The Preparation Of Thrombus-targeted Phospholipid-coated Microbubbles And Its Application In The Arterial Thrombus Detection

Background:The morbility of thromboembolic disease is increasing year by year due to the rapiddevelopment of the society and the intensified process of aging.It has been estimated thatabout 15 million people die of such disease each year globally,which has become the firstcause of the global mortality.Every year the morbility is about 10 million and the mortalityis 1 million in our country.In a word,we have faced with great challenges against thethromboembolic disease.Thus,detecting and treating thrombus exactly as early as possibleare of great significance for decreasing mortality and morbility of complications.Although DSA is the primary imaging modality for detecting vascular lesions,it isinvasive,expensive and may cause complications at 2%-4% possibility.MRA and CTAmay show vessels and the thrombus better,but they are limited in manifesting smallthrombus.As a widely clinically applied imaging detected means,ultrasound is importantin the early diagnosis and follow-up observation of thrombus diseases.However,it islimited in the fresh thrombus detection due to the similarity of resonance between freshthrombus and blood.In 1968,Gramiak first proposed the concept of ultrasound contrast imaging.Theyobserved blooming artifacts in echocardiogram after intracardial injection of indocyaninegreen dye and attributed the effect to the gas bubbles generated by injecting the green dye.Thus,indocyanine green dye has become the standard ultrasound contrast agent anddeveloped three generations currently.The first generation is a milestone in the contrastagent development,such microbubbles were filled with air and could be injected viaperipheral vein.But it could not be used clinically due to the short time maintenance in the blood.The second generation was filled with perfluorocarbon gas,has great stability andhas been widely applied clinically.The third generation is reported as targeted microbubbleand can be imaged in localized tissue.It is now in the stage of lab experiment domestically.Therefore,targeted contrast agent has become the hot spot in the few future and has broadprospect as well as potential market value.So we are expected to prepare suchthrombus-targeted contrast agent to improve the image effects of thrombus and increase theaccuracy and sensitivityObjective:The purpose of this study was designed to prepare a thrombus-targetedphospholipid-coated microbubbles,investigate its biologic activity and evaluate itsimaging effect on the enhancement on acute thrombus made in carotid artery of rabbitthrough visual examination and video analysis.Methods:1.To prepare common lipid-coated fluorocarbon microbubble and determinate itsbiologic activityDPPC,DPPG,DPPE,cholesterol were prepared as phospholipid-coated microbubblefollowing membrane-hydration method.The stability of microbubbles was observed bylight microscope,the diameter and surface potential were measured by Zetasizer NanoZS90 and the concentration was detected by blood cell count machines.2.To prepare thrombus-targeted lipid-coated fluorocarbon microbubbles anddeterminate its biologic activityNBD-PC,DPPG,biotinyl PE,cholesterol were prepared as phospholipid-coatedmicrobubble following membrane-hydration method.The stability and appearance ofmicrobubbles were observed by light and fluorescent microscope,the diameter and surfacepotential were measured by Zetasizer Nano ZS90 and the concentration were detected byblood cell count machines.3.To establish thrombus model and evaluate its stabilityRabbit thrombus model made by FeCl₃ infiltration in common carotid artery of rabbitwas set up.Six rabbits were randomly invided into three groups according to FeCl₃infiltration time:20min (the first group),40min (the second group),60min (the third group).After sacrificing the animals,vessels were obtained for HE staining to evaluate the thrombus model.4.To evaluate the thrombus model by various imaging meansThe establishment of thrombus model:Normal vessel ultrasound imaging wereobtained and saved by CDFI (Color Doppler Flow Imaging) before experiment.Rightcommon carotid artery of 20 healthy rabbits dissociated about 2cm were infiltrated byFeCl₃ about 30min to induce the formation of non-obstuctive thrombus.Thrombusultrasound imaging was obtained and saved by CDFI after the establishment of thrombusmodel.Ultrasound contrast imaging after contrast agent injection:common lipidmicrobubble through ear via intravenous bolus injection at a dose of 0.5ml/kg was observedconsecutively about 10 min,then Sono Vue at a dose of 0.5ml/kg was injected again andobserved 10min after clearing the residual microbubbles.Finally,we injected thebiotinylated rabbit anti-human fibrinogen (diluted as 1:200) 0.1ml,30min later,avidin(diluted as 1:20) 0.1ml and after 30 min,injected targeted lipid-coated contrast agent at adose of 0.5ml/kg,then we observed about 10min consecutively.MRI evaluation:thethrombus model was imaged and saved by 3D-TOF-MRA (3D-Time-Of-Flight MagneticResonance Angiography).The video images were assessed qualitatively by fiveindependent observers.5.Thrombus-targeted lipid-coated microbubbles' experiment in vivo and in vitroFour rabbits were established non-obstructive thrombus model following the same way.Two rabbits' artery were placed in 2ml saline and we added the biotinylated rabbitanti-human fibrinogen (diluted as 1:200) 0.1ml,30min later avidin (diluted as 1:20) 0.1mland after 30min,added targeted lipid-coated contrast agent at a dose of 0.5ml/kg in theartery by order,then we frozen some of samples rapidly for fluorescence detection anddetected a part of samples by transmission electron microscope to observe the division ofmicrobubbles in the thrombus.Another two rabbits were injected the biotinylated rabbitanti-human fibrinogen (diluted as 1:200) 0.1 ml,30min late avidin (diluted as 1:20) 0.1mland after 30 min,injected targeted lipid-coated contrast agent at a dose of 0.5ml/kg via earvein by order,then we frozen some of samples rapidly for fluorescence detection anddetected a part of samples by transmission electron microscope to observe the division ofmicrobubble in the thrombus. Results:1.To prepare common lipid-coated fluorocarbon microbubble and determinate itsbiologic activityCommon lipid-coated fluorocarbon microbubble showed white appearance and theaverage diameter ranged from 0.8-2.5 um,some of which arrived at nm stage.The zetaelectric potential was about-11mv and the concentration was about 1.08×10¹⁰/mL.Thediameter and potentials were changed little,while concentration has decreased after a weekunder room temperature.Amounts of microbubbles were decreased obviously after abouthalf a month,while after about one month,just a few scattered microbubbles were observedunder the microscope.2.To prepare thrombus-targeted lipid-coated fluorocarbon microbubbles anddeterminate its biologic activityThrombus-targeted lipid-coated fluorocarbon microbubbles showed yellow appearanceand the average diameter ranged from 1.1-2.5 um.The zeta electric potential was about-11mv and the concentration was about 6.5×10⁹/ml.The diameter and potentials werechanged little,while concentration has decreased after a week under room temperature.Justa few scattered microbubbles were observed under the microscope after about a month andits stability was a little weak than common lipid-coated microbubble.3.To establish the thrombus model and evaluate its stabilityGross appearance:the first group:no significant thrombosis;the second group:non-obstructive thrombosis and thrombus holds about 1/3-1/4 of vessel;The third group:obstructive-occluding thrombosis and thrombus holds the total vessel.Microscope:Thesecond and third group could be imaged as mixed thrombus.4.To evaluate the thrombus model by various imaging meansCDFI showed smooth signals and completed lumens in the right common carotidartery of 20 rabbits before the establishment of thrombus model,while only 4 rabbits'arteries were detected as obvious filling defect and inconsecutive blood signals by CDFI,the rest showed worse image in the established thrombus model.Common lipid-coatedmicrobubble developed an image in the right common carotid artery 3 second afterintravenous bolus injection and such image persisted about 60 second,arrived at peak inabout 10 second.Just 10 rabbits' ultrasound showed obvious filling defect,inconsecutive blood signals and insufficient smooth vessel wall in local lumens.SonoVue developed animage in the right common carotid artery 3 second after intravenous bolus injection andsuch image persisted about 150 second,arrived at peak in about 20 second.Ultrasoundshowed the same image as above.Targeted lipid-coated microbubbles developed an imagein the right common carotid artery 3 second after intravenous bolus injection and suchimage persisted about 200 second,arrived at peak in about 25 second.Ultrasound showedthe same image as above.As compared with above,3D-TOF-MRA showed inconsecutiveblood signals and insufficient smooth vessel wall in local lumens in only 3 rabbits,the restshowed worse.5.Thrombus-targeted lipid-coated microbubbles' experiment in vivo and in vitroMassive targeted lipid-coated microbubbles flocked around and some of themaggregated within the fresh blood clots under light and fluorescence microscope after rapidfrozen sections both in vivo and in vitro.While amounts of aggregated microbubbles invitro were larger than that in vivo,the fluorescence ejected in vitro was more obvious thanin vivo.The electron microscope showed the same image.Conclusions:1.The preparation of thrombus-targeted phospholipid-coated microbubbles and itsapplication in the arterial thrombus-targeted detection have been successfully performed,which establish a sound foundation for exploitation of targeted contrast agent domesticallyas well as provide the potential market value.2.The imaging of thrombus-targeted phospholipid-coated microbubbles wasperformed more direct-viewing and longer time than common lipid microbubbles,CDFIand MRA.3.Thrombus-targeted phospholipid-coated microbubbles could aggregate rapidly withhigher concentration in fresh blood clot and deep into the internal part both in vitro and invivo.Thus,it may realize the targeted thrombus detection as well as provide the theoreticfoundation for investigating targeted thrombolysis therapy.