Reactive oxygen species-activated nanoprodrug of Ibuprofen for targeting traumatic brain injury in mice

Abstract

Traumatic brain injury (TBI) is an enormous public health problem, with 1.7 million new cases of TBI recorded annually by the Centers for Disease Control. However, TBI has proven to be an extremely challenging condition to treat. Here, we apply a nanoprodrug strategy in a mouse model of TBI. The novel nanoprodrug contains a derivative of the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen in an emulsion with the antioxidant α-tocopherol. The ibuprofen derivative, Ibu2TEG, contains a tetra ethylene glycol (TEG) spacer consisting of biodegradable ester bonds. The biodegradable ester bonds ensure that the prodrug molecules break down hydrolytically or enzymatically. The drug is labeled with the fluorescent reporter Cy5.5 using nonbiodegradable bonds to 1-octadecanethiol, allowing us to reliably track its accumulation in the brain after TBI. We delivered a moderate injury using a highly reproducible mouse model of closed-skull controlled cortical impact to the parietal region of the cortex, followed by an injection of the nanoprodrug at a dose of 0.2 mg per mouse. The blood brain barrier is known to exhibit increased permeability at the site of injury. We tested for accumulation of the fluorescent drug particles at the site of injury using confocal and bioluminescence imaging of whole brains and brain slices 36 hours after administration. We demonstrated that the drug does accumulate preferentially in the region of injured tissue, likely due to an enhanced permeability and retention (EPR) phenomenon. The use of a nanoprodrug approach to deliver therapeutics in TBI represents a promising potential therapeutic modality.

Figure 1. Nanoprodrug preparation and characterization.

This chemical schematic shows the molecular structures of the individual ibuprofen molecule, the Ibu₂TEG complex consisting of two ibuprofen molecules jointed by a tetraethylene glycol (TEG) spacer, the anti oxidant α-tocopherol, and the hydrophobic 1-octadecanethiol which is joined to Cy5.5 after emulsification. The final product is represented on the right hand side of the schematic.

Figure 2. Comparing accumulation for IV and IP administration.

The injection of nanoprodrug either IV or IP results in similar accumulation in animals with TBI, while normal animals given nanoprodrug and TBI animals do not show any background flourescence. Brains are oriented with the rostral portion toward the top of the image.

Figure 3. Drug accumulation in the area of injury.

Accumulation of the drug in the left parietal area is visualized (a) using fluorescent imaging in the top panels and (b) by traditional photography and hemotoxylin and eosin staining in the lower pannels.

Figure 4. Disorganized vascular structures at the region of nanoprodrug uptake.

Representative images from two brains showing nanoprodrug uptake on the left column and CD31 staining of vascular endothelial cells on the right. Outside of the TBI region, vascular structures exist in normal tubular arrangements, but these are disorganized within the region of injury. The nuclei are stained with DAPI are displayed in blue. Scale bar, 50 µm.

Figure 5. Behavioral testing of motor function using Open Field Test and Rotorod.

(a) The number of ambulatory movements over the course of one hour in the Open Field Test (OFT) was reduced for the mice in the IV group. (b) The number of rearing movements in the OFT was not significantly different between groups. (c) Rotorod performance demonstrates that all mice were able to balance on a rotating rod for similar amounts of time. Statistical comparison was performed using a two tailed Student’s t-test, with a level of p = 0.05 considered significant.

Figure 6. Behavioral testing of memory function using the Barnes Maze.

The Barnes Maze was conducted for five days of training followed by traumatic brain injury (TBI). One day after traumatic brain injury (Day 7 on graph), mice did not demonstrate any significant differences in time to find the escape box. Statistical comparison was performed using a two tailed Student’s t-test, with a level of p = 0.05 considered significant. Day 8 serves as a control in which the location of the box is changed to show that mice are not using other cues such as scent to locate the box.

Figure 7. Enhanced Permeability and Retention (EPR) at the site of TBI.

This schematic illustrates the difference between healthy brain and injured brain in terms of the structural organization of the blood vessels and how this influences drug delivery. In normal tissue, the blood brain barrier is intact and the nanoprodrug does not penetrate into the tissue. An injured vessel becomes leaky, and the disruption of the blood brain barrier allows for uptake and accumulation of the nanoprodrug particles.

Figure 8. The COX system regulates blood flow and platelet activity.

The COX1 enzyme acts in platelets to activate thromboxane A2, which leads to vasoconstriction and enhanced platelet aggregation. The COX2 enzyme acts in endothelial cells to stimulate vasorelaxation and platelet inhibition.