Cryoprotectants Disrupt Hydrogen-Bond Networks at the Lipid-Water Interface

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Cryoprotectant agents (CPAs) are mixtures of small organic compounds used to inhibit ice-crystal growth during cell and tissue freezing and storage, but these compounds can be themselves toxic to cells. Increased membrane permeability, caused by partial disruption of lipid-lipid interactions, combined with dehydration, have been hypothesized to be the main driver of cell toxicity, but the specific interactions between lipids and cryoprotectants are poorly understood. In this study, we investigate the effects of dimethyl sulfoxide (DMSO) on phosphatidylcholine bilayers. We have measured ultrafast two-dimensional infrared (2D IR) spectra of the ester carbonyl vibrations to directly probe the interfacial hydrogen bond networks. Our measurements directly quantify the picosecond hydrogen bond lifetimes at the ester carbonyl positions. Spectra are interpreted at an atomistic level using molecular dynamics simulations, which are in semi-quantitative agreement with experiment. Our results show that biologically relevant cryoprotectant concentrations (<20 mol%), partially dehydrate the interface, but surprisingly, hydrogen-bond lifetimes are non-monotonic with increasing concentration: 1. Low DMSO concentrations (<10%) severely disrupt the otherwise ice-like interfacial hydrogen bond networks, without altering the lipid-lipid interactions, leading to faster overall hydrogen bond dynamics at the ester carbonyls. 2. Above >10 mol%, dynamics become slower as a result of dehydration, more rigid H-bond conformations, trapped water, and stronger headgroup-headgroup interactions. Together, these results provide an atomistic foundation for understanding the complex effects of cryoprotectants on lipid membranes, showing that different effects dictate the properties of membranes within various cryoprotectant concentration regimes.