ABSTRACT
Objective To develop and validate a ultrasonographic (US)imaging agent with targeted microbubbles that attaches to chemokine receptor 2 (CCR2)and to compare the US single obtained from targeted microbubble with that from control microbubble in murine breast tumor model.Methods The microbubble which carried CCR2 antibody (MBCCR2 )and isotype-macthed immunoglobulin G-labled control microbubble (MBcontrol ) were prepared. The microbubble size and distribution were assessed by AccuSizer780.Binding specificities of targeted microbubble compared with control microbubble were tested with murine microvascular endothelial cells (bEnd.3 ).Orthotopic breast tumor model was estabished in BALB/c mice with mouse breast cancer 4T1 cell.In vivo imaging signals of contrast material-enhanced ultrasound by use these two different types of microbubble which were injected respectively into each mouse at random order and 30 min interval.Tumor tissue was stained for CCR2 and CD3 1 .Results Automatic Particle Sizer showed size uniform of two kinds of microbubbles,and narrow distribution of particle size (mean diameter of about 1 -2 μm),which were not significantly different (P >0.05).Adhension to bEnd. 3 endothelial cells was significantly higher (P < 0.001 )for MBCCR2 (mean,9.50 ± 1 .5 1 )than that for MBcontrol (mean,0.01 ±0.01).Imaging signal in the murine tumor model was significantly higher for MBCCR2 [mean,(6.76±0.26)dB]than that for MBcontrol [mean,(1 .06 ±0.62)dB,P <0.001 ].Immunofluorescence confirmed expression of CCR2 on tumor vasculature.Conclusions The targeted microbubbles with CCR2 monoclonal antibody had been successfully prepared,which precisely targeted to CCR2 of tumor angiogenesis in the murine breast cancer xenograft tumor models in vivo.These results suggest that the targeted microbubbles as a kind of ultrasound molecular imaging agent with a better specificity can be used for both evaluating tumor neovascularization and monitoring therapeutic effect of anti-angiogenesis.
ABSTRACT
Objective To prepare Integrin αvβ3/CCR2 dual targeted microbubble,test physical-chemical properties,enhancement effect and targeting ability in vitro.Methods The dual targeted microbubbles (MBdual-target )with FITC labled iRGD and PE labled CCR2 were prapared,and non-target microbubbles as control (MBcontrol ) were prapered.Physical and chemical properties of two groups of microbubbles were tested,connectivity of peptides/antibodies and microbubbles were detected by fluorescence microscope and flow cytometry instrument.Enhancement effect and the stability of two groups of microbubbles was observed and compared in vitro.The affinity of MBdual-target and MBcontrol for bEnd.3 cells was investigated with light and fluorescent microscope.Results ①The particle size of MBdual-target was (0.93±0.23)μm,with no statistically significant difference compared with MBcontrol (P >0.05).②MBcontrol showed no fluorescent,while MBdual-target showed both clear green and red light,under fluorescent microscope.③There was no significant difference of gray scale of enhancement between MBdual-target and MBcontrol in vitro.④ It was showed that MBdual-target adhered to bEnd.3 cells in vitro experiment.Conclusions Integrinαvβ3/CCR2 dual targeted targeted microbubbles was successfully prepared and proved having good enhancement effect and targeting ability in vitro.