Peptide Block Copolymers Research Articles

Preparation of Non-Toxic Fluorescent Peptide-Coated Silica/PEG Nanoparticles from Peptide-Block Copolymer Conjugates

Peptide-decorated nanosystems have demonstrated higher stability and improved cellular uptake as compared to bare NPs and appear highly promising in diagnostics and theranostics of cancer. Herein, we discuss the preparation and structural characterization of peptide-functionalized silica/PEG NPs, starting from peptide–block copolymers, prepared in turn by conjugation of the peptides to block copolymers before NP formation. This synthetic design allowed full control of density and composition of peptide surface coverage. Preliminary experiments support the low toxicity of the fluorescent peptide–NPs and their ability of cell internalization.

Poly(Ethylene Glycol)-Poly(l-Alanine)/Hyaluronic Acid Complex as a 3D Platform for Understanding Cancer Cell Migration in the Tumor Microenvironment.

Cancer progression and migration in the tumor microenvironment are related to cell types and three-dimensional (3D) matrices. Therefore, developing biomimetic tumor models, including co-culture systems and a tunable 3D matrix, could play an essential role in understanding the cancer environment. Here, multicellular spheroids using human adipose-derived mesenchymal stem cells (hADSCs) and breast cancer cells (MDA-MB-231) within the 3D matrix were used as a tumor microenvironment (TME) mimicking platform. The amphiphilic peptide block copolymer and hyaluronic acid (HA) formed a self-assembled structure, which provides a biocompatible 3D environment for the cells. Multicellular spheroids were formed on the optimized plate and were observed as cell migration from a spheroid within a 3D matrix, such as the invasive and metastatic cancer of TME. This study suggests a new 3D platform using polymer complexes and the importance of tumor complexities, including various cell types and microenvironments.

Self-Assembly-Assisted Kinetically Controlled Papain-Catalyzed Formation of mPEG-b-Phe(Leu)x.

Self-assembling peptide materials are promising next-generation materials with applications that include tissue engineering scaffolds, drug delivery, bionanomedicine, and enviro-responsive materials. Despite these advances, synthetic methods to form peptides and peptide-polymer conjugates still largely rely on solid-phase peptide synthesis (SPPS) and N-carboxyanhydride ring-opening polymerization (NCA-ROP), while green methods remain largely undeveloped. This work demonstrates a protease-catalyzed peptide synthesis (PCPS) capable of directly grafting leucine ethyl ester (Leu-OEt) from the C-terminus of a methoxy poly(ethylene glycol)-phenylalanine ethyl ester macroinitiator in a one-pot, aqueous reaction. By using the natural tendency of the growing hydrophobic peptide segment to self-assemble, a large narrowing of the (Leu)x distributions for both mPEG45-b-Phe(Leu)x and oligo(Leu)x coproducts, relative to oligo(Leu)x synthesized in the absence of a macroinitiator (mPEG45-Phe-OEt), was achieved. A mechanism is described where in situ β-sheet coassembly of mPEG45-b-Phe(Leu)x and oligo(Leu)x coproducts during polymerization prevents peptide hydrolysis, providing a means to control the degree of polymerization (DP) and dispersity of diblock (Leu)x segments (matrix-assisted laser desorption time-of-flight (MALDI-TOF) x = 5.1, dispersity ≤ 1.02). The use of self-assembly to control the uniformity of peptides synthesized by PCPS paves the way for precise peptide block copolymer architectures with various polymer backbones and amino acid compositions synthesized by a green process.

Strategies from nature: polycaprolactone-based mimetic antimicrobial peptide block copolymers with low cytotoxicity and excellent antibacterial efficiency

PCL16-b-Kn diblock copolymers exhibit excellent antibacterial activities and low cytotoxicity. Meanwhile, they act by the pore-forming bactericidal mechanism without inducement of drug resistance.

Thermoresponsive Elastin-b-Collagen-Like Peptide Bioconjugate Nanovesicles for Targeted Drug Delivery to Collagen-Containing Matrices.

Over the past few decades, (poly)peptide block copolymers have been widely employed in generating well-defined nanostructures as vehicles for targeted drug delivery applications. We previously reported the assembly of thermoresponsive nanoscale vesicles from an elastin-b-collagen-like peptide (ELP-CLP). The vesicles were observed to dissociate at elevated temperatures, despite the LCST-like behavior of the tethered ELP domain, which is suggested to be triggered by the unfolding of the CLP domain. Here, the potential of using the vesicles as drug delivery vehicles for targeting collagen-containing matrices is evaluated. The sustained release of an encapsulated model drug was achieved over a period of 3 weeks, following which complete release could be triggered via heating. The ELP-CLP vesicles show strong retention on a collagen substrate, presumably through collagen triple helix interactions. Cell viability and proliferation studies using fibroblasts and chondrocytes suggest that the vesicles are highly cytocompatible. Additionally, essentially no activation of a macrophage-like cell line is observed, suggesting that the vesicles do not initiate an inflammatory response. Endowed with thermally controlled delivery, the ability to bind collagen, and excellent cytocompatibility, these ELP-CLP nanovesicles are suggested to have significant potential in the controlled delivery of drugs to collagen-containing matrices and tissues.

Novel Self-Assembling Amino Acid-Derived Block Copolymer with Changeable Polymer Backbone Structure.

Block copolymers have attracted much attention as potentially interesting building blocks for the development of novel nanostructured materials in recent years. Herein, we report a new type of self-assembling block copolymer with changeable polymer backbone structure, poly(Fmoc-Ser)ester-b-PSt, which was synthesized by combining the polycondensation of 9-fluorenylmethoxycarbonyl-serine (Fmoc-Ser) with the reversible addition-fragmentation chain transfer (RAFT) polymerization of styrene (St). This block copolymer showed the direct conversion of the backbone structure from polyester to polypeptide through a multi O,N-acyl migration triggered by base-induced deprotection of Fmoc groups in organic solvent. Such polymer-to-polymer conversion was found to occur quantitatively without decrease in degree of polymerization and to cause a drastic change in self-assembling property of the block copolymer. On the basis of several morphological analyses using FTIR spectroscopy, atomic force, and transmission and scanning electron microscopies, the resulting peptide block copolymer was found to self-assemble into a vesicle-like hollow nanosphere with relatively uniform diameter of ca. 300 nm in toluene. In this case, the peptide block generated from polyester formed β-sheet structure, indicating the self-assembly via peptide-guided route. We believe the findings presented in this study offer a new concept for the development of self-assembling block copolymer system.

Co-assembly of cyclic peptide nanotubes and block copolymers in thin films: controlling the kinetic pathway.

Directed co-assembly of polymer-conjugated cyclic peptide nanotubes (CPNs) and block copolymers in thin films is a viable approach to fabricate sub-nanometer porous membranes without synthesizing nanotubes with identical length and vertical alignment. Here we show that the process is pathway dependent and successful co-assembly requires eliminating CPNs larger than 100 nm in solution. Optimizing polymer-solvent interactions can improve conjugate dispersion to a certain extent, but this limits thin film fabrication. Introduction of a trace amount of hydrogen-bond blockers, such as trifluoroacetic acid by vapor absorption, is more effective to reduce CPN aggregation in solution and circumvents issues of solvent immiscibility. This study provides critical insights into guided assemblies within nanoscopic frameworks toward sub-nanometer porous membranes.

Complexation‐Induced Biomimetic Long Range Fibrous Orientation in a Rigid‐Flexible Block Copolymer Thermogel

AbstractThe orientation of the organofibrous structure plays an important role not only in the biomineralization process but also in the extracellular matrix (ECM) of articular cartilage by providing cells with biomechanical cues. Here, it is reported that a long range nanofibrous orientation can be realized by a self‐assembling ionic complex between (+)‐charged amphiphilic peptide block copolymers with a rigid‐flexible block structure (polyalanine‐PLX‐polyalanine; PA‐PLX‐PA) and (‐)‐charged hyaluronic acid (HA). A biomimetic 3D culture system encapsulating chondrocytes is formed by a temperature‐sensitive sol‐to‐gel transition of the PA‐PLX‐PA/HA complex aqueous solution, which provides a compatible microenvironment for the cells. The cell proliferation and biomarker expression for articular cartilage are significantly improved in the PA‐PLX‐PA/HA complex system relative to the PA‐PLX‐PA or the commercially available Matrigel systems. In particular, noticeable cell clustering is observed in the PA‐PLX‐PA/HA complex system with the long range nanofibrous structure. This research suggests a new method for developing a nanofibrous structure using an amphiphilic peptide block copolymer and demonstrates its potential uses as a unique biomimetic cell‐culture matrix.

Peptide block copolymers by N -carboxyanhydride ring-opening polymerization and atom transfer radical polymerization: The effect of amide macroinitiators

The synthesis of polypeptide-containing block copolymers combining N-carboxyanhydride (NCA) ring-opening polymerization and atom transfer radical polymerization (ATRP) was investigated. An amide initiator comprising an amine function for the NCA polymerization and an activated bromide for ATRP was used. Well-defined polypeptide macroinitiators were obtained from γ-benzyl-L-glutamate NCA, O-benzyl-serine NCA, and N-benzyloxy-L-lysine. Subsequent ATRP macroinitiation from the polypeptides resulted in higher than expected molecular weights. Analysis of the reaction products and model reactions confirmed that this is due to the high frequency of termination reactions by disproportionation in the initial phase of the ATRP, which is inherent in the amide initiator structure. In some cases selective precipitation could be applied to remove unreacted macroinitiator to yield well-defined block copolymers. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2009

Orientational ordering in the nematic phase of a polyethylene glycol–peptide conjugate in aqueous solution

The orientational ordering of the nematic phase of a polyethylene glycol (PEG)-peptide block copolymer in aqueous solution is probed by small-angle neutron scattering (SANS), with the sample subjected to steady shear in a Couette cell. The PEG-peptide conjugate forms fibrils that behave as semiflexible rodlike chains. The orientational order parameters P2 and P4 are obtained by modeling the data using a series expansion approach to the form factor of uniform cylinders. The method used is independent of assumptions on the form of the singlet orientational distribution function. Good agreement with the anisotropic two-dimensional SANS patterns is obtained. The results show shear alignment starting at very low shear rates, and the orientational order parameters reach a plateau at higher shear rates with a pseudologarithmic dependence on shear rate. The most probable distribution functions correspond to fibrils parallel to the flow direction under shear, but a sample at rest shows a bimodal distribution with some of the rodlike peptide fibrils oriented perpendicular to the flow direction.

Self‐assembly of star‐shaped polystyrene‐block‐polypeptide copolymers synthesized by the combination of atom transfer radical polymerization and ring‐opening living polymerization of α‐amino acid‐N‐carboxyanhydrides

AbstractThe self‐assembling nature and phase‐transition behavior of a novel class of triarm, star‐shaped polymer–peptide block copolymers synthesized by the combination of atom transfer radical polymerization and living ring‐opening polymerization of α‐amino acid‐N‐carboxyanhydride are demonstrated. The two‐step synthesis strategy adopted here allows incorporating polypeptides into the usual synthetic polymers via an amido–amidate nickelacycle intermediate, which is used as the macroinitiator for the growth of poly(γ‐benzyl‐L‐glutamate). The characterization data are reported from analyses using gel permeation chromatography and infrared, 1H NMR, and 13C NMR spectroscopy. This synthetic scheme grants a facile way to prepare a wide range of polymer–peptide architectures with perfect microstructure control, preventing the formation of homopolypeptide contaminants. Studies regarding the supramolecular organization and phase‐transition behavior of this class of polymer‐block‐polypeptide copolymers have been accomplished with X‐ray diffraction, infrared spectroscopy, and thermal analyses. The conformational change of the peptide segment in the block copolymer has been investigated with variable‐temperature infrared spectroscopy. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 2774–2783, 2006

Double hydrophilic block copolymers with switchable secondary structure as additives for crystallization control

The synthesis and folding behavior of two different series of double hydrophilic block copolymers based on polyethyleneglycol and short acidic polypeptide blocks are described. The employment of those species in the crystallization of CaCO 3 and BaSO 4 shows that in contrast to the expectations from biomineral examples, a random conformation of the peptide is more advantageous in mineralization control than an α-helical arrangement of the same chain, which is contradictory to the epitaxial adsorption model in biomineralization. Otherwise, the peptide block copolymers support formation of superstructures similar to those already reported, e.g. well-defined spherical, prolate, and dumbbell morphologies are found.

Application of solid phase peptide synthesis to engineering PEO–peptide block copolymers for drug delivery

This work describes a simple, versatile solid-phase peptide-synthesis (SPPS) method for preparing micelle-forming poly(ethylene oxide)- block-peptide block copolymers for drug delivery. To demonstrate its utility, this SPPS method was used to construct two series of micelle-forming block copolymers (one of constant core-composition and variable length; the other of constant core length and variable composition). The block copolymers were then used to study in detail the effect of size and composition on micellization. The various block copolymers were prepared by a combination of SPPS for the peptide block, followed by solution–phase conjugation of the peptide block with a proprionic acid derivative of poly(ethylene oxide) (PEO) to form the PEO- b-peptide block copolymer. The composition of each block component was characterized by mass spectrometry (MALDI and ES-MS). Block copolymer compositions were characterized by 1H NMR. All the block copolymers were found to form micelles as judged by transmission electron microscopy (TEM) and light scattering analysis. To demonstrate their potential as drug delivery systems, micelles prepared from one member of the PEO- b-peptide block copolymer series were physically loaded with the anticancer drug doxorubicin (DOX). Micelle static and dynamic stability were found to correlate strongly with micelle core length. In contrast, these same micellization properties appear to be a complex function of core composition, and no clear trends could be identified from among the set of compositionally varying, fixed length block copolymer micelles. We conclude that SPPS can be used to construct biocompatible block copolymers with well-defined core lengths and compositions, which in turn can be used to study and to tailor the behavior of block copolymer micelles.

Facile synthesis of block copolypeptides of defined architecture.

Many natural polymeric materials (particularly structural proteins) display a hierarchy of structure over several length scales. Block copolymers are able to self-assemble into ordered nanostructures, but the random-coiled nature of their polymer chains usually suppresses any further levels of organization. The use of components with regular structures, such as rigid-rod polymers, can increase the extent of spatial organization in self-assembling materials. But the synthesis of such polymeric components typically involves complicated reaction steps that are not suitable for large-scale production. Proteins form hierarchically organized structures in which the fundamental motifs are generally alpha-helical coils and beta-sheets. Attempts to synthesize polypeptides with well-defined amino-acid sequences, which might adopt similar organized structures, have been plagued by unwanted side reactions that give rise to products with a wide range of molecular weights, hampering the formation of well-defined peptide block copolymers. Here I describe a polymerization strategy that overcomes these difficulties by using organonickel initiators which suppress chain-transfer and termination side reactions. This approach allows the facile synthesis of block copolypeptides with well-defined sequences, which might provide new peptide-based biomaterials with potential applications in tissue engineering, drug delivery and biomimetic composite formation.

Polypeptide Materials: New synthetic methods and applications

AbstractA review of recent developments in polypeptide synthesis is provided. This article is focused on development of new materials based on polypeptides rather than on reproduction or applications of natural protein‐based materials. Synthetic methods fall into two major categories: biological and chemical. The most successful biological approaches utilize cellular protein synthesis machinery to perform the task of assembling the polymeric molecules. Adaptation of this machinery for production of new, artificial polypeptide sequences and use of this machinery for incorporation of artificial amino acids into polypeptides have been the key recent contributions in this area. Advances in chemical polypeptide syntheses include new applications in solid and solution phase peptide coupling reactions as well as advances in the polymerization of a‐amino acid‐N‐carboxyan‐ hydride (NCA) monomers. The use of transition metal initiators in NCA polymerizations has allowed the preparation of very well defined homopolypeptides and may lead to facile routes into peptide block copolymer materials. The presence of stable, chiral structural elements in polypeptides results in self‐assembly into ordered films, composites, and liquid crystals. If their sequences are designed and constructed properly, artificial polypeptides have considerable potential for use in construction of artificial tissues and implants, ordered inorganic/organic composites, and in medical diagnostics and biosensors.