Study On Pharmacokinetics And Cytobiology Properties Of The Htdroxytyrosol Block Copolymer Nanoparticles

In this study, Hydroxytyrosol was used as model drug, which had been studied with exact curative effect on hepatitis B in Folium Syringae. It was prepared to be Hydroxytyrosol loaded mPEG-PLGA nanoparticles to prolong in vivo circulation and target to the liver. Pharmacokinetics and HepG2.2.15 were used to study biological effect of Hydroxytyrosol nanoparticles.1. Pharmacokinetic study of hydroxytyrosol nanoparticlesHealthy SD rats was used as experimental animal. HT saline solution was designed as control group to study dynamic parameters of HT loaded nanoparticles. Extract the blood of rat eyes at the time points designed before. The blood concentration of HT were measured by UPLC, using the method of drug concentration in blood. The pharmacokinetic parameters were calculated by DAS2.0 software, and the significant difference was analyzed relying on SPSS17.0 software. The results of the study show that T1/2(α) and T1/2(β) of HT nanoparticles is 1.13 times and 2.27 times respectively compared to control group, and AUC was 6.91 times, the difference was significant. HT nanoparticles changed its pharmacokinetic parameters, prolonged the retention time in rats and half-life in vivo of HT. The clearance rate reduced significantly, AUC increased, indicating that the HT made nanoparticles had certain long cycle effect.2. Effects of HT nanoparticles on HepG2.2.15 cellsThe toxic effects of HepG2.2.15 cells with different concentrations of HT nanoparticles was examine by the method of MTT. Cell cycle, Cell apoptosis and the change of mitochondrial membrane potential of HepG2.2.15 cells was analysised by flow cytometry. The results of the study show that both group HT and group HT nanoparticles can inhibit the proliferation of HepG2.2.15 cells, showing a significant time and dose dependent. Inhibition rate of group HT nanoparticles was decreased slightly compared with group HT, but still have the inhibitory effect on the growth of cells.The inhibitory rates of group HT nanoparticles in all concentrations were low at 24h later. After 48h inhibition rate gradually increased, showed obvious drug sustained-release property. Both HT and HT nanoparticles arrest cell cycle in S phase, and the effect of two groups of drugs on cell cycle had no significant difference. Cells in early and late apoptosis rate increased with the drug concentration, and it showed promote late apoptosis obviously. Percentage of polarized cells increased after adding HT and HT nanoparticles, indicating that the medicine is to promote the apoptosis of HepG2.2.15 cells through the mitochondrial membrane potential.3. Research on cellular uptake HT nanoparticles and the mechanism of uptake(1) Study on the cellular uptakeQualitative analysis of the HepG2.2.15 cellular uptake was analyzed by Fluorescence Microscopy and Fluorescence In Vivo Endomicroscopy, and quantitative analysis relaied on flow cytometry. There was no fluorescence detected in the two groups, group NPs which did not marked by FITC and group FITC solution incubated with cells. Exclude the interference of carries and free FITC. FITC-HT-NPs cells can be observed in the fluorescence, and showed the cells contour clearly. The density and intensity of spot decreases with the concentration of nanoparticles decreased, shows a dose dependent. This shows that HT-NPs can be uptake in HepG2.2.15 cells. Flow cytometry was used to analyze, it showed that the HepG2.2.15 cell uptake of HT-NPs had positive correlation with drug concentration, incubation time and incubation temperature as qualitative analysis, results.(2) Study on the cellular uptake mechanismColchicine and chloroquine were chose as endocytosis inhibitors. Add endocytosis inhibitors into cell culture medium. Compare the percentage of positive cells by flow cytometry, study on HT nanoparticles cellular uptake mechanism preliminary. As the results showed nanoparticles uptake can be inhibited by Colchicine and chloroquine, It could be concluded that the nanoparticles were internalized into the cells by non-specific adsorption of endocytosis.In summary, this paper explores biological characteristics of animal in vivo and in vitro of HT nanoparticles. In vivo pharmacokinetic experiments proved that the HT nanoparticles have effects of sustainied release and long circulating. The experiment of cellular uptake showed that HT nanoparticles can be uptake by liver cells. This article provided methods and techniques for the study of water soluble multicomponent of traditional Chinese medicine nano drug delivery system.