Study On The Tissue Distributions In Vivo,Anti-tumor Effects In Vitro And Celluar Uptake Mechanisms Of The Folate-modified Non-ionic Surfactant Vesicles Loading With Neogambogic Acid

Objective:On the basis of the previous study,this study was established to further evaluate the folate-modified non-ionic surfactant vesicles loading with neogambogic acid?FA-GNA-NISVs?.It also aims to study the pharmacokinetic behavior in rats as well as to explore the mechanism of tumor inhibition and uptake in vitro in order to study its reliability as an anti-tumor drug carrier,which lays a foundation for the development and design of new dosage form of neogambogic acid.Methods:?1?A HPLC method was developed to determine GNA in rats tissues;?2?SD rats were administered by tail vein injection to study the tissue distribution of GNA,GNA-NISVs and FA-GNA-NISVs in vivo,and the differences of pharmacokinetics parameters were analyzed;?3?MTT methods were used to detect the cytotoxicity of GNA,GNA-NISVs and FA-GNA-NISVs in A549 cells.Cell apoptosis was evaluated by flowing cytometry and DAPI staining;?4?The concentration of GNA in cells was determined by HPLC,and the protein concentration in cells was measured by BCA protein kit.Meanwhile,the amount of GNA in per microgram protein was presented through the cellular uptake.By studying the influence of different factors of the uptake of cells,endocytosis pathway and the mechanism of cellular uptake were discussed.Results:?1?The HPLC method for the determination of GNA in rats was established successfully and the method was validated;?2?The results indicated the concentration of GNA in liver and spleen was almost the highest after a single tail vein injection of GNA,GNA-NISVs and FA-GNA-NISVs.In comparison to free GNA,the GNA-NISVs and FA-GNA-NISVs increased the concentration of drug in the liver,spleen,lung,kidney,intestinal.FA-GNA-NISVs exhibit more obvious effects than GNA-NISVs,and the highest relative intake rate of FA-GNA-NISVs in lung demonstrated the ablility of targeting;?3?MTT assay showed that FA-GNA-NISVs significantly enhanced cytotoxicity against A549 cells and all groups in a dose-dependent cytotoxicity.The IC₅₀of GNA?GNA-NISVs and FA-GNA-NISVs were 13.143?g/mL?7.090?g/mL and4.279?g/mL with significant differences,respectively.In addition,FA-GNA-NISVs had a highly efficient in inducing cells apoptosis;?4?Cellular uptake experiments showed that FA-GNA-NISVs exhibited a superior performance in increasing uptake of drug in A549 cells.Compared with the GNA groups,some of GNA-NISVs and FA-GNA-NISVs were endocyzed by clathrin-mediated endocytosis,caveolae-mediated pathway and micropinocytosis-mediated endocytosis were the main routes for GNA,GNA-NISVs and FA-GNA-NISVs entered into cells.However,the amount of FA-GNA-NISVs were mediated by the caveolae pathway is significantly increased,which could escape from the lysosomal system.Conclusion:Folate-modified GNA nonionic surfactant vesicles can significantly change the distribution of GNA in the tissue,increase bioavailability,enhance drug anti-tumor effect in vitro and increase cellular uptake as well as change the pathway into the cell,which indicates that FA-GNA-NISVs has a potential application prospect in the treatment of tumor.