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.

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.

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

Glycopeptide-based hydrogelators with well-defined molecular structures and varied contents of sugar moieties were prepared via in vitro peptide glycosylation reactions. With systematic glucose modification, these glycopeptide hydrogelators exhibited diverse self-assembling behaviors in water and formed supramolecular hydrogels with enhanced thermostability and biostability, in comparison with their peptide analogue. Moreover, because of high water content and similar structural morphology and composition to extracellular matrixes (ECM) in tissues, these self-assembled hydrogels also exhibited great potential to act as new biomimetic scaffolds for mammalian cell growth. Therefore, peptide glycosylation proved to be an effective means for peptide modification and generation of novel supramolecular hydrogelators/hydrogels with improved biophysical properties (e.g., high biostability, increased thermostability, and cell adhesion) which could promise potential applications in regenerative medicine.Keywords: glycopeptide; hydrogel; peptide; self-assembly; supramolecular

Maintaining the protein activity and stability under acidic conditions is important in bioengineering and biomedical applications. Polyelectrolyte conjugation as a means of stabilizing proteins has received much recent attention. Retention of protein activity, and especially, improvement of protein stability by minimizing the number of polymer chains in the conjugate, as well as by choosing the optimal site for conjugation, is critical in practical applications. In this research, the cationic polyelectrolyte poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA) was conjugated to the inorganic pyrophosphatase (PPase) site specifically. Conjugation of pDMAEMA to the specific site N124 on the protein surface led to a significant increase in activity at acidic pH. At pH 4.0, the activity of the pDMAEMA-conjugated protein was increased 3-fold relative to the unconjugated one. Dynamic light scattering (DLS) measurements showed that the aggregation state of the protein depended on the polymer charge as the pH was varied. Protein aggregation at low pH was prevented by pDMAEMA conjugation, resulting in an increase in protein stability under acidic conditions. The conjugate retained 60% of its initial activity after 4 h at pH 4.0, whereas the unconjugated protein lost 40% of its initial activity within 15 min at this pH. These results suggest an approach for preserving the protein activity and stability at low pH based on site-specific polyelectrolyte conjugation to the protein surface, thereby providing a new strategy for expanding the use of proteins in an acidic environment.

The key property of protein–nanoparticle conjugates is the bioactivity of the protein. The ability to accurately modulate the activity of protein on the nanoparticles at the interfaces is important in many applications. In the work reported here, modulation of the activity of protein–gold nanoparticle (AuNP) conjugates by specifically orienting the protein and by varying the surface density of the protein was investigated. Different orientations were achieved by introducing cysteine (Cys) residues at specific sites for binding to gold. We chose Escherichia coli inorganic pyrophosphatase (PPase) as a model protein and used site-directed mutagenesis to generate two mutant types (MTs) with a single Cys residue on the surface: MT1 with Cys near the active center and MT2 with Cys far from the active center. The relative activities of AuNP conjugates with wild type (WT), MT1, and MT2 were found to be 44.8%, 68.8%, and 91.2% of native PPase in aqueous solution. Site-directed orientation with the binding site far from the active center thus allowed almost complete preservation of the protein activity. The relative activity of WT and MT2 conjugates did not change with the surface density of the protein, while that of MT1 increased significantly with increasing surface density. These results demonstrate that site-directed orientation and surface density can both modulate the activity of proteins conjugated to AuNP and that orientation has a greater effect than density. Furthermore, increasing the surface density of the specifically oriented protein MT2, while having no significant effect on the specific activity of the protein, still allowed increased protein loading on the AuNP and thus increased the total protein activity. This is of great importance in the study on the interface of protein and nanoparticle and the applications for enzyme immobilization, drug delivery, and biocatalysis.Keywords: Au−S bond; gold nanoparticles; protein; site-specific orientation; surface density

A new strategy for the fabrication of glycosaminoglycan (GAG) analogs was proposed by copolymerizing the sulfonated unit and the glyco unit, ‘splitted’ from the sulfated saccharide building blocks of GAGs. The synthetic polymers can promote cell proliferation and neural differentiation of embryonic stem cells with the effects even better than those of heparin.

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;

Efficient control of the self-renewal and pluripotency maintenance of embryonic stem cell (ESC) is a prerequisite for translating stem cell technologies to clinical applications. Surface topography is one of the most important factors that regulates cell behaviors. In the present study, micro/nano topographical structures composed of a gold nanoparticle layer (GNPL) with nano-, sub-micro-, and microscale surface roughnesses were used to study the roles of these structures in regulating the behaviors of mouse ESCs (mESCs) under feeder-free conditions. The distinctive results from Oct-4 immunofluorescence staining and quantitative real-time polymerase chain reaction (qPCR) demonstrate that nanoscale and low sub-microscale surface roughnesses (Rq less than 392 nm) are conducive to the long-term maintenance of mESC pluripotency, while high sub-microscale and microscale surface roughnesses (Rq greater than 573 nm) result in a significant loss of mESC pluripotency and a faster undirectional differentiation, particularly in long-term culture. Moreover, the likely signalling cascades engaged in the topological sensing of mESCs were investigated and their role in affecting the maintenance of the long-term cell pluripotency was discussed by analyzing the expression of proteins related to E-cadherin mediated cell–cell adhesions and integrin-mediated focal adhesions (FAs). Additionally, the conclusions from MTT, cell morphology staining and alkaline phosphatase (ALP) activity assays show that the surface roughness can provide a potent regulatory signal for various mESC behaviors, including cell attachment, proliferation and osteoinduction.

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

Embryonic stem cells (ESCs) can be induced to differentiate into nerve cells, endowing them with potential applications in the treatment of neurological diseases and neural repair. In this work, we report for the first time that sulfated chitosan can promote the neural differentiation of ESCs. As a type of sulfated glycosaminoglycan analog, sulfated chitosan with well-defined sulfation sites and a controlled degree of sulfation (DS) were prepared through simple procedures and the influence of sulfated glycosaminoglycan on neural differentiation of ESCs was investigated. Compared with other sulfation sites, 6-O-sulfated chitosan showed the most optimal effects. By monitoring the expression level of neural differentiation markers using immunofluorescence staining and PCR, it was found that neural differentiation was better enhanced by increasing the DS of 6-O-sulfated chitosan. However, increasing the DS by introducing another sulfation site in addition to the 6-O site to chitosan did not promote neural differentiation as much as 6-O-sulfated chitosan, indicating that compared with DS, the sulfation site is more important. Additionally, the optimal concentration and incubation time of 6-O-sulfated chitosan were investigated. Together, our results indicate that the sulfate site and the molecular structure in a sulfated polysaccharide are very important for inducing the differentiation of ESCs. Our findings may help to highlight the role of sulfated polysaccharide in inducing the neural differentiation of ESCs.Keywords: embryonic stem cells; heparin; neural differentiation; sulfated chitosan

•Patterning of silk fibroin films.•The influence of topography on HUVEC cell behavior.•HUVEC cells are aligned by topographic grooves and ridges.•The primary growth direction of filopodia is altered by pattern ridges.Silk fibroin is an ideal blood vessel substitute due to its advantageous qualities including variable size, good suture retention, low thrombogenicity, non-toxicity, non-immunogenicity, biocompatibility, and controllable biodegradation. In this study, silk fibroin films with a variety of surface patterns (e.g. square wells, round wells plus square pillars, square pillars, and gratings) were prepared for in vitro characterization of human umbilical vein endothelial cell's (HUVEC) response. The affects of biomimetic length-scale topographic cues on the cell orientation/elongation, proliferation, and cell-substrate interactions have been investigated. The density of cells is significantly decreased in response to the grating patterns (70 ± 3 nm depth, 600 ± 8 nm pitch) and the square pillars (333 ± 42 nm gap). Most notably, we observed the contact guidance response of filopodia of cells cultured on the surface of round wells plus square pillars. Overall, our data demonstrates that the patterned silk fibroin films have an impact on the behaviors of human umbilical vein endothelial cells.

The MTT (3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide) reduction method is widely used for measuring cell viability and proliferation. However, when MTT was used to study cells on silicon nanowire arrays (SiNWAs), the measured viability was much higher than normal values, resulting in a misleading estimate of cell viability. Our results demonstrated that the apparent high viability of cells is due to the fact that the SiNWAs itself was capable of reducing MTT in the absence of cells. In the presence of coenzyme, its reducing capacity was enhanced, thus showing the reductase-like function of SiNWAs. Furthermore, the chemical composition and nanostructure of Si surface had a strong influence on MTT reduction with the HF-treated SiNWAs (H-SiNWAs) showing significant reducing capacity. For example, the reduction capacity of H-SiNWAs samples was significantly higher than that of HF-treated planar silicon, whereas Piranha-treated SiNWAs and planar silicon did not reduce MTT. H-SiNWAs were also used for the reduction of azo dyes and showed a decolorization rate of more than 65% and as high as 90%. These findings suggest the potential use of SiNWAs as enzyme-mimics in biotechnology and environmental chemistry.Keywords: cell viability; MTT; reductant; silicon nanowire arrays;

The availability of techniques for the sensitive detection of early stage cancer is crucial for patient survival. Our previous research (Langmuir, 2011, 27, 2155–2158) showed that gold nanoparticle layers (GNPL) used in indirect format ELISA amplified the signal, and gave a lower limit of detection (LOD) compared with commercial ELISA plates. However, due to its intrinsic limitations, indirect ELISA is not suitable for samples of complex composition, such as serum, plasma, etc., thus limiting the clinical performance of this kind of ELISA. In the work reported here, a GNPL-based sandwich format ELISA was developed, which showed superiority in terms of detection limit and sensitivity in the determination of rabbit IgG in buffer. More importantly, experiments using plasma spiked with carcinoembryonic antigen (CEA) as a representative biomarker showed that our GNPL-based ELISA assay amplified the signal and lowered the LOD compared to other assays, including commercialized CEA ELISA kits. This simple and cost-effective GNPL-based sandwich ELISA holds promise in clinical applications.

The purpose of this research is to investigate the effects of the variously sulfated chitosans on lysozyme activity and structure. It was shown that the specific enzymatic activity of lysozyme remained almost similar to the native protein after being bound to 6-O-sulfated chitosan (6S-chitosan) and 3,6-O-sulfated chitosan (3,6S-chitosan), but decreased greatly after being bound to 2-N-6-O-sulfated chitosan (2,6S-chitosan). Meanwhile, among these sulfated chitosans, 2,6S-chitosan induced the greatest conformational change in lysozyme as indicated by the fluorescence spectra. These findings demonstrated that when sulfated chitosans of different structures bind to lysozyme, lysozyme undergoes conformational change of different magnitudes, which results in corresponding levels of lysozyme activity. Further study on the interaction of sulfated chitosans with lysozyme by surface plasmon resonance (SPR) suggested that their affinities might be determined by their molecular structures.

It is well known that adsorbed proteins play a major role in cell adhesion. However, it has also been reported that cells can adhere to a protein-resistant surface. In this work, the behavior of L02 and BEL-7402 cells on a protein-resistant, 3D topographical surface was investigated. The topographical gold nanoparticle layer (GNPL) surfaces were prepared by chemical gold plating, and the topography was described by roughness parameters acquired from a multiscale analysis. Both smooth Au and GNPL surfaces were modified with POEGMA polymer brushes using surface-initiated ATRP. The dry and hydrated thicknesses of POEGMA brushes on both smooth and rough surfaces were measured by AFM using a nanoindentation method. Protein adsorption experiments using 125I radiolabeling revealed similarly low levels of protein adsorption on smooth and GNPL surfaces modified with POEGMA, thus allowing an investigation of the effects of topography on cell behavior under conditions of minimal protein adsorption. The roles of VN and FN adsorption in both L02 cells and BEL-7402 cells adhesion were investigated using cell culturing with and without a serum supplement. It was found that initial cell adhesion occurred via proteins adsorbed from the cell culture medium, whereas subsequent durable cell adhesion could be attributed to the topographical structure of the surface. Although cell spreading on protein-resistant surfaces was constrained because of the lack of adsorbed proteins, we found that cells adherent to topographical surfaces were more firmly attached and thus were more durable compared to those on smooth surfaces. In general, however, we conclude that topography is more important for cell adhesion on a protein-resistant surface.

The control of protein adsorption and cell attachment to materials is of great importance in many fields, including biomaterials, tissue engineering, biosensors, drug delivery and bioseparations. The wettability of a material strongly affects the binding of proteins and cells. Thus, changes in wettability and, in particular, “jump-wise” and smaller “step-wise” changes, can be exploited to control these interactions. In this work, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) was grafted onto silicon nanowire arrays (SiNWAs) by surface-initiated atom transfer radical polymerization (SI-ATRP). The wettability of the modified material was shown to be tunable by varying the environmental pH and NaCl concentration. The water contact angle (WCA) response was different for these two variables. A sharp or “jump-wise” change of WCA between ∼10 and 110° was observed at a pH of about 5.0. With decreasing ionic strength (IS), the surface wettability changed gradually in a step-wise fashion from superhydrophilic (WCA <2°) to strongly hydrophobic (WCA >110°). Protein adsorption and bacterial attachment on the surface varied with wettability changes caused by varying the ionic strength at pH 7.0. Thus, variations in ionic strength can be used as a means of controlling these interactions. It is concluded that fine control of protein adsorption and bacterial attachment can be achieved on PDMAEMA-modified SiNWAs by tuning surface wettability via salt concentration. This approach also has potential applications in the control of adsorption and release of drugs and cells, in biosensors and in environmental treatments using microorganisms.

Surface modification with stimuli-responsive polymers leads to switchable wettability and bioadhesion that varies in response to environmental stimuli. The introduction of nanoscale structure onto surfaces also results in changes to the surface properties. However, the synergistic effects of stimuli-responsive polymers with nanoscale structures are unclear. In this work, two typical stimuli-responsive polymers, thermo-responsive poly(N-isopropylacrylamide) (poly(NIPAAm)) and pH-responsive poly(methacrylic acid) (poly(MAA)), were grafted from initiator-immobilized silicon nanowire arrays (SiNWAs) with nanoscale topography via surface-initiated atom transfer radical polymerization. Because of the synergistic effects of the stimuli-responsive conformation transition of polymer chains and the nano-effects of three-dimensional nanostructured SiNWAs, these new platforms possess several unique properties. Compared with their corresponding modified flat silicon surfaces, the introduction of nanoscale roughness enhanced the thermo-responsive wettability of SiNWAs-poly(NIPAAm) but weakened the pH-responsive wettability of SiNWAs-poly(MAA). More importantly, these surfaces exhibited special protein-adsorption behavior. The SiNWAs-poly(NIPAAm) surface showed good non-specific protein resistance regardless of temperature, suggesting a weakened thermo-responsivity to protein adsorption. The SiNWAs-poly(MAA) surface showed obvious enhancement of pH-dependent protein adsorption behavior.

Developing new technologies applicable to the sensitive detection of cancer in its early stages has always been attractive in diagnosis. A stable gold nanoparticle layer (GNPL)-modified high-binding ELISA plate was obtained via chemical plating and was proven to be more efficient in binding proteins while maintaining their activity. GNPL-based ELISA for the representative biomarker carcinoembryonic antigen (CEA) demonstrated that GNPL markedly amplified the ELISA signal and significantly improved the limit of detection (LOD). Antithrombin detection further confirms the effectiveness and universality of this GNPL-based platform. The entire assay procedure is simple and low in cost and does not require special facilities. All these virtues indicate that this GNPL platform holds great promise in clinical applications for the early diagnosis of cancer.

Contact guidance and external force field are two important factors that influence cell orientation. Microgroove-like patterns on PDMS substrates were fabricated using soft-lithography, and the effects of gravitational field direction relative to the pattern, on L929 cell orientation were investigated. The majority of cells were aligned along the grooves when gravity was parallel to the groove direction, because of contact guidance. When gravity was perpendicular to the groove direction, cell alignment decreased noticeably. Although contact guidance induced by the pattern played a dominant role in determining cell orientation, gravity also had an important influence when the field direction was perpendicular to the groove direction. No synergistic influence on cell alignment was observed when the field direction was parallel to the groove direction.

A new strategy for the fabrication of glycosaminoglycan (GAG) analogs was proposed by copolymerizing the sulfonated unit and the glyco unit, ‘splitted’ from the sulfated saccharide building blocks of GAGs. The synthetic polymers can promote cell proliferation and neural differentiation of embryonic stem cells with the effects even better than those of heparin.

The control of protein adsorption and cell attachment to materials is of great importance in many fields, including biomaterials, tissue engineering, biosensors, drug delivery and bioseparations. The wettability of a material strongly affects the binding of proteins and cells. Thus, changes in wettability and, in particular, “jump-wise” and smaller “step-wise” changes, can be exploited to control these interactions. In this work, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) was grafted onto silicon nanowire arrays (SiNWAs) by surface-initiated atom transfer radical polymerization (SI-ATRP). The wettability of the modified material was shown to be tunable by varying the environmental pH and NaCl concentration. The water contact angle (WCA) response was different for these two variables. A sharp or “jump-wise” change of WCA between ∼10 and 110° was observed at a pH of about 5.0. With decreasing ionic strength (IS), the surface wettability changed gradually in a step-wise fashion from superhydrophilic (WCA <2°) to strongly hydrophobic (WCA >110°). Protein adsorption and bacterial attachment on the surface varied with wettability changes caused by varying the ionic strength at pH 7.0. Thus, variations in ionic strength can be used as a means of controlling these interactions. It is concluded that fine control of protein adsorption and bacterial attachment can be achieved on PDMAEMA-modified SiNWAs by tuning surface wettability via salt concentration. This approach also has potential applications in the control of adsorption and release of drugs and cells, in biosensors and in environmental treatments using microorganisms.

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.

Maintaining the protein activity and stability under acidic conditions is important in bioengineering and biomedical applications. Polyelectrolyte conjugation as a means of stabilizing proteins has received much recent attention. Retention of protein activity, and especially, improvement of protein stability by minimizing the number of polymer chains in the conjugate, as well as by choosing the optimal site for conjugation, is critical in practical applications. In this research, the cationic polyelectrolyte poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA) was conjugated to the inorganic pyrophosphatase (PPase) site specifically. Conjugation of pDMAEMA to the specific site N124 on the protein surface led to a significant increase in activity at acidic pH. At pH 4.0, the activity of the pDMAEMA-conjugated protein was increased 3-fold relative to the unconjugated one. Dynamic light scattering (DLS) measurements showed that the aggregation state of the protein depended on the polymer charge as the pH was varied. Protein aggregation at low pH was prevented by pDMAEMA conjugation, resulting in an increase in protein stability under acidic conditions. The conjugate retained 60% of its initial activity after 4 h at pH 4.0, whereas the unconjugated protein lost 40% of its initial activity within 15 min at this pH. These results suggest an approach for preserving the protein activity and stability at low pH based on site-specific polyelectrolyte conjugation to the protein surface, thereby providing a new strategy for expanding the use of proteins in an acidic environment.

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.