Kumara, U. G. V. S. S. | Search bioRxiv

bioRxiv2026

Drying parameters and aging modulate protective properties of vitrified trehalose

Storage of biological materials is essential for medical, research, and biotechnological applications. While cold-chain preservation is effective, it is costly, infrastructure-dependent, and vulnerable to disruption. Room-temperature dry storage, inspired by desiccation-tolerant organisms, provides an alternative by stabilizing biomolecules in vitrified ("glass-like") matrices that limit molecular motion which if not reduced can lead to breakdown and loss of integrity. Trehalose is widely used as a vitrifying agent, but its protective capacity depends on glassy properties shaped by drying methods, environmental conditions, storage duration, and the type of preserved molecule. Systematic studies linking these factors to short and long-term stability remain limited. Here, we examine how drying conditions and storage duration influence the stability of DNA, RNA, and enzymes in vitrified trehalose systems. DNA remained stable under all conditions, independent of trehalose or drying parameters, reflecting its intrinsic resistance to desiccation-induced damage. RNA showed moderate sensitivity to drying without trehalose but was stabilized in its presence, although RNA integrity did not consistently correlate with measured vitrified properties. In contrast, enzymes were highly sensitive to drying without trehalose and were strongly protected under conditions that promoted favorable vitrified properties. Enzyme protection after 30 min correlated with high glass transition temperature. However, during prolonged drying, increased glass transition temperature was inversely correlated with enzyme protection and was a better indicator of detrimental physical aging of the vitrified system. These findings present the first insight into how drying methods, environmental conditions, and storage duration shape vitrified properties and stability. They guide optimization of room-temperature preservation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/700019v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@796fc0org.highwire.dtl.DTLVardef@97ca65org.highwire.dtl.DTLVardef@168d532org.highwire.dtl.DTLVardef@184ee58_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LINucleic acids are generally stable under various drying conditions/vitrified systems C_LIO_LIProtein preservation benefits from specific mediators and vitrified properties C_LIO_LIDrying method and parameters tune vitrified properties and protection C_LIO_LIVitrified systems age over time, changing properties and protectiveness C_LIO_LIInitially high Tg is protective, increasing Tg and fragility over time indicate aging C_LI

biochemistry↗

bioRxiv2023

Distinct material properties of sugar-glasses correlate with anhydrobiosis in vitro and in vivo

Water is essential for metabolism and all life processes. Despite this, many organisms distributed across the kingdoms of life survive near-complete desiccation or anhydrobiosis (Greek for "life without water"). Increased intracellular viscosity, leading to the formation of a vitrified state is necessary, but not sufficient, for survival while dry. 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 enzyme-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 three 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 also seen in all three organisms when considering reduced glass former 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 enzyme-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. 1.1 Statement of significanceFor the past three decades the anhydrobiosis field has lived with a paradox, while vitrification is necessary for survival in the dry state, it is not sufficient. Understanding what property(s) distinguishes a desiccation tolerant from an intolerant vitrified system and how anhydrobiotic organisms survive drying is one of the enduring mysteries of organismal physiology. Here we show in vitro the enzyme-protective capacity of different vitrifying sugars can be correlated with distinct material properties. However, in vivo, diverse desiccation tolerant organisms appear to combine these material properties to promote their survival in a dry state. 3.1 HighlightsO_LIThe enzyme-protective capacities of different glass forming sugars correlate with distinct material properties. C_LIO_LIMaterial properties of dried anhydrobiotic organisms differ dramatically when examined in desiccation tolerant and intolerant life stages. C_LIO_LIOrganismal desiccation tolerance is concomitant with changes in glassy properties including increased glass transition temperature and reduced glass former fragility. C_LI

biophysics↗