Molecular imaging with targeted perfluorocarbon nanoparticles: quantification of the concentration dependence of contrast enhancement for binding to sparse cellular epitopes

Abstract

Targeted, liquid perfluorocarbon nanoparticles are effective agents for acoustic contrast enhancement of abundant cellular epitopes (e.g., fibrin in thrombi) and for lower prevalence binding sites, such as integrins associated with tumor neovasculature. In this study, we sought to delineate the quantitative relationship between the extent of contrast enhancement of targeted surfaces and the density (and concentration) of bound perfluorocarbon (PFC) nanoparticles. Two dramatically different substrates were utilized for targeting. In one set of experiments, the surfaces of smooth, flat, avidin-coated agar disks were exposed to biotinylated nanoparticles to yield a thin layer of targeted contrast. For the second set of measurements, we targeted PFC nanoparticles applied in thicker layers to cultured smooth muscle cells expressing the transmembrane glycoprotein "tissue factor" at the cell surface. An acoustic microscope was used to characterize reflectivity for all samples as a function of bound PFC (determined via gas chromatography). We utilized a formulation of low-scattering nanoparticles having oil-based cores to compete against high-scattering PFC nanoparticles for binding, to elucidate the dependence of contrast enhancement on PFC concentration. The relationship between reflectivity enhancement and bound PFC content varied in a curvilinear fashion and exhibited an apparent asymptote (approximately 16 dB and 9 dB enhancement for agar and cell samples, respectively) at the maximum concentrations (approximately 150 microg and approximately 1000 microg PFOB for agar and cell samples, respectively). Samples targeted with only oil-based nanoparticles exhibited mean backscatter values that were nearly identical to untreated samples (<1 dB difference), confirming the oil particles' low-scattering behavior. The results of this study indicate that substantial contrast enhancement with liquid perfluorocarbon nanoparticles can be realized even in cases of partial surface coverage (as might be encountered when targeting sparsely populated epitopes) or when targeting surfaces with locally irregular topography. Furthermore, it may be possible to assess the quantity of bound cellular epitopes through acoustic means.

Figure 1

Experimental setup. Samples were scanned in a waterbath at room temperature for agar samples, and at 37°C for cell cultures.

Figure 2

Bottom: Example reflected RF data from surfaces of a) agar, and b) cultured smooth muscle cells grown on cellulose membranes. Waveforms are shown for untreated surfaces and surfaces targeted with high-scattering PFOB nanoparticles or low-scattering oil-based nanoparticles. In b), the time gate required for analysis is shown to illustrate the portion of the signal associated with the targeted, cell-covered upper surface of the cellulose substrate. The second echo is the reflection from the opposite surface of the cell culture substrate. Note in both a) and b) the greater RF magnitude from surfaces with bound PFOB nanoparticles.

Figure 3

“En face” example images of integrated backscatter from C-scanned sample surfaces under selected treatment conditions. a) Agar samples (white = −34 dB, black = −64 dB); b) Smooth muscle cell cultures (white = −10 dB, black = −28 dB).

Figure 4

Integrated backscatter vs. bound PFOB content for a) agar, and b) smooth muscle cells exposed to specific nanoparticle treatments.

Figure 5

Experimentally observed enhancement vs bound PFOB for a) agar, and b) smooth muscle cells exposed to PFOB nanoparticle mixtures (shown as data points), along with curve fits of the form y =A (1 − e^−Bx). Enhancement is defined as the difference in integrated backscatter of a treated sample from the mean value of untreated sample group. Values for adjustable parameters A and B are shown in the legend of each graph.

Figure 6

Scanning electron micrograph of untreated porcine aortic smooth muscle cells grown in culture at 300X magnification. Heterogeneous and overlapping distribution of cells is clearly visible, as is irregular, dendritic shape of individual cells.