Polymerization Of Α-amino Acid N-carboxyanhydrides Research Articles

Insight into the cross-linking of synthetic polypeptide gels prepared by ring-opening polymerization using l-cystine N-carboxyanhydride

Synthetic polypeptides have promising potential for various biomedical applications that often require a cross-linked network for stability against solvents or mechanical stress. Due to the natural occurrence and availability of the l-cystine α-amino acid, l-cystine N-carboxyanhydride (NCA) is commonly used as a cross-linker in the ring-opening (co)polymerization of α-amino acid NCAs to prepare covalently cross-linked synthetic polypeptides. In this work, we show that the l-cystine NCA tends to undergo undesired decomposition side reactions, i.e., β-elimination and formation of 2,5-diketopiperazine, which reduce the amount of the cross-linker available and make it difficult to control the final properties of the materials prepared from it. By using the analogous l-homocystine NCA instead, the decomposition side reactions can be completely avoided. We demonstrate the cross-linking performance of l-cystine NCA compared to l-homocystine NCA by studying the corresponding gels prepared by copolymerization with γ-benzyl-l-glutamate NCA.

Poly(l-cysteine) Peptide Amphiphile Derivatives Containing Disulfide Bonds: Synthesis, Self-Assembly-Induced β-Sheet Nanostructures, pH/Reduction Dual Response, and Drug Release

Three disulfide bond-containing peptide amphiphiles (PA1-3) with different lengths of alkyl tails (PA1 for C6, PA2 for C12, and PA3 for C18) were synthesized by ring-opening polymerization of α-amino acid N-carboxyanhydride followed by post-polymerization modification. The peptide segments were composed of 3-mercaptopropionic acid-modified poly(l-cysteine) [P(Cys-SS-CH2CH2COOH)]. We characterized the chemical structure and molecular parameters by 1H NMR, 13C NMR, gel permeation chromatography, Fourier transform infrared spectroscopy, and circular dichroism spectroscopy. It is found that alkyl-P(Cys-SS-CH2CH2COOH) mainly presents a β-sheet conformation at the solid state. However, the PAs present predominant random coils at pH 7.4 in aqueous solutions. The β-sheet conformation increased dramatically when the concentration of the PA exceeded its critical micelle concentration (ca. 0.3 mg/mL for PA3), indicating the formation of self-assembly-induced β-sheet nanostructures. Elongation of the alkyl chain length or a decrease of the pH of the PA solution can promote the formation of the β-sheet conformation. The three PAs can self-assemble into spherical micelles or nanofibrous hydrogels, which can be utilized as nanocarriers for systemic drug delivery or implants for localized drug delivery, respectively. Cisplatin (CDDP) was loaded as a model medicine to examine the drug delivery potential of PA3. We found that the CDDP-loaded PA3 micelles are stable at pH 7.4, have a spherical morphology with a hydrodynamic diameter of ca. 52 nm, and accomplish pH/reduction dual-responsive release of CDDP. In addition, alkyl-P(Cys-SS-CH2CH2COOH) can self-assemble into nanofibrous hydrogels at pH 5.0-6.0 or upon the addition of certain metal ions and show excellent dynamic reversibility. Moreover, the CDDP-loaded PA3 hydrogel exhibits a sustained release profile and a nearly linear release over 48 h. In vitro cytotoxicity of PA3 also indicates its nontoxicity. Thus, our findings suggest that alkyl-P(Cys-SS-CH2CH2COOH) has great potential for both systemic and localized delivery of therapeutics.

Two-tailed tadpole-shaped synthetic polymer polypeptide bioconjugate nanomicelles for enhanced chemo-photothermal therapy

A series of poly(N-isopropylacrylamide)30-SS-block-(cyclic-poly(L-lysine)n)-block-SS-poly(N-isopropylacrylamide)30 (n = 10, 20, 30) synthetic polymer polypeptide bio-conjugates have been synthesized for enhanced chemo-photothermal therapy. A combination of reversible addition-fragmentation chain transfer polymerization, click cycloaddition reaction, and ring-opening polymerization of α-amino acid N-carboxyanhydrides is utilized in the synthesis of a tadpole-shaped block copolymer. All the copolymers are self-assembled to nanoparticles in aqueous solutions, which are stabilized using a terephthalaldehyde crosslinker with successfully encapsulating doxorubicin (DOX) and IR780 dye. The polymer nanomicelles exhibited excellent drug loading capacity and efficiency. The in vitro drug release profiles demonstrate that the maximum release of DOX is achieved in pH 5.0 at 42 °C, in the presence of a glutathione intracellular mimic environment. IR780 dye significantly induces photothermal cytotoxicity to HT29 colon cancer lines under near-infrared laser irradiation. The new two-tailed tadpole-shaped synthetic polymer polypeptide bioconjugates exhibiting potential chemo-photothermal therapeutic effect thus appear to be a promising platform for anticancer therapy.

Nano Aggregate Formation Through Self-Assembly of Poly(L-lysine)-Block-Poly(γ-benzyl-L-glutamate)-Graft-Poly(ethylene glycol) Copolymer.

Polymeric nano aggregates based on poly(L-lysine)-block-PBLG-graft-poly(ethylene glycol) (PLL-b-PBLG-g-PEG) copolymers were prepared by a dialysis method in deionized water. PLL-b-PBLG-g-PEG copolymers with different degrees of PEG substitution were synthesized by combining the ring-opening polymerization of α-amino acid N-carboxyanhydrides and ester exchange reactions. Proton nuclear magnetic resonance spectroscopy and gel permeation chromatography were employed to confirm the polymer structures and molecular weights. Transmission electron microscopy and dynamic light scattering were used to observe the self-assembly behavior. The experimental results revealed that the volume content of deionized water in the mixture significantly affects the morphology and size of the aggregates formed by PLL-b-PBLG-g-PEG copolymers. Spherical, spindle-like, needle-like, and daisy-like aggregates were observed with an increase in the volume content of deionized water in the mixture.

Pure polylysine-based foamy scaffolds and their interaction with MC3T3-E1 cells and osteogenesis

Polypeptide-derived copolymers have widely been exploited for drug/gene delivery due to their pendant functional groups and non-toxic degradation products. However, fabrication of polypeptide-based scaffolds for tissue engineering has seldom been reported. In this study, foamy poly(Nε-benzyl formateoxycarbonyl-L-Lysine) (PZL) and poly(Nε-benzyl formateoxycarbonyl-L-lysine-co-L-phenylalanine) (PZLP) scaffolds were successfully prepared by a combination of ring-opening polymerization of α-amino acid N-carboxyanhydride and negative porous NaCl templating approach. The physicochemical properties of these scaffolds including glass transition temperature, contact angle, compression modulus and degradation behavior were characterized. Both in vitro and in vivo biocompatibility of the scaffolds were evaluated by MC3T3-E1 cell culture and SD subcutaneous model, respectively. The results from live-dead staining, MTT and ALP activity assays indicated that PZL scaffolds were more conducive to the adhesion, proliferation and osteoblastic differentiation of MC3T3-E1 cells compared to PZLP scaffolds in the initial culture period due to their specific surface properties. While porous structure rather than surface properties of scaffolds played a decisive role in the later stage of cell culture. The results of in vivo studies including H&E, Masson’s trichrome and CD34 staining further demonstrated that PZL scaffolds supported the ingrowth of microvessels than PZLP scaffolds due to their surface property difference. Collectively, PZL scaffolds displayed good biocompatibility and could be a promising candidate for tissue engineering application.

Mussel-Inspired Poly(3,4-dihydroxy-L-phenylalanine)-Block-Poly(γ-benzyl-L-glutamate) Bioconjugate-Assisted Green Synthesis of Silver Nanoparticles.

Hybrid polymeric micelles self-assembled from a mixture containing poly(3,4-dihydroxy-L-Phenylalanine)-Block-poly(γ-benzyl-L-glutamate) diblock copolymer bioconjugates and silver nanoparticles were reported. A series of amphiphilic copolymers with different molecular weights were synthesized via ring-opening polymerization of α-amino acid N-carboxyanhydrides. The polymer backbone was demonstrated via proton nuclear magnetic resonance spectroscopy and Fourier-transform infrared spectroscopy. It was observed that the resulting amphiphilic copolymer can self-assemble into rod-like micelles in dimethyl formamide/ethanol mixtures as confirmed via transmission electron microscopy. In addition, the formed rod-like micelles transformed to vesicular micelles upon introducing silver nanoparticles into the amphiphilic copolymer solution. The effect of silver nanoparticles content on the vesicle formation process was also studied. Spindle-like micelles were observed sequentially before the formation of the vesicles.

Investigation of α-amino acid N-carboxyanhydrides by X-ray diffraction for controlled ring-opening polymerization

The need for a scalable synthesis of not sequence defined polypeptides as biomaterials is met by the ring-opening polymerization of α-amino acid N-carboxyanhydrides (NCAs). Even though this polymerization technique appears straight forward, it holds pitfalls in terms of reproducibility and overall control over the polymerization conditions, which depends, beside choice of solvent or initiator, significantly on reagent purity. In addition, the synthesis of monomers can lead to the formation of racemic amino acids. Thus, in this work, we describe the benefits of highly pure monomers in order to control nucleophilic ring-opening polymerization NCAs. Hereby, monomer purity is investigated by relating melting points of NCAs with single-crystal and powder X-ray diffraction crystallography data, which further proves retained stereo-information of NCAs.

Lithium hexamethyldisilazide initiated superfast ring opening polymerization of alpha-amino acid N-carboxyanhydrides

Polypeptides have broad applications and can be prepared via ring-opening polymerization of α-amino acid N-carboxyanhydrides (NCAs). Conventional initiators, such as primary amines, give slow NCA polymerization, which requires multiple days to reach completion and can result in substantial side reactions, especially for very reactive NCAs. Moreover, current NCA polymerizations are very sensitive to moisture and must typically be conducted in a glove box. Here we show that lithium hexamethyldisilazide (LiHMDS) initiates an extremely rapid NCA polymerization process that is completed within minutes or hours and can be conducted in an open vessel. Polypeptides with variable chain length (DP = 20–1294) and narrow molecular weight distribution (Mw/Mn = 1.08–1.28) were readily prepared with this approach. Mechanistic studies support an anionic ring opening polymerization mechanism. This living NCA polymerization method allowed rapid synthesis of polypeptide libraries for high-throughput functional screening.

Nanoengineered Peptide-Grafted Hyperbranched Polymers for Killing of Bacteria Monitored in Real Time via Intrinsic Aggregation-Induced Emission.

Facing the global health crisis caused by drug-resistant bacteria, antimicrobial peptides and their analogues offer exciting solutions to this widespread problem. Without additionally introducing a fluorescent probe, novel nanoengineered peptide-grafted hyperbranched polymers (NPGHPs) are constructed for their combined outstanding antimicrobial activity and sensitive bacterial detection in real time. Hyperbranched polyamide amine (H-PAMAM) that exhibits aggregation-induced emission (AIE) effects is synthesized. Then, NPGHPs are prepared by ring-opening polymerization of α-amino acid N-carboxyanhydrides on the periphery of the H-PAMAM. The NPGHPs exhibit high-efficiency antibacterial properties against a wide spectrum of bacteria, especially against Gram-negative bacteria. On the basis of the AIE effect of NPGHPs, the interaction between NPGHPs and Escherichia coli is explored and the fluorescence intensity of NPGHPs is dependent on the number of E. coli present. Thus, a method for monitoring E. coli concentration is developed, and the detection limit is 1 × 104 CFU mL-1. Furthermore, NPGHPs are used as fluorescent probes to visualize antibacterial process via lighting-up bacteria. NPGHPs can penetrate the membrane of bacteria and cause cell rupture and apoptosis. In addition, the excellent selectivity of NPGHPs toward bacteria over mammalian cells makes them bright prospects for clinical applications.

Straightforward access to linear and cyclic polypeptides

Ring-opening polymerization of α-amino acid N-carboxyanhydrides (NCAs) is a powerful synthetic methodology for generating well-defined functional polypeptides. However, conventional procedures require a compromise between obtaining controlled microstructures and employing the optimized polymerization conditions. Specifically, a versatile method to access sequenced cyclic polypeptides remains challenging due to the difficulty in site-specific cyclization. Here we describe a general and straightforward method for the synthesis of both linear and cyclic polypeptides using organocatalytic living polymerization of NCAs. The use of an air-stable organocatalyst, imidazolium hydrogen carbonate, allows for the rapid and controlled polymerization of a variety of NCAs, leading to high conversion within a few minutes under mild conditions. Linear and cyclic block copolypeptides are also accessible simply by controlling the type of initiators and the order of addition of NCA monomers.

Preparation of water dispersible and biocompatible nanodiamond-poly(amino acid) composites through the ring-opening polymerization

Nanodiamond (ND) is one of the most fascinating carbon materials that have been extensively investigated for biomedical applications owing to its small size, high specific surface areas, chemical inert and desirable biocompatibility. It has been reported that surface modification of ND with polymers could not only improve the dispersibility of final ND based composites but also endow them novel functions to fulfill the requirement for biomedical applications. Although some strategies have been developed previously, surface modification of ND with poly(amino acid)s has not been reported previously. In this work, poly(amino acid)s functionalized ND composites were fabricated through a ring-opening polymerization of α-amino acid N-carboxyanhydrides (NCAs), which was synthesized by conjugation of hydrophilic ethylene glycol with glutamic acid. The successful preparation of ND-GluEG composites was confirmed by a series of characterization techniques. The results suggest that the water dispersibility of final ND-GluEG composites is obviously improved. Moreover, ND-GluEG composites show low toxicity and are of great potential for biomedical applications.

Controlled ring-opening polymerization of α-amino acid N-carboxyanhydrides in the presence of tertiary amines

The mechanism of the primary ammonium/tertiary amine-mediated ring-opening polymerization of γ-benzyl-l-glutamate N-carboxyanhydride (BlG-NCA) was investigated. Kinetic analyses revealed that the normal amine mechanism (NAM) together with a dormant-active chain end equilibrium were responsible for the controlled nature of this polymerization pathway, but that the polymerization also proceeded via the activated monomer mechanism (AMM). Mixtures of primary amines (1 equiv) and tertiary amines (0–1.5 equiv) were therefore tested to confirm the co-existence of the NAM and AMM and determine the limits for a controlled polymerization. For tertiary amine molar fractions smaller than 0.8 equiv, the reaction times were greatly reduced (compared to primary amine-initiated polymerization) without compromising the control of the reaction. Hence, the polymerization of NCA can proceed in a controlled manner even when the AMM contributes to the overall chain growth mechanism.

NAM-TMS Mechanism of α-Amino Acid N-Carboxyanhydride Polymerization: A DFT Study.

The normal amine mechanism via the proton-transfer route (NAM-H) is widely accepted for the synthesis of polypeptides with nonionic initiators. Besides proton transfer, the trimethylsilyl (TMS) group transfer process has been found in living/controlled polymerization initiated by N-TMS amine in experiments, but the corresponding mechanism has never been proposed. In this work, we employed density functional theory (DFT) with the solvation model to investigate the details of the TMS-transfer mechanism, defined as NAM-TMS, for the ring-opening polymerization of α-amino acid N-carboxyanhydride. The TMS transfer process of NAM-TMS is thermodynamically more favored than the NAM-H mechanism according to the lower addition energy barrier observed. The rate-determining step (RDS) in NAM-TMS is the decarboxylation step, i.e., the release of CO2, rather than carbonyl addition in NAM-H because of the low dipole stable precursor enlarged energy gap of decarboxylation. It is the first calculation evidence supporting decarboxylation as RDS in the NAM mechanism.

Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex

The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring.

Comb-like amphiphilic polypeptide-based copolymer nanomicelles for co-delivery of doxorubicin and P-gp siRNA into MCF-7 cells

A comb-like amphiphilic copolymer methoxypolyethylene glycol-graft-poly(l-lysine)-block-poly(l-phenylalanine) (mPEG-g-PLL-b-Phe) was successfully synthesized. To synthesize mPEG-g-PLL-b-Phe, diblock copolymer PLL-b-Phe was first synthesized by successive ring-opening polymerization of α-amino acid N-carboxyanhydrides followed by the removal of benzyloxycarbonyl protecting groups, and then mPEG was grafted onto PLL-b-Phe by reductive amination via Schiff's base formation. The chemical structures of the copolymers were identified by 1H NMR. mPEG-g-PLL-b-Phe copolymer had a critical micelle concentration of 6.0mg/L and could self-assemble in an aqueous solution into multicompartment nanomicelles with a mean diameter of approximately 78nm. The nanomicelles could encapsulate doxorubicin (DOX) through hydrophobic and π–π stacking interactions between DOX molecules and Phe blocks and simultaneously complex P-gp siRNA with cationic PLL blocks via electrostatic interactions. The DOX/P-gp siRNA-loaded nanomicelles showed spherical morphology, possessed narrow particle size distribution and had a mean particle size of 120nm. The DOX/P-gp siRNA-loaded nanomicelles exhibited pH-responsive release behaviors and displayed accelerated release under acidic conditions. The DOX/P-gp siRNA-loaded nanomicelles were efficiently internalized into MCF-7 cells, and DOX released could successfully reach nuclei. In vitro cytotoxicity assay demonstrated that the DOX/P-gp siRNA-loaded nanomicelles showed a much higher cytotoxicity in MCF-7 cells than DOX-loaded nanomicelles due to their synergistic killing effect and that the blank nanomicelles had good biocompatibility. Thus, the novel comb-like mPEG-g-PLL-b-Phe nanomicelles could be a promising vehicle for co-delivery of chemotherapeutic drug and genetic material.

Preparation and characterization of foamy poly(γ-benzyl-l-glutamate-co-l-phenylalanine)/bioglass composite scaffolds for bone tissue engineering

Novel foamy scaffolds of poly(γ-benzyl-l-glutamate) and poly(γ-benzyl-l-glutamate-co-l-phenylalanine) were fabricatedviaa combination of a sintered NaCl templating method and ring-opening polymerization of α-amino acid N-carboxyanhydrides.

Synthesis and Characterization of pH-Responsive Copolypeptides Vesicles for siRNA and Chemotherapeutic Drug Co-Delivery.

Novel diblock copolypeptides are synthesized and evaluated as a pH-sensitive drug delivery vector for anticancer drugs and siRNA co-delivery. The synthetic process involve the ring opening polymerization of α-amino acid N-carboxyanhydrides as well as aminolysis and deprotection reactions, and the structures are characterized. The copolypeptides can self-assemble into stable vesicles at neutral pH, and disassemble in acidic endosomal/lysosomal, which shown the pH-responsive behavior. The cell uptake results indicated that the vesicles can co-deliver DOX and siRNA inside the same cell and exhibit a pH-sensitive release behavior. Therefore, the novel polymeric vesicles are a promising carrier for co-delivery anticancer drug and siRNA to overcome the drug resistance and enhance cancer treatment.

Polypeptide brushes grown via surface-initiated ring-opening polymerization of α-amino acid N-carboxyanhydrides

Polypeptide brushes are attractive platforms to generate functional and responsive interfaces that are of potential interest due to their possible biodegradability, biocompatibility and tunable secondary structures. Surface-initiated ringopening polymerization (SI-ROP) of α-amino acid N-carboxyanhydrides represents a powerful and versatile strategy to generate polypeptide brushes. This review is an attempt to capture the state-of-the-art in this field and highlights the latest developments in several selected areas. In addition to presenting an overview of the synthetic methods that have been used to generate polypeptide brushes via SI-ROP, this article will discuss the preparation of patterned polypeptide brushes, the conformational properties of surface-tethered polypeptides, ways to control chain orientation at surfaces as well as properties and applications of these thin polymer films.

Organocatalysis by hydrogen-bonding: a new approach to controlled/living polymerization of α-amino acid N-carboxyanhydrides

Hydrogen-bonding organocatalytic controlled/living ROP of α-amino acid N-carboxyanhydrides.

Polymer-<I>Block</I>-Polypeptides and Polymer-Conjugated Hybrid Materials as Stimuli-Responsive Nanocarriers for Biomedical Applications

Stimuli-responsive nanocarriers are a class of soft materials that includes natural polymers, synthetic polymers, and polypeptides. Recently, modern synthesis tools such as atom transfer radical polymerization, reversible addition-fragmentation chain transfer polymerization, nitroxide-mediated radical polymerization, ring-opening polymerization of α-amino acid N-carboxyanhydrides, and various "click" chemistry strategies were simultaneously employed for the design and synthesis of nanosized drug delivery vehicles. Importantly, the research focused on the improvement of the nanocarrier targetability and the site-specific, triggered release of therapeutics with high drug loading efficiency and minimal drug leakage during the delivery to specific targets. In this context, nanocarriers responsive to common stimuli such as pH, temperature, redox potential, light, etc. have been widely used for the controlled delivery of therapeutics to pathological sites. Currently, different synthesis and self-assembly strategies improved the drug loading efficacy and targeted delivery of therapeutic agents to the desired site. In particular, polypeptide-containing hybrid materials have been developed for the controlled delivery of therapeutic agents. Therefore, stimuli-sensitive synthetic polypeptide-based materials have been extensively investigated in recent years. This review focuses on recent advances in the development of polymer-block-polypeptides and polymer-conjugated hybrid materials that have been designed and evaluated for various stimuli-responsive drug and gene delivery applications.