Preparation And Evaluation Of Thermosensitive Nanocarrier

Objectives: In order to provide experimental evidences to the development of novel vaccine carrier systems, the physicochemical properties of poloxamer grafted chitosan nanomicelles with cationtic and thermosensitive characteristics and factors affecting the uptake of macrophages and dendritic celles were investigated.Method: Poloxamer-g-chitosan was synthesised by EDC and NHS mediated reaction. The chemical structure of the intermediates and final products were confirmed using nuclear magnetic resonance spectra (1H-NMR) and infrared spectra (FT-IR). CMC and LCST of Poloxamer-g-chitosan micelles were measured by pyrene fluorescent probe method and visible spectrophotometry method, respectly. OVA loaded polymer micelles were prepared by dissolve directly, BCA method was used to determine the OVA concentration. The formulation and preparation process of micelles were evaluated in terms of the particle size, encapsulation efficiency and loading capacity. The release profiles of OVA loaded micelles in 37℃and 4℃were investigated, respectively. Subsequently, the cytotoxicity of poloxamer-g-chitosan micelles was detected by MTT method after incubating with macrophage (Mφ). Dendritic cells (DC) were generated from murine bone marrow cell by an established in vitro method. The uptakes of FITC-OVA loaded poloxamer-g-chitosan micelles by macrophage and DCs were observed using fluorescence microscope and flow cytometry.Result: The spectra of IR and 1H-NMR confirmed the chemical structures of intermediate products and poloxamer-g-chitosan, and further verified by thermal assay. The CMC and LCST of poloxamer-g-chitosan micelles was (3.24±0.06)×10^-6 mol·L^-1and around 37℃when we using the ratio of poloxamer and chitosan was 0.63 to 0.025, respectively. The preliminary optimized formulation and preparation process of micelles with the average size (219.1±2.3) nm was established. The encapsulation efficiency, loading capacity and zeta potential of OVA loaded micelles were (42.2±5.6) %, ?(21.4±2.3) % and (11.67±2.25) mV, respectively. The 4 h-cumulative release of OVA was 30 % at 37℃, but reached 98% at 4℃. The results of MTT assay indicated relative low cytotoxicity of poloxamer-g-chitosan. Both of the macrophage and dendritic cell uptake study showed the significant enhanced endocytosis phenomena of FITC-OVA loaded poloxamer-g-chitosan compared with FITC-OVA solution while investigated using fluorescence microscope and flow cytometry.Conclusion: Micelles formulated with poloxamer-g-chitosan displayed thermosensitive and cationic characteristis with low cytotoxicity, and enhances the endocytosis by antigen presentation cells. In order to further improve the specific targeting delivery of antigens to DCs, the monoantibody modification of poloxamer-g-chitosan is under going.