TITLE:
Active Transport of Therapeutic Triblock Amphiphilic Polymer Poloxamer 188 in Brain Endothelial Cells for Cellular Repair
AUTHORS:
Nagham Alatrash, Anne Alsup, Mia Grubbs, Vanessa Nomellini, Michael Cho
KEYWORDS:
Poloxamer 188, Transport Mechanisms, Computer Vision Pipeline, Blood-Brain Barrier, Cellular Repair Machinery
JOURNAL NAME:
Journal of Biomedical Science and Engineering,
Vol.18 No.7,
July
30,
2025
ABSTRACT: Amphiphilic triblock poloxamer 188 (P188) has demonstrated its therapeutic potential for muscle, cardiac and neurological injuries. While this surfactant is thought to primarily reseal the disrupted cell membrane, the specific mechanisms that mediate the reparative effect of P188 remain to be fully elucidated. Here, we investigated the transport mechanisms of P188 cellular uptake by fluorescently conjugating P188 with the fluorophore, Rhodamine 110 (Rh110). Fluorescent conjugation did not alter the P188 structure as characterized by nuclear magnetic resonance, Fourier Transform infrared spectroscopy, and acid-base titration, and the hydrophobicity was also quantified. In mouse brain endothelial cells, Rh110 alone was unable to accumulate inside the cells, while the P188 + Rh110 was rapidly transported across the cell membrane and became saturated in less than 1 hour. The transport dynamics were determined to be clathrin-dependent endocytosis, which was significantly altered in saponin-damaged cells or in cells with disrupted actin cytoskeletal organization; this suggests that transport via vesicle trafficking may be involved. Reparative effects of P188 appear to remodel the membrane organization and restore the transport properties. Instead of relying on manual image analysis, we utilized a machine learning pipeline that was recently developed in our laboratory to more rapidly and accurately analyze the cellular images of fluorescent P188 dynamics. This computer vision pipeline significantly reduced the time needed to segment, analyze, and perform statistical analyses. Finally, when injected into the mouse tail vein following a traumatic injury to the brain, we report for the first time that the P188 + Rh110 was observed in the brain tissue, indicating that P188 can cross the blood-brain barrier (BBB). Taken together, the dual therapeutic effects of P188 should include (1) resealing the disrupted cell membrane and (2) modulation of the intracellular cell repair machinery that might be involved in response to traumatic brain injury.