Multifunctional Liposomes Fabricated Microneedle Arrays As A Vaccine Adjuvant-dual Delivery System

To develop effective mucosal vaccines,two types of multifunctional liposomes,the mannosylated lipid A-liposomes(MLLs)with a size of 200 nm and the stealth lipid A-liposomes(SLLs)of 50 nm,both loaded with a model antigen and NH4HCO3,were fabricated together into microneedles,forming the proSLL/MLL-constituted microneedle array(proSMMA),which upon rehydration dissolved rapidly recovering the initial MLLs and SLLs.Further exploration demonstrated that the MLLs reconstituted from the administered proSMMAs were mostly taken up by vaginal mucosal APCs,whereas the recovered SLLs trafficked directly to draining lymph nodes wherein to be picked up by APCs.Moreover,the antigens delivered by either liposomes were also cross-presented for MHC-I displaying by APCs thanks to lysosome escape,both of which occurred when lysosomal acidifying the liposome-released NH4HCO3 into CO2 and NH4+/NH3 to rupture lysosomes by gas expansion.The tracing experiments in vivo showed that SLLs delivered the vaccine to the lymph node APCs,whereas MLLs delivered the vaccine to the inoculated mucosal APCs.Thus,the proSMMAs are in fact a vaccine adjuvant-dual delivery system capable of eliciting robust humoral and cellular immunity against the invading pathogens,especially,the sexually transmitted ones.In this experiment,OVA and ammonium bicarbonate were co-encapsulated in multifunctional liposomes to construct microneedle vaccine adjuvant-double delivery system,in which,ovalbumin is model antigen.Combined with microneedleadministration mechanism,to carry out a vaccine delivery system through transmucosal route vaccination.To study the preparation and characterization of liposome microneedles,the promotion of multifunctional liposomes on APCs uptake of antigen,and the effect of multifunctional liposomes on APCs function and its mechanism.Analysis of the characteristics of its immune response,the initial clarification of the immune response effect.This study is divided into two parts that will be briefly described.The first part: Different types of multifunctional liposome microneedle arrays were constructed and characterized.There are many kinds of preparation methods of liposomes.Here we had successfully prepared two kinds of multifunctional liposomes:SLLs and MLLs with thin film dispersion method,and the microneedle arrays had been successfully prepared mixing with microneedle matrix related substances by microneedle mold.In the follow experiments,liposomes were loaded with fluorescent dye calcein and sulfonyl rhodamine B(SRB)in order to track the trends of liposomes in vivo and intracellular.The liposomes and proSMMA were characterized by the detection of liposomes size,zeta potentials and binding efficiencies.The second part: Investigation of section immune mechanism of different types of multifunctional liposomes.The main mechanisms of microneedle mucosal administered were discussed,including the observation of the intracellular localization of liposomes by APCs uptake by confocal microscopy.The liposome stimulated APCs activation and maturation and their antigen presentation were analyzed by flow cytometry,and then analyzing the tends of the two types of multifunctional liposomes in vivo after administered the pro-SMMA throuh the vaginal mucosa.