Traumatic brain injury heterogeneity affects cell death and autophagy.

Traumatic brain injury (TBI) mechanism and severity are heterogenous clinically, resulting in a multitude of physical, cognitive, and behavioral deficits. Impact variability influences the origin, spread, and classification of molecular dysfunction which limits strategies for comprehensive clinical intervention. Indeed, there are currently no clinically approved therapeutics for treating the secondary consequences associated with TBI. Thus, examining pathophysiological changes from heterogeneous impacts is imperative for improving clinical translation and evaluating the efficacy of potential therapeutic strategies. Here we utilized TBI models that varied in both injury mechanism and severity including severe traditional controlled cortical impact (CCI), modified mild CCI (MTBI), and multiple severities of closed-head diffuse TBI (DTBI), and assessed pathophysiological changes. Severe CCI induced cortical lesions and necrosis, while both MTBI and DTBI lacked lesions or significant necrotic damage. Autophagy was activated in the ipsilateral cortex following CCI, but acutely impaired in the ipsilateral hippocampus. Additionally, autophagy was activated in the cortex following DTBI, and autophagic impairment was observed in either the cortex or hippocampus following impact from each DTBI severity. Thus, we provide evidence that autophagy is a therapeutic target for both mild and severe TBI. However, dramatic increases in necrosis following CCI may negatively impact the clinical translatability of therapeutics designed to treat acute dysfunction in TBI. Overall, these results provide evidence that injury sequalae affiliated with TBI heterogeneity is linked through autophagy activation and/or impaired autophagic flux. Thus, therapeutic strategies designed to intervene in autophagy may alleviate pathophysiological consequences, in addition to the cognitive and behavioral deficits observed in TBI.

Sex-based differences of antioxidant enzyme nanoparticle effects following traumatic brain injury.

Following traumatic brain injury (TBI), reactive oxygen species (ROS) are released in excess, causing oxidative stress, carbonyl stress, and cell death, which induce the additional release of ROS. The limited accumulation and retention of small molecule antioxidants commonly used in clinical trials likely limit the target engagement and therapeutic effect in reducing secondary injury. Small molecule drugs also need to be administered every several hours to maintain bioavailability in the brain. Therefore, there is a need for a burst and sustained release system with high accumulation and retention in the injured brain. Here, we utilized Pro-NP™ with a size of 200 nm, which was designed to have a burst and sustained release of encapsulated antioxidants, Cu/Zn superoxide dismutase (SOD1) and catalase (CAT), to scavenge ROS for >24 h post-injection. Here, we utilized a controlled cortical impact (CCI) mouse model of TBI and found the accumulation of Pro-NP™ in the brain lesion was highest when injected immediately after injury, with a reduction in the accumulation with delayed administration of 1 h or more post-injury. Pro-NP™ treatment with 9000 U/kg SOD1 and 9800 U/kg CAT gave the highest reduction in ROS in both male and female mice. We found that Pro-NP™ treatment was effective in reducing carbonyl stress and necrosis at 1 d post-injury in the contralateral hemisphere in male mice, which showed a similar trend to untreated female mice. Although we found that male and female mice similarly benefit from Pro-NP™ treatment in reducing ROS levels 4 h post-injury, Pro-NP™ treatment did not significantly affect markers of post-traumatic oxidative stress in female CCI mice as compared to male CCI mice. These findings of protection by Pro-NP™ in male mice did not extend to 7 d post-injury, which suggests subsequent treatments with Pro-NP™ may be needed to afford protection into the chronic phase of injury. Overall, these different treatment effects of Pro-NP™ between male and female mice suggest important sex-based differences in response to antioxidant nanoparticle delivery and that there may exist a maximal benefit from local antioxidant activity in injured brain.

Antioxidant theranostic copolymer-mediated reduction in oxidative stress following traumatic brain injury improves outcome in a mouse model.

Following a traumatic brain injury (TBI), excess reactive oxygen species (ROS) and lipid peroxidation products (LPOx) are generated and lead to secondary injury beyond the primary insult. A major limitation of current treatments is poor target engagement, which has prevented success in clinical trials. Thus, nanoparticle-based treatments have received recent attention because of their ability to increase accumulation and retention in damaged brain. Theranostic neuroprotective copolymers (NPC3) containing thiol functional groups can neutralize ROS and LPOx. Immediate administration of NPC3 following injury in a controlled cortical impact (CCI) mouse model provides a therapeutic window in reducing ROS levels at 2.08-20.83 mg/kg in males and 5.52-27.62 mg/kg in females. This NPC3-mediated reduction in oxidative stress improves spatial learning and memory in males, while females show minimal improvement. Notably, NPC3-mediated reduction in oxidative stress prevents the bilateral spread of necrosis in male mice, which was not observed in female mice and likely accounts for the sex-based spatial learning and memory differences. Overall, these findings suggest sex-based differences to oxidative stress scavenger nanoparticle treatments, and a possible upper threshold of antioxidant activity that provides therapeutic benefit in injured brain since female mice benefit from NPC3 treatment to a lesser extent than male mice.

Theranostic Copolymers Neutralize Reactive Oxygen Species and Lipid Peroxidation Products for the Combined Treatment of Traumatic Brain Injury.

Traumatic brain injury (TBI) results in the generation of reactive oxygen species (ROS) and lipid peroxidation product (LPOx), including acrolein and 4-hydroxynonenal (4HNE). The presence of these biochemical derangements results in neurodegeneration during the secondary phase of the injury. The ability to rapidly neutralize multiple species could significantly improve outcomes for TBI patients. However, the difficulty in creating therapies that target multiple biochemical derangements simultaneously has greatly limited therapeutic efficacy. Therefore, our goal was to design a material that could rapidly bind and neutralize both ROS and LPOx following TBI. To do this, a series of thiol-functionalized biocompatible copolymers based on lipoic acid methacrylate and polyethylene glycol monomethyl ether methacrylate (FW ∼ 950 Da) (O950) were prepared. A polymerizable gadolinium-DOTA methacrylate monomer (Gd-MA) was also synthesized starting from cyclen to facilitate direct magnetic resonance imaging and in vivo tracking of accumulation. These neuroprotective copolymers (NPCs) were shown to rapidly and effectively neutralize both ROS and LPOx. Horseradish peroxidase absorbance assays showed that the NPCs efficiently neutralized H2O2, while R-phycoerythrin protection assays demonstrated their ability to protect the fluorescent protein from oxidative damage. 1H NMR studies indicated that the thiol-functional NPCs rapidly form covalent bonds with acrolein, efficiently removing it from solution. In vitro cell studies with SH-SY5Y-differentiated neurons showed that NPCs provide unique protection against toxic concentrations of both H2O2 and acrolein. NPCs rapidly accumulate and are retained in the injured brain in controlled cortical impact mice and reduce post-traumatic oxidative stress. Therefore, these materials show promise for improved target engagement of multiple biochemical derangements in hopes of improving TBI therapeutic outcomes.

Claudin-1-Targeted Nanoparticles for Delivery to Aging-Induced Alterations in the Blood-Brain Barrier.

Aging-induced alterations to the blood-brain barrier (BBB) are increasingly being seen as a primary event in chronic progressive neurological disorders that lead to cognitive decline. With the goal of increasing delivery into the brain in hopes of effectively treating these diseases, a large focus has been placed on developing BBB permeable materials. However, these strategies have suffered from a lack of specificity toward regions of disease progression. Here, we report on the development of a nanoparticle (C1C2-NP) that targets regions of increased claudin-1 expression that reduces BBB integrity. Using dynamic contrast enhanced magnetic resonance imaging, we find that C1C2-NP accumulation and retention is significantly increased in brains from 12 month-old mice as compared to nontargeted NPs and brains from 2 month-old mice. Furthermore, we find C1C2-NP accumulation in brain endothelial cells with high claudin-1 expression, suggesting target-specific binding of the NPs, which was validated through fluorescence imaging, in vitro testing, and biophysical analyses. Our results further suggest a role of claudin-1 in reducing BBB integrity during aging and show altered expression of claudin-1 can be actively targeted with NPs. These findings could help develop strategies for longitudinal monitoring of tight junction protein expression changes during aging as well as be used as a delivery strategy for site-specific delivery of therapeutics at these early stages of disease development.

Antioxidant thioether core-crosslinked nanoparticles prevent the bilateral spread of secondary injury to protect spatial learning and memory in a controlled cortical impact mouse model of traumatic brain injury.

The secondary phase of traumatic brain injury (TBI) is partly caused by the release of excess reactive oxygen species (ROS) from the primary injury. However, there are currently no therapies that have been shown to reduce the secondary spread of injury beyond the primary insult. Nanoparticles offer the ability to rapidly accumulate and be retained in injured brain for improved target engagement. Here, we utilized systemically administered antioxidant thioether core-cross-linked nanoparticles (NP1) that scavenge and inactivate ROS to reduce this secondary spread of injury in a mild controlled cortical impact (CCI) mouse model of TBI. We found that NP1 treatment protected CCI mice from injury induced learning and memory deficits observed in the Morris water maze (MWM) test at 1-month post-CCI. This protection was likely a result of NP1-mediated reduction in oxidative stress in the ipsilateral hemisphere as determined by immunofluorescence imaging of markers of oxidative stress and the spread of neuroinflammation into the contralateral hippocampus as determined by immunofluorescence imaging of activated microglia and neuron-astrocyte-microglia triad formation. These data suggest NP1-mediated reduction in post-traumatic oxidative stress correlates with the reduction in the spread of injury to the contralateral hippocampus to protect spatial memory and learning in CCI mice. Therefore, these materials may offer an improved treatment strategy to reduce the secondary spread of TBI.

Ultrasmall Mixed Eu-Gd Oxide Nanoparticles for Multimodal Fluorescence and Magnetic Resonance Imaging of Passive Accumulation and Retention in TBI.

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. TBI can have a long-term impact on the quality of life for survivors of all ages. However, there remains no approved treatment that improves outcomes following TBI, which is partially due to poor delivery of therapies into the brain. Therefore, there is a significant unmet need to develop more effective delivery strategies that increase the accumulation and retention of potentially efficacious treatments in the injured brain. Recent work has revealed that nanoparticles (NPs) may offer a promising approach for site-specific delivery; however, a detailed understanding of the specific NP properties that promote brain accumulation and retention are still being developed. Multimodal imaging plays a vital role in the understanding of physicochemical properties that initiate the uptake and accumulation of NPs in the brain at both high spatial (e.g., fluorescence imaging) and temporal (e.g., magnetic resonance imaging, MRI) frequency. However, many NP systems that are currently used in TBI only provide contrast in a single imaging modality limiting the imaging data that can be obtained, and those that offer multimodal imaging capabilities have complicated multistep synthesis methods. Therefore, the goal of this work was to develop an ultrasmall NP with simple fabrication capable of multimodal imaging. Here, we describe the development, characterization, accumulation, and retention of poly(ethylene glycol) (PEG)-coated europium-gadolinium (Eu-Gd) mixed magnetic NPs (MNPs) in a controlled cortical impact mouse model of TBI. We find that these NPs having an ultrasmall core size of 2 nm and a small hydrodynamic size of 13.5 nm can be detected in both fluorescence and MR imaging modalities and rapidly accumulate and are retained in injured brain parenchyma. These NPs should allow for further testing of NP physicochemical properties that promote accumulation and retention in TBI and other disease models.

Evaluating differential nanoparticle accumulation and retention kinetics in a mouse model of traumatic brain injury via Ktrans mapping with MRI.

Traumatic brain injury (TBI) is a leading cause of injury-related death worldwide, yet there are no approved neuroprotective therapies that improve neurological outcome post-injury. Transient opening of the blood-brain barrier following injury provides an opportunity for passive accumulation of intravenously administered nanoparticles through an enhanced permeation and retention-like effect. However, a thorough understanding of physicochemical properties that promote optimal uptake and retention kinetics in TBI is still needed. In this study, we present a robust method for magnetic resonance imaging of nanoparticle uptake and retention kinetics following intravenous injection in a controlled cortical impact mouse model of TBI. Three contrast-enhancing nanoparticles with different hydrodynamic sizes and relaxivity properties were compared. Accumulation and retention were monitored by modelling the permeability coefficient, Ktrans, for each nanoparticle within the reproducible mouse model. Quantification of Ktrans for different nanoparticles allowed for non-invasive, multi-time point assessment of both accumulation and retention kinetics in the injured tissue. Using this method, we found that 80 nm poly(lactic-co-glycolic acid) nanoparticles had maximal Ktrans in a TBI when injected 3 hours post-injury, showing significantly higher accumulation kinetics than the small molecule, Gd-DTPA. This robust method will enable optimization of administration time and nanoparticle physicochemical properties to achieve maximum delivery.

Nanoparticle-mediated knockdown of DNA repair sensitizes cells to radiotherapy and extends survival in a genetic mouse model of glioblastoma.

Glioblastoma (GBM) remains incurable, and recurrent tumors rarely respond to standard-of-care radiation and chemo-therapies. Therefore, strategies that enhance the effects of these therapies should provide significant benefits to GBM patients. We have developed a nanoparticle delivery vehicle that can stably bind and protect nucleic acids for specific delivery into brain tumor cells. These nanoparticles can deliver therapeutic siRNAs to sensitize GBM cells to radiotherapy and improve GBM treatment via systemic administration. We show that nanoparticle-mediated knockdown of the DNA repair protein apurinic endonuclease 1 (Ape1) sensitizes GBM cells to radiotherapy and extend survival in a genetic mouse model of GBM. Specific knockdown of Ape1 activity by 30% in brain tumor tissue doubled the extended survival achieved with radiotherapy alone. Ape1 is a promising target for increasing the effectiveness of radiotherapy, and nanoparticle-mediated delivery of siRNA is a promising strategy for tumor specific knockdown of Ape1.