Influence of Poly(ethylene oxide)-b-Poly(propylene oxide) Block Copolymers and Poly(ethylene oxide) Homopolymer on Cellular Function

Abstract

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 are widely used for biological applications. In the 1990s, these polymers, specifically Poloxamer 188 (P188, 8.4 kDa, 80 wt% EO), were found to provide therapeutic effects through membrane stabilization. However, the complete mechanism by which the polymer interacts with the membrane to provide stabilization is unclear. To better understand the mechanisms behind the polymer-membrane interaction, this work aims to investigate (i) the role of different architectures, including the inverted (PPO-PEO-PPO) architecture, (ii) the effect of polymer dose, and (iii) the effects of polymer treatment and/or osmotic stress on transcription.Previous work in this field has shown that protective copolymers have a hydrophobic block to engage the molecule with the membrane and a hydrophilic block to solubilize the molecule in aqueous solution and stabilize the interaction with the membrane. To further expound on this proposed mechanism, an inverted poloxamer analog to P188 (PPO15-PEO200-PPO15) and a systematic variation of PPO-PEO diblocks were synthesized and tested in vitro to determine if increasing the number of hydrophobic ends would increase protection against enzyme leakage in osmotically stressed murine myoblasts. Through titrating the polymer dose, small, yet significant, differences showed enhancing the hydrophobicity through one or more hydrophobic ends, with possible addition of a non-polar end group, did increase the molecule’s potency. The titration of polymer dose also elucidated a concentration threshold where all the polymers tested reduced enzyme leakage by 50% compared to the untreated control between a dose of 0.8-4 µM. Through collaborative efforts, this trend was further seen in poly(butylene oxide)-PEO and bottlebrush poloxamers with a slightly larger threshold of 0.3-4 µM. Although enzyme leakage is a good metric to measure membrane permeability, the definition of cell health should be defined more broadly. To determine if the P188 or PEO181 (8 kDa) have an effect on the biochemical response of healthy or osmotically stressed murine myoblasts, RNA sequencing was used to quantify the transcriptome. Differential gene expression analysis showed that short-term exposure to 14 µM of P188 or PEO significantly changed expression of genes compared to the unstressed, untreated control. Subjecting the myoblasts to osmotic stress also impacted the transcriptome; however, the addition of polymer treatment to stressed cells did not result in significant differential expression compared to stress alone, which is consistent with a protective mechanism based on the physical polymer-membrane interaction.