Synthesis And In Vitro/in Vivo Evaluation Of Docetaxel-Loaded Nanomicelles

Objective:Docetaxel is a new alkaloid antineoplastic drugs, in order to improve its solubility inwater, we synthesized mPEG-b-PDLLA copolymers used as carrier materials of docetaxel, and has carried on the quality evaluation. Investigation the quality standard of Docetaxel-loaded Nanomicelles and Its curative effect on A549 cells of NSCLC.Methods:With mPEG and D, L-lactide as raw material, stannous octoate as catalyst, ring opening polymerization, the formation of block copolymers was synthesized by a ring opening polymerization.The characterization of mPEG-b-PDLLA was confirmed by FT-IR,1H-NMR, DSC, MALDI-TOF-TOF-MS, GC, inductively coupled and pH meter methods.Docetaxel-loaded Nanomicelles were prepared by solvent evaporation of thin film hydration method. Determination of its particle size and distribution by the Malvern Nano ZS laser particle size analyzer. The morphology of anomicelles was observed by transmission electron microscopy. The entrapment efficiency and drug loading were determined by High Performance Liquid Chromatography (HPLC), and studied its quality standard; The stability of DTX-loaded Nanomicelles was observed by the change of particle size and drug loading.Study inhibitory effect of docetaxel loaded nanomicelles on A549 cells by the cell migration test, MTT method and laser scanning confocal experiment.Tumor were inoculated into the subcutaneous of nude mice to establish a tumor-burdened nude mice model of lung cancer. Nude mices were injected by using the method of tail vein with glucose solution, DTX-PM solution and commercially available solution, respectively. Changes of subcutaneous tumor size and weight of nude mice were regularly observed.Result:1H-NMR showd that the molar content of lactide was 17～19% and the molar content of mPEG2000 was 81%-83%,the average molecular weight of the copolymer was about 3300.Through IR analysis Confirmed that lactide and mPEG successfully polymerized into the block polymer. MALDI-TOF-TOF-MS showed that peak detection mainly in 3300 Da, impurity peak decreased obviously after purification. DSC measurements obtained that melting point of mPEG-b-PDLLA polymer was about 41.8 ℃, melting enthalpy Hm=20.3 mJ/mg.The residue inspection showed that the residual amount of toluene, methylene chloride and diethyl ether were no residue, the residual content of lactide was 1.2%, the residual content of tin is 1.521E+05 μg/kg.The pH value of mPEG-b-PDLLA was 5.2.The size of DTX-PM was determined as 16nm, PDI were maintained at between 0.1-0.2. The shape of DTM-PM that observed by transmission electron microscope was regular spherical. Detected by HPLC, the encapsulation efficiency of Docetaxel-loaded Nanomicelles was (88.88%±0.61)%, and the drug loading was (15.00%±0.69)%. In the study of the quality standard, the experimental results of inter day precision, intra day precision, repeatability and recovery test were qualified. Study the reconstituted stability of DTX-PM, results showed that micellar stability was good enough for infusion.The results of MTT showed that, the commercially available docetaxel had stronger inhibition effect of cell proliferation than DTX-PM at high concentration, while at low concentration DTX-PM has stronger inhibition of cell proliferation. Cell migration test showed that, the commercially available docetaxel and DTX-PM can inhibit the proliferation of A549 cells. Confocal laser scanning test showed that the speed of micelles into the cell was very fast and the micelles can uptake into the nucleus, had better killing effect on A549 cells.After the nude mice were injected with glucose solution, commercially available docetaxel solution and DTX-PM solution, found that the tumor growth in DTX-PM group was slower than that in commercially available docetaxel group, less toxic than the commercially available docetaxel group. The results show that the anti-lung cancer effect of DTX-PM was stronger than the commercially available docetaxel.Conclusions:The structural characterization of mPEG-b-PDLLA copolymer can confirm that lactide and mPEG successfully polymerized into the block polymer.The residues of organic solvents, lactide and metal tin which in polymers were qualified.The content determination method in quality standard is simple, accurate, reproducible and can be used for the quality control of DTX-loaded Nanomicelles. Docetaxel-loaded nanomicelles can effectively inhibit The proliferation of A549 cells, has the effect of anti lung cancer.