Study On Preparation And Cellular Uptake Of Doxorubicin-loaded Lipid/Nanoparticles

The anticancer drug doxorubicin has capability of broad-spectrum whichi is wisdely used in clinical practice. However, the tumor cells produce drug resistance due to the repeated use of these drugs, which become one of the major reasons for chemotherapy failure. Looking for the low toxicity and efficient drug carries has been one of the primary tasks. We designd core-shell lipid/nanoparticles, and then investigated its efficiency of lethal to tumor cells. Lastly, we explored the machnism of its multidrug resistance which could provide basis in exploring more efficient and novel nanocarries.In this study, we synthesized DOX-PLGA polymer, and characterized it by1H-NMR and GPC. We prepared nanoparticles with the obtained polymer by emulsification-diffusion method, and investigated their diameter, zeta potential, release profile as well as morphology.DOX-PLGA nanoparticles, as the core, were physically wrapped by DPPC to prepare lipid/nanoparticles. Furthermore, we characterized the LNPs by zeta potential, diameter and TEM. The results indicated that LNPs mainly scattered in200～300nm, and distributed centralizedly. Pictures from TEM could demonstrate that LNPs was spherical and the diameter was250nm, which was accordant with the result from diameter, in addition, the nanoparticles were wrapped by the out layer of lipid. The zeta potential of LNPs was-10～5mV and distributed uniformLy, consistently with theoretical analysis.We investigated compatibility of vacant LNPs by MTT, it was shown that LNPs had a good ability of compatibility with no or little cell toxicity. Meantime, we inspected the cell toxicity of LNPs with four cell lines MCF-7, MCF-7/ADR, HL-60and HL-60/ADR. Compared with free drug DOX, LNPs loaded by DOX could reverse multidrug resistance of ADR cells. Pictures from CLSM indicated that the DOX released from LNPs could diffuse into nucleus, thus we can draw a conclusion that LNPs could control the tumor cells. Data from FACS demonstrated the ratio of drug entered into cells, it was shown that LNPs loaded DOX could stay in ADR cells in contrast with free DOX.Drug mainly entered into cells by endocysis, however, most of the drug cwould be assimilated by enzymes in endosomes which limited the application of nano drug delivery by decreasing the cure effect. Calcein is a small hydrophilic fluorescent needle, which was usually used for model drug in nano drug delivery to investigate the ratio of drug entered into cells. In this study, we prepared calcein nanoparticles used as the small model drug and investigated the endocytic process of in MCF-7, MCF-7/ADR, HL-60and HL-60/ADR cells as well as the mechanism. It was shown that, calcein could bypass the assimilation of endosomes and successfully entered into nucleus. The self-assemblies were internalized into the cells via lipid raft/caveolae (which is localized by Pgp) mediated endocytosis using Flow cytometry system (FACS) and endocytic inhibitors.All these results suggest that the LNPs are low cell toxicity and have good biocompatibility. LNPs exhibit potential as nano drug delivery for reversing multidrug resistance, and it is worth in depth study.