Preparation And Characterization Of Luteinising-hormone Releasing Hormone Nanoliposomal Microbubble Specifically Targeting Ovarian Cancer Cells In Vitro

Objective：To prepare luteinising-hormone releasing hormone (LHRH) nanoliposomalmicrobubble specifically targeting ovarian cancer cells and characterize its physioch-emical properties in vitro, for achieve the extravascular tumor targeting developingstudy provides a preliminary basis.Methods：1.Lyophilization/sonication method and Mechanical oscillations were used toprepare non-targeting liposomal microbubble (N-N-Mb).2.The morphology and shelf life of microbubbles was detected by opticalmicroscope, and the particle size range was measured by Zeta detector.3.Lyophilization/sonication method and Mechanical oscillations were used toprepare non-targeting nanoliposomal microbubble (N-N-Mb). Using the biotin-avidinbridge method, conjugated biotinylated LHRH antibodies to N-N-Mb generatedbiotinylated LHRH nanoliposomal microbubble (LHRH-N-Mb) targeting specificallyovarian cancer cells.4.The morphology and shelf life of microbubbles was detected by opticalmicroscope, and the particle size range and the surface potential were measured byZeta detector.5.The binding affinity between the secondary antibody and LHRH-N-Mb orN-N-Mb was determined by flow cytometry.6.The binding of LHRH-N-Mb to human OVCAR-3ovarian cancer cells, wasdetected by light microscopy.7.Whereas pre-incubation with LHRH antibody blocked human OVCAR-3ovarian cancer cells. The binding of LHRH-N-Mb to human OVCAR-3ovariancancer cells was detected by light microscopy. Results：1.Non-targeted lipid microbubbles is a milky suspension,400times the lightmicroscope and Zeta detector60s,90s,120s, microbubbles evenly distributed, noreunion, rounded appearance, size range from a single grain of microbubbles diameterranges were329~1008nm,295~468nm,369~618nm,157~268nm,400-fold lightmicroscope and Zeta detector150s uneven distribution of microbubbles, no reunion,the appearance of a single round of microbubbles, particle size range from range60~896nm2.The rounded and uniformly distributed N-N-Mb and LHRH-N-Mb weresuccessfully generated. The particle size ranged from295~468nm with a mean of360nm for N-N-Mb or369~618nm with a mean of508nm for LHRH-N-Mb. Therewas a significant size difference between the two groups (P<0.05) although thesurface potential of the two microbubbles remained the same (-14.6mv).3.By optical microscope, After being kept at room temperature for14days, nosignificant difference of the LHRH-N-Mb’s physiochemical properties was detectedcompared with that of freshly prepared microbubbles.4.The antibody binding rate of LHRH-N-Mb or N-N-Mb was75.6%or0.83%.5.Moreover, the rosette formation surrounding OVCAR-3cells was observedunder light microscopy after cells were incubated with LHRH-N-Mb.6.While pre-incubated biotinylated LHRH antibody blocked the rosetteformation. The binding of LHRH-N-Mb to human OVCAR-3ovarian cancer cellswas not detected by light microscopy.Conclusion：1.Mechanical oscillations90s for the manufacture of non-targeted lipidmicrobubbles best time, namely the preparation of non-targeted nano-lipid microb-ubbles mechanical oscillation time2.The LHRH nanoliposomal microbubble specifically targeting ovarian cancercells was successfully prepared through biotin-avidin mediation and lyophilization/sonication method. The key feature of LHRH nanoliposomal microbubble is itssmaller size, stability. 3.High efficiency of targeting human OVCAR-3ovarian cancer cells in vitro.4.Human OVCAR-3ovarian cancer cells was blocked by biotinylated LHRHantibody，did not connet LHRH-N-Mb by light microscopy.5. By LHRH, LHRH-N-Mb can connet Human OVCAR-3ovarian cancer cells.