Dilation and degradation of the brain extracellular matrix enhances penetration of infused polymer nanoparticles

Abstract

This study investigates methods of manipulating the brain extracellular matrix (ECM) to enhance the penetration of nanoparticle drug carriers in convection-enhanced delivery (CED). A probe was fabricated with two independent microfluidic channels to infuse, either simultaneously or sequentially, nanoparticles and ECM-modifying agents. Infusions were performed in the striatum of the normal rat brain. Monodisperse polystyrene particles with a diameter of 54 nm were used as a model nanoparticle system. Because the size of these particles is comparable to the effective pore size of the ECM, their transport may be significantly hindered compared with the transport of low molecular weight molecules. To enhance the transport of the infused nanoparticles, we attempted to increase the effective pore size of the ECM by two methods: dilating the extracellular space and degrading selected constituents of the ECM. Two methods of dilating the extracellular space were investigated: co-infusion of nanoparticles and a hyperosmolar solution of mannitol, and pre-infusion of an isotonic buffer solution followed by infusion of nanoparticles. These treatments resulted in an increase in the nanoparticle distribution volume of 51% and 123%, respectively. To degrade hyaluronan, a primary structural component of the brain ECM, a pre-infusion of hyaluronidase (20,000 U/mL) was followed after 30 min by infusion of nanoparticles. This treatment resulted in an increase in the nanoparticle distribution of 64%. Our results suggest that both dilation and enzymatic digestion can be incorporated into CED protocols to enhance nanoparticle penetration.

Figure 1

Upper: An electron micrograph of the probe showing the silicon shank and two parallel channels on top of the shank. The outlets of the channels are 0.5 mm from the shank tip. Lower: The probe is shown fixed to a custom holder by a screw and washer. Two small-bore polyimide tubes can be seen outside the holder. Each tube delivers fluid to a channel on the probe.

Figure 2

Left: Nanoparticle distribution was determined by capturing a fluorescent image of the right hemisphere of the brain. Right: The fluorescent images were converted into binary images with a threshold operation. Pixels exhibiting at least 10% the maximum fluorescence were included in calculating the nanoparticle distribution. The distribution volume in gray matter was calculated separately (area within the box) and compared to the total distribution volume. Infusions where the volume of nanoparticles exceeded 10% in the white matter were rejected from analysis so that the total distribution volume would not be biased by poor insertions of the microfluidic probe.

Figure 3

Representative distributions of fluorescently-labeled polystyrene nanoparticles in the striatum of the normal rat. A suspension of nanoparticles was infused at a flow rate 0.75 μL/min for a total volume 5 μL. Control: Infusion into normal tissue without any pretreatment or co-infusion. Mannitol: Co-infusion of nanoparticles in 25% mannitol into normal tissue. Enzyme: Infusion of nanoparticles into normal tissue 30 min after an infusion of hyaluronidase (20,000 U/ml). PBS: Infusion of nanoparticles into normal tissue 30 minutes after an infusion of 5 μL of PBS.

Figure 4

The distribution volume (V_d) of 5 μL of BSA coated polystyrene nanoparticles for each treatment represented as the mean, standard deviation, and p-values less than 0.05 (*) or 0.01 (**). Significant difference (p=0.0035, Kruskal-Wallis ANOVA) in distribution volume was found among the four groups; Control (n=7, 5 μL of nanoparticles in PBS), Mannitol (n=3, 5 μL of nanoparticles in 25% mannitol), Enzyme (n=3, 30 min digestion by 20,000 U/ml of hyaluronidase prior to nanoparticle infusion), PBS (n=4, 5 μL of PBS infused 30 min prior to nanoparticle infusion).

Figure 5

The area of fluorescence in each slice as a function of the AP distance in the region ±1.5 mm from the infusion site for each treatment; (+) untreated tissue, (▪) mannitol co-infusion into untreated tissue, (○) enzyme degraded tissue, and (▲) tissue dilated by pre-infusion PBS. Each symbol is the average area of fluorescence for each treatment at given position.

Figure 6

In the enzyme treatment group, hyaluronidase is infused 30 min prior to nanoparticle infusion to allow for degradation of the ECM. The effect of the enzyme treatment was determined by comparing (A) the distribution of hyaluronan in the untreated striatum to (B) the distribution of hyaluronan following a 30 min digestion with 20,000 U/mL of hyaluronidase. The absence of fluorescence indicates regions where hyaluronan has been degraded.

Figure 7

The area of each slice plotted as a function of the square of the AP; (+) untreated tissue, (▪) mannitol co-infusion into untreated tissue, (○) enzyme degraded tissue, and (▲) tissue dilated by pre-infusion PBS. Each symbol is the average area for each treatment at the given position. A line segment with a slope magnitude of π is drawn for reference. Data that fall on a line with slope of π or -π indicate regions where the infusion of nanoparticles is locally isotropic. Deviations from the slope indicate an anisotropic volume distribution.