| Skin is the natural barrier between human body and the external environment.Once damage occurs,it must be closed in time,otherwise acute and chronic infections of the wound and its corresponding complications will occur.As a temporary skin substitute,wound dressing can protect wounds,stop bleeding and prevent infections.The structure of nanofibers is similar to the extracellular matrix.It has the characteristics of absorbing fluid,permeable to water and oxygen,and antibacterial properties,which can improve the repair ability of damaged tissues.Electrospinning is an important technology for the preparation of biomimetic nanofiber membranes using biopolymers,which has the advantages of simplicity,efficiency,and low cost.Chitosan(CS)is a natural alkaline polysaccharide with good biocompatibility,antibacterial,bioadhesive and hemostatic properties.It is a potential wound dressing material.In addition,chitosan-based nanofibers can also be used as drug delivery vehicles to achieve drug release at the wound site and accelerate the wound healing process.Shikonin is a naphthoquinone compound extracted from the traditional Chinese medicine.It has various biological activities such as antibacterial,anti-inflammatory,promoting cell proliferation,and inhibiting scar formation.It has great application prospects in promoting wound healing.Shikonin-loaded preparations are currently used to treat skin wounds.Therefore,in this study,chitosan/polyvinyl alcohol(PVA)nanofibers loaded with shikonin were prepared by electrospinning technology,and the best prescription and preparation process were selected,characterizedand evaluated in vitro and in vivo.Combine the advantages of nanofibers and drugs to explore their application potential in the field of woundhealing.Part one Preparation and evaluation of CS/PVA nanofiber membranesObjective:Investigate the formulation and technology of CS/PVA nanofiber membranes and evaluate its potential as wound dressing.Methods:CS/PVA nanofiber membranes was prepared by electrospinning,and the best formulation and process were selected according to the morphology of nanofiber,and then crosslinked by glutaraldehyde vapor.The diameter,porosity,wettability,water vapor permeability and water absorption of CS/PVA nanofiber membranes were characterized and evaluated.Results:In this part,CS/PVA electrospun nanofiber membranes was prepared by optimizing the formulation and process.When the mass ratio of CS/PVA is 25/75,the voltage is 29 KV,and the speed of pushing liquid is0.0010 mm/s,the CS/PVA electrospun nanofiber obtained is smooth and without beads.The CS/PVA electrospun nanofiber membranes was crosslinked by glutaraldehyde vapor.Compared with the uncrosslinked nanofiber membranes,the water stability of the crosslinked nanofiber membranes for 3 hours increased,and the fiber diameter did not change significantly.The final CS/PVA electrospun nanofiber membrane has a diameter of 76.30±18.34 nm,a porosity of 51.87%,a contact angle of47.35±1.40°,good wettability,a water vapor transmission of 0.2587±0.0096g/(cm~2·24 h),a water absorption of 180.1±41.65%,and a water absorption capacity of 1.6?2.5 times of self weight.Conclusions:In this experiment,CS/PVA electrospun nanofiber membranes were prepared by electrospinning technology and cross-linked to increase water stability.The test results show that the nanofiber membrane is a hydrophilic dressing,which is conducive to cell adhesion and proliferation,can prevent the evaporation of certain water at the wound and absorb wound exudate,maintains a suitable humid environment,and has the potential as a wound dressing.Part two Preparation and evaluation of shikonin nanocrystalline suspensionObjective:Shikonin nanocrystalline suspension was prepared,its formulation and technology were investigated and evaluated.Methods:The suspension of shikonin nanocrystalline was prepared by anti-solvent precipitation-ultrasonic method,and the best preparation prescription and technology were selected by single factor test with particle size and polydispersity coefficient as evaluation index,and its particle size,PDI and appearance were evaluated.Results:The best preparation process of shikonin nanocrystalline suspension is as below:preparation temperature of 10?,ultrasound(250 W)for 15 minutes,drug concentration of 6 mg/m L,second cell crushing ultrasound(700 W)for 5 minutes.The particle size of the shikonin nanocrystal suspension prepared is 242.8±4.25 nm,and the PDI is0.146±0.024.Conclusion:The optimized preparation process is simple and feasible,and the particle size of the suspension is small and uniform.Part three Preparation and evaluation of shikonin-loaded nanofiber membranesObjective:The nanofiber membrane containing shikonin was prepared by electrospinning technology.The content determination method of shikonin was established.The characteristics of nanofiber membrane and its drug release performance were investigated.Methods:The shikonin-loaded CS/PVA nanofiber membranes was prepared,and the diameter and porosity of the nanofiber were characterized.The high-performance liquid chromatography was used to determine the shikonin content,and the drug release performance of the drug-loaded nanofibers was investigated.Results:The method for the determination of shikonin by high performance liquid chromatography was established,and the linearity,stability,precision and recovery were in line with requirements.The drug-loaded nanofiber membrane was successfully prepared.The SEM image showed that the diameter of the nanofiber was 82.60±22.71 nm,and the porosity was 50.92%.The drug-loaded nanofibers have an encapsulation rate of 90%,and the accumulated releasing amount of drug was more than90%in 5 hours.Conclusion:The morphology,diameter and porosity of the drug loaded nanofiber membranes are not significantly different from those of CS/PVA nanofiber membranes without drug loading,and the drug-loaded nanofiber membrane has good drug release performance and can be used as a functional wound dressing of nanofiber membrane.Part four In vitro and in vivo evaluationObjective:To investigate the antibacterial effect and wound healing effect of blank CS/PVA nanofiber membrane and drug-loaded nanofiber membrane.Methods:The antibacterial effects of nanofiber membrane on E.coli and S.aureus were investigated by inhibition zone test,and the effect of promoting wound healing was investigated by mouse skin wound repair test.Results:The blank and drug-loaded nanofiber membranes had no obvious antibacterial effect on E.coli.The drug-loaded nanofiber membranes had a slightly better antibacterial effect than the blank nanofiber membranes on S.aureus,but the antibacterial effect was not strong.Animal test results showed that compared with the control group,CS/PVA nanofiber membrane and drug-loaded nanofiber membrane could effectively promote wound closure in the early stage of wound healing.After the sixth day,the wound healing rate of the control group was 50.00%,the CS/PVA nanofiber membrane group was 63.64%,and the drug-loaded nanofiber membrane was as high as 78.87%.After loading the drug,the wound healing effect was better,and the wound could be completely closed within 12 days.By histological observation,CS/PVA nanofiber membrane and drug loaded nanofiber membrane could promote the formation of new skin tissue,and the tissue structure of drug loaded nanofiber membrane group was closer to normal skin tissue.Conclusion:Shikonin-loaded CS/PVA nanofiber membranes can accelerate the wound healing process and can be used as a new type of wound dressing. |
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