Study On Copolymer Micellar Delivery System Of Bletilla Striata Polysaccharide For Targeting Tumor Cells Mediated By Folic Acid

The stearic acid-modified Bletilla Striata polysaccharides?BSPs?copolymers?BSPs-SA?with pH sensitivity were prepared in our previous study.It was found to have a sustained release and solubilization effect on docetaxel?DTX?,in addition to increasing the concentration of drugs in the tumor site,thereby improving the anti-tumor effect of the drug.In this study,the preparation conditions of BSPs-SA were further optimized based on the previous research of our group.The effects of stearic acid?SA?amount,catalyst amount and reaction temperature on the degree of substitution of SA were investigated,and the optimum synthesis conditions were selected.Folic acid?FA?was grafted onto the BSPs-SA to further enhance the targeting ability of the copolymers.FA modified SA grafted Bletilla Striata polysaccharides?FA-BSPs-SA?copolymers with different FA substitution degrees were prepared by changing the amount of FA.Its structure was confirmed by hydrogen nuclear magnetic spectroscopy?¹H-NMR?,UV-vis spectrophotometry?UV-vis?and infrared spectroscopy?FT-IR?.The critical aggregation concentration?CAC?of FA-BSPs-SA copolymers was determined by fluorescence spectrometer.DTX was used as a model drug,and drug-loaded micelles?DTX/FA-BSPs-SA?were prepared by emulsification-solvent volatilization method.The particle size,particle size distribution and zeta potential of the micelles were determined by dynamic light scattering spectrometer.The drug loading and encapulation efficiency were determined by high performance liquid chromatography?HPLC?.The drug release behavior in vitro was studied by a dialysis method.The safety of FA-BSPs-SA copolymers was evaluated by hemolysis test and MTT assay.The cytotoxicity of FA-BSPs-SA copolymers and the drug-loaded micelles was measured by MTT assay.The results showed that when the amount of BSPs was 400 mg,the optimum conditions for the synthesis of BSPs-SA were as follows:the amount of SA was 0.45mmol,The amount of catalyst 1-?3-dimethylaminopropyl?-3-ethylcarbodiimide hydrochloride?EDC?and 4-dimethylaminopyridine?DMAP?is n _SA:n_DMAP:n_EDC=1:1:1.2,the reaction temperature was 38?.The degree of substitution?DS?of SA onto BSPs-SA is 11.93%under the synthesis condition.The results of ¹H-NMR,UV-vis and FT-IR demonstrated that FA has been successfully grafted onto BSPs-SA.When the amount of FA was 0.05 mmol,0.1 mmol and 0.2 mmol,the degree of FA substitution of the copolymer were 1.12%,2.4%and 5.6%,respectively.Moreover with the increase of FA substitution,CAC decreased gradually.DTX could be entrapped into micelles of FA-BSPs-SA copolymers.The particle sizes of copolymer micelles decreased whereas encapsulation efficiency and loading capacity increased with the substitution degree increase of FA.DTX/FA-BSPs-SA copolymer micelles displayed pH-dependent properties in the respects of in vitro release behavior ranging from pH 5.0 to pH 7.4 with greater release percentage in acid media.The release percentage of DTX from DTX/FA-BSPs-SA copolymer micelles were?61.12±0.53?%and?67.9±0.06?%under pH 5.0 and pH 7.4 conditions within 48 h,respectively.The drug in vitro release rate increased as the pH decreased.The hemolysis rates of FA-BSPs-SA copolymers were less than 5%in the range of 0.1-0.5mg/mL concentration.MTT results showed that the cell survival rates subjected to FA-BSPs-SA and BSPs-SA were still above 80%at the concentration of 40?g/mL.The results showed good biocompatibility of FA-BSPs-SA.The anti-tumor effects in vitro of DTX/FA-BSPs-SA and DTX/BSPs-SA micelles were superior to that of DTX solution at the same concentration.Moreover,DTX/FA-BSPs-SA micelles exhibited stronger inhibitory effects on tumor cells overexpressed folate receptor in comparison to DTX/BSPs-SA micelles.The above results indicate that FA-BSPs-SA copolymers are expected to be nanoscale materials for hydrophobic antitumor drugs.