Abstract
Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.
Highlights
Trehalose is a naturally occurring alpha-linked disaccharide formed by an α,α-1,1-glucosidic bond between two α-glucose units
We investigated an approach to increasing the membrane permeability of trehalose by conjugation with 2, 4 and 6 acetyl groups
We found that conjugation with 2 and 4 acetyl groups did not significantly enhanced the membrane permeability of trehalose (Fig 3)
Summary
Trehalose is a naturally occurring alpha-linked disaccharide formed by an α,α-1,1-glucosidic bond between two α-glucose units. It is proposed that trehalose contributes to the formation of a stable glassy state when dried, which prevents deleterious conformational changes in proteins and significantly impedes molecular mobility, reducing the rate of deteriorating biochemical reactions [10,11,12,13]. For these properties and the fact that it is generally nontoxic, trehalose is an attractive protective agent in biopreservation. It is used as a common additive in pharmaceutical formulations, contributing to the stabilization of compounds in frozen or dried states [14], and it has shown promise as a protective agent against freezing and desiccation-induced damages in some mammalian cells when delivered intracellularly [15,16,17,18,19,20,21,22]
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