Carboxylated nanodiamonds are neither cytotoxic nor genotoxic on liver, kidney, intestine and lung human cell lines

Abstract Although nanodiamonds (NDs) appear as one of the most promising nanocarbon materials available so far for biomedical applications, their risk for human health remains unknown. Our work was aimed at defining the cytotoxicity and genotoxicity of two sets of commercial carboxylated NDs with diameters below 20 and 100 nm, on six human cell lines chosen as representative of potential target organs: HepG2 and Hep3B (liver), Caki-1 and Hek-293 (kidney), HT29 (intestine) and A549 (lung). Cytotoxicity of NDs was assessed by measuring cell impedance (xCELLigence® system) and cell survival/death by flow cytometry while genotoxicity was assessed by γ-H2Ax foci detection, which is considered the most sensitive technique for studying DNA double-strand breaks. To validate and check the sensitivity of the techniques, aminated polystyrene nanobeads were used as positive control in all assays. Cell incorporation of NDs was also studied by flow cytometry and luminescent N–V center photoluminescence (confirmed by Raman microscopy), to ensure that nanoparticles entered the cells. Overall, we show that NDs effectively entered the cells but NDs do not induce any significant cytotoxic or genotoxic effects on the six cell lines up to an exposure dose of 250 µg/mL. Taken together these results strongly support the huge potential of NDs for human nanomedicine but also their potential as negative control in nanotoxicology studies.

[1]  C. van Hooijdonk,et al.  TO-PRO-3 iodide: a novel HeNe laser-excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry. , 1994, Cytometry.

[2]  Huan-Cheng Chang,et al.  Mass production and dynamic imaging of fluorescent nanodiamonds. , 2008, Nature nanotechnology.

[3]  Xiao Xu,et al.  The xCELLigence system for real-time and label-free monitoring of cell viability. , 2011, Methods in molecular biology.

[4]  A. Krüger.  Hard and soft: biofunctionalized diamond. , 2006, Angewandte Chemie.

[5]  A. El-Osta,et al.  γH2AX: a sensitive molecular marker of DNA damage and repair , 2010, Leukemia.

[6]  Saber M Hussain,et al.  Are diamond nanoparticles cytotoxic? , 2007, The journal of physical chemistry. B.

[7]  Fedor Jelezko,et al.  Single defect centres in diamond: A review , 2006 .

[8]  J. Harvey,et al.  Genotoxicity screening via the γH2AX by flow assay. , 2011, Mutation research.

[9]  Helinor Johnston,et al.  Development of in vitro systems for nanotoxicology: methodological considerations , 2009, Critical reviews in toxicology.

[10]  Erik Pierstorff,et al.  Active nanodiamond hydrogels for chemotherapeutic delivery. , 2007, Nano letters.

[11]  Huan-Cheng Chang,et al.  Adsorption and immobilization of cytochrome c on nanodiamonds. , 2004, Langmuir : the ACS journal of surfaces and colloids.

[12]  Li Lu,et al.  Biotinylated nanodiamond: simple and efficient functionalization of detonation diamond. , 2008, Langmuir : the ACS journal of surfaces and colloids.

[13]  Reyes Sierra-Alvarez,et al.  Application and validation of an impedance-based real time cell analyzer to measure the toxicity of nanoparticles impacting human bronchial epithelial cells. , 2012, Environmental science & technology.

[14]  Anne E Carpenter,et al.  CellProfiler: image analysis software for identifying and quantifying cell phenotypes , 2006, Genome Biology.

[15]  W. Boyes,et al.  Detection of TiO2 nanoparticles in cells by flow cytometry , 2010, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[16]  T. Petit,et al.  Surface chemical modifications and surface reactivity of nanodiamonds hydrogenated by CVD plasma. , 2011, Physical chemistry chemical physics : PCCP.

[17]  Katharina Landfester,et al.  Amino‐functionalized polystyrene nanoparticles activate the NLRP3 inflammasome in human macrophages , 2011, ACS nano.

[18]  J. Boudou,et al.  Peptide‐Grafted Nanodiamonds: Preparation, Cytotoxicity and Uptake in Cells , 2008, Chembiochem : a European journal of chemical biology.

[19]  C. Koshland,et al.  Particle-induced artifacts in the MTT and LDH viability assays. , 2012, Chemical research in toxicology.

[20]  Huan-Cheng Chang,et al.  Bright fluorescent nanodiamonds: no photobleaching and low cytotoxicity. , 2005, Journal of the American Chemical Society.

[21]  Alexandra Kroll,et al.  Interference of engineered nanoparticles with in vitro toxicity assays , 2012, Archives of Toxicology.

[22]  Elena Perevedentseva,et al.  Nanodiamond for intracellular imaging in the microorganisms in vivo , 2012, Journal of biophotonics.

[23]  Elena Perevedentseva,et al.  Direct and in vitro observation of growth hormone receptor molecules in A549 human lung epithelial cells by nanodiamond labeling , 2007 .

[24]  Andrea Zappe,et al.  Dynamics of diamond nanoparticles in solution and cells. , 2007, Nano letters.

[25]  P Bergonzo,et al.  Surface properties of hydrogenated nanodiamonds: a chemical investigation. , 2011, Physical chemistry chemical physics : PCCP.

[26]  Chia-Liang Cheng,et al.  The interaction of the protein lysozyme with bacteria E. coli observed using nanodiamond labelling , 2007 .

[27]  A. Krueger,et al.  Beyond the shine: recent progress in applications of nanodiamond , 2011 .

[28]  Tiancheng Wang,et al.  Pulmonary toxicity and translocation of nanodiamonds in mice , 2010 .

[29]  Jui‐I Chao,et al.  Covalent linkage of nanodiamond-paclitaxel for drug delivery and cancer therapy , 2010, Nanotechnology.

[30]  Huan-Cheng Chang,et al.  The long-term stability and biocompatibility of fluorescent nanodiamond as an in vivo contrast agent. , 2012, Biomaterials.

[31]  P. Bergonzo,et al.  Hydrogenation of nanodiamonds using MPCVD: A new route toward organic functionalization , 2010 .

[32]  Lei Tao,et al.  A comparative study of cellular uptake and cytotoxicity of multi-walled carbon nanotubes, graphene oxide, and nanodiamond , 2012 .

[33]  John J. Schlager,et al.  Differential biocompatibility of carbon nanotubes and nanodiamonds , 2007 .

[34]  V. Paget,et al.  Nano-silicon dioxide toxicological characterization on two human kidney cell lines , 2011 .

[35]  Thierry Gacoin,et al.  Nanodiamond as a vector for siRNA delivery to Ewing sarcoma cells. , 2011, Small.

[36]  K. Donaldson,et al.  Principal component and causal analysis of structural and acute in vitro toxicity data for nanoparticles , 2014, Nanotoxicology.

[37]  Kuang-Kai Liu,et al.  Endocytic carboxylated nanodiamond for the labeling and tracking of cell division and differentiation in cancer and stem cells. , 2009, Biomaterials.

[38]  M. Durante,et al.  Radiation Dose Detection by Imaging Response in Biological Targets , 2012, Radiation research.

[39]  A. Nakamura,et al.  Recent developments in the use of γ -H2AX as a quantitative DNA double-strand break biomarker , 2011, Aging.

[40]  Cheng-Chun Chang,et al.  Receptor-mediated cellular uptake of folate-conjugated fluorescent nanodiamonds: a combined ensemble and single-particle study. , 2009, Small.

[41]  Huan-Cheng Chang,et al.  In vivo imaging and toxicity assessments of fluorescent nanodiamonds in Caenorhabditis elegans. , 2010, Nano letters.

[42]  H-C Chang,et al.  Functionalized fluorescent nanodiamonds for biomedical applications. , 2009, Nanomedicine.

[43]  Huan-Cheng Chang,et al.  The biocompatibility of fluorescent nanodiamonds and their mechanism of cellular uptake , 2009, Nanotechnology.

[44]  R. Zucker,et al.  Detection of TiO2 nanoparticles in cells by flow cytometry. , 2012, Methods in molecular biology.

[45]  Hugh J. Byrne,et al.  Resonant Mie scattering (RMieS) correction of infrared spectra from highly scattering biological samples. , 2010, The Analyst.

[46]  Michael D. Abràmoff,et al.  Image processing with ImageJ , 2004 .

[47]  Wei Xiong,et al.  DNA damage in embryonic stem cells caused by nanodiamonds. , 2011, ACS nano.

[48]  V. Paget,et al.  Toxicity and genotoxicity of nano-SiO2 on human epithelial intestinal HT-29 cell line. , 2012, The Annals of occupational hygiene.

[49]  Julia Gorelik,et al.  Respiratory epithelial cytotoxicity and membrane damage (holes) caused by amine-modified nanoparticles , 2012, Nanotoxicology.