C-1005: A Novel and highly efficient method for intracellular delivery and accumulation of trehalose

Abstract

The presence of trehalose on both sides of the cell membrane contributes to protection against desiccation damage. Since mammalian cell membranes are naturally impermeable to trehalose, invention of novel methods for efficient intracellular trehalose delivery has been an ongoing investigation. It has been established that increasing small molecule lipophilicity enhances cellular uptake. Using this concept as a starting point, we designed a series of acetylated trehalose derivatives, with the 6-acetylated analogue (6-Ac-Tre) displaying the most promising properties in rat hepatocytes, among other cells. Once 6-Ac-Tre penetrated the cell membrane, endogenous nonspecific esterases cleave acetyl groups, resulting in release of de-acetylated trehalose in the cytoplasm. The total intracellular concentration of trehalose and its non-fully-de-acetylated variants was 5-10-fold higher than the extracellular concentration of 6-Ac-Tre, reaching concentrations as high as 300 mM within 6 h of incubation. Using a diffusion–reaction model, the permeability and reaction kinetics of 6-Ac-Tre conversion to trehalose was calculated and optimum incubation concentration and time were obtained. Further studies on cell metabolism suggest that there is only a minimal impact of intracellular trehalose on cellular function. This study demonstrates a novel and robust method for loading trehalose in primary mammalian cells. Despite the somewhat longer loading time compared to some alternative methods, it is an easy-to-perform method for trehalose loading in cells with unprecedented efficiency. The described method is especially suitable for biopreservation studies on primary cells and in cells which may not be synthetically/genetically manipulated for further therapeutic applications. This approach has been carried forward with comprehensive biopreservation studies in progress.