The biocompatibility of fluorescent nanodiamonds and their mechanism of cellular uptake

Abstract

The labeling of cells with fluorescent nanoparticles is promising for various biomedical applications. The objective of this study is to evaluate the biocompatibility and the mechanism of the cellular uptake of fluorescent nanodiamonds (FNDs) in cancer cells (HeLa) and pre-adipocytes (3T3-L1). With flow cytometry and the use of a battery of metabolic and cytoskeletal inhibitors, we found that the mechanism of the FND uptake in both cells is by energy-dependent clathrin-mediated endocytosis. In addition, the surface charge of FND influences its cellular uptake, as the uptake of poly-L-lysine-coated FNDs is better than that of oxidative-acid-purified FNDs at the same concentration in regular medium with or without serum. We also confirm that the proliferative potential of FND-treated and untreated cells does not exhibit any significant differences when measured at bulk cultures, and more stringently at clonal cell density. Further biocompatibility studies indicate that the in vitro differentiation of 3T3-L1 pre-adipocytes and 489-2 osteoprogenitors is not affected by the FND treatment. Our results show that FNDs are biocompatible and ideal candidates for potential applications in human stem cell research.

Luminescent nanodiamonds for biomedical applications

“…This can be done by atmospheric plasma treatment with nitrogen or hydrogen (Gibson et al 2009), annealing in hydrogen gas (Williams et al 2010) or treatment with NaCl or sodium oleate (Xu et al 2005). Functionalisation with some biomolecules, such as poly-L-lysine (Vaijayanthimala et al 2009) and bovine serum albumin (Yuan et al 2009), can also increase nanodiamond solubility and reduce re-aggregation. The Table 4 Various biological moieties which have been physisorbed to nanodiamonds…”

“…-apoobelin ) -aflatoxin B1 (AfB1) ) -DNA (Fu et al 2007) -luciferase Puzyr' et al 2004) -Alpha-bungarotoxin (Liu et al 2008 (Miller and Brown 1996;Knickerbocker et al 2003;Lu et al 2004;Strother et al 2002;Yang et al 2002;Takahashi et al 2003;Yeap et al 2008;Zhang et al 2009b;Zheng et al 2009;Chang et al 2010) (Strother et al 2002;Takahashi et al 2003;Ida et al 2003;Tsubota et al 2003;Härtl et al 2004;Christiaens et al 2006;Chang et al 2010) (Liu et al 2004;Ando et al 1996b;Khabashesku et al 2005;Ando et al 1996a;Ando et al 1993) (Ando et al 1996a;Ikeda et al 1998;Takahashi et al 2003;Miller and Brown 1996) Silanes Aryl Alkyl Amino (Zhang et al 2009b;Liang et al 2009;Krüger et al 2006;Yeap et al 2008 Zheng et al 2009) choice of biological buffer systems or media is also important, since both foetal bovine serum and phosphate buffered saline, two common biological buffer systems, tend to cause re-aggregation (Neugart et al 2007;Vaijayanthimala et al 2009). Despite the challenges involved in preparing and maintaining de-aggregated nanodiamonds, positive results strongly support their use in biological applications.…”

“…The measurement of mitochondrial damage using the methyl thiazolyl tetrazolium assay revealed that the toxicity of nanodiamonds (Schrand et al 2009) in a wide range of cell lines is extremely low (Table 6). In acute conditions, cells tolerate concentrations up to about 400 μg/mL of raw or surface functionalised nanodiamonds in vitro Chao et al 2007;Liu et al 2007;Schrand et al 2007;Faklaris et al 2008;Vaijayanthimala et al 2009), with a decrease by approximately 20% in cell viability in some cancer cells at concentrations of 4 × 10 7 nanodiamonds/mL (Burleson et al 2009). Protein expression ), rates of cellular proliferation and differentiation (Vaijayanthimala et al 2009), as well as rates of apoptosis ) have all been used to show nanodiamond in vitro biocompatibility.…”

“…In acute conditions, cells tolerate concentrations up to about 400 μg/mL of raw or surface functionalised nanodiamonds in vitro Chao et al 2007;Liu et al 2007;Schrand et al 2007;Faklaris et al 2008;Vaijayanthimala et al 2009), with a decrease by approximately 20% in cell viability in some cancer cells at concentrations of 4 × 10 7 nanodiamonds/mL (Burleson et al 2009). Protein expression ), rates of cellular proliferation and differentiation (Vaijayanthimala et al 2009), as well as rates of apoptosis ) have all been used to show nanodiamond in vitro biocompatibility. This is further borne out by the low levels of reactive oxygen species measurable in treated cells since high levels of reactive oxygen species are a common sign of cell stress (Schrand et al 2007).…”

“…In one study, the application of nanodiamonds to the skin of mice did not cause contact hypersensitivity or other allergic reactions (Burleson et al 2009). At a dose of 1mg/kg, Other -DNA (Christiaens et al 2006;Takahashi et al 2003;Knickerbocker et al 2003;Lu et al 2004;Rezek et al 2006;Yang et al 2002) -DNA (Ushizawa et al 2002) (ester linkage) Table 6 Cells showing good biocompatibility with nanodiamond in vitro HeLa cells (human cervical carcinoma) (Faklaris et al 2008;Vaijayanthimala et al 2009) Human lung adenocarcinoma cells Liu et al 2007;Liu et al 2009) Keratinocytes (Burleson et al 2009;Schrand et al 2007) Neuroblastomas (Schrand et al 2007) PC-12 cells (Schrand et al 2007) Lung fibroblasts Embryonic fibroblasts Vaijayanthimala et al 2009) Human kidney cells Osteoprogenitor cells (Vaijayanthimala et al 2009) Macrophages (Schrand et al 2007) 4 nm nanodiamonds instilled into the mouse trachea were phagocytosed by alveolar macrophages within 24 h, with a decrease in the number of nanodiamonds observed in the alveolar area throughout the study. Histopathological and ultrastructural investigations at 7, 14 and 28 days postexposure also showed no adverse effects in the lungs; nor did nanodiamonds evidently translocate into the bloodstream and/or other organs (Yuan et al 2010).…”