Dual-targeted Ultrasound Contrast Agent Carrying Both Anti-ICAM-1Monoclonal Antibody And Anti-CD34Monoclonal Antibody: Preparation And In Vitro Experiment

PART Ⅰ PREPARATION OF DUAL-TARGETEDULTRASOUND CONTRAST AGENT CARRYING BOTHANTI-ICAM-1MONOCLONAL ANTIBODY ANDANTI-CD34MONOCLONAL ANTIBODY AND INTROSTUDYObjective To prepare dual-targeted ultrasound contrast agent carryingboth anti-ICAM-1monoclonal antibody and anti-CD34monoclonalantibody, to identify its basic characteristics and antibody-conjugated ratesof ultrasound contrast agent， then observation of the effect ofultrasound imaging in vitro.Methods ICAM-1-targeted ultrasound contrast agent (MBICAM-1),CD34-targeted ultrasound contrast agent (MBCD34R) and dual-targeted(withboth monoclonal antibodies) ultrasound contrast agent (MBD) wereprepared by biotin-avidin bridging chemistry method. The targetedultrasound contrast agent were observed under optical microscrope and characterized by a Mastersizer3000, DFY software, laser confocalmicroscopy(LCM), and flow cytometry.Results The morphous, diameter, surface potential, concentration,and the antibody-binding rates of ultrasound contrast agent were notstatistically significant in MBRICAM-1, MBRRCD34RR, and MBD(P>0.05).Theresults of LCM showed that anti-ICAM-1antibody and anti-CD34antibody were distributed on the shell of dual-targeted ultrasound contrastagent. Echo intensity in MBRRDRRsignificantly increased compared withMBICAM-1, MBCD34and MBBiotin（P<0.01）.Conclusions Dual-targeted ultrasound contrast agent carrying bothanti-CD34monoclonal antibody and anti-ICAM-1monoclonal antibody aresuccessful prepared. PART Ⅱ IN VITRO PERFORMANCE OFDUAL-TARGETED ULTRASOUND CONTRAST AGENTCARRYING BOTH OF ANTI-ICAM-1ANTIBODY ANDANTI-CD34ANTIBODYSECTION Ⅰ SEPARATION CULTURE ANDIDENTIFICATION OF ENDOTHELIAL PROGENITORCELLS FROM SD-RAT BONE MARROW IN VITROObjective To separation culture and identification of endothelialprogenitor cells (EPCs) from marrow of rat’s bone, in order to supplysubsequently studies.Methods Using the method of density gradient centrifugation tocollect the mononuclear cells from SD rat bone marrow. The EPCs wereinduced culture in EGM-2MV. Observing the process of cell growth,immunofluorescence was used to detect the expressions of CD34、CD133、VEGFR-2, cells uptaking DiI-ac-LDL and combined with FITC-UEA-1.Then tested the abilities of cell’s migration.Results The sizes of fresh cells isolated from rat bone were different.The EPCs were expressing CD34、CD133、VEGFR-2and uptakingDiI-ac-LDL and combined with FITC-UEA-1was positively. The numberof migratory cells reached (16.6±1.05) field of view.Conclusions From the density gradient centrifugation we couldisolated mononuclear cells after culture in EGM-2MV,they coulddifferentiated to EPCs. SECTIONⅡ TARGETING STUDY OF DUAL-TARGETEDULTRASOUND CONTRAST AGENT CARRYING BOTHOF ANTI-ICAM-1ANTIBODY AND ANTI-CD34ANTIBODYObjective Investigated the ability of targeting in dual-targetedultrasound contrast agent carrying both anti-ICAM-1monoclonal antibodyand anti-CD34monoclonal antibody.Methods Using biotin-avidin bridging chemistry method to preparedual-targeted(with both monoclonal antibodies) contrast agent (MBDR)、ICAM-1-targeted contrast agent (MBICAM-1) and CD34-targeted contrastagent (MBCD34R);The human umbilical vein endothelial Cells (HUVECs)were treated by TNF-αto established damaged HUVECRSR; The targetingspecificity and attachment capability of dual-targeted contrast agent toendothelial progenitor cells (EPCs) and damaged HUVECs were assessedin vitro.Results The laser confocal microscopy showed there was much greenfluorescence in cytoplasm; The targeting experiment in vitro showed thatthe attachment rate of MBDto EPCs and damaged HUVECs significantlyincreased.Conclusions The study in vitro has proved that the dual-targetedcontrast agent can specifically bind to both EPCs and damaged HUVECs.