Ultrasound-guided Intratumor Injection Of Plga Microspheres Containing Docetaxel In Treatment Of Human Hepatoma-bearing Nude Mice

ObjectiveTo prepare sustained-release poly(lactic-co-glycolic acid)(PLGA) microspheres containing docetaxel, and investigate the physiochemical properties, in vitro release and drug stability; and to investigate the effect and mechanism in treatment of human hepatoma-bearing nude mice by ultrasound-guided intratumor injection of the drug-loaded microspheres.Methods1. Sustained-release PLGA microspheres containing docetaxel were prepared by solvent evaporation method, thereafter morphology and particle size were observed by scanning electron microscope, while drug loading, encapsulation efficiency, and in vitro release within 21days were examined by HPLC. The effect of irradiation steriliazation at three doses(5, 15 and 25kGy) was examined. Dichloromethane residue in microspheres was detected by gas chromatography. Post hypodermic administration on the mice back for 4 weeks, death of mice was recorded to calculate half-lethal dose by modified Karber's method.2. Established mude mice model of human heptoma cell line BEL-7407 transplanted subcutaneously. Ninety mice were randomly enrolled into a model(M), blank microspheres(BM), docetaxel intraperitoneal injection(DP), docetaxel intratumor injection(DT),low-dose(DMTL) and high-dose(DMTH) of docetaxel-loaded PLGA microspheres intratumor injection group with 15 animals in each group. Either group was given corresponding treatment under ultrasound guidance.3. Tumor volume, parenchyma echo and blood supply between six groups were compared by regular ultrasound follow-up every week for consecutive 3 weeks.4. At 3 weeks postinjection,5 nude mice randomly selected in each group were examined on contrast-enhanced ultrasound following by sacrification, and the tumors were excised, weighted to evaluate the inhibition rate.5. tumor tissues were examined on histopathology by microscopy, on microvessel density by immunohistochemical method, and on the gene expression of vascular endothelial growth factor(VEGF), basic fibroblast growth factor(bFGF) and Angiopoietin-2(Ang2) by real-time fluorescence quantitative PCR.6. Survival of remaining nude mice was observed for another 12 weeks.Results1. Mean diameter of drug-loaded microspheres were 23.1μm by solvent evaporation method; the optimal drug loading rate(4.82%) and encapsulation efficiency(96.3%) were gained when the feed ratio of PLGA to docetaxel was 100mg/5mg. Stably the cumulative release of docetaxel from microspheres was 82.5% within 21days without occurrence of burst release, and docetaxel encapsulated in microspheres, detected by HPLC, kept stability of structure. The limited amount of dichloromethane residue(0.026%) in microspheres accorded with general rule of Chinese Pharmacopoeia. No bacillus pumilus colony was cultured in microspheres after irradiation steriliazation at three doses.2. Half-lethal dose calculated in docetaxel-loaded microspheres injection group (483.6mg/kg) was far higher than docetaxel injection group (52.7mg/kg).3. The tumors of model and blank microspheres group were significantly increased after administration, with inhomogeneous internal echo and relatively rich blood flow signal on ultrasonography. The tumor growth was slower in the other docetaxel group, especially for DMTH group which inhibited tumor growth markedly with a little flow signal around tumors detected by color Doppler ultrasonography. DMTH displayed a potent antitumor effect on human hepatoma with a tumor inhibitory rate 66.01%, higher than that of DP(34.65%), DT(35.62%), and DMTL group(48.37%) significantly.4. At 3 weeks postinjection,contrast-enhanced ultrasound was founded that a mass of tumors parenchyma showed filling defect except slight enhancement in paratumor parenchyma in DMTH group, but tumors parenchyma of all the other groups was enhanced in various degrees. 5. Extensive necrosis of tumor tissue was found in the histopathologic examination of docetaxel-loaded microspheres injection group. And the expression of MVD, as well as VEGF, bFGF and Ang2, decreased compared with the other groups.6. For Survival analysis, survival rate in drug-loaded microspheres group(70%) was higher than DP group(30%), as well as DT group(30%) significantly(P<0.05).Conclusion : Sustained-release PLGA microspheres containing docetaxel were successfully prepared by solvent evaporation method with optimal particle size, distribution, dispersion, encapsulation efficiency and low drug degradation, whose release period was about 3 weeks; the effect of docetaxel in treatment of human hepatoma-bearing nude mice was promoted by ultrasound-guided intratumor injection of PLGA microspheres containing docetaxel, which depressed the gene expression of VEGF, bFGF and Ang2 resulting in inhibition of tumor angiogenesis and also developed survival rate of nude mice. This new method of hepatoma treatment has potential clinical value.