(460a) Developing an in Vitro Model to Study Amphiphilic Block Copolymer-Mediated Stabilization of the Blood-Brain Barrier Under Oxidative Stress

The blood-brain barrier (BBB) is comprised of brain microvascular endothelial cells where tight junction protein complexes form between the cells, creating a highly restrictive barrier at the interface of the brain and vascular system. The low paracellular diffusion across the BBB is crucial to maintain brain homeostasis. Accordingly, BBB dysfunction is a hallmark of many neurological injuries and disorders including ischemic stroke, traumatic brain injury, and Alzheimer’s disease. These conditions are caused or exacerbated by oxidative stress in which an excess of reactive oxygen species relative to antioxidants damages tight junction proteins, increasing BBB permeability. To address the many issues arising from barrier dysfunction, there is interest in investigating therapies that directly interact with the compromised BBB to restore its function. Poloxamer 188 (P188), a commercial water-soluble triblock copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), has mitigated BBB damage in mouse models of ischemic stroke, traumatic brain injury, and intracerebral hemorrhage. While the therapeutic efficacy of P188 has been demonstrated, the mechanism by which amphiphilic block copolymers grant barrier stabilization remains unknown. Many studies have utilized acellular models to probe polymer-cell interactions, but simplifying the cell as the phospholipid bilayer makes it difficult to relate findings from these models to the observed therapeutic function in vivo.

To enable the investigation of the interactions between polymers and the damaged BBB while maintaining biological complexity, we differentiated human induced pluripotent stem cells into brain microvascular endothelial cells (iBMECs) that exhibit key attributes of the in vivo BBB, including low paracellular diffusion and expression of endothelial and brain-specific proteins. We induced oxidative stress by exposing iBMECs to hydrogen peroxide, resulting in a 30% reduction in barrier function, as indicated by the transendothelial electrical resistance, and decreased continuity of tight junction proteins claudin-5 and occludin, as quantified by analysis of confocal fluorescence microscopy images. We further confirmed the activation of iBMECs under oxidative stress by visualizing F-actin cytoskeleton rearrangement from the intact cortical actin rim in undamaged iBMECs to increased formation of actin stress fibers upon exposure to hydrogen peroxide. We next treated iBMECs under oxidative stress with P188, resulting in a 10% increase in barrier function relative to the untreated iBMECs under oxidative stress. This result, that P188 mitigates BBB damage, affirms previous findings in mouse models of injuries related to oxidative stress. Given that P188 does not fully restore function to the damaged BBB, we tuned parameters in the amphiphilic block copolymer design to identify improved therapeutic molecules while elucidating the relationship between polymer composition and therapeutic function. We synthesized amphiphilic block copolymers while varying the number of chemically distinct blocks, PEO block length, and end group functionality of the PPO block. When studying a diblock copolymer analog to P188 with the same wt% PEO, we found that this molecule granted a 25% increase in barrier function relative to the untreated iBMECs under oxidative stress. We further evaluated novel diblock copolymers that differed in PEO block length and end group hydrophobicity and found that all three diblock copolymers were more efficacious than P188 in mitigating endothelial cell activation induced by hydrogen peroxide.

We have established a human-based in vitro model of the BBB under oxidative stress that reports damage and enables the evaluation of block copolymer efficacy. Through this work, we identified additional therapeutic molecules to P188 that are effective in improving function of the BBB under oxidative stress. This tissue engineered model of the BBB is highly tunable (i.e., can include physiological flow and co-culture with additional cell types in the neurovascular unit), enabling deeper investigation into the mechanism of polymer-mediated barrier stabilization while continuing to bridge an unfilled gap between acellular and in vivo models.