The protective effect of beta-casomorphin-7 via promoting Foxo1 activity and nuclear translocation in human lens epithelial cells.

PURPOSE: To investigate the protective effect of beta-casomorphin-7 (ß-CM-7) in oxidative stressed human lens epithelial cells (HLECs) and to explore the possible mechanism for oxidative stress in HLECs induced by high glucose. METHODS: We used HLECs to determine the effect of different concentrations of ß-CM-7 on cell viability by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolimol/L bromide (MTT) assay. We used flow cytometry to determine the content of reactive oxygen species (ROS) induced by oxidative stress and a bioassay kit to determine the oxidant malondialdehyde (MDA) and antioxidant enzyme superoxide dismutase (SOD) levels. We used Western blotting and an immunofluorescence assay to determine the expression of Forkhead box o1 (Foxo1), SP1, and the related protein glutathione peroxidase (GSH-px) at the molecular biology level as well as their intracellular localization. RESULTS: The expression of Foxo1 and SP1 was weakly expressed when the glucose concentration was 40 mM/L, but was highly expressed when cells were pre-treated with an appropriate concentration of ß-CM-7. After pre-treatment with ß-CM-7, the cells treated with 40 mM/L glucose for 48 h showed Foxo1 was transferred to the nucleus, and the expression of SP1 was increased. The content of ROS and MDA in the HLECs that were pre-treated with ß-CM-7 was lower than in those that was not pre-treated (p <0.05). Accordingly, SOD was elevated in the cells pre-treated with ß-CM-7. The relative expression of GSH-px increased with increases of Foxo1 and SP1. CONCLUSION: ß-CM-7 protects HLECs from oxidative damage by upregulating the relative expression of Foxo1 and promoting Foxo1 nuclear translocation.

Silver Inlaid with Gold Nanoparticle/Chitosan Wound Dressing Enhances Antibacterial Activity and Porosity, and Promotes Wound Healing.

Silver inlaid with gold nanoparticles (Au-Ag NPs) prepared by using egg white with an average sized of 10 nm and homogeneous dispersion were tested and presented red fluorescence. Au-Ag NPs were loaded into chitosan as wound dressing (CS-Au-Ag). CS-Au-Ag released silver ions faster, in higher amount, and in a more durable manner than chitosan dressing loaded with silver nanoparticles with the same silver content (CS-Ag), consequently, showing enhanced antibacterial activity. Cytotoxicity tests indicated that CS-Au-Ag showed low cytotoxicity to L929 cells similar to CS-Ag. These data suggest that cytotoxicity, which restricts further application of silver NPs, can be eliminated by decreasing the silver content. CS-Au-Ag presented rich and well-distributed pores, good mechanical properties, and enhanced swelling and retention properties, contributing to keeping the wound moist in the presence of residual egg white. Altogether, our results suggest that CS-Au-Ag greatly promoted wound healing compared to CS-Ag in vivo, demonstrating that CS-Au-Ag presents great potential for wound dressing, promoting wound healing.

Dissolution behavior of silk fibroin in a low concentration CaCl2-methanol solvent: From morphology to nanostructure.

Regenerated Silk biomaterials are usually pre-formed from silk fibroin solutions. However, the dissolution of silk fibroin in proper solvents by a simple and low cost way is still a challenge. Here, we employed a CaCl2-methanol solvent system with a very low CaCl2 concentration of 6wt% to dissolve silk fibroin. During the dissolution process, the evaporation of methanol cause the changing of solvation sheath of ions in the solvent. The remaining solvent with the incomplete solvation sheath is absorbed by the silk fiber and interacts with fibroin chains to complete the solvation sheath, which accounts for the dissolution of silk fibroin. Silk fibroin dissolution stops as all the solvation sheaths are complete. The final CaCl2 concentration is ca. 26% and silk fibroin is completely dissolved with a yield of about 90%. Silk fibroin is dissolved into multi-scale nanofibrils solution which is potential for producing regenerated silk fibroin materials for functional applications.

Self-assembly of natural protein and imidazole molecules on gold nanoparticles: Applications in wound healing against multi-drug resistant bacteria.

Developing highly active and green antibacterial agents for pathogens, especially multidrug-resistant superbugs, is vital for solving the problem of serious antibiotic resistance. Herein, we report a unique system of gold nanoparticles coated with chicken egg white (CEW) and 2-mercapto-1-methylimidazole (MMT) as a novel antibacterial agent. The CEW was used to prepare the gold nanoparticles as a commercially available reducing and stabilizing agent, and then the MMT self-assembled on the surface of nanoparticles. The resulting Au@CEW/MMT was found to be a highly efficient antibacterial agent, and the activity is mainly attributed to the synergistic effects of MMT and Au@CEW in undermining the bacterial membrane. Meanwhile, the studies of antibacterial activities and biocompatibility of Au@CEW/MMT with different ratios of MMT conjugation to Au@CEW confirmed that Au@CEW/MMT3 (MMT:HAuCl4 = 1:50) can maintain a balance between antibacterial properties and biocompatibility. Furthermore, in an in-vivo study using the rabbit model, gauze loaded with Au@CEW/MMT3 can effectively accelerate the healing of wounds infected with methicillin-resistant S. aureus and promote the formation of collagen. Therefore, this work illustrated a promising material with broad-spectrum antibacterial activities for preclinical applications in treating wound infections.

Imidazole-molecule-capped chitosan-gold nanocomposites with enhanced antimicrobial activity for treating biofilm-related infections.

Biofilms that are widely associated with persistent bacterial infections impose a heavy burden on patients primarily due to their formidable resistance to conventional antiseptic drugs and local immune defense. Here, we successfully synthesized functional gold nanocomposites (CS-Au@MMT) by reducing chloroauric acid in the presence of biocompatible chitosan polymers with cationic amine and the small molecule 2-mercapto-1-methylimidazole (MMT). The cationic amine allowed transport of the CS-Au@MMT to the negatively charged sites at the surface of bacterial cells though electrostatic adhesion, with synergistic effects from the gold nanoparticles and MMT then exerting a strong bactericidal effect to inhibit biofilm formation. For established mature biofilms, CS-Au@MMT was able to adhere to the biofilm surface to render nearby bacterial cells inactive, resulting in biofilm rupture. This allowed CS-Au@MMT to penetrate through the biofilm, leading to sustained damage and achieving biofilm elimination. Furthermore, the nanocomposites efficiently inhibited infections induced by mature biofilm in vivo. These findings indicated that the CS-Au@MMT nanocomposites provide ease of synthesis and fabrication, high bactericidal effect, and low toxicity; thus, they show potential as biofilm-disrupting agents for biomedical and industrial applications.

A novel wound dressing based on a Konjac glucomannan/silver nanoparticle composite sponge effectively kills bacteria and accelerates wound healing.

A novel Konjac glucomannan/silver nanoparticle (KGM/AgNP) composite sponge was successfully prepared via a simple 2-step method for biomedical applications as wound-healing materials. First, AgNPs were prepared with green deoxidizer egg white. Then, KGM powder was added to the AgNP solution and stirred vigorously, and the composite sponge was obtained by freeze-drying. The KGM/AgNP composite sponge showed excellent water absorption and water retention, and considerable mechanical properties. KGM/AgNP composite sponges displayed good antibacterial activity against test microorganisms. In vitro cytocompatibility testing showed that L929 cells could survive well in the presence of KGM/AgNPs, indicating that KGM/AgNPs have good cytocompatibility. Animal models showed that the KGM/AgNP composite sponges effectively accelerated wound healing, and histological findings showed that they promoted fibroblast growth and accelerated epithelialization. The experimental results showed that KGM/AgNP composite sponges have great potential in promoting wound healing.

Author Correction: In situ assembly of Ag nanoparticles (AgNPs) on porous silkworm cocoon-based wound film: enhanced antimicrobial and wound healing activity.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

In situ reduction of silver nanoparticles by chitosan-l-glutamic acid/hyaluronic acid: Enhancing antimicrobial and wound-healing activity.

Spongy composites with silver nanoparticles (AgNPs) were synthesized by freeze-drying a mixture of silver nitrate (AgNO3) and chitosan-l-glutamic acid (CG) derivative loaded with hyaluronic acid (HA) solution. CG/AgNP spongy composites had an interconnected porous structure and rough surfaces. When AgNPs (5-20nm) were immobilized on these spongy composites, AgNP aggregation was dependent on AgNO3 concentration. The spongy composites exhibited good mechanical properties, swelling, and water retention capacity. In vitro antibacterial activity showed that the CG/AgNP spongy composites effectively inhibited bacterial (Escherichia coli and Staphylococcus aureus) growth and penetration. Spongy composites containing low concentrations of AgNP were non-toxic to L929 cells, while CG/HA/AgNP spongy composites promoted wound healing, as determined by in vivo tests, wound contraction ratio, average healing time, and histological examination. These results indicate that the spongy composites can serve as effective antibacterial wound dressings.

Accelerated wound-healing capabilities of a dressing fabricated from silkworm cocoon.

Silk fibroin materials have shown some success in wound dressing applications; however, their use for this purpose remains limited by a complex production process and wasted sericin. In the present study, Bombyx mori cocoon materials are used because the protective function of the silkworm cocoon resembles the manner in which the skin protects the human body. A series of silkworm cocoon sol-gel film (SCSF) wound dressings are prepared by immersion in a CaCl2-ethanol-H2O solution for different treatment times. The accelerated wound-healing capabilities of SCSFs are systematically evaluated. Among them, the SCSF sample immersed for 90min exhibits stronger biocompatibility and antibacterial performance compared to other SCSFs. SCSF-90 also exhibits excellent transparency, a high swelling ratio, and good extensibility. Furthermore, in vivo experiments indicate that SCSF-90 can significantly accelerate the healing rate of wounds in New Zealand white rabbits, compared to the standard Mepitel® dressing, and histological examinations reveal that SCSF-90 aided in the successful reconstruction of intact and thickened epidermis. These results demonstrate that the proposed approach may be utilized in the design of antibacterial materials with promising applications in wound dressing.

In situ assembly of Ag nanoparticles (AgNPs) on porous silkworm cocoon-based wound film: enhanced antimicrobial and wound healing activity.

Preventing wound infection and retaining an appropriate level of moisture around wounds represent the most critical issues in wound treatment. Towards these ends, special focus has been placed on Bombyx mori cocoons because the protective function of the silkworm cocoon resembles the manner in which the skin protects the human body. We have designed a facile technique to develop a novel silkworm cocoon-based wound film (SCWF) wound dressing utilizing a CaCl2-ethanol-H2O solution. To improve the anti-bacterial performance of SCWF, we have incorporated the ability of silk sericin to act as a reducing agent for the conversion of Ag+ to Ag, yielding nanoparticles (AgNPs) linked together by peptide bonds of silkworm cocoon wound film (SCWF-AgNPs). SCWF-AgNP dressing exhibited excellent biocompatibility, anti-bacterial performance, and good extensibility. Furthermore, in vivo experiments indicated that SCWF-AgNP dressing was able to significantly accelerate the healing rate of infected wounds in New Zealand White rabbits and histological examination revealed that it aided in the successful reconstruction of intact and thickened epidermis during 14 days of healing of impaired wound tissue. These results demonstrate that the present approach might shed new light on the design of anti-bacterial materials such as SCWF-AgNPs with promising applications in wound dressing.

Preparation and properties of a novel thermo-sensitive hydrogel based on chitosan/hydroxypropyl methylcellulose/glycerol.

Chitosan-based thermosensitive hydrogels are known as injectable in situ gelling thermosensitive polymer solutions which are suitable for biomaterials. In this study, a novel thermosensitive hydrogel gelling under physiological conditions was prepared using chitosan together with hydroxypropyl methylcellulose and glycerol. Hydroxypropyl methylcellulose is to facilitate the thermogelation through large amounts of hydrophobic interactions. Glycerol in heavy concentration destroys the polymer water sheaths promoting the formation of the hydrophobic regions, and lowering the phase transition temperature. The thermosensitive hydrogels showed a physiological pH ranging from 6.8 to 6.9 and gelation time within 15min at 37°C. The prepared hydrogels were characterized by FT-IR, XRD, SEM, and rheological studies, mechanical studies and contact angle studies. The properties of degradability, cytotoxicity and protein release behaviors of the hydrogels were investigated. The results indicate this thermosensitive hydrogel possess good fluidity, thermosensitivity and biodegradability, as well as low-cytotoxicity and controlled release, showing the potential use in biomedical applications.

Preparation of a partially carboxymethylated cotton gauze and study of its hemostatic properties.

In this study, we attempted to modify cotton gauze by partial carboxymethylation by varying the reaction time and concentration of monochloroacetic acid and sodium hydroxide. For each experiment, the relative value of the degree of substitution (DS) of the modified cotton gauze was evaluated and the whole blood clotting time (WBCT) and water absorption property were compared with cotton gauze and Surgicel. This revealed that, following an initial decrease, WBCT gradually increased. Using rabbit ear artery and liver haemorrhage models, the performance of the optimal modified gauze was compared to that of Surgicel and unmodified cotton gauze. The average bleeding times in the presence of modified cotton gauze in the rabbit ear arteries and the liver were 51.7s and 60.6s, while those with Surgicel and the unmodified cotton gauze were 76.8s and 95.5s, and 93.2s and 129.2s, respectively. The hemostatic and biocompatibility properties were evaluated using in vivo degradation experiments. This revealed that the modified gauze and Surgicel were totally degraded within 6 weeks.

Preparation and characterization of N-chitosan as a wound healing accelerator.

Chitosan is insoluble in water due to its rigid crystalline structure, which has significantly restricted its application in wound healing. The objective of this study was to synthesize a water-soluble chitosan derivative, N-succinyl-chitosan (NSC), and evaluate its ability to accelerate the wound healing process. NSC was synthesized with succinic anhydride, hydrochloric acid, and alkaline chitosan under optimized conditions, and characterized using Fourier transform infrared, proton nuclear magnetic resonance, and X-ray diffraction spectroscopy; thermal gravimetric analysis; and a solubility test. The cytotoxicity of NSC was investigated in L929 cells, and its antibacterial activity was evaluated by the inhibition zone method and bacterial growth curves analysis. The results showed that the solubility of NSC was substantially improved compared to chitosan, and NSC was non-toxic with good antibacterial properties. An animal wound healing test indicated that NSC could significantly reduce the healing time compared to chitosan. Histopathological examination suggested that the underlying mechanisms of these effects were related to NSC's ability to promote the formation of granulation tissue and enhance epithelialization. Collectively, these results demonstrate the good potential for NSC to be applied as a wound dressing material.

Chitosan/gelatin composite sponge is an absorbable surgical hemostatic agent.

Chitosan is a versatile biological material that is very well known for its hemostatic properties. The purpose of this study was to test the hemostatic properties of a chitosan composite obtained from silkworm pupae and gelatin. This spongy porous material was cross-linked with tannins and then freeze-dried under vacuum to obtain composites containing chitosan and gelatin in different proportions. Results showed that the best blood-clotting index (BCI) was achieved in vitro by a chitosan/gelatin sponge (CG) ratio of 5/5 (W/W). Furthermore, CG had the best hemostatic effect in rabbit artery bleeding and liver model tests compared to the two components separately. The better hemostatic effect of CG may be due to its ability to absorb blood platelets easily and to the higher liquid adsorption ratio. However, no obvious differences were observed in thrombin generation with both aPTT and PT tests. Cell toxicity tests with L929 cells showed that CG caused no obvious cytotoxicity. In addition, subcutaneous transplantation of CG into rabbits resulted in almost complete degradation of CG after 6 weeks, together with rich vascular generation and proliferation in the transplanted region. Thus, CG can be considered an effective absorbable hemostatic material.

Ultraviolet A exposure induces reversible disruption of gap junction intercellular communication in lens epithelial cells.

Gap junction intercellular communication (GJIC) is essential for the proper function of many organs including the lens. Disruption of GJIC can cause lens metabolic disorder and can induce cataracts. The purpose of this study was to investigate the signal transduction pathways involved in GJIC disruption following ultraviolet A (UVA) exposure in lens epithelial cells. Following exposure of human lens epithelial cells to UVA, connexin 43 (Cx43), the main component of gap junctions, was down-regulated at both the mRNA and protein levels. Furthermore, we observed that UVA exposure can increase protein kinase C activity and stimulate reactive oxygen species generation and lipid peroxidation. Using scrape load dye transfer technique, we found that the GJIC is compromised by UVA exposure. In addition, we demonstrated that UVA-induced modulation of GJIC was associated with p38 mitogen-activated protein kinase activation. More importantly, at non-lethal doses (10 J/cm²), the UVA-induced GJIC disruption and the consequent alterations were reversible. Collectively, our data revealed a new signaling pathway in GJIC disruption following UVA exposure, suggesting that UVA-compromised gap junction activity may sensitize human lens to photoaging and cataract formation.

Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield.

Spider dragline silk is a unique fibrous protein with combination of tensile strength and elasticity, but the isolation of large amount of silk from spiders is not feasible. In this paper, we used a newly established Bac-to-Bac/BmNPV Baculovirus expression system to express the recombinant spider (Nephila clavata) dragline silk protein (MaSp1) fused EGFP in BmN cells and larvae of silkworm. A 70 kDa fusion protein was visualized after rBacmid/BmNPV/drag infection by SDS-PAGE and immunoblotting analysis. Fusion protein expressed in the BmN cells probably occupied five percent of the cell total protein; In a silkworm larva, approximately 6 mg fusion proteins were expressed. Solubility analysis of the expressed spider dragline silk protein indicated that 60% fusion protein is insoluble. EGFP fluorescence showed that fusion protein is tend to form aggregate by self assemblage. The results indicated the solubility is the primary limit for spider dragline proteins yield. It also suggested that directly produce fibrous spider silk in the secreting-silk organs of the transgenic silkworm larvae might be a better method.

No advantage of cell-penetrating peptides over receptor-specific antibodies in targeting antigen to human dendritic cells for cross-presentation.

Induction of CTL responses by dendritic cell (DC)-based vaccines requires efficient DC-loading strategies for class I Ags. Coupling Ags to cell-penetrating peptides (CPPs) or receptor-specific Abs improves Ag loading of DCs. In contrast to CPPs, receptor-specific Abs deliver conjugated Ags to DCs with high specificity, which is advantageous for in vivo strategies. It has, however, been speculated that CPPs facilitate uptake and endosomal escape of conjugated Ags, which would potently enhance cross-presentation. In this study, we directly compare the in vitro targeting efficiency of a humanized D1 Ab directed against the human DC surface receptor DC-SIGN hD1 to that of three CPPs. The three CPPs colocalized within endosomes when targeted to human monocyte-derived DCs simultaneously, whereas hD1 was present in a different set of endosomes. However, within 75 min after uptake CPPs and hD1 colocalized extensively within the lysosomal compartment. Ab-mediated targeting of class I-restricted peptides to DC-SIGN enhanced cross-presentation of the peptides, while only one of the CPPs enhanced peptide presentation. This CPP and hD1 enhanced cross-presentation with equal efficiencies. Thus, we found no evidence of CPP specifically favoring the delivery of conjugated Ag to the DC class I presentation pathway. Given the specificity with which Abs recognize their targets, this favors the use of DC receptor-specific Abs for in vivo vaccination strategies.

In vivo targeting of antigens to human dendritic cells through DC-SIGN elicits stimulatory immune responses and inhibits tumor growth in grafted mouse models.

Multiple cancer vaccine trials have been carried out using ex vivo generated autologous dendritic cells (DCs) loaded with tumor antigen before readministration into patients. Though promising, overall immunologic potency and clinical efficacy might be improved with more efficient DC-based therapies that avoid ex vivo manipulations, but are instead based on in vivo targeting of DCs. For initial in vivo proof of concept studies, we evaluated targeting of proteins or peptides to DCs through DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN). Because the biology of DC-SIGN is different between mice and humans, we assess human DC-SIGN targeting in the setting of elements of a human immune system in a mouse model. Administration of anti-DC-SIGN antibodies carrying either tetanus toxoid peptides or keyhole limpet hemocyanin (KLH) to Rag2gammaC mice reconstituted with human immune cells raised stimulatory human T-cell responses to the respective antigen without additional adjuvant requirements. Furthermore, administration of anti-DC-SIGN antibody-KLH conjugate enhanced the adjuvant properties of KLH resulting in inhibition of RAJI (Human Burkitt's Lymphoma Cell Line) cell tumor growth in Nonobese Diabetic/Severe Combined Immunodeficient mice transplanted with human immune cells. Thus, mouse models reconstituted with human immune cells seem to be suitable for evaluating DC-targeted vaccines, and furthermore, targeting to DCs in situ via DC-SIGN may provide a promising vaccine platform for inducing strong immune responses against cancer and infectious disease agents.

CD200 expression on tumor cells suppresses antitumor immunity: new approaches to cancer immunotherapy.

Although the immune system is capable of mounting a response against many cancers, that response is insufficient for tumor eradication in most patients due to factors in the tumor microenvironment that defeat tumor immunity. We previously identified the immune-suppressive molecule CD200 as up-regulated on primary B cell chronic lymphocytic leukemia (B-CLL) cells and demonstrated negative immune regulation by B-CLL and other tumor cells overexpressing CD200 in vitro. In this study we developed a novel animal model that incorporates human immune cells and human tumor cells to address the effects of CD200 overexpression on tumor cells in vivo and to assess the effect of targeting Abs in the presence of human immune cells. Although human mononuclear cells prevented tumor growth when tumor cells did not express CD200, tumor-expressed CD200 inhibited the ability of lymphocytes to eradicate tumor cells. Anti-CD200 Ab administration to mice bearing CD200-expressing tumors resulted in nearly complete tumor growth inhibition even in the context of established receptor-ligand interactions. Evaluation of an anti-CD200 Ab with abrogated effector function provided evidence that blocking of the receptor-ligand interaction was sufficient for control of CD200-mediated immune modulation and tumor growth inhibition in this model. Our data indicate that CD200 expression by tumor cells suppresses antitumor responses and suggest that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing cancers.

Internalizing antibodies to the C-type lectins, L-SIGN and DC-SIGN, inhibit viral glycoprotein binding and deliver antigen to human dendritic cells for the induction of T cell responses.

The C-type lectin L-SIGN is expressed on liver and lymph node endothelial cells, where it serves as a receptor for a variety of carbohydrate ligands, including ICAM-3, Ebola, and HIV. To consider targeting liver/lymph node-specific ICAM-3-grabbing nonintegrin (L-SIGN) for therapeutic purposes in autoimmunity and infectious disease, we isolated and characterized Fabs that bind strongly to L-SIGN, but to a lesser degree or not at all to dendritic cell-specific ICAM-grabbing nonintegrin (DC-SIGN). Six Fabs with distinct relative affinities and epitope specificities were characterized. The Fabs and those selected for conversion to IgG were tested for their ability to block ligand (HIV gp120, Ebola gp, and ICAM-3) binding. Receptor internalization upon Fab binding was evaluated on primary human liver sinusoidal endothelial cells by flow cytometry and confirmed by confocal microscopy. Although all six Fabs internalized, three Fabs that showed the most complete blocking of HIVgp120 and ICAM-3 binding to L-SIGN also internalized most efficiently. Differences among the Fab panel in the ability to efficiently block Ebola gp compared with HIVgp120 suggested distinct binding sites. As a first step to consider the potential of these Abs for Ab-mediated Ag delivery, we evaluated specific peptide delivery to human dendritic cells. A durable human T cell response was induced when a tetanus toxide epitope embedded into a L-SIGN/DC-SIGN-cross-reactive Ab was targeted to dendritic cells. We believe that the isolated Abs may be useful for selective delivery of Ags to DC-SIGN- or L-SIGN-bearing APCs for the modulation of immune responses and for blocking viral infections.