34. Designing next generation protectants

In order to advance towards the vitrification of complex organs, further advances in cryoprotectant compositions will be necessary. Systematic solution optimization has enabled the identification of superior combinations of cryoprotectants for various cell and tissue types, yet the range of chemicals used as protectants is still quite narrow and it is oftentimes not well understood why some compositions work better than others. The White House recently launched a “Materials Genome Initiative” to catalyze an integrated design process that uses feedback from computational studies to improve the efficiency of advanced materials development. This approach is very suitable to the design of Next Generation Protectants. For vitrification applications the main materials optimization parameters are cryoprotectant toxicity and glassy state properties. While advances have been made in understanding and predicting CPA toxicity, it is much harder to predict the glassy state characteristics of complex mixtures. Additive mixing rules provide little insight, as superior compositions often exhibit strong deviations from ideal solution behavior. In recent work we have combined advanced thermal analysis techniques, specifically Dynamic Mechanical Analysis (DMA) and Differential Scanning Calorimetry (DSC), together with molecular modelling to explore the underling molecular mechanisms that give rise to Tg enhancements in protectant compositions in an effort to better understand the design rules that lead to improved compositions. Using molecular dynamics (MD) simulation, we have recently shown that divalent cations can strengthen the interacting network in electrolyte/glycerol mixtures via strong cation-dipole attractions, leading to an increase in T g 1 . In contrast, monovalent cations were observed to have little effect on Tg as the cation-dipole attraction was only slightly stronger than the original hydrogen-bonding network amongst glycerol molecules. The precursor of salt crystallization was also observed in these monovalent ion compositions, potentially contributing to weak Tg-enhancing ability. Using both thermal analysis and MD simulation we were also able to better understand the effects of phosphate anions on the glassy state properties of trehalose2. We observed that the addition of NaH2PO4 decreased both the glass transition temperature and the α -relaxation temperature of the dehydrated trehalose/NaH2PO4 mixture compared to trehalose alone while both Tg and Tα were increased by adding Na2HPO4 to pure trehalose. The hydrogen-bonding interactions between trehalose and HPO 4 2 - were found to be stronger than both the trehalose-trehalose hydrogen bonds and those formed between trehalose and H2 PO 4 - . The HPO 4 2 - ions also aggregated into smaller clusters than H2 PO 4 - ions, contributing to a more homogeneous glass network. Using DMA techniques we also demonstrated that when pure trehalose, a fragile glass-former, was diluted with a small amount of the strong glass-former, glycerol, the overall mixture was strengthened significantly. The fragility index (m) was observed to be minimized when glycerol was added at a 5–10 wt% concentration, the same formulation that has been shown by others to be maximally effective for preserving proteins in the glassy state. The combination of MD simulation and thermal analysis should prove to be very valuable for the development of Next Generation Protectants.