Block Copolymer Stereoregularity and Its Impact on Polymeric Micellar

Review pubs.acs.org/molecularpharmaceutics

Block Copolymer Stereoregularity and Its Impact on Polymeric Micellar Nanodrug Delivery Hoda Soleymani Abyaneh,† Mohammad Reza Vakili,*,† Alireza Shafaati,†,‡ and Afsaneh Lavasanifar*,†,§ †

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2E1, Canada School of Pharmacy, Shaheed Beheshti Univ. of Med. Sci., P.O. Box 14155-6153, Tehran, Iran § Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta T6G 2V4, Canada ‡

ABSTRACT: Stereoregularity of polymers is known to influence their physicochemical and functional properties in the bulk form. Recent studies have also provided evidence for the effect of polymer stereoregularity on the physicochemical and functional properties of their self-assembled nanostructures. Research in this area has witnessed a relatively rapid pace in the past few years; however, to the best of our knowledge, a proper review of the literature has not been made to date. The goal of this review article was to fill this gap and provide a detailed overview on the current knowledge and understanding on the effect of block copolymer stereoregularity on the properties of their self-assembled nanocarriers such as size, morphology, thermodynamic and kinetic stability, and drug loading and release. Emphasis is placed on poly(ester) containing block copolymers because of their safe history of human use and extensive application in drug delivery research. KEYWORDS: stereoregularity, stereoactive polymers, polymeric micelles, tacticity, polymer crystallinity, stereocomplex micelles

1. INTRODUCTION Polymer based nanocarriers have been the subject of extensive research for application in drug, gene, and vaccine delivery. The results of research in this field clearly show that the chemical structure of the polymeric material has a great impact on the physiochemical as well as functional properties of the developed nanodelivery systems. One of the important structural characteristics of polymers that can impact their properties is their stereochemistry (i.e., three-dimensional properties). Stereoactive polymers are composed of repeating units of monomers bearing chiral centers. The type of chiral centers (R or S configuration), their number, and the sequential arrangement of these configurations are important structural features of stereoactive polymers that can affect their physiochemical characteristics, such as the crystalline or amorphous status of the polymer.1,2 Stereoactive polymers are divided to four main microstructure categories based on the sequential arrangement of their stereorepeating units: (1) isotactic polymers carry the same chiral centers along the backbone of the polymer; (2) syndiotactic polymers exhibit alternative opposite chiral centers (R and S) sequential arrangement of the backbone; (3) heterotactic polymers demonstrate alternating opposite pairs of chiral centers (RR and SS) along the backbone, and (4) atactic polymers bear a randomly sequential arrangement of different chiral centers in their backbone.3,4 An example for the structure of isotactic, syndiotactic, heterotactic, and atactic polymers of poly(lactide) is shown in Figure 1a,b. Isotactic, syndiotactic, and heterotactic polymers are stereoregular polymers with a © XXXX American Chemical Society

higher likelihood of crystallization in the polymer bulk. However, atactic polymers make polymeric materials with a higher percentage of amorphous structure. The stereoregularity of polymers as building blocks of nanocarriers is suspected to affect the arrangement and compactness of the polymer chains within the nanocarrier structure, hence affecting properties like morphology, stability, drug loading, and drug release profile.5−9 Polymeric micelles are one of the most researched polymer based nanocarriers for the solubilization of poorly water-soluble compounds, development of depots, and targeted drug delivery systems. The impact of the stereoregularity of the amphiphilic block copolymers, particularly the core-forming segment, on the functional properties of polymeric micelles has been the subject of several research articles, to date. The results, so far, show polymeric micelles consisting of block copolymers with stereoregular core-forming blocks to exhibit changes in their physicochemical and functional properties. The aim of the present review is to provide an overview on the impact of stereoregularity of the core-forming block in amphiphilic block copolymers on the properties of polymeric micellar nanodelivery systems illustrated in Scheme 1. The emphasis is Special Issue: Polymers in Drug Delivery: Chemistry and Applications Received: December 28, 2016 Revised: February 10, 2017 Accepted: February 21, 2017

A

DOI: 10.1021/acs.molpharmaceut.6b01169 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Review

Molecular Pharmaceutics

Figure 1. (a) Stereoisomers of lactides (LA). (b) Schematic model for the structure of isotactic, syndiotactic, heterotactic, atactic, and stereoblock polymers of poly(lactide) (PLA).

complexation of the core on the properties of polymeric micelles is also evaluated and summarized (Table 3).

Scheme 1. Different Physicochemical and Functional Properties of Polymeric Micelles That Are Affected by the Stereoregularity of the Core-Forming Block in SelfAssembling Block Copolymers

2. THE EFFECT OF STEREOREGULARITY OF CORE-FORMING BLOCK ON THE FUNCTIONAL PROPERTIES OF POLYMERIC MICELLES 2.1. Micellar Stability. 2.1.1. Thermodynamic Stability. The stability of block copolymer micelles includes two different concepts: thermodynamic stability and kinetic stability. The thermodynamic stability or tendency for self-assembly of block copolymers is reflected by their critical micelle concentration (CMC), i.e., the copolymer concentration below which only single chains exist but above which micelles are formed through self-assembly of block copolymers. Lower CMCs are indications of higher tendency for self-assembly and better thermodynamic stability of polymeric micelles. The selfassembly of block copolymers is believed to be an entropy driven phenomenon in water, and the CMC of block copolymers is largely determined by their hydrophilic/lipophilic balance (HLB).10 The stereoregularity in the hydrophobic block appears to affect the CMC of block copolymers, although the direction of the effect (whether stereoregularity leads to

placed on studies that have compared the characteristics of polymeric micelles made from block copolymer with stereoregular core-forming blocks to those with atactic core-forming blocks (illustrated in Scheme 2). The effect of stereoB


Review


more by the degree of branching rather than PLA stereoregularity.14 Similar results were also observed by Ding et al., who synthesized two types of triblock copolymers containing PEG and L- or -DL poly(leucine) (PLeu) blocks as P(LLeu)− PEG−P(LLeu) and P(DLLeu)−PEG−P(DLLeu), respectively, and measured their CMC using pyrene. The higher CMC of PLLeu-based micelles (bearing the semicrystalline core) compared with that of PDLLeu-based micelles (bearing the amorphous core) was attributed to the α-helical secondary conformation of the PLLeu blocks, which endowed the hydrophobic moiety with rigidity, and subsequently prevented the interaction between the pyrene and the polypeptide chains.15 Contrary to the above observations, an opposite correlation between the degree of stereoregularity in the core-forming block and CMC has been reported for diblock copolymers of poly(N-(2-hydroxypropyl)-methacrylamide)−PLLA (PHPMAPLLA) and PHPMA−P(DLLA). In this study, a higher CMC value for PDLLA-based micelles (i.e., the amorphous core) as compared to PLLA-based micelles (i.e., the semicrystalline core) was measured using pyrene.16 Similar results were obtained in another study using PEG-based block copolymers with hydrophobic blocks constructed from ε-caprolactone (CL), L-lactide (LLA), and ε-decalactone (ε-DL), either as homopolymers of ε-DL, CL, or LLA or random copolymers of CL/ε-DL, CL/LLA, and LLA/εDL. Block copolymers with amorphous cores (PEG−P(CL/εDL), PEG−P(CL/LLA), PEG−P(LLA/εDL), and PEG−P(εDL)) exhibited considerably higher CMCs than those with semicrystalline cores (PEG−PCL and PEG−PLLA).17 Our group has also prepared diblock copolymers of methoxy poly(ethylene oxide) (mPEO) and PLAs of L-lactide, D-lactide, DL-lactide or racemic mixture of 8 D- and L-lactides. Our CMC measurement by the pyrene and 1 dynamic light scattering (DLS) methods showed PLLA- or PDLA-based micelles to have a significantly lower CMC. This observation was attributed to the facilitation of micelle formation by polymeric cores capable of forming semicrystalline structure. In addition to the formation of micelles from single-type block copolymers with stereoregular core-forming blocks, many groups have investigated the stereocomplexation of two oppositely oriented stereoregular chains of block copolymers within the micellar structure (Scheme 2). Stereocomplexation between PLLA and PDLA segments within the micellar core has resulted in high crystallinity and stability of the micelles. This originates from the higher chance of hydrogen bonds and dipole−dipole interactions between the complementary chain structures within the core in stereocomplex micelles.18 Similar to what we found for CMC of micelles formed from single stereoregular block copolymer, stereocomplexation has also produced conflicting results on CMC value. For instance, in a study by Kang et al., stereocomplex micelles of mPEG−PLLA and mPEG−PDLA at a molar ratio of 1:1 did not show differences in CMC when compared to micelles prepared from a single type of block copolymers, i.e., mPEG−PLLA or mPEG−PDLA.19 Our group has also measured similar CMC values by DLS for stereocomplex micelles of mPEG−PLLA and mPEG−PDLA compared to single block copolymers.1 Similar results were obtained in another study where a series of PEG− PLA stereocomplex micelles with different PLA lengths were tried.20 Opposite to the above observations, several studies reported on a lower CMC for stereocomplex micelles compared to

Scheme 2. Schematic Structure of Polymeric Micelles Prepared from Block Copolymers of Different Stereoregularitya

a

Stereoregular micelles composed of stereoregular core-forming blocks as well as stereocomplex micelles (formed from 1:1 molar ratio of oppositely orientated stereoregular block copolymers) have a higher likelihood of forming semicrystalline core, as opposed to amorphous micelles composed of atactic core-forming block.

higher or lower CMCs) is a matter of controversy in the literature. Critical micellar concentration of block copolymers is measured by different means. The most widely used method for determination of CMC is the use of a hydrophobic fluorescent probe like pyrene, which shows changes in its excitation and emission spectra based on the polarity of its microenvironment. In addition to fluorescence spectroscopy (pyrene method), other experimental measurements such as light scattering, tensiometry (surface tension), and conductometry have been used to measure CMC of micelle.11 The CMC of different diblock copolymers of methoxy poly(ethylene glycol)-poly(lactic acid) (mPEG−PLA) composed of D-, L-, DL-lactide or a blend of DL- and L-lactide have been measured using pyrene.12 Using NMR analysis, the authors quantitatively compared the stereoregularity of different block copolymers by calculating the percentage of isotacticity (Pm) for each block copolymer and then proposed a positive correlation between degree of stereoregularity and CMC values.12 A similar correlation between stereoregularity and CMC was seen for polymeric micelles from amphiphilic block copolymers of mPEG−poly(lactide-caprolactone) mPEG− P(LA/CL). Using pyrene method, the measured CMCs for polymers with semicrystalline cores of P(LLA/CL) were higher than that of amorphous P(DLLA/CL) core. Interestingly, the measured partition equilibrium coefficient (Kv) of pyrene in the micellar solution of mPEG−P(LLA/CL), with stereoregular cores, was lower than that of mPEG−P(DLLA/CL), which had atactic cores. This finding was attributed to differences in the physical state of the core-forming blocks (i.e., the crystalline P(LLA/CL) versus the amorphous P(DLLA/CL)).13 Nevertheless, the observation pointed to the possibility of pyrene core distribution as a potential factor affecting the measured CMC values by this method. This trend has also been reported for linear block copolymers of PEG−PLA where the CMC of mPEG−P(DLLA) with an amorphous core was lower than that of mPEG−P(LLA) with a semicrystalline core; however, the results were inconclusive in Y/tree-shaped block copolymers. Apparently, CMCs of branched copolymers were influenced C


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Molecular Pharmaceutics Table 1. Effects of Core-Forming Block Stereoregularity on CMC single-type micelles mPEG2K−P(LLA)3.3K mPEG2K−P(L80%/DL20%LA)3.2K mPEG2K−P(L60%/DL40%LA)3.2K mPEG2K−P(L40%/DL60%LA)3.2K mPEG2K−P(L20%/DL80%LA)3.2K mPEG2K−P(DLLA)3.2Kb mPEG2K−P(DLLA)3.3Kc mPEG2K−P(DLLA)3.3Kd mPEG1.1K−P(CL/LLA)7.7K mPEG1.9K−P(CL/LLA)7K mPEG5.4K−P(CL/LLA)4.9K mPEG1.1K−P(CL/DLLA)8.2K mPEG1.9K−P(CL/DLLA)7.5K mPEG5.4K−P(CL/DLLA)4.9K mPEG2K−P(LLA)2.2K mPEG2K−P(DLLA)2K mPEG5K−P(LLA)5.1K mPEG5K−P(DLLA)5.4K Tree-Shaped Block Copolymer mPEG5K−P(LLA2.6K)2 mPEG5K−P(DLLA1.4K)2 mPEG2K−P(DLLA1.4K)2 mPEG5K−P(LLA1.7K)4 mPEG5K−P(DLLA1.8K)4 P(LLeu)1.2K−PEG4K−P(LLeu)1.2K P(DLLeu)1.3K−PEG4K−P (DLLeu)1.3K mPEG5K−P(LLA)9.5K mPEG5K−P(DLA)8.4K mPEG5K−P(DLLA)8.1K mPEG5K−P(L50%/D50%LA)8.6K mPEG5K−P(LLA)3.5K mPEG5K−P(L50%/D50%LA)2.6K PHPMA12.2K−P(LLA)2.5K PHPMA12.5K−P(DLLA)2.5K PEG2k−P(CL)1.8k PEG2k−P(εDL)2k PEG2k−P(LLA)2k PEG2k−P(CL/εDL)2.2k PEG2k−P(LLA/εDL)2.1k PEG2k−P(CL/LLA)1.9k Stereocomplex Micelles mPEG5K−P(LLA)9.5K mPEG5K−P(DLA)8.4K stereocomplex mPEG5.4K−P(LLA)2.1K mPEG5.4K−P(DLA)2.1K stereocomplex mPEG5.4K−P(LLA)3.7K mPEG5.4K−P(DLA)3.7K stereocomplex mPEG5.4K−P(LLA)5.2K mPEG5.4K−P(DLA)5.2K stereocomplex mPEG5.4K−P(LLA)7.5K mPEG5.4K−P(DLA)7.5K stereocomplex mPEG2K−P(LLA)1.6K mPEG2K−P(DLA)1.5K stereocomplex

CMC (mg/L) 5 2.5 0.8 0.5 0.5 0.5 2.5 0.5 1.7 2.3 3.5 0.43 0.50 1.5 10.5 18 11.1 39.5

method of measurement

ref

pyrene

12

relation between stereoregularity and CMC stereoregularity ↑ CMC ↑

13

14

46.3 35.1 18.6 11.9 12.3 4.5 ± 0.3 3.3 ± 0.4 3.83 ± 0.01 3.67 ± 0.02 7.58 ± 0.01 7.59 ± 0.01 0.625 ± 0.01 0.952 ± 0.01 1.8 3.8 12 × 103 28 × 103 16 × 103 27 × 103 26 × 103 34 × 103 3.83 3.67 4.05 3.7 ± 4.6 ± 5.1 ± 2.9 ± 2.9 ± 3.9 ± 2.1 ± 2.1 ± 2.0 ± 1.5 ± 1.7 ± 1.3 ± 6

a

0.8 0.6 0.5 0.4 0.6 0.4 0.5 0.6 0.3 0.3 0.4 0.3

15

DLS

1

Pyrene

8

stereoregularity ↑ CMC ↓

16 DPH (hydrophobic UV-probe)

17

DLS

1

pyrene

19

stereocomplex versus single micelles no difference in CMC of stereocomplex micelles with the single-type micelles

20

6

D


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Molecular Pharmaceutics Table 1. continued single-type micelles PEG5K−P(LLA)2K PEG5K−P(DLA)2K stereocomplex Y-Shaped Block Copolymer PEG5K−P(DLA)2K−P(DLA)2K PEG5K−P(LLA)2K−P(LLA)2K stereocomplex PEG5K−P(LLA)2K−P(DLA)2K PEG5K−P(DLA)2K−P(DLA)2K PEG5K−P(LLA)2K−P(LLA)2K Y-Shaped Block Copolymer mPEG1.9K−P(DLA2.5K)2 mPEG5K−P(DLA2.5K)2 zwitterionic-P(LLA2.5K)2+ mPEG1.9K−P(DLA2.5K)2 zwitterionic-P(LLA2.5K)2+ mPEG5K−P(DLA2.5K)2 DEX6K−P(LLA)2K DEX6K−P(DLA)2K stereocomplex DEX6K−P(LLA)4K DEX6K−P(DLA)4K stereocomplex PNIPAAM6K−P(LLA)2.3K PEG5K−P(DLA)1.9K stereocomplex mPEG5K−P(LLA)2.3K mPEG5K−P(DLA)2.4K stereocomplex mPEG5K−P(LLA)4.7K mPEG5K−P(DLA)5.2K stereocomplex mPEG5K−P(LLA)11.2K mPEG5K−P(DLA)11.5K stereocomplex mPEG2.5K−P(DLA)4.6K mPEG2.5K−P(DLA)4.6K+ mPEG2.5K−P(LLA)4.6K Stereotriblock Copolymers mPEG5.2K−P(DLA)1.3K−P (LLA)3.8K mPEG5.2K−P(DLA)2.4K−P (LLA)2.3K mPEG5.2K−P(DLA)3.6K−P (LLA)1.4K P(DLA)0.8K−PEG4.6K−P(LLA)0.8K P(DLA)0.9K −PEG4.6K−P (DLA)0.9K stereocomplex P(DLA)0.8K−PEG20K−P(LLA)0.8K P(DLA)0.9K−PEG20K−P(DLA)0.9K stereocomplex PEG2K−P(LLA)0.8K PEG2K−P(DLA)0.7K stereocomplex PEG2K−P(LLA)1.3K PEG2K−P(DLA)1.2K stereocomplex PEG5K−P(LLA)1.2K PEG5K−P(DLA)1.2K stereocomplex PEG5K−P(LLA)1.8K

CMC (mg/L)


24 25 15.8

ref

relation between stereoregularity and CMC

23

lower CMC of the stereocomplex micelles as compared to the singletype micelles

20 19.1 10 15 20 19.1 24 46.6 126.5 0.0213 68.8 6.63 6.87 1.53 3.87 3.99 0.63 7.9 (11.2) 25.1 5.0 (7.5) 4.3 ± 0.3 4.8 ± 0.4 3.2 ± 0.3 2.6 ± 0.4 2.8 ± 0.5 1.6 ± 0.4 1.2 ± 0.6 1.3 ± 0.3 0.8 ± 0.3 2.34 2.61

27

28

7

25

1.66 1.67 1.73 50 50

surface tension (γ)

22

40 106 110 90 73 80 68 50 50 45 100 97 86 90 E


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Molecular Pharmaceutics Table 1. continued single-type micelles PEG5K−P(DLA)1.8K stereocomplex PEG5K−P(LLA)4.6K PEG5K−P(DLA)5.1K stereocomplex PEG5K−P(LLA)4.6K PEG5K−P(DLA)5.1K stereocomplex

CMC (mg/L)


ref

90 80 13 12 8.9 13.8 14.1 13.0

relation between stereoregularity and CMC

26

a

() Denotes a block in a given block copolymer. / Separate different monomers used within a block of a given block copolymer. A series of mPEG− PLA block copolymers was prepared using DL-lactide as monomer of PLA block by employing different catalysts (Sn or Al series), producing polymers with different tacticity of batactic, cisotactic stereoblock, and dheterotactic for PLA blocks. The different tacticity of block copolymers was translated to different CMC values of related micelles, in spite of having similar MWt for the consisting blocks.

repulsion between the shell-forming PEG and PNIPAAM blocks can be compensated for, using the strong stereocomplexation between PDLA and PLLA blocks constituting the hydrophobic core of mixed micelles.28 Overall, several studies point to the stabilizing effect of coreforming block stereoregularity and/or core stereocomplexation, but contradicting reports also exist in the literature. This contradiction makes it difficult to make a definite conclusion in this regard. The divisive results also point to a need for further systematic studies to elucidate the effect of core stereoregularity and stereocomplexation on the CMC of block copolymers. In this regard, contributing factors such as the molecular weight of the core and/or hydrophilic/hydrophobic Wt/Wt ratios may swing the results one way or another. Therefore, the effect of these structural parameters should be considered. In addition, it appears that the difference in pyrene partitioning the micellar core with semicrystalline versus amorphous structures could also affect the results of CMC measurements and lead to the over- or underestimation of CMC from its actual value. It is therefore likely that the measured CMC using pyrene partitioning is not an ideal method for these intended comparisons and that another method of measurement (e.g., DLS or surface tension) should be adopted for a systematic comparison. Overall, upon evaluation of the data presented above and summarized in Table 1, it appears that other factors, such as polymer average molecular weight, hydrophilic/lipophilic balance, polydispersity, level of isotacticity, degree of crystallinity, and method of measurement can influence the direction of observed changes in CMC when we move from block copolymers with atactic, to syndiotactic to isotactic coreforming structures. These properties are not characterized and taken into account in several reports made to date. Thus, for now, in the absence of such data and until the role of these contributing factors is delineated, the results of one study cannot be extrapolated to others and become the foundation for a general rule for CMC. 2.1.2. Kinetic Stability. At concentrations below CMC, polymeric micelles are still expected to maintain their core/ shell structure at least for a short period of time, or in other words demonstrate kinetic stability.29 Studies on the effect of stereoregularity of block copolymers on kinetic stability of micelles are scarce. DLS following incubation of polymeric micelles in the presence of sodium dodecyl sulfate (SDS), which acts as a destabilizing agent, has been used as an indication of micellar kinetic stability. Under these conditions, stereocomplex micelles of mPEG−PLLA and mPEG−PDLA have shown higher kinetic stability as compared to single type

micelles prepared from a single type of block copolymers with stereoregular segments. This includes a study where lower CMC values were measured for stereocomplex micelles of mPEG−PLLA and mPEG−PDLA using pyrene compared to micelles from each of this block copolymers.7 Similarly, for amphiphilic block copolymers comprising PEG and poly(methylcarboxytrimethylene carbonate) (PMTC) backbone branched with either PLLA or PDLA, formation of stereocomplexes polymeric micelles reduced the CMC as measured by pyrene.21 Lower CMC values for stereocomplex micelles were also observed in aqueous solutions of PEG−PLA diblock and triblock copolymers using surface tension (γ) measurements.22 In a series of linear and Y-shaped block co/terpolymers of PEG−PLA-PLA, stereocomplexation resulted in reduced CMC values as compared to the corresponding micelles from single linear or Y-shaped copolymer as measured by pyrene.23 For Y-shaped polymers containing zwitterionic sulfobetaine−(PLLA)2 and PEG−(PDLA)2 with different molecular weights (MWts) for the PEG block, a lower CMC value for stereocomplex micelles was observed as compared to micelles of the single type PEG−(PDLA)2, irrespective of the PEG MWt.24 Similar results were obtained in another study when a set of block copolymers, i.e., diblock copolymers of PEG−PLLA and PEG−PDLA, enantiomeric copolymer blend of diblock copolymers (PEG−PDLA/PEG−PLLA), and stereotriblock copolymers of PEG−PDLA-PLLA25 and PEG−PLLAPDLA, was used.26 The obtained data revealed that micelles of the stereotriblock copolymers had lower CMC values than those of single-type diblock copolymers as well as the enantiomeric copolymer blends, as measured using pyrene25 and surface tension methods.26 This observation was attributed to the easier formation of stereocomplex crystals in the PLA core of micelles from triblock copolymers. Similarly, stereocomplex micelles of dextran-PLLA (Dex-PLLA) and Dex-PDLA, containing equimolar mixture of diblock polymers exhibited lower CMC values compared to micelles of single-type block copolymers, as measured by pyrene.27 In an interesting approach, the possibility of the formation of stable mixed micelles through PLA stereocomplexation was investigated using PEG−PDLA and poly(N-isopropylacrylamide)−PLLA (PNIPAAM−PLLA) block copolymers. Mixed micelles of PNIPAAM−PLLA and PEG−PDLA were prepared by directly dissolving the two block polymers in deionized water at a mass ratio of 1:1. The measurement of CMC values using pyrene showed that the CMC for the PNIPAAM-PLLA and PEG− PDLA mixture is much lower than those of single type block copolymers. This low CMC value for mixed micelles of PNIPAAM−P LLA and PEG−P DLA confirmed that the F


Review

Molecular Pharmaceutics PLLA- or PDLA-based micelles. Of note, PDLLA-based micelles with amorphous cores have shown the least kinetic stability and were more prone to dissociation upon incubation with SDS. Dissociation of a large fraction of SDS-treated PDLLA-based micelles, exhibited by drastic decrease in scattered light intensity within 2 h, was associated with an increase in polydispersity index (PI). The same trend, but to a much smaller extent, has been observed with PLLA- or PDLA-based micelles. In contrast, the stereocomplex micellar solutions were substantially more stable as they showed only a minimal decrease in scattered light intensity. Importantly, this phenomenon was reproducible in the presence of a drug. A 0.5 Wt% paclitaxel (PTX) loaded stereocomplex polymeric micelles showed enhanced kinetic stability.19 This may be an indication that loading a drug in stereocomplex micelles did not interfere with the crystallization of the core-forming block and/ or contributed in the semicrystalline structure formation itself. As a result the stereocomplex system maintained its superior stability as compared to drug-loaded micelles of single-type block copolymer. Our research group studied the effect of method of polymerization on micellar kinetic stability by synthesizing different PEO−PLA diblock copolymers of L-lactide, DL-lactide or racemic mixture of D- and L-lactides via two methods of bulk or solution polymerization. In our study, SDS-treated micelles prepared from polymers of DL-lactide or racemic mixture of Dand L-lactide exhibited a drastic decrease in scattered light intensity and an increase in PI. The same trend, but to a reduced extent, was observed with the same group of micelles prepared by solution polymerization. In contrast, micellar structures of PEO−PLLA, particularly those prepared by solution polymerization, showed an enhanced kinetic stability. In general, our results indicated that the polymeric micelles prepared from copolymers synthesized via the solution method were more stable, with PEO−PLLA micelles being the most stable ones.8 Overall, a correlation between higher degree of crystallinity of the core-forming block and kinetic stability of micelles under this experimental condition was observed.8 In an elegant study, Mochida et al. investigated the stability of polymeric micelles of PEG−poly(glutamic acid) (PEG− P(Glu)) of L-, D-, and DL-P(Glu) complexed with cisplatin on the P(Glu) segments. They found that L- and D-P(Glu)-based micelles to form regular secondary structures, whereas DLP(Glu)-based micelles adopt mainly random conformation. In this case, the release of cisplatin was expected to destabilize the polymeric micelles as it was assumed to form cross-links between polymer chains within the micellar structure (Figure 2). In this paper, they followed the disassembly of D-, L- or DLP(Glu)-based micelles, upon cisplatin release, by monitoring the scattered light intensity over time. For L- and D-P(Glu)based micelles, the scattered light intensity gradually decreased over 96 h. In contrast, DL-P(Glu)-based micelles showed a drastic decrease in the scattered light intensity only after 10 h. The contrasting profiles of the intensity−time curves was attributed to the ordered core structure based on α-helix bundles in D- and L-P(Glu)-based micelles, which is in contrast to the random aggregation of P(Glu) strands in DL-P(Glu)based micelles. Moreover, in this study the pathway of disassembly was examined by applying the sedimentation velocity (SV) method of analytical ultracentrifugation to the micelle solutions. The results showed a gradual and relatively constant discharge of unimers and dimers over time range of 72 h for L- and D-P(Glu)-based micelles. In contrast, there was an

Figure 2. Proposed hierarchical structure of cisplatin (CDDP)-loaded micelles (CDDP/m) prepared through the self-assembly of poly(ethylene glycol)-poly(L-glutamic acid) (PEG−P(Glu)) after polymer−metal complex formation with cisplatin. The core of the micelles is composed of 18 P(Glu) blocks, which form associated R-helix bundles, being surrounded by a PEG shell. The carboxylates in the P(Glu) blocks coordinate with cisplatin. Reprinted with permission from ref 30.

accelerated discharge of unimers and dimers from DL-P(Glu)based micelles even after 10 h, which correlated with the starting time point of the progressive decline in the scattered light intensity. A decrease in the hydrophobicity of the core in DL-P(Glu)-based micelles due to cisplatin release within the first 10 h of experiment was suggested to promote the penetration of water and chloride ions into the micellar core, leading to an accelerated release of cisplatin and an abrupt disassembly of micelles. Conversely, the presence of robust αhelix bundles in the core of L- and D-P(Glu)-based micelles was suggested to further stabilize the structure by close association of hydrophobic α-helices, maintaining the hydrophobicity of the micellar core even after release of cisplatin, leading to gradual disassembly of these micelles.30 In addition to following the scattered light intensity of polymeric micelles by DLS as a method of stability measurement, the Foerster Resonance Energy Transfer (FRET) based assay is being used as a valuable tool in evaluating and screening stability of polymeric micelles;31−34 however, to our knowledge there is no report on using this technique for following the stability of stereoregular micelles. Measuring micellar degradation rate is another approach that was recently employed for testing system stability. In a recent study, enzymatic degradation of micelles of PEG−PLLA-PDLA stereotriblock copolymers, PEG−PLA diblock copolymers of L-, D-, and DL-lactides, and PEG−PLLA/PEG−PDLA enantiomeric mixtures were investigated in aqueous solution with the presence of proteinase K. After partial degradation, the micelle samples were analyzed by 1H NMR to calculate the LA/EG molar ratio as an indicator of degradation rate. Based on this measurement, the PEG−PDLLA micelles compriseing amorphous cores degraded 3−6 times faster than that of stereocomplex micelles.26 2.2. Micellar Morphology. The physical state of the coreforming block is shown to affect the morphology of polymeric micelles. High surface tension in the core−water interface favors aggregation of more copolymers into one micelle so that the interfacial area per copolymer chain can be reduced. As the aggregation number grows, the dense corona chains become more stretched because of an increase in their repulsion. This leads to a free energy penalty, restricting the aggregate growth. The micellar equilibrium state is, therefore, determined by a balance between these two forces: the surface free energy associated with the core−water interface, and the free energy penalty due to the stretching of the dense corona chains. In the G


Review


depending on the PLA block length. It was suggested that the crystallization tendency of the core-forming PLLA blocks to be the main factor governing the smaller size of micelles in this case.38 Similar results were observed for micelles based on PHPMA−PLA copolymer, where PDLLA-based micelles with amorphous cores showed slightly larger diameters compared to PLLA-based micelles with semicrystalline cores.16 In contrast to previous studies, the presence of different stereoregularity in P(Glu) backbone of cisplatin-loaded polymeric micelles of PEG−P(Glu) did not affect the micellar size, and DLS analysis revealed a comparable hydrodynamic diameter of approximately 25 nm for L-, D-, and DL-P(Glu)based micelles. It is worth mentioning that the distribution of the core diameter of DL-P(Glu)-based micelles was relatively broader than that of L- and D-P(Glu)-based micelles.30 Investigation on the effect of hydrophobicity on the size in micelles with different status of core crystallinity has been the topic of another study. Here, micelles with semicrystalline cores (PEG−PCL) exhibited distinctly different trends in size as a function of the hydrophobic ratio (defined as Mn,hydrophobic/ Mn,hydrophilic) as compared to those with amorphous cores (PEG−Pε−DL), measured by DLS. Amorphous PEG−Pε−DL micelles increased in size with increasing hydrophobic ratio, presumably due to an increase in the core volume. By contrast, the semicrystalline PEG−PCL micelles decreased in size as the hydrophobic ratio increased. These observations are most likely due to changes in core crystallinity, which influences the core size. However, no discernible difference was observed between the semicrystalline and amorphous micelles when the hydrophobic ratio was held constant.17 For stereocomplex micelles, depending on the structure of the polymers, smaller or larger micelles after stereocomplexation were reported as compared to those from single-type stereoregular polymers. For instance, measurement of the size of micelles of PAA−poly(N-acryloyl-D-leucine methyl ester) (PAA−PDLeuOMe) and PAA−PLLeuOMe by DLS showed similar particle sizes with reasonably narrow PIs. Nonetheless, the average size of the stereocomplex system was approximately 20 nm larger and had a broader dispersity. Results obtained by TEM were in agreement with DLS data, indicating that the stereocomplex micelles were relatively larger.39 Formation of stereocomplex micelles with larger size was also observed in another study where the effect of the addition of homopolymers of PLLA, PDLA, and PDLLA on the size of lactosome (a core−shell type polymeric micelle composed of amphiphilic polydepsipeptide of helical PLLA and a hydrophilic block of poly(sarcosine) (N-methylated glycine)) was examined by DLS. With an increase of the mixing PLLA amount to 25 mol %, the hydrodynamic diameters of lactosome (35 nm) remained within 20% of its original size. Similar results were obtained with the addition of PDLLA, which was in contrast to results obtained by the addition of PDLA. PDLA induced a drastic increase of the hydrodynamic diameters, which became over 100 nm at 25 mol %. PLLA is known to form hexagonal crystalline structures. In the hydrophobic core of lactosomes, PLLA blocks are considered to be regularly aligned and molecularly packed to locally form the semicrystalline structure. When PLLA or PDLLA were added to the lactosomes up to 25 mol %, the assembly sizes remained similar to original ones. However, PDLA that forms stereocomplex with the matrix polydepsipeptide of lactosome directly increased the size of polymeric micelles perhaps by interfering with the original hexagonal crystalline structure in the core.40

case of copolymers with semicrystalline core-forming block, strong competition between the energy of the semicrystalline core and chain stretching of the dense corona makes formation of nonspherical micelles possible because the low-curvature flat (i.e., nonspherical) surface with more area per corona chain is thermodynamically more stable than the spherical surface with less area per corona chain.13,35 In line with this explanation, in a study performed by Zhang et al., different morphologies have been reported for amphiphilic block copolymers of PEG−P(CL-DLLA) and PEG−P(CL-LLA). Transmission electron microscopy (TEM) images showed that micelles with amorphous cores of PDLLA adopted a spherical shape, whereas micelles with semicrystalline core of PLLA exhibited a cylindrical shape.13 In concert with this observation, in another study, it was shown that D- or Lbased micelles of PEO−PLA or poly(acrylic acid)−PLA (PAA−PLA) copolymers adopted cylindrical shapes. However, stereocomplexation triggered the reorganization of these cylindrical micelles to form spherical stereocomplex micelles. This phenomenon is in contrast to that usually observed in crystallization-driven self-assembly systems and was an unexpected observation given that under thermodynamic control, stereocomplex micelle formation between two polymers of such structural similarity (i.e., D- and L- mixture) would be expected to retain the cylindrical morphology. Nonetheless, the stereocomplexation driving force that exists in this system changed the crystallization behavior of the coreforming blocks and hence was able to drive this morphology switch for the reasons that are not fully understood.36 Similar results were also reported in the study of Y-shaped polymers containing PEG−(PLLA)2 and PEG−(PDLA)2 with different MWt for PEG block, where TEM images revealed a worm-like morphology for micelles of single-type copolymers and spherical shape for stereocomplex micelles independent of MWt of PEG.37 Furthermore, a similar study using Y-shaped polymers containing zwitterionic sulfobetaine−PLAs was conducted by the same research group. In concert with their previous results, TEM images revealed the formation of spherical stereocomplexes micelles. However, different morphologies were observed with single-type PEG−(PDLA)2 micelles depending on hydrophilic/lipophilic ratio, with the formation of worm-like micelles containing a low MWt of PEG as opposed to spherical micelles containing a high MWt of PEG.24 Coexistence of several morphologies was also observed in another study of PEG−PLA diblock copolymers of L-, D-, and DL-lactides for different length of PEG and PLA blocks. In this study, micellar morphology turned from spherical micelles to filomicelles by decreasing the weight fraction of PEG. Of note, the mixture of filomicelles of PEG−PLLA and PEG−PDLA also yielded stereocomplex filomicelles. Comparisons of the micellar morphologies with L- and DL-lactide content at similar PLA block lengths revealed spherical forms for DL-based and mainly filomicelles for L-based micelles. This observation was attributed to the more rigid nature of L-based micelles with higher tendencies to form filomicelles as compared to DL-based micelles containing randomly distributed L- and D-lactide units along their backbone. These findings were confirmed by atomic force microscopy (AFM) analysis, as well.20 2.3. Micellar Size. In a study performed by Agrawal et al., triblock copolymers of PDLLA−PEG−PDLLA were shown to form micelles ranging from 18 to 20 nm in diameter; while the PLLA−PEG−PLLA copolymers formed micelles of 7−16 nm H


Review


the incorporated MCT was too low to be considered as a reason for increase in drug loading capacity. Therefore, it was concluded that the reduced core crystallinity played the major role in improving drug loading. OCMs with MCT contents of 40 Wt % showed decreased release rate for all tested drugs. Reduced core crystallinity would enlarge the volume of the amorphous region, causing more widespread dispersion of the loaded drug in the micelle cores and subsequently increase the diffusional path and reduce drug release rate. Nonetheless, the extent of reduction of release rate was still majorly determined by drug−polymer compatibility.43 In contrast to previous observations, which showed the effect of micellar core crystallinity on the level of drug loading, in another study, triblock copolymers of PLA−PEG−PLA with either semicrystalline PLLA or amorphous PDLLA core exhibited similar level of drug loading for sulindac and tetracaine. Of note, drug release was much faster for polymers with semicrystalline PLLA cores as compared to those with amorphous PDLLA cores. For instance, the maximum release of sulindac occurred in 4−8 days from the PLLA-based micelles in comparison to 18 days for PDLLA-based micelles. As for tetracaine, the maximum release occurred over 2−4 versus 8−9 days for PLLA and PDLLA-based micelles, respectively. In this study, the release rate of sulindac increased as the PLLA or PDLLA block lengths were increased. This observation may be attributed to the tighter packing of the core. For tetracaine, the rate of drug release from PLLA-based micelles of increased length appeared similar but was again much faster than the PDLLA-based micelles. However, with an increase in block length of PDLLA, a slower release was observed. In this case, increasing PLA block length likely resulted in a better polymer−drug interaction, thereby slowing the rate of tetracaine release.38 In alliance with these results, our research group also observed a more rapid release of nimodipine from polymeric micelles with semicrystalline cores, i.e., PEO−PLLA micelles, at high loaded drug levels. We attributed this observation to the fast formation of PEO-PLLA micelles due to presence of semicrystalline PLLA core-forming blocks, which prevent the uniform dispersion of the drug in the micellar core. In this case, the drug may be squeezed out of the core and eventually accumulate in the surrounding regions (core/shell interface or the shell) and release with a rapid rate from micelles. In contrast, PEO−PDLLA copolymers that have less crystalline core-forming structure may allow for better drug loading within the micellar core. As for the effect of polymerization method on rate of drug release, higher release of nimodipine from micelles was observed when polymers were prepared by solution polymerization compared to those prepared by the bulk method. This was also attributed to higher stereoregularity of core-forming blocks in polymers prepared in solution than bulk.8 Similar to our results, Ding et al. also reported a higher rate of drug release from micelles containing a semicrystalline core. Their results revealed a faster release of doxorubicin (DOX) from PLLeu−PEG−PLLeu micelles as compared to PDLLeu−PEG−PDLLeu micelles with slightly higher loading of doxorubicin (DOX) for PLLeu-based micelles.15 In the case of cisplatin-loaded polymeric micelles of PEG− P(Glu), encapsulation efficiency was comparable in L- and DP(Glu)-based micelles and lower than that of DL-P(Glu)-based micelles. However, release profiles of cisplatin from L- and DP(Glu)-based micelles showed a sustained release, while cisplatin release from DL-P(Glu)-based micelles was accel-

In contrast to previous studies, Chen et al. have observed smaller size for stereocomplex micelles prepared from equimolar mixtures of PEG−PLLA and PEG−PDLA when compared with the single-type micelles of PEG−PLLA or PEG−PDLA. In this study, environmental scanning electron microscope (ESEM) images showed an average diameter in line with the data measured by DLS.7 Similarly, in another study, stereocomplex micelles of Dex-PLLA and Dex-PDLA, containing equimolar mixture of diblock polymers, exhibited a smaller size as measured by DLS.27 Of note, in a study by Kang et al., stereocomplexation had no effect on the micellar size. In this study, data obtained by DLS for stereocomplex micelles of PEG−PLLA and PEG−PDLA copolymers at a molar ratio of 1:1 versus D-, L-, and DL-based micelles showed no significant difference in size at a fixed lactide content.19 The controversy observed in the above-mentioned studies may be due to other contributing factors such as the length of the hydrophobic/hydrophilic blocks. Sites of stereocomplex crystallization can also influence the micellar compactness and size. This was the subject of a study where the effect of the lengths of the middle block of stereotriblock copolymers of PEG−PDLA-PLLA on stereocomplex crystallinity and its location was investigated. Obtained data revealed that crystallization was lower in the triblock copolymer having a short middle PLA block because of the influence of the PEG block. Furthermore, in this type of micelles, stereocomplex crystals were formed in the outer layer of the core, i.e., in the neighborhood of the core−shell interface, which made the micelles more compact.25 2.4. Micellar Drug Loading and Release. The interaction of a drug with the core of polymeric micelles has a significant impact on the level of drug loading and the release behavior.41 The physical state of the core can, however, affect this drug− polymer interaction. Micellar cores with a highly packed semicrystalline structure are less likely to accommodate an even distribution of drug. Instead, drug molecules may be pushed to the periphery of the core and eventually show rapid drug release. Micelles with amorphous cores allow better drug− micellar core interaction, but might be less efficient than semicrystalline cores in restricting the diffusion path of the incorporated drug.38 Studying the effect of core stereoregularity and crystallinity on the level of drug loading in polymer micelles as well as drug release can provide clues toward the design of optimized micellar carriers that simultaneously maintain high drug content and slow release properties. This was a matter of investigation in a study where block copolymers of PEG−P(CL-LLA) with semicrystalline cores and PEG− P(CL-DLLA) with an amorphous core were used for the encapsulation of two model drugs, indomethacin and vitamin E. Much more drug was entrapped in the micelles with the amorphous core compared to the micelles with semicrystalline cores. Since in both carriers the chemical structure of the core was identical, the difference in drug loading was attributed to the differences in the physical state of the core in terms of crystallinity.42 Preparation of oil-containing micelles (OCMs) of medium chain triglyceride (MCT) was another method used to inhibit crystallization in the PCL core of PEG−PCL micelles and subsequently to improve drug loading. Incorporation of MCT at low levels (5 Wt %) improved drug loading marginally and by increasing MCT content to 25 Wt %, the drug loading of three model drugs of disulfiram (DSF), cabazitaxel (CTX), and TM-2 (a taxane derivative) increased. Of note, considering the solubility of drugs in MCT, the amount of drug dissolved in I


Review

Molecular Pharmaceutics erated.30 Of note, the results are due to the difference in the method of drug encapsulation for these micelles where cisplatin is complexed to the block copolymer core-forming block, and its release may be governed by micellar disassembly more than drug diffusion. Stereocomplex formation between blends of enantiomeric structures is another approach that can alter the level of drug loading and release. The loading and release of rifampin from micelles was studied after stereocomplexation between equimolar mixtures of PEG−PLLA and PEG−PDLA block copolymers in water. Stereocomplex polymeric micelle revealed higher levels of drug loading and encapsulation efficiency and slower rate of drug release as compared to the single-type copolymer micelles, possibly due to the higher stability caused by stereocomplex crystallization.7 Similar results were achieved by PTX-loaded micelles prepared from a stereocomplex mixture of Y-shaped PEG−PDLA−PDLA and PEG−PLLA− PLLA, where stereocomplex micelles yielded higher level of drug loading (11.6 Wt % in stereocomplex micelles compared to 7.9 Wt % for PEG−PDLA−PDLA micelles) and slower release. For instance, the amount of PTX released from PEG− PDLA−PDLA micelles after 10 days was more than 50%, whereas only 30% of the PTX was released from stereocomplex micelles. The slower rate of drug release from stereocomplex micelles was speculated to be the result of stronger interactions between the stereocomplexes and PTX molecules.23 In line with these observations, in another study, stereocomplex micelles of PEG−PDLA and PEG−PLLA presented slightly higher drug encapsulation than micelles prepared from either of the single-type polymers. However, the rate of PTX release from stereocomplex micelles was only significantly slower when compared to PLLA-based micelles.44 Similar results were also reported in another study of PTX-loaded micelles of PEG− PLA diblock copolymers where stereocomplex micelles showed better PTX loading and slower rate of drug release as compared to PLLA-based micelles. The more compact core structure of stereocomplex micelles, which disfavors drug diffusion, accounted for the observed profile. Of note, in this study, PDLLA-based micelles showed lower drug loading as compared to PLLA-based micelles. The higher drug loading of PLLA-based micelles was attributed to the formation of filomicelles that present higher drug loading as compared to spherical micelles formed by PDLLA-based micelles. The in vitro release behaviors of these micelles in PBS showed that less than 35% of PTX was released after 71 days in all cases.20 Similar to PTX, DOXloaded stereocomplex micelles of cholesterol-enhanced PEG− PLA copolymers exhibited higher drug loading and encapsulation efficiency and slower drug release. The cumulative DOX release from single-type PLLA- and PDLA-based micelles was about 90% in 72 h, while that of stereocomplex micelles was about 75%.45 In contrast to the results of this study, stereocomplexation decreased loading of DOX in micelles of Dex-PLAs as compared to single type PLLA- and PDLA-based micelles. Nonetheless, DOX-loaded stereocomplex micelles still exhibited a slower release rate.27 Level of DOX loading and its in vitro release behavior were also extensively investigated in a separate study from micelles of PEG−PLLA−PDLA triblock copolymers, single-type PEG−PLLA or PEG−PDLA diblock copolymers (with semicrystalline cores) as well as their enantiomeric mixture (forming interchain stereocomplexes in the core), and PEG−P DL LA block copolymers (with amorphous cores). Data revealed that drug loading was highest

in PEG−PLLA−PDLA triblock copolymer micelles and lowest in PEG−PDLLA (with amorphous cores). Larger drug loading of PEG−PLLA−PDLA micelles was attributed to the bigger size in micelle cores for these structures. As for drug release, fastest release was observed for PEG−PDLLA with amorphous cores, which was followed by PEG−PLLA micelles and PEG−PLLA− PDLA triblock copolymers of short PLLA chains. The slowest release in this study was observed for PEG−PLLA−PDLA triblock copolymer micelles of longer PLLA chains and stereocomplex micelles formed from enantiomeric mixtures of PEG−PLLA and PEG−PDLA. This was attributed to the interactions between stereocomplex crystals and the drugs. PEG−PDLLA micelles comprising amorphous cores had the smallest drug loading, even with the looser packing of PLA in the micelle core; they also showed much faster drug release. This was explained by the weak drug/polymer interactions in the micelle core, but the reason for this weak interaction in this specific structure was not elaborated on.26 The stereoactivity of the copolymer can also determine their capacity for the encapsulation of a certain enantiomer, although studies on this aspect are rather scarce in the literature. We only found one study where the effect of compatibility of chirality between drug and polymer on level of drug loading was investigated. In this study, the loading of drugs with different chirality (cymarin, D-genistin and L-peruvoside) was tested in PEG−PLLA and PEG−PDLA copolymers. In general, micelles containing PDLA core showed slightly higher entrapment efficiency for compounds with D-sugars compared to micelles with core of PLLA. For peruvoside with L-sugars, no significant difference was observed between copolymers containing PLLA and PDLA block in the terms of drug loading.46 Overall, very much like the effect of stereoregularity on micellar thermodynamic stability, controversial results on the effect of this factor on drug loading and release behavior is seen. The difference can be attributed to the interfering contribution of micellar size as well as polymer−drug pair interactions in this case. In general for compatible and interacting polymer−drug pairs, an increase in crystallinity seems to enhance drug loading and slowdown drug release. This may not be true for some polymer−drug pairs where an increase in polymer stereoregularity and crystallinity can lead to nonuniform distribution or squeezing of the drug out of the core structure in polymeric micelles. 2.5. Biological Behavior of Polymeric Micelles. Polymer stereoregularity can directly influence the kinetics of cellular uptake and represent a key feature in determining the in vivo fate of polymeric micellar carriers. 2.5.1. In Vitro Cell Interactions. Considering the above reports, it is not surprising that the observations on the effect of core-forming block stereoregularity and crystallinity on biological behavior of polymeric micelles seem contradicting in the literature. In general, two methods were used to follow the cellular uptake of polymeric micelles. In the first approach, a fluorescent dye was chemically conjugated to the polymer backbone.14,16 In the second approach a fluorescent anticancer drug DOX (or probe) was physically loaded in the micelles.15,27,45 When chemical conjugation of a fluorescent dye to micellar components was used, a lower cell uptake for polymeric micelles with stereoregular core-forming blocks has been reported.14,16 For instance, in the study by Garofalo et al., the cellular uptake of the “tree-shaped” copolymers of PEG− (PLA)n with one, two, or four atactic or isotactic PLA arms was J


Review

Molecular Pharmaceutics Table 2. Effects of Core-Forming Block Stereoregularity on in Vitro Cell Interactions single-type micelles mPEG2K−P(LLA)2.2K mPEG2K−P(DLLA)2K mPEG5K−P(LLA)5.1K mPEG5K−P(DLLA)5.4K Tree-Shaped Block Copolymer mPEG5K−P(LLA2.6K)2 mPEG5K−P(DLLA1.4K)2 mPEG2K−P(DLLA1.4K)2 mPEG5K−P(LLA1.7K)4 mPEG5K−P(DLLA1.8K)4 PHPMA12.2K−P(LLA)2.5K PHPMA12.5K−P(DLLA)2.5K P(LLeu)1.2K−PEG4K− P(LLeu)1.2K

method of micellar tracing fluorescently labeled with FITC conjugation at terminal chain of PLA

loaded DOX

cholesterol enhanced-PEG10K−P(LLA)4.6K cholesterol enhanced-PEG10K−P(DLA)4.6K stereocomplex DEX6K−P(LLA)2K DEX6K−P(DLA)2K stereocomplex DEX6K−P(LLA)4K DEX6K−P(DLA)4K stereocomplex

method of tracing

size (nm) DLS

no report

fluorescently labeled with Oregon Green conjugation at terminal chain of PHPMA

P(DLLeu)1.3K−PEG4K− P(DLLeu)1.3K stereocomplex micelles

a

dye/drug content

two two two two two two 879 267 two

OG mole% 0.9 0.9 Dox Wt% 18.3 ± 0.4 19.7 ± 0.4

DL%

peaksa peaks peaks peaks peaks peaks ± 212 ± 77 peaks

shape

spherical

charge (zeta potential) −29 −30 −31 −27 −24 −20 −41 −24 −26

± ± ± ± ± ± ± ± ±

10 7 8 16 6 7 11 7 12

effect of stereoregularity on cell uptake stereoregularity ↑ toxicity ↓

17.0

ref 14

16

20.4 211 ± 5.9 179 ± 7.8

all spherical

stereoregularity ↑ toxicity ↑

15

size (nm) DLS

shape

stereocomplex versus single micelles

ref

loaded DOX

8.8 8.3 9.5

104 ± 4.3 118 ± 5.2 96 ± 4.8

all spherical

higher

45

loaded DOX

9.57 9.34 5.70 12.43 12.60 10.54

158 164 103 182 179 121

± ± ± ± ± ±

all spherical

lower

27

8.7 9.6 6.7 8.3 7.9 7.8

DLS measurement showed formation of polydispersed micelles as evidenced by existence of two peaks for size distribution.

formulations within the time frame of toxicity or cell uptake assay of PTX-loaded micelles in the HeLa cells. In the second group of studies, where a physical loading of the fluorescent probe (or DOX) rather than its conjugation is used, the content of loaded drug and rate of release during the time frame of uptake study should be similar to be able to make direct comparisons between polymeric micellar structures. Otherwise, the degree of cell associated fluorescence and/or cytotoxicity can be influenced by the content of loaded drug and/or release rate from the micellar carrier and will not be a true indication of their cellular interaction. For instance, in the study by Ding et al.,15 both DOX-loaded PLLeu- and PDLLeubased micelles showed a similar rate of release within a 2 h time frame of a cell uptake study. The PLLeu-based micelle had slightly lower DOX content and larger size (Table 2), but showed higher cell uptake in MG63 and Saos-2 cells (two types of human osteosarcoma cell lines). In this study, the release of DOX from both micellar carriers was similar within the time frame of the uptake study, but the media in the cell uptake study was different from the release experiment. In addition, the contribution of nanoparticles size in the observations is not clear. Cell associated DOX fluorescence intensities were higher for stereocomplex micelles of cholesterol-enhanced DOX-loaded PEG−PLA micelles as compared to single type PLLA- and PDLA-based micelles in renal carcinoma cells. DOX-loaded stereocomplex micelles showed better cytotoxicity as compared

compared, and the micelles of PEG−(PDLLA)2 with amorphous cores showed enhanced cellular uptake compared to the other formulations in kidney (HEK293t) and uterine (HeLa) derived tumor cell lines.14 In this study, micelles were covalently tagged with the fluorescein isothiocyanate isomer I (FITC) dye; however, no comparison was made on the conjugation efficiency of FITC among different polymers. Since the FITC conjugation happens at the PLA terminal, it is important to know the content/conjugation efficiency of the FITC, especially in the branched copolymers, which have more than one PLA terminal. In another study where another florescent dye, Oregon Green 488 cadaverin (OG), was covalently conjugated to the micelles of PHPMA−PLA micelles at PHPMA terminal, higher cellular uptake was seen for PDLLA-based micelles with amorphous cores compared to PLLA-based micelles with crystalline cores in HeLa cells.16 In this study, PDLLA-based micelles showed a slightly larger size than the PLLA-based micelles (20.4 nm versus 17.0 nm, respectively). The higher cellular uptake observed with the PDLLA-based micelles was tentatively attributed to the amorphous nature of the core and subsequently the different size and stability. In line with higher cellular uptake observed with the PDLLA-based micelles, the PTX containing micelles with the PDLLA core showed significantly higher cytotoxicity. Both formulations showed similar levels of drug loading; however, no release data were presented to compare the release of PTX from these K


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Figure 3. Biological performance of cisplatin-loaded micelles (CDDP/m) after i.v. administration to BALB/c nu/nu mice bearing a BxPC3 tumor. Time course of the concentration of platinum in (a) plasma and (b) blood cells, and the accumulation of platinum in (c) kidneys, (d) liver, (e) spleen, and (f) tumor after i.v. administration of CDDP/m at a dose of 100 μg/mouse on a cisplatin basis. (g) Antitumor efficacy after three administrations of CDDP/m on days 0, 2, and 4 at a dose of 4 mg/kg on a cisplatin basis. The arrows indicate the day of drug administration. The volume of the tumor was normalized to the initial day (day 0). Data are the mean ± SEM, n = 5 [n = 6 for (g)]. *p < 0.05, **p < 0.01, ***p < 0.001. Reprinted with permission from ref 30.

to single-type micelles, as well.45 These observations were speculated to be the result of the higher drug loading/ encapsulation efficiency, and more efficient intracellular DOX release from stereocomplex micelles, and are not true indications of enhanced cell uptake.45 In contrast, in another study, DOX-loaded stereocomplex micelles of Dex−PLLA and Dex−PDLA exhibited significantly lower toxicity as compared to DOX-loaded micelles of single type against human liver carcinoma (HepG2) cells.27 This was attributed to the lower drug loading/encapsulation efficiency, slower DOX release, and/or lower cell uptake for stereocomplex micelles. 2.5.2. In Vivo Fate of the Micellar Drug Delivery System. As for in vivo behavior, the most extensive reported studies in this context were completed by Mochida et al. on polymeric micellar formulations of cisplatin based on PEG−P(Glu) block copolymer. Cisplatin-loaded micelles of L-, D-, and DL-P(Glu) were intravenously injected to mice bearing subcutaneous xenografts of human pancreatic adenocarcinoma. Blood and tissue samples were collected at defined time points, and the concentration of platinum was quantified by inductively coupled plasma mass spectrometry (ICP-MS). The obtained data showed that both L- and D-P(Glu)-based micelles had prolonged and comparable blood circulation time, as approximately 20 and 3% of the injected dose remained in the plasma at 24 and 48 h, respectively. However, cisplatin as part of DL-P(Glu)-based micelles was rapidly eliminated from the bloodstream, showing only 0.7 and 0.2% of the injected dose in the plasma at the corresponding time points,

respectively. The distribution of platinum drugs to blood cells was significantly low throughout the experiment compared to their concentration in plasma, confirming that blood cells did not affect the biodistribution of micelles. Moreover, the accumulation in kidneys for all micelles stayed low and slightly decreased over 48 h, indicating that the kidney clearance was not responsible for the significant drop of cisplatin as part of DLP(Glu)-based micelles in plasma. Conversely, in the liver and spleen, while cisplatin concentration as part of L- and D-P(Glu)based micelles was relatively low over 48 h, DL-P(Glu)-based micelles displayed an abrupt increase in the accumulation of cisplatin at 8 h after administration. This accumulation of DLP(Glu)-based micelles in liver and spleen corresponded with their decreasing profile in plasma concentration, that is, from approximately 71% of the injected dose per milliliter of plasma at 4 h to 29% at 8 h. Presumably, the loss of integrity in the structure of DL-P(Glu)-based micelles, which synchronized with discharging unimers and dimers, altered the surface properties of DL-P(Glu)-based micelles leading to impaired stealth efficacy during circulation, and abrupt accumulation in the liver and spleen through opsonisation. In contrast, L- and D-P(Glu)based micelles underwent a well-controlled erosion-like disassembly process keeping the integrity of the PEG palisade intact, owing to their ordered-core structure stabilized by αhelix bundles. In line with longer circulation of L- and DP(Glu)-based micelles, an increase in the cumulative amount of platinum in pancreatic tumor xenograft over 24 h was observed for these micelles (Figure 3). Conversely, cisplatin as part of DLL


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Figure 4. (a) In vivo imaging of subcutaneous pancreatic cancer-bearing mouse using three types of ICG-lactosomes (n = 5). Images were taken at 48 h after the lactosome administration. For the near-infrared fluorescence (NIRF) imaging was used. (b) Graphical image of three types of ICGpoly(lactic acid)s used for lactosome labeling. (c) Time-courses of NIRF signal intensities detected at three region of interests (ROIs) as illustrated in Figure 2a. Reprinted with permission from ref 47. Copyright 2012 Elsevier.

were made.44 In a similar manner, the in vivo antitumor activity of DOX-loaded of PLLeu−PEG−PLLeu, PDLLeu−PEG− PDLLeu micelles, and free DOX in nude mice bearing osteosarcoma tumors xenografts has been reported. The in vivo antitumor efficacies were in the order of DOX-loaded PDLLeu-based micelles > DOX-loaded PLLeu-based micelles > free DOX. However, in the absence of pharmacokinetic and biodistribution data, the reason behind better antitumor activity of PDLLeu- over PLLeu-based micelles is not clear.15 Development of new probes for tumor detection using nanocarriers is a major field of investigation. For this purpose, in an interesting study, in vivo disposition of lactosomes (which contains PLLA in its composition) mixed with indocyanine green (ICG) labeled PLLA, PDLA, and PDLLA homopolymers at 1.5 mol % was examined. The aim was to find an imaging probe with improved signal ratio in the tumor against background by controlling the in vivo blood clearance time of lactosome. Lactosomes containing ICG-PLLA, ICG-PDLA, and ICG-PDLLA are abbreviated as ICG-lactosome(L), ICGlactosome(D), and ICG-lactosome(DL), respectively, which were administered intravenously to nude mouse bearing human pancreatic cells. In the case of ICG-lactosome(L), ICG fluorescence signal spread over the whole body soon after administration, and gradually accumulated at the tumor region, where it peaked 24 h after administration. In the case of ICGlactosome(D), a similar observation was made, but the signal intensity remained high for 48 h. This was in contrast to a gradual decrease of ICG-lactosome(L) at 48 h. ICG-lactosome(D) thus shows a longer retention property at the tumor region than ICG-lactosome(L), which might be due to the formation of stereocomplexes with PLLA in the lactosome. However, ICG-lactosome(DL) spread quickly over the whole body and showed diminished fluorescence intensity. The fluorescence

P(Glu)-based micelles revealed no increase in platinum accumulation after 8 h in tumor, which was consistent with the significant drop in cisplatin plasma concentration. The level of cisplatin as part of DL-P(Glu)-based micelles considerably decreased at 48 h, whereas L- and D-P(GLu)-based micelles maintained their cisplatin accumulation in the tumor even at 48 h, achieving 2-fold higher drug levels than their DL-P(Glu) counterparts at this time point. It is reasonable to assume that the faster dissociation of DL-P(Glu)-based micelles into unimer/dimer than L- and D-P(Glu)-based micelles may result in their lower retention in tumor tissues. As expected, cisplatinloaded PEG−P(Glu) micelles with L- or D-configuration in the core, effectively suppressed pancreatic tumor growth and maintaining the initial size of the tumors for more than 20 days. The therapeutic efficacy of cisplatin-loaded PEG−P(Glu)based micelles with DL-configuration in the core was significantly lower than L- and D-P(GLu)-based micelles, yet significantly better than that of control (PBS). The in vitro cytotoxicity of cisplatin micelles was similar regardless of the core conformation; therefore, the observed difference in the in vivo antitumor activity can be correlated with the enhanced cisplatin exposure to tumor tissue by micelles with isotactic core structure.30 In another study, the in vivo biodistribution and antitumor efficacy of PTX-loaded micelles was investigated in mice bearing human Lewis lung cancer cells. Obtained results showed that stereocomplex micellar solutions of PLLA−PEG− PLLA and PDLA−PEG−PDLA copolymers had higher plasma PTX concentrations compared to the currently available clinical formulation of this drug. Nonetheless, in this study no comparison between in vivo pharmacokinetic, biodistribution, and/or antitumor activity of stereocomplex micellar formulations of PTX over those prepared from single-type polymers M


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those with atactic (or amorphous) cores. Self-assembly of block copolymers containing stereoregular core-forming segments is also shown to lead to formation of cylindrical (micelles) as opposed to spherical ones. In contrast, the effect of introduction of stereoregular core-forming blocks on the direction of the change on micellar thermodynamic stability (CMC), drug loading, drug release, and cell interaction has been unpredictable. Most studies conducted so far also point to higher micellar stability, slower drug release, higher cellular uptake, and enhanced tumor accumulation, for stereocomplex micelles compared to micelles prepared from single-type stereoregular block copolymers. However, the direction of the change in micellar thermodynamic stability, drug loading, micellar size, and morphology by introduction of stereocomplexes to the core is still inconclusive. The identified controversy in the literature, by itself, indicates a need for the conduction of more systematic studies using appropriate methods and controls enabling the elucidation of other contributing factors and interactive parameters pertaining to structure−activity relationships in this regard. This could include conduction of studies on block copolymers with well controlled methods of polymerization, ratios of hydrophobic to hydrophilic blocks, and molecular weight polydispersity. In addition, instead of dividing the polymers to general subgroups of stereoregular and atactic, we need to be more deliberate at the structural level. The stereoregularity of the block copolymers should be reported as a percentage of isotactic enchainment (Pm) using NMR analyses to show what is the exact percentage of isotactic, syndiotactic, heterotactic, and/or atactic segments in a hydrophobic backbone of a given block copolymer.12,48 Having this information will provide us with a better foundation to make a better correlation between the degree of polymer isotacticity and micellar properties. Moreover, as the stereoregularity of the polymer backbone may translate to the formation of crystalline regions, in future studies the percentage of crystallinity in the polymer and related micelle should be identified using different techniques such as DSC and XRD;12,19 then correlated with micellar properties. Finally, the methods used for the characterization of micellar properties should be analyzed for potential artifact effects during the comparisons.

intensity at the tumor region remained nearly the same from 4 to 24 h after administration of ICG-lactosome(DL), however. These results suggested that the stability of these lactosomes in the bloodstream changes depending on the stereoregularity in the polymeric micelle core. Interestingly, the amount of lactosomes accumulated at tumor region in the time range from 2 to 9 h after administration was highest for ICGlactosome(DL). However, at 48 h after administration, the level for ICG-lactosome(D) was the highest. The half-lives of ICGlactosome(D), ICG-lactosome(L), and ICG-lactosome(DL) in the blood were calculated to be 54, 24, and 14 h, respectively, showing a positive correlation with the order of tumor accumulated lactosomes 48 h after administration (Figure 4).47

3. CONCLUSION AND FUTURE PROSPECTS In summary, the stereoregularity of the core-forming block in self-associating block copolymers appears to affect the physiochemical properties of polymeric micelles and their functional behavior both in vitro and in vivo. The direction of the effect on each characteristic is not resolved well, however, and is a matter of controversy, as summarized in Table 3. The results of the conducted studies so far appear to point to a better kinetic stability and tumor accumulation for block copolymers of higher stereoregularity in the core-forming block (which can form cores of higher crystallinity) compared to Table 3. Effects of Core-Forming Block Stereoregularity on Different Micellar Properties micellar property thermodynamic stability (CMC)

kinetic stability morphology

size

drug loading

release profile

cellular uptake

in vitro toxicity

tumor accumulation antitumor efficacy

in single type polymeric micelles stereoregularity ↑ CMC ↑12−15 stereoregularity ↑ CMC ↓1,8,16,17 stereoregularity ↑ stability ↑8,19,26,30 stereoregularity ↑ cylindrical shape8,13,20,24,36,37 coexistence of several shapes20,24 stereoregularity ↑ size↓16,38 independent of stereoregularity17,30,39 stereoregularity ↑ loading ↑15,20,26 stereoregularity ↑ loading ↓30,42,43 independent of stereoregularity38,46 stereoregularity ↑ release ↑8,15,38,43 stereoregularity ↑ release ↓26,30 stereoregularity ↑ uptake ↑15 stereoregularity ↑ uptake ↓14,16 stereoregularity ↑ toxicity ↑ 15

stereoregularity ↑ toxicity ↓14,16 independent of stereoregularity30 stereoregularity ↑ accumulation ↑30,47 stereoregularity ↑efficacy↑30 stereoregularity ↑efficacy↓15

stereocomplex versus single micelles lower7,21−28 equal1,19,20 higher8,19,26,30 cylindrical shape20 spherical shape24,36,37 smaller7,27 equal19 larger39,40 higher7,20,23,44,45

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AUTHOR INFORMATION

Corresponding Authors lower27

*Phone: 587-920-4349. Fax: 780-492-1217. E-mail: vakili@ ualberta.ca. *Phone: 780-492-2742. Fax: 780-492-1217. E-mail: afsaneh@ ualberta.ca.

slower7,20,23,26,27,44,45

ORCID

Afsaneh Lavasanifar: 0000-0001-5108-7124 higher

45

higher

45

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by grants from Natural Sciences and Engineering Research Council of Canada (NSERC). H.S.A. acknowledges funding from Alberta Cancer Foundation (ACF) and Women and Children Health Research Institute (WCHRI). The authors would like to thank Amir Soleimani, Department of Pharmacy and Pharmaceutical Sciences, University of Alberta, for critical reading of the manuscript.

lower27

higher47 comparable to clinical formulation44 N


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Block Copolymer Stereoregularity and Its Impact on Polymeric Micellar

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