Glycosaminoglycans (GAGs), especially heparin and heparan sulfate (HS), hold great potential for inducing the neural differentiation of embryonic stem cells (ESCs) and have brought new hope for the treatment of neurological diseases. However, the disadvantages of natural heparin/HS, such as difficulty in isolating them with a sufficient amount, highly heterogeneous structure, and the risk of immune responses, have limited their further therapeutic applications. Thus, there is a great demand for stable, controllable, and well-defined synthetic alternatives of heparin/HS with more effective biological functions. In this study, based upon a previously proposed unit-recombination strategy, several heparin-mimicking polymers were synthesized by integrating glucosamine-like 2-methacrylamido glucopyranose monomers (MAG) with three sulfonated units in different structural forms, and their effects on cell proliferation, the pluripotency, and the differentiation of ESCs were carefully studied. The results showed that all the copolymers had good cytocompatibility and displayed much better bioactivity in promoting the neural differentiation of ESCs as compared to natural heparin; copolymers with different sulfonated units exhibited different levels of promoting ability; among them, copolymer with 3-sulfopropyl acrylate (SPA) as a sulfonated unit was the most potent in promoting the neural differentiation of ESCs; the promoting effect is dependent on the molecular weight and concentration of P(MAG-co-SPA), with the highest levels occurring at the intermediate molecular weight and concentration. These results clearly demonstrated that the sulfonated unit in the copolymers played an important role in determining the promoting effect on ESCs’ neural differentiation; SPA was identified as the most potent sulfonated unit for copolymer with the strongest promoting ability. The possible reason for sulfonated unit structure as a vital factor influencing the ability of the copolymers may be attributed to the difference in electrostatic and steric hindrance effect. The synthetic heparin-mimicking polymers obtained here can offer an effective alternative to heparin/HS and have great therapeutic potential for nervous system diseases.Keywords: embryonic stem cells (ESCs); heparin-mimicking polymer; neural differentiation; sulfonated unit; unit-recombination strategy;

Protein modified functional surfaces have been applied extensively in the field of biomaterials and medicine. Regulation of the amount and activity of proteins on the material surface is always a challenge and a key research issue. A multifunctional micro/nano-composite based surface system for efficient controllable capture and release of proteins is proposed and studied in the present paper. This novel system contains (1) gold nanoparticles (AuNPs) co-modified with an enzyme and poly(methacrylic acid) (PMAA), e.g., AuNP-pyrophosphatase (PPase)-PMAA, as nanostructured protein carriers; (2) gold nanoparticle layers (GNPLs) modified with poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), i.e., GNPL-PDMAEMA, as a micro/nano-structured support platform for surface bioactivity regulation. The capture–release of proteins and the regulation of surface bioactivity in this composite surface system were investigated under different conditions. The results showed that the proposed system is capable of protein capture and release with simple adjustment of the pH value from neutral pH to basic pH. When the pH of the system is stabilized at 7.0, the GNPL-PDMAEMA surface could adsorb plenty of AuNP-PPase-PMAA conjugates and maximum surface bioactivity occurred, but when the pH of the system is adjusted to 10.0, the GNPL-PDMAEMA surface could liberate almost all the AuNP-PPase-PMAA conjugates and thus surface bioactivity disappeared. Meanwhile, by cyclical variations between pH 7.0 and pH 10.0, this surface protein capture/release system could realize recycling and reuse of one certain protein multiple times, a series of proteins acting sequentially in accordance with pre-designed procedures, and a functional combination of multiple proteins. This recyclable multifunctional surface with the capability of protein capture/release has great potential in many applications, such as biomonitoring and biomolecule immobilization.

The modulation of protein activity is of significance for disease therapy, molecular diagnostics, and tissue engineering. Nanoparticles offer a new platform for the preparation of protein conjugates with improved protein properties. In the present work, Escherichia coli (E. coli) inorganic pyrophosphatase (PPase) and poly(methacrylic acid) (PMAA) were attached together to gold nanoparticles (AuNPs), forming AuNP–PPase–PMAA conjugates having controllable multi-biofunctionalities and responsiveness to pH. By treating with poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) and regulating the pH, the bioactivity of the conjugate becomes “on/off”-switchable. In addition, by taking advantage of the ability of AuNPs to undergo reversible aggregation/dispersion, the conjugates can be recycled and reused multiple times; and due to the shielding effect of the PMAA, the conjugated enzyme has high resistance to protease digestion. This approach has considerable potential in areas such as controlled delivery and release of drugs, biosensing, and biocatalysis.

Glycopolymer–protein biomimetic materials have been receiving considerable attention in various biomedical and pharmaceutical studies and applications. However, owing to the easily induced inactivation of protein and the complexity of preparation, efficient in vitro synthesis of highly active and stable glycopolymer–protein conjugates is considerably difficult. Here, an ideal and facile one-step method using an analogue of N-acetyl-D-glucosamine (GlcNAc), the key unit of the universal biological polysaccharide, is proposed to prepare glycopolymer–protein conjugates with improved protein activity and stability. Inorganic pyrophosphatase (PPase) was conjugated through a one-step thiol-disulfide exchange reaction with a series of well-defined and molecular weight-controlled glycopolymers, poly(2-methacrylamido glucopyranose) (PMAG), prepared via reversible addition–fragmentation chain transfer (RAFT) polymerization. The results showed that the PMAG–PPase conjugate with a polymer chain of comparatively lower molecular weight (8.0 kDa) exhibited excellent ability to improve enzymatic activity by about 50%; it can maintain the activity at extreme pH and high salt concentration. The glycopolymer–protein conjugates also exhibited excellent resistance to both protease and glycosidase. Moreover, the glycopolymer–protein conjugates could efficiently catalyze the hydrolysis reaction of calcium pyrophosphate (CPP), exhibiting great potential in the treatment of the CPP deposition disease (CPDD). All the results revealed that the glycopolymer–protein materials described in this work, especially the conjugates with small molecular weight PMAG, possess significantly enhanced capacity to improve enzymatic activity, that the structure and property of PMAG play important roles in the tolerance of the conjugates to both environmental and biological stresses, and that the PMAG–PPase conjugate could potentially assist in the therapy of pseudogout. It can be predicted that this type of glycoprotein mimic will be favorable and valuable for applications in biological detection and clinical treatment.

Escherichia coli plays a crucial role in various inflammatory diseases and infections that pose significant threats to both human health and the global environment. Specifically inhibiting the growth of pathogenic E. coli is of great and urgent concern. By modifying gold nanoparticles (AuNPs) with both poly[2-(methacrylamido)glucopyranose] (pMAG) and poly[2-(methacryloyloxy)ethyl trimethylammonium iodide] (pMETAI), a novel recyclable E. coli-specific-killing AuNP–polymer (ESKAP) nanocomposite is proposed in this study, which based on both the high affinity of glycopolymers toward E. coli pili and the merits of antibacterial quaternized polymers attached to gold nanoparticles. The properties of nanocomposites with different ratios of pMAG to pMETAI grafted onto AuNPs are studied. With a pMAG:pMETAI feed ratio of 1:3, the nanocomposite appeared to specifically adhere to E. coli and highly inhibit the bacterial cells. After addition of mannose, which possesses higher affinity for the lectin on bacterial pili and has a competitive advantage over pMAG for adhesion to pili, the nanocomposite was able to escape from dead E. coli cells, becoming available for repeat use. The recycled nanocomposite retained good antibacterial activity for at least three cycles. Thus, this novel ESKAP nanocomposite is a promising, highly effective, and readily recyclable antibacterial agent that specifically kills E. coli. This nanocomposite has potential applications in biological sensing, biomedical diagnostics, biomedical imaging, drug delivery, and therapeutics.Keywords: antibacterial polymer; Escherichia coli; glycopolymer; gold nanoparticle; specific-killing

The major bottleneck for gene delivery lies in the lack of safe and efficient gene vectors and delivery systems. In order to develop a much safer and efficient transfection system, a novel strategy of combining traditional Ca2+-dependent transfection with cationic polymer poly(N,N-dimethylamino)ethyl methacrylate (PDMAEMA) modified silicon nanowire arrays (SiNWAs) was proposed in this work. Detailed studies were carried out on the effects of the PDMAEMA polymerization time, the Ca2+ concentration, and the incubation time of Ca2+@DNA complex with PDMAEMA-modified SiNWAs (SN-PDM) on the gene transfection in the cells. The results demonstrated that the transfection efficiency of SN-PDM assisted traditional Ca2+-dependent transfection was significantly enhanced compared to those without any surface assistance, and SN-PDM with polymerization time 24 h exhibited the highest efficiency. Moreover, the optimal transfection efficiency was found at the system of a complex containing Ca2+ (100 mM) and plasmid DNA (pDNA) incubated on SN-PDM for 20 min. Compared with unmodified SiNWAs, SN-PDM has little cytotoxicity and can improve cell attachment. All of these results demonstrated that SN-PDM could significantly enhance Ca2+-dependent transfection; this process depends on the amino groups’ density of PDMAEMA on the surface, the Ca2+ concentration, and the available Ca2+@DNA complex. Our study provides a potential novel and excellent means of gene delivery for therapeutic applications.

Protein has been widely applied in biotechnology and biomedicine thanks to its unique properties of high catalytic activity, outstanding receptor–ligand specificity, and controllable sequence mutability. Owing to the easily induced structural variation and thus the inactivation of protein, there has been much effort to improve the structural stability and biological activity of proteins by the use of polymers to modify protein to construct protein–polymer conjugates. However, during the conjugation of polymer to protein active center, the great loss in the original biological activity of the protein is still a serious and so far unsolved question. Here, for the purpose of preparing site-directed and highly structurally stable protein–polymer conjugate, which would possess at least a substantially similar level of biological activity as the original unmodified protein, we proposed a new strategy by using a pyridine chain-transfer agent (CTA-Py) with a soft pyridine-terminated chain for visible-light-induced reversible addition–fragmentation chain transfer (RAFT) polymerization specifically on a number of sites close to the protein active center. The results showed that all the intermediate conjugates PPa–CTA-Py at different modification sites could retain full enzymatic activities (about 110–130% of the unmodified PPa). It was demonstrated by dynamic computer simulation that introducing of CTA-Py had little interference to the protein spatial structure as compared to the popular maleimide chain-transfer agent (CTA-Ma) with rigid maleimide-terminated. Moreover, intermediate conjugates PPa–CTA-Py is facile and ready for further light polymerization under mild conditions. Final PPa–PNIPAAm conjugate produced from CTA-Py exhibited excellent temperature responsiveness and maintained its enzymatic activity even at high temperature. These highly stable and responsive protein–polymer conjugates have great potential and could be widely used in various industrial, chemical, biological, and pharmaceutical applications.

Polymerase chain reaction (PCR) is a powerful method for nucleic acid amplification. However, the PCR is inhibited in its yield due to its byproduct, pyrophosphate (PPi), a byproduct of the reaction; the yield is thereby limited. The conventional method for hydrolysis of PPi by pyrophosphatase (PPase) is not well adapted for operation at elevated temperatures over long times as required during the PCR. In this work, we reported a strategy to improve the PCR yield using a conjugate of the enzyme with the thermally responsive polymer poly(N-isopropylacrylamide) (PNIPAM). Pyrophosphatase (PPase) was conjugated to PNIPAM site-specifically near the active center. As compared to the free enzyme, the optimum temperature of the conjugate was shown to increase from 45 to 60 °C. For the conjugate, about 77% enzyme activity was retained after incubation at 60 °C for 3 h, representing a 6.8-fold increase as compared to the unconjugated enzyme. For the PCR using the conjugate, the yield was 1.5-fold greater than using the unconjugated enzyme. As well as improving the yield of the PCR (and possibly other biological reactions) at elevated temperature, polymer conjugation may also provide a strategy to improve the heat resistance of proteins more generally.Keywords: PCR enhancement; poly(N-isopropylacrylamide); protein activity; pyrophosphatase; thermal stability

It is important to effectively maintain and modulate the bioactivity of protein-nanoparticle conjugates for their further and intensive applications. The strategies of controlling protein activity via “tailor-made surfaces” still have some limitations, such as the difficulties in further modulation of the bioactivity and the proteolysis by some proteases. Thus, it is essential to establish a responsive protein-nanoparticle conjugate system to realize not only controllable modulations of protein activity in the conjugates by incorporating sensitivity to environmental cues but also high resistance to proteases. In the work reported here, Escherichia coli (E. coli) inorganic pyrophosphatase (PPase) and poly(N-isopropylacrylamide) (pNIPAM) were both fabricated onto gold nanoparticles (AuNPs), forming AuNP-PPase-pNIPAM conjugates. The bioactivity-modulating capability of the conjugates with changes in temperature was systematically investigated by varying the molecular weight of pNIPAM, the PPase/pNIPAM molar ratio on AuNP, and the orientation of the proteins. Under proper conditions, the activity of the conjugate at 45 °C was approximately 270% of that at 25 °C. In the presence of trypsin digestion, much less conjugate activity than protein activity was lost. These findings indicate that the fabrication of AuNP-protein-pNIPAM conjugates can both modulate protein activity on a large scale and show much higher resistance to protease digestion, exhibiting great potential in targeted delivery, controllable biocatalysis, and molecular/cellular recognition.Keywords: gold nanoparticle; inorganic pyrophosphatase; poly(N-isopropylacrylamide); protein activity; thermoresponsive;

The use of functional material surfaces in antibacterial applications shows great potential. This study proposes a new strategy to evade the decrease in surface antibacterial activity associated with physical adsorption or the covalent modification of antibacterial substances onto material surfaces. 3,6-O-Sulfated chitosan (3,6S-chitosan), as a specific ligand for lysozyme, was synthesized and grafted onto a silicon wafer. The specific adsorption of lysozyme onto a 3,6S-chitosan-modified surface was detected, and the activity of the surface-bound lysozyme was measured. The results showed that the 3,6S-chitosan-modified surface exhibited very high hydrolysis activity for bacterial cell wall components, over 16 times greater than that on the other surfaces. The specific activity of the lysozyme bound on the 3,6S-chitosan-modified surface was approximately 37000 U mg−1, close to the activity of native lysozyme. The 3,6S-chitosan-modified surface loaded with lysozyme was capable of killing almost all of the E. coli cells attached to the material. Moreover, the surface could be regenerated and its antibacterial activity could be regained through high-salinity treatment. The results suggest that a 3,6S-chitosan-modified surface could be useful for the preparation of new recyclable antibacterial materials through the specific adsorption of lysozyme with 3,6S-chitosan.

In this work, a novel gene delivery strategy was proposed based on silicon nanowire arrays modified with high-molecular-weight 25 kDa branched polyethylenimine (SN-PEI). Both the plasmid DNA (pDNA) binding capacity and the in vitro gene transfection efficiency of silicon nanowire arrays (SiNWAs) were significantly enhanced after modification with high-molecular-weight bPEI. Moreover, the transfection efficiency was substantially further increased by the introduction of free pDNA/PEI complexes formed by low-molecular-weight branched PEI (bPEI, 2 kDa). Additionally, factors affecting the in vitro transfection efficiency of the novel gene delivery system were investigated in detail, and the transfection efficiency was optimized on SN-PEI with a bPEI grafting time of 3 h, an incubation time of 10 min for tethered pDNA/PEI complexes consisting of high-molecular-weight bPEI grafted onto SiNWAs, and with an N/P ratio of 80 for free pDNA/PEI complexes made of low-molecular-weight bPEI. Together, our results indicate that high-molecular-weight bPEI modified SiNWAs can serve as an efficient platform for gene delivery.Keywords: cytotoxicity; gene transfection; polyethylenimine; SiNWAs; SN-PEI; surface modification

A new strategy for accurate and reversible modulation of protein activity via simple conjugation of the sulfhydryl modifier and polymer with the introduced Cys residue in protein was developed in this study. With Escherichia coli inorganic pyrophosphatase (PPase) as a model protein, we used site-directed mutagenesis to generate a mutant PPase (PPC) with a substituted Cys residue at the specific Lys-148 site, which is within a conserved sequence near the active site and exposed to the surface of the PPC for chemical reaction. The site-specific conjugation of the mutated Cys residue in PPC with sulfhydryl modifier p-chloromercuribenzoate (PCMB) and pyridyl disulfide-functionalized poly(2-hydroxyethyl methacrylate) (pHEMA) resulted in obvious decrease or complete loss of the catalytic activity of PPC, due to the conformational change of PPC. Compared with the effect of small molecule modification (PCMB), the pHEMA conjugation led to greater inhibitory effect on protein activity due to the significant change of the tertiary structure of PPC after conjugation. Moreover, the protein activity can be restored to different extents by the treatment with different amount of reductive reagents, which can result in the dissociation between PPC and PCMB or pHEMA to recover the protein conformation. This study provides a new strategy for efficient control of protein activity at different levels by site-specific conjugation of a small molecule and polymer.

Silicon nanowire arrays (SiNWAs) were found to have catalytic activities similar to those of biological enzymes catalase and peroxidase. Thus not only can these materials catalyze the decomposition reaction of H2O2 into water and oxygen, but they can also catalyze the oxidation of o-phenylenediamine (OPD), a common substrate for peroxidases, by H2O2. The presence of Si–H bonds and the morphology of the SiNWAs are found to be crucial to the occurrence of such catalytic activity. When the SiNWAs are reacted with H2O2, the data from Raman spectroscopy suggests the formation of (Si–H)2···(O species) ((Si–H)2···Os), which is presumably responsible for the catalytic activity. These findings suggest the potential use of SiNWAs as enzyme mimics in medicine, biotechnology, and environmental chemistry.

The use of functional material surfaces in antibacterial applications shows great potential. This study proposes a new strategy to evade the decrease in surface antibacterial activity associated with physical adsorption or the covalent modification of antibacterial substances onto material surfaces. 3,6-O-Sulfated chitosan (3,6S-chitosan), as a specific ligand for lysozyme, was synthesized and grafted onto a silicon wafer. The specific adsorption of lysozyme onto a 3,6S-chitosan-modified surface was detected, and the activity of the surface-bound lysozyme was measured. The results showed that the 3,6S-chitosan-modified surface exhibited very high hydrolysis activity for bacterial cell wall components, over 16 times greater than that on the other surfaces. The specific activity of the lysozyme bound on the 3,6S-chitosan-modified surface was approximately 37000 U mg−1, close to the activity of native lysozyme. The 3,6S-chitosan-modified surface loaded with lysozyme was capable of killing almost all of the E. coli cells attached to the material. Moreover, the surface could be regenerated and its antibacterial activity could be regained through high-salinity treatment. The results suggest that a 3,6S-chitosan-modified surface could be useful for the preparation of new recyclable antibacterial materials through the specific adsorption of lysozyme with 3,6S-chitosan.

Glycopolymer–protein biomimetic materials have been receiving considerable attention in various biomedical and pharmaceutical studies and applications. However, owing to the easily induced inactivation of protein and the complexity of preparation, efficient in vitro synthesis of highly active and stable glycopolymer–protein conjugates is considerably difficult. Here, an ideal and facile one-step method using an analogue of N-acetyl-D-glucosamine (GlcNAc), the key unit of the universal biological polysaccharide, is proposed to prepare glycopolymer–protein conjugates with improved protein activity and stability. Inorganic pyrophosphatase (PPase) was conjugated through a one-step thiol-disulfide exchange reaction with a series of well-defined and molecular weight-controlled glycopolymers, poly(2-methacrylamido glucopyranose) (PMAG), prepared via reversible addition–fragmentation chain transfer (RAFT) polymerization. The results showed that the PMAG–PPase conjugate with a polymer chain of comparatively lower molecular weight (8.0 kDa) exhibited excellent ability to improve enzymatic activity by about 50%; it can maintain the activity at extreme pH and high salt concentration. The glycopolymer–protein conjugates also exhibited excellent resistance to both protease and glycosidase. Moreover, the glycopolymer–protein conjugates could efficiently catalyze the hydrolysis reaction of calcium pyrophosphate (CPP), exhibiting great potential in the treatment of the CPP deposition disease (CPDD). All the results revealed that the glycopolymer–protein materials described in this work, especially the conjugates with small molecular weight PMAG, possess significantly enhanced capacity to improve enzymatic activity, that the structure and property of PMAG play important roles in the tolerance of the conjugates to both environmental and biological stresses, and that the PMAG–PPase conjugate could potentially assist in the therapy of pseudogout. It can be predicted that this type of glycoprotein mimic will be favorable and valuable for applications in biological detection and clinical treatment.