Silver nanoplate contrast agents for in vivo molecular photoacoustic imaging

Abstract

Silver nanoplates are introduced as a new photoacoustic contrast agent that can be easily functionalized for molecular photoacoustic imaging in vivo. Methods are described for synthesis, functionalization, and stabilization of silver nanoplates using biocompatible ("green") reagents. Directional antibody conjugation to the nanoplate surface is presented along with proof of molecular sensitivity in vitro with pancreatic cancer cells. Cell viability tests show the antibody-conjugated silver nanoplates to be nontoxic at concentrations up to 1 mg/mL. Furthermore, the silver nanoplates' potential for in vivo application as a molecularly sensitive photoacoustic contrast agent is demonstrated using an orthotopic mouse model of pancreatic cancer. Results of these studies suggest that the synthesized silver nanoplates are well suited for a host of biomedical imaging and sensing applications.

Figure 1

Silver nanoplates directly after step-wise growth adding edge length from B1 to B4 (a–d) with their corresponding extinction spectra (e) and color change in solution (f). Scale bars are 100 nm.

Figure 2

Demonstration of the biostability of PEGylated Ag nanoplates suspended in saline solution over a period of 62 days.

Figure 3

Darkfield microscopy of pancreatic cancer cells after incubation with no nanoplates (a), PEGylated nanoplates (b), and a-EGFR conjugated nanoplates (c). All scale bars are 20 μm.

Figure 4

Results of the MTS assay for cell viability on three cell lines after incubation for 24 hr with a-EGFR conjugated Ag nanoplates at various concentrations.

Figure 5

2-D cross sections of an orthotopic pancreatic tumor in a nude mouse model. US image (a) shows the anatomical features, while the USPA image (b) shows molecular accumulation of a-EGFR conjugated nanoplates (yellow), oxygenated blood (red), and deoxygenated blood (blue). Image dimensions are 14.5 mm by 11.8 mm.

Figure 6

Reconstructed 3-D USPA image of an orthotopic pancreatic tumor in a mouse model. The PA visualization of accumulated a-EGFR-conjugated nanoplates (yellow), oxygenated blood (red), and deoxygenated blood (blue) is shown overlaid on the US image of the mouse anatomy. Image dimensions are 8 mm by 17.5 mm by 11.8 mm.

Figure 7

Histological cross-section of the orthotopic pancreatic tumor imaged using USPA imaging. The section is stained with nuclear fast red (pink areas) and silver stain (dark areas in the slice that indicate the presence of Ag nanoplates). The distribution of the nanoplates is heterogeneous and correlates with the distribution of nanoplates displayed in USPA imaging.