Molecular photoacoustic imaging of angiogenesis with integrin-targeted gold nanobeacons

Photoacoustic tomography (PAT) combines optical and acoustic imaging to generate highresolution images of microvasculature. Inherent sensitivity to hemoglobin permits PAT to image blood vessels but precludes discriminating neovascular from maturing microvasculature. α_vβ ₃-Gold nanobeacons (α_vβ₃-GNBs) for neovascular molecular PAT were developed, characterized, and demonstrated in vivo using a mouse Matrigel-plug model of angiogenesis. PAT results were microscopically corroborated with fluorescent α_vβ ₃-GNB localization and supporting immunohistology in Rag1 ^tm1Mom Tg(Tie-2-lacZ)182-Sato mice. α_vβ ₃-GNBs (154 nm) had 10-fold greater contrast than blood on an equivolume basis when imaged at 740 nm to 810 nm in blood. The lowest detectable concentration in buffer was 290 nM at 780 nm. Noninvasive PAT of angiogenesis using a 10-MHz ultrasound receiver with α_vβ₃- GNBs produced a 600% increase in signal in a Matrigel-plug mouse model relative to the inherent hemoglobin contrast pretreatment. In addition to increasing the contrast of neovessels detected at baseline, α_vβ ₃-GNBs allowed visualization of numerous angiogenic sprouts and bridges that were undetectable before contrast injection. Competitive inhibition of α_vβ₃-GNBs with α_vβ ₃-NBs (no gold particles) almost completely blocked contrast enhancement to pretreatment levels, similar to the signal from animals receiving saline only. Consistent with other studies, nontargeted GNBs passively accumulated in the tortuous neovascular but provided less than half of the contrast enhancement of the targeted agent. Microscopic studies revealed that the vascular constrained, rhodamine-labeled α_vβ ₃-GNBs homed specifically to immature neovasculature (PECAM ⁺, Tie-2^-) along the immediate tumor periphery, but not to nearby mature microvasculature (PECAM⁺, Tie-2⁺). The combination of PAT and α_vβ₃-GNBs offered sensitive and specific discrimination and quantification of angiogenesis in vivo, which may be clinically applicable to a variety of highly prevalent diseases, including cancer and cardiovascular disease.