| Background: Anti-tumor immune mechanisms of human are complex, involving a variety of immune components, including humoral immunity and cellular immunity. They are mutual synergistic reaction and kill the tumor cells. It is generally considered that T-cells, NK cells, macrophages and dendritic cell-mediated immunity play important roles. Specific targeted drug delivery system will be expected to enhance antigen targeting dendritic cells or macrophages, increase antigen presenting, promote the immune response, and enhance anti-tumor effects. Based on the surface expression of mannose receptor on dendritic cells and macrophages, the mannose-modified chitosan coated PLGA nanoparticles (PLGA-NPs) were prepared and characterized.Method: After synthesis of mannose-modified chitosan, the OVA loaded PLGA-NPs, chitosan (CS) or mannose modified chitosan (MAN-CS) coated PLGA-NPs were prepared by double emulsification method, respectively. The effects of different factors on the size of PLGA particle, encapsulation efficiency of OVA and release profile were investigated. Subsequently, the effects of nanoparticles concentration and incubation time on the macrophage (Mφ) cytotoxicity were observed. Furthermore, the effects of incubation temperature, period, NPs concentration and mannose on the uptake and location of Mφwere confirmed by fluorescence microplate reader, flow cytometry and fluorescence microscopy, and confocal laser scanning microscope (CLSM). Dendritic cells (DC) were generated from murine bone marrow cell by an established in vitro method. Nanoparticles uptaken by DC was measured using flow cytometry, and the localization of nanoparticles in DC was observed by CLSM.Result: The structure of mannose-modified chitosan and intermediate products were confirmed by IR, NMR and elemental analysis. The preliminary optimized formulation and preparation process of nanoparticles with the average size about 255nm was established. The loading efficiency and entrapment efficiency of OVA were 8% and 70%, respectively. The size and zeta potential of the CS coated nanoparticles increased as the concentration of CS improved. The additive of PEG1000 to the inner-phase, sucrose to the out-phase, as well as CS or MAN-CS treatment could retard the release of OVA, and reduce initial burst release phenomenon. Low cytotoxicity was measured by FITC-OVA nanoparticles. The phagocytosis by Mφwas inhibited at 4℃, and Mφuptaken of nanoparticle closed to saturation at 8h. At given concentration range, the uptake of nanoparticles by Mφgradually increased. The previous treatment by mannose could inhibit the phagocytosis of the MAN-CS coated PLGA nanoparticles. In addition, the highest uptaken of MAN-CS-PLGA nanoparticles was confirmed by flow cytometry, and the nanoparticles were mainly located in the cytoplasm was checked by CLSM. DC were generated in vitro from mouse bone marrow cell culture in the presence of GM-CSF and IL-4, and immature dendritic cells expressed high level of CD11c. It showed that the endocytosis of the nanoparticles containing FITC-OVA was more than FITC-OVA solution when checked by flow cytometry. Finally, the endocytosis of MAN-CS-PLGA nanoparticles showed more satisfied result.Conclusion: The formulation of PLGA nanoparticles was well established through a single factor design, and preparation method for nanoparticles also showed a good reproducibility. Based on the present formulation, only the CS coated and the MAN-CS-PLGA nanoparticles containing OVA with relatively low toxicity were successfully prepared using double emulsion method. The results indicated apparently that the phagocytosis of MAN-CS-PLGA nanoparticles by DC and Mφin vitro was more effective.
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