Fundamental Study On The Mesoporous Silica Nanoparticles With Large Pore Size(MSNLP)as Oral Vaccine Carriers

Oral vaccine is an effective strategy to control infectious diseases by inducing both systemic and mucosal immune responses.However,the new generation of vaccines obtained by recombinant DNA technology and chemical synthesis has poor immunogenic and is easy to degrade in the gastrointestinal environment due to the lack of active ingredient such as the DNA of bacteria and lipopolysaccharide,which usually contained in traditional vaccines,therefore limits its practical application to a large extent.The carrier of oral vaccine can provide an effective method to improve the antigen immune response.However,the traditional oral vaccine carrier such as liposomes,chitosan and PLGA microspheres is easy to degrade and leakage and have a poor stability in the gastrointestinal environment,which limits its practical application.In resent years,mesoporous silica nanoparticles(MSNs)has been paid widespread attention as a novel oral pharmaceutical carrier due to their characters of non-toxic,good biocompatibility,large surface area,tunable pore size and controllable morphology.However,the report about taking it as an oral vaccine carrier is rare.The purpose of this study is to prepare oral vaccine by taking prepared mesoporous silica nanoparticles with large pore size(MSNLP)as a carrier and BSA as a model antigen for their following popential advantages.?MSNLP have a large aperture(up to 40 nm)in favor of antigenic molecules into the duct,therefore the chemically stable and rigid skeleton of MSNLP can keep activity of the antigen in the gastrointestinal tract.?MSNLP have a controllable morphology.The release rate of the antigen can be slowed by control the particle size and pore structure of MSNLP,thereby achieving the "storage" function of the carrier.?MSNLP have a adjustable particle size.Preparation of MSNLP with a smaller particle size can improve the interaction between antigens and immune related cells,thereby enhancing immune responses.In order to examine the usefulness of MSNLP as an oral vaccine carrier and examine the relationship between the characteristic of MSNLP and immune responses,this paper makes the following studies.MSNLP was successfully prepared by sol-gel method.Honeycombed MSNLP(S1)has a particle diameter of bout 430 nm and a pore size of about 40 nm.Vermiculate MSNLP(S2)has a particle size of about 130 nm and a pore size of 25-30 nm.The classic mesoporous silica SBA-15 was prepared with length of about 1?m,width of about 400 nm and a two-dimension pipe-like structure with pore size of about 7 nm.In order to examine the effect of hydrophilic-hydrophobic property of the carrier on immune responses,honeycombed mesoporous carbon nanoparticles with large pore size(MCNLP)was prepared(C1)with particle size of about 430 nm and pore size of about 130 nm.Then,the above four kinds of nanoparticles were taken as a carrier and BSA was taken as a model antigen to prepare oral vaccine.The in vitro release behavior of BSA loaded carrier was investigated in simulated gastrointestinal solution.The BCA detection method for protein quantitation was established.The results show that the adsorption method obtained a better antigen loading effect and the percentage content of antigen adsorption is 3.96%,4.92%,1.97%and 7.32%for S1,S2,SBA-15 and Cl respectively.The morphology and structure of the BSA loaded system was investigated by SEM and nitrogen adsorption.The release behavior of the BSA loaded system was evaluated in simulated gastrointestinal solution.At last,ELISA was used to examine the IgG IgG1 and IgG2a in the serum and IgA in the salivary and intestinal secretions after the mice were gastrointestinal immunized.The results shows that oral immunization with free BSA induced almost no immune responses and the IgG titer of serum and IgA titer of salivary and intestinal secretions is(1.5±0.1,0.58±0.2 and 1.58±0.15)respectively.A robust humoral immunity and a negligible mucosal immunity were observed following i.m.immunization of BSA emulsified in FCA and the IgG titer of serum and the IgA titer of salivary and intestinal secretions is(4.6 ± 0.2,0.78 ± 0.2 and 1.58 ± 0.15).Following oral immunization with BSA loaded in S1,S2 SBA-15 and C1,a significantly higher level of immune response was observed and systemic and mucosal immune responses were successfully induced.The IgG titer of serum and IgA titer of salivary and intestinal secretions is(4.3±0.2,2.35±0.2 and 3.76±0.3)in S1 group,(4±0.2,1.95±0.2 ? 3.28±0.3)in S2 group,(3.6±0.2,1.5±0.2 and 2.76±0.1)in SBA-15 group and(4±0.2,1.8±0.1 and 2.35±0.2)in C1 group respectively.The usefulness of MSNLP used to improve both the humora and mucosal immune responses was validated.The effect of the structure and hydrophilic-hydrophobic property of the carrier on immune response was also investigated.The combination nature of a right size,a large honeycombed mesopores(40 nm)and a property of hydrophility for S1 can provide an effective profect,slow down the release rate and increase the uptake of immune cells,which can improve both humoral and mucosal immune responses significantly.