PEO–PPO Diblock Copolymers Protect Myoblasts from Hypo-Osmotic Stress In Vitro Dependent on Copolymer Size, Composition, and Architecture

Abstract

Poloxamer 188, a triblock copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), protects cellular membranes from various stresses. Though numerous block copolymer variants exist, evaluation of alternative architecture, composition, and size has been minimal. Herein, cultured murine myoblasts are exposed to the stresses of hypotonic shock and isotonic recovery, and membrane integrity was evaluated by quantifying release of lactate dehydrogenase. Comparative evaluation of a systematic set of PEO-PPO diblock and PEO-PPO-PEO triblock copolymers demonstrates that the diblock architecture can be protective in vitro. Short PPO blocks hinder protection with >9 PPO units needed for protection at 150 µM and >16 units needed at 14 µM. Addition of a tert-butyl end group enhances protection at reduced concentration. When the end group and PPO length are fixed, increasing the PEO length improves protection. This systematic evaluation establishes a new in vitro screening tool for evaluating membrane-sealing amphiphiles and provides mechanistic insight to guide future copolymer design for membrane stabilization in vivo.

Muscle membrane integrity in Duchenne muscular dystrophy: recent advances in copolymer-based muscle membrane stabilizers

“…MD studies feature a simplified phospholipid bilayer as a basic model of the biological membrane, which is comprised of proteins, complex mixtures of lipid types, and other macromolecules, all organized in a tightly regulated manner. Nonetheless, all atom MD results are qualitatively comparable to results derived in cells and animals [ 156 , 183 , 184 ]. Complementation of findings from in silico to in vivo methods underscores MD simulations as a powerful tool to further mechanistic understanding of copolymer-bilayer interactions and to ultimately guide design and optimization of copolymers for physiological membrane stabilization.…”

“…This strategy has precedence in the surfactancy literature where novel terminal functional groups have been shown to influence solution and bulk phase behavior [ 187 , 188 ]. Diblock copolymers have never been investigated for biological membrane stabilization until a recent report demonstrating that diblock PEO–PPO architectures can confer membrane stabilization in both in vitro and in vivo DMD models [ 156 , 171 , 183 , 184 ]. This establishes that specific PPO end group chemistries play a critical role in defining muscle membrane stabilization [ 183 , 184 ].…”

“…Recent diblock studies have advanced an “anchor and chain” model of membrane stabilization (Fig. 4 ) [ 156 , 171 , 183 , 184 ]. Here, the addition of a small hydrophobic end group “anchor,” as demonstrated by tert-butoxy to the PPO block, discretely increases the hydrophobic character of the end of the PPO block, without significantly increasing the overall mass of the copolymer.…”

Quantifying Binding of Ethylene Oxide–Propylene Oxide Block Copolymers with Lipid Bilayers

“…29 Recently, on the basis of an in-vitro cellular assay it was found that the protection efficacy of PPO-PEO block copolymers on cell membranes is highly affected by the polymer composition as well as the overall molecular weight. 35 In this context, the polymer structure plays a critical role in the nature of the interactions between PPO-PEO block copolymers and lipid membranes, which therefore determines the performance of polymers in both membrane stabilization and permeabilization. However, few studies have been able to quantify the associations between polymers and membranes directly.…”

Modeling and rescue of defective blood–brain barrier function of induced brain microvascular endothelial cells from childhood cerebral adrenoleukodystrophy patients

“…PEO–PPO diblock copolymers offer an opportunity to tune the end group on the PPO block, which recent work suggests to be an important molecular parameter in the ability of a PEO–PPO copolymer to confer protection [ 73 ]. A systematic in vitro screening of PEO–PPO diblock copolymers identified the polymer E 182 P 16 t (number average molar mass = 9 kg/mol and 90 wt% PEO; numerical subscripts indicate number of repeat units), which has a hydrophobic tert -butyl ( t ) end group on the PPO block, to be the most efficacious in stabilizing myoblasts under hypo-osmotic stress and isotonic recovery [ 74 ]. Thus, in addition to the commonly used Poloxamer 188, we hypothesized that E 182 P 16 t could also improve iBMEC function.…”