Unimolecular Cascaded Multienzyme Conjugates Modulate the Microenvironment of Diabetic Wound to Promote Healing.

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.

Glycan-selective in-situ growth of thermoresponsive polymers for thermoprecipitation and enrichment of N-glycoprotein/glycopeptides.

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.

Preliminary Study on Hepatoprotective Effect and Mechanism of (-)-Epigallocatechin-3-gallate against Acetaminophen-induced Liver Injury in Rats.

Antipyretic acetaminophen (APAP) is a commonly used drug that generally associates with liver injury. (-)-Epigallocatechin-3-gallate (EGCG), an active polyphenol extracted from green tea, is extensively reported to have the potential to impact a variety of human diseases. However, few studies were reported regarding the protective effect of EGCG on APAP-induced liver injury and the mechanism is still unclear. In this study, in-vitro and in-vivo experiments were carried out to verify the hepatoprotective effect of EGCG against APAP-induced liver injury and explore the potential mechanism. Results indicated that EGCG effectively relieved the liver injury caused by APAP, as well as APAP-induced mitochondrial dysfunction. The protective role of EGCG was not only attributed to its antioxidant capacity; but also might be related to the protective effect on hepatic mitochondrial impairment; based on that, EGCG could improve the membrane potential and activities of the respiratory chain complexes in liver mitochondria. Our study casts a new light on the mechanism of EGCG's hepatoprotective effect and suggests that EGCG has considerable potential in developing tonics for relieving APAP-induced liver injury.

Site-Selective and Biocompatible Growth of Polymers from Glycan Moieties of Glycoproteins and Living Cells.

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.

Partially Hydrolyzed Guar Gum Modulates Gut Microbiota, Regulates the Levels of Neurotransmitters, and Prevents CUMS-Induced Depressive-Like Behavior in Mice.

SCOPE: Depression is the leading cause of disability around the world; however, most antidepressants have drug tolerance and serious side effects. In this study, it is explored whether partially hydrolyzed guar gum (PHGG) is a safe food that exhibits protection in a mouse model of depression. METHODS AND RESULTS: PHGG is orally administered to mice with depression induced by chronic unpredictable mild stress (CUMS) in two animal experiments (prevention trial and intervention trial) to characterize the potentially protective effect of PHGG. The results in the prevention trial show that PHGG significantly inhibits the loss of body weight, and prevents CUMS-induced depressive-like behavior in mice. The beneficial effects may be associated with PHGG modulating the gut microbiota structure and then increasing the levels of short-chain fatty acids in mice feces and the levels of 5-hydroxytryptamine and dopamine in serum, striatum, and hippocampus. Besides, PHGG in the intervention trial is less effective than that in the prevention trial, but it may have a synergistic effect on improving depression with fluoxetine. CONCLUSIONS: This study suggests that moderate daily intake of PHGG can contribute to relieving depressive-like behavior.

Nano-micelles based on hydroxyethyl starch-curcumin conjugates for improved stability, antioxidant and anticancer activity of curcumin.

In this study, amphiphilic conjugates were synthesized by conjugating curcumin (CUR) to a food-derived hydrophilic hydroxyethyl starch (HES) via an acid-labile ester linker. The self-assembly of the conjugates formed uniform micellar nanoparticles (HES-CUR NPs) with a desirable drug loading efficiency, excellent colloidal and storage stability, as well as acid-responsive release manner. Besides, the formation of the nanoparticles increased the solubility of CUR to thousands times higher than free CUR, and effectively protected the loaded CUR from degradation upon exposure to UV light and high temperature. In vitro cytotoxicity assay and radical scavenging experiments demonstrated that the HES-CUR NPs significantly improved the cytocompatibility, anticancer and antioxidant activity of CUR due to the enhanced solubility, stability, and bioavailability. The HES-CUR NPs reported herein have a great potential in developing functional food or pharmaceutical formulations for preventing or treating various diseases such as inflammatory diseases and cancer.

Polyphenol extract from superheated steam processed tea waste attenuates the oxidative damage in vivo and in vitro.

In this study, tea polyphenols (TPs) was first extracted from tea waste by superheated steam (SS) pretreated ultrasonic-assisted hydrothermal extraction (UAH). The optimized strategy presented extracts with the extraction yield up to 21.19% with a significantly higher antioxidant ability, compared with the one without SS pretreatment. Further investigation proved that the SS suppressed the polyphenol oxidase activity of the TPs extract. The ability to scavenge the free radicals were compared in mouse liver mitochondria. Mitochondrial swelling, mitochondrial membrane potential (MMP), cardiolipin peroxidation, and respiratory chain complex (RCC) I-V activities were also evaluated as the index of the mitochondrial oxidative damage. The study supports evidence that the TPs extract exhibited significant protection against oxidative damage on mitochondrial. Furthermore, the effect of TPs on antioxidant ability in zebrafish embryo was evaluated. After TPs pretreatment for 1 day, zebrafish embryos showed a significantly higher survival rate as well as heart rate when facing the oxidative stress. PRACTICAL APPLICATIONS: Polyphenols from tea leaves have been viewed as an antioxidant additive in food, mainly due to the ability of scavenging free radicals and reactive oxygen species. The results of this study suggest that the SS pretreatment could be used as an efficient method to extract TPs from the tea waste for the prevention of oxidative damage in the mouse liver mitochondria and zebrafish embryos.

A fluorescent biosensor based on catalytic activity of platinum nanoparticles for freshness evaluation of aquatic products.

In this study, a fluorescence biosensor based on the peroxidase mimicking activity of platinum nanoparticles (Pt NPs) was fabricated for rapid detection of hypoxanthine (Hx), which is a sensitive indicator of the freshness of aquatic products. The fluorescence intensity of the sensing system had a linear relationship with the concentration of Hx in the range of 8-2500 µM, and the limit of detection was as low as 2.88 µM (S/N = 3). Moreover, benefiting from the excellent selectivity of the biosensor, Hx content in fish, shrimp and squid samples could be quickly detected with good recovery rates (103.94-109.00%). And the Pt NPs used in the biosensor was reusable, which was proved by the recovery rate was only slightly decreased to 91% after three cycles. In addition to the advantages of facile preparation and low cost, the proposed biosensor will be a promising candidate for rapid and convenient freshness evaluation of aquatic products.

Cytocompatible Modification of Thermoresponsive Polymers on Living Cells for Membrane Proteomic Isolation and Analysis.

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.

Facile fabrication of microfluidic surface-enhanced Raman scattering devices via lift-up lithography.

We describe a facile and low-cost approach for a flexibly integrated surface-enhanced Raman scattering (SERS) substrate in microfluidic chips. Briefly, a SERS substrate was fabricated by the electrostatic assembling of gold nanoparticles, and shaped into designed patterns by subsequent lift-up soft lithography. The SERS micro-pattern could be further integrated within microfluidic channels conveniently. The resulting microfluidic SERS chip allowed ultrasensitive in situ SERS monitoring from the transparent glass window. With its advantages in simplicity, functionality and cost-effectiveness, this method could be readily expanded into optical microfluidic fabrication for biochemical applications.

Protein purification by chemo-selective precipitation using thermoresponsive polymers.

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.

Surface confined retro Diels-Alder reaction driven by the swelling of weak polyelectrolytes.

Recently, the type of reactions driven by mechanical force has increased significantly; however, the number of methods for activating those mechanochemical reactions stays relatively limited. Furthermore, in situ characterization of a reaction is usually hampered by the inherent properties of conventional methods. In this study, we report a new platform that utilizes mechanical force generated by the swelling of surface tethered weak polyelectrolytes. An initiator with Diels-Alder (DA) adduct structure was applied to prepare the polyelectrolyte-carboxylated poly(OEGMA-r-HEMA), so that the force could trigger the retro DA reaction. The reaction was monitored in real time by quartz crystal microbalance and confirmed with atomic force microscopy and X-ray photoelectron spectroscopy. Compared with the conventional heating method, the swelling-induced retro DA reaction proceeded rapidly with high conversion ratio and selectivity. A 23.61 kcal/mol theoretical energy barrier supported the practicability of this retro DA reaction being triggered mechanically at ambient temperature. During swelling, the tensile force was controllable and persistent. This unique feature imparts this mechanochemical platform the potential to "freeze" an intermediate state of a reaction for in situ spectroscopic observations, such as surface-enhanced Raman spectroscopy and frequency generation spectroscopy.

Molecular composition, grafting density and film area affect the swelling-induced Au-S bond breakage.

In previous studies, we reported the first observation of the Au-S bond breakage induced mechanically by the swelling of the surface-tethered weak polyelectrolyte brushes in phosphate buffered saline (PBS), a phenomenon with broad applications in the fields of biosensors and functional surfaces. In this study, three factors, namely the molecular composition, grafting density and film area of the weak polyelectrolyte, carboxylated poly(oligo(ethylene glycol) methacrylate-random-2-hydroxyethyl methacrylate) (poly(OEGMA-r-HEMA)), were studied systematically on how they affected the swelling-induced Au-S bond breakage (ABB). The results showed that, first, the swelling-induced ABB is applicable to a range of molecular compositions and grafting densities; but the critical thickness (Tcritical,dry) varied with both of the two factors. An analysis on the swelling ratio further revealed that the difference in the Tcritical,dry arose from the difference in the swelling ability. A film needed to swell to ∼250 nm to induce ABB regardless of its composition or structure, thus a higher swelling ratio would lead to a lower Tcritical,dry value. Then, the impact of the film area was studied in micrometer- and sub-micrometer-scale brush patterns, which showed that only partial, rather than complete ABB was induced in these microscopic films, resulting in buckling instead of film detaching. These results demonstrated that the ABB is suitable to be used in the design of biosensors, stimulus-responsive materials and mechanochemical devices. Although the >160 µm(2) required area for uniform ABB hinders the application of ABB in nanolithography, the irreversible buckling provides a facile method of generating rough surfaces.

A proton shelter inspired by the sugar coating of acidophilic archaea.

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.

Fibronectin and bone morphogenetic protein-2-decorated poly(OEGMA-r-HEMA) brushes promote osseointegration of titanium surfaces.

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.

Integrated poly(dimethysiloxane) with an intrinsic nonfouling property approaching "absolute" zero background in immunoassays.

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.

Poly(dimethylsiloxane) elastomers with tethered peptide ligands for cell adhesion studies.

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.