Consequences of poly(ethylene oxide) and poloxamer P188 on transcription in healthy and stressed myoblasts.

Poly(ethylene oxide) (PEO) and poloxamers, a class of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers, have many personal and medical care applications, including the stabilization of stressed cellular membranes. Despite the widespread use, the cellular transcriptional response to these molecules is relatively unknown. C2C12 myoblasts, a model muscle cell, were subjected to short-term Poloxamer 188 (P188) and PEO181 (8,000 g/mol) treatment in culture. RNA was extracted and sequenced to quantify transcriptomic impact. The addition of moderate concentrations (14 µM) of either polymer to unstressed cells caused substantial differential gene expression, including at least twofold modulation of 357 and 588 genes, respectively. In addition, evaluation of the transcriptome response to osmotic stress without polymer treatment revealed dramatic change in RNA expression. Interestingly, the addition of polymer to stressed cells-at concentrations that provide physiological protection-did not yield a significant difference in expression of any gene relative to stress alone. Genome-scale expression analysis was corroborated by single-gene quantitative real-time PCR. Changes in protein expression were measured via western blot, which revealed partial alignment with the RNA results. Collectively, the significant changes to expression of multiple genes and resultant protein translation demonstrates an unexpectedly broad biochemical response to these polymers in healthy myoblasts in vitro. Meanwhile, the lack of substantial transcriptional response to polymer treatment in stressed cells highlights the physical nature of that protective mechanism.

Effect of Bottlebrush Poloxamer Architecture on Binding to Liposomes.

Poloxamers─triblock copolymers consisting of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO)─have demonstrated cell membrane stabilization efficacy against numerous types of stress. However, the mechanism responsible for this stabilizing effect remains elusive, hindering engineering of more effective therapeutics. Bottlebrush polymers have a wide parameter space and known relationships between architectural parameters and polymer properties, enabling their use as a tool for mechanistic investigations of polymer-lipid bilayer interactions. In this work, we utilized a versatile synthetic platform to create novel bottlebrush analogues to poloxamers and then employed pulsed-field-gradient NMR and an in vitro osmotic stress assay to explore the effect of bottlebrush architectural parameters on binding to, and protection of, model phospholipid bilayers. We found that the binding affinity of a bottlebrush poloxamer (BBP) (B-E1043P515, Mn ≈ 26 kDa) is about 3 times higher than a linear poloxamer with a similar composition and number of PPO units (L-E93P54E93, Mn ≈ 11 kDa). Furthermore, BBP binding is sensitive to overall molecular weight, side-chain length, and architecture (statistical versus block). Finally, all tested BBPs exhibit a protective effect on cell membranes under stress at sub-µM concentrations. As the factors controlling membrane affinity and protection efficacy of bottlebrush poloxamers are not understood, these results provide important insight into how they adhere to and stabilize a lipid bilayer surface.

Lipid Membrane Binding and Cell Protection Efficacy of Poly(1,2-butylene oxide)-b-poly(ethylene oxide) Copolymers.

Poloxamers consisting of poly(ethylene oxide) (PEO) and poly(propylene oxide) segments can protect cell membranes against various forms of stress. We investigated the role of the hydrophobic block chemistry on polymer/membrane binding and cell membrane protection by comparing a series of poly(butylene oxide)-b-PEO (PBO-b-PEO) copolymers to poloxamer analogues, using a combination of pulsed-field-gradient (PFG) NMR experiments and a lactate dehydrogenase (LDH) cell assay. We found that the more hydrophobic PBO-b-PEO copolymers bound more significantly to model liposomes composed of 1-palmitol-2-oleoyl-glycero-3-phosphocholine (POPC) compared to poly(propylene oxide) (PPO)/PEO copolymers. However, both classes of polymers performed similarly when compared by an LDH assay. These results present an important comparison between polymers with similar structures but with different binding affinities. They also provide mechanistic insight as enhanced polymer/lipid membrane binding did not directly translate to increased cell protection in the LDH assay, and therefore, additional factors need to be considered when trying to achieve greater membrane protection efficacy.

Effect of chain architecture on the structure, dynamics, and rheology of thermoresponsive poloxamer hydrogels and associated blends.

Poloxamers, ABA triblock polymers composed of a poly(propylene oxide) (PPO) midblock (B) and poly(ethylene oxide) (PEO) endblocks (A), are widely studied for biomedical applications. Aqueous poloxamer 407 (P407; also referred to as F127) undergoes a solution-to-gel transition with increasing temperature, driven by the formation and ordering of micelles onto periodic lattices; however, the gel temperature and resulting modulus has limited tunability. Here, reverse P407 (RP407), a BAB polymer of the same composition and molar mass but the inverted architecture, is synthesized via anionic polymerization. The micellization and gelation temperatures of RP407 are higher than that of P407 and the PPO endblocks allow for intermicelle bridging; however, both single-component solutions favor body-centered cubic (BCC) packings. Further, aqueous RP407 displays a "soft gel" region with interesting rheological behavior, including viscoelastic aging and thermal hysteresis. Combining P407 and RP407 yields solutions with intermediate transition temperatures and alters the size and micelle packing. While the single-component solutions produce BCC packings, the blends form close-packed structures and larger micelles of higher aggregation numbers. Blends of P407 with an analogous AB diblock (E111P32) display similar behavior, whereas RP407/diblock blends form intermediate-sized BCC-packed micelles. These differences in packing and aggregation alter the local environments within the gels, which could have implications for applications such as drug delivery and protein stabilization.

Rapid restitution of contractile dysfunction by synthetic copolymers in dystrophin-deficient single live skeletal muscle fibers.

Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin, a cytoskeletal protein essential for the preservation of the structural integrity of the muscle cell membrane. DMD patients develop severe skeletal muscle weakness, degeneration, and early death. We tested here amphiphilic synthetic membrane stabilizers in mdx skeletal muscle fibers (flexor digitorum brevis; FDB) to determine their effectiveness in restoring contractile function in dystrophin-deficient live skeletal muscle fibers. After isolating FDB fibers via enzymatic digestion and trituration from thirty-three adult male mice (9 C57BL10, 24 mdx), these were plated on a laminin-coated coverslip and treated with poloxamer 188 (P188; PEO75-PPO30-PEO75; 8400 g/mol), architecturally inverted triblock (PPO15-PEO200-PPO15, 10,700 g/mol), and diblock (PEO75-PPO16-C4, 4200 g/mol) copolymers. We assessed the twitch kinetics of sarcomere length (SL) and intracellular Ca2+ transient by Fura-2AM by field stimulation (25 V, 0.2 Hz, 25 °C). Twitch contraction peak SL shortening of mdx FDB fibers was markedly depressed to 30% of the dystrophin-replete control FDB fibers from C57BL10 (P < 0.001). Compared to vehicle-treated mdx FDB fibers, copolymer treatment robustly and rapidly restored the twitch peak SL shortening (all P < 0.05) by P188 (15 µM = + 110%, 150 µM = + 220%), diblock (15 µM = + 50%, 150 µM = + 50%), and inverted triblock copolymer (15 µM = + 180%, 150 µM = + 90%). Twitch peak Ca2+ transient from mdx FDB fibers was also depressed compared to C57BL10 FDB fibers (P < 0.001). P188 and inverted triblock copolymer treatment of mdx FDB fibers increased the twitch peak Ca2+ transient (P < 0.001). This study shows synthetic block copolymers with varied architectures can rapidly and highly effectively enhance contractile function in live dystrophin-deficient skeletal muscle fibers.

Concentration Threshold for Membrane Protection by PEO-PPO Block Copolymers with Variable Molecular Architectures.

Poloxamer 188, a poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer, protects cell membranes in several injury models. However, the nature of the copolymer/membrane interaction and the mechanism of membrane protection remain unknown. Systematic variations of the block copolymer architecture - including PPO-PEO-PPO triblocks and PPO-PEO diblocks - were used to probe the mechanism and evaluate the potential for alternative architectures to yield superior protection. To test the polymers, murine myoblasts were subjected to an osmotic stress, and membrane integrity was quantified by measuring lactate dehydrogenase (LDH) leakage. These experiments exposed a concentration threshold effect where all tested polymers reach 50% leakage of LDH compared to a non-treated buffer only control over a narrow concentration range of 0.8-4 µM. Differences in polymer protection at lower concentrations indicate that protection increases with the PPO-PEO-PPO molecular architecture and increasing hydrophobicity.

Synthesis and Micellization of Bottlebrush Poloxamers.

Bottlebrush polymers are characterized by an expansive parameter space, including graft length and spacing along the backbone, and these features impact various structural and physical properties such as molecular diffusion and bulk viscosity. In this work, we report a synthetic strategy for making grafted block polymers with poly(propylene oxide) and poly(ethylene oxide) side chains, bottlebrush analogues of poloxamers. Combined anionic and sequential ring-opening metathesis polymerization yielded low dispersity polymers, at full conversion of the macromonomers, with control over graft length, graft end-groups, and overall molecular weight. A set of bottlebrush poloxamers (BBPs), with identical graft lengths and composition, was synthesized over a range of molecular weights. Dynamic light scattering and transmission electron microscopy were used to characterize micelle formation in aqueous buffer. The critical micelle concentration scales exponentially with overall molecular weight for both linear and bottlebrush poloxamers; however, the bottlebrush architecture shifts micelle formation to a much higher concentration at a comparable molecular weight. Consequently, BBPs can exist in solution as unimers at significantly higher molecular weights and concentrations than the linear analogues.