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.

Block copolymers composed of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) have been widely used in cell membrane stabilization and permeabilization. To explore the mechanism of interaction between PPO-PEO block copolymers and lipid membranes, we have investigated how polymer structure influences the polymer-lipid bilayer association by varying the overall molecular weight, the hydrophobic and hydrophilic block lengths, and the endgroup structure systematically, using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) unilamellar liposomes as model membranes. Pulsed-field-gradient NMR (PFG-NMR) was employed to probe polymer diffusion in the absence and presence of liposomes. The echo decay curves of free polymers in the absence of liposomes are single exponentials, indicative of simple translational diffusion, while in the presence of liposomes, the decays were biexponential, with the slower decay corresponding to polymers bound to liposomes. The binding percentage of polymer to the liposome was quantified by fitting the echo decay curves to a biexponential model. The NMR experiments showed that increasing the total molecular weight and hydrophobicity of the polymer can significantly enhance the polymer-lipid bilayer association, as the binding percentage and liposome surface coverage both increase. We hypothesize that the hydrophobic PPO block inserts into the lipid bilayer, due to the fact that little molecular exchange between bound and free polymers occurs on the time scale of the diffusion experiments. Additionally, as polymer concentration increases, the liposome surface coverage increases and approaches a limit. These results demonstrate that PFG-NMR is a simple yet powerful method to quantify interactions between polymers and lipid bilayers.

Block copolymers can be synthesized in an array of architectures and compositions to yield diverse chemical properties. The triblock copolymer Poloxamer 188 (P188), the family archetype, consisting of a hydrophobic poly(propylene oxide) core flanked by hydrophilic poly(ethylene oxide) chains, can stabilize cellular membranes during stress. However, little is known regarding the molecular basis of membrane interaction by copolymers in living organisms. By leveraging diblock architectural design, discrete end-group chemistry modifications can be tested. Here we show evidence of an anchor and chain mechanism of interaction wherein titrating poly(propylene oxide) block end group hydrophobicity directly dictates membrane interaction and stabilization. These findings, obtained in cells and animals in vivo, together with molecular dynamics simulations, provide new insights into copolymer–membrane interactions and establish the diblock copolymer molecular architecture as a valuable platform to inform copolymer–biological membrane interactions. These results have implications for membrane stabilizers in muscular dystrophy and for other biological applications involving damaged cell membranes.

Duchenne muscular dystrophy (DMD) is a fatal disease of striated muscle deterioration. A unique therapeutic approach for DMD is the use of synthetic membrane stabilizers to protect the fragile dystrophic sarcolemma against contraction-induced mechanical stress. Block copolymer-based membrane stabilizer poloxamer 188 (P188) has been shown to protect the dystrophic myocardium. In comparison, the ability of synthetic membrane stabilizers to protect fragile DMD skeletal muscles has been less clear. Because cardiac and skeletal muscles have distinct structural and functional features, including differences in the mechanism of activation, variance in sarcolemma phospholipid composition, and differences in the magnitude and types of forces generated, we speculated that optimized membrane stabilization could be inherently different. Our objective here is to use principles of pharmacodynamics to evaluate membrane stabilization therapy for DMD skeletal muscles. Results show a dramatic differential effect of membrane stabilization by optimization of pharmacodynamic-guided route of poloxamer delivery. Data show that subcutaneous P188 delivery, but not intravascular or intraperitoneal routes, conferred significant protection to dystrophic limb skeletal muscles undergoing mechanical stress in vivo. In addition, structure-function examination of synthetic membrane stabilizers further underscores the importance of copolymer composition, molecular weight, and dosage in optimization of poloxamer pharmacodynamics in vivo.

SummaryPoloxamer 188 (P188) is a nonionic triblock copolymer believed to prevent cellular injury after ischemia and reperfusion. This study compared intracoronary (IC) infusion of P188 immediately after reperfusion with delayed infusion through a peripheral intravenous catheter in a porcine model of ST-segment elevation myocardial infarction (STEMI). STEMI was induced in 55 pigs using 45 min of endovascular coronary artery occlusion. Pigs were then randomized to 4 groups: control, immediate IC P188, delayed peripheral P188, and polyethylene glycol infusion. Heart tissue was collected after 4 h of reperfusion. Assessment of mitochondrial function or infarct size was performed. Mitochondrial yield improved significantly with IC P188 treatment compared with control animals (0.25% vs. 0.13%), suggesting improved mitochondrial morphology and survival. Mitochondrial respiration and calcium retention were also significantly improved with immediate IC P188 compared with control animals (complex I respiratory control index: 7.4 vs. 3.7; calcium retention: 1,152 nmol vs. 386 nmol). This benefit was only observed with activation of complex I of the mitochondrial respiratory chain, suggesting a specific effect from ischemia and reperfusion on this complex. Infarct size and serum troponin I were significantly reduced by immediate IC P188 infusion (infarct size: 13.9% vs. 41.1%; troponin I: 19.2 μg/l vs. 77.4 μg/l). Delayed P188 and polyethylene glycol infusion did not provide a significant benefit. These results demonstrate that intracoronary infusion of P188 immediately upon reperfusion significantly reduces cellular and mitochondrial injury after ischemia and reperfusion in this clinically relevant porcine model of STEMI. The timing and route of delivery were critical to achieve the benefit.