Water content, transition temperature and fragility influence protection and anhydrobiotic capacity

Abstract

Water is essential for metabolism and all life processes. Despite this, a number of organisms distributed across the kingdoms of life can survive near-complete desiccation or anhydrobiosis (life without water). Increases in intracellular viscosity, leading to the formation of a vitrified (glassy) state is necessary, but not sufficient, for conferring desiccation tolerance. What properties of a vitrified system make it desiccation-tolerant or -sensitive are unknown. We have analyzed 18 different in vitro vitrified systems, composed of one of three protective disaccharides (trehalose, sucrose, or maltose) and varying amounts of glycerol, quantifying their protective capacity and their material properties in a dry state. We find that protection conferred by mixtures containing maltose correlates strongly with increased water content, increased glass-transition temperature, and reduced glass former fragility, while the protection of glasses formed with sucrose correlates with increased glass transition temperature and the protection conferred by trehalose glasses correlates with reduced glass former fragility. Thus, in vitro different vitrified sugars confer protection through distinct material properties. Extending on this, we have examined the material properties of a dry desiccation tolerant and intolerant life stage from four different organisms. In all cases, the dried desiccation tolerant life stage of an organism had an increased glass transition temperature relative to its dried desiccation intolerant life stage, and this trend is seen in three of four organisms when considering reduced glass fragility. These results suggest that while drying of different protective sugars in vitro results in vitrified systems with distinct material properties that correlate with their protective capacity, in nature organismal desiccation tolerance relies on a combination of these properties. This study advances our understanding of how protective and non-protective glasses differ in terms of material properties that promote anhydrobiosis. This knowledge presents avenues to develop novel stabilization technologies for pharmaceuticals that currently rely on the cold-chain.

Protecting Proteins from Desiccation Stress Using Molecular Glasses and Gels

“…246 In other studies, adding glycerol to a sugar glass matrix improved its ability to protect proteins. 247,248…”

“…Mixing polyethylene glycol (PEG) with sugar alcohols or sugars (trehalose, lactose, or mannitol) in a freeze-dried matrix preserves lactate dehydrogenase or phosphofructokinase better than the respective single-component matrices . In other studies, adding glycerol to a sugar glass matrix improved its ability to protect proteins. , …”

“…This duality makes it challenging to determine which property leads to trehalose’s efficacy compared to other sugars. The inconsistent relationships between glass composition, including hydration level, sugar, and polyol content and the extent of glass-induced stability increase highlight some of the unresolved questions concerning glasses and dry protein stability …”