Preparation Of Chitosan Nanoparticles Loaded Hydrogel And Its Effect On Skin Wound Repair

ObjectiveIn clinical practice,most wound infections are caused by antibiotics and antibacterial drugs,which are difficult to control and have no function of promoting wound healing.However,the dressings commonly used for wound healing do not have antibacterial ability,so a wound healing dressings with excellent antibacterial effect is particularly important.The purpose of this study was to prepare an antimicrobial dressing for wound healing using chitosan nanoparticles(CS NPs)and sodium alginate as raw materials.Antibacterial test,cytological test and pharmacodynamic test were conducted to study the antibacterial effect of the dressing and the effect and mechanism of promoting wound healing.MethodsChitosan nanoparticles were prepared using chitosan and polysodium phosphate as raw materials by ionic cross-linking method.CS NPs loaded CaAlg hydrogel was prepared using sodium alginate,chitosan nanoparticles and calcium chloride as raw materials.The formulation was optimized according to the release of chitosan nanoparticles.The surface morphology,structure and rheological properties were characterized by scanning electron microscope(SEM)and infrared spectroscopy(IR),rheological analysis and swelling rate,and the antibacterial effect and mechanism of the dressing were explored through bacterial experiments.In the cytological evaluation,thiazolyl blue tetrazolium bromide(determined by MTT method)makes a research on the chitosan nanoparticles hydrogel Human umbilical vein endothelial cells(Human umbilical vein endothelial cells,HUVECs)proliferation;the changes of activated oxygen content in HUVECs were detected by chitosan nanoparticle hydrogel and reactive oxygen inhibitor.Scratch and Transwell experiments were conducted to investigate the effect of chitosan nanoparticles hydrogels on the migration ability of HUVECs.To explore the effect of chitosan nanoparticles hydrogel on angiogenesis through angiogenesis experiment;Finally by Western Blot experiments testing chitosan nanoparticles hydrogel effect on the protein expression level in cells,including inflammatory factor interleukin-6(IL-6),vascular endothelial growth factor(VEGF),matrix metalloproteinase(MMP-2),antiapoptoic factor(Bcl-2),etc.In the pharmacodynamic experiment,the wound healing of different groups was observed using the rat skin wound model,and the wound tissue was stained with H&E to evaluate the wound healing quality.ResultsA fluorescence spectrophotometric method was established to analyze the content of chitosan nanoparticles.The formulation was optimized according to the release of chitosan nanoparticles from the gel.The surface porosity of chitosan nanoparticles hydrogels is low and film forming.Chitosan nanoparticles are uniformly dispersed in the hydrogels.The average particle size of chitosan nanoparticles was about 208.4±15.7 nm,the Zeta potential was 24.2±3.9 mV,the gel showed shear thinning characteristics,the gel viscosity decreased with the increase of shear rate,the swelling rate of hydrogels increased with time,and increased with the increase of pH value.In bacteriostatic experiment,the higher the concentration of chitosan nanoparticles in the hydrogels bactericidal effect,the better,extracellsular nucleic acid testing results show that the load chitosan nanoparticles extracellsular nucleic acid amount of hydrogels group were significantly higher than the load chitosan hydrogels group,and with the increase of concentration of nanoparticles,extracellsular nucleic acid quantity will increase,coated board experiment along with the increase of the concentration of chitosan nanoparticles can completely kill two kinds of bacteria.In cells experiments,through determined by MTT method,chitosan nanoparticles hydrogels on HUVECs has no cytotoxicity,effect is not obvious,but the proliferation of chitosan nanoparticles hydrogels can promote the generation of reactive oxygen species HUVECs,and can promote its migration,invasion and angiogenesis,and after joining ros inhibitors,migration,invasion and angiogenesis significantly reduced.Then we by Western Blot examines the load chitosan nanoparticles hydrogels influence on cells protein expression levels,according to the results,at the time of 12 h,can promote the expression of IL-6 and cause inflammation factors of acute inflammation cells associated in wound area to raise all sorts of wound healing,at 48 h can reduce inflammatory factor expression of IL-6 anti-inflammatory effect.At 48 h,MMP-2 and VEGF were promoted,and there was no significant difference in anti-apoptotic protein Bcl-2.In vivo experiments,chitosan loaded nanoparticles improved wound healing speed and quality compared with the control group and chitosan loaded hydrogels.ConclusionsChitosan nanoparticles have suitable size,and good stability.Chitosan nanoparticle hydrogels have low porosity and they are film-like.Nanoparticles are evenly dispersed in the hydrogels and have good antibacterial effects.They are not cytotoxic to HUVECs.From the mechanism through the regulation of inflammatory factors,metalloproteinases,growth factors,and so on,promote cell migration and invasion,as well as angiogenesis.Animal experiments have shown that chitosan nanoparticles hydrogel can accelerate wound healing and improve the quality of wound healing.