Construction Of 3DOM As BSA Carrier And Its Preliminary Evaluation For Oral Immunization

Oral delivery is the preferred route for drug administration due to its good patient compliance and safety nature.Oral delivery of antigens have several advantages,for example,it can induce not only mucosal immunity,but also a robust systemic immune response which making it to be an effective strategy to control infectious diseases.However,proteins and other biological macromolecules trend to inactivation/aggregation due to changes of external environment and will degradation when exposed to the proteolytic enzymes in the gastrointestinal tract,it is difficulty to induce sufficiently immune response just rely on antigen itself.In recent years,some inorganic carrier was successfully developed as an oral antigen delivery system.The three-dimensional ordered macroporous(3DOM)carrier has high porosity and interconnected huge pore space which can used to load protein in order to prevent the protein from degradation in the gastrointestinal tract.Its skeletal structure to endow it with high protein loadings,so the 3DOM carrier is expected to be an ideal antigen carriers for oral immune.On this view,according to the moleculae size of BSA,we constructed a 3DOM carrier with an 50 nm internal pore in order to acieve internal load and play a blockade effect during the release procedure of internal proteins.It is designed to take advantages of 3DOM carrier such as high protein loading ability and protect the protein from degradation when exposed to enzyme to verify the potential application of 3DOM carrier as BSA oral immune carrier.In order to prepare the 3DOM carrier that meeting the requirement,monodisperse PS spheres with particle size of about 300 nm were synthesized using a soap-free emulsion polymerization first.Then the 3DOM materials are synthesized using the PS spheres as the colloidal crystal template.In our research,a surface oxidation reaction was used as the surface modification method and we found that the carboxylated 3DOMC(S-3DOMC)carrier maintained its morphology after the successful surface modification.The external pore of our S-3DOMC is about 230 nm and internal pore is 50 nm.The bimodal pore system of 3DOMA is 260 nm and 60 nm,respectively.The adsorption equilibrium method was employed as our protein loading method and 3DOM carrier was used to construct the protein loading system.BSA/3DOMA and BSA/S-3DOMA system were obtained and the dissolution behavior was investigated.The release rate of protein was defferent between the high protein loading system and low protein loading system and a "plateau" appears in the dissolution curve.Both the 3DOMA and S-3DOMC carrier showed a high protein loading ability The S-3DOMC carrier has a high adsorption capacity of about 396 mg/g with our model biomacromolecules(BSA,the major axis was about 12 nm)and the 3DOMA carrier with has an adsorption capacity of about 325 mg/g.In our research,we found that our carrier showed a double-plateau adsorption behavior.SEM,Zeta potential and cumulative dissolution data were used to study the protein loading mechanism.The result showed that the emegence of platform was affected by the steric hindrance between BSA molecule and internal pore of 3DOM..Since the protein was adsorbed on different surface of carrier,the different release-rate between high and low protein loading system emerged.That meanings we could obtain different protein release rate by adjusting the amount of protein that adsorbed on the internal/external surface.Using this feature of our carrire,we also studied the relationship between the release rate of protein and immune strength in latter part of our study,and the results showed that a slow release of protein is benefitted to oral immune.On the basis of our previous research,we adopted a strategy of organic-polymer coating for our repository-type carrier and investigate the efficiency in inducing the mucosal immune response.The Zeta potential of CTS/BSA/S-3DOMC system was about-17.3 mv,PDDA/BSA/S-3DOMC and PEI/BSA/S-3DOMC was about-1.56 mv and 4.28 mv.Fluorescence spectroscopy and CD spectropolarimetry method was used to evaluate the conformational changes of protein.The result suggested that our carrier did not lead to a irreversible structural changes to BSA and the conformational properties did not change significantly for most protein molecules.Two kinds of 3DOM carrier and three kinds of polymer coated 3DOM carrier were used to immunized mice through oral route.Compared with free BSA alone,a significant level of serum IgG and IgA was induced by BSA loaded in both carrier and indicated that this customized carrier effectively induce immunity following oral administration.The IgG1 and IgG2a titers suggesting that both the cell and humoral immune response were induced.