The Effect Of Ultrasound Mediated LHRHa-targted Paclitaxel Liposomes-microbubbles Complex On Ovarian Cancer Cell In Vitro

PART ONEPreparation of LHRHa-targeted PaclitaxelLiposomes-Microbubbles complex and targeting study invitroObjective: To prepare a LHRHa targeted paclitaxel loaded liposome-microbubbles complex and and detect its targeting ability to ovariancancer A2780/DDP cells in vitro.Methods:1、Paclitaxel liposomes were prepared by thin-film ultrasonic dispersiontechnique, and its morphology was observed by transmission electronmicroscope. The encapsulation efficiency of different paclitaxel liposomeswere quantified by high-performance liquid chromatography(HPLC).Thefluorescent biotinylated liposomes was prepared by adding the NBDfluorescein labled cholesterol for the fluorescent identification experiment. 2、The biotinylated microbubbles were prepared by mechanic vibrationtechnique, the concentration,size and potential of the biotinylatedmicrobubbles were detected also.3、 The LHRHa-targeted paclitaxel loaded liposomes-microbubblescomplex was prepared by biotin-streptavidin bridge method.Themorphylogy of the complex was observed by laser confocalmicroscope,the conjugation efficiency were determined by flowcytometer(FCM),the targeting ability of the complex to ovarianA2780/DDP cells was observed by fluorescence microscope.Results:1、The paclitaxel liposomes were prepared by thin-film ultrasonicdispersion technique have good morphylogy shape, The biotinylatedpaclitaxel liposomes with average size of120.5nm.The encapsulationefficiency of these paclitaxel liposomes were above90%.2、The biotinylated microbubbles have good configuration, with averagesize of1208nm.3、The LHRHa-targeted paclitaxel lipoosmes-microbubbles complexwere prepared by biotin-streptavidin linkage with good shape. The flowcytometer showed that adding100μl liposomes to approximately2×107microbubbles is an optimal dose for liposome loading on the microbubblesshells. Approximately1.6mg paclitaxel was loaded on1×109 liposomes-microbubbles complex Under the optimal condition.TheLHRHa-targeted paclitaxel liposomes-microbubbles complex could adhereto the A2780/DDP cells efficiently in vitro.Conclusion: The LHRHa targeted liposomes-microbubbles complexcould be successfully prepared by biotin-streptavidin linkage method. Thiscomplex has targeting ability in vitro.PART TWOThe effection of ulrasound mediated LHRHa-targetedpaclitaxel liposomes-microbubbles complex on ovarian cancercell in vitroObjective: To Investigate effect of ultrasound mediatedLHRHa-targeted paclitaxel liposomes-microbubbles complex on thegrowth and apoptosis of ovarian cancer A2780/DDP cells.Methods: The A2780/DDP cells were cultivated in vitro and dividedinto the following groups:1.Control,2.Lipo-MB+US,3.PTX,4.PTX-Lipo,5.LHRHa-PTX-Lipo,6.LHRHa-PTX-Lipo-MB,7.LHRHa-PTX-Lipo+US,8.LHRHa-PTX-Lipo-MB+US. The survival rate of the cell in each groupwere detected by MTT24,48,72hours after treatment. The apoptosis rate ofeach group were assayed by flow cytometry. The ultrastructure of the cell in control group and LHRHa-PTX-Lipo-MB+US group were observed bytransmission electron microscopy (TEM).Results: MTT assay showed that the survival rate decreased graduallywith the time in all the treatment groups. The cells survival rate inLHRHa-PTX-Lipo-MB+US group reduce to (52.32±2.85)%、(35.30±1.27)%and (23.22±2.11)%after24h,48h,72h respectively, which wassignificantly lower than other groups (P<0.05).The cells apoptosis rate inLHRHa-PTX-Lipo-MB+US group is（37.15±1.01）%24h after treatment，which was significantly higher than other groups (P<0.05). The apoptoticbodies were observed in the LHRHa-PTX-Lipo-MB+US group24h aftertreatment.Conclusion: Ultrasound mediated LHRHa-targeted paclitaxelliposomes-microbubbles complex could inhibite proliferation and induceapoptosis of A2780/DDP ovarian cancer cells.This study may provide anew approach for drug delivery in tumor targeted therapy.