| Oral immunization can induce both mucosal as well as systemic immune responses, mucosal responses are not generally observed following systemic immunization. Orally administered vaccines have a number of other potential advantages in terms of improved safety, patient compliance and acceptance and economy. It has been established that mucosal immune system plays an important role to protect animals and humans from viral, bacterial or parasitic pathogens initiate infections, independent of systemic immunity.However, there are a number of problems encountered when delivering antigen via the gastrointestinal tract, in particular the substantial degradation of antigens by gastric hydrochloric acid and proteolytic enzymes, and the poor absorption of antigens by the gut associated lymphoid tissue (GALT). The purposes of the present study were to develop the novel vaccine delivery system to overcome the two main obstacles. Therefore, two kinds of liposomes, including mannose-modified bile salt enclosed liposomes (OPM-coated NaDC-Lip) and TMC-coated multivesicular liposomes (TMC-coated MVLs), were developed to improve the oral delivery efficiency of vaccine.Taking into account that the lipid component could be dissolved in ethanol, and BSA would be precipitated due to its lower dissolubility in ethanol, so ethanol precipitation-BCA method was developed to determine the content of protein in lipid samples.NaDC-Lip were prepared by the method of reverse-phase evaporation. The optimum amount of total lipid, CH to PC ratio (w/w) and NaDC to total lipid molar ratio were 225 mg,0.5 and 0.5, respectively. NaDC-Lip were characterized for shape, size and zeta potential with the optimal formulation. The interaction of NaDC with PC was also investigated by turbidity, pinacyanol chloride (PIN) spectral characteristics, and the distribution of NaDC in NaDC-Lip, which showed that NaDC can be incorporated into the lipid bilayer, and effective surfactant to lipid molar ratio Re and the partition coefficient K were 0.26 and 0.21 mmol-1, respectively.O-palmitoyl mannan (OPM) was synthesized by esterification of mannan by the reaction of palmitoyl chloride in dimethyl formamide under anhydrous catalytic conditions. The OPM characterization revealed the existence of an ester bond between manna and the palmitoyl group, indicating that the two compounds were covalently bonded, and the substitution degree of palmitoyl residues in OPM was 0.37. The NaDC-Lip coated with OPM were prepared and the characteristics were evaluated. The results indicated as follows:the OPM coating procedure did not modify the vesicular structure. At 1:1 weight ratio of OPM to PC, the integration of OPM with the liposomal bilayer membrane occurred at saturation level. The absolute value of zeta potential decreased on addition of OPM. The particle size distribution of NaDC-Lip coated with OPM become uniform when the weight ratio of OPM to PC was greater than 1:1. The preparation conditions did not cause any significant irreversible degradation of the antigen protein. Both NaDC-Lip and OPM coated NaDC-Lip were stable in buffer pH 7.4, buffer pH 2.0, 20 mmol·mL-1 bile salt and 1.0% pancreatin.A stable, rapid and accurate method was developed for the determination of antibody levels in mice. The intramuscular standard of BSA produced the highest titer value (6.8±0.6) after 4 weeks. The IgG titer value achieved after 4 weeks of oral immunization using Lip, NaDC-Lip and OPM-coated NaDC-Lip were 4.2±0.3,5.7±0.4 and 6.7±0.5, respectively. Very low titer was obtained with orally administered BSA solution (oral control) and empty NaDC-Lip coated with OPM (1.9±0.4 and 2.1±0.4, respectively). Negligible sIgA response was observed with intramuscularly administered vaccine. Both NaDC-Lip and OPM-coated NaDC-Lip more efficiently elicited mucosal immune response (slgA level) than traditional liposomes (p<0.01). Serum IgG2a/IgG1 ratio using the formulation of OPM-coated NaDC-Lip was higher than other formulations, reflecting that oral administration of antigens in mannosylated NaDC-Lip could elicit both humoral and cellular immune response.MVLs containing BSA were prepared, and the formulation was optimized taken entrapment efficiency and particle size as the index. TMC with a degree of quaternization (DQ) of 70.2% was synthesized from chitosan. MVLs coated with TMC were prepared and characterized in vitro and in vivo. The results indicated as follows:0.5%(w/v) of TMC was not only enough to coat MVLs, but also made the particles stable. The preparation conditions did not cause any degradation of the antigen. TMC-coated MVLs were more stable in the simulated gastric fluid, compared with uncoated MVLs. It was observed that TMC-coated MVLs produced better IgG levels upon oral administration (5.7±0.4). Oral TMC-coated MVLs also elicited a significant mucosal immune response (sIgA levels in mucosal secretions).OPM-coated NaDC-Lip and TMC-coated MVLs could be considered as the potential oral vaccine delivery carriers and adjuvants. |
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