Resveratrol protects against lipopolysaccharide-induced cardiac dysfunction by enhancing SERCA2a activity through promoting the phospholamban oligomerization.

The bacterial endotoxin lipopolysaccharide (LPS) is a main culprit responsible for cardiac dysfunction in sepsis. This study examined whether resveratrol could protect against LPS-induced cardiac dysfunction by improving the sarcoplasmic endoplasmic reticulum Ca2+-ATPase (SERCA2a) activity. Echocardiographic parameters, cardiomyocyte contractile and Ca2+ transient properties, markers for cardiac inflammation, cell death, and oxidative stress, SERCA2a activity, and the ratios of phospholamban (PLB) monomer to oligomer were measured. Cardiac function was decreased >50% after LPS challenge (6 mg/kg for 6 h), which was improved by resveratrol. There was neither difference in plasma tumor necrosis factor-α and troponin I levels nor in infiltration of CD45+ cells in cardiac tissue between resveratrol-treated and untreated groups. In cardiomyocytes, LPS significantly decreased contractile amplitude, elongated relengthening time, diminished Ca2+ transient, reduced SERCA2a activity, and increased superoxide generation. These pathological alterations were attenuated by resveratrol treatment. Immunoblot analysis showed that LPS-treated mice had increased levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and the monomer form of PLB, along with decreases in the levels of SERCA2a, the oligomer form of PLB and nuclear factor erythroid 2-related factor (Nrf-2). Resveratrol treatment upregulated SERCA2a, the oligomer form of PLB, and Nrf-2 expression and function, and downregulated MDA, 4-HNE, and the monomer form of PLB. Our data suggest that the activity of SERCA2a in endotoxemia is inhibited, possibly due to increases in the monomer form of PLB. Resveratrol protects the heart from LPS-induced injuries at least in part through promoting the oligomerization of PLB that leads to enhanced SERCA2a activity.

Restraint of the differentiation of mesenchymal stem cells by a nonfouling zwitterionic hydrogel.

The success of human mesenchymal stem cell (hMSC) therapies is largely dependent on the ability to maintain the multipotency of cells and control their differentiation. External biochemical and biophysical cues can readily trigger hMSCs to spontaneously differentiate, thus resulting in a rapid decrease in the multipotent cell population and compromising their regenerative capacity. Herein, we demonstrate that nonfouling hydrogels composed of pure poly(carboxybetaine) (PCB) enable hMSCs to retain their stem-cell phenotype and multipotency, independent of differentiation-promoting media, cytoskeletal-manipulation agents, and the stiffness of the hydrogel matrix. Moreover, encapsulated hMSCs can be specifically induced to differentiate down osteogenic or adipogenic pathways by controlling the content of fouling moieties in the PCB hydrogel. This study examines the critical role of nonspecific interactions in stem-cell differentiation and highlights the importance of materials chemistry in maintaining stem-cell multipotency and controlling differentiation.

Zwitterionic Nanocages Overcome the Efficacy Loss of Biologic Drugs.

For biotherapeutics that require multiple administrations to fully cure diseases, the induction of undesirable immune response is one common cause for the failure of their treatment. Covalent binding of hydrophilic polymers to proteins is commonly employed to mitigate potential immune responses. However, while this technique is proved to partially reduce the antibodies (Abs) reactive to proteins, it may induce Abs toward their associated polymers and thus result in the loss of efficacy. Zwitterionic poly(carboxybetaine) (PCB) is recently shown to improve the immunologic properties of proteins without inducing any antipolymer Abs against itself. However, it is unclear if the improved immunologic profiles can translate to better clinical outcomes since improved immunogenicity cannot directly reflect amelioration in efficacy. Here, a PCB nanocage (PCB NC) is developed, which can physically encase proteins while keeping their structure intact. PCB NC encapsulation of uricase, a highly immunogenic enzyme, is demonstrated to eradicate all the immune responses. To bridge the gap between immunogenicity and efficacy studies, the therapeutic performance of PCB NC uricase is evaluated and compared with its PEGylated counterpart in a clinical-mimicking gouty rat model to determine any loss of efficacy evoked after five administrations.

Achieving low-fouling surfaces with oppositely charged polysaccharides via LBL assembly.

UNLABELLED: The aim of this work is to understand and achieve low fouling surfaces by mixing two oppositely charged polysaccharides through layer-by-layer (LBL) assembly. Diethylaminoethyl-dextran hydrochloride and alginate were employed as a model system to build LBL films. A surface plasmon resonance (SPR) biosensor was used to measure quantitatively the adsorption behavior of charged macromolecules during LBL buildup and the protein adsorption behavior of each deposited bilayer in situ in real time accordingly. Results show that LBL films have lower protein adsorption as the films are constructed above the substrate surface. These LBL films eventually reach very low fouling when they are sufficiently far from the substrate surface, where the substrate surface effect is minimized and bilayers consisting of positively and negatively charged marcromolecules are uniformly mixed. Single proteins, undiluted human blood serum and plasma and cells were tested for adsorption to LBL films with similar trends. To verify the generality of these findings, alginates of low and high molecular weights and carboxymethylcellulose as a substitute to alginate were studied with similar trends observed. These results demonstrate that oppositely charged polymers, when uniformly mixed, are able to achieve low fouling properties. Findings from this work will provide a fundamental understanding of and design principles on how to build nonfouling LBL films. STATEMENT OF SIGNIFICANCE: We demonstrate that protein adsorption decreases with the increase of bilayer numbers. Results indicate that oppositely charged components tend to be uniformly mixed and distinct change layers in classical layer-by-layer (LBL) theories no longer exist as LBL films are sufficiently far from the substrate surface. Findings from this work provide a fundamental understanding of and design principles on how to build nonfouling LBL films.

Ultra-low fouling and high antibody loading zwitterionic hydrogel coatings for sensing and detection in complex media.

UNLABELLED: For surface-based diagnostic devices to achieve reliable biomarker detection in complex media such as blood, preventing nonspecific protein adsorption and incorporating high loading of biorecognition elements are paramount. In this work, a novel method to produce nonfouling zwitterionic hydrogel coatings was developed to achieve these goals. Poly(carboxybetaine acrylamide) (pCBAA) hydrogel thin films (CBHTFs) prepared with a carboxybetaine diacrylamide crosslinker (CBAAX) were coated on gold and silicon dioxide surfaces via a simple spin coating process. The thickness of CBHTFs could be precisely controlled between 15 and 150nm by varying the crosslinker concentration, and the films demonstrated excellent long-term stability. Protein adsorption from undiluted human blood serum onto the CBHTFs was measured with surface plasmon resonance (SPR). Hydrogel thin films greater than 20nm exhibited ultra-low fouling (<5ng/cm(2)). In addition, the CBHTFs were capable of high antibody functionalization for specific biomarker detection without compromising their nonfouling performance. This strategy provides a facile method to modify SPR biosensor chips with an advanced nonfouling material, and can be potentially expanded to a variety of implantable medical devices and diagnostic biosensors. STATEMENT OF SIGNIFICANCE: In this work, we developed an approach to realize ultra-low fouling and high ligand loading with a highly-crosslinked, purely zwitterionic, carboxybetaine thin film hydrogel (CBHTF) coating platform. The CBHTF on a hydrophilic surface demonstrated long-term stability. By varying the crosslinker content in the spin-coated hydrogel solution, the thickness of CBHTFs could be precisely controlled. Optimized CBHTFs exhibited ultra-low nonspecific protein adsorption below 5ng/cm(2) measured by a surface plasmon resonance (SPR) sensor, and their 3D architecture allowed antibody loading to reach 693ng/cm(2). This strategy provides a facile method to modify SPR biosensor chips with an advanced nonfouling material, and can be potentially expanded to a variety of implantable medical devices and diagnostic biosensors.

Co-delivery of doxorubicin and tumor-suppressing p53 gene using a POSS-based star-shaped polymer for cancer therapy.

In this work, a star-shaped polymer consisting of a cationic poly[2-(dimethylamino) ethyl methacrylate] (PDMAEMA) shell and a zwitterionic poly[N-(3-(methacryloylamino) propyl)-N,N-dimethyl-N-(3-sulfopropyl) ammonium hydroxide] (PMPD) corona was grafted from a polyhedral oligomeric silsesquioxanes (POSS)-based initiator via atomic transfer radical polymerization (ATRP). The reported star-shaped polymer could form stable micelles in aqueous solutions even in the presence of serum. In addition, anti-cancer drug doxorubicin and tumor-suppressing p53 gene were loaded in the process of micelle formation. The formed polyplex was biocompatible and highly efficient for both drug and gene delivery. Furthermore, the polyplex was able to cause a high apoptotic rate of tumor cells both in vitro and in vivo. This combination delivery strategy offers a promising method for cancer therapy and can be used for further clinical applications.

Zwitterionic fusion in hydrogels and spontaneous and time-independent self-healing under physiological conditions.

The biomedical applications of current self-healing materials are largely impeded by their healing conditions, which usually require heating, UV exposure or harsh pH environments. At the same time, for very few existing spontaneously self-healing materials, healing can only be achieved immediately after rupture occurs. Here, we developed a spontaneously healing material, driven by a new mechanism, "zwitterionic fusion", which is repairable independent of time after damage under physiological conditions. We also tested the anti-fatigue property of this zwitterionic hydrogel. Furthermore, we utilized this zwitterionic fusion to link different cell-hydrogel constructs together.

Differences in cationic and anionic charge densities dictate zwitterionic associations and stimuli responses.

Zwitterionic materials have shown their excellent performance in many biological and chemical applications. Zwitterionic materials possess moieties that own both cationic and anionic groups. The associations among zwitterionic moieties through electrostatic interactions play an important role in properties of zwitterionic materials. However, the relationship between the molecular structures and associations of zwitterionic moieties are still not well understood. This work compared thermal- and salt-responsive behaviors of sulfobetaine and carboxybetaine polymers by examining their rheological properties as a function of temperature and their hydrodynamic sizes as a function of salt concentration. Results showed that carboxybetaine polymers do not exhibit stimuli responses as expected from the antipolyelectrolyte behavior of zwitterionic polymers as observed in sulfobetaine polymers. We studied and compared the associations among zwitterionic moieties in these two zwitterionic polymers using molecular dynamic simulations. Simulation results show that the charge-density difference between cationic and anionic groups determines the associations among zwitterionic moieties, which are responsible for different stimuli responses of carboxybetaine and sulfobetaine polymers.

Zwitterionic hydrogels implanted in mice resist the foreign-body reaction.

The performance of implantable biomedical devices is impeded by the foreign-body reaction, which results in formation of a dense collagenous capsule that blocks mass transport and/or electric communication between the implant and the body. No known materials or coatings can completely prevent capsule formation. Here we demonstrate that ultra-low-fouling zwitterionic hydrogels can resist the formation of a capsule for at least 3 months after subcutaneous implantation in mice. Zwitterionic hydrogels also promote angiogenesis in surrounding tissue, perhaps owing to the presence of macrophages exhibiting phenotypes associated with anti-inflammatory, pro-healing functions. Thus, zwitterionic hydrogels may be useful in a broad range of applications, including generation of biocompatible implantable medical devices and tissue scaffolds.

Softer zwitterionic nanogels for longer circulation and lower splenic accumulation.

Zwitterionic nanogels of varying stiffness were prepared by tuning their cross-linking densities and reactant contents. In vivo studies of these nanogels show that softer nanogels pass through physiological barriers, especially the splenic filtration, more easily than their stiffer counterparts, consequently leading to longer circulation half-life and lower splenic accumulation. Results from this work emphasize the role of stiffness in designing long-circulating nanoparticles.

High-strength hydrogel as a reusable adsorbent of copper ions.

A mechanically strong hydrogel was prepared by photoinitiated polymerization of oligo(ethylene glycol) methacrylate (OEGMA), 2-vinyl-4,6-diamino-1,3,5-triazine (VDT) and cross-linker N,N'-methylenebisacrylamide (MBAA). Introduction of the monomer VDT considerably strengthened the mechanical properties of the hydrogel by self-hydrogen bonding of diaminotriazine, and enhanced the adsorption of copper ion onto the hydrogel by chelation between amino groups and metal ion. Adsorption studies were carried out by varying the OEGMA/VDT ratio, contact time, pH value, counterion and initial concentration of Cu(2+) ions. The evaluation of adsorption properties showed that the hydrogel exhibited better correlation with Langmuir isotherm model. This adsorbent could be used repeatedly with little loss in adsorption capacity.

The effect of lightly crosslinked poly(carboxybetaine) hydrogel coating on the performance of sensors in whole blood.

Surface coatings of high packing densities have been routinely used to prevent nonspecific biomolecular and microorganism attachment. Hydrogels are another class of low fouling materials used to create three-dimensional matrixes for the free diffusion of small analytes or drugs and the high-loading of bio-recognition elements. However, biomolecules are subject to being entrapped within hydrogel matrixes or adhered onto hydrogel surfaces, making them questionable for use in whole blood. Here, we demonstrate the feasibility of a lightly crosslinked poly(carboxybetaine) hydrogel for use in whole blood, as opposite to the conventional wisdom of high packing density in surface coatings. Proteins are able to diffuse in and out of the matrix freely without being altered from their native conformations. In order to demonstrate its long-term performance in whole blood, this hydrogel was used as the surface coating of a glucose sensor. This work paves a new way for the development of surface coatings and sensors to achieve long-term stability and high performance in whole blood.

Robust MeO2MA/vinyl-4,6-diamino-1,3,5-triazine copolymer hydrogels-mediated reverse gene transfection and thermo-induced cell detachment.

We have fabricated a robust temperature sensitive hydrogel by photoinitiated copolymerization of 2-(2-methoxyethoxy)ethyl methacrylate (MeO(2)MA), 2-vinyl-4,6-diamino-1,3,5-triazine (VDT) and crosslinker polyethylene glycol diacrylate (PEGDA). It was shown that self-hydrogen bondings of VDT moieties in the bulk considerably strengthened the mechanical properties of gels, which was dependent on the weight ratio of MeO(2)/VDT and the initial monomer concentration; the VDT motifs on the surface could efficiently bind DNA for reverse gene transfection. On this soft-wet platform, gene expression lasted 7 days and the re-treated gels could be reused for new cycle of transfection. The gene modified cells could be detached by thermo-triggered switchable surface hydrophilicity of PMEO(2)MA in hydrogel. MTT assay showed low cytotoxicity of hydrogels. The results suggested that this type of mechanically strong H-bonded and thermoresponsive hydrogels hold a great potential as an integrated functional soft-wet platform for the unharmful harvest of gene modified seed cells for tissue engineering or as implantable scaffold for gene therapy and regenerative medicine applications.

[Experimental research on repair of rabbit articular cartilage deffects with composite of autologous cell-carriers].

OBJECTIVE: To study the effect of the repair of rabbit articular cartilage defects by the composite of chondrogenic induction of autologous MSCs and autologous "two-phase" bone matrix gelatin (BMG). METHODS: Twenty-four healthy adult New Zealand rabbits weighing 2 to 3 kg were divided into group A, B and C with 8 in each. Autologous MSCs derived from group A were cultured in vitro and observed under inverted phase contrast microscope when enough cells through trypsinization transferring in vitro were obtained. Then the growth curves of 1, 3 and 5 passage culture of MSCs were drawn. The 3rd passage MSCs were induced into chondrogenic differentiation by adding TGF-beta1 (10 ng/mL), IGF-1 (10 ng/mL) and vitamin C (50 ng/mL) in vitro. At 8 days after induction, the features of chondrocytes were observed under inverted phase contrast microscope,and immunohistochemical staining and Mallory staining were made. Getting out part of the ilium of group A and B, according to the method of Urist, the "two-phase" BMG was acquired. Chondrogenic induction of autologous MSCs was inoculated into the corresponding BMG to set up a composite of cell-carrier, and then it was observed through scanning electric microscope after 3 days of culture. The model of articular cartilage defects of rabbits was made: in group A,autologous cell-carriers were implanted; in group B, there only existed autologous BMG; in group C,there was nothing. At 8, 12 weeks after operation, the gross, HE staining and immunohistochemical staining were made,and grading scales were evaluated according to Wakitani histological grading method. RESULTS: Features of MSCs were as follows: the shape of primary cells was shot-spindled and of passage cells was long. As to the growth curves of 1, 3 and 5 passage culture of MSCs,passage cells grew slowly for 3 days after being passaged and went into log-growth during the 3rd and the 7th days and into plateau later, but the 3rd passage cells grew best. Observation of MSCs after chondrogenic induction was performed: the shape of cells was elliptical and the effect of induction was verified by the positive results of collagen type II, S-100 and Mallory staining. Under scanning electric microscope,the structure of BMG was good and cells were observed growing in it well. As far as repair of articular cartilage defects are concerned at 8, 12 weeks after transplantation, the defects in group A were repaired by the hyline-like tissue and the structures of the cartilage surface and normal cartilage were in integrity,and immunohistochemical staining of collagen type II was positive, while those in group B and C were repaired by the fibrous-like tissues and the surfaces were irregular. In Wakitanni histological score, at 8 weeks after operation, group A was (3.50 +/- 1.51) points, group B was (10.00 +/- 1.41) points and group C was (12.00 +/- 0.93) points; at 12 weeks, group A was (1.13 +/- 0.99) points, group B was (8.38+/-1.30) points, and group C was (10.13 +/- 1.64) points. At different time points,group A was significantly better than group B and C, showing significant differences (P < 0.05). CONCLUSION: Induced autologous MSCs and the composite withautologous "two-phase" BMG have the function to repair articular cartilage defects, and they are better than autologous BMG transplanted only or nothing transplanted.