(F-210) Repurposing Poloxamer P188 for TBI Treatment

Introduction:: Traumatic brain injury (TBI) continues to be a major cause of death and disability in the U.S. (CDC.gov). It can result from mechanical, chemical, or thermal damage to the tissue. However, there appear to be no proven efficacious treatments or preventive measures. Our laboratory recently demonstrated that FDA-approved triblock amphiphilic poloxamer 188 (P188) can repair cellular damages in response to traumatic injury [1], suggesting P188 may be repurposed for potential treatment of TBI. Therapeutic effects of P188 now include resealing the damaged cell membrane and enhancing the intrinsic cellular repair machinery. Successful conjugation of P188 with fluorophores allows us to monitor the dynamics of P188 uptake and co-localization with organelles inside the cell. Elucidation of the transport mechanisms and injury repair processes has been greatly improved by incorporating a machine learning pipeline to accurately analyze a growing number of subcellular images that are generated in our laboratory and accelerate the time required to perform such tasks from hours to less than 15 minutes.

Materials and Methods:: Mouse brain endothelial cells (BECs) were commercially purchased and cultured to produce confluent monolayers. P188 were purchased either from Sigma or Maroon Biotech (Chicago, IL). Cellular damages were created by either saponin-induced cell membrane permeabilization or fluid microjet (estimated 50 kPa). P188 was conjugated with Rhodamine 110 (Rh-110) fluorophore and incubated with BECs (10 uM). Accumulation of P188 inside the cell was monitored for 30 min at typically 1 min intervals. Co-localization was monitored by fluorescently labeling the cell membrane and organelle-specific fluorophores. A computer vision machine learning algorithm (Cellpose) [2] was adopted and modified to auto-segment confluent brain endothelial cells. Each segmented cell was registered and tracked over time.

Results, Conclusions, and Discussions:: Zwitterionic Rh-110 fluorophore alone did not penetrate the cell membrane. However, when conjugated to P188, its accumulation inside the cell was rapid and saturated in ~ 30 min but was cell type-dependent. In control BECs, excess accumulation of P188 was expeditiously exocytosed in less than 1 hr. A cell surface fluorophore (Membrite, Biotium, Freemont, CA) was used to demonstrate that saponin-induced permeabilization of the cell membrane was restored by P188 (Fig. 1). Furthermore, in injured cells, P188 was found to co-localize with the lysosomes. Interestingly, previous studies [3] have demonstrated the role of lysosomes that mediates the cell membrane repair. It appears likely the lysosome could be one of the organelles that P188 intercalates to promote restoration of the cell membrane structure and function. Development of a machine learning pipeline (PoloxamerPal) accelerated characterization and analysis of the dynamic P188 accumulation which, in turn, led to formulation of additional testable hypotheses.

Evidence is growing to support the hypothesis that P188 reseals the membrane defects and pores at the cell surface and enhances the cellular repair machinery. While the specific interactions inside the cell remain to be fully elucidated, therapeutic effects of P188 in response to traumatic injuries are becoming better understood and likely involve subcellular organelles (e.g., lysosomes and mitochondria). Detailed analyses are greatly facilitated by the development of machine-learning pipelines that foster the discovery efforts to repurpose FDA-approved drugs. Preclinical studies are underway to demonstrate efficacious reparative treatment of P188 in response to TBI.

Acknowledgements (Optional): : Authors are grateful for financial support from the Office of Naval Research (N00014-21-1-2039) and from the Alfred R. and Janet H. Potvin Endowment.

References (Optional): : 1. Inyang, E., Kuriakose, A., Nguyen, K., Cho, M. ACS Omega. 2020
2. Stringer, C., Wang, T., Michaelos, M., Pachitariu, M. Nature Methods. 2021
3. Reddy, A., Caler, E., Andrews, N. Cell. 2001