Experimental Study On Preparation And Targeting Function In Vitro Of Targeted Nano-Lipid Ultrasound Contrast Agent

PARTⅠEXPERIMENTAL STUDY ON PREPARATION AND ENHANCEMENT EFFECTS OF NANO-LIPID ULTRASOUND-ENHANCED CONTRAST AGENTObjective: To prepare a novel nano-lipid ultrasound-enhanced contrast agent, then compare it with common-lipid microbubbles by animal experiment to evaluate the enhancement effects.Methods: A certain proportion of dipalmitoylphosphatidylcholine (DPPC),distearoylphosphatidylcholine(DSPE),diphenylphosphoryl azide (DPPA) and mannitol were mixed in a tube,incubated,freezed and thawed some times repeatedly,then the nano-lipid microbubbles was prepared by mechanical vibration, its base physical properties like morphous,size and surface potential were observed and detected; before and after intravenous injecting 0.25 ml/kg(3.0×109 /ml) of the two contrast agents(nano-lipid and common-lipid) in succession,the dynamic enhancement effects of the grey scale imaging of the healthy rabbit livers were observed by the Ultrasonographic Quantitative analisis system in type DFY for detecting and analysing the time-activity curve of the gray scale of the rabbit livers to evaluate the enhancement effects.Results: The mean diameter of the nano-lipid microbubbles was (415.4±51.8) nm and the surface potential was -(13.7±1.6) mV;the nano-lipid microbubbles could greatly enhance the grey scale imaging of the liver,compared with the common-lipid microbubbles,its peak intensity was lower,the to-peak-time was later,but the enhancement effects persistence time was longer than the common-lipid microbubbles(P<0.05).Conclusions: The self-made nano-lipid microbubbles has a good shape and uniform distribution,it can enhance the grey scale imaging of the liver greatly and provide a protophase foundation for targeting research of tumors. PARTⅡEXPERIMENTAL STUDY ON PREPARATION AND TARGETING FUNCTION IN VITRO OF TARGETED NANO-LIPID ULTRASOUND-ENHANCED CONTRAST AGENTObjective: To prepare a hunman breast carcinoma-targeted nano-lipid ultrasound-enhanced contrast agent and investgate the targeting function in vitro.Methods: After the biotinylated monoclonal antibody Neu(F-11) was prepared, the biotinylated degree and immunological activity were detected by ultraviolet spectrophotometry and cell enzyme-labeled immunosorbent assay(ELISA); then biotinylated microbubbles was prepared by mechanical vibration and biotinylated antibody was attached to the surface of biotinylated nano-lipid contrast agents by avidin-biotin interaction to made the targeted nano-lipid contrast agents, its base physical properties like size and surface potential were detected,the combination of Neu(F-11) with nano-lipid contrast agents was proved by immunofluorescent assay qualitatively,while the targeting function was investgated by observing the combined effection of the targeted nano-lipid contrast agents with different cells in vitro.Results: About 7 biotin molecules were coupled to each antibody molecule in average,the immunological activity of the biotinylated antibody didn't decrease compared with the free one;the mean diameter of the targeted nano-lipid microbubbles was (573.4±65.8) nm and the mean surface potential was -(31.7±1.6) mV,red fluorescence was observed at the edge of the targeted nano-lipid contrast agents in immunofluorescent assay, and only MCF-7 cells were surrounded by targeted nano-lipid contrast agents while control groups were negative.Conclusion: Contrast agents and antibodys can be combined firmly by avidin-biotin system, the targeted nano-lipid contrast agents prepared by this system has strong and especial targeting function in vitro.