Investigating Structure-Activity Relationships and the Mechanism of Action of Small-Molecule Ice Recrystallization Inhibitors

Abstract

The cryopreservation of biological material is of critical importance to the success of numerous medical and industrial technologies, including supporting biobanks for immunotherapy cancer treatments and reproductive assistance, global food supply chains, as well as biodiversity and ecological preservation campaigns. However, current cryopreservation methods are complicated by the occurrence of ice recrystallization, a phenomenon of ice crystal growth, that has detrimental effects on the post-thaw recovery, viability, and functionality of cryopreserved materials. Over the last decade the Ben Laboratory has reported the discovery of several small-molecule ice recrystallization inhibitors (IRIs) that can be used during the cryopreservation of cells and tissues to control ice crystal growth and protect them against mechanical damage. While a vast library - consisting of thousands of IRIs - has been developed, only a handful have proven to be effective at improving cryopreservation outcomes in an in vitro or in vivo model. In part, this is due to a lack of knowledge on the mechanism by which these small-molecule IRIs work.

The research described throughout this thesis critically investigates the mechanism of action and structure activity relationships (SAR) of carbohydrate-based small-molecule IRIs as it relates to improving cryopreservation outcomes. Specifically, a study investigating mesenchymal stromal cell (MSC) cryopreservation was continued, where inhibition of ice recrystallization was reported to improve proliferation and immunotherapeutic potential (chapter 2). Then, nitrile- and amide-containing IRIs were investigated for the cryopreservation of red blood cells and the nitrile‑containing IRIs were further studied as live-cell probes to monitor cellular uptake and internalization using novel Raman imaging techniques (chapter 3). Furthermore, several O-aryl-β-D-glucopyranoside compounds were rationally designed, synthesized, and tested for ice recrystallization inhibition activity by the 5-minute splat‑cooling assay to aid future development and optimization of this class of compounds by correlating their activity with various structural features and parameters (chapter 4). In general, correlations are found with respect to a compound's polar surface area to molecular surface area ratio and predicted logP values; these correlations could be used to identify whether a compound would be active or inactive given a specific IC₅₀ threshold but are unable to differentiate between compounds of similar structural features or activity. Given this limitation, a more traditional SAR approach was undertaken that investigated modifications to the hydroxyls of the carbohydrate core of the O-aryl-β-D-glucopyranoside 4‑methoxyphenyl-β-D-glucopyranoside (PMP‑Glc) resulted in the discovery of a library of novel C₄ O‑methylated compounds with high nanomolar ice recrystallization inhibition activity (chapter 5). Finally, interactions between small-molecule IRIs and water molecules in both the liquid and solid states were investigated using proton NMR relaxation time measurements that provided insight into the mechanism responsible for inhibiting ice recrystallization (chapter 6).

Collectively, this research provides detailed insight into the mechanism of action of small-molecule carbohydrate-based ice recrystallization inhibitors to inform downstream rational design campaigns. Furthermore, the addition of IRIs into standard cryopreservation protocols has the potential transform the landscape for translating clinical research into human health technology and ultimately improve the accessibility of cell-based therapeutics.