Star-Shaped Peptide-Polymer Hybrids as Fast pH-Responsive Supramolecular Hydrogels.

Significant challenges have gone into the design of smart hydrogels, with numerous potential applications in the industrial, cosmetic, and biomedical fields. Herein, we report the synthesis of novel 4-arm self-assembling peptide-polyethylene glycol (PEG) hybrid star-shaped polymers and their comprehensive hydrogel properties. ß-sheet-forming oligopeptides with alternating hydrophobic Leu/ionizable Glu repeats and Cys residues were successfully conjugated to 4-arm PEG via a thiol-maleimide click reaction. The hybrid star-shaped polymers demonstrated good cytocompatibility and reversible ß-sheet (lightly acidic pH)-to-random coil (neutral and basic pH) transition in dilute aqueous solutions. At increasing polymer concentrations up to 0.5 wt %, the star-shaped polymers formed transparent hydrogels with shear-thinning and self-healing behaviors via ß-sheet self-assembly, as well as a conformation-dependent gel-sol transition. Interestingly, the star-shaped polymers responded rapidly to pH changes, causing gelation to occur rapidly within a few seconds from the change in pH. Hydrogel characteristics could be modulated by manipulating the length and net charge of the peptide blocks. Furthermore, these star-shaped polymers served as satisfactory network scaffolds that could respond to dynamic environmental changes in the pH-oscillation system, owing to their excellent gelation capability and pH sensitivity. As such, they are highly favorable for diverse applications, such as pH-responsive controlled release.

Narcissistic Self-Sorting of Amphiphilic Collagen-Inspired Peptides in Supramolecular Vesicular Assembly.

Herein, we describe the hierarchical self-assembly accompanying self-sorting of collagen-inspired peptides (CPs). The two amphiphilic CPs used in this study contained an azobenzene (Az) moiety at the N-terminal, connected through a flexible spacer, but with different lengths of the (Gly-Pro-Hyp)n triplet (n = 5 and 7). When the CP aqueous solution (60 °C) was cooled to 4 °C, both CPs formed a triple helix structure and the pre-organized helices subsequently self-assembled into highly ordered vesicles with a diameter of 50-200 nm. Interestingly, narcissistic self-sorting was observed in both triple helix- and matured vesicle-formation processes, when the two CPs were mixed. Owing to the difference in the propensity for triple helix formation with temperature, the two CPs discriminate each other in response to a temperature change and form two kinds of triple helix foldamers, each containing a single component. The resulting differences in the amphiphilic balance and molecular length between the foldamers appear to allow individual self-sorting to form distinct vesicles. Furthermore, such vesicular assemblies were found to disassemble upon UV irradiation via trans-cis isomerization of the Az-groups. These findings offer important insights into the design of new complex but ordered, peptide self-assembly systems with potential applications in nanobiotechnology.

Supramolecular Nanofibers from Collagen-Mimetic Peptides Bearing Various Aromatic Groups at N-Termini via Hierarchical Self-Assembly.

Self-assembly of artificial peptides has been widely studied for constructing nanostructured materials, with numerous potential applications in the nanobiotechnology field. Herein, we report the synthesis and hierarchical self-assembly of collagen-mimetic peptides (CMPs) bearing various aromatic groups at the N-termini, including 2-naphthyl, 1-naphtyl, anthracenyl, and pyrenyl groups, into nanofibers. The CMPs (R-(GPO)n: n > 4) formed a triple helix structure in water at 4 °C, as confirmed via CD analyses, and their conformations were more stable with increasing hydrophobicity of the terminal aromatic group and peptide chain length. The resulting pre-organized triple helical CMPs showed diverse self-assembly into highly ordered nanofibers, reflecting their slight differences in hydrophobic/hydrophilic balance and configuration of aromatic templates. TEM analysis demonstrated that 2Np-CMPn (n = 6 and 7) and Py-CMP6 provided well-developed natural collagen-like nanofibers and An-CMPn (n = 5-7) self-assembled into rod-like micelle fibers. On the other hand, 2Np-CMP5 and 1Np-CMP6 were unable to form nanofibers under the same conditions. Furthermore, the Py-CMP6 nanofiber was found to encapsulate a guest hydrophobic molecule, Nile red, and exhibited unique emission behavior based on the specific nanostructure. In addition to the ability of CMPs to bind small molecules, their controlled self-assembly enables their versatile utilization in drug delivery and wavelength-conversion nanomaterials.

Unique Self-Assembly of Sequence-Controlled Amino Acid Derived Vinyl Polymer with Gradient Thermoresponsiveness along a Chain.

A novel water-soluble amino acid derived vinyl polymer whose block sequence was designed to achieve a gradient thermoresponsiveness along a chain was accurately prepared through an ultrarapid reversible addition-fragmentation chain-transfer polymerization. The polymer exhibited unique temperature-regulated self-assembly in water, leading to multiple nanostructural transformations including disassembly-to-ordered and ordered-to-ordered transitions. The morphologies were drastically changed by heating the solution from 4 °C (soluble form) to 20 °C (spherical micelle) to 70 °C (vesicle). Moreover, such transitions exhibited hysteresis upon cooling, namely, from 70 °C (vesicle) to 20 °C (wormlike micelle) to 4 °C (soluble form). In this polymer system, the specific monomer sequence contributed to the self-assembly behavior. These findings provide significant insight into the design of new thermoresponsive nanomaterials with potential applications in biomedical chemistry.

Transparent, High-Strength, and Shape Memory Hydrogels from Thermo-Responsive Amino Acid-Derived Vinyl Polymer Networks.

Herein, the formation of unique shape-memory hydrogels that are composed of thermo-responsive amino-acid-derived vinyl polymer networks is reported; these are readily prepared by radical copolymerization of N-acryloyl glycinamide with commercially available cross-linkers, namely, methylenebis(acrylamide) and poly(ethylene glycol) diacrylate. These hydrogels are transparent (>90% transmittance at 600 nm) and are comprised of 97-70 wt% water. Furthermore, these contain both chemical and physical cross-linkages that are based on the multiple hydrogen bonds attained via amino acid units; this composition is aimed at generating opposing stimuli-responsive characters, namely, chemically stable and thermo-sensitive properties. A cooperative interplay of these two networks enables the hydrogels to exhibit a decent mechanical toughness (breaking strength ≈0.3 MPa and breaking elongation >600%) and a shape fix/memory capability. The temporary shape is easily fixed by cooling at 4 °C after deformation at high temperature, and it instantly recovers its original shape through reheating. Furthermore, a multi-shape memory effect is achieved by incorporating the pH-responsive N-acryloyl alanine unit into the hydrogel system as a comonomer; in this system, three distinct shapes can be fixed through temperature and pH manipulations. This facilely attainable shape memory hydrogel has significant potential in various fields, such as soft actuators, sensors, and biomedical materials.

Precisely Synthesized Sequence-Controlled Amino Acid-Derived Vinyl Polymers: New Insights into Thermo-Responsive Polymer Design.

Thermo-responsive block copolymers are of great interest in biomedical and nanotechnological fields. These polymers achieve a versatile and complex responsiveness through a sophisticated and intricate combination of different thermo-responsive blocks. While their utility is clear, the fundamental design principles of such vinyl polymers are not yet thoroughly understood. Herein, a precise synthesis of sequence-controlled amino-acid-derived vinyl polymers and their unique thermal response in water are reported. Seven distinct block (random) copolymers that contain two kinds of amino acid blocks (poly(N-acryloyl alanine(A)- or glycine(G)-methyl ester)) with the same total chain length (degree of polymerization [DP] ≈30) and chemical composition (A/G ≈1), but with systematic variations in the block sequence and length, with an accuracy target of DP ± 1, are prepared. By specifying the primary structure, the thermal responses including transition temperature, thermo-sensitivity, and microenvironment in the dehydrated state can be finely tuned. These findings offer new directions in the design of structurally and functionally diverse thermo-responsive vinyl polymers.

Photo-responsive azobenzene interactions promote hierarchical self-assembly of collagen triple-helical peptides to various higher-order structures.

Collagen is an essential structural protein in animal tissues and plays key roles in cellular modulation. We investigated methods to discover collagen model peptides (CMPs) that would self-assemble into triple helices and then grow into supramolecular organizations with diverse morphological features, which would be valuable as biomaterials. This challenging undertaking was achieved by placing azobenzene groups on the ends of the CMPs, (GPO) n (n = 3-10), Azo-(GPO) n . In a dilute aqueous solution (80 µM), CD spectra indicated that the Azo-(GPO) n (n > 4) formed triple helices due to strong hydrophobic azobenzene interactions, and that helix stability was increased with the peptide segment length. The resulting triple helices induced a specific azobenzene orientation through turned and twisted configurations as shown by CD spectra. TEM observations for the same solutions disclosed the morphologies for the Azo-CMPs. Azo-(GPO)3, having the shortest peptide segment, showed no nanostructure, both Azo-(GPO)4 and Azo-(GPO)5 provided consistent well-developed nanofiber structures resembling the natural collagen fibers, and Azo-(GPO) n s (n = 6-10) grew into flexible rod-like micelle fibers. In addition, alkyl chain-attached C m Azo-(GPO)5 displayed a toroidal morphology, and Azp-deg-(GPO)5 having a hydrophilic spacer assembled into a bilayer vesicle structure. These diverse morphological features are considered to be due to the characteristics of the pre-organized triple helix units. Photo-isomerization of the azobenzene moiety brought about the disappearance of such characteristic nano-architectures. When the solution concentration was increased up to 1 wt%, only Azo-(GPO)4 and Azo-(GPO)5 spontaneously formed hydrogels exhibiting a satisfactory gel-to-sol transition upon UV irradiation.

Spontaneous Formation of Nanoparticles from Peptide-Vinyl Polymer Diblock Hybrids Prepared by RAFT Polymerization and Their Interactions with Cells.

Novel polymeric nanoparticles (NPs) with uniform sizes were prepared from peptide-vinyl polymer diblock hybrids by the self-organized precipitation method. Hybrid polymers of polystyrene (PSt) and tetrapeptide (cell-binding epitope RGDS, reverse SDGR, cationic KKKK, and anionic DDDD) were successfully synthesized by combining solid-phase peptide synthesis and reversible addition fragmentation chain transfer polymerization methods. Narrowly dispersed hybrid polymers (polydispersity index < 1.25, M n 14 000-17 000) were obtained. Altering the preparation conditions easily tuned the size and size distribution of the NPs. When the ζ-potentials for the NP suspensions were measured at pH 6.0, the obtained values corresponded to the net charge of each peptide segment. More importantly, the NPs could encapsulate fluorescent Nile red (NR) and magnetic iron oxide NP (MNP), which might be suitable for fluorescent imaging and magnet-induced patterning of cells, respectively. The interactions of NPs with cells (NIH/3T3 fibroblast) and the magnetic effects were examined for NR/MNP-loaded PSt-RGDS and -SDGR NPs. Both NPs were readily incorporated into cells, but only NR/MNP-loaded PSt-RGDS NP showed magnetic responsiveness in cell adhesion and cultures.

Photocleavable Peptide-Poly(2-hydroxyethyl methacrylate) Hybrid Graft Copolymer via Postpolymerization Modification by Click Chemistry To Modulate the Cell Affinities of 2D and 3D Materials.

Controlling the surface properties of engineered materials to enhance or reduce their cellular affinities remains a significant challenge in the field of biomaterials. We describe a universal technique for modulating the cytocompatibilities of two-dimensional (2D) and three-dimensional (3D) materials using a novel photocleavable peptide-grafted poly(2-hydroxyethyl methacrylate) (PHEMA) hybrid. The reversible addition-fragmentation chain transfer copolymerization of HEMA and propargyl acrylate was successfully controlled. The resultant alkyne-containing PHEMA was then used to modify the azide-terminated oligopeptides [Arg-Gly-Asp-Ser (RGDS)] with a photolabile 3-amino-3-(2-nitrophenyl)propanoic acid moiety via the copper-catalyzed alkyne-azide click chemistry. This strategy was readily used to decorate the surfaces of both hydrophilic and hydrophobic materials with RGDS peptides due to the high film-forming abilities of the PHEMA unit. The resultant thin film acted as an effective scaffold for improving cell adhesion and growth of NIH/3T3 fibroblasts and MC3T3-E1 osteoblast-like cells in vitro. In addition, UV irradiation of the surface led to the detachment of cells from the material surface accompanied by the photocleavage of RGDS grafts and enabled the 2D-patterning of cells and cell sheet engineering. The applicability of this system to 3D materials was investigated, and the cell adhesion was remarkably enhanced on a 3D-printed poly(lactic acid) object. This facile, biocompatible, and photoprocessable peptide-vinyl polymer hybrid system is valuable for its ability to advance the fields of tissue engineering, cell chips, and regenerative medicine.

A novel thermo-responsive multiblock architecture composed of a sequential peptide and an amino acid-derived vinyl polymer: toward protein-mimicking single-chain folding.

A novel multiblock architecture composed of an alternating ß-sheet forming oligopeptide and a thermo-responsive glycine-derived vinyl polymer was synthesized. The polymer exhibited lower critical solution temperature (LCST) behavior in water, unlike the behavior of a glycine-derived homopolymer, and formed nanoparticles through protein-mimicking folding driven by thermal cycle-induced ß-sheet formation.

Stepwise Thermo-Responsive Amino Acid-Derived Triblock Vinyl Polymers: ATRP Synthesis of Polymers, Aggregation, and Gelation Properties via Flower-Like Micelle Formation.

Novel thermo-responsive ABA-type triblock copolymers (poly(NAAMen-b-NAGMe240-b-NAAMen), n = 18-72) composed of naturally occurring amino acid-based vinyl polymer blocks such as poly(N-acryloyl-l-alanine methyl ester (poly(NAAMe)) as the A segment and poly(N-acryloyl-glycine methylester)(poly(NAGMe)) as the B segment have been synthesized by the atom transfer radical polymerization (ATRP). Their thermal behaviors were analyzed in dilute aqueous solutions by turbidimetry. The turbidity curves provided two-step LCST transitions, and a flower-like micelle formation was confirmed at the temperature region between the first and second LCST transitions by dynamic light scattering, AFM and TEM. At higher copolymer concentrations, hydrogels were obtained at temperatures above the first LCST due to network formation induced with the flower-like micelles as cross-linker. The hydrogels were found to be switched to a sol state when cooled below the first LCST. These hydrogels also exhibited self-healable and injectable capabilities, which were evaluated by rheological measurements.

Thermo-responsive polymer brushes on glass plate prepared from a new class of amino acid-derived vinyl monomers and their applications in cell-sheet engineering.

In this study, we present a novel thermo-responsive polymer platform that is based on the alanine methyl ester-containing homopolymer (PNAAMe) and the copolymer with glycine methyl ester-based vinyl monomer (P(NAAMe-co-NAGMe)) brushes prepared via surface-initiated atom transfer radical polymerization. Water contact angles for these brushes measured at different temperatures reveal that the polymer brushes collapse and dehydrate around 13°C and 25°C (TTs), respectively, upon elevating the temperature. At 37°C, seeded fibroblasts (NIH/3T3) adhere to and spread well onto these brush surfaces although the copolymer brush of P(NAAMe-co-NAGMe) depresses the number of adherent cells less than half of that for the homopolymer of PNAAMe after 24h of cell culture due to increment in hydrophilicity. To prepare the cell-sheet, the cells are seeded on both polymer brushes and cultured at 37°C in the presence of serum. After 4days, the cells proliferated confluently on these brush surfaces. Lowering the temperature to 4°C and 20°C below TT of each brush led to the cell-sheet detachment as a monolayer form from the polymer brushes accompanying with the switching of surface affinity.

Facile Synthesis of Multiblock Copolymers Containing Sequence-Controlled Peptides and Well-Defined Vinyl Polymers by Nitroxide-Mediated Polymerization.

Precisely incorporating a wide range of structural and functional multiblocks along a polymer backbone is a significant challenge in polymer chemistry and offers promising opportunities to design highly ordered materials, including controlled polymer folding. Herein, a facile and versatile strategy for preparing functional multiblock copolymers composed of sequential peptides and well-defined vinyl polymers with a narrow polydispersity is reported. Cyclic oligopeptides have been developed that contain an alkoxyamine bond in the framework. By using this type of cyclic initiator, peptide-containing multiblock copolymers are successfully synthesized by nitroxide-mediated polymerization of styrene. To demonstrate the versatility of this method, radical (co)polymerizations were carried out for different monomers (p-chlorostyrene, 4-vinylpyridine, and styrene/acrylonitrile) and by three different cyclic peptide initiators with specific amino acid sequences. The resultant multiblock copolymer is foldable through intramolecular interactions between peptide blocks. It is believed that this approach will significantly advance the field of controlled polymer synthesis for complex structures and single-chain folding.

Temperature induced self-assembly of amino acid-derived vinyl block copolymers via dual phase transitions.

The unique thermoresponsive phase behaviors of diblock copolymers from amino acid-derived vinyl monomers have been demonstrated in view of variation in the aggregation state in water. Amino acid-based block copolymers composed of N-acryloyl-Ala-methylester (NAAMe) and N-acryloyl-ßAla-methylester (NAßAMe) are successfully synthesized by RAFT polymerization. The resultant block copolymers poly(NAAMe48-b-NAßAMem) contain a constant degree of polymerization (DP=48) of the poly(NAAMe) block, but the DP of the poly(NAßAMe) block varies (m=80-122). The turbidimetry subjected to these copolymer aqueous solutions exhibits two LCST transitions upon heating. In the 1st LCST region, the block copolymer forms a relatively loose-molecular packing, while large aggregates due to partial dehydration of polymer molecules, which subsequently transform into a stable micelle structure in a region of 30-39°C. Finally, a tight aggregate composed of the dehydrated micelles is formed. Temperature-dependent 1H NMR spectroscopy of the diblock copolymers also supports such a postulation for the dual phase transitions and stable micelle structure formation. In addition, a typical salting-out effect is observed in the thermal behavior of the polymer, but a serious cytotoxic effect is not observed in NIH/3T3 cells, suggesting that the novel diblock copolymers are relevant for biomedical applications.

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 ß-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.

Preparation and self-assembly behavior of ß-sheet peptide-inserted amphiphilic block copolymer as a useful polymeric surfactant.

A hybridization of structurally regulated biopolymers and conventional synthetic polymers offers promising opportunities to design novel polymeric nanomaterials. In this study, we newly prepared an amphiphilic triblock copolymer with ß-sheet forming peptide as a central block, polystyrene-block-tetra(leucine)-block-poly(ethylene glycol) (PS-L4-PEG), by combining the solid phase peptide synthesis with the atom transfer radical polymerization (ATRP). On the basis of several morphological and structural analyses using atomic force microscopy, transmission electron microscopy, FTIR spectroscopy, and contact angle measurement, the PS-L4-PEG was found to form PEG-shell spherical and/or elliptical vesicles with a diameter of 30-100 nm in aqueous medium. By contrast, the PS-L4-PEG self-assembled into PS-shell spherical aggregates in toluene, which is good solvent for both PEG and PS. In both cases, the central peptide formed a ß-sheet network in the nanoassemblies. Furthermore, the PS-L4-PEG was found to stabilize a water-in-oil emulsion remarkably in comparison with the PS-PEG diblock copolymer, demonstrating the potential of this peptide-polymer hybrid as a useful polymeric surfactant.

Novel peptide-shelled dendrimer with dramatically changeable thermo-responsive character.

The preparation of a novel peptide/dendrimer hybrid is reported in which an elastin-like oligopeptide is successfully assembled onto a poly(amidoamine) dendrimer surface (G4-ELP), and its unique thermo-responsive behavior is discussed. As a result, the G4-ELP is found to exhibit LCST behavior in the pH range 3-10 including physiological temperature range under neutral-pH conditions. Moreover, cooperative interplay between the folding state of the ELP shell and the ionization state of the dendrimer core enables the G4-ELP to control its LCST widely by pH variation. This achievement provides a new insight for the design of dual-responsive materials with a potential in biological applications.

Shape-specific nanofibers via self-assembly of three-branched peptide.

A novel amphiphilic branched peptide (1), in which three ß-sheet formable peptides (L(4)K(8)L(4)) were connected by Lys residue, was newly prepared as a building block for self-assembly. A detailed analysis of the conformation and self-assembling property of 1 in water at various pH conditions was performed by using circular dichroism, FTIR, atomic force and transmission electron microscopies. The experimental results revealed that the branched peptide showed a pH-dependent conformation forming a shape-specific ß-sheet-based nanofiber with morphologically kinked structures under specific pH conditions. Exploring a novel peptide building unit that has the ability to self-assemble into designed and complicated nano-objects is valuable to facilitate a bottom-up nanotechnology.

Fabrication of nucleobase-functionalized supramolecular nanofiber through peptide self-assembly.

In this study, we describe a novel molecular self-assembling system of a nucleobase-functionalized oligopeptide. An adenine-terminated amphiphilic triblock peptide (1), which consists of hydrophobic Leu and hydrophilic Lys, was newly synthesized as a building block for self-assembly. The conformational and self-assembling properties of 1 in water at various conditions were examined by means of circular dichroism, FTIR, UV, atomic force microscopy, and transmission electron microscopy measurements. The experimental results revealed that the peptide 1 self-assembled into beta-sheet nanofiber under certain conditions, in which the nucleobase moieties were stacked with each other. The nanofiber possessed a diameter of ca. 6 nm and clearly visible left-handed twist with a periodicity of ca. 50 nm. Such secondary and quaternary structures of 1 could be easily controlled by manipulating the solution pH. Furthermore, the addition of complemental nucleoside, thymidine, was found to obviously prevent and/or retard the nanofiber formation of 1.