Real-time imaging of perivascular transport of nanoparticles during convection-enhanced delivery in the rat cortex

Abstract

Convection-enhanced delivery (CED) is a promising technique for administering large therapeutics that do not readily cross the blood brain barrier to neural tissue. It is of vital importance to understand how large drug constructs move through neural tissue during CED to optimize construct and delivery parameters so that drugs are concentrated in the targeted tissue, with minimal leakage outside the targeted zone. Experiments have shown that liposomes, viral vectors, high molecular weight tracers, and nanoparticles infused into neural tissue localize in the perivascular spaces of blood vessels within the brain parenchyma. In this work, we used two-photon excited fluorescence microscopy to monitor the real-time distribution of nanoparticles infused in the cortex of live, anesthetized rats via CED. Fluorescent nanoparticles of 24 and 100 nm nominal diameters were infused into rat cortex through microfluidic probes. We found that perivascular spaces provide a high permeability path for rapid convective transport of large nanoparticles through tissue, and that the effects of perivascular spaces on transport are more significant for larger particles that undergo hindered transport through the extracellular matrix. This suggests that the vascular topology of the target tissue volume must be considered when delivering large therapeutic constructs via CED.

Figure 1

Experimental set-up for real-time two-photon excited fluorescence imaging of perivascular transport of nanoparticles.

Figure 2

Time course showing transport of fluorescent nanoparticles (red, 24-nm nominal diameter) through perivascular spaces during CED. Fluorescently-labeled blood vessels are shown in green. Images were captured 243 μm above the outlet of the microfluidic device. Image A is at infusion time = 0 s; B = 30 s; C = 90 s; D = 150 s. Note appearance of nanoparticles around vessels in panel B, and gradual filling in of background ECS in panels C and D. The dark band across the image from top right to bottom left is due to a large blood vessel on the surface of the brain that obscures the imaging below.

Figure 3

Image showing 24-nm nanoparticles (red) in perivascular spaces around a penetrating arteriole and branching capillary (blood vessels shown in green). BSA-coated red fluorescent nanoparticles were infused into the rat cortex, and the vasculature was labeled with FITC-dextran. The image shows the distribution of fluorescent nanoparticle around a branching capillary (∼10-μm diameter). Nanoparticles extend for ∼50 μm along the capillary after branching from the arteriole.

Figure 4

Time course showing transport of fluorescent nanoparticles (red, 24-nm nominal diameter) along a vessel in the imaging plane. The vasculature is labeled with FITC-dextran. Times represent duration of the infusion (A = 14.5 s, B = 34.8 s, C = 41.0 s and D = 65.5 s). Images were captured at a plane 360 μm above the outlet of the microfluidic probe, 240 μm below the surface of the brain.

Figure 5

Sections from a post-infusion imaging stack in the dorsoventral direction, showing red fluorescent 100-nm nanoparticles constrained in the perivascular space (vessels shown in green). Frame A is an optical section 50 μm below the brain surface; B = 100 μm; C = 150 μm; D = 200 μm. Images show that the nanoparticles are distributed in the perivascular space of the vessel over a distance of several hundred micrometers. The outlet of the microfluidic device was 550 μm below the brain surface.

Figure 6

a) Schematic showing geometry used in determining the flow rate of nanoparticles. The blue sphere represents the volume of infusion, and the red plane represents the area measured in our imaging stacks. b) Plot of ${\mathit{\text{R}}}_{\mathit{\text{INF}}}^3$ (see Equation 2) as a function of time, for infusion of 24-nm nanoparticles (infusion 1). The slope of 0.0096 μl/min allows us to determine the nanoparticle flow rate to be 0.008 μl/min.