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1 Self-Assembly and Responsiveness of Polypeptide-Based Block Copolymers: How Smart Behavior and Topological Complexity Yield Unique Assembly in Aqueous Media Jacob G. Ray, Ashley J. Johnson, Daniel A. Savin School of Polymers and High Performance Materials, University of Southern Mississippi, Hattiesburg, MS Correspondence to: D. A. Savin ( Received 17 December 2012; accepted 11 January 2013; published online 12 February 2013 DOI: /polb ABSTRACT: Polypeptide-based amphiphilic block copolymers are an attractive class of materials given their ability to form welldefined aqueous nanoassemblies that respond to external stimulus through secondary structure transitions. This report will highlight recent literature in the area of polypeptide-based block copolymer self-assembly, with the major focus being on how the responsive nature and structural complexity of the polypeptide blocks can be incorporated into systems with complex topologies such as ABA/BAB/ABC triblock copolymers, AB 2 and A 2 B star copolymers, and miktoarm l-abc star terpolymers. In particular, the role of interfacial curvature changes and how they result in morphology transitions will be discussed. The smart assembly properties of peptides in complex block copolymer topologies can lead to enhanced responsiveness, morphological complexity, and unique morphological transitions with varying solution conditions. VC 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2013, 51, KEYWORDS: amphiphiles; biomimetic; block copolymers; selfassembly INTRODUCTION Amphiphilic block copolymers are able to self-assemble into well-defined nanostructures in aqueous solution. 1 5 The equilibrium morphology and aggregation number of diblock copolymer assemblies is primarily determined by the balance of three energetic factors: (1) interfacial tension, (2) corona chain crowding, and (3) core chain stretching. The balance of these three factors dictates an equilibrium curvature for the aggregate. 6 8 Typically, solution morphologies formed by amphiphilic block copolymers follow a trend of increasing interfacial curvature. As the hydrophilic fraction is increased in the copolymer, vesicles, cylindrical micelles, and spherical micelles (in the order of increasing interfacial curvature,) are the most common morphologies (Figure 1). 5,9,10 Physically, this is explained as a balance between entropic freedom of the hydrophilic coronal chains and shielding of the hydrophobic blocks from the aqueous solution; as the hydrophilic fraction increases, the chains are more able to effectively stabilize these assemblies without close-packing, and the free energy of the system is lowered when the coronal chains are provided more entropic freedom/mobility through increased curvature. For diblock copolymers, the size of spherical micelles can be predicted based on the aggregation number and degree of polymerization of the coronal chains. 11 The theoretical sizes of vesicles and worm-like micelles are difficult to predict, yet the thickness of these types of assemblies is often dictated by the chain length of the hydrophobic blocks. 12 Other non-typical morphologies such as multi-compartment micelles, disk micelles, nanotubes, 20 toroidal micelles, 21,22 bicontinuous micelles, 23,24 and corkscrew micelles 25 have been observed, often arising through structural complexity, solvent composition, or specific interactions. Although there is much known about the solution behavior of diblock copolymer aggregates, a new and increasingly useful class of amphiphilic materials based on polypeptides has been shown to form aggregates where the size and morphology is responsive to solution ph and temperature. These morphological changes are partially the result of changes in chain density and/or interfacial curvature as the peptide block shifts from a rod-coil type of block copolymer to a coil-coil block copolymer. 26,27 Some recent studies have addressed this behavior theoretically. 28 In addition, the diversity of synthetic polymer chemistries and click" reactions offer the ability to covalently link a variety of different polymers together, and the potential for biocompatibility from peptides creates possible applications in drug/gene delivery, imaging, and nanocatalysis Nanostructures from block copolymers have many advantages over small molecule surfactants such as enhanced stability with increasing molecular weight, tuning assembly size or membrane thickness (in the case of bilayer formation) with molecular weight, and synthetic variability allowing integration of specific chemistries/ physical properties within the assemblies. In particular, the synthetic variability specifically allows for the tunability of VC 2013 Wiley Periodicals, Inc. 508, PART B: POLYMER PHYSICS 2013, 51,

2 Daniel A. Savin received a BS in chemistry from Harvey Mudd College in 1995 and a PhD in chemistry with Prof. Gary Patterson at Carnegie Mellon University in After a postdoctoral position with Prof. Timothy Lodge at the University of Minnesota, he began his independent career at the University of Vermont. He joined the School of Polymers and High Performance Materials at The University of Southern Mississippi (USM) in Research in the Savin group is focused in three primary areas: self-assembly and responsiveness of topologically complex peptide-based block copolymers, energy damping nanocomposites, and polymers for environmental applications. Ashley J. Johnson received a BS in biochemistry from Mississippi College in She is currently a 5th year PhD student at USM. Her research interests include the self-assembly of polypeptide amphiphilic block copolymers and tunable stimuli responsive peptides for use as delivery vehicles. From 2009 to 2011, Ashley was a GAANN fellow where she spent time promoting the importance of science and mathematics to young students. Jacob G. Ray received a BS in polymer science from USM in 2008 working under Prof. Charles McCormick. He received a PhD in polymer science and engineering from USM in 2013 working under Savin, where his research focused on understanding the aqueous selfassembly behavior of polypeptide hybrid copolymers with complex topologies. He was a GK-12 fellow at USM, and he recently accepted a position as an Advanced Applications Development Scientist at Eastman Chemical in Kingsport, TN. chemical/physical properties to access precise applications (i.e., attachment or complexation of a drug or gene molecule within solution aggregates); additionally, the rich variety of synthetic reactions allows for the creation of complex molecular topologies that can allow for unique, advantageous assembly properties. With great interest in biotechnology, block copolymer assemblies that respond to stimuli such as ph, salt, and temperature allow for greater function in biological environments. Pioneering research on ph and salt responsiveness involved synthetic polyelectrolytes such as poly(acrylic acid) and poly(vinyl pyridine) coupled to neutral, hydrophobic blocks (i.e., poly(butadiene)), where changes in ph or salt concentration impacted the ionic nature of the hydrophilic block Common examples of temperature responsiveness are a result of the incorporation of blocks that contain a lower critical solution temperature (LCST) such as poly(n-isopropyl acrylamide) 41 or poly(propylene oxide) 11 or the incorporation of polypeptides where the secondary structure transitions with temperature (i.e., a-helix to b-sheet). 42 Polypeptide-based block copolymers offer many advantages in the realm of responsive block copolymer self-assembly such as biocompatibility and enhanced biofunctionality. Drawing from proteins shape-specific function, we can exploit the differential, organized behavior of the peptides secondary structure and its three adaptable structural motifs of random coil, a-helix and b-sheet. The a-helix is a tightly coiled, rod like structure stabilized by intramolecular hydrogen bonding of main chain NH and CO groups that are situated 4 residues apart from one another creating a helix with a pitch of 0.54 nm and 3.6 amino acids residues per turn. Conversely, b- strands adopt a more extended conformation where the distance of adjacent amino acids is approximately 0.35 nm, wherein the a-helix the adjacent residue distance is 0.15 nm. Within b-strands, adjacent amino acids side chains adopt opposite directions along the strand, this orientation allows for the intermolecular hydrogen bonding to occur creating b- sheets of several b-strands. These systems have been classified as smart" materials given their ability to reversibly change their well-defined secondary structure in response to an external stimulus such as ph (a-helix to random coil) or temperature (a-helix to b-sheet) where the change in secondary structure drastically alters the physical behavior of the self-assembled morphologies. Aqueous self-assembly of polypeptide-based hybrid block copolymers, often called molecular chimeras, and copolypeptides will be of main focus in this review. While there have been recent reviews in this area, this article attempts to highlight the role of interfacial curvature changes and how they result in morphology transitions. This discussion will then lead into an overview of polypeptide-based copolymers of higher topological complexity such as ABA/BAB triblock copolymers, AB 2 star copolymers, ABC triblock terpolymers, and l-abc star terpolymers. As will be discussed, the smart" assembly properties that are offered in diblock systems can be of benefit in advanced topologies leading to PART B: POLYMER PHYSICS 2013, 51,

3 FIGURE 1 Cyro-TEM images of (a) vesicles, (b) worm-like micelles, and (c) spherical micelles showing morphological progression to higher curvature assemblies with increasing hydrophilic fraction (f) of amphiphilic block copolymers. Reproduced from Ref. 10. Copyright Reprinted with permission from AAAS. enhanced responsiveness, morphological complexity, and morphological transitions. ASSEMBLY AND RESPONSIVENESS OF PEPTIDE-BASED DIBLOCK COPOLYMERS This section will be devoted to peptide-based hybrid diblock copolymers with the main focus being on how the complex three-dimensional (3D) secondary structures of the polypeptide impact assembly properties. Several examples of responsiveness governed by secondary structure transitions of peptide coronal chains will be highlighted, and this will illustrate that these transitions can lead to size changes with ph/t, physical changes at the core/corona interface, and even morphological transitions. Synthetic Techniques for Polypeptide-Based Diblock Copolymers For nearly 70 years, the most common, efficient, and costeffective method to produce polypeptides of sufficient molecular weight has been through the polymerization of N-carboxyanhydrides (NCAs). 48 NCAs are easily synthesized through a reaction between amino acids and phosgene or a phosgene derivative (i.e., triphosgene), and the polymerization can be achieved through initiation using an amine-functional starting material. Recently, Cheng and Deming 49 reviewed different synthetic strategies that can be used to produce polypeptides using NCA polymerization, whereby they outlined some potential side reactions and ways of controlling the polymerization to diminish side reactions and result in peptides with low molecular weight distributions. Among the ways to control the polymerization include using transition metal initiator complexes (i.e. cobalt or nickel) or TMS protected amine initiators, 53,54 both of which lead to faster kinetics without undesired side reactions. Furthermore, unwanted side reactions have also been shown to effectively be diminished by simply running the polymerization at 0 C instead of room temperature, or at reduced pressures Assemblies with Polypeptide Coronas The past two decades have seen several literature reports of aqueous assembly of block copolymers with peptide corona chains, whereby responsiveness is governed in part by changes in the secondary structure. 11,42,58 72 Much of this research focus has been devoted to ph-responsive block copolymers containing peptides such as poly(l-lysine) (PLys) (pka 10.3) 73 and poly(l-glutamic acid) (PLGA) (pka 4.3) 70 which exhibit helix-coil transitions associated with the charged state of the amino or carboxylic acid side chains of PLys and PGlu respectively. Our group recently reported spherical micelle formation for poly(propylene oxide) 44 -b-poly(l-lys) 217 and poly(butadiene) 107 -b-poly(llys) 200 block copolymers. 11,62 By performing in-depth light scattering studies, we were able to calculate a hydrophobic core size based on the aggregation number (N agg ) determined from Zimm analysis, assuming a fully dehydrated core. A theoretical change in assembly dimensions can be estimated from the change in the rise/ repeat unit for the peptide as a result of the helix-coil transition (random coil ¼ 0.37 nm, a-helix ¼ 0.15 nm). 74 This dimension change was found to agree well with the secondary structure of the peptide at the extremes of ph. This suggests that: (1) the responsiveness comes purely from the helix-coil transition and (2) there is negligible chain exchange as a function of ph. Similarly, Lecomandoux and coworkers reported spherical micelles from poly(isoprene) 49 -b-plys 123 which showed a size change from 23 nm to 44 nm from ph 11 to 6 in a saline solution. 65 Interestingly, they also reported a similar system of poly(butadiene) 48 -b-poly (LGA) 114 (PB-b-PLGA), that resulted in spherical micelle formation that only showed a size change of ca. 8 nm through the helix-coil transition; they mention that this smaller size change could be a result of folding or disruption of the PLGA helices. 59 Schlaad and coworkers reported large vesicle formation for PB 165 -b-plys 88 under physiological saline conditions. 71 They reported a change in average radius from 364 nm at ph 7.0 (100% protonation) to 215 nm at ph 10.3 (35% protonation). In contrast to the spherical micelles above, the dimension change in the vesicle cannot simply be a result of the helix-coil transition. Instead, they report that the interfacial (bilayer membrane) properties are changing dramatically 510, PART B: POLYMER PHYSICS 2013, 51,

4 FIGURE 2 Schematic images from Schlaad et al. that illustrates changes in chain density at the vesicle interface as a result of differences in secondary structure and charge at ph 7.0 and 10.3 for PB 165 -b-plys 88 vesicles. Reproduced from Ref. 71. Copyright Reprinted with permission American Chemical Society. while the aggregation number of the assembly is remaining relatively constant. In particular, the interfacial chain density, calculated based on Kratky analysis of the static light scattering, changes dramatically when the coronal chains go from charged at ph 7 to mainly charge neutral at ph This was explained in terms of colloid stabilization; charge charge repulsions at ph 7 cause the interfacial density to be lower (i.e., chains need more space to avoid charge charge interactions) than when the helices prefer to pack closely at ph 10.3 (Fig. 2). 71 Around the same time, a related PB 107 -b- PLys 27 vesicle-forming system from our group showed a similar result in both the larger-than-expected dimension change and in the decreased interfacial chain density at high ph observed from Kratky analysis. Temperature responsiveness of peptide-based assemblies has also been achieved through secondary structure transitions of PLys with temperature. 75 Our group reported the temperature-responsiveness of the PB 107 -b-plys 27 vesicle-forming system as a function of temperature at high ph. 42 It was found that a-helix to b-sheet was observed at ph 10.9 over the temperature range from 20 to 60 C. Figure 3 shows R h versus temperature data at ph 2, 10.3, and 10.9; these data show that the peptide must be fully helical (above the pk a of Lys) for the helix-sheet transition to be observed. At ph 10.9, the vesicles nearly double in size (ca nm) when going from an intramolecular a-helix to an intermolecular antiparallel b-sheet. This size increase is attributed to the flattening of the vesicle interface from intermolecular hydrogen bonding between corona chains. The inset of Figure 3 confirms the transition by circular dichroism. Physically, this type of transition is interesting because it not only FIGURE 3 R h vs. temp. for PB-PLys vesicles at ph2 (green triangles), 10.3 (red circles), and 10.9 (black squares). The inset is a CD spectrum at ph 11.2 at 20 o C (squares) and 60 C (circles). from Reproduced from Ref. 42, Copyright 2008, Reprinted with permission from Elsevier. changes the size of the vesicles but also the interfacial packing of the molecules, leading to potential changes in porosity and wall thickness. In addition to temperature responsiveness through secondary structure changes of a polypeptide, synthetic polymers with LCST behavior have been incorporated into block copolymers with polypeptides to yield thermoresponsiveness and schizophrenic behavior. 11,67,76 Agut et al. studied the assembly behavior of a Jeffamine-b-PLGA block copolymer that showed double hydrophilic properties at temperatures below the LCST of the Jeffamine (ca. 30 C) under neutral ph conditions. Upon heating the sample above the LCST, self-assembly was observed. Interestingly, the overall size of the micelles increased slightly with elevated temperatures above the LCST, a result of increasing aggregation number with increasing temperature. However, through the use of small angle neutron scattering, it was determined that the hydrophobic core radius decreased with increasing temperature through dehydration for the Jeffamine core. 67 Similarly, our group recently reported temperature-responsive micelles and vesicles of poly(propylene oxide)-b-plys (PPO-b-PLys) block copolymers (PPO LCST ¼ 10 C). 11 Through the use of dynamic light scattering, we observed a sharp increase in scattering intensity above the LCST denoting a unimer to micelle/vesicle transition. Furthermore, we illustrated the potential for schizophrenic behavior of these assemblies at low temperature and high ph (Fig. 4). Liu and coworkers also reported schizophrenic assembly behavior of a dual responsive diblock copolymer, poly(n-isopropyl acrylamide) 65 -b-poly(lga) 110 (PNIPAM 65 -b- PLGA 110 ). 76 At ph below 4 and at 25 C, they report spherical micelles formation with PNIPAM as the corona and the hydrophobic PLGA as the core. However, inversion of these micelles was realized going to alkaline conditions and PART B: POLYMER PHYSICS 2013, 51,

5 FIGURE 4 Schematic of schizophrenic assembly behaivor of ph and temperature responsive PPO-PLys. heating the sample to 45 C, well above the LCST of PNIPAM (ca. 35 C). 76 Lecommandoux and coworkers have reported schizophrenic behavior of double hydrophilic block copolymers (DHBCs) with two ph-responsive blocks. 68,72 In one report, they demonstrate reversible vesicle formation for a symmetric, zwitterionic PLGA 15 -b-plys 15 copolypeptide. 72 Under mild ph conditions (5 9), conditions where both blocks have some degree of charged side groups, the chains existed as individual unimers. At low ph, the vesicles were comprised of charged PLys coronas and hydrophobic PLGA cores, whereas the corona and core blocks were reversed under alkaline conditions. They additionally discuss the importance of the rod-like character in the hydrophobic peptide block (at either ph extreme) serving to drive the formation of flatter interface assemblies. 72 Deviation from Theoretical Morphologies and Morphology Transitions in Diblocks There have been a few examples of peptide-based diblock copolymers that self-assemble into morphologies that deviate from theoretical morphologies based simply on hydrophilic fraction. For example, vesicles have been observed for PB 40 - b-plga 100 containing ca. 85 wt % glutamate (in the range where spherical micelles would be expected). 60,61 Our group reported vesicle formation of PPO-b-PLys with high hydrophilic fractions. It is known that PPO is mildly hydrophobic at ambient temperature, and vesicle formation (rather than micellization) may be a result of partial hydration of the hydrophobic block leading to an increased hydrophobic volume, thus forcing a lower interfacial curvature assembly. 11 Another report demonstrated non-spherical micelle formation from poly(styrene) (PS)-b-PLys amphiphiles, and the morphological behavior was shown to be independent of the block length of PLys. As noted from neutron and light scattering data, the assemblies were likely rod-like micelles. 66 It is possible that polydispersity plays a role in dictating the morphology of these assemblies. For example, it is known that short hydrophilic chains are able to selectively partition into the core of a vesicle (with higher curvature). In addition, it is likely that longer chains in a polydisperse sample can stabilize the higher interfacial curvature in the end cap of a rod-like micelle. Our group reported rod-like micelle formation for PB-b-PLys block copolymers with a PLys fraction in the vicinity of either a rod-like or spherical micelle assembly behavior. 62 Figure 5 shows a plot of R h versus ph for PB b-plys 100 and PB 60 -b-plys 50. At ph above 4, the assemblies exist as large rod-like micelles or a mixture of rod-like and spherical micelles, with the size decreasing from low to high ph from deprotonation of the PLys side chains. However, as the ph is decreased from 4, we observed that the average hydrodynamic radii decreased. This was attributed to the formation of spherical micelles; as the protonation of the PLys chains increases, the corona chains enter a region with a higher effective interfacial curvature due to charge charge interactions. The concentration of spherical micelles continues to increase at lower ph values (as seen from the continued decrease in the radius). Physically, this shows that the helix-coil transition can lead to slight changes in overall hydrophilic volume which can initiate morphology transitions from assemblies of higher and lower interfacial curvature. Assemblies Composed of Hydrophobic Peptide Cores As shown above, polypeptides form well-defined secondary structures that can influence the morphological properties of polypeptide-based amphiphiles. When the hydrophobic portion of the amphiphile is a helical peptide, the structurally complex nature of the peptide strongly influences or dictates the assembly properties. In general, the helical, rod-like nature of the peptide results in a flatter interface, which favors vesicle of fibril formation FIGURE 5 R h vs. ph plots for PB 107 -b-plys 100 (top) and PB 60 -b- PLys 50 (bottom). The insets of these figures are CONTIN analyses from dynamic light scattering at a 90 scattering angle at the highest ph values (PB 60 -b-plys 50 data shows a small distribution of smaller sizes at high ph denoting a small concentration of spherical micelles). Reproduced from Ref. 62. Copyright Reprinted with permission from American Chemical Society. 512, PART B: POLYMER PHYSICS 2013, 51,

6 FIGURE 6 Illustration of molecular arrangement of helix-coil PBLG-b-HYA within flat interface vesicles. Reproduced from Ref. 83. Copyright Reprinted with permission from American Chemical Society. Lecommandoux et al. have reported vesicle formation based on polypeptide hybrid compolymers In their research, they studied assemblies formed from poly(c-benzyl-l-glutamate) 23 -b-hyaluronan 10 (PBLG 23 -b-hya 10, polypeptide-bpolysaccharide) polysaccharide. They denote that the PBLG block was chosen to guarantee the formation of vesicles, whereby its rigid, helical nature leads to close packing of helices which serves to favor flatter interface morphologies (Fig. 6). 83 Given the formation of well-defined vesicles and biofunctionality of the hydrophilic HYA, they were further able to illustrate the ability for these systems to effectively serve as controlled drug delivery vehicles for the hydrophilic chemotherapeutic, doxorubicin. 83,84 In another report, they illustrated vesicle formation for a similar system, dextran-b- PBLG. 85 More recently, Lecommandoux and coworkers detailed vesicle formation of copolypeptide-based amphiphiles comprised of hydrophobic PBLG and hydrophilic glycosylated propragyl (glycine) (PGC). The PBLG-b-PGC block copolymers were synthesized by sequential addition N-carboxy anhydrides (NCAs), and the PG blocks were glycosylated through azide-alkyne cycloaddition. 81 Interestingly, it was shown that vesicle formation was seen throughout the range of synthesized PBLG-b-PGC copolymers of ranging hydrophilic fraction. However, at the highest hydrophilic weight fraction, 68%, a mixture of vesicles and spherical micelles were observed denoting an upper limit of hydrophilic fraction to result in polymersome formation for these copolypeptides. These examples of vesicle formation were contingent on proper nanoprecipitation, whereby the molecules were directly dissolved in DMSO and assembly formation was brought induced by addition to water. In general, amphiphiles with rod-like core blocks often require nanoprecipitation to form well-equilibrated assemblies. Physically, rod-like hydrophobic blocks have a lower dynamic mobility than flexible, hydrophobic coil blocks. As such, the order of nanoprecipitation (organic solution to water or vice versa) and speed of addition can impact the assembly size, size dispersity, or even the observed morphology for these kinetically trapped assemblies. 81,86 Other reports in literature have shown vesicle formation for copolypeptide amphiphiles that contain two blocks of naturally occurring amino acids; one block being a rod-like, hydrophobic helix and the other a polyelectrolyte serving to stabilize the assembly through electrostatic interaction with water. 75,78 80,82,87 Deming and coworkers have performed seminal work in this area, and the physical behavior of these vesicle-forming copolypeptides is well illustrated through their work. For example, they studied the aggregation behavior of PLys-b-poly(L-leucine) (PLys-b-PLLeu) copolymers; the PLeu was employed as due to its hydrophobicity and its ability to form stable helices. Early reports by their group showed that the PLys block lengths must be kept modestly low to avoid hydrogel formation through interchain charge charge repulsion resulting in twisted fibril networks. 88,89 They reported vesicle formation for PLys b-plleu and PLGA 60 -b-plleu 20 copolypeptides, whereby vesicle formation was first established through nanoprecipitation and dialysis methods. Interestingly, they employed liposomebased extrusion and were able to show the ability to trap small molecule solutes, thus providing a method for efficiently encapsulating therapeutics. They later reported vesicle formation for an analogous poly(l-arginine) 60 -b-poly(lleucine) 20 PLArg 60 -b-plleu 20 copolypeptide. 80 Finally they reveal that certain block ratios formed morphologies other than vesicles. For example, highly asymmetric PLys 60 -b- PLLeu 10 formed spherical micelles. 79 UTILIZING TOPOLOGICAL COMPLEXITY FOR UNIQUE ASSEM- BLY IN POLYPEPTIDE-BASED COPOLYMERS The solution self-assembly of peptide diblock copolymers is fairly well-established. This next section will discuss the PART B: POLYMER PHYSICS 2013, 51,

7 SCHEME 1 Synthesis of PLys-PPO-PLys ABA linear triblock coplymers using a diamine-functional PPO macroinitiator. Reproduced from Ref. 16. Copyright 2012, with permission from Wiley-VCH Verlag GmbH & Co. solution assembly of more complex triblock copolymers. These may be ABA, BAB, or ABC linear copolymers or AB 2, A 2 B, or l-abc star polymers, where we indicate the A block as the peptide. This small addition of architectural complexity produces intriguing responsiveness and the potential for morphology transitions that are dictated more by the actual topology or the secondary structure of the polypeptide rather than by the hydrophilic fraction. This relatively new area of research is opening doors to applications such as biomimetics, triggered drug release, and compartmentalized micellization. Synthetic Techniques for Polypeptide-Based Copolymers with Topological Complexity Synthesis of more complex architectures utilizes controlled polymerization techniques for the peptide component as described above. In addition, it is possible to exploit the use of click" reactions for more complex structures in a modular fashion. ABA triblock copolymers have been synthesized using the direct approach of initiating polymerization of an NCA using a diamino-functional macroinitiator (Scheme 1) 16,20,90 This topology contains hydrophilic polypeptide outer A blocks and a synthetic hydrophobic inner B block. In many of these cases, commercially available JeffamineVR polymers are used as the diamine. ABA copolypeptides have been synthesized by Hadjichristidis et al. using 1,6-diamino hexane and high-vacuum techniques to initiate ring opening polymerization of c-benyl-l-glutamate NCA followed by subsequent addition of e-tert-butyloxycarbonyl-l-lysine NCA (BLLys NCA). 75,91,92 They showed that the boc protecting group could be selectively removed to yield ph-responsive PLys-PBLG-PLys triblocks. 75 Having a polypeptide center block, BAB triblock copolymers are not as trivial to synthesize, and typically require multi step strategies. Kiick and coworkers recently reported the synthesis of BAB systems containing a collagen mimicking sequenced peptide. 93 Here, they utilized the telechelic amine peptide in a coupling strategy with activated ester terminated poly(diethylene glycol methyl ether methacrylate) (PDEGMEMA.) Similar strategies could be used to create this topology with a hydrophilic homopeptide, synthesized through ROP of NCAs, employing a small molecule diamine initiator as the center block and either a hydrophobic synthetic polymer/polypeptide as the outer blocks. In addition to the coupling chemistry used by Kiick, Deming et al. 94 have used amine-isocyanate chemistries to proficiently create polypeptide multiblock hydrid polymers. Depending on the desired placement of blocks within the molecule, ABC triblock terpolymers can be synthesized using a number of strategies. Cai et al. showed the synthesis of ABCs using poly(ethylene glycol)-nh 2 to initiate ROP of poly(z-lysine) followed by subsequent polymerization of a second monomer, L-leucine NCA. 95 Jing et al. synthesized a PEG-poly(L-Lactide)-PLGA (PEG-PLA-PLGA) terpolymer again using a macrointiator approach, whereby PEG-PLA-NH 2 initiated polymerization of c-benzyl-l-glutamate NCA. 96 Ten Cate and Borner employed solid state peptide synthesis and a solid-supported PEG-NH 2 to synthesize PEG-poly(arginine)- NH 2. The solid support was later removed and the amine group was reacted with dithiobenzoic acid to yield a dithioester end group that was used to initiate polymerization of butyl acrylate by reversible addition-fragmentation chain-transfer polymerization; therefore, this resulted in a PEG-PArg-PBA terpolymer. 97 ABC triblock terpolymers can also be synthesized by using an amine macroinitiator for ROP of the NCA followed by the use of accessible coupling chemistry with the amine chain end (Scheme 2). For the synthesis of AB 2 stars, our group has employed efficient thiol-yne coupling chemistry to create lipid-mimetic 514, PART B: POLYMER PHYSICS 2013, 51,

8 SCHEME 2 Model synthesis of ABC/BAB/ABA peptide hybrid triblocks through a two step process. The first step involves macroinitiation to yield a diblock copolymer. The second step involves efficient coupling chemistry (amine-isocyanate is presented here) to yield a triblock copolymer/terpolymer. structures. 98,99 As illustrated in Scheme 3, both convergent and divergent synthetic strategies can be used to create this topology. Starting with propargyl amine, the convergent approach first uses the amine to initiate ROP of the NCA followed by UV-initiated, radical-mediated thiol-yne chemistry between the acetylene group and hydrophobic thiols. 100 Alternatively, in the divergent approach, propargyl amine is first reacted with the hydrophobic thiols followed by ROP of the NCA. Both synthetic routes have proven to be effective in achieving the desired AB 2 topology. 98,99 A 2 B star copolymers have been synthesized containing two hydrophilic peptide blocks attached to a hydrophobic polymer. 59,101 Both of these reports took advantage of controlled radical techniques to create synthetic polymers that could be efficiently used to couple to synthesized polypeptide blocks or used as macroinitiators for ROP of the NCA. For example, Lecommandoux and coworkers modified a bromine-terminated PS with a trifunctional amine to afford PS-(NH 2 ) 2. They then used this difunctional PS to initiate ROP of c-benzyl-l-glutamate NCA. 101 Similar to A 2 B star polymers, three independent polymers can be covalently linked at a common junction to yield l-abc star terpolymers. Hadjichristidis and coworkers report the synthesis of molecular chimeras of this topology as well as other complex topologies Assembly Behavior of ABA Linear Triblock Copolymers with Polypeptide Outer Blocks One of the first examples for the self-assembly of a peptidebased ABA triblock copolymer was published by Wang. They characterized the ph-responsive behavior of PLGA-b-poly (propylene oxide)-b-plga (PLGA-b-PPO-b-PLGA) triblock SCHEME 3 Divergent (top) and convergent (bottom) strategies to creating AB 2 star copolymers with two lipophilic chains attached to a polar, hydrophilic peptide. In this case, PLGA was covalently linked to two dodecane thiol chains. Reproduced from Ref. 98. Copyright 2011, with permission from Royal Society of Chemistry. PART B: POLYMER PHYSICS 2013, 51,

9 FIGURE 7 R h vs. ph of PLGA-b-PPO-b-PLGA (GPG) assemblies. Reproduced from Ref. 90. Copyright Reprinted with permission from American Chemical Society. copolymers with a set hydrophobic block of 4000 g/mol (DP ¼ 69) and various block lengths of PLGA: DP ¼ 18 (GPG18), 47 (GPG47), and 99 (GPG99.) Figure 7 shows the measured R h versus ph for these self-assembled structures. GPG18, having a hydrophilic weight fraction of 60%, was shown by TEM and LS to form vesicles over the range form ph The vesicles showed an increase in size from ph 4.1 to a maximum size at ph 6.7, but the assembly sizes decrease significantly at higher ph. GPG47 (hydrophilic fraction of 75%) assemblies showed a mixture of spherical micelles and vesicles at low ph, and the average sizes of these assemblies decreased as the ph was increased. It was observed through TEM that the assemblies had transformed into spherical micelles at higher ph denoting a vesicle-to-sperhical micelle transition with ph. Only GPG99, being highly asymmetric given a hydrophilic fraction of 86%, showed the theoretically expected spherical micelle assemblies over the entire ph range. 90 This work illustrates that polypeptide-based triblock copolymers may show unique behavior that differs from analogous diblock copolymers, and our group aimed to look at similar systems to get a more in-depth understanding of these types of systems. Recently, we reported on the aqueous self-assembly behavior of PLys-b-poly(propylene oxide)-b-plys (PLys-b-PPO-b-PLys, abbreviated KPK). 16 Within our studies, we looked at three triblock copolymers: KPK27, KPK46, and KPK52. In-depth light scattering (DLS and SLS) and microscopy (TEM and AFM) techniques as well as circular dichroism spectroscopy were used to fully characterize the behavior of these systems. KPK27 and 52 copolymers are structurally the same, having a hydrophilic fraction of around 75%, but KPK52 has double the total molecular weight. Figure 8(a,b) show the R h, R g, and q (q ¼ R g /R h ) for KPK 27 and 52 respectively. These data, and the associated TEM, suggest a sphere-to-vesicle transition. This was further supported by SLS data that showed a large increase in aggregate effective molecular weight (M agg w ) from ph 3 to 4, which is anticipated because vesicles are expected to have a much higher aggregation number than spherical micelles. KPK46 has a hydrophilic fraction of 85% and light scattering results again show a morphology transition. Figure 8(c) shows the corresponding SLS and DLS results for KPK46, where it is observed that the assemblies increase in size with increasing ph. TEM [Fig. 8(h,i)] and AFM results suggest that the morphological transition occurring here is a spherical micelle-to-disk micelle transition. We hypothesized (Fig. 9) that disk micelles were observed for this sample from close packing of the PLys corona chains upon deprotonation. SLS data show a steady large increase in the effective molecular weight of the aggregate M agg w over the ph range, suggesting an open association assembly process. Similar to our recent work, Tian et al. reported vesicle formation for PLys-b-poly(tetrahydrofuran)-b-PLys (PLys-b- PTHF-b-PLys) ABA triblocks. 20 Both PLys 18 -b-pthf 14 -b- PLys 18 and PLys 30 -b-pthf 14 -b-plys 30 showed vesicle formation in aqueous media with R h values around 80 and 130 nm respectively. They found that the assembly properties changed above and below the melting temperature (T m )of the hydrophobic PTHF block (19.5 C). When the solution temperature was taken below the T m, nanotube formation was observed through TEM analysis. (Fig. 10) They hypothesize that the vesicle to tube transition is a result of vesicle fusion, whereby crystallization of the PTHF block drives the formation of the flatter interface nanotube assemblies. Moreover, both block lengths of PLys showed the vesicle to nanotube transition, with some individual transient morphologies for each respective system (i.e., pearl necklace structures for PLys 18 -b-pthf 14 -b-plys 18 and branching sites on tubes for PLys 30 -b-pthf 14 -b-plys 30 ). 20 Hadjichristidis and coworkers reported vesicle formation for an amphiphilic triblock copolypeptides, poly(llys)-b-poly(cbenzyl-d7-l-glutamate)-b-poly(llys) (PLys-b-PBLG-b-PLys), with hydrophilic fraction ranging from 25 to 80%. 75 Vesicle formation was aided by the molecular topology and by the rigidity of the rod-like (helical), hydrophobic middle block. Similar to our earlier work on PB-b-PLys vesicles, these systems were reported to show responsiveness to ph and temperature through a-helix-to-random coil and a-helix-to-bsheet transitions, thus changing the interfacial properties and sizes of the vesicles. Finally, in addition to solution selfassembly, solid-state assembly of polypeptide-based ABA triblock copolymers has been described. 105,106 Assembly Behavior of BAB Linear Triblock Copolymers with a Responsive Polypeptide Inner Block The aqueous self-assembly behavior of linear triblock BAB copolymers has been evaluated widely for synthetic coil coil coil type systems These type of systems are reported to aggregate into sunflower micelles, enabled by molecular bending of a flexible middle block, in dilute solution. 108,111 At higher concentrations, these systems often form network structures from the association of corona chains between micelles resulting in hydrogelation. 107,109,110,112,113 A few studies have been devoted to self-assembly of coil-rod-coil BAB triblock copolymers containing a functionalized, hydrophilic gold nanorod center 516, PART B: POLYMER PHYSICS 2013, 51,

10 FIGURE 8 Size (Rg and Rh) and q (Rg/Rh) vs. ph for (a) KPK 27, (b) KPK 46, and (c) KPK 52. TEM images of KPK aggregates: (d) KPK27 at ph 3.1, (e) KPK 27 at ph 6.9, (f) KPK 52 at ph 3, (g) KPK52 at ph 6.9, (h) KPK46 at ph 3.1, (i) KPK 46 at ph 6.8; all samples were stained with 1% w/w phosphotungstic acid in water. Reproduced from Ref. 16. Copyright 2012, with permission Wiley-VCH Verlag GmbH & Co. block and hydrophobic coil outer blocks This category of amphiphilic molecules exhibited self-assembly into doughnut-like" supramolecular barrels (similar to toroid micelles), as a result of stiffness from the center block. Similar to these gold nanorod systems, Kiick and coworkers recently reported the aqueous assembly of fully organic coilrod-coil BAB copolymers.93 Their model copolymer for this study was poly(diethylene glycol methyl ether methacrylate) (PDEGMEMA) attached to the outside of a triple-helix forming collagen-like peptide, PDEGMEMA(coil)-collagen-peptide(rod)-PDEGMEMA(coil). As in the case of gold nanorod systems, these molecules were found to form 3D toroid-type spherical assemblies through the bending constraint that the triple-helix forming peptide imposes. They report that this type of assembly definitely imposes water-hydrophobic interactions, however, the multilayering and rigidity of the peptide leads to steric stability of the supramolecular assembly. It was further shown that increasing the temperature led to the formation of larger spherical assemblies, a result of the conformational change of the peptide, as partial unfolding of the helix leads to larger sizes, as confirmed by cryo-sem and TEM. Upon heating to 75 C, the peptide helix unfolds into a coiled secondary structure (triple-helix-to-coil transition), determined by CD spectroscopy. This gives rise to a morphological transition into fibrils, whereby bending/flexibility of the peptide block drives disassembly of the spherical aggregates and reformation into fibrils which allows for shielding hydrophobic-water interactions. To our knowledge, no, PART B: POLYMER PHYSICS 2013, 51,

11 FIGURE 9 Schematic of responsive morphology transitions for KPK polypeptide-based triblock copolymer assemblies. Reproduced from Ref. 16. Copyright 2012, with permission from Wiley-VCH Verlag GmbH & Co. studies have focused on ph-responsive self-assembly of polypeptide-based BAB copolymers with a middle polypeptide block capable of undergoing helix-coil transitions with ph (i.e., PLys or PLGA). These types of systems could allow for complex hierarchical assembly such as those reported by Kiick, where changes in ph could stimulate morphological transitions based on the rigidity of the center peptide block. Assembly Behavior of ABC Linear Triblock Copolymers Containing a Responsive Polypeptide Block ABC linear triblock terpolymers contain three separate block components within one molecule, and this topology can be utilized to give a permanently hydrophilic block, a permanently hydrophobic block, and a tunable block such as a pep- tide. A few studies have been devoted to studying the aqueous self-assembly of peptide-containing ABC triblocks Jing and coworkers demonstrated ph-induced morphology transitions for amphiphilic poly(ethylene glycol)17-poly(l-lactide)23-plga60 (PEG17-PLL23-PLGA60).96 A sphere-to-connected rod transition was driven by the hydrophilicity of the PLGA block. At ph > 4, the PLGA is hydrophilic and the hydrophilic fraction of the molecule is conducive for spherical micelle formation. However, at ph below 3.2, the PLGA has completely shielded all interactions with water making it hydrophobic. As such, they observed the transition to rodlike micelles. They further showed that the transition between different morphologies was reversible. Recently, spherical micelle formation was described for PEG-PLys- FIGURE 10 Schematic describing self-assembly of PLys-b-PTHF-b-PLys triblock copolymers into vesicles and nanotubes. Reproduced from Ref. 20. Copyright Reprinted with permission from American Chemical Society. 518, PART B: POLYMER PHYSICS 2013, 51,

12 FIGURE 11 Schematic illustrating attachment of different lipophilic thiols being conjugated to a polar, helix-forming peptide; the results of such a lipid-like" topology leads to the formation of polymersomes. Reproduced from Ref. 99. Copyright 2011, with permission from Royal Society of Chemistry. poly(l-leucine) triblock terpolymers. 95 The sizes of the spherical micelles were effectively tuned between 40 and 90 nm by changing the block lengths of lysine and leucine. This model terpolymer was found to effectively complex DNA through association with the positively charged PLys middle block, whereby the placement of the PLys block promoted internalization of the DNA allowing for protection against lysosomes. Their results proved that these micellar-dna systems efficiently supported gene expression, having both in vitro and in vivo data, and the transfection efficiency/biocompatibility was influenced by PLys and PLLeu block compositions. Recently, Ambade and Sen Gupta studied a complex linear-dendritic ABC terpolymer consisting of a glycopeptide (A), poly(ethylene glycol) (B) and a dendron end cap (C). 117 This complex topology was able to self-assemble into organogels in DMSO, or into micelles and nanotubes in aqueous solution, depending on composition of the component blocks. Polypeptide-Based AB 2 Star Copolymers AB 2 star copolymers containing a hydrophilic peptide (A) block covalently linked to two hydrophobic B blocks structurally resemble phospholipids and are therefore often referred to as lipid mimetics. As is the case for phospholipids, AB 2 star copolymers should be expected to form bilayer assemblies (i.e., vesicles) due to geometrical constraints and the topology serving to maximize the hydrophobic volume. Recently, our group utilized thiol-yne coupling chemistry to create polypeptide-based AB 2 star copolymers. 98,99 In our earliest report, we studied the kinetics and self-assembly of AB 2 star copolymers composed of dodecane thiol doubly conjugated (either convergently or divergently) with PLys. These materials were shown to produce ph-responsive vesicles. Given the modularity of this approach, we further utilized the convergent thiol-yne synthesis to create AB 2 systems with various hydrophobic moieties (Fig. 11); three different thiol functionalized lipophilic moieties [octadecane (ODT), cholesterol (Chol), and polyhedral oligomeric silsequioxane (POSS)] were conjugated to PLGA (DP ¼11, 16, and 36). 99 We explored the ph-responsive self-assembly of these copolymers with DLS, SLS, and TEM as a function of ph. Figure 12 shows R h versus ph plots of these various assemblies as well as representative TEM images. All systems demonstrated large size changes when the PLGA went from an uncharged helical conformation at ph 4 5 to a charged, random coil at ph > 6. Similar to the diblock vesicles discussed above, it was determined that changes in chain density at the interface cause the enhanced ph responsiveness compared with what would be expected from the helix-coil transition. Specifically, we determined that (1) the ph transitions were immediate, faster than chain exchange would occur for a morphology transition, (2) the assembly size was independent of concentration, suggesting no interassembly exchange, and (3) the effective molecular weight of the assemblies remained constant as a function of ph, suggesting a closed association process. As such, the ph transitions are result of chain density changes at the interface of the vesicle. Finally, the effective M agg w, as well as the vesicle size dispersities showed depend on the hydrophobic moiety. The Chol-based vesicles showed substantially higher M agg w values and more uniform size distributions; we hypothesized that this behavior is a result of liquid crystallinity of the Chol within the vesicle membrane leading to a high degree PART B: POLYMER PHYSICS 2013, 51,

13 hydrophilic peptide chains attached to a hydrophobic B block. 59,119 Ding and coworkers studied poly(n-isopropyl acrylamide)-b-(plys) 2 (PNIPAM-b- (PLys) 2 ),coupled through azide-alkyne chemistry. 59 Above the LCST of PNIPAM (ca. 35 C), the molecules were reported to form spherical micelles. This morphology was locked in by shell crosslinking the amino groups of PLys with glutaraldehyde, and it was shown that these assemblies were maintained when cooling the solution below the LCST, with micelle size changes resulting from changes in ph. 59 The utility of this approach was illustrated through loading of a hydrophobic drug into the hydrophobic NIPAM core, whereby an increase in % crosslinking slightly decreased the release rate from the shell crosslinked micelle. Other groups have reported on the nanophase-separated bulk assembly properties of A 2 B polypeptide hybrid star copolymers, and the behaviors of these rod-coil star copolymer systems was found to differ from AB diblock systems. 90,107 Polypeptide-Based l-abc Star Copolymers Another emerging polymer topology are miktoarm l-abc star terpolymers. Lodge has demonstrated multicompatment micelle formation in aqueous media for synthetic star terpolymers, 15, yet research in the area of peptide-containing l-abc systems has been limited to bulk self-assembly properties. 103,104 In one such study, PS-PI-poly(e-tert-butyloxycarbonyl-L-Lysine) (PS-PI-PBLL) was found to self-assemble into lamellar nano-domains of amorphous PS and PI oriented perpendicular to liquid crystalline (smectic) PBLL domains (Fig. 13). 104 Given this sophisticated bulk morphology produced by these star terpolymer systems, one may anticipate interesting self-assembled, responsive structures in aqueous solution. CONCLUSIONS AND OUTLOOK Polypeptide-based amphiphilic block copolymers are an attractive class of materials given (1) their ability to form well-defined aqueous nanoassemblies with complex morphologies, (2) the biomimetic secondary structures similar with natural proteins, and (3) responsiveness to external stimulus through secondary structure transitions. Diblock FIGURE 12 R h versus ph plots for ODT 2 -PLGA 16 (top) (inset is TEM image at ph 6.5), Chol 2 PE 11 and Chol 2 PE 11 (middle) (inset is TEM image for Chol 2 PE 11 at ph 6.5), and POSS 2 PE 16 and POSS 2 PE 36 (bottom) (inset is TEM image for POSS 2 PE 36 at ph 7.2). Reproduced from Ref. 99. Copyright Reprinted with permission from Royal Society of Chemistry. of order and close packing of molecules. We believe this assembly motif will be an effective platform for the design of polymer vehicles for hydrophilic drugs whereby there are no concerns about designing molecules with a certain hydrophilic fraction to facilitate vesicle formation. 98,99,118 Polypeptide-Based A 2 B Star Copolymers There have been a few recent reports on the aqueous self-assembly of peptide-based A 2 B star copolymers, with two FIGURE 13 Illustration of the nanophase-separated morphology produced by a PS-PI-PBLL miktoarm star terpolymers. Reproduced from Ref Copyright Reprinted with permission from American Chemical Society. 520, PART B: POLYMER PHYSICS 2013, 51,