RESUMEN
An abnormal microenvironment underlies poor healing in chronic diabetic chronic wounds. However, effectively modulating the microenvironment of the diabetic wound remains a great challenge due to sustained oxidative stress and chronic inflammation. Here, we present a unimolecular enzyme-polymer conjugate that demonstrates excellent multienzymatic cascade activities. The cascaded enzyme conjugates (CECs) were synthesized by grafting poly(N-acryloyl-lysine) (pLAAm) from the glycan moieties of glucose oxidase (GOx) via glycan-initiated polymerization. The resulting CECs exhibited multiple enzymatic properties of GOx, superoxide dismutase mimic, and catalase mimic activities simultaneously. The CECs facilitated the depletion of high blood glucose, ROS scavenging, bacteria-killing, anti-inflammatory effects, and sustained oxygen generation, which restored the microenvironment in diabetic wounds. In vivo results from a diabetic mouse model confirmed the capacity and efficiency of the cascade reaction for diabetic wound healing. Our findings demonstrate that the three-in-one enzyme-polymer conjugates alone can modulate the diabetic microenvironment for wound healing.
Asunto(s)
Diabetes Mellitus , Glucosa Oxidasa , Animales , Ratones , Modelos Animales de Enfermedad , Polímeros , Cicatrización de Heridas , Polisacáridos , Especies Reactivas de Oxígeno , HidrogelesRESUMEN
Formation of protein-polymer conjugates (PPCs) is critical for many studies in chemical biology, biomedicine, and enzymatic catalysis. Polymers with coordinated physicochemical properties confer synergistic functions to PPCs that overcome the inherent limitation of proteins. However, application of PPCs has been synthetically restricted by the limited modification sites and polymer grafting method. Here, we present a versatile strategy for site-selective PPC synthesis. The initiator was specifically tethered to the preoxidized glycan moieties through oxime chemistry. Polymer brushes were grown in situ from the glycan by atom-transfer radical polymerization to generate well-controlled PPCs. Notably, the modification is site-specific, multivalent, and alterable depending on protein glycosylation. Additionally, we demonstrated that the cytocompatible method enabled the growth of polymer chains from the surface of living yeast cells. These results verified a facile technology for surface modification of biomacromolecules by desired polymers for various biomedical applications.
Asunto(s)
Polímeros , Polisacáridos , Glicoproteínas , Polimerizacion , Propiedades de SuperficieRESUMEN
Efficient strategies for enriching and separating proteins are important and challenging for membrane proteomics. Many existing methods are caught in the dilemma of preserving maximal membrane proteins while avoiding the contamination of cytoplasmic proteins and organelles. Here, we report a polymer anchoring strategy for the selective preparation of membrane proteins through cell surface-initiated atom transfer radical polymerization. The cytocompatible polymerization strategy enables thermoresponsive poly( N-isopropylacrylamide) (pNIPPAm) chains to be grown from a specific protein on the surface of living cells. The polymer tagged membrane protein could be easily separated and enriched by thermoprecipitation. This method led to the identification of 1825 proteins of which 1036 (71.7%) were specific membrane proteins in E. coli. The separated proteins were identified by 2-DE and mass spectrometry. Among the 12 protein spots from the gel slice, eight were identified as outer membrane proteins. The described strategy opens up a new avenue for membrane protein enrichment and separation and may expedite the future development of membrane proteomics.
Asunto(s)
Resinas Acrílicas/química , Materiales Biocompatibles/química , Espectrometría de Masas , Proteínas de la Membrana/análisis , Electroforesis en Gel Bidimensional , Escherichia coli/metabolismo , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , TemperaturaRESUMEN
A recoverable and thermoresponsive polymer-protein bioconjugate is synthesized and employed in the purification of protein with free sulfhydryl groups. Initiator with disulphide was modified on the cysteine residue of the target protein. Poly(N-isopropylacrylamide) exhibiting a lower critical solution temperature was grown from the protein. The resulting protein-polymer conjugate was successfully thermoprecipitated and separated from other proteins. The approach was demonstrated with bovine serum albumin with the recycling yield of 76.4%. Enzyme activity test with papain verified the reversible polymer modification protected protein under extreme environments without affecting the functionality of the protein. This study implies the favorable potential of chemo-selective enriching and purification of proteins.
Asunto(s)
Precipitación Química , Polímeros/química , Proteínas/aislamiento & purificación , Temperatura , Bromelaínas/metabolismo , Electroforesis en Gel de Poliacrilamida , Papaína/metabolismoRESUMEN
In view of the biological significance and micro-heterogeneity of protein glycosylation for human health, specific enrichment of N-glycosylated proteins/peptides from complex biological samples is a prerequisite for the discovery of disease biomarkers and clinical diagnosis. In this work, we propose a "grafting-from" N-glycoprotein enriching method based on the in-situ growth of thermoresponsive polymer brushes from the N-glycosylated site of proteins. The initiator was first attached to the pre-oxidized glycan moieties by hydrazide chemistry, from which the thermoresponsive polymers can be grown to form giant protein-polymer conjugates (PPC). The thermosensitive PPC can be precipitated and separated by raising the temperature to above its lower critical solubility temperature (LCST). Mass spectrometry verified 210 N-glycopeptides corresponding to 136 N-glycoproteins in the rabbit serum. These results demonstrate the capability of the tandem thermoprecipitation strategy to enrich and separate N-glycoprotein/glycopeptide. Due to its simplicity and efficiency specifically, this method holds the potential for identifying biomarkers from biological samples in N-glycoproteome analysis.
Asunto(s)
Glicopéptidos , Polímeros , Humanos , Animales , Conejos , GlicoproteínasRESUMEN
To be better used as medical implants in orthopedic and dental clinical applications, titanium and titanium-based alloys need to be capable of inducing osteogenesis. Here we describe a method that allows the facile decoration of titanium surfaces to impart an osteogenesis capacity. A Ti surface was first deposited on a poly(OEGMA-r-HEMA) film using surface-initiated atom-transfer radical polymerization (SI-ATRP) with the further step of carboxylation. The modified surfaces were resistant to cell adhesion. Fibronectin (FN) and recombinant human bone morphogenetic protein-2 (rhBMP-2) were further immobilized onto p(OEGMA-r-HEMA) matrices. Our results demonstrate that the FN- and rhBMP-2-conjugated polymer surfaces could induce the adhesion of MC3T3 cells on Ti surfaces. Moreover, the protein-tethered surface exhibited enhanced cell differentiation in terms of alkaline phosphatase activity compared to that of the pristine Ti surface at similar cell proliferation rates. This research establishes a simple modification method of Ti surfaces via Ti-thiolate self-assembled monolayers (SAMs) and SI-ATRP and identifies a dual-functional Ti surface that combines antifouling and osseointegration promotion.
Asunto(s)
Proteína Morfogenética Ósea 2/química , Fibronectinas/química , Metacrilatos/química , Osteogénesis , Polietilenglicoles/química , Titanio/química , Humanos , Ácidos Polimetacrílicos , Proteínas Recombinantes/química , Propiedades de SuperficieRESUMEN
The key to achieve a highly sensitive and specific protein microarray assay is to prevent nonspecific protein adsorption to an "absolute" zero level because any signal amplification method will simultaneously amplify signal and noise. Here, we develop a novel solid supporting material, namely, polymer coated initiator integrated poly(dimethysiloxane) (iPDMS), which was able to achieve such "absolute" zero (i.e., below the detection limit of instrument). The implementation of this iPDMS enables practical and high-quality multiplexed enzyme-linked immunosorbent assay (ELISA) of 11 tumor markers. This iPDMS does not need any blocking steps and only require mild washing conditions. It also uses on an average 8-fold less capture antibodies compared with the mainstream nitrocellulose (NC) film. Besides saving time and materials, iPDMS achieved a limit-of-detection (LOD) as low as 19 pg mL(-1), which is sufficiently low for most current clinical diagnostic applications. We expect to see an immediate impact of this iPDMS on the realization of the great potential of protein microarray in research and practical uses such as large scale and high-throughput screening, clinical diagnosis, inspection, and quarantine.
Asunto(s)
Biomarcadores de Tumor/análisis , Dimetilpolisiloxanos/química , Ensayo de Inmunoadsorción Enzimática/métodos , Anticuerpos/inmunología , Análisis por Matrices de Proteínas/métodos , Proteínas/químicaRESUMEN
The acidophilic archaeons are a group of single-celled microorganisms that flourish in hot acid springs (usually pH < 3) but maintain their internal pH near neutral. Although there is a lack of direct evidence, the abundance of sugar modifications on the cell surface has been suggested to provide the acidophiles with protection against proton invasion. In this study, a hydroxyl (OH)-rich polymer brush layer was prepared to mimic the OH-rich sugar coating. Using a novel pH-sensitive dithioacetal molecule as a probe, we studied the proton-resisting property and found that a 10-nm-thick polymer layer was able to raise the pH from 1.0 to > 5.0, indicating that the densely packed OH-rich layer is a proton shelter. As strong evidence for the role of sugar coatings as proton barriers, this biomimetic study provides insight into evolutionary biology, and the results also could be expanded for the development of biocompatible anti-acid materials.
Asunto(s)
Archaea/metabolismo , Carbohidratos/química , Materiales Biomiméticos/química , Membrana Celular/química , Membrana Celular/metabolismo , Manantiales de Aguas Termales , Concentración de Iones de Hidrógeno , Metacrilatos/química , Polietilenglicoles/química , Ácidos Polimetacrílicos , ProtonesRESUMEN
Poly(dimethylsiloxane) (PDMS) is the choice of material for a wide range of biological and non-biological applications because of its chemical inertness, non-toxicity, ease of handling and commercial availability. However, PDMS exhibits uncontrolled protein adsorption and cell adhesion and it has proved difficult to functionalize to present bioactive ligands. We present a facile strategy for functional surface modification of PDMS using commercial reagents to engineer polymer brushes of oligo(ethylene glycol) methacrylate that prevent cell adhesion and can be functionalized to display bioadhesive ligands. The polymer brushes resist biofouling and prevent cell adhesion and bioadhesive peptides can be tethered either uniformly or constrained to micropatterned domains using standard peptide chemistry approaches. This approach is relevant to various biomedical and biotechnological applications.