Combinations of synthetic polymers and natural proteins provide a route to the synthesis of new biomaterials. The bioconjugates combining tunable properties of polymers with functionalities of proteins have found broad applications. One of the most challenging problems in this research field is the self-assembly behaviors of responsive polymer–protein bioconjugates. In this research, synthesis and self-assembly of bioconjugates composed of zwitterionic block copolymer and streptavidin were investigated. Block copolymers of poly(ethylene glycol) (PEG) and poly[3-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate] (PDMAPS) with cleavable biotin groups at the junction points were synthesized. The zwitterionic block copolymers exhibit phase transitions with upper critical solution temperatures (UCSTs) in aqueous solutions. The concentration of sodium chloride exerts a significant influence on the UCST. The zwitterionic block copolymer chains self-assemble into vesicles in aqueous solution at a temperature below UCST. Bioconjugates comprising of streptavidin molecules and zwitterionic block copolymer chains were fabricated based on biotin–streptavidin coupling. Upon conjugation to the protein molecules, the UCST of the zwitterionic block copolymer decreases due to the screening effect of the protein molecules. The bioconjugates are able to make self-assembly into different structures, depending on the average number of block copolymer chains on a protein molecule. The bioconjugate molecules with average 1.3 block copolymer chains on a streptavidin self-assemble into rodlike structures, while those with average 2.9 chains on a streptavidin self-assemble into spherical micelles.

Recently, the synthesis of bioconjugates combining tunable properties of polymers with the functionalities of proteins has aroused great interest in the biomaterials community. In this research, thermally-responsive polymer chains with pendant lysozyme molecules were prepared via a “grafting to” approach. The enzyme molecules were anchored to the polymer backbones through covalent bonds. The influences of the number of pendant enzyme molecules and the salt concentration on the thermal-responsive properties of the bioconjugates were investigated. With an increase in the number of pendant lysozyme molecules, the cloud point (TCP) shifts to a higher temperature. The TCPs of the bioconjugates decrease with an increase in the salt concentration. In aqueous solutions, the bioconjugate molecules make core–shell structures at a temperature below TCP due to the unfavorable interaction between the polymer chains and the enzyme molecules. The bioconjugates are able to self-assemble into mesoglobules at temperatures above TCP. The secondary structure and bioactivity of the enzyme on the assembled structures were basically maintained.

AbstractThe development in the synthesis and self-assembly of patchy nanoparticles has resulted in the creation of complex hierarchical structures. Co-assembly of polymeric nanoparticles and protein molecules combines the advantages of polymeric materials and biomolecules, and will produce new functional materials. Co-assembly of positively charged patchy micelles and negatively charged bovine serum albumin (BSA) molecules is investigated. The patchy micelles, which were synthesized using block copolymer brushes as templates, leads to co-assembly with protein molecules into vesicular structures. The average size of the assembled structures can be controlled by the molar ratio of BSA to patchy micelles. The assembled structures are dissociated in the presence of trypsin. The protein–polymer hybrid vesicles could find potential applications in medicine.

AbstractThe development in the synthesis and self-assembly of patchy nanoparticles has resulted in the creation of complex hierarchical structures. Co-assembly of polymeric nanoparticles and protein molecules combines the advantages of polymeric materials and biomolecules, and will produce new functional materials. Co-assembly of positively charged patchy micelles and negatively charged bovine serum albumin (BSA) molecules is investigated. The patchy micelles, which were synthesized using block copolymer brushes as templates, leads to co-assembly with protein molecules into vesicular structures. The average size of the assembled structures can be controlled by the molar ratio of BSA to patchy micelles. The assembled structures are dissociated in the presence of trypsin. The protein–polymer hybrid vesicles could find potential applications in medicine.

A thermoresponsive polymer-protein biodynamer was prepared via the bioconjugation of an aliphatic aldehyde-functionalized copolymer to hydrazine-modified bovine serum albumin (BSA) through reversible pyridylhydrazone linkages. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) results indicated that the pyridylhydrazone linkages cleaved in an intracellular-mimicking acidic milieu, thus leading to the release of BSA. The dynamic character of the protein biodynamer was demonstrated by exchange reactions with aldehyde-containing molecules. The biodynamer self-assembled into spherical micelles at a temperature above its lower critical solution temperature (LCST). Subsequently, BSA molecules within the hydrophilic coronae of the micelles were readily cross-linked via reaction with cystamine at 45 °C, and multi-stimuli-responsive nanoparticles were generated. The biohybrid nanoparticles reversibly swelled and shrank as the cores of the nanoparticles were solvated below the LCST and desolvated above the LCST. The accessible reversibility of the pyridylhydrazone bonds imparts pH-responsive and dynamic characteristics to the nanoparticles. The nanoparticles displayed glutathione (GSH) responsiveness, and the synergistic effects of pH and GSH resulted in complete disintegration of the nanoparticles under the intracellular-mimicking acidic and reductive conditions. The nanoparticles were also enzyme-responsive and disintegrated rapidly in the presence of protease. In vitro cytotoxicity and cell uptake assays demonstrated that the nanoparticles were highly biocompatible and effectively internalized by HepG2 cells, which make them interesting candidates as vehicles for drug delivery application and biomimetic platforms to investigate the biological process in nature.Significance StatementIn this research, we report the synthesis of a temperature and pH dual-responsive polymer-protein biodynamer through reversible pyridylhydrazone formation. The prepared biodynamer can offer a potential platform for intracellular protein delivery. The multi-stimuli-responsive biohybrid nanoparticles containing disulfide functionalities are constructed by cross-linking albumin coronae of the biodynamer micelles. With the combination of a thermoresponsive polymer, protein and reversible covalent bonds, the biohybrid nanoparticles are endowed with highly biocompatible, environmentally responsive and adaptive features. These nanoparticles present the ability to undergo changes in their constitution, hydrodynamic size and nanostructure in response to physical, chemical and biological stimuli, which make them interesting candidates as vehicles for drug delivery application and a biomimetic platform to investigate the biological process in nature.Download high-res image (108KB)Download full-size image

Proteinosomes are a type of protein-based spherical capsules, which have potential applications in drug delivery, cell imaging, gene expression, and biocatalysis. In this research, a novel approach to the fabrication of proteinosomes entirely composed of protein molecules based on self-assembly of a supramolecular protein–polymer conjugate is proposed. A supramolecular protein–polymer conjugate was prepared by mixing βCD-modified bovine serum albumin (BSA) and adamantane-terminated poly(N-isopropylamide) (Ad-PNIPAM) in aqueous solution. The BSA-PNIPAM bioconjugate self-assembled into micelles with PNIPAM cores and BSA coronae at a temperature above the lower critical solution temperature (LCST) of PNIPAM. After cross-linking of BSA in the coronae, and followed by addition of excess βCD, PNIPAM chains were cleaved from the micellar structures, and nanoscale proteinosomes were prepared. The dual-responsive proteinosomes dissociated in the presence of trypsin or glutathione.

•Enzyme-polymer nanogels are prepared by thiol-disulfide exchange reactions.•Morphology changes at different temperatures are investigated.•The hybrid nanogels show thermal responsiveness, and LCSTs are determined.•Enzyme molecules immobilized on the nanogels show enhanced heat resistance.In this paper a novel method for the fabrication of hybrid nanogels based on thiol-disulfide exchange reaction is reported. Poly(oligo(ethylene glycol) monomethyl ether methacrylate-co-di(ethylene glycol) methyl ether methacrylate-co-2-(2-pyridyldisulfide) ethyl methacrylate) (POEGMA-co-PDEGMA-co-PDSMA) was synthesized by reversible addition-fragmentation chain transfer polymerization. Pyridyl disulfide functionalized porcine pancreatic lipase (PPL-S-S-Py) was prepared by treatment of PPL with Traut’s reagent (2-iminothiolane) and 2,2′-dithiodipyridine. Upon addition of meso-2,3-dimercaptosuccinic acid into aqueous solutions of PPL-S-S-Py and POEGMA-co-PDEGMA-co-PDSMA, enzyme-polymer hybrid nanogels were prepared. The hybrid nanogels show thermal responsiveness. With an increase in the content of PPL in the nanogels, the lower critical solution temperature (LCST) shifts to the higher temperature. At a temperature below LCST, PPL molecules are in the shells of the nanogels, and at a temperature above LCST, PPL molecules are embedded inside the nanosized structures. The immobilized PPL show enhanced heat resistance and good reusability.

Thioester chemistry was applied to construct the “reduction” responsive thymine-conjugated biodynamer by a transthioesterification reaction. The copolymers of N,N-dimethylacrylamide (DMA) and pyridyldisulfide ethylacrylamide (PDSEA) were obtained by reversible addition–fragmentation chain transfer (RAFT) radical polymerization, and used as a handle for incorporating thiol-reactive groups onto the copolymers. Thymine thioester reacted with the pendent thiol group produced in situ by the cleavage of PDS. The obtained thymine-conjugated biodynamer containing reversible thioester linkages can interact with the adenine-modified molecule mediated by hydrogen bonding, and demonstrate L-glutathione (GSH)-responsive feature by a thiol–thioester exchange reaction. The biodynamer can also interact with melamine and adenosine-5′-triphosphate (ATP), and form spherical aggregates in water. It is expected that this nucleobase-containing biodynamer has potential applications in the thiol-responsive controlled drug delivery and the construction of complex nanostructures and advanced materials as building blocks.

Biodynamers display the properties of biomolecules with the adaptive nature of dynamic covalent polymers and have great potential as “smart” materials. The bioconjugation of L-tyrosine hydrazide to the end of bisaldehyde-functionalized thermoresponsive copolymer chains via reversible acylhydrazone linkage generated a biodynamer with thermo/pH-responsive and adaptive features. Because of the introduction of amino acid end groups, the biodynamer had an isoelectric point (IEP) at pH 4.70 and possessed a pH-dependent LCST. The properties of the biodynamer were tunable through chain exchange reactions with other hydrazine or hydrazide molecules. The biodynamer endowed biological recognition by exchange with biotin hydrazide. The complex formed by the biotinylated dynamer and streptavidin demonstrated pH-responsive features. Because of the versatile reactivity of the phenolic moiety, this biodynamer provided a reactive scaffold for further modification. The cyclic diazodicarboxamide was attached to the biodynamer through the tyrosine-click reaction. Protein bioconjugate with pH-responsive and adaptive features was prepared via HRP-catalyzed coupling reaction.

On the basis of an active ester monomer methacryloylacetone oxime (MAO), a well-defined hydrazide-containing block copolymer poly(poly(ethylene glycol methacrylate))-b-poly(methylacryloylhydrazide) (P(PEGMA)-b-PMAH) was synthesized by RAFT polymerization and subsequent aminolysis in the presence of hydrazine hydrate. The dynamic covalent copolymer was generated by the bioconjugation of pyridoxal phosphate (PLP) to the pendant hydrazide groups through reversible acylhydrazone linkages. An in vitro study confirmed that the block copolymer and the PLP-conjugated dynamer were nontoxic to HeLa cells. The PLP-conjugated dynamer was negatively charged at physiological pH. Polyion complex (PIC) micelles were formed through electrostatic interaction between lysozyme and the PLP-conjugated dynamer. These PIC micelles demonstrated pH-, salt-, and enzyme-responsive features. The enzymatic activity of PIC micelles toward the hydrolysis of the bacterial substrate Micrococcus luteus cells was evaluated. A reduced activity was observed after lysozyme was entrapped in the PIC micelles because of the shielding effect of the P(PEGMA) corona. However, the dissociation of micelles, triggered by the increase in ionic strength of the milieu, led to the recovery of lysozyme activity. These PIC micelles formed by the PLP-conjugated dynamer and protein have potential applications in biomedical and bioengineering areas.

Well-defined hydrazide-containing copolymers poly(poly(ethylene glycol) methacrylate-co-methacryoyl hydrazide) (P(PEG-co-MAH)) via reversible addition–fragmentation chain transfer radical polymerization were used as a reactive scaffold for bioconjugations to prepare polymers for protein recognition. The nucleophilic reaction of hydrazide and glucose generated glycoconjuagted copolymer that can recognize Con A. Biotinylated copolymer was prepared by the conjugation of aldehyde-functionalized biotin to the copolymer via hydrazone bond. Subsequently, dynamic covalent cross-linked nanoparticles were constructed via reversible acylhydrazone linkages by the reaction of copolymer and terephthaldicarboxaldehyde. The cross-linked nanoparticles demonstrated reversible pH-dependent formation/disintegration and adaptive characters. The cross-linked nanoparticles were further adorned through successive reactions of their remaining hydrazide units with aldehyde-functionalized biotin and fluorescein isothiocyanate to generate multifunctional nanoparticles. An in vitro study confirmed that the cross-linked nanoparticles were nontoxic to HeLa cells. These nanoparticles can encapsulate a cargo of small hydrophobic molecules, Nile red. The dye-loaded nanoparticles exhibited pH-triggered release behavior around the acidic tumoral environment, implying that these nanoparticles via hydrazone linkages have promise as therapeutic nanocarriers in a drug delivery system. Therefore, these dynamic covalent nanoparticles generated from hydrazide-containing copolymers can be utilized not only as building blocks for the construction of multifunctional materials with pH-responsive and adaptive characters but also as smart nanocarriers in biomedicine.

A bisaldehyde-containing trithiocarbonate chain transfer agent was prepared and mediated the synthesis of polymers with bisaldehyde-functionalized α-termini by reversible addition–fragmentation chain transfer (RAFT) radical polymerization. The α-termini of RAFT-derived thermoresponsive poly(N-isopropylacrylamide) was conjugated with hydrazides via reversible acylhydrazone bonds. Introduction of hydrophilic end-groups generated a dynamic covalent polymer with “isothermal” lower critical solution temperature (LCST) response to medium pH and dynamic chain exchange character. A biofunctional dynamic covalent polymer was prepared by conjugation with biotin hydrazide. Under appropriate reaction conditions, a dynamic covalent block copolymer with an aldehyde group at the junction point was constructed through a reversible arylhydrazone linkage by coupling with acylhydrazide-terminated poly(ethylene glycol). The dynamic covalent block copolymer exhibited pH-dependent LCST. The remaining aldehyde group was used to react with amino-containing molecules. Bioconjugated dynamic covalent block copolymers containing reversible imine and arylhydrazone linkages were constructed by conjugation of the block copolymer to glucosamine and protein.