The authors have declared that no competing interests exist.
Conceived and designed the experiments: MAC BSL LEJ JSY. Performed the experiments: MAC BSL MS TA AWL. Analyzed the data: MAC BSL MS TA AWL DD BK EJL JSY. Contributed reagents/materials/analysis tools: EJL JSY. Wrote the paper: MAC BSL JY MS BK EJL JSY.
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.
In industrialized countries, traumatic brain injury (TBI) is the leading cause of death in those under the age of 45
For both patients and society at large, traumatic brain injury carries a large cost burden estimated for the United States to be $60–100 billion per year due to resulting healthcare costs and lost productivity
Decades of clinical and basic science trials have attempted to improve outcomes of traumatic brain injury using a wide variety of novel treatment strategies. The most recent trials have investigated drugs such as calcium channel inhibitors
Among the animal models available for traumatic brain injury, the controlled cortical impact (CCI) method represents a refined and highly reproducible means of producing several gradations of injury
CCI recapitulates the characteristics of human traumatic brain injury such as edema, hemorrhage, contusion, altered cerebral metabolism and inflammation
Non-steroidal anti-inflammatory drugs (NSAIDs) are a promising candidate for controlling the deleterious effects of inflammation after TBI. Post injury inflammation leads to degradation of the blood brain barrier, edema, increased intracranial pressure, metabolic disturbances, activation of microglia and infiltration of peripheral immune cells
NSAIDs possess well-documented analgesic, antipyretic, and anti-inflammatory effects
Ibu2TEG was stabilized by hydrophobic interactions with the antioxidant α-tocopherol. α-tocopherol is the most biologically active form of vitamin E and is believed to be the most potent lipid-soluble antioxidant because it is capable of breaking the chain of propagation of free radical mediated lipid peroxidation
The blood brain barrier (BBB) is a tightly regulated interface between the central nervous system and the circulating blood, formed by CD31+ vascular endothelial tissue. The BBB protects the CNS from edema and neurotoxic macromolecules. When the BBB is functioning normally, it also often blocks the delivery of therapeutics that would be used to treat conditions such as neurodegenerative diseases, CNS infections, and brain tumors. However, in TBI the integrity of the BBB is known to be severely compromised at the site of injury
The phenomenon of failing vascular barrier activity has been described in oncology literature as the enhanced permeability and retention (EPR) effect
All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the Cedars-Sinai Medical Center (protocol #2620). All efforts were made to minimize suffering through the use of anesthesia, analgesia, and post-injury care and monitoring.
Male 12-week old C57BL/6 wild type mice (strain #000664) were obtained from Jackson Laboratory (Bar Harbor, Maine). On the day of injury, the placebo group and the treatment group had similar weights (26.01±1.18 g v, 25.48±1.64 g, p = 0.42). Mice were anesthetized with inhalation isoflurane (4% to induction, and 2% maintenance), shaved in the region of cortical impact, and secured in a stereotaxic frame. Mice were then subjected to TBI using electromagnetic controlled cortical impact (CCI)
Eight mice (n = 8) were randomized to placebo group, undergoing CCI and then an intraperitoneal injection of phosphate buffered saline (PBS) after injury. Twelve mice (n = 12) were assigned to the intraperitoneal (IP) treatment group and were given an immediate IP injection of nanoprodrug (100 µl, 0.2 mg/mouse). Six mice (n = 6) were assigned to the intravenous (IV) group, recovered from anesthesia for five minutes, and injected with nanoprodrug (100 µl, 0.2 mg/mouse) via tail vein. All mice were recovered on a warming pad until ambulatory, returned to their cages, and housed in groups of two with a 14∶10 hour light-dark cycle with water and softened chow available
The nanoprodrug is constructed of ibuprofen molecules joined by a tetra ethylene glycol (TEG) spacer in an emulsion with the antioxidant α-tocopherol and 1-octadecanethiol which is irreversibly bonded to the Cy5.5 fluorescent tracer. The combination of two ibuprofen molecules joined by the TEG spacer is referred to as Ibu2TEG (
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.
The details of Ibu2TEG synthesis are previously described
Behavioral testing was determined using the Barnes Maze for cognitive function, and the open field and rotorod tests of motor function. The Barnes Maze assessed spatial reference and working memory retention. Ten animals were tested in the Barnes Maze (n = 6 IP, n = 4 placebo). Prior to injury animals received five days of training to locate and enter a hide box within a two-minute time limit. Injury occurred on day 6, and memory retention of the task was assessed on day 7. On day 8, a probe test was conducted as a control, in which the box was moved to a new location to determine if the animals were not using non-memory associated cues (such as the scent of the box) to locate the hide box.
The open field and rotorod tests were conducted twenty-four hours after TBI to assess gross motor function. In the open field test, mice were placed in a plexiglass box, with motion monitored by lasers over the course of one hour. Ambulation is defined as more than two consecutive laser beam breaks. The rotarod test assesses coordination and strength by measuring the time the animal can balance on a rod rotating at constantly increasing angular velocity.
Post mortem brain tissue was imaged using Xenogen 200 Imaging System (Caliper Life Sciences) to localize accumulation of the fluorescent nanoprodrug within the brain. Intact whole brains were imaged and then sectioned for repeat imaging. Frozen tissue was mounted in OCT compound, cryosectioned using a cryotomb (10µm), and stained with hematoxylin and eosin. For fluorescent confocal microscopy, brains were cryosectioned (10µm) and mounted and coverslipped with one drop of mounting medium with DAPI (Prolong Gold, Invitrogen). A fluorescent microscope (Model Upright Zeiss) and a confocal laser-scanning microscope (Leica Microsystem SP5) equipped with a digital camera were used for microscopic analysis.
After behavioral testing and imaging procedures, mice were sacrificed three days after injury using carbon dioxide inhalation followed by cervical dislocation. Brains were then immediately harvested by peeling the skull away and extracting the whole brain onto dry ice for snap freezing. Tissues were stored at −80 degrees Celsius until processing.
Groups are described as means with standard deviations and compared using a two tailed Student’s t-test, with a level of p = 0.05 considered significant.
Whole brains were collected from mice 36 hours after injury and nanoprodrug administration. Using Xenogen bioluminescence imaging, the Cy5.5 fluorescent marker was detected at the site of injury on the left parietal region of whole brain (
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.
Confocal microscopy of sectioned brains revealed accumulation of the nanoprodrug (pink, Cy5.5) at 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.
To investigate the cerebral vasculature, we stained for CD31+ vascular endothelial cells (
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.
On the open field test, mice treated with the nanoprodrug IP had no significant reduction in ambulation events compared to placebo (nanoprodrug IP 2024±484 v. placebo 1865±302, p = 0.47) or rearing (nanoprodrug IP 174±136 v. placebo 188±90, p = 0.82) (
(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.
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.
Each year, approximately 1.5 million Americans sustain a traumatic brain injury (TBI), resulting in over 50,000 deaths and 80,000 long-term disabilities
The enhanced permeability and retention (EPR) effect was first described in reference to rapidly growing solid tumors, which often exhibit disorganized angiogenesis
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.
NSAIDs have widely known analgesic, antipyretic, and anti-inflammatory effects. Their mechanism of effect is through COX inhibition. COX enzymes are produced as two isoforms, COX-1 and COX-2. In endothelial tissue, the constitutive production of COX-2 leads to the production of PGI2, which causes vasorelaxation and inhibits platelet aggregation. Normal hemostasis is maintained by a balance between this epithelial effect and a COX-1 catalyzed thromboxane A2 activity in platelets, which mediates a vasoconstrictive and pro-aggregation effect
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.
In the brain, COX-2 induction is known to be upregulated after traumatic brain injury in rats starting at 3 hours and lasting for at least 12 days
The nanoprodrug contains α-tocopherol (vitamin E) as an antioxidant component and stabilizing structural component. Increasing evidence suggests that vitamin E may play a promising role in the prevention and treatment of oxidative damage-related neurodegenerative diseases
Bioluminescence imaging reveals that the novel NSAID nanoprodrug accumulates at the area of injury, possibly due to an enhanced permeability and retention (EPR) effect. The nanoprodrug targets the injury with high specificity, which may potentially reduce off target effects on other organs. Behavioral testing indicates that only animals receiving the drug intravenously demonstrated a significant reduction in ambulation, whereas animals receiving the drug IP showed equivalent motor function to animals receiving PBS control. Rotorod and Barnes Maze showed no significant differences in outcome with the use of the NSAID nanoprodrug.
We believe this study demonstrates the feasibility of using the NSAID nanoprodrug to target TBI. Further testing will explore off target organ toxicities, nanoprodrug half-life, and determine the minimal effective dose. Further preclinical studies may investigate the use of the nanoprodrug in a blast injury model in addition to controlled cortical impact. The novel combination of the nanoprodrug delivery strategy and NSAID therapy represents a promising therapeutic modality for the treatment of many types of TBI.