| PART ONEIDENTIFICATION OF LHRHA-MODIFIED BRUCEAJAVANICA OIL LIPOSOMES AND TARGETING STUDYIN VITROObjective: To investigate the reliability of connecting the bruceajavanica oil liposomes with Luteinzing hormone releasing hormoneanalogue (LHRHa) by biotin-streptavidin linkage and evaluate theirtargeting ability in vitro.Method: Colloidal gold immunoassay was used to confirm whetherLHRHa was connected to brucea javanica oil liposomes. A fluorescent probe,coumarin-6was incorporated into LHRHa-modified liposomes(LHRHa-C6-Lipo) and their uptake by A2780/DDP cells which expressedpositive LHRH receptors and SKOV3cells which expressed negative LHRHreceptors were compared to that of unconjugated liposomes (C6-Lipo) byconfocal laser scanning microscopy. The mean fluorescence intensity wasdetermined by flow cytometry quantitatively after A2780/DDP cells and SKOV3cells were treated with LHRHa-C6-Lipo, LHRHa-C6-Lipo and freeLHRHa, C6-Lipo respectively.Results: LHRHa-modified brucea javanica oil liposomes (LHRHa-BJOL) group was positive in colloidal gold immunoassay while the BJOLgroup was negative. The results from the qualitative experiment showedthat the uptake amount of LHRHa-C6-Lipo by A2780/DDP cells was morethan that of C6-Lipo; but for SKOV3cells, the uptake of LHRHa-C6-Lipowas made little difference from C6-Lipo. The results from the quantitativeexperiment showed that A2780/DDP cells had a higher uptake forLHRHa-C6-Lipo than LHRHa-C6-Lipo with free LHRHa and C6-Lipo（P<0.05）, but there was no significant difference among the uptake ofthree liposomes by SKOV3cells (P＞0.05). Besides, by comparing withSKOV3cells, A2780/DDP cells had a higher uptake for LHRHa-C6-Lipoand LHRHa-C6-Lipo with free LHRHa （P<0.05）, but there was nosignificant difference between the uptake of C6-Lipo by A2780/DDP cellsand SKOV3cells (P＞0.05).Conclusions: LHRHa-Lipo was successfully developed as a targeteddrug delivery system, and this targeted liposome could enter A2780/DDPcells via receptor-mediated endocytosis in vitro. PART TWOTHE PROLIFERATIVE AND APOPTOSIS EFFECTS OFLHRHA-MODIFIED BRUCEA JAVANICA OILLIPOSOMES ON OVARIAN CANCER A2780/DDP CELLSObjective: To evaluate the proliferative and apoptosis effects ofLHRHa-modified brucea javanica oil liposomes on ovarian cancerA2780/DDP cells.Method: The survival rate of the cultured A2780/DDP cells wasdetected by MTT assays after the cells were treated with a series ofLHRHa-Lipo concentration for24h,48h and72h respectively. TheA2780/DDP cells were randomly divided into four groups: control group,brucea javanica oil emulsion group, brucea javanica oil liposomes group andLHRHa-modified brucea javanica oil liposomes group. The antiproliferativeeffects of A2780/DDP cells were measured by MTT assays at24h,48h and72h after the above treatments. Apoptotic phenotype of A2780/DDP cellswas observed by Hoechst33258staining, the cell apoptosis andmitochondrial membrane potential dissipation were analyzed by flowcytometry. The expression of apoptosis-associated proteins was analyzed bywestern blot.Results: Even though the concentration of LHRHa-Lipo increased from0.5mg/ml to8mg/ml and the time lasted from24h to72h, the cells viabilitywere all above90%. The three brucea javanica oil drugs could all inhibit the proliferation of A2780/DDP cells and the inhibitory rate ofLHRHa-modified brucea javanica oil liposomes group were(37.66±1.73)%,(51.26±3.46)%and (65.45±4.42)%respectively at24h,48hand72h, significantly higher than those in the other groups（P<0.05）．Hoechst33258staining showed that three kinds of bruceajavanica oil drugs could make chromatic agglutination in A2780/DDP cells,LHRHa-BJOL group had the most severely morphological change ofnucleus, and the apoptosis efficiency of A2780/DDP cells in this group was(33.36±1.31)%, significantly higher than other groups（P<0.05）.Compared with the control group, all the treatments could lowermitochondrial membrane potential（P<0.05）, but there was a most markeddrop in the LHRHa-modified brucea javanica oil liposomes group（P<0.05）.The LHRHa-BJOL could lead to increase in the expression of bax protein,caspase-3protein and caspase-8protein significantly while lead to decreasein the expression of bcl-2protein.Conclusions: LHRHa-BJOL could inhibit proliferation and induceapoptosis of A2780/DDP cells. The mechanism may be related to deathreceptor pathway and mitochondrial pathway. |
