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The Preparation Of Lipid-shelled Targeted Microbubbles And Their Application In Early Diagnosis Of Thrombosis

Posted on:2018-01-14Degree:MasterType:Thesis
Country:ChinaCandidate:T T WangFull Text:PDF
GTID:2334330542451996Subject:Biomedical engineering
Abstract/Summary:PDF Full Text Request
Thromboembolic disease is a serious threat to human life and health due to the highest incidence.Therefore,the targeting and accurate theranostic approach has been attracted more and more attentions in recent years.Bioorthogonal coupling chemistry has been studied as one significant advantage for molecular imaging as it offers rapid,efficient,and strong binding,which may also benefit in stability,production,and chemical conjugation.The inverse-electron-demand Diels-Alder reaction between s-tetrazine and trans-cyclooctene(TCO)is an example of a highly selective and rapid bioorthogonal coupling reaction to be used successfully to prepare targeted molecular imaging probes.The aim of this study is to design a two-step microbubble thrombus targeting strategy based on the principle of tetrazines and trans-cyclooctene(TCO)bioconjugate orthogonal reactions.Tetrazine modified microbubbles(Tetra-MBs)could be uniquely and rapidly captured by subsequent click chemistry of trans-cyclooctene tagged p-selectin antibody(antiCD62p)pre-treated thrombus.Such Tetra-MBs showed great long-term stability under physiological condition to maintain the ability to monitor thrombus dynamic changes in real-time.It is demonstrated that Tetra-MBs based bioorthogonal targeting molecular ultrasound imaging strategy could be a simple but powerful tool for rapid diagnosis of acute thrombosis.The following points are mainly involved in the dissertation:(1)Firstly,DSPE-PEG2k-PEG5-Tetrazine was synthesized by the amide reaction between 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000](DSPE-PEG2k-NH2)and the orthogonal-molecular Tetrazine-PEG5-NHS.Then the tetrazine modified MBs were prepared by shaking the sulfur hexafluoride(SF6)gas saturated mixture of DSPE-PEG2k-PEG5-Tetrazine and phosphatidylcholine(DSPC)lipid solution.Furthermore,TCO-PEG4-antiCD62p was synthesized by the antiCD62p molecule chemically coupled with the trans-cyclooctene-polyethylene glycol-N-hydroxysuccinimide ester(TCO-PEG4-NHS)molecule.The Tetra-MBs was characterized and the TCO-PEG4-antiCD62p was confirmed by matrix assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF)and SDS-PAGE.(2)Secondly,the targeting efficiency of Tetra-MBs to the TCO-PEG4-antiCD62p pre-tagged activated platelets was verified by in vitro parallel plate flow chamber experiments.The results showed that about 17%of Tetra-MBs were able to accumulate on the activated platelets in vitro.In vivo rat deep vein thrombosis model experiment further verified the high targeting efficiency of Tetra-MBs on early deep vein thrombosis area.Results indicated that the rapid Tetra-MBs accumulation in the thrombus can maintain ultrasound contrast enhanced imaging ability ranging from 5 min to 30 min and following within the next 2 h timeframe.(3)Finally,positive charged tetrazine modified microbubbles(Dotap-Tetra-MBs)were prepared and loaded with low molecular weight heparin on the surface of Tetra-MBs by electrostatic interaction.The heparin contents loaded in Dotap-Tetra-MBs were determined by UV absorption method.The thrombin-induced plasma anticoagulant effects of heparin-modified MBs have been evaluated in vitro.And the preliminary therapeutic effect for rat deep vein thrombus also has been demonstrated in vivo.
Keywords/Search Tags:orthogonal reactions, targeted microbubbles, thrombus, p-selectin, ultrasound molecular imaging
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