Study On The Treatment Of Subcutaneous Transplanted Ovarian Carcinoma Using LHRHa-targeted Brucea Javanica Oil Liposomes

PART ONEPREPATION AND EVALUATION OF LHRHa-TARGETEDBRUCEA JAVANICA OIL LIPOSOMESObjective: To prepare LHRHa-Targeted Brucea Javanica oilLiposomes (LHRHa-BJO-Lipo) and observe its physical characteristicproperties and stability.Methods: The LHRHa-BJO-Lipo were prepared by conjugatingavidinylated Brucea Javanica oil Liposomes (BJO-Lipo) with biotinylatedLHRHa.The partical size and Zeta potential as well as the entrapmentefficiency of the liposomes were determined. The leakage rate of liposomeswere evaluated by centrifugal acceleration experiment. The stability of theliposomes was investigated by different storage conditions (40C、250C).Results: The best prescription preparation of LHRHa-BJO-Lipo wasselected by means of orthogonal design.The optimum formula was asfollows: the ratio of lecithin to cholesterol was4:1, Brucea Javanica oil： lipid was3:10, DSPE-PEG （2000）-Biotin: lecithin was3%,Ultrasonic-homogenized for8minutes. The average diameter and zetapotential of LHRHa-BJO-Lipo were (155.1±14.5) nm and-(24.1±0.54) mV,respectively. The average entrapment efficiency was （92.2±1.59）%.Steady centrifugal acceleration test confirmed that the liposome stabilityparameters of KE was stable.There were no phenomenons ofstratification,precipitation at40C for15days.However,there werephenmenons of stratification,precipitation at250C for one week.Conclusions: LHRHa-BJO-Lipo were prepared successfully bybiotin-streptavidin bridge method. The partical size was well-distributedwith high drug entrapment efficiency. The stability of this liposomes can beimproved at40C.PART TWOSTUDY OF THE TARGETING ABILITY OFLHRHa-TARGETED BRUCEA JAVANICA OILLIPOSOMES IN VIVOObjective: To observe the targeting ability of LHRHa-BJO-Lipo onsubcutaneous transplantation Ovarian carcinoma tumor in nude mice.Methods:The cell suspension derived from A2780/DDP were injectedintraperitoneally in nude mice to establish human epithelial ovariancarcinoma models.Four weeks after injection of A2780/DDP cells,6nude mices were randomly divided into3groups:Blank liposomes group, LHRHa-targeted Coumarin-6liposomesgroup, Coumarin-6liposomes group.2mice in each group were treatedwith different liposomes by tail vein injection.All nude mice were observedby fluorescence imaging system in vivo4hours after injection ofliposomes.Results: The human ovarian carcinoma subcutaneous model wereestablished successfully. It was found that the tumor fluorescence intensityof LHRHa-targeted Coumarin-6liposomes was more than that ofCoumarin-6liposomes group.Tumor fluorescence was not observed atblank liposomes group.Conclusions: The model of subcutaneous ovarian cancer xenografts innude mice are easy to establish. The LHRHa-BJO-Lipo has targetingability to ovarian cancer model in vivo.PART THREESTUDY ON THE THERAPEUTIC EFFECT ANDMECHANISM OF SUBCUTANEOUS TRANSPLANTEDOVARIAN CARCINOMA USING LHRHa-TARGETEDBRUCEA JAVANICA OIL LIPOSOMESObjective: To explore the inhibiting effects and the mechanism ofLHRHa-BJO-Lipo on ovarian carcinoma subcutaneous xenograft in nudemice. Methods: The cell suspension derived from A2780/DDP were injectedintraperitoneally in nude mice to establish human epithelial ovariancarcinoma models.28nude mices were randomly divided into4groups(I:PBS,II: Brucea Javanica Oil Emulsion; III: BJO-Lipo; IV:LHRHa-BJO-Lipo),7mice in each group were treated correspondingly.Two mice in each group were sacrificed randomly after the last treatmentand tumors were obtained for HE pathology. The expression of Caspase3,Caspase8were detected by immunohistochemical technique, and Theexpression of apoptosis related factors Caspase3, Caspase8, Bax, Bcl-2were detected by Western-blot respectively.The survival time and the tumorgrowth were observed among the other mice.Results: The median survival time of group I, II, III, IV were(42.4±1.47)days,(46±1.18)days,(52±1.40)days,(58.6±1.03)days,respectively. The median survival time of the group III, IV were longer than that of thegroup I(P＜0.05), group IV (LHRHa-BJO-Lipo) was the longest(P＜0.05).The tumor volume growth rate of the group IV significantly lower than thegroup I, II, III. The expression of the apoptosis related factors Caspase3,Caspase8and Bax of three different treatment groups were much higherthan that of the group I(P＜0.05). While the expression of Bcl-2proteinwas significantly lower than that of the group I, group IV was the lowest (P＜0.05).Conclusions: The survival time of ovarian carcinoma bearing mice was significantly enhanced and the growth of tumor was obviouslyinhibited by LHRHa-BJO-Lipo. Its mechanism may be related withincreasing of the expression of apoptosis related factors Caspase3,Caspase8,Bax and inhibiting of Bcl-2protein expression. It was promisedto be an effective new targeting method for the treatment of ovarian cancer.