Transdermal Immune System Based On Ethosome/Silk Fibroin Composite Nanofibrous:Construction And Evaluation

Currently,vaccines are mainly administered subcutaneously or intramuscularly which can provide good vaccination effect for most people.However,these conventional methods are deficient in the aspects including suffering from needle contamination,causing pain,and need for professional medical staff to operate,as well as harsh conditions for storage and transportation of vaccine.Therefore,looking for an efficient and convenient needle-free vaccine delivery system has become a hot spot in pharmacy and immunology.Transcutaneous immunization(TCI)is a novel immunization strategy developed in recent years.It is a new method of eliciting a systemic immune response a by topically applying the antigen and adjuvant to the skin,which can overcome the shortcomings of current vaccination methods.As it is difficult for macromolecule antigen protein to pass through the barriers of cuticle,how to make the macromolecule break through the skin barrier into the active epidermis and dermis layers which is rich of antigen-presenting cells(Antigen-presenting cells,APCs)is the key to TCI.Liposomes have a similar structure with cell membrane and thus can be easily fused with cell membrane,which make it has good transdermal performance.Ethosome(Eth)is a special liposome which is more flexible and has better transdermal properties than liposome due to its containing of a certain amount of alcohol.Silk fibroin(SF)nanofibers have excellent biocompatibility and skin affinity.Our previous study confirmed that transdermal drug delivery system based on Eth/SF composite nanofiber membrane could promote the transdermal delivery of bovine serum albumin and plasmid effectively.In this study,we constructed a novel TCI system based on Eth/SF nanofibrous matrices and using OVA as the model drug,which was able to actively target the dendritic cells(Dendritic cells,DCs).This system was evaluated comprehensively.The main research contents and results are as follows:(1)Constructtion of ethosomes which can actively target DCs.Cationic ethosomes were prepared by film dispersion method.The Eth-HA was obtained by modifying a HA layer to the Eths and the Eth-GC was obtained by modifying a galactosylated chitosan(GC)layer on Eth-HA.The average particle size of the unmodified ethosomes was 135.4 nm.The Eth-HA was negatively charged and its particle size became bigger than that of the unmodified ones.The Eth-GC was positively charged,with a particle size between the former two.In vitro transdermal test showed that the modified ethosomes could deliver the protein molecules into the dermis layer effectively.(2)Evaluative study on the TCI performance of Eth-GC.The DCs precursor cells were extracted from C57BL/6 mice for evaluating the uptake of Eth-GC.The results showed that Eth-GC not only target DCs specifically but aslo promote the expression of CD11 c,CD80 and CD86.The ethosomes could also promote the secretion of TNF-?,IL-2 and IL-6,which indicated that they could promote the maturation of DCs.Further animal experiments showed that OVA-loaded Eth-GC(OVA@Eth-GC)could induce more anti-OVA-specific antibodies in transdermal-elicited mice,indicating that the OVA@Eth-GC effectively stimulated the antigen-specific humoral immune response in mouse.(3)Construction of Eth-GC/SF composite nanofibrous membrane and evaluation of its TCI performance.The OVA@Eth-GC/SF composite nanofibrous membrane was prepared by green electrospinning technology.Scanning electron microscopy,microfluorescence and MTT assays confirmed that the membrane had smooth surface,uniform fiber distribution and good cell compatibility.The results showed that the cell uptake rate of this system was higher,and the expression of CD11 c,CD80 and CD86 on the cell surface was also enhanced,which indicated that the structure of Eth-GC was not destroyed,the Eth-GC released from the nanofibers could still target DCs.Animal experiments confirmed that the system can stimulate mice to produce more anti-OVA specific antibodies,IFN-?,IL-2 and IL-6,and can inhibit the growth of the tumor.All this results indicated that this TCI system could induce effective humoral and cellular immunity.In conclusion,the Eth-GC and Eth-GC/SF composite nanofiber TCI system can not only effectively deliver antigenic proteins to the dermal layer of skin but aslo stimulate an effective immune response,which suggestting a broad application of this system in transdermal immunization and other biomedical related fields.