Study On The Treatment Of Transplanted Ovarian Carcinoma Using Ultrasound Mediated LHRHa-Targeted Paclitaxel Lipid Microbubbles

PART ONE PREPARATION AND DETECTION OF LHRHa-TARGETED PACLITAXEL LIPID MICROBUBBLESObjective:To synthesize LHRHa-targeted paclitaxel lipid microbubbles(PLMTs) and compare its properties with paclitaxel loaded lipid microbubbles(PLMs).Methods:The PLMTs and PLMs were made using mechanic vibration technique.Properties were studied containing size distribution, zeta potential,drug entrapment efficiency and drug-loading amount.Primary stability were compared under different storage condition.Results:PLMTs had a mean size of1864.5±245.4nm and a zeta potential of-(9.59±3.23)mV. Drug entrapment efficiency was (73.1±1.6)%and drug-loading amount was (20.8±1.2)%.Properties were not changed after being stored at4℃and-20℃for48hours,but the numbers of microbubbles decreased obviously after72h. PLMs had a mean size of1442.3±296.5nm and a zeta potential of-(8.45±2.02)mV. Drug entrapment efficiency was (96.5±1.4)%and drug-loading amount was (26.8±0.93)%. Properties were not changed after being stored at4℃and-20℃for14days,but the numbers of microbubbles decreased after21days.Conclusions:LHRHa-targeted paclitaxel lipid microbubbles(PLMTs) were successfully prepared.This kind of microbubbles had high drug entrapment efficiency.The size was well-distributed but it was not so stable as paclitaxel loaded lipid microbubbles(PLMs). Objective:To establish intraperitoneal model of human ovarian carcinoma in nude mice and to evaluate the targeting ability of LHRHa-targeted paclitaxel lipid microbubbles in vivo.Methods:The cell suspension derived from A2780/DDP were injected intraperitoneally in nude mice to establish human epithelial ovarian carcinoma models, and their growth behavior were observed. Expression of LHRH-R were investigated by immunohistochemistry.Three weeks after injection of A2780/DDP cells,4nude mice were divided into2groups randomly:PLMT group and PLM group, mice were sacrificed 20min after injection of fluorescence-labeled PLMTs or fluorescence-labeled PLMs, tumors of each mouse were obtained to make cryostat section and the cryostat sections were observed by microscope.Results:The human ovarian carcinoma intraperitoneal model were established successfully.The success rate of transplantation were100%. The latency period were13.8±1.3days, the median survival time were30±1.095days and the mean ascites volume were1.075±0.714ml. Immunohistochemistry staining of LHRH-R was positive.Tumor biopsy showed red fluorescein distribution was more obvious in PLMT group than PLM group.Conclusions:Human ovarian carcinoma intraperitoneal injection model in nude mice are easy to establish,and it may be an ideal model for study of ovarian carcinoma. The PLMTs has targeting function in vivo. Objective:To explore the anti-tumor effects and the mechanism of PLMTs on A2780/DDP ovarian carcinoma cells-bearing nude mice using the technique of ultrasound mediated drug release. Methods:49human ovarian carcinoma intraperitoneal model were established by injecting A2780/DDP cells into the peritoneal cavity of nude mice.15days after the model was established, the mice were divided into7groups(Ⅰ:PBS, Ⅱ:PTX,Ⅲ:PTX+US, IV:PLM,V:PLM+US,VI: PLMT,Ⅶ:PLMT+US),7mice in each group were treated correspondingly. Two mice in each group were sacrificed24hours after treatment and tumors were obtained. Apoptosis was detected by using TdT mediated UTP nick end labeling (TUNEL) method.The expression of the apoptosis related protein Caspase3was detected by immunohistochemical technique and western blot.The expression of vascular endothelial growth factor(VEGF) and MVD were detected by immunohistochemical technique.The survival time was observed among the other mice.Results:Compared with the control group, the survival time was longer in the other4groups(PLMT+US,PLM+US,PTX+US,PTX)(p<0.05), and the survival time in group PLMT+US was the longest (P<0.05), which was47±2.191d. The apoptosis rate and the expression of apoptosis related protein Caspase3were increased significantly in group PLMT+US,PLM+US,PTX+US and group PTX(P<0.05).The AI and Caspase3expression in group PLMT+US were much higher compared with the other3groups (PLM+US,PTX+US,PTX). The VEGF expression and MVD were decreased significantly in group PLMT+US,PLM+US,PTX+US and group PTX.When the expression of VEGF and MVD were compared between each group,those in group PLMT+US were significantly lower (P<0.05).Conclusions:The survival time of ovarian carcinoma bearing mice was significantly enhanced by Ultrasound mediated LHRHa-targeted paclitaxel lipid microbubbles. Its mechanism may be related with up-regulating of apoptosis related protein and inhibiting of angiogenesis.