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.
Inyang, E., Abhyankar, V., Chen, B. and Cho, M. (2020) Modulation of in Vitro Brain Endothelium by Mechanical Trauma: Structural and Functional Restoration by Poloxamer 188. Scientific Reports , 10, Article No. 3054. https://doi.org/10.1038/s41598-020-59888-2
Wu, D., Chen, Q., Chen, X., Han, F., Chen, Z. and Wang, Y. (2023) The Blood-Brain Barrier: Structure, Regulation and Drug Delivery. Signal Transduction and Targeted Therapy , 8, Article No. 217. https://doi.org/10.1038/s41392-023-01481-w
Archie, S.R., Al Shoyaib, A. and Cucullo, L. (2021) Blood-Brain Barrier Dysfunction in CNS Disorders and Putative Therapeutic Targets: An Overview. Pharmaceutics , 13, Article 1779. https://doi.org/10.3390/pharmaceutics13111779
Andrews, A.M., Lutton, E.M., Merkel, S.F., Razmpour, R. and Ramirez, S.H. (2016) Mechanical Injury Induces Brain Endothelial-Derived Microvesicle Release: Implications for Cerebral Vascular Injury during Traumatic Brain Injury. Frontiers in Cellular Neuroscience , 10, Article 43. https://doi.org/10.3389/fncel.2016.00043
Chodobski, A., Zink, B.J. and Szmydynger-Chodobska, J. (2011) Blood-Brain Barrier Pathophysiology in Traumatic Brain Injury. Translational Stroke Research , 2, 492-516. https://doi.org/10.1007/s12975-011-0125-x
Yuan, Y., Sun, J., Dong, Q. and Cui, M. (2023) Blood-Brain Barrier Endothelial Cells in Neurodegenerative Diseases: Signals from the “Barrier”. Frontiers in Neuroscience , 17, Article 1047778. https://doi.org/10.3389/fnins.2023.1047778
Bollenbach, L., Buske, J., Mäder, K. and Garidel, P. (2022) Poloxamer 188 as Surfactant in Biological Formulations—An Alternative for Polysorbate 20/80? International Journal of Pharmaceutics , 620, Article ID: 121706. https://doi.org/10.1016/j.ijpharm.2022.121706
Han, S. and Mallampalli, R.K. (2015) The Role of Surfactant in Lung Disease and Host Defense against Pulmonary Infections. Annals of the American Thoracic Society , 12, 765-774. https://doi.org/10.1513/annalsats.201411-507fr
Plataki, M., Lee, Y.D., Rasmussen, D.L. and Hubmayr, R.D. (2011) Poloxamer 188 Facilitates the Repair of Alveolus Resident Cells in Ventilator-Injured Lungs. American Journal of Respiratory and Critical Care Medicine , 184, 939-947. https://doi.org/10.1164/rccm.201104-0647oc
Salzman, M.M., Bartos, J.A., Yannopoulos, D. and Riess, M.L. (2020) Poloxamer 188 Protects Isolated Adult Mouse Cardiomyocytes from Reoxygenation Injury. Pharmacology Research & Perspectives , 8, e00639. https://doi.org/10.1002/prp2.639
Tang, S., Liao, W., Pao, H., Hsu, C., Wu, S., Huang, K., et al. (2021) Poloxamer 188 Attenuates Ischemia-Reperfusion-Induced Lung Injury by Maintaining Cell Membrane Integrity and Inhibiting Multiple Signaling Pathways. Frontiers in Pharmacology , 12, Article 650573. https://doi.org/10.3389/fphar.2021.650573
G. Moloughney, J. and Weisleder, N. (2012) Poloxamer 188 (P188) as a Membrane Resealing Reagent in Biomedical Applications. Recent Patents on Biotechnology , 6, 200-211. https://doi.org/10.2174/1872208311206030200
Alsup, A.M., Fowlds, K., Cho, M. and Luber, J.M. (2024) β Buddy: An Automated End-To-End Computer Vision Pipeline for Analysis of Calcium Fluorescence Dynamics in Β-cells. PLOS ONE , 19, e0299549. https://doi.org/10.1371/journal.pone.0299549
Fowlds, K., Alsup, A.M., Kunwar, A., Darden, C.M., Luber, J.M., Lawrence, M.C., et al. (2025) Wavelength-Dependent Calcium Signaling Response to Photobiomodulation in Pancreatic Cells. Photonics , 12, Article 99. https://doi.org/10.3390/photonics12020099
Kim, O., Cho, Y., Chung, D.Y., Kim, M.J., Yoo, J.M., Park, J.E., et al. (2015) Facile and Gram-Scale Synthesis of Metal-Free Catalysts: Toward Realistic Applications for Fuel Cells. Scientific Reports , 5, Article No. 8376. https://doi.org/10.1038/srep08376
Jeannot, V., Salmon, J., Deumié, M. and Viallet, P. (1997) Intracellular Accumulation of Rhodamine 110 in Single Living Cells. Journal of Histochemistry & Cytochemistry , 45, 403-412. https://doi.org/10.1177/002215549704500308
Foroozandeh, P. and Aziz, A.A. (2018) Insight into Cellular Uptake and Intracellular Trafficking of Nanoparticles. Nanoscale Research Letters , 13, Article No. 339. https://doi.org/10.1186/s11671-018-2728-6
Alatrash, N., Issa, F.H., Bawazir, N.S., West, S.J., Van Manen-Brush, K.E., Shelor, C.P., et al. (2020) Disruption of Microtubule Function in Cultured Human Cells by a Cytotoxic Ruthenium(II) Polypyridyl Complex. Chemical Science , 11, 264-275. https://doi.org/10.1039/c9sc05671h
Sun, X., Gan, Q. and Ouyang, J. (2017) Size-Dependent Cellular Uptake Mechanism and Cytotoxicity toward Calcium Oxalate on Vero Cells. Scientific Reports , 7, Article No. 41949. https://doi.org/10.1038/srep41949
Puckett, C.A. and Barton, J.K. (2008) Mechanism of Cellular Uptake of a Ruthenium Polypyridyl Complex. Biochemistry , 47, 11711-11716. https://doi.org/10.1021/bi800856t
Schmid, S.L. and Carter, L.L. (1990) ATP Is Required for Receptor-Mediated Endocytosis in Intact Cells. The Journal of Cell Biology , 111, 2307-2318. https://doi.org/10.1083/jcb.111.6.2307
Slater, E.C. (1967) Application of Inhibitors and Uncouplers for a Study of Oxidative Phosphorylation. Methods in Enzymology , 10, 48-57. https://doi.org/10.1016/0076-6879(67)10011-6
Zhang, J., Jiang, C., Figueiró Longo, J.P., Azevedo, R.B., Zhang, H. and Muehlmann, L.A. (2018) An Updated Overview on the Development of New Photosensitizers for Anticancer Photodynamic Therapy. Acta Pharmaceutica Sinica B , 8, 137-146. https://doi.org/10.1016/j.apsb.2017.09.003
Chen, C., Hou, W., Liu, I., Hsiao, G., Huang, S.S. and Huang, J.S. (2009) Inhibitors of Clathrin-Dependent Endocytosis Enhance TGF β Signaling and Responses. Journal of Cell Science , 122, 1863-1871. https://doi.org/10.1242/jcs.038729
Chen, Y., Wang, S., Lu, X., Zhang, H., Fu, Y. and Luo, Y. (2011) Cholesterol Sequestration by Nystatin Enhances the Uptake and Activity of Endostatin in Endothelium via Regulating Distinct Endocytic Pathways. Blood , 117, 6392-6403. https://doi.org/10.1182/blood-2010-12-322867
Kirchhausen, T., Macia, E. and Pelish, H.E. (2008) Use of Dynasore, the Small Molecule Inhibitor of Dynamin, in the Regulation of Endocytosis. Methods in Enzymology , 438, 77-93. https://doi.org/10.1016/s0076-6879(07)38006-3
Chen, W.N., Shaikh, M.F., Bhuvanendran, S., Date, A., Ansari, M.T., Radhakrishnan, A.K., et al. (2022) Poloxamer 188 (P188), a Potential Polymeric Protective Agent for Central Nervous System Disorders: A Systematic Review. Current Neuropharmacology , 20, 799-808. https://doi.org/10.2174/1570159x19666210528155801
Ding, W., Lin, H., Hong, X., Ji, D. and Wu, F. (2020) Poloxamer 188-Mediated Anti-Inflammatory Effect Rescues Cognitive Deficits in Paraquat and Maneb-Induced Mouse Model of Parkinson’s Disease. Toxicology , 436, Article ID: 152437. https://doi.org/10.1016/j.tox.2020.152437
Riehm, J.J., Wang, L., Ghadge, G., Teng, M., Correa, A.M., Marks, J.D., et al. (2018) Poloxamer 188 Decreases Membrane Toxicity of Mutant SOD1 and Ameliorates Pathology Observed in SOD1 Mouse Model for Als. Neurobiology of Disease , 115, 115-126. https://doi.org/10.1016/j.nbd.2018.03.014
Mustafi, D., Smith, C.M., Makinen, M.W. and Lee, R.C. (2008) Multi-Block Poloxamer Surfactants Suppress Aggregation of Denatured Proteins. Biochimica et Biophysica Acta ( BBA )— General Subjects , 1780, 7-15. https://doi.org/10.1016/j.bbagen.2007.08.017
Maskarinec, S.A., Hannig, J., Lee, R.C. and Lee, K.Y.C. (2002) Direct Observation of Poloxamer 188 Insertion into Lipid Monolayers. Biophysical Journal , 82, 1453-1459. https://doi.org/10.1016/s0006-3495(02)75499-4
Schütt, F., Aretz, S., Auffarth, G.U. and Kopitz, J. (2012) Moderately Reduced ATP Levels Promote Oxidative Stress and Debilitate Autophagic and Phagocytic Capacities in Human RPE Cells. Investigative Opthalmology & Visual Science , 53, 5354-5361. https://doi.org/10.1167/iovs.12-9845
Bonam, S.R., Wang, F. and Muller, S. (2019) Lysosomes as a Therapeutic Target. Nature Reviews Drug Discovery , 18, 923-948. https://doi.org/10.1038/s41573-019-0036-1
Settembre, C., Fraldi, A., Medina, D.L. and Ballabio, A. (2013) Signals from the Lysosome: A Control Centre for Cellular Clearance and Energy Metabolism. Nature Reviews Molecular Cell Biology , 14, 283-296. https://doi.org/10.1038/nrm3565
Perera, R.M. and Zoncu, R. (2016) The Lysosome as a Regulatory Hub. Annual Review of Cell and Developmental Biology , 32, 223-253. https://doi.org/10.1146/annurev-cellbio-111315-125125
Poüs, C. and Codogno, P. (2011) Lysosome Positioning Coordinates Mtorc1 Activity and Autophagy. Nature Cell Biology , 13, 342-344. https://doi.org/10.1038/ncb0411-342
Kaushik, S. and Cuervo, A.M. (2018) The Coming of Age of Chaperone-Mediated Autophagy. Nature Reviews Molecular Cell Biology , 19, 365-381. https://doi.org/10.1038/s41580-018-0001-6
Davidson, S.M. and Vander Heiden, M.G. (2017) Critical Functions of the Lysosome in Cancer Biology. Annual Review of Pharmacology and Toxicology , 57, 481-507. https://doi.org/10.1146/annurev-pharmtox-010715-103101
Eriksson, I., Wäster, P. and Öllinger, K. (2020) Restoration of Lysosomal Function after Damage Is Accompanied by Recycling of Lysosomal Membrane Proteins. Cell Death & Disease , 11, Article No. 370. https://doi.org/10.1038/s41419-020-2527-8
Serbest, G., Horwitz, J., Jost, M. and Barbee, K.A. (2005) Mechanisms of Cell Death and Neuroprotection by Poloxamer 188 after Mechanical Trauma. The FASEB Journal , 20, 308-310. https://doi.org/10.1096/fj.05-4024fje
Zeng, J., Shirihai, O.S. and Grinstaff, M.W. (2020) Modulating Lysosomal pH: A Molecular and Nanoscale Materials Design Perspective. JOLS , Journal of Life Sciences , 2, 25-37. https://doi.org/10.36069/jols/20201204
Webb, B.A., Aloisio, F.M., Charafeddine, R.A., Cook, J., Wittmann, T. and Barber, D.L. (2021) pHLARE: A New Biosensor Reveals Decreased Lysosome pH in Cancer Cells. Molecular Biology of the Cell , 32, 131-142. https://doi.org/10.1091/mbc.e20-06-0383
Feng, X., Liu, S. and Xu, H. (2023) Not Just Protons: Chloride Also Activates Lysosomal Acidic Hydrolases. Journal of Cell Biology , 222, e202305007. https://doi.org/10.1083/jcb.202305007
Luo, C., Chen, X., Li, L., Li, Q., Li, B., Xue, A., et al. (2013) Poloxamer 188 Attenuates in Vitro Traumatic Brain Injury-Induced Mitochondrial and Lysosomal Membrane Permeabilization Damage in Cultured Primary Neurons. Journal of Neurotrauma , 30, 597-607. https://doi.org/10.1089/neu.2012.2425