KEYWORDS: Skin, Microscopes, Green fluorescent protein, In vivo imaging, Confocal microscopy, 3D image processing, Visualization, Mouse models, Molecular imaging, Luminescence
Quantum dots (Qdots) have become ubiquitous in biomedical research due to their excellent
brightness, photostability, monodispersity, and fluorescent yield. Furthermore, they have
become increasingly useful as imaging agents which are valuable for answering molecular
questions in living subjects. However, little is currently known about how nanoparticles such
as Qdots interact at the microscale within the vasculature and tumor microenvironments in
living subjects. In order to further our understanding of the dynamic processes involved in
Qdot targeting in the intact tumor, we developed an in vivo binding assay to visualize and fully
elucidate this approach using a variety of animal models and tumor types. We employed
argine-glycine-aspartic acid (RGD) peptides to specifically target the αvβ3 integrins which are
expressed on the surface of endothelial cells comprising newly formed or forming blood
vessels; RGD peptides were conjugated to the Qdot surface. Exploiting intravital microscopy
with subcellular-level resolution, we directly observed and recorded the binding of
nanoparticle conjugates in two different murine models, using three different tumor cell lines.
Using this generalizable approach, we learned that RGD-qdots unexpectedly bind to tumor
blood vessels in all models tested only as aggregates rather than individually. Understanding
these issues on the microscale using such techniques will provide a platform for the rational
design of molecularly-targeted nanoparticles including Qdots. This is critical for
nanoparticles to become a valuable research tool with the potential to become clinically
valuable imaging and therapeutic agents, particularly for ensuring regulatory approval of
such nanoparticles.
Photoacoustic molecular imaging is an emerging technology offering non-invasive high resolution imaging of the molecular expressions of a disease using a photoacoustic imaging agent. Here we demonstrate for the first time the utility of single walled carbon nanotubes (SWNTs) as targeted imaging agents in living mice bearing tumor xenografts. SWNTs were conjugated with polyethylene-glycol-5000 connected to Arg-Gly-Asp (RGD) peptide to target the αvβ3 integrin that is associated with tumor angiogenesis. In-vitro, we characterized the photoacoustic spectra of the particles, their signal linearity and tested their uptake by αvβ3-expressing cells (U87MG). The photoacoustic signal of SWNTs was found not to be affected by the RGD conjugation to the SWNTs and was also found to be highly linear with concentration (R2 = 0.9997 for 25-400nM). The cell uptake studies showed that RGD-targeted SWNTs gave 75% higher photoacoustic signal than non-targeted SWNTs when incubated with U87MG cells. In-vivo, we measured the minimal detectable concentration of SWNTs in living mice by subcutaneously injecting SWNTs at increasing concentrations. The lowest detectable concentration of SWNTs in living mice was found to be 50nM. Finally, we administered RGDtargeted and non-targeted SWNTs via the tail-vein to U87MG tumor-bearing mice (n=4 for each group) and measured the signal from the tumor before and up to 4 hours post-injection. At 4 hours post-injection, tumors of mice injected with RGD-targeted SWNTs showed 8 times higher photoacoustic signal compared with mice injected with non-targeted SWNTs. These results were verified ex-vivo using a Raman microscope that is sensitive to the SWNTs Raman signal.
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