Preparation Of Squalene-based Cationic Nanostructured Lipid Carriers And Preliminary Study On The Effect Of Immune Adjuvant

New vaccines need immune adjuvants to improve the level of immune response due to their low immunogenicity,so nanoparticles have been widely studied as the potential immune adjuvants.Among them,nanostructured lipid carriers(NLCs),can efficiently encapsulate antigenic substances or immunostimulatory substances,which can enhance the stability of the antigens and protect them from degradation.Squalene,as the main component of the marketed safe adjuvant MF59,has the great potential as the immunologic adjuvants,and the surface modification of nanoparticles by chitosan can enhance the ability of cells to absorb nanoparticles.Therefore,this study intends to combine the advantages of the above substances to prepare squalene-based NLCs and investigate its immune adjuvant effect.Firstly,the high-shear-ultrasonic dispersion method was used to prepare csNLCs to screen the ratio of solid lipids to liquid lipids,the type and weight of emulsifiers,and the concentration of chitosan.The optimal formulation was determined according to a series of characterized results,and 1:9 ratio of palmitic acid to squalene,1:1 of Tween 80 and Span 85(total 30%of mixed lipids)as emulsifiers,and 0.1%chitosan solution.The hydrodynamic diameter was 235.80±5.991 nm,the zeta potential was 34.90 ± 6.954 mV,and the PDI was 0.283± 0.015.The particles were spherical and non-agglomerated under a transmission electron microscope.Then chicken egg white albumin(OVA)was successfully encapsulated to prepare the OVA-csNLCs for subsequent experiments.The hydrodynamic diameter was 222.1±14.22 nm,PDI was 0.351±0.024,zeta potential was 19.03±0.306 mV,and the encapsulation efficiency reached 83.36%.The results of SDS-PAGE electrophoresis showed that the free OVA bands contained in the OVA-csNLCs filtrate were in the same position and lighter in color than that of pure OVA solution with the same concentration,suggesting the inclusion of OVA in csNLCs.In order to maintain the long-term activity of OVA-csNLCs during production and storage,a lyophilized protectant agent was selected to prepare the lyophilized powder.Based on the morphology after lyophilization and the quality after redispersion,10%sucrose was selected as protective agent,the redispersion solution of lyophilized powder had particle size of 218.5 ± 5.59 nm,PDI of 0.256 ± 0.036,and zeta potential of 14.17 ± 0.153 mV.Then,RAW264.7 cells and Balb/c mice were used for in vitro and in vivo experiments to evaluate the effect of csNLCs as an adjuvant.The safe concentrations of csNLCs and OVAcsNLCs were first screened by the MTT method,showing that csNLCs had no obvious toxicity and had good biocompatibility.Subsequently,the FITC-OVA and FITC-OVAcsNLCs,were co-cultured with RAW264.7 cells.The fluorescence intensity of cells in FITCOVA group and FITC-OVA-csNLCs group was analyzed by flow cytometiy to compare the uptake ability against antigens.The result showd that the fluorescence intensity increases as the increment of the csNLCs concentration,while the fluorescence intensity of FITC-OVA did not change significanty in the same concentration,indicating that csNLCs can promote the uptake of antigen by macrophages.The mice were then divided into blank group,OVA group,csNLCs group and OVA-csNLCs group.Each group of 10 mice was immunized twice,and the body weight of the mice was recorded during the immunization period.The serum and spleen cells of mice were extracted 7 days after the second immunization.ELISA was used to test the concentration of IgG,IL-4 and IFN-y in mice serum.The results showed the levels of IgG antibodies in mice serum in the OVA-csNLCs group were much higher than the other groups,indicating that csNLCs could stimulate the body’s humoral immune response;In addition,the content of IL-4 showed the csNLCs and OVA-csNLCs groups were higher than the other groups,but the levels of IFN-y except for the blank group were comparable,suggesting the immune response caused by OVA-csNLCs was more biased towards the Th2 type.During the entire immunization process,the mice in each group have the similar weight change trend with,no dead individuals,indicating the safety of csNLCs.Similarly,when the spleen lymphocytes of each group were stimulated and recultured with the same concentration of antigen OVA solution in vitro,the proliferation ratio of spleen lymphocytes of mice in the OVA-csNLCs group was greater than that in the other groups,further proving that csNLCs had the function of enhancing immune response.In the current study,we have successfully prepared csNLCs with immune adjuvant effect,which provides a nano-plateform for developing novel adjuvants,but it also needs more improvement and investigation.