Temporal and mechanistic tracking of cellular uptake dynamics with novel surface fluorophore-bound nanodiamonds

Abstract

Nanoparticles (NPs) offer promise for a multitude of biological applications including cellular probes at the bio-interface for targeted delivery of anticancer substances, Raman and fluorescent-based imaging and directed cell growth. Nanodiamonds (NDs), in particular, have several advantages compared to other carbon-based nanomaterials - including a rich surface chemistry useful for chemical conjugation, high biocompatibility with little reactive oxygen species (ROS) generation, physical and chemical stability that affords sterilization, high surface area to volume ratio, transparency and a high index of refraction. The visualization of ND internalization into cells is possible via photoluminescence, which is produced by direct dye conjugation or high energy irradiation that creates nitrogen vacancy centers. Here, we explore the kinetics and mechanisms involved in the intracellular uptake and localization of novel, highly-stable, fluorophore-conjugated NDs. Examination in a neuronal cell line (N2A) shows ND localization to early endosomes and lysosomes with eventual release into the cytoplasm. The addition of endocytosis and exocytosis inhibitors allows for diminished uptake and increased accumulation, respectively, which further corroborates cellular behavior in response to NDs. Ultimately, the ability of the NDs to travel throughout cellular compartments of varying pH without degradation of the surface-conjugated fluorophore or alteration of cell viability over extended periods of time is promising for their use in biomedical applications as stable, biocompatible, fluorescent probes.

Surface Modifications of Nanodiamonds and Current Issues for Their Biomedical Applications

“…Although NV centers are promising for in vitro imaging applications [203,224,225], their limited photoluminescence intensity is an obstacle to their detection in the body. In the literature, several groups reported the efficient tracking of NDs into cells by grafting of fluorophores [24,98,202,226]. Bright blue luminescence was even obtained by octadecylamine covalently attached to NDs-COOH [227].…”

“…Efficient surface treatments also permit to conjugate ND surfaces with fluorescent molecules [202][203][204][205], with DNA [206], siRNA [27], proteins [207,208], lysozymes [208], growth hormones [209], antibodies [210,211], anti-cancer drugs [30][31][32], as well as with dopamine derivatives [212].…”

Nanodiamonds as Intracellular Probes for Imaging in Biology and Medicine

“…Inside the cell, endosomes containing NDs can either transform into lysosomes or fuse with existing lysosomes, as has been shown by colocalization experiments with dyed lysosomes and NDs (Fig. 8) (Schrand et al 2011;Lin et al 2012;Alhaddad et al 2012;Faklaris et al 2009a). The interaction between endosomes and lysosomes during the early stages of the ND uptake pathway was observed in an experiment with brefeldin A (a known lactone antibiotic that interferes with the merging of endosomes and lysosomes).…”

“…Some studies imply that a fraction of NDs is entrapped and localized in membrane-bound compartments called endosomes (Schrand et al 2011;Horie et al 2012;Alhaddad et al 2012;Faklaris et al 2009a). These vesicles are responsible for transport of extracellular materials.…”

Nanocarbon materials fabricated using plasmas

“…Then, fluorescent particles can also be produced by linking or adsorbing various fluorophores onto NDs. Fluorophore-conjugated NDs can travel through cellular compartments of varying pH without degradation of the surface-conjugated fluorophore or alteration of cell viability over extended periods of time (Schrand et al 2011). Bright blue fluorescent ND was produced by covalent linking of octadecylamine {ODA: CH 3 (CH 2 ) 17 NH 2 } to carboxylic groups on ND surface (ND-ODA), as summarized in Fig.…”