Polypeptide-based Block Copolymers Research Articles

Block Sequence Effects on the Self-Assembly Behaviors of Polypeptide-Based Penta-Block Copolymer Hydrogels.

Peptide-based hydrogels have great potential for applications in tissue engineering, drug delivery, and so on. We systematically synthesize, characterize, and investigate the self-assembly behaviors of a series of polypeptide-based penta-block copolymers by varying block sequences and lengths. The copolymers contain hydrophobic blocks of poly(γ-benzyl-l-glutamate) (PBG, Bx) and two kinds of hydrophilic blocks, poly(l-lysine) (PLL, Ky) and poly(ethylene glycol) (PEG, EG34), where x and y are the number of repeating units of each block, where PBG and PLL blocks have unique functions for nerve regeneration and cell adhesion. It shows that a sufficient length of the middle hydrophilic segment capped with hydrophobic end PBG blocks is required. They first self-assemble into flower-like micelles and sequentially form transparent hydrogels (as low as 2.3 wt %) with increased polymer concentration. The hydrogels contain a microscale porous structure, a desired property for tissue engineering to facilitate the access of nutrient flow for cell growth and drug delivery systems with high efficiency of drug storage. We hypothesize that the structure of Bx-Ky-EG34-Ky-Bx agglomerates is beyond micron size (transparent), while that of Ky-Bx-EG34-Bx-Ky is on the submicron scale (opaque). We establish a working strategy to synthesize a polypeptide-based block copolymer with a wide window of sol-gel transition. The study offers insight into rational polypeptide hydrogel design with specific morphology, exploring the novel materials as potential candidates for neural tissue engineering.

Thermo-induced physically crosslinked polypeptide-based block copolymer hydrogels for biomedical applications.

Stimuli-responsive synthetic polypeptide-containing block copolymers have received considerable attention in recent years. Especially, unique thermo-induced sol-gel phase transitions were observed for elaborately-designed amphiphilic diblock copolypeptides and a range of poly(ethylene glycol) (PEG)-polypeptide block copolymers. The thermo-induced gelation mechanisms involve the evolution of secondary conformation, enhanced intramolecular interactions, as well as reduced hydration and increased chain entanglement of PEG blocks. The physical parameters, including polymer concentrations, sol-gel transition temperatures and storage moduli, were investigated. The polypeptide hydrogels exhibited good biocompatibility in vitro and in vivo, and displayed biodegradation periods ranging from 1 to 5 weeks. The unique thermo-induced sol-gel phase transitions offer the feasibility of minimal-invasive injection of the precursor aqueous solutions into body, followed by in situ hydrogel formation driven by physiological temperature. These advantages make polypeptide hydrogels interesting candidates for diverse biomedical applications, especially as injectable scaffolds for 3D cell culture and tissue regeneration as well as depots for local drug delivery. This review focuses on recent advances in the design and preparation of injectable, thermo-induced physically crosslinked polypeptide hydrogels. The influence of composition, secondary structure and chirality of polypeptide segments on the physical properties and biodegradation of the hydrogels are emphasized. Moreover, the studies on biomedical applications of the hydrogels are intensively discussed. Finally, the major challenges in the further development of polypeptide hydrogels for practical applications are proposed.

Supramolecular Polypeptide Self-Assembly Mediated In Situ Elicitation of Robust Innate and Adaptive Immune Responses Boosts Immunogenic Photothermal Therapy toward "Cold" Tumor.

As a promising cancer treatment modality that has emerged, photothermal therapy can harness antitumor immunity by triggering immunogenic cell death (ICD) in addition to direct cell ablation. However, the immuno-stimulation induced by PTT alone is insufficient to achieve satisfactory cancer eradication, especially in immunologically "cold" tumors due to their harsh immunosuppressive microenvironment. Effective activation of the innate immune system is indispensable to boost a robust adaptive antitumor immune response typically initiated by dendritic cells (DCs). Herein the above issues are addressed by constructing an environmentally responsive supramolecular nanoself-assembly (PSAs) derived from a novel polypeptide-based block copolymer, which is capable of co-load photothermal immunomodulators efficiently under structure-guided π-π stacking interactions. In the murine model of 4T1 xenograft tumors, the fabricated PSAs with payloads trigger both adaptive and innate immune responses in situ through activation of ICD as well as STING-dependent signal pathway. The findings reveal a new mechanism of harnessing photothermal therapy toward immunologically "cold" tumors.

Crucial Impact of Residue Chirality on the Gelation Process and Biodegradability of Thermoresponsive Polypeptide Hydrogels.

Thermosensitive polypeptide hydrogels have gained considerable attention in potential biomedical applications, of which the polymer structure may be tuned by residue chirality. In this study, polypeptide-based block copolymers with different chiralities were synthesized by ring-opening polymerization of γ-ethyl-l-glutamate N-carboxyanhydride and/or γ-ethyl-d-glutamate N-carboxyanhydride using amino-terminated monomethoxy poly(ethylene glycol) as a macroinitiator. All mPEG-polypeptide copolymers underwent sol-gel transition with an increase in temperature. The block copolymers with mixed enantiomeric residues of γ-ethyl-l-glutamate (ELG) and γ-ethyl-d-glutamate (EDG) in the polypeptide blocks exhibited lower critical gelation concentrations and lower critical gelation temperatures compared with those composed of pure ELG or EDG residues. We established that the difference in gelation properties between the copolymers was derived from the distinction of the secondary structures. We further demonstrated the influence of polypeptide chirality on the degradability and biocompatibility of hydrogels in vivo. Our findings provide insights into the design of hydrogels having tailored secondary conformation, gelation property, and biodegradability.

Synthesis of polypeptide bearing 1,4-dithiane pendants for ROS-responsive drug release

Stimuli-responsive polypeptides have been intensively investigated for controlled drug release, owing to their favorable biocompatibility and biodegradability. In this work, we designed and synthesized a new kind of polypeptide bearing 1,4-dithiane pendants for reactive oxygen species (ROS)-responsive drug release. The polypeptide-based block copolymer was facilely synthesized by ring-opening polymerization (ROP) of 1,4-dithian-substituted l-glutamate N-carboxyanhydride (DTG-NCA) monomer using an amino-terminated poly(ethylene glycol) methyl ether (mPEG-NH2) as the macromolecular initiator. The resultant block copolymer, mPEG-b-PDTG, could self-assemble into uniform micelles in aqueous medium owing to its amphiphilic structure. Then, the H2O2-triggered oxidation behaviors of the mPEG-b-PDTG micelles were studied by dynamic light scattering (DLS), FT-IR and turbidimetric assay. It was revealed that the oxidation of thioether into sulfoxide in the side chains would result in disassembly of the micelles. Furthermore, the ROS-responsive drug release behavior of the mPEG-b-PDTG micelles was verified by using Nile Red as a model drug. MTT assay also proved that mPEG-b-PDTG was non-toxic in B16F10 and L929 cells. Therefore, such a new class of oxidation-responsive polypeptide might provide a promising platform for ROS-responsive drug delivery.

Micelle-Coated, Hierarchically Structured Nanofibers with Dual-Release Capability for Accelerated Wound Healing and Infection Control.

Tailoring nanofibrous matrices-a material with much promise for wound healing applications-to simultaneously mitigate bacterial colonization and stimulate wound closure of infected wounds is highly desirable. To that end, a dual-releasing, multiscale system of biodegradable electrospun nanofibers coated with biocompatible micellar nanocarriers is reported. For wound healing, transforming growth factor-β1 is incorporated into polycaprolactone/collagen (PCL/Coll) nanofibers via electrospinning and the myofibroblastic differentiation of human dermal fibroblasts is locally stimulated. To prevent infection, biocompatible nanocarriers of polypeptide-based block copolymer micelles are deposited onto the surfaces of PCL/Coll nanofibers using tannic acid as a binding partner. Micelle-modified fibrous scaffolds are favorable for wound healing, not only supporting the attachment and spreading of fibroblasts comparable to those on noncoated nanofibers, but also significantly enhancing fibroblast migration. Micellar coatings can be loaded with gentamicin or clindamycin and exhibit antibacterial activity as measured by Petrifilm and zone of inhibition assays as well as time-dependent reduction of cellular counts of Staphylococcus aureus cultures. Moreover, delivery time of antibiotic dosage is tunable through the application of a novel modular approach. Altogether, this system holds great promise as an infection-mitigating, cell-stimulating, biodegradable skin graft for wound management and tissue engineering.

PH-responsive poly(ethylene glycol)-poly(ϵ-caprolactone)-poly(glutamic acid) polymersome as an efficient doxorubicin carrier for cancer therapy

The synthesis, characterization and potential application in doxorubicin (Dox) delivery system of an biodegradable polypeptide-based block copolymer: poly(ethylene glycol)2000-poly(e-caprolactone)6000-poly(glutamic acid)1000 (PEG2000-PCL6000-PGA1000) were investigated. The copolymer was synthesized via ring-opening polymerization (ROP) and characterized by 1H-NMR and FTIR. The synthesized copolymer could self-assemble into aggregates and the critical aggregation concentration was 0.23 mg/mL. Transmission electron microscope (TEM) indicated that the spherical polymersomes formed with a desirable size about 180 nm. Therefore Dox was encapsulated into these polymersomes, and then investigated its applications in drug delivery system. This Dox-loaded polymersomes (PolyDox) were characterized by dynamic light scattering (DLS), zeta potential and pH responsiveness measurements. In vitro drug release indicated the release rate of drug from PolyDox was pH-responsive and significantly decreased. The drug pharmacokinetic parameters were improved in comparsion to the group treated with free Dox, which proved the prolonged Dox release from PolyDox. WST-1 assay indicated a low toxicity and good compatibility of copolymer to cells within 48 h. The results also showed PolyDox appeared to induce higher antitumor effect. Cell uptake results indicated that PolyDox displayed higher cellular uptaken in A549 cells. Endocytosis inhibition results demonstrated the internalization of PolyDox was mostly mediated by fluid-phase endocytosis pathway.

Ring-Opening Polymerization of N-Carboxyanhydrides Initiated by a Hydroxyl Group.

We report on a method for preparation of well-defined synthetic polypeptides by ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCA) initiated by a hydroxyl group. To overcome the issue of slow initiation by hydroxyl group, an acid catalyst was used in the initiation step to catalyze opening of the NCA ring by the hydroxyl group and to simultaneously suppress further chain propagation by protonation of the formed amine group. In this way, we have separated slow initiation from the fast chain propagation, since such a combination leads to poorly defined products, and instead performed them in a successive manner. Only after completion of the initiation, the propagation was started by the addition of a base to deprotonate the ammonium group. This method was successfully applied for the synthesis of homopolypeptides by using alcohol as an initiator as well as polypeptide-based block copolymers by using poly(ethylene glycol) or poly(styrene) macroinitiator terminated with the hydroxyl group. This approach not only expands the pool of possible initiators, but also significantly facilities the preparation of polypeptide-based hybrid polymers.

A novel self-assembled hybrid organogel of polypeptide-based block copolymers with inclusion of polypeptide-functionalized graphene

Self-assembled hybrid organogels of polypeptide-based BCPs with incorporation of functionalized graphene were generated, and showed enhanced mechanical performance for the potential nanomaterials application.

A comparative study of linear, Y-shaped and linear-dendritic methoxy poly(ethylene glycol)-block-polyamidoamine-block-poly(l-glutamic acid) block copolymers for doxorubicin delivery in vitro and in vivo

The linear, Y-shaped, and linear-dendritic block copolymers of methoxy poly(ethylene glycol)-block-polyamidoamine-block-poly(l-glutamic acid) (MPEG-b-PAMAM-b-PGA) with one, two, four, and eight PGA arms but similar MPEG/PGA weight ratios (W/W) (named as P1PA, P2PA, P4PA and P8PA, respectively) were synthesized and comparatively investigated for doxorubicin hydrochloride (DOX) delivery. All the obtained block copolymers were highly biocompatible and could efficiently load DOX into nanoparticles (NPs) through electrostatic interaction. The NPs formed by linear (P1PA) or Y-shaped (P2PA) block copolymers and DOX were spherically shaped with smaller sizes, while the NPs formed from linear-dendritic block copolymers (P4PA and P8PA) were irregular in shape and larger in size. The P1PA/DOX and P2PA/DOX NPs exhibited better DOX protection and slower DOX release profile. However, cell cytotoxicity assays indicated that all the DOX-loaded NPs exhibited similar cytotoxicities with free DOX, indicating effective DOX release after cellular uptake. The NPs from linear and Y-shaped block copolymers greatly extended the blood circulation time, and displayed more accumulation in tumor site and less accumulation in the liver and kidney compared with the linear-dendritic counterparts. In addition, the P1PA/DOX and P2PA/DOX NPs also exhibited higher anti-tumor efficacy and less toxicity than the other DOX formulations. All these results indicated that the linear and Y-shaped MPEG-b-PAMAM-b-PGA block copolymers displayed better DOX delivery ability in anti-tumor treatment than the linear-dendritic copolymers. Statement of SignificancePolymeric NPs derived from block copolymers have emerged as effective vehicles for drug delivery. However, the majority of the researches in this field have involved simple linear block copolymers and there are very few comparative studies on the self-assembly, in vitro, and in vivo drug delivery by the block copolymers with similar composition but different architectures. In this study, a series of linear, Y-shaped, and linear-dendritic polypeptide-based block copolymers were prepared and thoroughly investigated for DOX delivery. These block polymers loaded DOX into NPs with different sizes and morphologies, and exhibited different anti-tumor capabilities both in vitro and in vivo. The results indicated that the architecture of the block copolymers played an important role in their drug delivery behaviors.

Insulin/poly(ethylene glycol)-block-poly(L-lysine) Complexes: Physicochemical Properties and Protein Encapsulation.

Insulin (INS) was encapsulated into complexes with poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLys), which is a polypeptide-based block copolymer (a neutral-cationic block polyelectrolyte). The particular cationic-neutral block copolymer can complex INS molecules in aqueous media via electrostatic interactions. Light-scattering techniques are used to study the complexation process and structure of the hybrid nanoparticles in a series of buffers, as a function of protein concentration. The physicochemical and structural characteristics of the complexes depend on the ionic strength of the aqueous medium, while the concentration of PEG-b-PLys was constant through the series of solutions. As INS concentration increased the size distribution of the complexes decreased, especially at the highest ionic strength. The size/structure of complexes diluted in biological medium indicated that the copolymer imparts stealth properties and colloidal and biological stability to the complexes, features that could in turn affect the clearance properties in vivo. Therefore, these studies could be a rational roadmap for designing the optimum complexes/effective nanocarriers for proteins and peptides.

PEG-polypeptide block copolymers as pH-responsive endosome-solubilizing drug nanocarriers.

Herein we report the potential of click chemistry-modified polypeptide-based block copolymers for the facile fabrication of pH-sensitive nanoscale drug delivery systems. PEG–polypeptide copolymers with pendant amine chains were synthesized by combining N-carboxyanhydride-based ring-opening polymerization with post-functionalization using azide–alkyne cycloaddition. The synthesized block copolymers contain a polypeptide block with amine-functional side groups and were found to self-assemble into stable polymersomes and disassemble in a pH-responsive manner under a range of biologically relevant conditions. The self-assembly of these block copolymers yields nanometer-scale vesicular structures that are able to encapsulate hydrophilic cytotoxic agents like doxorubicin at physiological pH but that fall apart spontaneously at endosomal pH levels after cellular uptake. When drug-encapsulated copolymer assemblies were delivered systemically, significant levels of tumor accumulation were achieved, with efficacy against the triple-negative breast cancer cell line, MDA-MB-468, and suppression of tumor growth in an in vivo mouse model.

Simulation‐Assisted Self‐Assembly of Multicomponent Polymers into Hierarchical Assemblies with Varied Morphologies

As you like it: The synthesis of supramolecular hierarchical nanostructures with designed morphologies has been realized through computer-simulation-guided multicomponent assembly of polypeptide-based block copolymers and homopolymers. By adjusting the attraction between hydrophobic polypeptide rods, as well as other parameters such as the molar ratio of copolymers and the rigidity of polymers, a variety of morphologies were obtained.

An Efficient Approach for Preparing Giant Polypeptide Triblock Copolymers by Protein Dimerization

An efficient and convenient approach for preparing a giant polypeptide-poly(ethylene oxide) triblock copolymer architecture of defined structure and composition is reported. This copolymer consists of two long polypeptide chains derived from bovine serum albumin of distinct lengths with stabilizing poly(ethylene oxide) side-chains, a connecting poly(ethylene oxide) block, and the presence of secondary structure elements along the polypeptide backbone. It is synthesized from the abundant plasma protein serum albumin and the polypeptide backbone is fully biodegradable. This approach represents a convenient and efficient strategy for preparing giant polypeptide-based block copolymers of defined structure via a semi-synthetic strategy. Such high-molecular-weight, biodegradable copolymers are attractive for various biomedical applications.

Photoresponsive Poly(S-(o-nitrobenzyl)-l-cysteine)-b-PEO from a l-Cysteine N-Carboxyanhydride Monomer: Synthesis, Self-Assembly, and Phototriggered Drug Release

A photoresponsive S-(o-nitrobenzyl)-l-cysteine N-carboxyanhydride (NBC-NCA) monomer was for the first time designed, and the related poly(S-(o-nitrobenzyl)-l-cysteine)-b-poly(ethylene glycol) (PNBC-b-PEO) block copolymers were synthesized from the ring-opening polymerization (ROP) of NBC-NCA in DMF solution at 25 °C. Their molecular structures, physical properties, photoresponsive self-assembly, and drug release of PNBC-b-PEO were thoroughly investigated. The β-sheet conformational PNBC block within copolymers presented a thermotropic liquid crystal phase behavior, and the crystallinity of PEO block was progressively suppressed over the PNBC composition. The characteristic absorption peaks of these copolymers at about 310 and 350 nm increased over UV irradiation time and then leveled off, indicating that the o-nitrobenzyl groups were gradually photocleaved from copolymers until the completion of photocleavage. The PNBC-b-PEO copolymers self-assembled into spherical nanoparticles in aqueous solution, presenting a photoresponsive self-assembly behavior, together with a size reduction of nanoparticles after irradiation. The anticancer drug doxorubicin can be released in a controlled manner by changing the light irradiation time, which was induced by gradually photocleaving the PNBC core of nanoparticles. This work provides a facile strategy not only for the synthesis of photoresponsive polypeptide-based block copolymers but also for the fabrication of photoresponsive nanomedicine potential for anticancer therapy.

Surface segregation of polypeptide-based block copolymer micelles: An approach to engineer nanostructured and stimuli responsive surfaces

We describe the surface segregation of polypeptide-based block copolymer micelles to produce stimuli-responsive nanostructures at the polymer blend/air interface. Such structures were obtained by simultaneous surface migration and self assembly at the surface of diblock copolymer/homopolymer blends. We employed blends composed of homopolymer (PS) and an amphiphilic block copolymer polystyrene-b-poly(l-glutamic acid) (PS-b-PGA). The surface was functionalized based on the preferential segregation to the polymer blend/air interface of the hydrophilic PGA block of the diblock copolymer upon annealing to water vapor. The surface migration of the diblock copolymer to the interface was demonstrated both by XPS and contact angle measurements. As a consequence, the PGA interfacial attraction leads to a large surface excess on diblock copolymer which in turn, through macrophase and microphase separation, produced separated domains at the surface with regions composed either of homo or block copolymer. Herein we demonstrate that the use of asymmetric diblock copolymers with a higher content in PS lead to spherical micellar assemblies randomly distributed at the surface. As observed by AFM imaging the blend composition, i.e. the amount of block copolymer within the blend influences the density of micelles at the surface. Finally, when exposed to water, the pH affects the surface morphology. The PGA segments are collapsed at low pH values and extended at pH values above 4.8, thus inducing variations on the topography of the films at the nanometer scale.

Organic/inorganic hybrid materials from polypeptide-based block copolymers

Novel organic/inorganic hybrid material was synthesized from poly(acrylazapropyl-trimethoxysilane)- b-poly( N ε -trifluoroacetyl- l-lysine) (P(AAPTMS)- b-P(TLL)) block copolymer using the sol–gel process. The treatment with ammonia solution permitted the hydrolysis of both trifluoro acetyl and trimethoxysilyl groups of the hydride copolymer followed by the condensation of the silanol groups, leading to cross-linked copolymers. The resulting cross-linked hybrid material was characterized using FT-IR, 29Si NMR, DSC, TGA, and environmental scanning electron microscopy. These techniques evidenced that a siloxane network was obtained. Amphiphilic polypeptide/inorganic hybrid copolymers were achieved and self-organized as particles in water.

Role of secondary structure changes on the morphology of polypeptide-based block copolymer vesicles

The pH and temperature responsive properties of poly(butadiene) 107–poly( l-lysine) 27 (PB 107–P(Lys) 27) block copolymer vesicles in aqueous solution were studied using dynamic and static light scattering, circular dichroism spectroscopy and transmission electron microscopy. In this material, the responsiveness comes partially from secondary structure changes within the polypeptide chain. These studies seek to elucidate the effect of these different polypeptide secondary structure changes on the morphology of self-assembled vesicles. It was found that as pH decreases, protonation of P(Lys) side-chain amine groups causes swelling in the vesicles due to the helix–coil transition and associated charge–charge interactions within the corona chains. At high pH and high temperature, P(Lys) corona chains undergo a secondary structure change from α-helix to β-sheet which causes an increase in vesicle size due to the relief of interfacial curvature. This study represents one of the first instances whereby different secondary structure transitions within the same polypeptide have been incorporated into a block copolymer assembly that can be used to produce dual-responsive materials.

Synthesis of hierarchically structured silica with polypeptide-based triblock copolymer as a template

Hierarchically structured silica was synthesized at near-neutral pH and under ambient conditions, utilizing synthetic polypeptide-based triblock copolymer poly( l-phenylalanine)- b-poly(ethylene glycol)- b-poly( l-phenylalanine) (Phe 7–PEG 135–Phe 7) as a template. In the synthesis procedure, anilino-methyl triethoxy silane (AMTS) was used as an intermedium, i.e., on one hand it interacts with polypeptide-based triblock copolymer through π–π interaction between the phenyl groups of Phe segments and AMTS; on the other hand, AMTS can co-condense with tetraethoxylsilane (TEOS) through hydrolysis process. The prepared silica possesses mesoscale short-range-order and exhibits hierarchical structure with both supermicropores and interconnected layers of macropores. It is proposed that the supermicropores are templated by the polypeptide segments, while the open 3D interconnected macroporous networks are presumably ascribed to both PEG segments and organic solvent. It is proved that both polypeptide-based block copolymer and AMTS play important roles in the formation of mesoscale short-range-order and hierarchical structure.