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Stimuli-Sensitive Synthetic Polypeptide-Based Materials for Drug and Gene Delivery

Chaoliang He, Xiuli Zhuang, Zhaohui Tang, Huayu Tian, and Xuesi Chen*
Stimuli-sensitive synthetic polypeptides are unique biodegradable and biocompatible synthetic polymers with structures mimicking natural proteins. These polymers exhibit reversible secondary conformation transitions and/or hydrophilichydrophobic transitions in response to changes in environmental conditions such as pH and temperature. The stimuli-triggered conformation and/or phase transitions lead to unique self-assembly behaviors, making these materials interesting for controlled drug and gene delivery applications. Therefore, stimuli-sensitive synthetic polypeptide-based materials have been extensively investigatid in recent years. Various polypeptide-based materials, including micelles, vesicles, nanogels, and hydrogels, have been developed and tested for drug- and gene-delivery applications. In addition, the presence of reactive side groups in some polypeptides facilitates the incorporation of various functional moieties to the polypeptides. This Review focuses on recent advances in stimuli-sensitive polypeptide-based materials that have been designed and evaluated for drug and gene delivery applications. In addition, recent developments in the preparation of stimulisensitive functionalized polypeptides are discussed.
biocompatible synthetic polymers with structures mimicking natural proteins.[8] In comparison with conventional biodegradable polymers, synthetic polypeptides may form stable secondary structures, such as -helix and -sheet, due to cooperative hydrogen-bonding, leading to unique selfassembly behaviors.[9,10] In addition, the self-assembly structures of some polypeptides exhibit transitions in response to external stimuli, such as pH, salt, and temperature. Especially, some polypeptides with ionizable side groups can form electrostatic interactions with oppositely charged drugs and bioactive macromolecules, such as DNAs, RNAs, and proteins, and functional moieties can be incorporated into the side chains of the polypeptides. Therefore, polypeptide-based materials have attracted increasing attention for their great potential in biomedical and pharmaceutical applications.[1114] It is worth mentioning that polymers that exhibit sharp phase and structure transitions in response to physiologically relevant stimuli have advantages in drugdelivery applications. Typical physiologically relevant stimuli include the difference in microenvironment between normal tissues and some pathological sites, e.g., tumor sites,[5,15] the difference in microenvironment, such as pH and redox environment,[5,1517] between the extracellular and intracellular space, as well as the pH shift between the stomach (pH 2.0) and the intestine (pH 7.0).[6] In addition, polymers that exhibit temperature-dependent phase transitions at around physiological temperature, such as 1042 C,[18,19] are also interesting for controlled drug delivery. Consequently, polypeptide-based materials capable of responding to physiologically relevant stimuli, including pH, temperature, redox environment, and dual stimuli, have been extensively developed for drug delivery applications in recent years, and many promising results have been reported. Based on the polypeptides with different architectures and compositions, intelligent polymeric materials, including micelles, vesicles, nanogels, and hydrogels, have been developed, as schematically illustrated in Figure 1. This Review focuses on recent progress in stimuli-responsive polypeptide-based materials that have been designed and evaluated for drug- and genedelivery applications, including polypeptide-based micelles, vesicles, nanogels, and hydrogels. In addition, recent developments in the preparation of stimuli-sensitive functionalized polypeptides are discussed.
1. Introduction
Controlled drug release systems are designed to deliver therapeutical drugs to desirable sites at appropriate time.[13] In recent years, stimuli-sensitive polymers that respond to changes in environmental conditions,[3,4] such as pH, temperature, salt, light, biomolecules, and electromagnetic eld, have received extensive attention for their unique advantages in applications for controlled drug delivery. The release of drugs from the smart delivery systems can be triggered by the specic environments of some organs, intracellular space, or pathological sites, leading to an enhanced specicity of drug delivery and less side effects.[5,6] In addition to intelligent drug-release behaviors, drug carriers with good biocompatibility and appropriate biodegradability are preferred in practical applications.[7] Synthetic polypeptides, which are poly(amino acid)s linked by peptide bonds, are unique biodegradable and
Dr. C. He, X. Zhuang, Z. Tang, H. Tian, Prof. X. Chen Key Laboratory of Polymer Ecomaterials Changchun Institute of Applied Chemistry Chinese Academy of Sciences Changchun 130022, P. R. China E-mail: xschen@ciac.jl.cn
DOI: 10.1002/adhm.201100008
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2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Healthcare Mater. 2012, 1, 4878
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Xuesi Chen received his Ph. D. at Waseda University, Japan, in 1997, and completed his post-doctoral fellowship at the University of Pennsylvania, USA, in 1999. He has been a full Professor at Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, since 1999. His research interests focus on the development and biomedical applications of biodegradable polymers and intelligent biomaterials, mainly based on polyesters, polypeptides, polycarbonates, and their copolymers.
Figure 1. Schematic illustration of intelligent polypeptide-based materials, including micelles, vesicles, nanogels, and hydrogels, as well as functionalized polypeptides.
2. Synthesis of Polypeptides
Polypeptides are mainly prepared via ring opening polymerization (ROP) of -amino acid N-carboxyanhydrides (NCAs) by using amine-based initiators.[8,20,21] The typical mechanisms of amine-initiated ROP of NCAs include amine mechanism (AM) and activated monomer mechanism (AMM).[20,21] The ROP of NCAs via AM is believed to be performed by attacking CO-5 of the NCAs using primary amines or some highly nucleophilic secondary amines (Scheme 1a).[20] On the other hand, the ROP via activated monomer mechanism is achieved by deprotonation of NH-3 of the NCAs using tertiary amines or some secondary amines with bulkyl substitution groups (Scheme 1b).[20,21] The ROP of NCAs initiated by primary amines leads to the successful preparation of different kinds of polypeptides and polypeptide-based copolymers with desired molecular weights (MW) and architectures, while that initiated via AMM usually results in the formation of polypeptides with high MW. Even though some side reactions, e.g., end-group cyclization, may occur during the primary amine-initiate-polymerization, causing contamination of polypeptide products, considerable approaches have been developed recently to prepare polypeptides with controlled MW and polydispersities (PDIs) as well as welldened topological structures. Deming and co-workers have used a series of transition metal complexes as initiators to obtain welldened polypeptides and block copolypeptides.[8] Hadjichristidis and co-workers have obtained well-dened polypeptides using conventional amines as initiators under high vacuum conditions.[22] Schlaad et al. and Lutz et al. have reported that ammonium chloride-functionalized macro-initiators can effectively suppress side reactions,[23,24] leading to the formation of well-dened polypeptide-based block copolymers. In addition, controlled ROP of NCAs has been achieved by using organosilicon.[25] In the past decade, amphiphilic polypeptide-based block copolymers have been extensively studied for biomedical applications, attributed to the fact that they can self-assemble into different stimuli-sensitive nanometer-sized or micrometer-
sized structures in aqueous solution and the size and morphology of these materials can be readily tuned by varying the composition of the block copolymers.[2628] Accordingly, different amphiphilic polypeptide-based block copolymers have been synthesized for biomedical applications. The most widely used method to synthesize polypeptide-based block copolymers is the ROP of NCAs by using polymeric macro-initiators. In addition, block copolymers are also prepared by highly effective coupling reactions, such as click chemistry and carbondiimide chemistry, between functionalized polypeptide blocks and other polymer blocks. For instance, Deming and co-workers have synthesized different amphiphilic diblock copolypeptides containing a charged block and a hydrophobic block by using transition metal complexes as initiators.[27] Kataoka and coworkers have prepared poly(ethylene glycol) (PEG)/polypeptide diblock copolymers by using amino-functionalized methoxypoly(ethylene glycol)s (mPEG) as macro-initiators.[12] Chen and co-workers have developed a series of polyester/polypeptide block copolymers by using amino-terminated polyester blocks as macro-initiators.[2934] Lecommandoux and co-workers have synthesized polybutandiene-polypeptite diblock copolymers by using monoamino-terminated polybutandienes as macroinitiators.[35,36] In addition, Bae and co-workers have developed a class of poly(L-histidine) (PLHis)-based block copolymers by coupling PLHis with other synthetic polymers.[37]
3. Stimuli-Sensitive Polypeptide-Based Materials

3.1. Micelles Polymeric micelles are generally formed by self-assembly of amphiphilic polymers in aqueous solution.[38] They have nano-sized amphiphilic structures composed of a hydrophobic inner core and a hydrophilic outer shell. The micellar cores are commonly consisted of hydrophobic segments or complexes of oppositely charged polyelectrolytes, which are shielded and stabilized by hydrophilic shells, such as an antifouling PEG shell. The coreshell nano-structures of polymeric micelles make them suitable for drug loading and delivery. The hydrophobic or ion complex cores of micelles facilitate the loading of hydrophobic drugs or ionic biopharmaceuticals, such as
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(a)
R2-NH2
O 1 O 2 5 NH O 4 3 R1 O
R2
H N
O R1
H N COOH
R2
H N
O NH2 R1 + CO2
O n O NH R1 R2 H N O R1 H N O R1 H N H + n CO2
O O O O + Et3N O O O NH R3 O O N R3 R3 O
(b)
O O NH R3 O O O NH R3
N
R3
H N COO
O O O N R3
O O NH2 R3 + O O
O O + CO2 O N R3
N
R3
O R3
H N
O R3
H N COO
O O O NH R3 O O N R3 R3 O O O H N O NH2 R3 + O O
N
R3
+ CO2
NCA Propagation
Scheme 1. The ring opening polymerization of NCAs via amine mechanism (a) and activated monomer mechanism (b).[20,21]
DNAs, RNAs, and proteins, while the hydrophilic antifouling shells improve the stability and biocompatibility of the micelles in circulation system in vivo. Additionally, nanometersized particles (10200 nm) have been shown to passively accumulate in tumor sites due to the enhance permeation and retention (EPR) effect.[5] Hence, nanomedicine approaches have emerged as the most popular methods for cancer therapy.[39,40] As a result, nanometer-sized polymeric micelles have received extensive investigation as drug delivery systems. Among them, micelles based on stimuli-sensitive synthetic polypeptides are interesting due to their stimuli-responsive self-assembly behaviors and conformation transitions as well as good biocompatibility and biodegradability.[914] In recent years, polypeptide-based micelles comprising pH-sensitive polypeptide blocks, acid-labile polypeptidedrug conjugates, polypeptide containing ion complexes and dual stimuli-sensitive segments have been developed for controlled drug and gene delivery. 3.1.1. PEG-Poly(L-histidine) Block Copolymers Drug nano-carriers may encounter different environmental stimuli, such as pH change and change in redox environment, after they are administrated in vivo. For example, weak acidic environments (pH 6.07.0) are formed in tumor sites,
compared to the neutral pH (pH 7.4) of normal tissues and blood.[5,15] In addition, more acidic environments are formed as the nano-carriers are entrapped in endosomes (pH 5.06.5) and lysozomes (pH 4.05.0).[5,15] Therefore, pH-sensitive polymers have attracted considerable interest for their potential applications in anticancer and intracellular drug delivery. In order to avoid enzymatic degradation within lysosomes, rapid endosomal/lysosomal drug release plays a key role in achieving ideal therapeutical efciency. It has been pointed out that endosomal pH-triggered drug release may lead to a high intracellular drug dose and therefore result in improved efciency in killing multi-drug resistance (MDR) tumor cells.[41] Therefore, drug-loaded nanocarriers capable of responding to endosomal pH (5.06.5) may exhibit enhanced antitumor efcacy. Synthetic polypeptides containing ionizable pendant groups, such as poly(L-glutamic acid) (PLGlu), poly(aspartic acid) (PAsp), poly(L-lysine) (PLLys) and PLHis exhibit pH-induced hydrophilic-hydrophobic transitions at pH around their pKa values. Among the above polyelectrolytes, PLHis containing imidazole groups with a pKa of 6.0 has a sharp buffering capacity at endosomal pH.[42] Additionally, hydrophilic PEG has been widely incorporated in biomaterials due to its good biocompatibility, non-immunogenicty and antifouling property, and PEG with relatively low molecular weights (MW < 50 000) can be excreted from the body through renal clearance.[11,12]
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above its pKa with the deprotonated PLHis segments as a hydrophobic core and PEG O O O H H as a hydrophilic shell. The micelles showed O N H O HO N NH C6H13 O N O an average diameter of 114 nm at pH 8.0. m z n y x H O O The PEG-b-PLHis micelles displayed a pHinduced destabilization behavior at pH below O N NH 7.4. With reducing the solution pH from 8.0 HO PEG-b-PLHis to below 7.2, the critical micelle concentration PEG-b-PLLA-b-PLGlu (a) (b) (CMC) of the copolymer increased obviously and the stability of the micelles decreased O markedly, due to the protonation-deprotonaH O O N H tion transition of the PLHis block. As doxoO N H O H N m n H O n H rubicin (Dox) loaded micelles were cultured mN H O O with A2780 cells, the in vitro uptake of Dox O NH by A2780 cells was enhanced more than HO O OH N ve times by reducing pH from 7.4 to 6.8, PAsp- b-PLLA OH resulting in an increased cytotoxicity against (c) A2780 cells at pH 6.8.[43] The enhanced Dox OH cellular uptake and cytotoxicity at pH 6.8 O O O OH was believed to be due to the acid-triggered O O O H3C release of Dox. After administration of the H H N N O Dox-loaded micelles into mice, the DoxNH 2 N OH m nH loaded micelles showed markedly increased O O PEG-b-P(Asp-Hyd-ADR) half life time in vivo compared to free Dox. HN O (d) HN After intravenous injection into nude mice bearing human ovarian A2780 tumor, the NH O pH-sensitive drug loaded micelles exhibited H H NH N N H signicantly higher antitumor efciency in H S n m comparison with free Dox. O H2N HN In order to improve the stability of the H2N O PEG-b-PLHis micelles, mixed micelles comPEG-b-P(Asp-DET) HO posed of PEG-b-PLHis and an amphiphilic (e) PNIAPM-b-PLGlu PEG-b-poly(L-lactide) (PEG-b-PLLA) diblock (f) copolymers have been subsequently developed.[44] The mixed micelles maintained O obvious pH-sensitivity provided that O O H H H H O H N N H the weight ratio of PEG-b-PLLA within N H H N N N S nH n m mH m the blends is less than 50 wt%.[45] With O HN increasing the weight ratio of PEG-b-PLLA from 0 to 40 wt%, the destabilization pH of O O the mixed micelles was shifted from 7.2 to HO HO NH2 6.6.[44,45] A formulation containing 25 wt% PLGlu-b-PPO-b-PLGlu PNIAPM-b-PLLys PEG-b-PLLA, i.e. mixed micelle (75/25), was (h) (g) selected to be the optimal formulation, for Scheme 2. Chemical structures of some representative polypeptide-based block copolymers its transition pH (6.8) is close to the pH of that form stimuli-sensitive micelles. the extracellular space of solid tumors. The mixed micelles (75/25) displayed a constant hydrodynamic diameter of around 129 nm in the pH range of Consequently, pH-sensitive micelles based on PEGPLHis 7.07.4. In contrast, the micelle size increased signicantly to amphiphilic block copolymers have been intensively developed 195 nm as the pH was decreased from 7.0 to 6.8, indicating for drug delivery. swelling of the mixed micelles caused by the protonation of Bae and co-workers have developed a PEG-b-PLHis diblock the PLHis segments. In vitro cytotoxicity tests against human copolymer by ROP of Nim-2,4-dinitrobenzene-L-histidinebreast adenocarcinoma (MCF-7) cells indicated that the mixed NCA using n-hexylamine or isopropylamine as an initiator, micelles exhibited good biocompatibility.[44] In vivo cytotoxicity followed by coupling monoamino-terminated PLHis with [ 37 ] test demonstrated that the micelles caused no apparent system monocarboxylic PEG (Scheme 2a). The pKa values of PLHis toxicity in mice at concentrations up to 2400 mg kg1.[41] The and PEG-b-PLHis were determined to be 6.5 and 7.0, respecDox containing mixed micelles (75/25) showed a stable diamtively (Table 1). The relatively higher pKa of PEG-b-PLHis was eter of 183 nm at pH 7.4.[46] In contrast, a signicant increase believed to be attributed to the hydration effect of hydrophilic in particle size was observed when the pH was reduced to 6.8, PEG. The diblock copolymer formed core-shell micelles at pH
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Table 1. pKa values of some typical pH-sensitive peptide-based polymers.

Polymer Poly(L-histidine) (PLHis) Poly(ethylene glycol)-b-poly(L-histidine) (PEG-b-PLHis) PEG-b-poly(L-histidine-co-L-phenylalanine) (PEG-b-P(LHis-coLPhe)) Poly(L-lactide)-b-PEG-b-PLHis (PLLA-b-PEG-b-PLHis) PEG-b-PLHis-b-PLLA-b-PEG PEG-b-poly(aspartic acid) (PEG-b-PAsp) PEG-b-poly(L-lysine) (PEG-b-PLLys) PAsp-b-PLLA PEG-b-poly(3-morpholinopropyl aspartamide)-b-PLLys (PEG-b -PMPA-b-PLLys) PEG-b-poly(N-(2-aminoethyl)-2-aminoethyl aspartamide) (PEG-b-(PAsp-DET)) PEG-poly(2-aminopentyl-,-aspartamide) (PEG-P(Asp-AP)) Methylamine functionalized poly(L-glutamate) Diethylamine functionalized poly(L-glutamate) Diisopropylamine functionalized poly(L-glutamate) Poly(L-glutamate)-g-oligo(2-aminoethyl methacrylate) (PLGlu-g -OAEMA) p Ka 6.5 7.0 6.47.0 7.0 6.26.8 4.88 9.54 6.77.2 6.2 and 9.4 6.0 and 9.5 10.47 7.3 6.5 5.25 7.3 Ref. [37] [37] [52] [54] [63] [75] [75] [56] [84] [85] [157] [255] [255] [255] [240]
in the cytosol, likely caused by the pH-triggered micelle destabilitzation and endosomal disruption. The rapid distribution of Dox within the tumor cells led to increased drug retention in both wild type and MDR tumor cells, and therefore resulted in an improved anti-MDR tumor efcacy.[49,50] The Dox-loaded Fol-PHSMM effectively suppressed the growth of MDR ovarian tumor in nude mice for 50 days by 3 intravenous injections at a dose of 10 mg kg1 at a 3-day interval. In vivo optical imaging tests conrmed that the Cy5.5-labeled pH-sensitive micelles accumulated at tumor sites at 1 hour post-injection. A further in vivo study indicated that nude mice bearing metastatic 4T1 murine breast tumors treated with the Dox-loaded Fol-PHSMM exhibited marked suppression of tumor growth and no apparent metastasis at 28 days, compared to signicant metastasis for the group treated with Dox solution in PBS, Dox-loaded PEGb-PLLA micelles or Dox-loaded PEG-b-PLHis micelles without folate.[51] In addition, the pKa of the PLHis-based copolymer was found to be inuenced by introduction of hydrophobic unit, e.g., L-phenylalanine, to the PLHis segment.[52] PEG-bpoly(L-histidine-co-L-phenylalanine) (PEG-b-P(LHis-co-LPhe)) diblock copolymers showed pKa values varying from 7.0 to 6.4, depending on the L-phenylalanine content within the P(LHis-coLPhe) block. 3.1.2. PolypeptidePolyester Block Copolymers Biodegradable synthetic polymers, especially aliphatic polyesters and polycarbonates, have received extensive investigation in the past several decades, owing to their vast potential in biomedical applications, such as drug delivery and tissue engineering. In recent years, amphiphilic block copolymers containing a polypeptide block and a polyester block have attracted considerable attention,[2934,5358] due to their stimuli-responsiveness, good biocompatibility, adjustable biodegradability, tunable hydrophobicity, and the ability to incorporate functional groups.[7] The above advantages make these materials interesting for biomedical applications. As discussed in Section 3.1.1, micelles containing both polypeptide and polyester segments can be obtained by physically mixing two kinds of block copolymers, such as PEG-bPLHis and PEG-b-PLLA.[44] Additionally, micelles formed by polypeptide-polyester block copolymers have also been fabricated. A PLLA-b-PEG-b-PLHis triblock copolymer was fabricated via Michael-addition reaction between a thiol-terminated PLLAb-PEG and a maleimide-functionalized PLHis.[54] The triblock copolymer showed a pKa (7.0) close to that of PEG-b-PLHis. At pH above its pKa, ower-like micelles with a PLLA/PLHis mixed core and a PEG shell are formed, whereas swollen micelles with a PLLA core and a PEG/PLHis shell are obtained at lower pH. The triblock copolymer micelles showed a constant size of 80 nm at pH 7.27.4, but increased drastically with decreasing pH to 6.8. The release of Dox from the micelles was obviously accelerated with reducing pH from 7.4 to 6.0, attributed to the decrease in micellar stability caused by the ionization of PLHis segments. Some bioactive moieties, such as biotin and cell penetrating peptides (CPP),[59] have been shown to markedly increase cellular uptake of drug nanocarriers. Nevertheless, nonspecicity of some bioactive molecules limits their applications in vivo. Due to its hydrophilichydrophobic transition at
accompanying with a markedly accelerated drug release. A lyophilized micelles formulation has been prepared by using excipients.[41] The reconstituted micelles showed a particle size, pH sensitivity and toxicity against MDR tumor cells comparable to the freshly made micelles. In order to impart active tumor-targeting ability to the pH-sensitive micelles, a tumortargeting ligand, i.e., folic acid, was introduced into the surface of the micelles by mixing folate-conjugated PEG-b-PLHis (FolPEG-b-PLHis) with folate-conjugated PEG-b-PLLA (Fol-PEG-bPLLA).[47] It was found that the folate-conjugated pH-sensitive mixed micelles (Fol-PHSMMs) containing Dox exhibited significantly higher efciency in killing MDR MCF-7 cells (MCF-7/ DoxR) compared to free Dox and Dox-loaded micelles without folate. The enhanced efcacy in killing MDR tumor cells was proposed to be due to improved intracellular uptake via folatereceptor mediated endocytosis and subsequent endosomal escape. After injection into nude mice bearing Dox resistant MCF-7 (MCF-7/DoxR) xenografts through the tail vein, the Doxloaded Fol-PHSMMs displayed markedly higher efciency in suppressing tumor growth than either free Dox or Dox-loaded micelles without folate. The Dox accumulation level in tumors of the group treated with folate-conjugated micelles was twenty times higher than that of free Dox treated group and three times higher than that of the group treated with the micelles without folate. In addition, Dox-loaded Fol-PHSMMs have also been shown to have higher in vitro cytotoxicity against both wild type and MDR tumor cells than the folate-conjugated PEGPLLA (Fol-PEG-b-PLLA) micelles.[48] It was found that both micelles showed comparable cellular uptake levels of Dox due to the folate receptor-mediated encytosis, whereas cells treated with Fol-PHSMMs resulted in a widespread distribution of Dox
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tumor extracellular pH (pH 6.07.0),[5,15] PLHis can be used as an onoff switch for selectively exposing these molecules at tumor sites. Biotin and CPP have been separately conjugated to the end of the PLHis block of PLLA-b-PEG-b-PLHis.[60,61] Mixed micelles comprising biotin conjugated PLLA-b-PEG-bPLHis (PLLA-b-PEG-b-PLHis-Bio) and PEG-b-PLHis exhibited an acid-triggered avidin binding ability and improved cellular uptake by MCF-7 cells expressing biotin receptor at pH < 7.2.[60] The pH-dependent avidin-binding behavior of the micelles is illustrated in Figure 2. The biotin was tethered at the core-shell interface of the micelles at pH > 7.0 due to the deprotonation and hydrophobic nature of the PLHis spacer, leading to a low avidin-binding ability and lower uptake of the micelles by MCF-7 cells. In contrast, biotin was exposed to the surface of the micelles at 6.5 < pH < 7.0 caused by the partial protonation of PLHis, resulting in signicant increase in cellular internalization of the micelles. At more acidic endosomal pH (<6.5), the drug loaded micelles destabilized, attributed to the further protonation of PLHis and the electrostatic repulsion between the PLHis segments. Mixed micelles based on a PLLA-b-PEGb-PLHis linked with a CPP, i.e., transactivator of transcription (TAT) peptide, (PLLA-b-PEG-b-PLHis-TAT) were subsequently reported.[61] The TAT-PLHis-conjugated micelles showed similar pH-dependent cell killing efciency. The cytotoxicity was signicantly increased as the pH decreased from 7.4 to 6.8, as compared with high cytotoxicities at all experimental pH for the micelles containing TAT linked through a PEG spacer. The pHdependent cytotoxicity of the micelles with PLHis-linked TAT was believed to be due to the selective exposure of TAT at acidic pH values. Since slightly acidic microenvironments are formed in tumor sites,[5,15] it is envisioned that the PLHis spacer can be used to enhance tumor-targeting ability of the linked bioactive moieties. Additionally, PLHis/PLLA block copolymers with different architectures, such as PEG-b-PLHis-b-PLLA triblock copolymers and PEG-b-PLHis-b-PLLA-b-PEG tetrablock copolymers,[62,63] have been developed by combination of ROP of L-lactide (LLA) and coupling reactions. These block copolymer micelles showed similar pH-dependent micellar destabilization and accelerated drug release proles at acidic pH. In addition to coupling approaches, polypeptidepolyester block copolymers are largely synthesized via ROP of NCAs by using amino-functionalized polyesters as macroinitiators. Chen and co-workers have synthesized a series of block copolymers comprising polypeptide blocks and aliphatic polyester blocks,[2934] such as PLLA and poly(-carpolactone) (PCL), via ROP of NCAs by using mono- or diamino-functionalized polyesters as macro-initiators. Different bioactive moieties, such as RGD,[3032] biotin and sugar residues,[64,65] have been incorporated into the polypeptide segments through the pendent functional groups. The pH-dependent self-assembly behaviors of these block copolymers have been investigated.[66] A series of PEGb-poly(L-lactide)-b-poly(L-glutamic acid) (PEG-b-PLLA-b-PLGlu, Scheme 2b) were synthesized by ROP of -benzyl-L-glutamate NCA (BLG-NCA) using amino-terminated PEG-b-PLLA as a macro-initiator, followed by the deprotection of the benzyl groups.[29] PLGlu shows a random coil-to--helix transition, associated with a hydrophilic-hydrophobic transition, as the pH decreases from neutral pH to the pH below the pKa (4.1) of the L-glutamic acid (LGlu) residues.[4] The triblock copolymers
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Figure 2. Schematic diagram depicting the central concept of pH-induced vitamin repositioning on the micelle. While above pH 7.0, biotin that is anchored on the micelle core via a pH-sensitive molecular chain actuator (polyHis) is shielded by PEG shell of the micelle; biotin is exposed on the micelle surface (6.5 < pH < 7.0) and can interact with cells, which facilitates biotin receptor-mediated endocytosis. When the pH is further lowered (pH < 6.5), the micelle destabilizes, resulting in enhanced drug release and disrupting cell membranes such as endosomal membrane. Reproduced with permission.[60] Copyright 2005, American Chemical Society.
formed micelles in water with the size and morphology depending on the pH and the PLGlu block length.[66] For a triblock copolymer of PEG17-b-PLLA23-b-PLGlu60 (where the subscript numbers refer to the average number of the repeat unit for each segment), spherical micelles with an average diameter of 58 nm were observed at pH 4.5. The size of the spherical micelles decreased with decreasing pH to 3.9. Notably, when the pH was decreased to 3.2, rod-like micelles with a mean diameter of 28 nm were formed. pH-dependent 1H NMR tests revealed that the PLGlu segments were located in the outer shell part of the micelles at pH 4.5, whereas they were dehydrated and encapsulated by the PEG segments at pH 3.2. A three-layer structure was proposed for the rod-like micelles, which are composed of a PLLA core, a PLGlu intermediate layer and a PEG shell. The formation of the rod-like micelles was assumed to be attributed to the fact that the relatively short PEG block (degree of polymerization (DP) = 17) was not long enough to stabilize the spherical micelles at pH 3.2. On the other hand, only spherical micelles were detected within the experimental pH region for a triblock copolymer with a shorter PLGlu block (DP = 10). Additionally, the CMC of the triblock copolymer was found to increase with increasing the PLGlu block length or pH. AB-type diblock copolymers with PLGlu or poly(aspartic acid) (PAsp) as the A block and PLLA as the B block have been synthesized by ROP of NCA using amino-terminated PLLA as a macroinitiator.[5558] Spherical micelles with a PLLA core and an ionizable polypeptide shell were formed by the diblock copolymers in water. The morphology and size of the micelles were found to be inuenced by the relative block length and the pH.[56] The pKa values (6.77.2) of PAsp-b-PLLA (Scheme 2c) were signicantly higher than that of the pendant carboxylic
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groups of the aspartic acid (Asp) residues (3.9).[4] The formation of hydrogen bonds between the PAsp segments was thoughted to be responsive for the increase in pKa.[56] The size of the micelles increased markedly with increasing pH from 4 to 7 when the PAsp block is comparable or longer than the PLLA block. In contrast, when the PLLA block is dominant in the diblock copolymer, no obvious change in size was detected with increasing pH. In addition, in vitro cytotoxicity tests indicated that the polypeptide-polyester diblock copolymers displayed good biocompatibility. 3.1.3. Drug Conjugated Micelles via Acid-Labile Linkers Amphiphilic PEG-polypeptide diblock copolymers, with anticancer drugs covalently conjugated to the polypeptide block, have advantages in anticancer drug delivery for their high stability in blood stream, increased solubility of hydrophobic drugs, less systematic toxicity, as well as improved accumulation at tumor sites owing to EPR effect.[14,15] Polypeptidedrug conjugates based on PLGlu and PEG-b-PAsp have been developed and entered clinical trails.[14,67,68] It has been pointed out that the release of conjugated drugs from the polymerdrug conjugates after they are uptaken by tumor cells plays a crucial role in achieving desirable antitumor efcacy.[16] Therefore, drugs have been linked to polymers via covalent bonds cleavable by physiologically relevant stimuli, such as acidic pH,[15] reducing environment and enzymes.[16] The stimuli-labile linkers may facilitate the release of the conjugated drugs from polymer backbones at desirable sites. Different polypeptide drug conjugate micelles containing acid-labile linkers have been developed in recent years. Kataoka and coworkers have developed a PEG-poly(aspartate-hydrozone-adriamycin) (PEGb-P(Asp-Hyd-ADR)) diblock copolymer, in which a anti-cancer drug, adriamycin (ADR), was conjugated to the PAsp block via a acid-cleavable hydrazone bond with a substitution ratio of 67.6 mol% (Scheme 2d).[69] The block copolymer formed micelles in aqueous solution with the drug-conjugated PAsp segments as a core and PEG blocks as a shell. The micelles showed an average diameter of about 65 nm. The drug was tethered to the polypeptide backbone and loaded within the micelles at pH > 6.5, whereas the release of ADR was triggered at acidic pH. When cultured with human small cell lung cancer cell SBC-3, micelles trapped in endocytic compartments were observed by confocal laser scanning microscopy (CLSM), and cytotoxicity tests conrmed the antitumor efcacy of the ADRconjugated micelles. In contrast to free ADR, the micelles displayed a time-dependent cytotoxicity, probably due to a delayed acid-triggered drug release process.[70] Both in vitro tests using a multicellular tumor spheroid (MCTS) model and subsequent in vivo antitumor tests suggested the penetration of the ADRconjugated micelles into tumor tissues.[70] The ADR-conjugated micelles exhibited enhanced antitumor efcacy and lower toxicity compared to free ADR. A tumor-targeting polypeptide drug conjugate was subsequently prepared by incorporation of folate to the PEG block of PEG-b-P(Asp-Hyd-ADR).[71] Mix micelles composed of folate-functionalized PEG-b-P(Asp-HydADR) (Fol-PEG-b-P(Asp-Hyd-ADR)) and PEG-b-P(Asp-HydADR) were prepared and displayed an average size varying from 60 nm to 90 nm, depending on the substitution ratio of
folate.[72] Surface plasmon resonance (SPR) measurements conrmed the binding ability between the folate-conjugated micelles and folate-binding proteins (FBP). A folate conjugation ratio of 10% on the micelles was found to be enough to effectively bind FBP. The selective binding effect resulted in an enhanced intracellular uptake of the micelles and hence an increased efciency in suppressing the growth of tumor cells.[71] In vivo tests indicated that an increase in folate substitution ratio from 10% did not cause an increase in tumor accumulation of the micelles, attributed to the increased accumulation of micelles in liver. After intravenous injection into tumor-bearing mice, the folate-conjugated micelles with a folate conjugation ratio of 10% exhibited higher tumor suppression efcacy than either free ADR or micelles without folate. In addition, anticancer drugs have also been covalently conjugated to dendritic polypeptides via acid-labile linkers. Dox has been conjugated to a dendritic PLGlu containing biotin via hydrazone bonds by Gu and co-workers.[73] The dendritic PLGlu-Dox conjugate formed spherical nanoparticles with an average diameter of around 50 nm in aqueous solution. The release of Dox from the nanoparticles was slow at pH 7.4, whereas it was dramatically accelerated at pH 5.0. In vitro cytotoxicity tests indicated that the Dox-conjugated nanoparticles showed an enhanced intracellular uptake and higher efciency in killing of tumor cells. 3.1.4. pH-Sensitive Polyion Complex Micelles Polyion complex (PIC) micelles are mainly formed by a charged amphiphilic block copolymer with an oppositely charged polymer or ionic biopharmaceuticals, such as DNA, RNA, and proteins. These materials are interesting for controlled delivery of ionic bioactive molecules, attributed to the fact that ionic biopharmaceuticals can be efciently encapsulated in the micelles through electrostatic complexation.[74] PIC micelles exhibit sensitivity to pH, salt and other competing polyelectrolytes. Since the commonly used biopharmaceuticals, such as DNA, RNA, and some proteins, are negatively charged at physiological condition, different positively charged polypeptides have been used for fabrication of PIC micelles. Kaotaka and co-workers have developed PIC micelles based on PEG-b-poly(L-lysine) (PEG-PLLys) diblock copolymers and DNA. Zeta () potential tests conrmed the formation of core-shell micelles with ionic complexes as a core and hydrophilic PEG segments as a shell.[75,76] The PIC micelles showed a constant diameter with the salt concentration varying from 0 to 300 mM. The shielding effect of the PEG shell led to increased stability of the complexes in the presence of nuclease or serum,[76,77] and reduced immunogenicity in vivo.[78] The PIC micelles exhibited higher cellular uptake and gene transfection efciency compared to a commercially available lipoplex system. After hydrodynamic injection into mice via limb vein, PIC micelles containing plasmid DNA (pDNA) displayed enhanced transgene expression than naked pDNA. After administration to tumor-bearing mice, PIC micelles containing pDNA encoding soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1) showed effective suppression of tumor growth. Active targeting ability has been imparted to the PIC micelles by introduction of cyclic RGD (cRGD),[79] a moiety capable of recognizing v3 and v5 integrins overexpressed in tumor vasculature and malignant
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tumor cells.[80] When cultured with HeLa cells expressing v3 and v5 integrins, the cRGD-conjugated PIC micelles showed higher transfection efciency and enhanced cellular uptake than PIC micelles without cRGD. In contrast, for 293T cells without v3 and v5 integrins, both systems displayed comparable transfection efciency. The selective increase in transfection efciency suggested a receptor-mediated endocytosis mechanism. Additionally, PIC nano-carriers based on PLLys multiblock copolymers, graft copolymers, and hydrophobic segment containing triblock copolymers have also been developed.[8183] Due to the relatively high pKa (10) of PLLys,[75] PLLys-based PIC micelles show no effective proton buffering capacity within the acidic endosomal pH range. Therefore, poly(3-morpholinopropyl aspartamide) (PMPA) with a pKa of 6.2 has been incorporated with PLLys to improve the buffering capacity of the material at endosomal pH.[84] A PEG-b-PMPA-b-PLLys triblock copolymer was fabricated by sequential ROP of -benzyl-Laspartate NCA (BLA-NCA) and -(benzyloxylcarbonyl)-L-lysine NCA (ZLL-NCA), followed by aminolysis of benzyl ester of PBLA using 4-(3-aminopropyl)morpholine and deprotection of the benzyloxylcarbonyl groups of poly(-(benzyloxylcarbonyl)-Llysine) (PZLL). PEG-b-PMPA-b-PLLys exhibited a two-step protonation process, and the triblock copolymer/pDNA complex formed a three-layer structure in aqueous solution, as illustrated in Figure 3. At pH 7.4, the PLLys segments with a higher pKa (9.4) condensed pDNA in the inner core and the PMPA segments with a lower pKa (6.2) formed the intermediate layer. In comparison with PEG-b-PLLys/pDNA systems, the triblock copolymer-based PIC micelles displayed markedly increased transfection efciency, due to the proton buffering capacity of PMPA segments at endosomal pH. Similarly, gene delivery systems showing two-step protonation process have been developed based on ethylenediamine-modied polyaspartamides,[85] PLLys/PLHis graft copolymers,[86] chitosan/PLLys graft copolymers,[82] and aminoethyl-substituted PLHis.[87] A N-(2aminoethyl)-2-aminoethyl substituted PEG-polyaspartamide (PEG-P(Asp-DET), Scheme 2e) was synthesized by aminolysis of PEG-poly(-benzyl-L-aspartate) (PEG-PBLA) using diethyenetriamine (DET).[85] 1H NMR test indicated the co-existence of -aspartamide and -aspartamide units within the poly(amino acid) backbone, caused by an intramolecular isomerization of aspartamide during the aminolysis. PEG-P(Asp-DET) showed a two-step protonation process with two pKa values of 6.0 and 9.5, respectively.[85,88] It was found that the pKa and buffering capacity of the N-substituted polyaspatamide were inuenced markedly by the structure of the diamine side groups.[85,88] The two-step protonation process facilitates both DNA condensation at physiological pH (7.4) and endosomal escape at endosomal pH. DLS tests suggested that polyion complex (PIC) micelles consisting of PEG-P(Asp-DET) and pDNA revealed diameters of 7090 nm with the N/P ratio ranging from 1 to 20. potential measurements suggested that the ionic complex was shielded by a PEG shell. The PEG-P(Asp-DET)/pDNA micelles showed a pH-dependent membrane destabilization behavior, causing a relatively low cytotoxicity at pH 7.4 and an increased endosomal disruption at acidic environments.[88,89] In vitro transfection and cytotoxicity tests indicated that the PEG-P(Asp-DET)based PIC micelles showed lower cytotoxicity and comparable or higher transfection efciency to primary cells compared to
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Figure 3. Chemical structure of PEG-b-PMPA-b-PLLys triblock copolymers and schematic illustration of the hypothesized three-layered polyplex micelles with spatially regulated structure. Reproduced with permission.[84] Copyright 2005, American Chemical Society.
either linear or branched polyethylenimine (PEI).[85,88,89] The PIC micelles incorporated in calcium phosphate cement scaffolds were found to be released from the scaffolds in a sustained manner and successfully transfected surrounding cells.[90] After administration of the scaffolds containing pDNAs expressing caALK6 and Runx2 to bone defects of mice, bone formation at the lower surface of the implants was observed for the mice treated with PIC micelles incorporated scaffolds, compared to no signicant bone formation for the groups treated with commercially available linear PEI and FuGENE6. It was found that the low toxicity of P(Asp-DET) in vitro and in vivo is related to the rapid degradation of P(Asp-DET) segments caused by a selfcatalytic process.[91] P(Asp-DET) exhibited a decrease in cytotoxicity as the culture time increased, compared to no marked change in cytotoxicity of either linear PEI or DET-substitutetd poly(L-glutamide) (P(LGlu-DET)), which showed no obvious degradation in PBS during the experimental period. In order to further improve the stability of PIC micelles containing biopharmaceuticals, such as DNA and siRNA, PEG-P(Asp-DET) has been modied by different hydrophobic moieties, i.e., cholesterol and stearoyl groups.[92,93] The introduction of hydrophobic groups to the P(Asp-DET) block led to an increase in association force between the polymers and hence resulted in an improved micellar stability. Subsequent in vitro and in vivo studies suggested that the hydrophobically modied PIC micelles displayed increased transfection efciency. Additionally, siRNA has also been conjugated to the poly(amino acid) backbone via disulde bond, which is cleavable under intracellular reducing environment.[94] The hydrophilic PEG shell of micelles plays an important role in improving biocompatibility and the blood circulation time of drug nanocarriers in vivo. On the other hand, the shielding effect of antifouling PEG segments may also lead to a decrease in cellular uptake and endosomal disruption.[95,96] Hence, drug and gene carriers with PEG segments cleavable under the microenvironments of tumor tissues and intracellular space have been fabricated.[9598] Detachable PEG blocks have been linked with P(Asp-DET) blocks via reduction-cleavable disulde bonds or acid-cleavable hydrazone bonds.[96,97] It was found that the PIC micelles with a detachable PEG shell exhibited higher in vitro gene transfection efciency than their counterparts with an undetachable PEG shell, likely due to an increased endosomal disruption and escape.[96,97] Stimuli-triggered desheilding
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systems have also been achieved by using ternary polyplex systems, in which ion complexes with excess positive charge are covered by a detachable negatively charged shell.[99101] A charge-reversal shielding polymer, cis-aconitic amide conjugated P(Asp-DET) (P(Asp-(DET-Aco))), has been synthesized by modication of P(Asp-DET) using an acid-labile cis-aconitic amide group.[95,100] After DNAs were condensed by excess cationic polymers, such as P(Asp-DET) and PLL, negatively charged P(Asp-(DET-Aco)) was incorporated to the surface of the ion complexes by elextrostatic interactions to form ternary polyion complexes. At acidic endosomal pH, the ourter layer detached from the ionic complexes due to the charge conversion of P(Asp-(DET-Aco)) caused by acidic hydrolysis of cis-aconitic amide. Besides their applications in gene delivery, pH-sensitive charge-conversional polypeptides have been investigated for controlled delivery of anticancer drugs and proteins.[99,102104] Other examples of pH-sensitive shielding systems include carboxymethyl-substituted PLHis (CM-PLHis) and hydrophobically modied PLGlu,[101,105,106] which both exhibited sharp response to endosomal pH. It is noteworthy that the ternary systems with a negatively charged surface exhibited no detectable cytotoxicity at physiological pH; on the other hand, excessive shielding polymers can also cause a decrease in transfection efciency, due to a decrease in intracellular uptake.[101,106] Additionally, polyion complexes based on negatively charged polypeptides,[107110] including PEG-b-PAsp and PEG-b-PLGlu, and positively charged proteins or polysaccharides have been studied. 3.1.5 pH- and Temperature-Sensitive Micelles Dual stimuli-sensitive polymers have attracted considerable attention due to their advantages in practical applications.[3,15] Especially, polymeric materials that sharply respond to physiologically relevant stimuli, especially pH and temperature, are interesting for controlled self-assembly and drug delivery.[111] pH- and temperature-sensitive polypeptide-based copolymers are mainly fabricated by combination of pH-sensitive polypeptide segments with temperature-responsive synthetic polymers. In addition, dual stimuli-sensitive block copolymers may selfassemble into so-called Schizophrenic micelles by varying temperature or pH.[112] Very recently, pH- and temperaturesensitive polymers containing PLGlu, PLLys, and zwitterionic polypeptides have been synthesized and their micellization behaviors have been investigated. Poly(N-isopropylacylamide) (PNIPAM) is one of the most widely studied temperature-sensitive polymers. It exhibits a sharp coil-to-globule transition at its low critical solution temperature (LCST) of around 32C.[18] Poly(N-isopropylacylamide)b-PLGlu (PNIPAM-b-PLGlu, Scheme 2f) diblock copolymers have been synthesized via ROP of BLG-NCA by using monoanimo-terminated PNIPAM as a macroinitiator, followed by deprotection of the BLG groups.[113116] In order to obtain well-dened PNIPAMpolypeptide diblock copolymers, PNIPAM was synthesized via controlled radical polymerization,[115117] and ammonium chloride-functionalized PNIPAM was used as macroinitiators for ROP of NCAs.[113,118] PNIPAMb-PLGlu showed a pH- and temperature-dependent micellization behavior in aqueous solution. Interesting schizophrenic
micellization behaviors were observed by adjusting the pH and temperature, as illusrated in Figure 4. The copolymer dissolves in PBS at neutral or alkaline pH and a temperature lower than its LCST. As the temperature is increased to above the LCST of PNIPAM, micelles with a PNIPAM core and an ionized PLGlu shell are formed, due to the dehydration of PNIPAM. In contrast, micelles with an -helical PLGlu core and a PNIPAM shell are obtained with decreasing the pH to below the pKa (4.1) of LGlu residues.[4] It was found that the LCST of the PNIPAM block showed a decrease as the PLGlu segment dehydrated at pH around the pKa of LGlu (4.1).[113,119] It is noteworthy that PLGlu does not exhibit a sharp hydrophilichydrophobic transition at endosomal pH, due to the relatively low pKa (4.1) of the pendant carboxylic groups and a gradual deprotonation process in a broad pH range.[4,120] To obtain polypeptides capable of responding to endosomal pH, Chen and co-workers synthesized a series of PNIPAM-poly[(L-glutamic acid)-co-(-benzyl L-glutamate)] (PNIPAM-b-P(LGlu-co-BLG)) diblock copolymers by partial deprotection of PNIPAM-b-PBLG.[113] The existence of hydrophobic BLG within the polypeptide block led to an increase in the critical pH for the hydrophilic-hydrophobic transition of the polypeptide block. Interestingly, as the BLG content within the polypeptide block was higher than 30 mol%, the copolymers displayed sharp hydrophilichydrophobic transitions in response to a narrow pH change in the pH region of pH 5.57.4. Additionally, pH- and temperature-sensitive PLGlu-g-PNIPAM grafted copolymers have been synthesized by grafting monoamino-terminated PNIPAM to the pendant carboxylic groups of PLGlu.[119] Amphiphilic aggregates were formed at neutral pH and a temperature above the LCST of PNIPAM. The size of the aggregates signicantly decreased with reducing pH from 7.4 to 4.5, due to the gradual protonation and shrinkage of the PLGlu shell. In contrast, larger aggregates were detected with further decreasing pH to 4.2, caused by hydrophobic aggregation of PLGlu segments. Cationic PNIPAM-b-PLLys diblock copolymers have also been synthesized via ROP of ZLL-NCA by using ammonium chloride-substituted PNIPAM as a macroinitiator by Chen and co-workers.[118,121] The chemical structure of PNIPAM-b-PLLys is shown in Scheme 2g. Schizophrenic micellization behaviors of the diblock copolymers were also observed by varying pH and temperature. PLLys exhibits a random coil-to--helix transition with increasing pH, and a hydrophilichydrophobic transition at pH around its pKa (10).[75] Accordingly, PNIPAMb-PLLys formed micelles with a hydrophobic PNIPAM core and a PLLys shell at lower pH and temperatures above its LCST,
Figure 4. Schematic illustration of the pH- and temperature-induced micellization behaviors of the PNIPAM-b-PLGlu diblock copolymer solution in PBS: micelles with an -helical PlGlu core are formed at pH < 4 and 15 C, whereas micelles with a PNIPAM core are formed at pH 7.4 and 37 C.[113,114]
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whereas micelles with a PLLys core and a PNIPAM shell are obtained at higher pH and lower temperatures. As shown in Figure 5, environmental scanning electron microscopy (ESEM) measurements revealed that spherical PNIPAM-core micelles with a diameter of 150 nm were formed at pH 5.0 and 45 C, while PLLys-core micelles with a diameter of 125 nm were observed at pH 12.5 and 25 C. Similarly, zwitterionic PNIPAMb-P(LLys-co-LGlu) diblock copolymers were synthesized by ROP of the mixture of ZLL-NCA and BLG-NCA using amino-terminated PNIPAM as a macroinitiator.[122] The copolymers formed large aggregates in water at pH ranging from 6.0 to 10.0, due to strong intramolecular electrostatic interactions. In contrast, at pH < 6 or pH > 10, the electrostatic interactions were suppressed due to the deionization of LGlu or LLys residues, leading to a marked increase in solubility of the copolymer. In addition, a zwitterionic AB2 Y-shaped star polypeptide copolymer with PLLys as the A block and PLGlu as the B block was synthesized via click chemistry, and its pH-dependent schizophrenic micellization behavior was investigated.[123] The zwitterionic copolymers may be interesting for some applications where dual stimuli-sensitivities at both acidic and alkaline conditions are needed.[124] Other examples of pH- and temperature-sensitive polypeptide copolymers include copolymers based on PLGlu and poly(propylene oxide) (PPO) or poly(2-ethyl-2-oxazoline) (PEOZ).[125127] PLGlu-b-PPO-b-PLGlu triblock copolymers (Scheme 2(h)) were synthesized via ROP of BLG-NCA using bis(amino)-end-capped PPO as a macroinitiator, followed by deprotection of the BLG groups.[125,126] Schizophrenic micellization behavior was observed for a PLGlu35-PPO33-PLGlu35 triblock copolymer with the PLGlu content of 78 wt%.[125] At pH 10 and a temperature above the LCST of PPO (15 C),[128] PPOcore micelles with a diameter of 20 nm were formed, while PLGlu-core micelles were detected at pH 2 and 5 C. Mixed micelles composed of PLGlu-b-PPO-b-PLGlu and a PEG-b-PPO diblock copolymer have been designed for controlled drug delivery.[126] The hydrophobic PPO core was encapsulated by a PEG/PLGlu mixed shell at 25 C and neutral pH. The release of loaded Dox from the micelles was found to be dependent on pH, temperature and the composition of the mixture. Drug release was accelerated at acidic pH (5.5). The increased release rate at acidic pH was proposed to be due to the formation of channels within the shell, caused by partial dehydration of PLGlu segments. 3.2. Vesicles Polymer vesicles are nanometer-sized or micrometer-sized hollow spheres composed of a hydrophobic bilayer or interdigitated layer and hydrophilic internal and external shells.[129131] In contrast to the solid hydrophobic core of polymer micelles, polymer vesicles can envelop aqueous solution by their semipermeable membranes, making these materials ideal candidates for delivery of hydrophilic drugs and bioactive molecules.[130] Additionally, hydrophobic drugs can also be loaded within the hydrophobic domain of the vesicle membrane. The size and permeability of the vesicle membrane can be tuned by adjusting the composition and chain length of the amphiphilic polymer.
Figure 5. ESEM image of spherical micelles formed by PNIPAM-b-PLLys (a) pH 5.0 and 45 C, and (b) pH 12.5 and 25 C, respectively. Reproduced with permission.[118]
Especially, functionality and stimuli-sensitivity may be incorporated into the corona of vesicles, leading to intelligent systems for controlled drug delivery. Therefore, stimuli-sensitive vesicles based on different polypeptide block copolymers have been developed and tested for drug delivery applications. 3.2.1. Block Copolypeptides A series of amphiphilic diblock copolypeptides with welldened structures have been synthesized via successive ROP of NCAs by using transitional metal complexes as initiators by Deming and co-workers.[27,132] The diblock copolymers are constituted of a hydrophobic polypeptide block, such as poly(Lleucine) (PLLeu) and poly(L-valine) (PLVal), and a charged or hydrophilically modied polypeptide block, such as PLGlu, PLLys and poly(N-2-(2-(2-methoxyethoxy)ethoxy)acetyl-L-lysine) (P(EO2)LL). Hydrophobic PLLeu and PLVal are interesting for self-assembly due to their ordered conformation in aqueous solution.[132] (P(EO2)LL)-b-PLLeu diblock copolymers with appropriate composition and chain length formed micrometer-sized vesicles (125 m).[133] It was found that the morphology of the assemblies was signicantly inuenced by the hydrophobic block content and the chain length of the whole copolymer. Spherical vesicles were formed at lower PLLeu
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contents and block lengths, while curved sheets were observed at higher PLLeu fractions and block lengths. As the PLLeu content was higher than 35 mol%, insoluble aggregates were formed. A pH-sensitive diblock copolypeptide, i.e., P((EO2)LL)160-b-P(LLeu0.3co-LLys0.7)40, were fabricated by incorporation of LLys residues into the hydrophobic PLLeu block. As shown in Figure 6, at a high pH (10.6), the diblock copolymer formed stable vesicles with a hydrophobic P(LLeu0.3co-LLys0.7)40 layer and hydrophilic P((EO2) LL)160 inner and outer shells. Hydrophilic Fura-2 dye was enveloped within the vesicles during the self-assembly process. As the pH was reduced by addition of acid, the vesicle membranes were disrupted rapidly, resulting in the release of the encapsulated dye. It is noteworthy that the oligo(ethylene glycol) modied PLLys, i.e., P(EO2)LL, acts as a biodegradable PEG analogy and may exhibit good biocompatibility, making this material interesting for biomedical applications. It was found charged PLLys60-b-PLLeu20 (Scheme 3a) and PLGlu60-b-PLLeu20 formed vesicles, and the size of the vesicles could be adjusted from nano-meter to micro-meter scale by a liposome-based extrusion technique.[134] The vesicles were stable in 0.1 M PBS solution, and negatively charged PLGlu- Figure 6. pH-responsive vesicles. a) Schematic drawing of P((EO )LL) -b-P(LLeu -co-LL ) 2 160 0.3 0.7 40 b-PLLeu vesicles exhibited good stability in (denoted as K160p(L0.3/K0.7)40), its change in conformation with pH, and release of entrapped the presence of serum containing anionic Fura-2 dye on pH change. b) Fluorescence emission as a function of excitation wavelength for proteins. In addition, vesicles have been Fura-2 dye (50 nM) entrapped in vesicles of K160p(L0.3/K0.7)40 in the presence of external calcium developed based on an amphiphilic diblock (5.0 mM) at pH 10.6 and 3.0. The frequency shift for maximum emission intensity at pH 3.0 is [133] Copyright 2004, copolypeptide consisting of PLLeu and pol- characteristic of calcium binding by Fura-2. Reproduced with permission. Nature Publishing Group. yarginine (PArg), which is a CPP analogy.[135] The dextran-loaded vesicles showed enhanced cellular uptake of dextran with minimal cytotoxicity compared copolymer. The vesicle size also exhibited a dependence on the to free dextran. initial polymer concentration and the solution pH. The average Vesicles based on block copolypeptides containing other vesicle size decreased as the pH was increased from 2.5 to 9, hydrophobic polypeptide blocks have also been reported. Jing due to a gradual decrease in electrostatic repulsion between the and co-workers have synthesized a series of PLLys-b-poly(LPLLys segments. Protein-encapsulated PLLys-b-PLPhe vesicles phenylalanine) (PLLys-b-PLPhe, Scheme 3b) by successive were prepared by direct dissolving the copolymer in PBS soluROP of ZLL-NCA and L-phenylalanine NCA (LPhe-NCA) tion containing FTIC-BSA or carbonylated hemoglobin (CO-Hb) using n-hexylamine as an initiator, followed by deprotection of (Figure 7B).[137] The encapsulation efciency was found to be the -benzyloxycarbonyl groups.[136] Interestingly, the diblock signicantly affected by the interactions between the positively copolypeptides with relatively shorter PLPhe blocks could be charged PLLys and the proteins. At a pH (5.8) slightly above the directly dissolved in water and spontaneously form giant vesiisoelectric point (PI) of FITC-BSA (PI = 4.8), negatively charged cles. Atom force microscopy (AFM) measurements conrmed BSA was absorbed on the surface of the positively charged vesithe formation of vesicles, as shown in Figure 7A. The height cles. In contrast, at a pH (3.8) below the PI, BSA was encapsuof the central part of the vesicle was lower than that of the lated within the inner compartments of the vesicles, due to the peripheral part, due to the collapse of the hollow vesicles. Conelectrostatic repulsion between PLLys segments and positively focal laser scanning microscopy (CLSM) tests indicated that a charged BSA. Similarly, CO-Hb was enveloped in the vesicles hydrophilic dye, Rhodamine B, could be encapsulated within at a pH (5.8) slightly below its PI (6.68). The encapsulation efthe interior aqueous compartments of the vesicles. 1H NMR ciency of CO-Hb was about 40 wt% and the loading content measurements in D2O suggested that the PLPhe segments was 32 wt%. It was found that CO-Hb encapsulated within the were surrounded by the hydrophilic PLLys blocks. Similar to the vesicles retained its bioactivity and showed enhanced stability aforementioned P(EO2)LL-b-PLLeu system, a higher content of as compared with free CO-Hb. Aqueous solution containing O2 the rigid PLPhe, e.g., >35 mol%, led to a water-insoluble diblock diffused into the interior compartments, causing the conversion
O H2NC5H10 NH H N n O H N H C6H13 N O H N O n
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H m
H N H m
H2N PLLys-b-PLLeu (a) H C6H13 N O H N O n H N H m O O O O HO H2N PLLys- b-PLGlu (c) O H O O n O N H O H N O
H2N PLLys-b-PLPhe (b) O O n O HO PLGA-b-PLGlu (d) O H m O x y N H H N O H N C6H13 m O
H N z O
PTMC-b-PLGlu (e)
HO
PB-b-PLGlu (f) O H N
HO
Br O O
n O
N N N
N H
H m O
HO PDMAEMA-b-PLGlu (g)
Scheme 3. Chemical structures of some typical polypeptide-based block copolymers that form stimuli-sensitive vesicles.
of CO-Hb to oxygen-binding hemoglobin under irradiation of visible light. An anionic PLGlu-b-PLPhe diblock copolymer has also been synthesized through consecutive ROP of BLG-NCA and LPheNCA using n-butylamineHCl as an initiator.[138] At pH > 6.0, vesicles with an average diameter of about 150 nm were formed by dissolving the diblock copolymer in deionized water. With decreasing pH from 6.0 to 5.0, the size of particles increased due to the gradual aggregation of the particles, and larger aggregates were formed at pH below 5.0. The self-assembly behavior of diblock copolypeptides containing a hydrophobic polypeptide block without an ordered secondary conformation, e.g., polyglycine (PGly), has also been investigated. The vesicle membrane formed by PLLys-b-PGly diblock copolymers was found to exhibit a high uidity and exibility.[139] In addition to the diblock copolypeptides, amphiphilic triblock copolymers comprising a rigid hydrophobic central block anked by two charged blocks, i.e., PLLys-b-PBLG-b-PLLys, have been developed. The PLLys-b-PBLG-b-PLLys triblock copolymers were prepared via sequential ROP of BLG-NCA and -Boc-L-
lysine NCA (BocLL-NCA) by using 1,6-diaminohexane as a difunctional initiator under high vacuum, followed by selective deprotection of the Boc groups.[140] Interestingly, in comparison with a relatively small composition range for the formation of vesicular structures by diblock copolypeptides containing a rigid hydrophobic polypeptide block (e.g., <35 mol%),[133,136] vesicles were formed by the triblock copolypeptides with the rigid hydrophobic block content of 1974 mol%. The difference was believed to be attributed to the fact that a monolayer membrane can be easily formed by the triblock architecture, whereas an antiparallel orientation is needed for the formation of the vesicle bilayer membranes by amphiphilic diblock copolymers.[140] It was found that an increase in the content of the middle PBLG block led to a more robust vesicular structure. The vesicles displayed a pH- and temperature-responsive structure conformation. The vesicle size decreased with reducing pH from 7.4 to 11.7 at 25 C, due to the conformation change of the PLLys block from extended random coil to compact -helix. In addition, the size showed an increase with increasing temperature from 25 to 37 C at pH 11.7, resulted from the conformation
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Vesicles based on a zwitterionic diblock copolypeptide have also reported. A zwitterionic PLGlu-b-PLLys (Scheme 3c) diblock copolymer with equal lengths of PLGlu and PLLys was synthesized via successive ROP of triuoroacetyl-L-lysine NCA (TFA-LL-NCA) and BLG-NCA, followed by deprotection of TFA and BLG groups by using KOH.[141] Spherical vesicles with a uniform diameter of 110 nm were formed at acidic pH (pH < 4) with -helical PLGlu blocks as the intermediate hydrophobic layer and PLLys as the inner and out shells, whereas vesicles with a size of 175 nm composed of reverse insideout components were formed at alkaline pH (pH > 10). At near neutral pH, the diblock copolymers formed precipitates due to the electrostatic interactions between the oppositely charged segments. The formation of vesicles was believed to be related to the presence of a rod-like conformation in the hydrophobic segment, leading to a low interfacial curvature and a hollow structure. 3.2.2. PolypeptidePolyester Block Copolymers Some polypeptide-polyester block copolymers containing an ionizable polypeptide block and a hydrophobic polyester have been shown to form pH-sensitive micelles in water, as discussed in Section 3.1.2.[5458,66] In addition, vesicular structures have also been observed for some polypeptide-polyester diblock copolymers with short polypeptide blocks. A poly(lactic-co-glycolic acid)-b-PLGlu (PLGA-b-PLGlu) diblock copolymer with a short PLGlu block (DP = 15, f = 18 wt%) was prepared by coupling carboxylic group functionalized PLGA with amino-terminated PBLG, followed by deprotection of the benzyl groups (Scheme 3d).[142] The diblock copolymer exhibited a pH-dependent self-assembly behavior. At a pH (3.0) below the pKa (4.1) of LGlu, the diblock copolymer formed large hydrophobic aggregates, due to the hydrophobic nature of both blocks. As the pH was increased to 5.0, spherical micelles with a mean hydrodynamic diameter of 81.5 nm Figure 7. A) AFM image of vesicles formed in a PLLys-b-PLPhe aqueous solution at pH 3.5: A-a) AFM height image, A-b) AFM phase image, A-c) height prole along the line indicated in were observed. The micelles were believed (A-a). B) ESEM image of CO-Hb-encapsulated PLLys-b-PLPhe vesicles. A,B) Reproduced with to be composed of a hydrophobic PLGA core and a relatively hydrophilic PLGlu shell. With permission.[136,137] Copyright 2007 and 2009, respectively, American Chemical Society. further increasing the pH to 7.0, uniform vesicular structures with an average size of about 160 nm were obtained. The vesicles were constituted of transition of the PLLys block from -helix to an elongated a PLGA intermediate layer and ionic PLGlu inner and outer sheet conformation. When mixed with pDNA, the triblock shells. The formation of vesicles was believed to be driven by copolypeptides also formed vesicles with higher size compared an increase in the degree of ionization of PLGlu and the electo the vesicles without pDNA, and it was found that pDNA was trostatic repulsion between the short polypeptide segments. As both complexed on the PLLys corona and encapsulated within the pH was further increased to 9.0, the vesicle size increased, the vesicles.
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likely due to the further increase in the electrostatic repulsion between PLGlu segments caused by the further deprotonation of PLGlu in water.[120] 3.2.3. Polypeptide-Polycarbonate Block Copolymers Besides polyesters, polycarbonates are another important class of biodegradable synthetic polymers. Amphiphilic block copolymers composed of a polypeptide block and a polycarbonate block have been reported recently. Poly(trimethylene carbonate)b-PLGlu (PTMC-b-PLGlu, Scheme 3e) diblock copolymers with PLGlu contents of 39 wt% and 46 wt%, respectively, were synthesized by ROP of BLG-NCA using a monoamino-functionalized PTMC, followed by deprotection of the BLG groups.[143,144] Vesicles comprising a PTMC intermediate layer as well as inner and outer PLGlu shells were prepared by either direct dissolution or solvent displacement method (nano-precipitation). The vesicles exhibited an almost constant size at pH not less than 7, whereas the size decreased with reducing pH from 7, due to a decrease in the electrostatic repulsion between the PLGlu blocks and a random coil-to--helix transition of the PLGlu chains. Additionally, the vesicles exhibited high stability against nonionic surfactant and nonpermeability to water, due to the high thickness of the vesicle membrane (30 nm). In contrast, the vesicles were rapidly disrupted in the presence of enzyme. The encapsulation of an ionizable anticancer drug, i.e., Dox, by the PTMC-b-PLGlu vesicles was found to be signicantly inuenced by the loading pH, likely due to the ionization of Dox at pH below its pKa (8.25).[145] When loaded at pH 7.4, positively charged Dox was partially absorbed in the PLGlu shell; in contrast, when loaded at pH 10.5, neutral Dox was encapsulated inside the vesicles. The Dox-loaded vesicles displayed a loading content and loading efciency of 47 wt% and 67 wt%, respectively, at pH 10.5. It was found that the release of Dox from the vesicles was affected by both pH and temperature. An obviously faster release prole was observed at pH 5.5 in comparison with the release pattern at pH 7.4. The higher release rate at pH 5.5 was thought to be resulted from a higher hydrophilicity of Dox at lower pH. Additionally, an increase in temperature from 25 to 45C resulted in an increase in drug release rate, probably due to an increase in permeability and mobility of the membrane. 3.2.4. Polydiene-Polypeptide Block Copolymers Polypeptide-based block copolymers containing a polyvinyl block or a polydiene block have also been fabricated, and their stimuli-sensitive self-assembly behaviors have been investigated. Gallot et al. and Klok et al. have investigated the self-assembly of different polystyrenepolypeptide block copolymers with a rodcoil structure.[146148] Especially, polypeptide-based block copolymers containing a polydiene block have attracted considerable interest, attributed to the fact that the self-assembled nanoaggregates can be further developed into shape-persistent nanostructures by photocrosslinking of the 1,2-vinyl bonds remained within the polydiene blocks.[35] In two separate studies, polybutadiene-b-PLGlu (PB-b-PLGlu, Scheme 3f) diblock copolymers were synthesized by anionic polymerization of butadiene and subsequent ROP of BLG-NCA.[35,149] It was
found that the diblock copolymers formed spherical micelles or vesicles after direct dissolution in water, depending on the composition and overall chain length.[149,150] Generally, spherical micelles should form when the fraction of the hydrophilic block is much higher than that of the hydrophobic block.[151] The PB-b-PLGlu micelles and vesicles exhibited almost constant sizes at pH between 7 and 12, whereas their sizes showed a marked decrease as the pH was decreased from 7, due to the conformation transition of PLGlu.[36] The vesicles formed by PB40-b-PLGlu100 showed a hydrodynamic radii ranging from 100150 nm, depending on the pH.[152] After irradiated by UV for 1h, the vesicles showed an enhanced stability and a reduced swelling ratio in THF, due to the covalent crosslinking of the vesicle membrane. In addition to PB-b-PLGlu, positively charged PLLys has also been incorporated with diene polymers to fabricate amphiphilic diblock copolymers. Savin et al. and Schlaad et al. have prepared PB-b-PLLys diblock copolymers and investigated the solution properties of the vesicles formed by PB-b-PLLys.[153,154] It was found that the vesicles responded to both pH and temperature. The pH-dependent size change of the aggregates exhibited a manner opposite to the PB-b-PLGlu systems. At higher pH, the vesicles showed a smaller size and chains were packed more compactly at the corecorona interface.[154,155] With decreasing pH to less than 5.5, the size increased signicantly. On the other hand, at a pH (10.9) slightly above the pKa (10.5) of the LLys residues,[4] the vesicle size increased obviously as the temperature was increased from 25 C to above 40 C, caused by an -helix-to--sheet transition.[152] Lecommandoux and co-workers have synthesized a series of polyisoprene-b-PLLys (PI-b-PLLys) diblock copolymers via ROP of ZLL-NCA using monoamino-functionalized PI, followed by deprotection of the ZLL groups.[155] It was found that the formation of micelles and vesicles by the diblock copolymers was dependent on the molar ratio of the polypeptide block to the PI block. A general conclusion was proposed that, to form a vesicular structure, the molar ratio of the charged polypeptide block within the PI-b-PLLys diblock copolymers is usually lower than 65 mol%.[155] 3.2.5. Polyion Complex Vesicles A series of polyionic complex (PIC) micelles based on positively charged PEG-polypeptide block copolymers and negatively charged biopharmaceuticals, such as DNA, RNA and some proteins, have been designed for gene and protein delivery, as discussed in Section 3.1.4. In addition to the PIC micelles containing biopharmaceuticals, PIC vesicles based on two oppositely charged polypeptide block copolymers have also developed. These vesicular structures are composed of a PIC intermediate layer and hydrophilic inner and outer shells. Kataoka and co-workers have prepared a PIC vesicle with a diameter up to 10 m by negatively charged PEG-PAsp and positively charged PEG-poly(2-aminoethyl-,-aspartamide) (PEG-P(Asp-AE)) or PEG-poly(5-aminopentyl-,-aspartamide) (PEG-P(Asp-AP)).[156] It was found that the formation of PIC vesicles was inuenced by both the charge ratio and relative block length. PEG-P(Asp-AP) and PEG-PAsp have pKa values of 10.47 and 4.88, respectively, and hence are almost equally charged at physiological pH.[157] The morphology of the PIC
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vesicles formed by PEG-P(Asp-AP) and PEG-PAsp displayed a pH-dependence. Stable vesicles were formed at pH > 5.7; in contrast, the vesicles were disassociated into smaller particles as the pH was decreased to 5.7 or less, caused by a decrease in association force of the complexes. Dextran encapsulated vesicles were prepared by simple mixing of PEG-P(Asp-AP) and PEG-PAsp in the presence of FITC-dextran (FITC-Dex). The FITC-Dex encapsulated vesicles showed good stability even in the presence of 10% fetal bovine serum. The permeability of the PIC vesicles was found to be dependent on both pH and the MW of the guest macromolecules. The permeation of dextran labeled with tetramethylrhodamine isothiocyanate (TRITC-Dex, Mn = 70 000) from the vesicles was signicantly faster at pH 5.8 than at pH 6.27.4, resulted from a reduced association force of the PIC layer at acidic pH. Additionally, a smaller guest molecule led to a higher permeation rate. Myoglobin (Mb) encapsulated vesicles with sizes ranging from 500 nm to 5 m were prepared by mixing of an PEG-PAsp aqueous solution containing Mb with an PEG-P(Asp-AP) aqueous solution.[158] Mb encapsulated inside the vesicles retained its bioactivity, and reversible oxygenation/deoxygenation of Mb was achieved in the presence of trypsin in the outer medium. In addition, polypeptide-based hollow capsules have also been prepared by layer-by-layer (LbL) assembly of PLLys and PLGlu on colloidal supports, followed by crosslinking of the lm via click chemistry.[159] 3.2.6. Polypeptide-Based A2B Lipid Mimetics A2B amphiphilic copolymers comprising two lipophilic A segments and a hydrophilic B block may facilitate the formation of vesicular structures, because the hydrophobic volume is maximized and hence the formation of a bilayer structure is favored.[160] Very recently, pH-sensitive A2B lipid mimetics with PLGlu as the B block and different lipophilic moieties, including octadecane, cholesterol and polyhedral oligomeric silsesquioxane, as the A segment have been developed via thiol-alkyne click chemistry.[160,161] All the above lipid mimetics were found to form vesicular structures. CD, DLS and SLS tests indicated that the vesicle size increased as the pH increased from 4 to 10, while the aggregation number of the vesicles showed an almost constant value within the experimental pH range, suggesting that the pH-induced size change of the vesicles was mainly caused by an -helix-coil transition of PLGlu and a change in chain packing. Additionally, A2B lipid mimetics with hydrophobic polypeptide or polyester as the A blocks have been developed.[162,163] 3.2.7. pH- and Temperature-Sensitive Vesicles Vesicles formed by pH- and temperature-sensitive copolymers are interesting for controlled self-assembly and drug delivery, because the formation and disruption of the vesicular structures can be controlled by varying pH or temperature. In addition, schizophrenic vesicles may be formed by some pH- and temperature-responsive block polymers with appropriate polymer architectures and block length. Recently, vesicles based on pHand temperature-sensitive polypeptide copolymers have been investigated. Savin and co-workers prepared a series of PPO-bPLLys diblock copolymers via click reaction between azide-terminated PPO and acetylene-terminated PLLys.[164] The diblock
copolymers displayed a random coil-to--helix transition at pH (78) lower than the pKa of the PLLys homopolymers (10),[75] owing to segmental charge repulsion. At pH 3 and a temperature (25 C) higher than the LCST (15 C) of PPO, vesicles with diameters ranging from 48 to 100 nm were formed by PPO-bPLLys with the PLLys weight contents of 34 wt%76 wt%, while spherical micelles were observed for the sample with the PLLys content of 92 wt%. In contrast, as the pH was increased from 3 to 7 at 0 C, aggregates with an -helical PLLys core and a PPO corona were detected. It was found that the presence of a triazole ring did not show signicant effect on the morphology of the aggregates, in contrast to an increase in interfacial curvature for the systems formed by polypeptide-driven self-assembly.[165] The loading of a water-soluble anticancer drug, DoxHCl, by the PPO-b-PLLys micelles and vesicles were compared. At pH 6.0, DoxHCl encapsulated in vesicles exhibited a slow and sustained release prole with an initial burst, in comparison with a rapid release pattern of DoxHCl from the PPO-b-PLLys micelles. This fast release of DoxHCl from the PPO-b-PLLys micelles was assumed to be attributed to the fact that the drug was mainly loaded within the hydrophilic shell. In addition to the above positively charged systems, anionic pH- and temperature-sensitive vesicles have also been investigated. Lin and co-workers have found that the formation of vesicular or micellar structures by a PLGlu-b-PPO-b-PLGlu triblock copolymer could be controlled not only by polymer composition but also by pH.[166] Only spherical micelles were observed for PLGlu-bPPO-b-PLGlu with the PLGlu content of 74.5 mol%, whereas only vesicles were formed for a sample with the PLGlu content of 34 mol% at both acidic (pH 4.1) and basic pH (pH 11.5). On the other hand, for a sample with an intermediate PLGlu content, spherical micelles were observed at pH 11.5, whereas a mixture of vesicles and micelles were detected at pH 4.1. The self-consistent eld theory (SCFT) simulation results suggested that the morphology change from micelles to vesicles was driven by a decrease in the hydrophilic-hydrophobic interfacial area caused by a decrease in the hydrophilic PLGlu block length or by the pH-induced coil-to-helix transition of the PLGlu block. Tertiary amine-modied polymethacrylates, such as poly(2dimethylamino)ethyl methacrylate) (PDMAEMA) and poly(2diethylamino)ethyl methacrylate) (PDEAEMA), are unique pH- and temperature-sensitive polymers that exhibit LCST behaviors at pH slightly above the pKa of the pendant tertiary amines.[167] For instance, PDMAEMA has a pKa of 7.07.8, and deionized PDMAEMA exhibits LCST of 3246 C, depending on the MW.[3,167] pH- and temperature-responsive vesicles based on PDMAEMA-b-PLGlu diblock copolymers have been reported recently.[168,169] The PDMAEMA-b-PLGlu (Scheme 3g) diblock copolymers were synthesized by coupling PDMAEMA and PBLG via Cu(I)-catalyzed azide-alkyne click chemistry, followed by deprotection of the benzyl groups.[168,169] The diblock copolymers exhibited PI values ranging from 4 to 8.5. At pH 6 and 25 C, vesicles with a size of 300 nm were formed by a diblock copolymer, i.e., PDMAEMA85-b-PLGlu77, due to the electrostatic interactions between PDMAEMA blocks and PLGlu blocks. The vesicles were stabilized by excessive amino groups. As the pH decreased from 6 to 4, the vesicle size decreased to 138 nm. In addition, temperature-induced formation of vesicles
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by the diblock copolymers was also observed at a pH (pH 11) higher than the pKa of PDMAEMA. It was found the formation of micelles and vesicles driven by the dehydration of the PDMAEMA block was dependent on the relative block length. At pH 11 and 60 C, vesicular stuctures were formed by the diblock copolymers with PLGlu contents of 26 wt% and 43 wt%, whereas micelles were obtained for the diblock copolymer with a PLGlu content of 64 wt%. 3.3. Crosslinked Nanoparticles Stimuli-sensitive crosslinked nanoparticles, such as crosslinked micelles and hydrogel nanoparticles, have unique advantages including high stability, high drug loading capacity, and ability to respond to environmental stimuli.[170172] Hydrogel nanoparticles, i.e., nanogels, are swollen nanometer-sized networks comprising hydrophilic or amphiphilic polymers.[170,171] Nanogels may be composed entirely of a polymeric network or a core-shell structure with a hydrogel core or shell.[171] Stimuli-sensitive nanogels with an hydrophilic and biocompatible shell, such as stimuli-dependent swellable crosslinked micelles, have received much attention due to their smart swellingdeswelling transitions and improved biocompatibility. Drugs can be loaded in the temporarily stable nanocarriers and the release of the drugs can be triggered by environmental stimuli at desirable sites. In the past decade, polypeptide-based crosslinked nanoparticles, such as crosslinked micelles and nanogels, which respond to external stimuli, such as pH, reducing environment, and dual stimuli, have been developed for different drug delivery applications. These systems have been fabricated through various methods including crosslinking of preformed polymer chains or selfassembled nanoaggregates, precipitation polymerization, and one-step ring-opening polymerization. 3.3.1. Reduction-Sensitive Crosslinked Nanoparticles Nanoparticles crosslinked by covalent bonds capable of responding to intracellular microenvironment, in particular the acidic endosomal pH and the reducing environment of the cytoplasm, are interesting for intracellular drug delivery.[5,17] The development of reduction-sensitive nanocarriers is commonly achieved by incorporation of disulde bonds to the drug delivery systems, due to selective cleavage of the disulde bonds in the cytoplasm. The disulde bonds are stable in the relative oxidation environment in vivo, such as body uids and extracellular space, resulted from a low concentration (220 M) of glutathione (GSH).[17] In contrast, the disulde bonds can be cleaved in the relative reducing environment of the cytoplasm due to a relatively high GSH concentration (0.510 mM). Consequently, disulde-crosslinked nanoparticles based on polypeptides have also been developed for intracellular drug and gene delivery.[173175] Reversible shell crosslinked micelles based on a series of poly(L-cysteine)-b-PLLA diblock copolymers (PLCys-bPLLA, Scheme 4a) have been synthesized.[173] PLCys-b-PLLA was synthesized via ROP of -benzyloxycarbonyl-L-cysteine N-carboxyanhydride (ZLC-NCA) by using amino-terminated
PLLA as a macro-initiator, followed by deprotection of the benzyloxycarbonyl groups using HBr. Micelles with a PLLA core and a PLCys shell were rstly formed in the presence of DTT, and then shell crosslinked micelles containing disulde bonds were obtained by removing DTT and aerial oxidation. Ellmans assay suggested that less than 6% free thiols remained after oxidation. The decrosslinking of the micelles was achieved by addition of DTT. DLS and ESEM measurements revealed that the particle size increased slightly from 41.7 nm to 55.1 nm after adding DTT, and decreased to 47.1 nm again following removal of DTT, suggesting a reversible crosslinkingdecrosslinking process of the micelles. Additionally, no intermicellar crosslinking was observed by DLS and ESEM tests. A hydrophobic model drug, rifampicin, was loaded into the shell crosslinked micelles by self-assembly of PLCys-b-PLLA in the presence of rifampicin, followed by aerial oxidation.[176] The drug-loading content and loading efciency were 15.0% and 17.5, respectively. The drugloaded crosslinked micelles showed a size (65 nm) slightly higher than the particles without drug. A faster release prole was observed in the presence of DTT, due to decrosslinking of the particles caused by cleavage of the disulde bonds by DTT. In addition, a model protein containing a free cysteine residue, i.e.; BSA, was successfully conjugated to the diblock copolymer via oxidation, and the release of BSA from the copolymer was triggered by addition of reducing agent. Reversible-core crosslinked micelles containing disulde bonds have also been developed. Kataoka and co-workers have prepared a series of core-crosslinked polyion complex (PIC) micelles via disulde bonds through mixing of thiolated PEG-b-PLLys and PAsp, followed by crosslinking of the micellar core via aerial oxidation.[174] The thiolation of PEG-PLL was performed via the coupling reaction between PEG-b-PLLys and N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or 2-iminothiolane (IM).[177] The IM-conjugated PLLys block has a higher charge density than the SPDP-modied PLLys block,
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H N
O mN H SH O O H On O n
H O N
H O N x
H N
H ym
PLCys-b -PLLA (a)
HS
NH
NH2
O O O NH S HN S
O O O
O PEG- b -P(LLys-MP) (b) H N O H N H m O O H2N PEG-b -PkSer (e)
O L-Cystine NCA (c) O O NH2
H2N
Ketal cross-linker (d)
Scheme 4. Chemical structures of some representative polypeptidebased block copolymers and crosslinkers that are used for the preparation of crosslinked micelles and nanogels.
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due to the introduction of positively charged imino moieties. The chemical structure of a thiolated PEG-b-PLLys (denoted as PEG-b-P(LLys-MP)) obtained by treating the SPDP-modied PEG-b-PLLys using DTT is shown in Scheme 4b. The corecrosslinked PIC micelles showed an almost constant diameter with the salt concentration ranging from 0 to 0.5 M, in contrast to a signicant decrease in stability of the parent uncrosslinked PIC micelles.[174] On the other hand, the disulde crosslinked micelles were found to be dissociated rapidly in the presence of a reducing agent, DTT, indicating a reduction-induced cleavage of the disulde bonds. It is noteworthy that the reductive stability of disulde bonds can be affected by the substitution groups close to the disulde bonds.[178] In subsequent studies, disulde core-crosslinked PIC micelles based on thiolated PEG-PLLys and negatively charged biopharmaceuticals, including antisense oligonucleotide (ODN), pDNA, and siRNA, were developed.[177,179,180] It was found that the core-crosslinked PIC micelles with thiolation degrees of 10 and 26% displayed sizes comparable to that of the uncrosslinked micelles.[179] The core-crosslinked PIC micelles exhibited a high stability in the presence of a competing polyanion, i.e., poly(vinyl sulfate) (PVS), and showed a markedly enhanced resistance to nuclease compared to free ODN and uncrosslinked micelles. The release of loaded biopharmaceuticals from the crosslinked PIC micelles was triggered by addition of reducing agents, such as GSH. A higher GSH concentration and a lower crosslinking density resulted in a faster release rate. The disulde crosslinked PIC micelles containing pDNA with a size of 100 nm showed enhanced in vitro transfection efciency than uncrosslinked micelles.[177] This was believed to be due to a higher stability of the crosslinked micelles in culture medium, leading to an increased cellular uptake.[181] In addition, the gene transfection efciency was also inuenced by a balance between the charge density and thiolation ratio. A freeze-dried formulation of the core-crosslinked PIC micelles containing pDNA was subsequently developed.[182] The micelles with a thiolation degree higher than 13% retained the original size and shape and showed comparable gene transfection efciency after a freezedrying/reconstitution process. After intravenous injection of the crosslinked micelles with a thiolation degree of 37% into mice via the orbital vein, a gene expression was observed in the liver, compared to no gene expression detected for the group treated with naked pDNA. Crosslinked PIC micelles conjugated with a targeting ligand, i.e., cRGDfk, have also been prepared by thiolated cRGDfk-PEG-PLLys and pDNA.[181] The introduction of cRGDfk ligand led to an increase in transfection efciency against HeLa cells expressing v3 integrin receptors. Based on CLSM observation, the increase in transfection efciency was proposed to be due to cellular internalization through caveolae-mediated endocytosis. Additionally, a series of disulde crosslinked PIC nanoparticles based on oligopeptides and glycopeptides containing both LLys and cysteine residues have been developed by Rice and co-workers.[178,183,184] Interestingly, disulde core-crosslinked nanoparticles (CCLNPs) based on polypeptides have been developed by a simple one-step method using a difunctional NCA comonomer. In two separate studies, a difuctional NCA containing a disulde bond and two NCA rings, i.e., L-cystine-NCA (Scheme 4c), was synthesized.[185,186] Accordingly, disulde CCLNPs based on
PEG and polypeptide was fabricated by one-step ring opening copolymerization of L-cystine-NCA with LPhe-NCA or BLGNCA using amino-terminated mPEG as macro-initiators. 1H NMR tests indicated that the typical signals of polypeptides were suppressed, suggesting a coreshell structure with a disulde crosslinked polypeptide core and a PEG shell.[185,186] As shown in Figure 8, transmission electron microsopy (TEM) observation revealed that the PEG-poly(LPhe-co-L-cystine) CCLNPs exhibited a uniform spherical morphology with sizes ranging from 50150 nm, which are smaller than those determined by DLS measurements, likely due to the shrinkage of the nanoparticles during the sample drying process.[185] The size of the CCLNPs increased with increasing the overall polypeptide fraction or the content of L-cystine residues (Figure 8A). The CCLNPs remained stable in PBS (pH 7.4) even at an extremely low concentration (1.53 105 mg mL1). In contrast, the disulde crosslinking bonds were cleaved in the presence of 10 mM GSH, rendering an obvious increase in the size of the nanoparticles. Dox-loaded CCLNPs showed a reduction-dependent drug release behavior, as shown in Figure 8B. In PBS without GSH, less than 20% of loaded Dox was released from the CCLNPs at 93.5 h. In contrast, the release was markedly accelerated by addition of GSH, and over 90% Dox was released at 93.5 h in the presence of 10 mM GSH. In addition, an increase in the content of the L-cystine crosslinks or an increase in the overall polypeptide fraction led to a decrease in drug release rate, resulted from an increase in crosslinking density and the formation of a more compact nano-structure. The subsequent CLSM observation revealed effective intracellular delivery of Dox by the Dox-loaded CCLNPs. Strong intracellular uorescence of Dox was observed within the HeLa cells cultured with the Dox-loaded CCLNPs for 2 h, suggesting intracellular release of Dox from the CCLNPs triggered by GSH-mediated cleavage of the disulde crosslinks. MTT assays revealed that the polypeptide-based CCLNPs exhibited no signicant cytotoxicity and the Dox-loaded CCLNPs showed lower cytotoxicity than free Dox. In addition, a similar GSH-accelerated release of indometacin from a PEG-poly(BLG-co-L-cystine) CCLNP was observed.[186] Because the PEG-polypeptide CCLNPs that are prepared through facile one-step ROP of NCAs exhibit an intelligent drug release pattern in response to intracellular reducing environment, it is envisioned that these materials may have potential applications in intracellular drug delivery. In addition, an interlayer-crosslinked micelle has also been reported.[187] A PEG-b-PAsp(MEA)-b-PAsp(DIP) triblock copolymer was synthesized by Cu(I)-catalyzed alkyne-azide click reaction between azido-terminated PEG-b-poly(N-(2mercaptoethyl) aspartamide) (PEG-b-PAsp(MEA) and alkynefunctionalized poly(N-(2-(diisopropylamino)ethyl) aspartamide) (PAsp(DIP). At pH 10, the triblock copolymer self-assembled into micelles with a three-layer structure including a PAsp(DIP) core, a PEG outer shell and a PAsp(MEA) intermediated layer (Figure 9A-a). Due to the effect of aerial oxidation, disulde crosslinking bonds were further formed within the PAsp(MEA) interlayer, leading to the formation of interlayer crosslinked micelles. Because of the presence of both tertiary amino groups in the core and disulde crosslinks in the interlayer, the micelles showed pH- and reduction-sensitive behaviors. At pH 5.0 without adding DTT, an interesting nanocage structure
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was observed (Figure 9A-b). The nanocages showed a markedly enhanced size than the crosslinked micelles, due to complete dissolution of the inner PAsp(DIP) core that is constrained by the crosslinked interlayer. In addition, with addition of DTT (10 mM) at pH 7.4, highly swollen micelles were observed due to cleavage of the disulde crosslinks and the swelling of partially dehydrated PAsp(DIP) segments (Figure 9A-c). Notably, the nanoparticles disassembled at pH 5.0 with the presence of 10 mM DTT (Figure 9A-d). In vitro drug release measurements revealed that Dox-loaded interlayer crosslinked micelles retained stable in PBS at pH 7.4 without DTT (Figure 9B). In contrast, the release of Dox was signicantly accelerated by either reducing the pH to 5.0 or addition of DTT (10 mM), and highest release rate was observed in the presence of dual stimuli. CLSM observation demonstrated that fast accumulation of Dox in the nuclei of Bel-7402 cells was detected for free Dox and the Doxloaded crosslinked micelles, compared to the distribution of Dox mainly in the cytoplasm for PEG-b-PCL micelles (Figure 9C), indicating an enhanced endosomal/lysosomal escape of Dox for the Dox-loaded crosslinked micelles. Further in vivo studies revealed that the Dox-loaded crosslinked micelles showed higher antitumor efcacy than either free Dox or the Dox-loaded PEG-b-PCL micelles. 3.3.2. pH-Sensitive Crosslinked Nanogels Nanometer-sized polypeptide networks that undergo swellingdeswelling transitions in response to pH change, i.e., pH-sensitive nanogels,[170] are interesting for pH-triggered drugdelivery systems. PEG-b-PAsp nanogels were synthesized by crosslinking of the PAsp blocks using 1,6-hexanediamine (HDA) and N,Ndiisopropylcarbodiimide (DIC) as a crosslinker and a coupling agent, respectively.[188] A coreshell structure with a crosslinked PAsp Figure 8. A) TEM image and the hydrodynamic radii (Rh) of the PEG-poly(LPhe- co -L-cystine) hydrogel core and a PEG shell was formed CCLNPs: a) CCLNPs-1 (1/2/19, mPEG/LCys/LPhe (molar ratio)), b) CCLNPs-2 (1/6/23), and c) CCLNPs-3 (1/9/32). B) In vitro release of Dox from the Dox-loaded CCLNPs in PBS at pH during the crosslinking. The nanogels exhib- 7.4 and 37 C: traces (a), (b) and (c) refer to the release results of CCLNPs-1, CCLNPs-2 and ited a pH-dependent swellingdeswelling tran- CCLNPs-3, respectively, without the presence of GSH; traces (d) and (e) represent the results sition, as schematically illustrated in Figure 10. of CCLNPs-3 with the presence of 2.5 mM and 5 mM GSH, respectively; traces (fh) refer to the As the pH increase from 4 to 9, the size of results of CCLNPs-1, CCLNPs-2 and CCLNPs-3, respectively, in the presence of 10 mM GSH. [185] the nanogels increased from below 20 nm to Reproduced with permission. Copyright 2011, Royal Society of Chemistry. above 40 nm, attributed to the swelling of the loading capacity (26.6 wt%) was obtained, likely due to the electropolypeptide core caused by the gradual ionization of the Asp resistatic interactions between oppositely charged Asp residues and dues at pH above its pKa (3.9).[4] The nanogels showed a conDox. The loading of drug showed no obvious inuence on the stant diameter in 0.15 M PBS (pH 7.4) as the polymer solution particle size. It was found that Dox was rapidly released from the was diluted from 5 to 0.2 mg mL1, compared to the dissociation nanogels at both pH 5.0 and 7.4, and a slightly faster drug release of PEG-b-PAsp/Ca2+ ion complex micelles at concentrations less prole was observed at pH 5.0 than at pH 7.4. It is noteworthy than 1 mg mL1. Dox-loaded nanogels were obtained by mixing that the nanogels are in a swollen state at 7.4, which may facilitate Dox with nanogels in deionized water. A relatively high drug 65
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Figure 9. A) TEM images of the nanoassembly at pH values of a) 7.4, b) 5.0, c) 7.4 with addition of DTT, and d) 5.0 with addition of DTT. The interlayer crosslinked micelles shown in (a) were decorated with Au. In TEM measurements, the Au-decorated crosslinked micelles were not stained and other samples were stained with uranyl acetate. The arrows in (b) indicate the watermark of staining agent formed as a result of nanocage shrinkage in sample drying. DTT concentration (if added): 10 mM. B) Quantitative Dox release from the dual-sensitive crosslinked micelles (mean standard deviation (SD), n = 3). C) Intracellular Dox release and migration into nuclei observed by confocal laser scanning microscopy (CLSM). Bel-7402 cells were incubated (37 C) for 6 h at a Dox-equivalent dosage of 10 g per dish. Dox loading contents: 10.5% in the interlayer crosslinked micelles and 5.1% in PEG3k-PCL3k micelles. Nuclei were stained with Hoechst 33342. Reproduced with permission.[187]
drug diffusion. The faster release pattern of Dox at acidic pH was assumed to be attributed to an increased solubility of Dox (pKa = 8.25) and reduced interactions between Asp and Dox. On the other hand, it was found that the change in the watersolubility of Dox with decreasing pH showed no signicant inuence on the drug release behavior of a PEG-b-PAsp-b-PLPhe ternary copolymer nanogel.[189] PEG-b-PAsp-b-PLPhe triblock
Figure 10. Schematic illustration of the pH-dependent swelling deswelling transition of a core-crosslinked PEG-b-PAsp nanogel.
copolymers were synthesized via successive ROP of BLAspNCA and LPhe-NCA using amino-terminated mPEG as a macro-initiator. Reversible or irreversible nanogels were then prepared by crosslinking the micelles of PEG-b-PAsp-b-PLPhe using an acid-labile ketal-containing crosslinker (Scheme 4d) or a nondegradable crosslinker. The Dox-loaded nanogels containing nondegradable crosslinks exhibited similar drug release proles at pH 7.4 and 5.0. In contrast, the nanogels crosslinked by acid-cleavable crosslinks showed a markedly enhanced drug release rate as the pH was reduced from 7.4 to 5.0. Hence, an acid-catalyzed hydrolysis of the ketal-containing crosslinks was proposed to be responsible for the accelerated drug release at acidic pH.[189] When cultured with MCF-7 cells, the Dox-loaded nanogels with ketal crosslinks resulted in a higher Dox uorescence intensity within the nuclei than the Dox-loaded nanogels with non-biodegradable crosslinks, indicating an enhanced Dox release from the former nanogels at acidic endosomal/ lysosomal environment. Ketal linkages have also been incorporated to the side chains of a polypeptide diblock copolymer, i.e., PEG-b-poly(ketalized serine) (PEG-b-PkSer, Scheme 4e), to fabricate a PEG-b-PLLys analogue with acid-cleavable side chains.[190] After crosslinking of PEG-b-PkSer/DNA polyplexes by bis(sulfosuccinimidyl)suberate, crosslinked PIC
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micelles with both acid-cleavable crosslinks and side chains were obtained. The resulting crosslinked micelles exhibited increased transfection efciency in the presence of serum than either uncrosslinked PEG-b-PLLys/DNA polyplexes or PEI/ DNA polyplexes. CLSM observation revealed an improved dissociation of PEG-b-PkSer and DNA in the cytoplasm compared to the uncrosslinked PEG-b-PLLys/DNA polyplexes, implying an acid-triggered cleavage of the ketal linkages. Besides the chemical crosslinking methods, photocrosslinking is another commonly used method for in situ crosslinking, for the crosslinking can be performed in a controllable manner with or without the presence of photo-initiators. Chen and co-workers have reported a series of pH-sensitive polypeptide nanogels developed through photo-crosslinking. Photocrosslinkable PEG-b-poly(LGlu-co--cinnamyl L-glutamate) (PEG-b-P(LGlu-co-CLG)) diblock copolymers were synthesized by ROP of BLG-NCA using amino-terminated mPEG as an initiator, followed by grafting cinnamyl alcohol to the LGlu residues.[191] PEG-b-P(LGlu-co-CLG) self-assembled into nanoparticles in aqueous solution due to the hydrophobic interactions between CLG segments. Under UV irradiation at 254 nm, the pendant cinnamyl groups within the polypeptide block underwent dimerization, resulting in in situ crosslinking of the polypeptides segments and hence the formation of nanogels. The nanogels showed hydrodynamic radii ranging from 80135 nm at pH 7.4, depending on the polypeptide block length and the substitution degree of cinnamyl groups. As the pH increased from 4.0 to 7.4, the nanogels exhibited a signicant increase in size, due to the swelling of the polypeptide cores caused by gradual ionization of the LGlu residues. MTT assays indicated that both the block copolymers and the nanogels showed no detectable cytotoxicity at concentrations up to 0.1 mg mL1. Drug-loaded nanogels were prepared by mixing PEG-b-P(LGlu-co-CLG) with a model drug, rifampicin, in aqueous solution, followed by in situ photocrosslinking. The drug-loaded nanogels showed pH-dependent release proles in vitro. A fast drug release pattern was observed at pH 7.4, compared to only small amount of drug released at pH 4.0, due to the swelling of the nanogels at higher pH. 3.3.3. pH- and Temperature-Sensitive Nanogels In addition to the nanogels that respond to single stimulus, polypeptide-based nanogels that exhibit swellingdeswelling transitions in response to dual stimuli, such as pH and temperature, have also been investigated for controlled drug delivery. Nanogels based on PNIPAM and PLGlu were synthesized via free radical polymerization of 2-hydroxyethyl methacrylate (HEMA) and PNIPAM grafted PLGlu (PLGlu-g-(HEMA/ PNIPAM)) by Chen and co-workers.[192] PLGlu-g-(HEMA/ PNIPAM) was rst prepared by means of conjugating aminoterminated PNIPAM and HEMA to the PLGlu side chains. The nanogels were then obtained by increasing the temperature from 25 C to 60 C at pH 8 to form a dispersion of nanoparticles, followed by free radical polymerization of the HEMA residues using ammonium peroxydisulde (APS) as an initiator. The polymerization of the HEMA residues led to a sharp decrease in the hydrodynamic diameter of the aggregates from 262 nm to about 60 nm, due to the formation of
crosslinked nanogels with a more compact structure. The nanogels exhibited pH- and temperature-dependent swelling deswelling behaviors. At 27 C, the nanogels showed a decrease in size from 70 nm to 60 nm as the pH was reduced from 10.0 to 6.0, caused by the gradual protonation of the LGlu segments. Notably, as the pH was further decreased to below 5.5, the particle size increased markedly and large aggregates were detected due to the hydrophobic aggregation of the PLGlu segments. In addition, at pH 7.0, the nanogel size showed a sharp decrease as the temperature was increased to above 36 C, attributed to hydration-dehydration transitions of the PNIPAM segments. PLGlu/PNIPAM hybrid microgels have also been prepared via free radical copolymerization of HEMA-grafted PLGlu and N-isopropylacylamide (NIPAM) in 0.05 M PBS (pH 7.0) at 60 C by using APS as an initiator.[193] In comparison with the above PLG-g-(HEMA/PNIPAM) nanogels, the microgels exhibited signicantly higher size (570 nm) at pH 7.0 and 25 C. The microgels displayed pH- and temperature-sensitive swellingdeswelling behaviors similar to the nanogels. Because of the dual-stimuli responsive swelling behaviors of the nanosized and micrometer-sized hydrogel particles, it is envisioned that these materials may be interesting for drug delivery systems, such as oral drug delivery systems. 3.4. Hydrogels Hydrogels are three-dimentional hydrophilic or amphiphilic polymer networks formed by chemical or physical crosslinking.[2,3] Stimuli-responsive macroscopic hydrogels exhibit volume phase transitions or solgel phase transitions in response to changes in environmental conditions.[3] Permanent polymer networks can be formed by chemically crosslinking and display volume phase transitions under environmental stimuli, whereas physically crosslinked hydrogels are usually reversible networks and can be reversibly change to an uncrosslinked sol state in respond to external stimuli.[3] Therefore, covalently crosslinked hydrogels that undergo volume phase transitions in respond to environmental stimuli have been designed for controlled drug delivery through stimuli-induced swellingdeswelling transitions. On the other hand, in situ forming physically crosslinked hydrogels, such as thermo-gelling hydrogels, have been developed for localized drug delivery.[3] Recently, polypeptide-based chemically crosslinked hydrogels and in situ gelling hydrogels have been fabricated and tested for different drug-delivery applications. 3.4.1. Chemically Crosslinked Stimuli-Sensitive Hydrogels Chemically crosslinked pH-sensitive bulk hydrogels are interesting for pH-triggered drug delivery systems. For example, after being crushed and sieved into microparticles, hydrogels that respond to the pH shift between the stomach (pH 2) and the small intestine (pH 7) can be used for oral drug delivery of peptides and proteins.[6] Thus, hydrogels based on PLGlu and PAsp containing pendant carboxylic groups have been developed for intestinal drug delivery. Yang and co-workers have synthesized a series of pH-responsive hydrogels based on PLGlu by using diamino-capped PEG as a crosslinker.[194] The
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equilibrium swelling ratios (SR) of the hydrogels exhibited a marked increase with increasing the pH from 4 to 6, due to gradual ionization of the carboxylic groups as well as a secondary conformation transition. An increase in ionic strength led to a decrease in SR at higher pH, owing to the charge screening effect of salt. CD measurements indicated that an ordered secondary structure, i.e., -sheet, was formed within the hydrogel at pH 4.5, whereas a random coil structure was observed at higher pH.[194] In a subsequent study, PLGlu-based hydrogels were prepared by photoinduced polymerization of PEG-methacrylate substituted PLGlu using PEG dimethacrylate (PEG-DA) as a crosslinker.[195] It was found that SR decreased with increasing the crosslinking density, and an increase in the MW of the PEG-DA crosslinker resulted in a decrease in SR at higher pH, probably due to a decrease in the relative charge density. The degradation of the hydrogels was signicantly accelerated as the pH was increased. Protein-loaded hydrogels containing insulin, lysozyme or albumin were prepared by a swelling-diffusion method. Initial burst releases were observed for all the protein-loaded hydrogels owing to a rapid diffusion of the proteins located at the hydrogel surface. The in vitro release behavior of the proteins was found to be inuenced by the crosslinking density of the hydrogels and the MW of the loaded protein.[195] A higher crosslinking density or a higher MW of the protein resulted in a lower release rate at pH 7.4. In addition, pH-sensitive hydrogels based on PLGlu/poly(acrylic acid) (PLGlu/PAA) hybrid systems and PAsp have also been investigated.[196198] pH- and temperature-sensitive hydrogels composed of PLGlu and a thermo-sensitive component have been developed. A series of hybrid hydrogels based on PLGlu and poly(NIPAMco-HEMA) have been developed by Chen and co-workers.[199] The hybrid hydrogels were prepared by coupling PLGlu with poly(NIPAM-co-HEMA) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl) as a coupling agent. The hybrid hydrogels exhibited swelling behaviors dependent on pH and the hydrogel composition. An increase in the PLGlu content led to an enhanced swelling ratio (SR), a higher pore size and an increased enzymatic degradation rate at pH 7.4. All the hydrogels showed fast swelling-deswelling transitions with varying the pH from 7.0 to 4.0 at 37 C, indicating potential applications in oral drug delivery. It is noteworthy that the change in temperature showed less inuence on the SR of the hydrogels compared to the pH change, even though sharp thermo-induced phase transitions were observed for the hydrogels at around 32 C. This was believed to be due to the highly hydrophilic nature and electrostatic repulsion of the PLGlu segments at higher pH. A model protein, lysozyme, was loaded in the hydrogels by a swelling-diffusion method. After an initial burst, the in vitro release rate of lysozyme was signicantly increased as the pH was increased from 4.0 to 7.0, suggesting that the release of lysozyme was retarded by shrunk hydrogels at acidic pH but accelerated from swollen hydrogels at neutral pH. Very recently, pH- and temperature-sensitive hybrid hydrogels composed of PLGlu and a naturally derived polysaccharide, i.e., hydroxypropylcellulose (HPC), have been prepared by the same group.[200,201] The PLGlu/HPC hybrid hydrogels were synthesized via free-radical copolymerization of HEMA modied PLGlu (PLGlu-g-HEMA) and acrylate substituted HPC.[200]
Due to the LCST behavior of HPC (LCST 41 C),[202] the hybrid hydrogels exhibited pH- and temperature-dependent swelling behaviors. The SR of the hydrogels increased markedly with increasing pH from 4.0 to 6.8 at 37 C, whereas the SR decreased gradually as the temperature was increased from 25 C to 48 C at pH 6.8. Interestingly, the HPC content within the hydrogel showed an inuence on the SR in an opposite manner at pH 4.0 and pH 5.0, respectively. A higher PLGlu content resulted in a higher SR of the swollen hydrogel at pH 5.0, due to stronger electrostatic repulsion between the PLGlu segments. In contrast, a higher HPC content led to a higher SR of the shrunk hydrogel at pH 4.0, likely attributed to the fact that a higher HPC content resulted in an enhanced hydrophilicity of the shrunk hydrogel and/or caused a steric hindrance to intermolecular hydrogen bonding.[194] Similar to the PLGlu/P(NIPAM-co-HEMA) hydrogels,[199] the inuence of pH on the swelling behavior of the PLGlu/HPC hydrogels was more marked than that of temperature. As shown in Figure 11, a clear swollen hydrogel was observed at pH 6.8 and 25 C. With increasing temperature to 42 C at pH 6.8, a turbid hydrogel with slightly reduced SR was observed due to the dehydration of HPC. In contrast, an opaque and shrunk gel with dramatically reduced SR was formed at pH 1.2 and 25 C, caused by deionization of the PLGlu segments and the formation of strong intermolecular hydrogen bonding. Interestingly, the enzymatic degradation of the hybrid hydrogels was markedly affected by pH and HPC content. The hydrogels with a HPC content of 45 wt% or higher exhibited no obvious degradation in articial gastric juice (pH 1.2) with the presence of 3.2 mg mL1 pepsin for 2 h, compared to rapid degradation of the hydrogels with a HPC content of 27 wt% or less. On the other hand, the degradation rate of the hydrogels with the HPC content of 45 wt% or higher was signicantly enhanced in articial intestinal liquid (pH 6.8) with the presence of 10 mg mL1 pancreatin, due to the swelling of the hydrogels and an increase in enzyme accessibility to the polypeptide backbones. The in vitro release of BSA from the hydrogels in articial gastric juice (pH 1.2) was suppressed after an initial burst release of BSA located at the hydrogel surface, whereas the release was obviously accelerated in articial intestinal liquid (pH 6.8). 3.4.2. In Situ Forming Physically Crosslinked Hydrogels In situ forming hydrogels or injectable hydrogels can be formed by the in situ formation of physical interactions in response to environmental stimuli,[3,203,204] or by in situ chemical reactions
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Figure 11. pH- and temperature-dependent swelling behaviors of the PLGlu/HPC (45/55, w/w) hybrid hydrogels. Reproduced with permission.[200]
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such as Michael addition reactions,[205] enzymatically catalyzed reactions,[206,207] and the reactions of aldehyde with amines.[208] In comparison with the permanent polymer networks crosslinked by covalent bonds, physically crosslinked in situ forming hydrogels are reversible networks and can be developed by simply varying the environmental conditions, such as temperature and pH. Due to their unique advantages, such as minimal invasion, no organic solvent, site-specicity, less systematic toxicity and ability to deliver both hydrophobic and hydrophilic drugs, stimuli-sensitve in situ forming hydrogels have received extensive investigation for drug delivery in the past decade.[3] For instance, drugs can be mixed with a thermosensitive polymer in aqueous solutions at a lower temperature, and drug-loaded hydrogels can be formed in situ after injection of the mixed solutions into body. Recently, stimuli-sensitive in situ gelling hydrogels based on polypeptides have been developed. Deming and co-workers have developed a series of in situ forming hydrogels by diblock copolypeptides comprising a charged PLLys or PLGlu block and a hydrophobic PLLeu or PLVal block.[28,132] It was found that rigid hydrogels were formed by these diblock copolypeptides at very low polymer concentrations (0.252.0wt%). The charged block was found to contribute to gelation at low concentrations. Replacement of positively charged PLLys block with a negatively charged PLGlu block did no affect gel formation. However, addition of salt led to a weaker hydrogel, due to charge screening effect. In addition, the ordered secondary structures, such as -helix and -sheet, of the hydrophobic block promoted gel formation. The strength of the hydrogels can be tuned by varying the composition and concentration of the polypeptides. The physically crosslinked polypeptide hydrogels can be deformed and thinned by stress and injected through small-bore cannulae.[209] LSCM, ultra SANS, and cryogenic TEM measurements revealed that the hydrogels were composed of an interconnected porous network.[210] In vitro cytotoxicity tests indicated that hydrogels containing both PLGlu and PLLys exhibited good cytocompatibility, even though free diblock copolymers containing PLLys are cytotoxic.[210] The low cytotoxicity of the hydrogels was proposed to be attributed to the fact that the polypeptide segments were tethered within the hydrogels and not available in solution to cause cytotoxicity. After injection of the PLLys-b-PLLeu hydrogels into the forebrain of mice, the hydrogels exhibited good biocompatibility comparable to physiological saline.[209] The hydrogels injected in vivo were found to integrate well with brain tissue, and time-dependent in-growth of blood vessels, certain glia and some nerve bers into the hydrogels were observed. In situ thermo-gelling hydrogels are the most widely studied in situ forming systems.[3,203,204] Biodegradable amphiphilic thermo-gelling hydrogels were rstly developed based on PEG/aliphatic polyesters block copolymers by Kim and coworkers.[203] In subsequent studies, biodegradable in situ gelling hydrogels based on different polyesters, polycarbonates and oligo(amidoamine)s were reported.[3,211,212] The stimuli-induced gelation was believed to be resulted from the synergistic effect of an increase in physical interactions between the hydrophobic segments and partial dehydration of the PEG block.[3,203,204] Very recently, thermo-gelling systems based on polypeptides
O n
H N
H N H m
O n
H N
H N
O x
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H N
H ym
PEG-b-PLAla (a) PEGb-P(LAlaco-LPhe) (b)
Scheme 5. Chemical structures of representative thermo-gelling polypeptide-based block copolymers.
have also been reported. Jeong and co-workers synthesized a PEG-b-poly(L-alanine) (PEG-b-PLAla, Scheme 5a) diblock copolymer via ROP of L-alanine NCA (LAla-NCA) by using amino-terminated mPEG as a macro-initiator.[19] The PEG-bPLAla aqueous solutions were found to exhibit sol-to-gel transitions with increasing temperature at a polymer concentration of 612 wt%. The solgel-transition temperatures were in the range of 2040 C depending on the polymer concentration, making these materials interesting for in situ forming drugdelivery systems. In contrast, the aqueous solutions of a PEG-bpoly(D,L-alanine) (PEG-b-PAla) diblock copolymer without a secondary conformation only formed hydrogels at higher polymer concentrations (16 wt%) and higher temperatures (>60C). This suggested that the secondary conformation of the PLAla block played an important role in the thermo-induced gelation process. CD and FTIR measurements indicated that an increase in temperature or polymer concentration caused a slight increase in -sheet content of the PLAla block. Accordingly, the thermo-induced gelation was proposed to be due to an increase in intermicellar aggregation caused by the synergistic effects of the increase in -sheet content of the polypeptide block and partial dehydration of the PEG block.[213] The self-assembled nanostructure and gelation behavior were found to be inuenced by the block sequence of the block copolymers.[214] The gelation behavior of a PEG-b-PLAla-b-PAla triblock copolymer with a central rigid block was compared with that of a PEGb-PAla-b-PLAla triblock copolymer. The PEG-b-PAla-b-PLAla aqueous solutions showed sol-to-gel-to-squeezed gel transitions at lower concentrations (4.09.0 wt%), compared to only sol-togel transitions observed for the PEG-b-PLAla-b-PAla aqueous solutions at higher polymer concentrations, as shown in Figure 12a. CD measurements suggested that the PLAla block within both triblock copolymers adopted an -helical structure at low concentrations (0.01 wt% and 0.025 wt%) and a lower temperature. As the temperature was increased above 40 C, the secondary conformation of the PLAla block within PEGb-PAla-b-PLAla exhibited a transition from -helix to random coil. In contrast, the secondary structure of the PLAla block in PEG-b-PLAla-b-PAla exhibited no signicant change within the experimental temperature range (450 C). The -helixto-random coil transition was assumed to be responsible for the gel-to-squeezed gel transition of PEG-b-PAla-b-PLAla. As shown in Figure 12b, hydrogels of both triblock copolymers formed at 37 C displayed highly porous structures, whereas the squeezed gel of PEG-b-PAla-b-PLAla at 60 C exhibited a collapsed pore structure. In addition, a PEG-b-poly(LAlaco-LPhe) diblock copolymer was also found to show thermoinduced sol-to-gel transitions at concentrations of 3.07.0 wt%
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copolymer showed inuence on the nanostructure of the hydrogels as well as the proliferation and differentiation of the encapsulated chondrocytes.[217] Block copolymers based on poloxamer end-capped by two poly(LAla-coLPhe) or poly(LAla-co-LLeu) blocks were found to exhibit similar sol-to-gel transitions.[218,219] As compared with the -sheet dominant secondary conformations of the copolymers based on PLAla and poly(LAla-co-LPhe), the poly(LAla-co-LLeu)-based copolymer adopted an -helical structure within the temperature range of 2050 C. In addition, in situ gelling PEG/peptide systems containing dipeptides end groups,[220] PEG/-cyclodextrin (-CD) complexes,[221] or an adhesive precursor, i.e., L-3,4-dihydroxylphenylalanine (DOPA),[222] have been investigated. It is noteworthy that some amphiphilic poly(amino acid) derivatives containing both Figure 12. a) Photos of sol (5 C)/gel (37 C or 60 C) states of PEG-b-PLAla-b-PAla (20.0 wt% hydrophilic and hydrophobic side groups in water) (A) and sol (5 C)/gel (37 C)/squeezed gel (60 C) states of PEG-b-PAla-b-PLAla also show thermo-induced gelation behav(5.0 wt% in water) (B). b) SEM images of gels. Images of the gel of PEG-b-PLAla-b-PAla at 37 C iors. Uyama and co-workers synthesized a (A), the gel of PEG-b-PAla-b-PLAla at 37 C (B-1), and the gel of PEG-b-PAla-b-PLAla at 60 C series of poly(/-aspatamide) (PAspAm) (B-2) are compared. The scale bar is 10 m. Reproduced with permission.[214] Copyright 2011, derivatives by successive aminolysis of polyRoyal Society of Chemistry. succinimide (PSI) using dodecylamine and amino alcohols.[223,224] The amphiphilic [ 213 ] (Scheme 5b). PAspAm derivatives exhibited solgel transitions in PBS. The A similar thermo-induced strengthening of sol-gel transition temperature was found to be inuenced by sheet structure of the polypeptide block was observed for the graft composition, the side chain length, polymer concentradiblock copolymer. The hydrogels were stable in PBS without tion and additives. Kim and co-workers developed a series of enzymes in vitro, whereas the degradation of the hydrogels thermo- and pH-sensitive amphiphilic PAspAm through sucwas signicantly accelerated after the hydrogels were subcutacessive aminolysis of PSI by N-alkylamines and N-isopropylneously injected into rats, due to the presence of enzymes in ethylenediamine.[225,226] The copolymers showed pH-dependent the subcutaneous layer. In vitro release of insulin from insulinencapsulated hydrogels showed an initial burst release at the thermo-sensitivity, and the phase transition temperature was rst day, followed by a diffusion-controlled release prole over inuenced by the alkyl chain length and graft composition. The 16 days. After a single subcutaneous injection of the polymer concentrated copolymer solutions in PBS (pH 7.4) displayed solution containing insulin into diabetic mice, a signicant thermo-induced solgel transitions in vitro and in vivo. The in hypoglycemic effect was maintained over 18 days. vitro release of a hydrophobic model drug, chlorambucil, from the hydrogels followed near zero-order kinetics over 16 days. In addition, thermo-gelling block copolymers based on poloxamer anked by two polyalanine blocks were synthesized by ROP of the mixture of LAla-NCA and DL-Alanine NCA (DLAla-NCA) using diamino-terminated poloxamer as 4. Stimuli-Sensitive Functionalized Polypeptides a macro-initiator.[215] It was found that the solgel transition temperature of the copolymer solution was affected by the relaSome polypeptides exhibit responsiveness to environmental tive block length and the LAla content within the polypeptide stimuli, such as pH and temperature, due to ionizationblock. Generally, an increase in the polypeptide block length, a deionization transitions of the ionizable pendant groups and/or decrease in the poloxamer block length, or an increase in the stimuli-induced conformation transitions. However, polypepLAla content led to a decrease in the phase transition temperatide-based materials that exhibit sharp phase-transitions in ture. The moduli of the in situ fomed hydrogels increased markresponse to physiologically relevant stimuli, such as a narrow edly with increasing the polymer concentration.[216] Additionally, pH change in the region of pH 5.07.4 and a temperature change in the range of 1041 C, may have advantages in drug the polymer concentration showed a marked inuence on the and gene delivery applications.[5,15,18,19] In addition, the incorpobiocompatibility of the hydrogels. Hydrogels with polymer concentrations of 7.0 wt% and 10.0 wt% were found to show good ration of bioactive molecules to polypeptide-based biomaterials biocompatibility and promote the proliferation and differentiais required for promoting cellmaterials interactions. Hence, tion of chondrocytes in vitro and in vivo.[216,217] In contrast, the the functionalization of polypeptides has attracted increasing attention in recent years. Polypeptides have been functionalized viability, proliferation and differentiation of chondrocytes culby introduction of various functional groups or stimuli-sensitive tured within the 15 wt% hydrogels were markedly decreased.[216] moieties to the side chains of the polypeptides. It was also found that the secondary structure of the polypeptide
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4.1. Functionalization of Polypeptides Functionalized polypeptides are mainly obtained through two approaches: ROP of the NCA monomers containing desired functional moieties and postpolymerization modication of polypeptides containing reactive side groups. The rst approach leads to the possibility of obtaining functionalized polypeptides
O
via one-step ROP. In contrast, the secondary method facilitates the preparation of polypeptides with different functional moieties. For instance, polypeptides containing oligo(ethylene glycol) (OEG) side chains or sugar residues have been synthesized via ROP of NCAs containing OEG or sugar residues, as shown in Scheme 6a.[227232] Recently, more attention has been paid to the approach of postpolymerization modication, such as click
(a)
O HN R1 O *
H N * n R1
-R1:
O O O HN O O x O AcO OAc OAc O BzO BzO OBz OBz O OH O HO OH OH O OAc HN O HN O
O x O O * O O O O O H N * n O *
(b)
O HN
N3-R2 Cu(I)
H N * n
-R2:
O O N N O HO HO O *
O x
H2N
N R2 O O O R4 *
OH OH
(c)
O HN
H N * n R4
HS-R5 UV
H N * n R4 S R5
-R5:
O HO OH O x OH OH H2N O OH
R4 : O O O O O
O O O HN O O O O Cl Cl Cl O O * H N * n O R3 Me6TREN/CuCl O O O m O R3 O * H N * n
(d)
-R3:
O x
H2N
Scheme 6. Synthesis of functionalized polypeptides by the ROP of functionalized NCAs (a), alkyne-azide click chemistry (b), thiol-ene click chemistry (c), and grafting from approaches (d).
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chemistry,[233237] controlled free-radical polymerization,[238240] aminolysis, and transesterications.[85,241] Click chemistry has emerged as one of the most widely used methods for modication and functionalization of synthetic polymers, due to its high efciency and high specicity. Very recently, clickable groups such as alkyne, azido and vinyl groups have been incorporated to the side chains of polypeptides. As illustrated in Scheme 6bd, the functionalization of polypeptides can be effectively achieved by incorporation of reactive groups to the NCA monomers, followed by ROP of the NCAs. In two separate studies, Chen et al. and Hammond et al. synthesized alkyne containing poly(-propargyl-L-glutamate) (PPLG) via the ROP of -propargyl-L-glutamate NCA (PLG-NCA) by using an alkyl amine as an initiator (Scheme 6b).[233,234] PLG-NCA was prepared by the reaction between LGlu and propargyl alcohol in the presence of sulfuric acid or chlorotrimethylsilane, followed by the reaction with triphosgene. Azido-terminated PEG or azido-substituted sugar molecules were then grafted to the side chains of PPLG via Cu(I)catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition reaction. The grafting efciencies were found to be near 100%. In addition, clickable azido-functionalized PLGlu were also developed by Zhang and co-workers.[235,242] Poly(-3-azidopropanyl-L-glutamate) (PAPLG) was obtained by the reaction between sodium azide and Poly(-3-chloropropanyl-L-glutamate) (PCPLG), which was synthesized via the ROP of CPLG-NCA by using hexamethyldisilazane (HMDS) as an initiator. Similarly, alkyne-substituted sugar moieties and alkyne-terminated PEG-b-PLA were then grafted to the polypeptide side chains via azide-alkyne click chemistry. It is noteworthy that the chloride-substituted side groups of polypeptides can also be used as initiators for atom transfer radical polymerization (ATRP). A highly efcient grafting from method by using the chloride-functionalized side groups as initiators for the modication of polypeptides has been developed by Chen and co-workers (Scheme 6d).[238240] Thiol-ene click chemistry is another versatile and efcient method for the functionalizaiton of polymers and biomolecules. Polypeptides containing vinyl side groups have been developed for the functionalization of side chains. Schlaad and co-workers developed a poly(D,L-allylglycine) (PAGly) via ROP of D,L-allylglycine NCA.[232] Glycopolypeptides were obtained via the thiol-ene click reaction between PAGly and 1-thio--D-glucopyranose under UV irradiation. A similar allyl containing polypeptide, i.e., poly(-allyl-L-glutamate) (PALG), was reported by Zhang and co-worker.[243] PALG was synthesized via ROP of -allyl-Lglutamate NCA (ALG-NCA) (Scheme 6c). In addition, multifunctional polypeptides with both allyl and azido side groups were obtained via successive ROP of ALG-NCA and CPLGNCA, followed by replacement of chloride with azide. As a result, functional moieties can be further introduced to the polypeptides by either thiol-ene or azide-alkyne click reaction. Cheng and co-workers synthesized a vinyl containing poly(-4-vinylbenzyl-L-glutamate) (PVBLG) via ROP of -4vinylbenzyl-L-glutamate NCA (VBLG-NCA) by using HMDA as an initiator.[237,244,245] In addition to the click reactions with thiol-substituted molecules,[237] the vinyl groups of PVBLG can also be converted to other functional groups, such as alcohol, aldehyde, carboxylic acid, or bishydroxyl groups.[244,245] Other popular approaches for modication of polypeptides include aminolysis and transesterications.[85,241]
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Poly(aspartamide)s containing various functional groups have been obtianed via aminolysis of PBLA or PSI.[85,223,224] PLGlu derivatives have been fabricated via aminolysis of PBLG.[246,247] In addition, PBLG modied with different reactive groups, such as azido group, propargyl group, allyl group and chlorosubstituted group, have also been developed via ester-exchange reactions, and the degree of substitution of the functional groups was found to be dependent on the feed ratio of alcohol to PBLG.[241] 4.2. Thermo-Sensitive Functionalized Polypeptides Although some polypeptides show thermo-responsiveness, such as thermo-induced change in secondary conformation,[153] intelligent polypeptides that exhibit sharp thermo-dependent phase-transitions at temperatures near the physiological condition (such as 1042 C) may have advantages in practical drug delivery applications.[18,19] Consequently, polypeptides functionalized by thermo-sensitive moieties have been developed. Oligo(ethylene glycol) (OEG) grafted polymers, e.g., MEGxgrafted polymethacrylate (denoted as P(MEGxMA), where the subscript numbers represent the number of EG residues within each oligomer segment), have been shown to be thermosensitive, and their LCST can be adjusted by varying the MEGx side chain length. Polypeptides with various MEGx side chains have also been synthesized recently. Chen and co-workers have synthesized a seres of MEGx-grafted PLGlu (PPLG-g-MEGx) via the click reaction between poly(-propargyl-L-glutamate) (PPLG) and azido-substituted MEGx (Scheme 6b).[248] PPLG-gMEGx exhibited sharp thermal phase transitions with the LCST depending on the polypeptide backbone length, MEGx sidechain length, polymer concentration and salt concentration. An increase in the MEGx side-chain length or a decrease in the polypeptide backbone length led to an increase in LCST. As a result, the LCST of the polypeptide derivative could be tuned from 22 C to 74 C, as shown in Figure 13. CD measurements suggested that the polypeptide derivatives adopted an -helical structure. MTT assays indicated that the polypeptide derivatives exhibited no detectable cytotoxicity. In vitro degradation of the
Figure 13. Thermal phase transitions of 10 mg mL1 aqueous solutions of oligo(ethylene glycol) functionalized poly(L-glutamate)s (denoted as PPLGn-g-MEOx, where the subscript numbers refer to the average number of the repeat units within each segment). Reproduced with permission.[248] Copyright 2011, Royal Society of Chemistry.
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polypeptides was observed in the presence of proteinase K. In a separate investigation by Li and co-workers, poly(-MEGx-Lglutamate) (P(MEGxLG)) was obtained by synthesis of NCAs containing MEGx, followed by the ROP of the NCA monomers (Scheme 6a).[228] P(MEGxLG) with mixed MEGx side chains, i.e., MEG2 and MEG3, were fabricated via ROP of the mixture of MEG2-L-glutamate NCA and MEG3-L-glutamate NCA. It was found that the LCST could be tuned from 32 C to 57 C by adjusting the NCA feed ratio. Temperature-dependent CD measurements indicated that the secondary structures of the polypeptides contributed to the LCST behaviors, even though the secondary structures were not disrupted at temperatures above the LCST. It is noteworthy that P(MEG2LG) predominantly existed as a -strand structure and eventually lost its LCST behavior, compared to an -helical conformation of PPLG-g-MEG2.[248] The difference is likely due to the effect of the triazole ring within the side chains of PPLG-g-MEG2. Thermo-sensitive PLGlu derivatives have also been developed via a grafting from strategy by Chen and co-workers.[238,239] PLGlu-g-P(MEGxMA) was synthesized via ATRP of MEGx methacrylate (MEGxMA) by using poly(-2-chloroethyl-L-glutamate) (PCELG) as a macro-initiator (Scheme 6d). The LCST of the graft copolymers was inuenced by both the MEGx chain length and the degree of polymerization of P(MEGxMA). CD measurements indicated that the copolymers adopted an -helical conformation in aqueous solutions, and the secondary conformation was not disrupted with the increase in temperature. It is noteworthy that some poly(amino acid) derivatives functionalized by amphiphilic pendant groups exhibit LCST behaviors. Kobayashi and co-workers have fabricated poly(N-substituted /-aspartamide)s by aminolysis of PSI using a mixture of 5-aminopentanol and 6-aminohexanol.[249] The LCST of the poly(N-substituted /-asparagine)s increased from 23 to 44 C as the content of pentanol side groups increased from 50% to 80%. A range of pH- and thermo-responsive poly(N-substituted /-aspartamide) derivatives have been obtained by incorporation of both alkylalcohol and alkylamino pendant groups.[250,251] In addition, thermo-responsive poly(-glutamic acid) derivatives functionalized by amino alcohols or hydrophobic side groups have also been reported.[252254] Because of the presence of carboxylic side groups, the LCST of the poly(-glutamic acid) derivatives showed dependence on pH and salt concentration. 4.3. pH-sensitive Functionalized Polypeptides Smart polymers that sharply respond to a narrow pH change within the pH region of pH 5.07.4 are interesting for intracellular and anticancer drug-delivery systems. Therefore, the development of pH-sensitive polypeptide derivatives capable of responding to a narrow pH change near the physiological pH has received increasing investigation. Hammond and coworkers synthesized a series of PLGlu derivatives containing various amine side groups, including primary, secondary and tertiary amine groups, via the click reactions between poly(propargyl-L-glutamate) (PPLG) and azido-substituted alkylamines (Scheme 6b).[255,256] It was found that all the polypeptide derivatives containing primary, secondary and tertiary amine pendant groups showed proton buffering capacity in the pH range of
5.07.4. The primary and secondary amine-functionalized polypeptides displayed broad proton buffering behavior in the pH range of 5.510 with a midpoint at 7.25. The buffering behavior of the primary and secondary amine-functionalized PPLG at pH lower than the pKa (911) of the primary and secondary amines was believed to be due to segmental charge repulsion. For the tertiary amine-functionalized PPLG, the buffering behavior was found to be inuenced by the hydrophobicity of the pendant group. The diisopropylamine-functionalized PPLG displayed a sharper buffering transition and a lower pKa compared to the diethylamine- and dimethylamine-functionalized PPLG, as listed in Table 1. It is notable that the triazole ring showed slight effect on the buffering behavior at pH 34. CD measurements revealed that the polypeptides adopted an -helical conformation at high pH values, whereas random coil structure was detected with decreasing pH to below the pKa. The pH-dependent water solubility of the amino-functionalized polypeptides was tested. The primary amine-, secondary amine-, and dimethylaminesubstituted PPLG show no detectable phase transition within the experimental pH range. This is different from the solution behavior of amino-functionalized poly(2-hydroxyethyl methacrylate)s (PHEMA) that are also synthesized by grafting the amino groups to PHEMA via click chemistry.[257] Sharp pH-induced phase-transitions were observed for the primary amine- and dimethylamine-functionalized PHEMA at pH 12.3 and 9.7, respectively. On the other hand, the diethylamine- and diisopropylamine-functionalized PPLG exhibited sharp pH-dependent phase-transitions, as shown in Figure 14.[255] The phase transition pH decreased with the increase in the hydrophobicity of the tertiary amine group or the DP of the polypeptide. The phase transition pH can also be tuned by incorporation of both tertiary amines to the polypeptides at a given ratio. The diblock copolymers comprising PEG and a tertiary amine-functionalized PPLG showed pH-dependent micellizationdemicellization transitions at pH near the physiological condition, making these copolymers interesting for pH-triggered drug release systems. In addition, the hydrolysis of the ester bonds in the side chains was found to be accelerated with increasing pH, but it was markedly reduced by the incorporation of the PEG block, due to the formation of a polypeptide core encapsulated by PEG at higher pH.
Figure 14. Transmission as a function of pH for all diethylamine and diisopropylamine functionalized polymers. Diethylamine is abbreviated DE and diisopropylamine is abbreviated DI. Reproduced with permission.[255] Copyright 2011, Royal Society of Chemistry.
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Amino-functionalized polypeptides have also been developed via grafting from approach and aminolysis. Chen and co-workers have synthesized an oligo(2-aminoethyl methacrylate) (OAEMA) grafted PLGlu (denoted as PLGlu-gOAEMA) by ATRP of 2-aminoethyl methacrylate (AEMA) using PCELG as an initiator (Scheme 6d).[240] Similar to the primary amine-functionalized PPLG,[255] PLGlu-g-OAEMA exhibited a pKa (7.3) much lower than that of primary amines, due to the segmental Coulombic repulsion. In vitro cytotoxicity assay indicated that PLGlu-g-OAEMA showed a lower cytotoxicity than PEI (MW 25,000), and gel retardation assay displayed that PLGlu-g-OAEMA effectively condensed DNA at a polymer/DNA weight ratio of 0.3 or higher. In addition, typical pH-responsive polypeptide derivatives prepared by aminolysis include diamine-functionalized poly(/-aspartamide)s and poly(Lglutamine)s containing two types of amino side groups.[85,246] Due to the presence of two types of amins in the side chains, the polypeptide derivatives exhibited a combined buffering behavior, leading to gene delivery systems capable of both forming complexes with DNAs and promoting endosomal escape.
can improve endosomal disruption and transfection efciency. pH- and thermo-sensitive hydrogels can realize drug loading and release by adjusting either pH or temperature. Besides the ability to respond to physical and chemical stimuli, incorporation of biofunctionality to drug-delivery systems plays a crucial role in promoting cellmaterial interactions. Owing to the presence of functional and/or ionizable groups in some polypeptides, multifunctional polypeptide-based materials can be readily obtained by incorporation of different functional moieties to the polypeptides. On the other hand, it is worth mentioning that even though polypeptides and their derivatives containing not more than two kinds of amino acid residues exhibit no or less immunogenicity, some synthetic random copolypeptides comprising three (or more) kinds of amino acid may be immunogenic in vivo.[258260] Therefore, the amino acid composition of synthetic polypeptides needs to be considered during the molecular design of polypeptide-based materials for in vivo applications. Additionally, incorporation of hydrophilic and biocompatible segments, e.g., PEG, can markedly reduce the immunogenicity and signicantly improve the biocompatibility of the synthetic polymeric materials.
5. Conclusions and Perspectives

In this Review, we have presented a summary of the recent stimuli-sensitive synthetic polypeptide-based materials that have been designed and tested for drug- and gene-delivery applications. Stimuli-sensitive synthetic polypeptides exhibit unique secondary conformation transitions in response to external stimuli, such as pH and temperature. The unique stimuli-responsive secondary conformations of polypeptides lead to interesting self-assembly behaviors. In addition, the presence of functional groups within the polypeptide side chain facilitates the incorporation of different stimuli-sensitive moieties, such as acid-labile linkers and reduction-sensitive bonds. Besides the above properties, the biodegradability, nontoxicity and the ability to form complexes with biopharmaceuticals and ionic molecules of polypeptides make these materials promising candidates for drug- and gene-delivery applications. Polypeptide-based materials including micelles, vesicles, nanogels, and hydrogels have been developed based on the polymers with different structures. Structure and/or phase transitions of these materials, such as micellizationdemicellization transitions, crosslinkingdecrosslinking reactions, swelling deswelling transitions, and solgel transitions, can be triggered by varying environmental conditions, such as pH, temperature, redox environment, and dual stimuli. Accordingly, these materials have been evaluated for different drug-delivery applications, including anticancer drug and gene delivery, oral delivery of proteins, and localized drug delivery. The basic reqirements for applying these materials in drug and gene delivery include efciency, specicity, and safety.[74] To achieve desirable therapeutical objectives, drug-delivery systems with multifunctionalities and the ability to respond to multiple physiologically relevant stimuli have received increasing attention. For instance, disulde-crosslinked pH-sensitive PEGpolypeptide nanocarriers have advantages including high stability in extracellular environment, improved endosomal escape and enhanced intracellular delivery. Gene carriers with both DNA condensation ability and buffering capacity at endosomal pH
Acknowledgements
The authors are grateful for the nancial support from the National Natural Science Foundation of China (projects 51003103, 21174142, 50973108, 20904053 and 21074129), the Ministry of Science and technology of China (International cooperation and communication program 2010DFB50890), and the Scientic and Technological Development Projects of Jilin Province (201101082 & 20110332). Received: October 14, 2011 Published online: December 5, 2011
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Stimuli-Sensitive Synthetic Polypeptide-Based Materials for Drug and Gene Delivery

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Healthcare Mater. 2012, 1, 4878

3. Stimuli-Sensitive Polypeptide-Based Materials

Adv. Healthcare Mater. 2012, 1, 4878

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Healthcare Mater. 2012, 1, 4878

Adv. Healthcare Mater. 2012, 1, 4878

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Table 1. pKa values of some typical pH-sensitive peptide-based polymers.

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Healthcare Mater. 2012, 1, 4878

Adv. Healthcare Mater. 2012, 1, 4878

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Healthcare Mater. 2012, 1, 4878

Adv. Healthcare Mater. 2012, 1, 4878

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Healthcare Mater. 2012, 1, 4878

Adv. Healthcare Mater. 2012, 1, 4878

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Healthcare Mater. 2012, 1, 4878

H2N PLLys-b-PLLeu (a) H C6H13 N O H N O n H N H m O O O O HO H2N PLLys- b-PLGlu (c) O H O O n O N H O H N O

H2N PLLys-b-PLPhe (b) O O n O HO PLGA-b-PLGlu (d) O H m O x y N H H N O H N C6H13 m O

Adv. Healthcare Mater. 2012, 1, 4878

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Healthcare Mater. 2012, 1, 4878

Adv. Healthcare Mater. 2012, 1, 4878

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Healthcare Mater. 2012, 1, 4878

PLCys-b -PLLA (a)

O PEG- b -P(LLys-MP) (b) H N O H N H m O O H2N PEG-b -PkSer (e)

O L-Cystine NCA (c) O O NH2

Ketal cross-linker (d)

Adv. Healthcare Mater. 2012, 1, 4878

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Healthcare Mater. 2012, 1, 4878

Adv. Healthcare Mater. 2012, 1, 4878

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Healthcare Mater. 2012, 1, 4878

Adv. Healthcare Mater. 2012, 1, 4878

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Healthcare Mater. 2012, 1, 4878

PEG-b-PLAla (a) PEGb-P(LAlaco-LPhe) (b)

Scheme 5. Chemical structures of representative thermo-gelling polypeptide-based block copolymers.

Adv. Healthcare Mater. 2012, 1, 4878

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Healthcare Mater. 2012, 1, 4878

Adv. Healthcare Mater. 2012, 1, 4878

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Healthcare Mater. 2012, 1, 4878