Shape matters: intravital microscopy reveals surprising geometrical dependence for nanoparticles in tumor models of extravasation

Abstract

Delivery is one of the most critical obstacles confronting nanoparticle use in cancer diagnosis and therapy. For most oncological applications, nanoparticles must extravasate in order to reach tumor cells and perform their designated task. However, little understanding exists regarding the effect of nanoparticle shape on extravasation. Herein we use real-time intravital microscopic imaging to meticulously examine how two different nanoparticles behave across three different murine tumor models. The study quantitatively demonstrates that high-aspect ratio single-walled carbon nanotubes (SWNTs) display extravasational behavior surprisingly different from, and counterintuitive to, spherical nanoparticles although the nanoparticles have similar surface coatings, area, and charge. This work quantitatively indicates that nanoscale extravasational competence is highly dependent on nanoparticle geometry and is heterogeneous.

Figure 1

Experimental set-up. (a) Our intravital microscope set-up is shown, with an image of an ear tumor of an anesthetized mouse being imaged. (b) The two nanoparticles we employ in this work are illustrated as cartoons. The quantum dot is approximately spherical and has a hydrodynamic diameter of ~20 nm. High aspect ratio single-walled carbon nanotubes are ~2–3 nm in diameter and ~200 nm in length. The quantum dot on the left and the nanotube cartoon on the far right extending across the height of the figure are drawn to scale with respect to one another. The clear differences in size and scale are more easily grasped by visualization with this graphic.

Figure 2

SKOV-3 tumor extravasation of qdots and SWNTs. Images on the left display nanoparticles (grayscale) within blood vessels in SKOV-3 tumor within minutes of nanoparticle injection (the tumor channel was removed for ease of viewing the vasculature). The location of vessels is specified by the dashed lines. Each set of images is accompanied by a graph on the right of the change in fluorescence intensity over time, in both the extravascular space (i.e., extravasated nanoparticles, illustrated by the blue curve) and nanoparticles in the intravascular space (illustrated by the black curve). The estimated slope (assuming a linear fit) is indicated for the extravasation curves to provide an approximate, quantitative appreciation for the rate of nanoparticle leakage. (a) Qdots (gray) can be easily observed in the blood vessels soon after injection. By one hour postinjection, no qdots are visible in the vessels nor outside the vessels, in the tumor interstitium. Scale bars represent 20 µm. (b) SWNTs (gray) are visible minutes after injection. The arrows point to hair follicles which are autofluorescent in their center; furthermore, general autofluorescence pervades the image. However, it is clear that by one hour post-injection, the fluorescence has not increased. This is quantified in graphs. Scale bars represent 50 µm.

Figure 3

LS174T and U87MG tumor extravasation of qdots and SWNTs. As in Figure 2, blood vessels are represented by dashed lines and nanoparticles are in grayscale. Each set of images is accompanied by a fluorescence intensity graph on the right, for extravasated nanoparticles (blue curve) and nanoparticles still circulating within the blood vessels (black curve). The estimated slope (assuming a linear fit) is indicated for the extravasation curves to provide an approximate, quantitative appreciation for the rate of nanoparticle leakage. (a) Qdots (grayscale) in LS174T tumor extravasate robustly out of the blood vessels within one hour of injection (see graph for the rapidity and monotonic increase over 50 minutes). It is notable that the very high signal produced by extravasated qdots in this image (due to the high level of extravasation) somewhat convolutes measurements of fluorescence intensity within the blood vessels, explaining the low absolute value of the slope in the black (intravascular) curve on the right. (b) On the other hand, SWNTs do not extravasate nearly as much as qdots. Note that they do extravasate minimally (by reference to the minor signal increase in the interstitium outside the vasculature between the two time-points). All scale bars in (a) and (b) represent 20 µm. (c) Qdots in U87MG tumor do not extravasate out of the vasculature, as can be observed in the tumor interstitium outside the blood vessels since there is no apparent change in fluorescence intensity. The graph shows this quantitatively. Scale bar represents 10 µm. (d) In the images in the SWNT condition, autofluorescence is visible in the condition immediately after injection (both diffuse autofluorescence as well as intense, punctate autofluorescence). Unexpectedly, as observed by the clear increase in extravascular intensity, SWNTs extravasate in U87MG tumors. This is supported by the graph quantifying the fluorescence increase. Scale bar is 50 µm.

Figure 4

The extravasation of qdots and SWNTS from the vasculature of murine tumor models. (a) The extravasation (averaged over all mice per nanoparticle/tumor group) of qdots is compared with that of SWNTs for each tumor type. There is a significant difference between the extravasation of the two nanoparticle types for U87MG tumors. The extravasation difference is also significant for LS174T tumors, but surprisingly the nanoparticle types are reversed in terms of their extravasational competence compared with U87MG tumors. Note that qdots displayed nearly zero average extravasation for both U87MG and SKOV-3 tumors, as did SWNTs in the SKOV-3 condition. Note also that SWNTs do appear to extravasate from LS174T, though qdots extravasate much better. * Denotes significance, with p<0.05. (b) Overview depiction of nanoparticle extravasation. The schematic shows that qdots extravasate from LS174T tumor but not U87MG tumor, while SWNTs extravasate from U87MG tumors but only minimally from LS174T.

Figure 5

Scanning Emission Microscopy on Tumor and Normal Blood Vessels. SEM images show pores in U87MG and LS174T tumor vasculature on the apparent boundary between endothelial cells. Pores are not observed on the boundary in the vasculature of a mouse ear without tumor. Scale bars: U87MG (500nm), Normal (1 µm), and LS174T (1 µm).