Phase coexistence in multilayer phospholipid films treated with certain cryoprotectants

The interaction of cryoprotective agents (CPAs) with complex phospholipid bilayers, crucial for cryopreservation, remains understudied beyond single-component models. We applied Raman mapping to multilayer films of DOPC/DPPC-d62/Chol = 1/3/1 exposed to the vapor phase of dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), ethylene glycol (EG), and their deuterated analogues. Raman data allowed us to evaluate the chemical content, conformational ordering of DPPC-d62, and the CPA incorporation into coexisting phases. It was found that under dry conditions, DMSO can extract Chol from the phospholipid film to form a co-crystalline phase. When added to water, DMSO caused dehydration of the film, making the chemical and conformational content of the coexisting phases similar to the dry film. Films treated with glycerol showed properties that are most similar to those of hydrated films. For EG, we found a decrease in the coexistence region in the phase diagram. PG further decreased the size of the region of phase coexistence and increased the conformational disorder of phospholipids. Finally, our data show that domains with conformationally disordered lipids contain more CPA than those with ordered ones. Overall, our research demonstrates that CPAs can significantly impact the organization and properties of complex phospholipid bilayers.