Preparation、Characterization And Bioactivity Of FA-CS-EGCG Nanoparticles

In the history of human development of the diseases, worldwide, cancer has become a serious threat to human health and one of the major diseases. Currently the drug selectivity of the main method of treatment is low, not only kills tumor cells, but also damages some normal cells in vivo, thus apparent toxicity often affect efficacy in the treatment. Studies have shown that folic acid, a large number of tumor cells surface have folate receptors which targeting folate role in cancer cells can be positioned. EGCG is a kind of polyphenolic substances with lipid-lowering, anti-oxidation, scavenging free radicals and other special physiological functions efficacy. However, due to its instability, vulnerability to changes in the external environment degradation or generate byproducts, which limits its application in various fields. Chitosan has good biocompatibility, safe, non-toxic, and has broad application prospects in the field of medical drug carrierd. At present, there is less studies on folate-conjugated chitosan embedded EGCG. In view of this, the objective was to use the folate-targeted and preparation the folate conjugated chitosan gels prepared using the obtained ion folate-chitosan-EGCG nanospheres. It can increase the stability of EGCG and the bioavailability rate of EGCG, while optimized the preparation and encapsulation process to EGCG, the basic characteristics of nanoparticles were characterized in vitro as well as its anti-tumor effect and mechanism research. Main results are listed as follows:1. Extraction Technology of folate-conjugated chitosanUsing homogeneous method to synthesis FA-CS. Folic acid active ester was first prepared and then coupled with chitosan, and the product was purified by ultraviolet spectroscopy. Use infrared spectroscopy, MS mass spectrometry to determine the structure of the product and characterization were analyzed to determine the the successful coupling of folic acid and chitosan.Select CS concentration, CS initial pH and reaction time three factors, respectively, single factor experiments. On this basis, select the concentration of CS, the initial pH of CS and reaction time for three factors and three levels of central composite design experiments to optimize the combination of experiments, optimum conditions for the preparation of folate-conjugated chitosan were obtained as follows:concentration of CS is 7.00 mg/mL, initial pH of CS is 5.00, the reaction time is 24 h and the resulting connection rate reached 38.72%.2. Process of folate-conjugated chitosan encapsulate EGCGIonization gel method were used to preparation the nanospheres. Concentration of FA-CS, pH of FA-CS, concentration of EGCG and the value of FA-CS/TPP were selected in the single factor experiments. On the basis of single factor experiments, through positive further optimization of experimental conditions for preparing FA-CS-EGCG-TPP. Preparation of folate-conjugated chitosan derived optimum conditions were as follows: FA-CS concentration 1.5 mg/mL, pH of FA-CS for 4, EGCG concentration 0.8 mg/mL, FA-CS/TPP ratio of 5, folic acid conjugated chitosan obtained for EGCG encapsulation efficiency was 66.64%.FA-CS-EGCG-TPP nanoparticles were observed by TEM characterization of its particle size distribution, the distribution and its live situations. The results showed that, FA-CS-EGCG-TPP nanoparticles form stable, regularly spherical in shape, and a typical size distribution profile of the nanoparticles with a mean diameter of 457.0 nm, the dispersion coefficient of the polymerization (PDI) of 0.560, Zeta potential of the surface of the microspheres was about+46.9 mV.3. The anti-tumor activity in vitro of FA-CS-EGCG-TPP and preliminary research on its mechanismProliferation inhibitory activities of FA-CS-EGCG-TPP-N for three tumor cell lines (MCF-7,4T1 and A549) were evaluated by MTT assay. It is found that human breast cancer cell line MCF-7 is the most sensitive one for FA-CS-EGCG-TPP-N dose relationship.Study on the confocal microscope showed that FA-CS-EGCG-TPP-N with FITC-labeled ratio of CS-EGCG-TPP-N FITC-labeled with the targeting of tumor cells by laser.Wound healing assay showed that after treatment 24 h, the cellular motility was evidently inhibited by FA-CS-EGCG-TPP-N, it could significantly decrease the motility and migration of human breast cancer MCF-7 cells.Use Annexin V-FITC double staining and PI single staining of FA-CS-EGCG-TPP-N on apoptosis and cell cycle. The results show that, in a certain range of concentrations and time certain role, FA-CS-EGCG-TPP-N was significantly induced apoptosis on human breast cancer MCF-7 cells and major blocked human breast cancer MCF-7 cell cycle inhibition in G0/G1 phase.