Effect of ice recrystallization inhibition on hydrogen bonding interactions and membrane leakage of liposomes

In this study, effects of ice recrystallization on membrane stability of liposomes were investigated using liposomes encapsulating a fluorescent dye. Membrane leakage was studied after freezing and storage at varying temperatures in solutions supplemented with polyvinyl alcohol (PVA), polyethylene glycol (PEG), dimethyl sulfoxide (DMSO) and combinations thereof. Leakage studies were corroborated with studies on ice crystal growth and hydrogen bonding interactions during holding at temperatures just below the ice melting temperature, i.e., at -10 °C. Cryomicroscopic observations confirmed that PVA exhibits ice recrystallization inhibition activity, whereas PEG did not. Both PVA and PEG reduced freezing-induced liposome leakage, alone and in combination with low DMSO concentrations. Temperature-scanning infrared spectroscopy (FTIR) combined with principal component analysis (PCA) was used as a novel approach to probe differences in hydrogen bonding interactions between frozen buffered saline (PBS) containing PVA and PEG. Score and loading plots show that symmetric hydrogen bonds are predominant with addition of PVA, and that the cluster of principal component data points remain compact during holding under ice recrystallization conditions. By contrast, PBS supplemented with PEG and PBS control solutions are characterized by weak hydrogen bonding interactions and more disperse clusters of principal component data points denoting rearrangements in hydrogen bonding interactions associated with ice crystal growth during holding. In conclusion, beneficial effects of adding PVA or PEG in cryopreservation solutions for liposomes are most evident under suboptimal cryopreservation conditions, e.g., during storage at elevated subzero temperatures, and when low concentrations of DMSO are used.