Real-time intravital imaging of RGD-quantum dot binding to luminal endothelium in mouse tumor neovasculature

Abstract

Nanoscale materials have increasingly become subject to intense investigation for use in cancer diagnosis and therapy. However, there is a fundamental dearth in cellular-level understanding of how nanoparticles interact within the tumor environment in living subjects. Adopting quantum dots (qdots) for their excellent brightness, photostability, monodispersity, and fluorescent yield, we link arginine-glycine-aspartic acid (RGD) peptides to target qdots specifically to newly formed/forming blood vessels expressing alpha vbeta 3 integrins. Using this model nanoparticle system, we exploit intravital microscopy with subcellular ( approximately 0.5 microm) resolution to directly observe and record, for the first time, the binding of nanoparticle conjugates to tumor blood vessels in living subjects. This generalizable method enabled us to show that in this model qdots do not extravasate and, unexpectedly, that they only bind as aggregates rather than individually. This level of understanding is critical on the path toward ensuring regulatory approval of nanoparticles in humans for disease diagnostics and therapeutics. Equally vital, the work provides a platform by which to design and optimize molecularly targeted nanoparticles including quantum dots for applications in living subjects.

Figure 1

Intravital microscopy used to investigate derivatized nanoparticles. (a) Our intravital microscope setup, with callout of a mouse being imaged under a 10× objective. Ears were affixed to double-sided tape for motion stabilization. (b) The chemistry used to conjugate cyclic RGD and RAD peptides to qdots for injection into mice.

Figure 2

TEM and fluorescence of RGD–qdots. RGD–qdots dispersed in PBS (left) and in mouse serum (middle) as shown by TEM. Overall concentration of qdots was identical in both conditions. Random (typical) fields-of-view were chosen. No aggregates were observed. Fluorescence microscopy (right), by contrast, displays various aggregates.

Figure 3

Direct visualization of binding of RGD–qdots to tumor vessel endothelium and controls. (a) Panel displays different output channels of the identical imaging plane along the row with scale bars. In the green channel, individual EGFP-expressing cancer cells are visible (marked by thick horizontal blue arrows; vertical blue arrow points to a hair follicle), while the red channel outlines the tumor's vasculature via injection of Angiosense dye. The NIR channel shows intravascularly administered qdots which remain in the vessels (i.e., they do not extravasate). Binding events are visible by reference to bright white signal. These are demarcated by arrows in the rightmost merged image, in which all three channels have been overlaid. (b) Displays the same as (a) in a different mouse, except that 6 times the RGD–qdot dose has been injected. Individual cells are not generally visible. Six binding events are observed in this FOV, as marked by arrows in the merged image at right. White arrows in the bottom merged image designate areas of tissue autofluorescence. Typical images of no binding in each control condition are shown in (c–f). Tumor neovasculature containing unconjugated qdots (c), normal vasculature containing RGD–qdots (d), and tumor neovasculature containing RAD–qdots (e). (f) Tumor vasculature shortly after Cy5.5 injection (left) and ∼20 min post-Cy5.5 injection (right). Individual cancer cells are visible before (left) and after dye extravasates (right, dyed red). Also see movie S6 in Supporting Information. Horizontal white arrows indicate tissue autofluorescence, vertical blue arrows denote hair follicles (which generally display autofluorescence in their center), and thick horizontal blue arrows indicate individual cancer cells.

Figure 4

Comparison of binding rates between conditions tested. Displays the binding rate (calculated as events/FOV) for each experimental condition with 95% confidence intervals.