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

Abstract

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 nontreated 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.

Effect of Bottlebrush Poloxamer Architecture on Binding to Liposomes

“…However, when the PEO side-chain length was shortened, the statistical copolymer performed significantly better than the corresponding block polymer at very low concentrations (red vs blue points, p < 0.01). Interestingly, it remained protective even down to 0.014 μM, which is the lowest protective polymer concentration of any polymer tested to date. , All other molecules follow the recently reported threshold effect, where polymers lose protective efficacy somewhere between 0.8 and 4 μM . Future experiments will be conducted on a tissue-level protection assay with dystrophic mouse muscle fibers, which is a step closer to a physiological situation …”

“…Finally, we evaluated the cellular protection efficacy of BBPs and compared them to linear analogues using an established osmotic stress assay. ,,, Briefly, this assay subjects C2C12 myoblast cells to a hypoosmotic stress followed by isotonic recovery. If the membrane is damaged, macromolecules, such as the protein LDH, leak out of the cell and can then be detected via enzymatic colorimetry.…”

“…Previously, linear block copolymer amphiphiles have only demonstrated protection efficacy up to ca. 20–30 kDa. , The dramatic extension in molecular weight of protective BBPs is notable given the well-established dependence of renal clearance on molecule size . Finally, unlike liposome binding, cell membrane protection is not unique to the block architecture as bottlebrush PEO homopolymers and statistical copolymers with PEO and PPO side chains do outperform L -E 87 P 31 E 87 (P188) in an osmotic stress assay on a molar basis.…”

“…Interestingly, it remained protective even down to 0.014 μM, which is the lowest protective polymer concentration of any polymer tested to date. 25,58 All other molecules follow the recently reported threshold effect, where polymers lose protective efficacy somewhere between 0.8 and 4 μM. 58 Future experiments will be conducted on a tissue-level protection assay with dystrophic mouse muscle fibers, which is a step closer to a physiological situation.…”

“…25,58 All other molecules follow the recently reported threshold effect, where polymers lose protective efficacy somewhere between 0.8 and 4 μM. 58 Future experiments will be conducted on a tissue-level protection assay with dystrophic mouse muscle fibers, which is a step closer to a physiological situation. 14 Finally, Figure 6d shows that bottlebrush PEO polymers can be protective even without the PPO block.…”