The Preparation And Evaluation Of Anti-infection Wound Dressing Based On Silver Nanoparticles

Infections and complications are the main causes for the death of patients with trauma, posing a tremendous obstacle in clinical medicine. Currently, synthetic antibiotics are the most commonly-used means to prevent and heal the infection effectively. However, the overuse of antibiotics may result in the increasing antibiotic resistance of pathogenic bacteria."Superbugs"is a result of such resistance created by an ingenious mechanism. In order to resolve the problem of antibiotic resistance, researchers have attempted for new antibacterial agents to replace antibiotics.Silver has been used as an antimicrobial agent for centuries in the form of silver ions, metallic silver and silver compounds. The silver nanoparticles demonstrated more efficient antimicrobial property than other forms due to their extremely large surface-to-volume ratio which increased their contacts with microorganisms. Moreover, sliver nanoparticles could release silver ions persistently in the bacterial cells, making their bactericidal activity more durable. The application of silver nanoparticles is clinically appealing for anti-infection treatment. The effect of their applications was mainly determined by dimensions of the particles, their size distribution, morphology, stability, state of their surface from the point of view of physics (e.g. surface charge) and chemistry (surface modification) as well. The combination of silver nanoparticles with stabilizing agents may change the intrinsic physiochemical properties of silver to influence the cellular uptake, interaction with biological macromolecules and translocation within the human body. As a bacterial polysaccharide, dextran has excellent biocompatibility, biodegradability, and anti-infection activities. However, no report focused on whether the modification by dextran could improve the characteristics and bioactivities of silver nanoparticles.Chitosan is a polysaccharide biopolymer derived from naturally occurring chitin, which has good biocompatibility, controllable biodegradability, non-toxicity, nonantigenicity. The chitosan has a broad application prospects, such as biomedicine, tissue engineering, drug delivery. Recently, the research on chitosan sponge dressing has been reported. The sponge dressing can absorb wound exudate and maintain a moist environment for the wound healing. Chitosan has hemostatic and antibacterial properties, which can promote the formation of blood vessels and epithelial cells for wound healing. Therefore, the combine of nano-silver and chitosan sponge may display a more excellent effect for wound repair. However, the further experiments are needed for this assumption.In this study, the dextran-capped silver nanoparticles were prepared by a modified chemical reduction method, and their physical properties were evaluated. On this basis, the antibacterial properties of silver nanoparticles were tested, and their cytotoxicity was evaluated. At the same time, we tried to explore the interactions between nano-silver and cells. Secondly, the anti-infective chitosan-gelatin biological materials were prepared using dextran-capped silver nanoparticles as an antimicrobial agent, and the performance of the sponges were tested and evaluated. Finally, the SD rats were used as the wound infection models to evaluate the anti-infective and wound healing effects.This study can be divided into four parts:PartⅠ: The preparation and characteristics of dextran-modified silver nanoparticlesThe synthesis of dextran-capped silver colloid was carried out by a modified chemical reduction method. In order to demonstrate their stability, the PVP-capped silver nanoparticles prepared by the same reaction system were used as a control. The nanoparticles were characterized by ultraviolet–visible (UV–Vis) spectrometry, nano-grainsize analyzer, X-ray diffraction (XRD) and transmission electron microscopy (TEM).The results showed that the silver nanoparticles capped with dextran were in uniform shape and narrow size distribution. Dextran-capped silver nanoparticles showed a spherical shape, and their average size was about 8nm. Compared with polyvinylpyrrolidone-capped silver nanoparticles, the dextran-capped ones possessed better stability.Part II: The biological properties of dextran-capped silver nanoparticlesIn this study, the antibacterial properties and cytotoxicity of dextran-capped silver nanoparticles were evaluated. Antibacterial tests of these nanocomposites were carried out for Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The in vitro cytotoxicity tests of dextran-capped silver nanoparticles were performed using mouse fibrosarcoma cells (L929). The toxicity was evaluated by the changes of cell morphology, Live/Dead staining and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide assay. In addition, the TEM tests were performed to study the interaction mechanism between silver nanoparticles and cells.The dextran-capped silver nanoparticles were demonstrated to have highly antibacterial activity against both Gram-positive and Gram-negative bacteria. The minimum inhibitory concentration was about 5.0μg/mL, and the minimal bactericidal concentration was about 10.0~20.0μg/mL. Cytotoxicity tests showed that silver nanoparticle doses of 10.0μg/mL could cause obvious toxicity to L929 cells, including morphology changes and the suppression of proliferation. The nanoparticles entered into the cells through endocytosis, and interacted with organelles to induce cellular damage.Part III: The preparation of chitosan-gelatin sponges composited with silver nanoparticlesIn this study, the chitosan-gelatin sponges composited with silver nanoparticles were prepared by freeze-dried method. The surface topography and internal structure of the sponges were detected by scanning electron microscopy (SEM), and their swelling ratio was evaluated. In addition, the antibacterial properties of the sponges were tested through bactericidal test.The results showed that chitosan-gelatin sponges with multilayer porous structure could absorb water effectively. Antimicrobial test results showed that the chitosan-gelatin sponges had good antibacterial properties. Their antibacterial effect was significantly improved after the silver nanoparticles incorporated.Part IV: The preparation of rat wound infection model and wound healing experimentsIn this study, the wound infection models were prepared by 1.5×1.5cm full-thickness skin remove in the back of SD rats. And then the wound sites were inoculated with Staphylococcus aureus and Pseudomonas aeruginosa (107cfu). The wound infection models of rat were divided into 4 treatment groups: pure chitosan-gelatin sponge group, chitosan-gelatin sponge composited with silver nanoparticles, general wound dressing group and the control group. The status of wound healing were observed and recorded continuously. The blood perfusion of wound was analyzed by using Laser Doppler. Finally, the effects of sponge dressing on wound healing were evaluated by histological examination.The results showed that the chitosan-gelatin sponge composited with silver nanoparticles could behave anti-infection property effectively. The sponge cound promote the formation of scab, and the healing time was shortened by 3~5 days. Laser Doppler perfusion analysis indicated that the formation of blood perfusion around the wound. The strongest blood perfusion could promote wound healing through the compensatory supply of blood for the wound. Histological results showed that chitosan-gelatin sponge composited with silver nanoparticles could promote the regeneration of blood vessels and hair follicle structures in the wound.In summary, we successfully prepared dextran modified nano-silver colloids with good homogeneity and stability. The dextran-capped silver nanoparticles showed a significant inhibitory effect on Gram-positive bacteria and Gram-negative bacteria. This study can provide some reference for the development of new antibacterial agents. In addition, this study found that the dextran-capped silver nanoparticles could cause toxicity to the cells in a concentration-dependent manner, which provides an experimental basis for the safety evaluation of silver nanoparticles in the process of clinical application. Finally, the chitosan-gelatin sponges composited with silver nanoparticles were prepared by freeze-dried method, and used for the wound infection treatment in rats. Then the sponges were demonstrated to have excellent properties in the anti-infection and wound healing aspects.