Characterization and internalization of nanodiamond–trehalose conjugates into mammalian fibroblast cells of naked mole rat

Abstract

Trehalose is a natural compound produced by certain organisms to cope with freezing and thawing. It, therefore, has a potential for use as a cryoprotective agent, but the absence of specific receptors requires alternative mechanisms of intracellular delivery. Nanodiamonds are a non-toxic, carbon-based technology, and when conjugated with trehalose, they may offer a suitable delivery system for trehalose internalization. The characteristics of commercially available detonation nanodiamonds with carboxylated (ND-COOH) or hydrogenated (ND-H) surface structures were characterized using dynamic light scattering (DLS) to determine particle size and zeta potential. Trehalose adsorption to the two nanodiamonds was examined at varying concentrations and pH values using Fourier-transform infrared spectroscopy (FT-IR). Naked mole rat fibroblasts were used to study internalization of three different suspensions of trehalose-conjugated nanodiamonds: nanodiamond alone (T1); nanodiamond + trehalose intense wash (T2); and nanodiamond + trehalose moderate wash (T3) at concentrations of 10 or 50 μg/ml (6 or 36 μg/cm2) and exposure durations of 2 or 24 h. Internalization was evaluated by flow cytometry analysis and dark-field microscopy. Cells incubated with the three nanodiamond suspensions were examined by phase-contrast microscopy to evaluate cell morphology after the addition of propidium iodide dye which detects necrotic cells. ND-COOH and ND-H suspensions had mean particle diameters of 8 nm and 32 nm, and zeta potentials of − 49 mv and + 53 mv, respectively. FT-IR absorption spectra showed that trehalose adsorption to ND-COOH and ND-H was greatest at a trehalose concentration of 50 mM and at a neutral pH. ND-COOH particles displayed the greatest affinity with trehalose. Flow cytometry analysis indicated dose- and exposure-dependent increases in light scattering (correlating with increased internal complexity) for all nanodiamond suspensions (T1, T2, and T3), with the highest levels of internalization observed with the 50 μg/ml concentration at exposure durations of 24 h. Untreated cells exhibited low light scattering. There were no significant differences between T1, T2, and T3 in the number of cells internalized. Cell size was not altered by internalization. Internalization of the nanodiamond suspensions was confirmed by dark-field microscopy. Toxicology experiments indicated some alterations to cell morphology, but no effect of the nanodiamond suspensions on cell counts or cell death. Nanodiamond delivery systems offer potential for the internalization of trehalose in mammalian cells. Further experiments should investigate the efficacy of trehalose–nanodiamond particles as cryoprotective agents.