Surfactant Copolymer Annealing of Chemically Permeabilized Cell Membranes

Structural breakdown of the cell membrane is a primary mediator in trauma-induced tissue necrosis. When membrane disruption exceeds intrinsic membrane sealing processes, biocompatible multi-block amphiphilic copolymer surfactants such as Poloxamer 188 (P188) have been found to be effective in catalyze or augment sealing. Although in living cells copolymer-induced sealing of membrane defects has been detected by changes in membrane transport properties, it has not been directly imaged. In this project, we used Atomic force microscopy (AFM) to directly image saponin permeabilized and poloxamer-sealed plasma membranes of monolayer-cultured MDCK and 3T3 fibroblasts. AFM image analysis resulted in the density and diameter ranges for membrane indentations per 5 × 5 μm area. For control, saponin lysed, and P188 treatment of saponin-lysed membranes, the supra-threshold indentation density was 3.6 ± 2.8, 13.8 ± 6.7, and 4.9 ± 3.3/cell, respectively. These results indicated that P188 catalyzed reduction in size of AFM indentations which correlated with increase cell survival. This evidence confirms that biocompatible surfactant P188 augments natural cell membrane sealing capability when intrinsic processes are incapable alone. Lay Summary and Future Studies The fundamental purpose of this study was to compare the topography of living mammalian cell membranes before and after detergent disruption using direct atomic force microscope (AFM) measurement membrane surface topography. Because increased roughness was observed, as reflected in greater depth and diameter of AFM tip indentations, the effect of Poloxamer 188 (P188, ~ 9 kDa) on the membrane was investigated. P188 is a copolymer surfactant that is known to seal disrupted pure lipid bilayer membranes. We observed that P188 reduced the roughness disrupted cell membranes consistent with defect sealing. The pure hydrophilic (10 kDa) neutral polyethylene glycol did not induce sealing. Future investigation will focus on optimizing the molecular design of block copolymer surfactants to maximize their membrane sealing efficacy. The optimal structure or membrane sealing is likely to be cell phenotype specific.