Study On Gallic Acid-Chitosan-D-α-tocopherol Polyethylene Glycol 1000 Succinate（TPGS） For Paclitaxel Oral Delivery

Objective:To obtain a good oral delivery carrier for insoluble drugs and improve the solubility and bioavailability of paclitaxel,a drug-loaded micelle system established by a kind of amphiphilic copolymer,Gallic acid-chitosan-D-α-tocopherol polyethylene glycol 1000 succinate（GA-CS-TPGS）,was prepared by grafting Methyl gallate and D-α-tocopherol polyethyleneglycol 1000 succinate（TPGS）respectively,as the donor of the micelle hydrophobic group,on chitosan（CS）as bioadhesive material,and loading paclitaxel as model drug.Methods:Gallic acid-chitosan（GA-CS）was prepared by Methyl gallate and chitosan.TPGS was activated by its hydroxy-terminal carboxylation with succinic anhydride（SA）and 4-dimethylaminopyridine（DMAP）,and the activity of carboxyl reaction was enhanced under the condensation of 1-ethyl-3-dimethyl-aminopropyl carbodiimide hydrochloride（EDC·HCL）and N-hydroxy-succinimide（NHS）.Finally,the GA-CS-TPGS copolymer was prepared by the amidation of free amino groups on chitosan.The chemical structure of the GA-CS-TPGS grafted copolymer was characterized by Fourier transform-infrared spectroscopy（FT-IR）and Nuclear magnetic resonance spectroscopy（NMR）.The polymer micelle loading paclitaxel as model drug was prepared by ultrasonic emulsification method.The encapsulation efficacy（EE）and drug loading（DL）were determined by High performance liquid chromatography（HPLC）.The Pyrene fluorescent probe method was employed to determine the critical micelle concentration（CMC）of the copolymer.The particle size,Zeta potential and size distribution of the micelle system were measured by Dynamic light scattering（DLS）.The surface morphology of the micelles was investigated by Transmission electron microscopy（TEM）.The encapsulation pattern of drugs in micelles was investigated by X-ray diffractometer（XRD）.The oral pharmacokinetic characteristics of the paclitaxel-loaded GA-CS-TPGS micelle and the reference preparation were investigated in rats by the intragastric administration.And evaluated the bioadhesion and the cellular biological mechanism of the GA-CS-TPGS micelle.Evaluated the inhibitory of GA-CS-TPGS on CYP3A enzyme and the anti-tumor effect of GA-CS-TPGS/PTX on human lung cancer cell A549.Results:The results of FT-IR and ~1H-NMR indicated that the copolymer（GA-CS-TPGS）was synthesized.The ultrasonic emulsification method was chose to prepare the paclitaxel-loaded micelles.The as-prepared micelle exhibited a small particle size of approximate 140 nm and obtained narrow size distribution.The encapsulation efficiency and drug loading can reach 80%and 8.2%respectively.The TEM result showed that the formed particles were uniform in shape without aggregation.The XRD result indicated that paclitaxel was well encapsulated in the polymer micelles.The optimized protective agent for freeze-drying focused on trehalose with concentration of 8%,which would obtain a full,uniform and dispersible freeze-dried product.The pharmacokinetic results manifested that the area under the concentration-time curves（AUCs）were increased from 0.883μg/m L·h to1.592μg/m L·h in comparison to the reference preparation,and the peak time(T_max)was prolonged from 1 h to 3 h.Mucin was used as experimental model to study the biological adhesion.The results showed that GA-CS-TPGS polymer micelles could interact with mucin,when the mucin’s concentration was 1 mg/m L,the amount of adsorption reached695μg/2 mg,and the amount of adsorption increased without increasing the concentration.In vivo tests have shown that drug-loaded polymer micelles increase the absorption constant.The intestinal absorption mechanism indicates that various proteins mediate the endocytosis of the micelle solution.This product has a certain inhibitory effect on CYP3A enzyme,IC50 is 2.977 mg/m L.this product has a certain inhibitory effect on human lung cancer A549.The relative tumor inhibition rate（TGI）of PTX、GA-CS-TPGS/PTX（5 mg/kg）、GA-CS-TPGS/PTX（10 mg/kg）、GA-CS-TPGS/PTX（20 mg/kg）is 48.15%、40.74%、51.85%and 66.67%,respectively.Conclusions:The proposed GA-CS-TPGS copolymer was successfully synthesized in this experiment,and the drug-loaded micelles prepared by ultrasonic emulsification exhibited good characteristics.Compared with the reference preparation,the oral bioavailability of the insoluble paclitaxel could be improved.The polymer micelles significantly increase the bioadhesion and increase the absorption constant.The endocytic process is mediated by clathrin,non-gliodin,caveolin,non-cavegin,giant cell drink and the like.Cellular experiments showed that the synthesized material had no obvious cytotoxicity,and the p-gp inhibitor increased the uptake and permeability of the cells.This product has a certain inhibitory effect on CYP3A enzyme and the human lung cancer A549.