In vitro acoustic molecular imaging of tissue factor expressed by smooth muscle cells with stable liquid perfluorocarbon nanoparticle contrast agents

Liquid perfluorocarbon nanoparticle contrast agents can be used to target specific tissue types through incorporation of appropriate ligands into the nanoparticles\´ outer lipid monolayer. In this study we sought to characterize the specificity of targeting of perfluorooctyl bromide (PFOB) nanoparticles to tissue factor, a transmembrane glycoprotein expressed by smooth muscle cells as part of inflammatory response after vessel injury (e.g., angioplasty). Porcine smooth muscle cells constitutively expressing tissue factor on the cell surface were cultured on microporous membranes and targeted with PFOB nanoparticles by avidin-biotin binding techniques. Treatment groups included: 1) "targeted" cell samples exposed sequentially to biotinylated tissue factor antibody/avidin/biotinlyated nanoparticles, 2) "non-targeted" smooth muscle cells exposed to avidin/biotinlyated nanoparticles; 3) "control" smooth muscle cells left untreated. Ultrasonic imaging and quantification of surface reflectivity for each cell group was accomplished by scanning a 25MHz transducer in a grid over the membrane inserts and analyzing the reflected RF signal from the cell-covered surfaces. Total PFOB content for each sample (directly related to nanoparticle concentration) was determined independently by gas chromatography. Targeted cells exhibited significantly enhanced surface reflectivity (-22.6±2.0 dB referenced to steel reflector, p<0.002) relative to either non-targeted (-29.6±1.1 dB) or control groups (-29.3±1.4 dB). Gas chromatography data indicated that bound PFOB was present in targeted cells at levels nearly six times greater than for nontargeted cells. Additionally, bound PFOB levels for the targeted samples Correlated directly with the magnitude of surface reflectivity (r=0.99; p<0.03). In conclusion, PFOB nanoparticles targeted to smooth muscle cell tissue factor specifically bind to such cells at levels nearly six times greater than to nontargeted cells. The targeted cells exhibit substantially enhanced acoustic reflectivity, thereby confirming the utility of quantitative ultrasonic molecular imaging with the use of a nongaseous nanoparticle contrast agent.