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Preparation And In Vitro Assay Of Microbubbles Targeting Hepatocellular Carcinoma With Biotin-avidin System

Posted on:2009-08-17Degree:MasterType:Thesis
Country:ChinaCandidate:F LiuFull Text:PDF
GTID:2144360272961986Subject:Digestive medicine
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Background:Targeted ultrasound techniques combine ultrasound imaging technology with specific contrast agents for the assessment of molecular or genetic signatures for disease. The idea of the targeted ultrasound agent centers around the use of gas-filled microbubbles with ligands attached to them.These microbubbles could circulate through the organism and accumulate at the surface of target tissue,thereby 'marking' the target in the ultrasound images.According to different chemical compositions of the shell of MBs,various methods have been developed using different specific linked ligands,We summarize several familiar ways for preparing of targeted MBs as below:simple-desorption,staticelectro-adsorption and covalence couple.Biotin- avidin system was extensive recognized for its stability and high performance.Klibanov firstly successfully applied biotin-avidin system to liposomes. Subsequently,Brekken demonstrated that biotin- avidin system can be used to perfluorocarbon-exposed sonicated dextrose albumin(PESDA) MBs(PESDA-MBs). However,their preparation used only biotin or avidin as one of native microbubble material,which needs many resources and has less flexibility.Purpose:To determine a optimized setting which is more stabe and effective to couple albumen microbubbles(MBs) with monoclonal antibodies(mAbs) by biotin-avidin system.Method: Ready-made albumen MBs modified with biotin were linked with Avidin(AV).And then biotinylated mAbs were conjugate to the MBs prepared above sequentially, synthesized targeted MBs eventually.The every step of process would be monitored by flow cytometry.The hepatocellular carcinoma cell(HCC) line is used to examine the novel linking strategy for target labeling.Results:1.Our experiments resulted in 96.03%of MBs bound to their shells.2.Agglutination test:the targeted MBs was seen be aggregated together,and there is on aggregating between control group.3.The Av-Bio-MBs was used to conjugate to mAbs specific for Hab18G/CD147.In vitro,MBs targeted to CD147 specifically bound to HCC cells,but not to HSC. After being dealt with Hab18G MAbs solution for rosette-blocking,few targeting MBs would bind to HCC cells.4.The best yield of mAb-linked MBs was achieved by using Bio 24μg,AV 9μg, and mAbs 9μg for 100μL of MBs(approximately 10~8 MBs).Conclusions:The method stated here is economic,effective and flexible. AV-Bio-MBs system is a novel and attractive tool to conjugate MBs with biotinylated mAbs for the purposes of HCC targeting and molecular imaging.
Keywords/Search Tags:targeted microbubbles, biotin - avidin, hepatocellular carcinoma
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