov: NCT02240238) [309]. NC-6004 was administered to named SKC1049. Overall, SP1049C may have several applications: as a
patients at 60 mg/m2 to 180 mg/m2 (on day 1) with gemcitabine new agent with novel mechanism of action for combination therapy;
(1250 mg/m2, on days 1 and 8) every 3 weeks. A dose of 135 mg/m2 salvage therapy; and as the first line single agent in doxorubicin
of NC-6004 was determined as the MTD and common hematologic indications.

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6. Conclusions and future directions of polymeric micelle formulations to help the researcher expedite
their preclinical formulation and PK analysis.
Polymeric micelle formulations for poorly soluble small molecules Clinical investigations of polymeric micelle formulations have re-
have been extensively studied over three decades as a versatile platform vealed promising therapeutic outcomes for human use, while we have
for drug delivery. Their ability to be customized and tailored to specific also witnessed a number of clinical trial failures from other polymeric
needs is a distinct advantage over other drug delivery systems. While micelle formulations. Section 5 provided the reader with an introduc-
simple at first pass, it is clear that polymeric micelles represent a tion to the regulatory pathway and challenges facing the approval of
much more complex system than early understanding suggests. With polymeric micelles and nanomedicines in general. We hope that our
some successful preclinical results, several polymeric micelle formula- section, in concert with the provided references, can help the reader
tions have entered clinical trials, but only a few have received regulatory better prepare for regulatory approval early and often throughout the
approval for human use. There have been many challenges affecting formulation development process. Previous sections provide the scien-
their ability to navigate the regulatory pathway, which we have worked tist with some of the tools they need to gather CQAs in preparation for
to address in this review. regulatory approval, which can hopefully expedite translation and im-
In Section 2, we have focused on the types of polymeric materials prove clinical outcomes.
that are used for the manufacturing of polymeric micelles. Block copol- It is also important to point out significant areas of research and fu-
ymer segments required for forming amphiphilic block copolymers ture directions that were deliberately left outside of the focused consid-
were described in order to aid in the proper selection of block copoly- eration of the current review. One such area is the use of ionic block
mer components for the efficient solubilization of target molecules. copolymers for the drug delivery of biopolymers. The introduction of
Although many polymeric materials were studied during the previous cationic block copolymers that contain polycation blocks to bind nega-
three decades, there are only a few such materials used for the tively charged nucleic acids and water soluble anionic blocks to ensure
manufacturing of micelles which have reached the clinical stage of de- micelle stability in solution [312–314] have resulted in a myriad of stud-
velopment. These include several hydrophobic polymers that are used ies focusing on the use of polymeric micelles for the delivery of a variety
to design the core-forming blocks of the polymeric micelles and just of therapeutic molecules including plasmid DNA (pDNA), antisense oli-
one hydrophilic polymer, PEG, used to manufacture the shell of the mi- gonucleotides, messenger RNA (mRNA), small interfering RNA (siRNA)
celles, which is applied in several nanoformulations in clinical studies. as well as negatively charged drug molecules such as nucleoside tri-
There are limitations to the use of the current materials including rela- phosphates [314–317]. Proteins, being polyampholytes, can also be for-
tively poor drug incorporation in some cases, toxicity, and, in the case mulated into polymeric micelles with either cationic or anionic block
of PEG, unfavorable immunological interactions such as antibody re- copolymers [318,319]. This technology can also be expanded for the de-
sponses. Therefore, the development of novel materials that are safe, livery of supramolecular biopolymer complexes including oligomeric
enable high drug loading, and are “immunologically inert” is needed. enzymes, multienzyme complexes [320], or protein and nucleic acid
In our opinion POx- and POzi- based block copolymers satisfy these re- complexes, such as Cas9 and guide RNA [321,322]. In all these cases,
quirements and deserve future research, and more useful materials are the polyelectrolyte blocks of the block copolymers bind electrostatically
likely to emerge in the future. with the oppositely charged molecules forming a polyion complex,
In Section 3, we have extensively described polymeric micelle for- which is usually insoluble and becomes segregated within the core of
mulations for the delivery of poorly soluble small drug molecules. polymeric micelles. The hydrophilic blocks of these block copolymers
While initially thought to solubilize based on simple hydrophobic inter- form a shell around the core that stabilizes the micelles in aqueous dis-
actions, recent advances in analytical techniques have revealed new in- persion. These structures sometimes are called “polyion complex mi-
sights into these drug delivery systems. For example, drug-polymer celles” or “block ionomer complexes” [323–325]. In selected cases, in
interactions are not simply limited to the hydrophobic blocks. In fact, addition to the electrostatic interactions, the hydrophobic interactions
the hydrophilic, shell-forming blocks also play an intimate roll in solubi- of the reacting molecules with each other, or the formation of hydrogen
lization. Hansen's solubility parameters and Flory-Huggins theory are bonds between block copolymer and therapeutic molecules, can play an
successful predictors of polymer-drug compatibility in certain cases, essential role in the self-assembly and stabilization of such polymeric
but in others a more complex analysis is needed to account for all the in- micelles [326]. Despite great advancements, none of these technologies
teractions taking place between polymers and drugs. To improve our have reached the clinical stage at this time and therefore we left them
understanding of drug solubilization, multi-disciplinary approaches for outside of the current consideration. These important technologies
investigating detailed molecular interactions between hydrophobic have some key features that are common with amphiphilic block copol-
segments of block copolymers and encapsulated drugs were described. ymer micelles discussed in this review but also are dissimilar in certain
Recent advances in ssNMR, fluorescence analysis, MD, and SANS tech- fundamental aspects, such as mechanisms of formation, stability, inter-
niques have proven effective in probing the intimate interactions taking actions with the components of body fluids, and cell entry which there-
place in the micelle core and beyond. Computational methods, like fore requires a separate update and review.
QSPR, have shown promise for predicting polymer-drug compatibility We also would like to point out that most, but not all, of the studies
and will surely be at the forefront of this field moving forward. discussed in this paper focus on the delivery of small molecules using
Section 3 provides the formulation chemist with a key set of theoretical polymeric micelles to treat cancer. All examples of clinically approved
and practical tools for characterizing novel formulations to better pre- polymeric micelle drugs or polymeric micelle drugs in clinical develop-
pare for pre-clinical formulation analysis. ment discussed here belong to the area of cancer therapy. In addition,
In Section 4, we discuss many of the intricacies of analyzing poly- numerous studies have been reported in preclinical animal models
meric micelle formulations. Due to their unique dynamic nature, we using small molecule drugs in polymeric micelles of amphiphilic block
must place additional considerations on their PK analysis. We layout copolymers for the treatment of various other diseases and conditions,
many of the considerations such as protein binding changes, concerns such as autoimmune diseases, cardiovascular disease, dementia, germ
with CMC, and morphology and size considerations among many infection, ocular disease, pain management, pulmonary arterial hyper-
others. A limited understanding of the complex dynamics of polymeric tension, skin disease, spinal cord injury, and wound healing. In these
micelle formulations has hindered their translation into clinics. In this studies, the polymeric micelles were shown to greatly improve drug sol-
review, we have highlighted some of the key problems still facing the ubility and formulation stability, improve PK and bioavailability of the
field today which, when addressed early in formulation development, drug at the target site (e.g., longer systemic circulation after IV injection,
may increase the translatability of these dynamic formulations. increased brain exposure, enhanced drug disposition in skin or corneal
Section 4 can be considered a basic guide to the design of and analysis permeability), decrease drug toxicity and unwanted side effects of

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Table 7
Examples of formulations of poorly soluble small molecule drugs in polymeric micelles of amphiphilic block copolymers evaluated for treatment of various diseases and other medical use.

Therapeutic agent Drug class/mechanism Block copolymers Main study results

Dexamethasone Corticosteroids/Anti-inflammatory PEG-b-PCL The polymeric micelles exhibited longer systemic circulation compared to conventional
drug format and accumulated preferentially in inflamed joints. Reduced joint swelling,
bone erosion, and inflammatory cytokine expression were observed in joint tissue and
serum for the micelle formulation treatments in a rat model [327].
Betamethasone Corticosteroids/Anti-inflammatory PLA and PEG-b-PLGA In in vivo studies, a 35% decrease in paw inflammation was observed in the first day of
phosphate treatment and the efficacy of the formulation maintained for 9 days with a single
injection. In AbIA mice, a single injection of the micelle formulation resulted in
complete remission of the inflammatory response after 1 week [328].
Cardiovascular disease
Andrographolide Labdane diterpenoids / PEG-b-poly(propylene Due to the reactive oxygen species (ROS)-responsive nature of the polymer, the micelle
Anti-inflammatory and sulphide) not only serves as a stimuli-responsive drug carrier to quickly release andrographolide,
anti-platelet aggregation but also consumes ROS at the pathologic sites, resulting in synchronical alleviation of
inflammation and oxidative stress [329].
Dimentia
Rivastigmine Acetylcholinesterase inhibitor PEG-b-PCL The PK study in rats indicated that the brain uptake of rivastigmine-loaded micelle was
significantly higher than that of the free drug. Pharmacodynamic studies using the
Morris water maze test confirmed that faster regain of memory loss with micellar
formulation when compared to the free drug solution [330].
Germ infection
Amphotericin B Aminoglycosides / Antifungal Pluronic F127 Amphotericin B-containing Poloxamer P407-based polymeric micelles were the most
efficient among the polymeric micelle, amphotericin B and Ambisome® in a mouse
model of Leishmania amazonensis. Low parasitism, Th1 immunity and no significant
toxicity were seen in the treated animals [331].
Protoporphyrin IX Porphyrin / Photosensitizer PEG-b -PCL and PCL-b- Bacterial killing of multidrug resistant Staphylococcus aureus biofilms by
PBAE protoporphyrin IX-loaded micelles was superior compared to the control formulation.
Staphylococcus aureus infection was eradicated by daily intravenous injection of the
micelle formulation combined with its light-activation at the infected site in the mouse
models of the bacteria infection [332].
Naphthoquinone Naphthoquinones / Antileishmanial Poloxamer 407 The polymeric micelle formulation was more effective to treat Leishmania amazonensis
derivative infected mice in comparison to amphotericin B and its liposomal formulation,
Ambisome®. No severe toxicity was identified in the treated animal model [333].
8-Hydroxyquinoline Quinolones / Antifungal Poloxamer 407 The 8-hydroxyquinoline loaded poloxamer 407 micelle showed significant reduction in
the average lesion diameter of the infected tissue and in the parasite burden in the skin
and all infected organs (spleen, liver and draining lymph nodes) compared to controls
in the mouse model of Leishmania amazonensis infection [334].
Itraconazole Azoles /Antifungal Linear-dendritic Itraconazole loaded micelle had similar efficacy against Candida albicans to that of the
PEG-b-PCL free drug in in vitro. In vivo PK study in healthy mice demonstrated that the micelle
formulation could improve tissue distribution of itraconazole [335].
PDLLA and PEG-b-PDLLA Single or repeated doses of itraconazole in rats and dogs equivalent to the drug clinical
doses, were well tolerated. The plasma levels of the micellar itraconazole and its major
metabolite were comparable to those of the control formulations (Sporanox® Injection
and Oral Solution) [336].
Efavirenz Benzoxazines / Antiretrovirals Pluronic F127 and The polymeric micelle formulation exhibited a dramatic drug solubility increase (over
Tetronic® T904 8400-fold) and superior stability in comparison with the control formulations [337].
Ocular disease
α-Lipoic acid Heterocyclic thia fatty acids / Polyvinyl The polymeric micelle formulation improved solubility and accumulation of enhanced
Antioxidant caprolactam-b-poly α-lipoic acid into the bovine cornea compared to regular eye drops format of this agent.
(vinyl acetate)-b-PEG Drug containing polymeric micelles tolerated dilution in lachrymal fluid and
freeze-drying [338].
Diclofenac Phenylacetic acid derivatives PEG-b-PCL The polymeric micelles increased diclofenac permeability up to 17-fold compared to
/Anti-inflammatory that of conventional diclofenac eye drops in the rabbit cornea. In vivo PK study revealed
that the diclofenac in the polymeric micelles has 2-fold greater drug exposure than the
eye drops [339].
Pain management
Propofol Cumenes / Anesthetics PVP-b-PDLLA Propofol loaded polymeric micelles induced anesthesia effect in healthy rats. PK profile
of the drug in the micelles was not significantly different from that of an emulsion
formulation (Diprivan®) [340].
Pulmonary arterial hypertension
Rapamycin Macrolides / mTOR inhibitor PEG-b-PCL In a rat model of pulmonary arterial hypertension of mTOR inhibitor Rapamycin in
polymeric micelles administered intravenously displayed lower toxicity, increased
accumulation in lungs, and similar efficacy as the free drug in DMSO administered
intraperitoneally [341].
Skin disease
Tacrolimus Macrolides / Immunosuppressant mPEG-b-dihexyl Tacrolimus in polymeric micelle formulation showed improved drug solubility (by
substituted polylactide 400-fold), and drug disposition in porcine and human skin compared to tacrolimus
ointment formulation (Protopic®) [342].
Retinoic acid Vitamers of vitamin A / Anti-acne mPEG-b-dihexyl Retinoic acid in polymeric micelles showed improved drug disposition in porcine and
substituted polylactide human skin compared to commercially available gel formulation (Retin-A micro) [343].
Spinal cord injury
Methylprednisolone Steroids / Anti-inflammatory PEG-b-PPO-b-PEG Polymeric micelles increased the methylprednisolone level in plasma and spinal cord
compared to the free drug solution in rabbit model. Increased expression of
anti-apoptotic proteins was seen in animal group treated with micellar [344].
Dexamethasone Corticosteroids/Anti-inflammatory mPEG-b-PCL Polymeric micelles improved dexamethasone solubility and the drug neuroprotective
acetate effect in the hemisection spinal cord injury model of rats [345].

(continued on next page)

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Table 7 (continued)

Therapeutic agent Drug class/mechanism Block copolymers Main study results

Zonisamide Benzisoxazoles/Anticonvulsant mPEG-b-PLLA-poly In the hemisection spinal cord injury rat model IV injection of zonisamide in polymeric
(trimethylene micelles improved the motor function and neuron density compared to the effect of the
carbonate) drug in dimethyl sulfoxide [346].
Wound healing
Curcumin Curcuminoids / Anti-inflammatory PEG-b-PCL Curcumin loaded polymeric micelle exhibited good tissue adhesion and could release
and antioxidant activities curcumin for an extended period in vitro. In animal excision model and histopathologic
examination in rats, the formulation exhibited enhancement of cutaneous wound
repair [347].

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Tim Gershon at UNC-Chapel Hill. Montgomery, H. Yuan, Z. Li, D. Alakhova, M. Sokolsky, D.B. Darr, C.M. Perou, R.
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