Interference of engineered nanoparticles with in vitro toxicity assays

Accurate in vitro assessment of nanoparticle cytotoxicity requires a careful selection of the test systems. Due to high adsorption capacity and optical activity, engineered nanoparticles are highly potential in influencing classical cytotoxicity assays. Here, four common in vitro assays for oxidative stress, cell viability, cell death and inflammatory cytokine production (DCF, MTT, LDH and IL-8 ELISA) were assessed for validity using 24 well-characterized engineered nanoparticles. For all nanoparticles, the possible interference with the optical detection methods, the ability to convert the substrates, the influence on enzymatic activity and the potential to bind proinflammatory cytokines were analyzed in detail. Results varied considerably depending on the assay system used. All nanoparticles tested were found to interfere with the optical measurement at concentrations of 50 μg cm−2 and above when DCF, MTT and LDH assays were performed. Except for Carbon Black, particle interference could be prevented by altering assay protocols and lowering particle concentrations to 10 μg cm−2. Carbon Black was also found to oxidize H2DCF-DA in a cell-free system, whereas only ZnO nanoparticles significantly decreased LDH activity. A dramatic loss of immunoreactive IL-8 was observed for only one of the three TiO2 particle types tested. Our results demonstrate that engineered nanoparticles interfere with classic cytotoxicity assays in a highly concentration-, particle- and assay-specific manner. These findings strongly suggest that each in vitro test system has to be evaluated for each single nanoparticle type to accurately assess the nanoparticle toxicity.

[1]  J. Mauderly,et al.  Diesel Particulate Material Binds and Concentrates a Proinflammatory Cytokine That Causes Neutrophil Migration , 2004, Inhalation toxicology.

[2]  François Huaux,et al.  Influence of size, surface area and microporosity on the in vitro cytotoxic activity of amorphous silica nanoparticles in different cell types , 2010, Nanotoxicology.

[3]  Sara Linse,et al.  Understanding the nanoparticle–protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles , 2007, Proceedings of the National Academy of Sciences.

[4]  H. Byrne,et al.  Spectroscopic analysis confirms the interactions between single walled carbon nanotubes and various dyes commonly used to assess cytotoxicity , 2007 .

[5]  Kenneth A. Dawson,et al.  Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts , 2008, Proceedings of the National Academy of Sciences.

[6]  N. Monteiro-Riviere,et al.  Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line. , 2009, Toxicology and applied pharmacology.

[7]  G. Oberdörster,et al.  Nanotoxicology: An Emerging Discipline Evolving from Studies of Ultrafine Particles , 2005, Environmental health perspectives.

[8]  C. Korzeniewski,et al.  An enzyme-release assay for natural cytotoxicity. , 1983, Journal of immunological methods.

[9]  Vicki Stone,et al.  The biological mechanisms and physicochemical characteristics responsible for driving fullerene toxicity. , 2010, Toxicological sciences : an official journal of the Society of Toxicology.

[10]  Steffen Hackbarth,et al.  Long-term exposure to CdTe quantum dots causes functional impairments in live cells. , 2007, Langmuir : the ACS journal of surfaces and colloids.

[11]  V. Grassian,et al.  Inhalation Exposure Study of Titanium Dioxide Nanoparticles with a Primary Particle Size of 2 to 5 nm , 2006, Environmental health perspectives.

[12]  D. Romberger,et al.  Ultrafine carbon black particles inhibit human lung fibroblast-mediated collagen gel contraction. , 2003, American journal of respiratory cell and molecular biology.

[13]  M. Dickerhof,et al.  NanoCare : Health related aspects of nanomaterials , 2009 .

[14]  Alexandra Kroll,et al.  Testing Metal‐Oxide Nanomaterials for Human Safety , 2010 .

[15]  J. Paulauskis,et al.  Endocytosis of ultrafine particles by A549 cells. , 2001, American journal of respiratory cell and molecular biology.

[16]  Zhi Pan,et al.  Adverse effects of titanium dioxide nanoparticles on human dermal fibroblasts and how to protect cells. , 2009, Small.

[17]  Wei Li,et al.  Potential neurological lesion after nasal instillation of TiO(2) nanoparticles in the anatase and rutile crystal phases. , 2008, Toxicology letters.

[18]  Hari Singh Nalwa,et al.  Nanotechnology and health safety--toxicity and risk assessments of nanostructured materials on human health. , 2007, Journal of nanoscience and nanotechnology.

[19]  Alexandra Kroll,et al.  Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays , 2011, Particle and Fibre Toxicology.

[20]  Ronni Wolf,et al.  Percutaneous absorption and delivery systems3 , 2001 .

[21]  A. Seligman,et al.  The determination of lactic dehydrogenase with a tetrazolium salt. , 1960, Analytical biochemistry.

[22]  M. Gehlen,et al.  Fluorescence Modulation of Acridine and Coumarin Dyes by Silver Nanoparticles , 2007, Journal of Fluorescence.

[23]  David S. Ensor,et al.  Endotoxin contamination of engineered nanomaterials , 2010, Nanotoxicology.

[24]  P. M. Williams,et al.  Confounding experimental considerations in nanogenotoxicology. , 2009, Mutagenesis.

[25]  Nancy A. Monteiro-Riviere,et al.  Challenges for assessing carbon nanomaterial toxicity to the skin , 2006 .

[26]  Agnes B Kane,et al.  Adsorption of essential micronutrients by carbon nanotubes and the implications for nanotoxicity testing. , 2008, Small.

[27]  G. Bartosz,et al.  2,7‐DICHLOROFLUORESCIN OXIDATION AND REACTIVE OXYGEN SPECIES: WHAT DOES IT MEASURE? , 2000, Cell biology international.

[28]  Peter Wick,et al.  The reliability and limits of the MTT reduction assay for carbon nanotubes-cell interaction , 2007 .

[29]  H. Krug,et al.  Oops they did it again! Carbon nanotubes hoax scientists in viability assays. , 2006, Nano letters.

[30]  Albert Duschl,et al.  The suitability of different cellular in vitro immunotoxicity and genotoxicity methods for the analysis of nanoparticle-induced events , 2010, Nanotoxicology.

[31]  Yong-Keun Lee,et al.  Influence of TiO2 nanoparticles on the optical properties of resin composites. , 2009, Dental materials : official publication of the Academy of Dental Materials.

[32]  David M. Brown,et al.  Proinflammogenic Effects of Low-Toxicity and Metal Nanoparticles In Vivo and In Vitro: Highlighting the Role of Particle Surface Area and Surface Reactivity , 2007, Inhalation toxicology.

[33]  M. Saboungi,et al.  Mesoporous silica nanoparticles enhance MTT formazan exocytosis in HeLa cells and astrocytes. , 2009, Toxicology in vitro : an international journal published in association with BIBRA.

[34]  J. Veranth,et al.  Cytokine responses of human lung cells (BEAS-2B) treated with micron-sized and nanoparticles of metal oxides compared to soil dusts , 2007, Particle and Fibre Toxicology.

[35]  Vicki Stone,et al.  Relating the physicochemical characteristics and dispersion of multiwalled carbon nanotubes in different suspension media to their oxidative reactivity in vitro and inflammation in vivo , 2010, Nanotoxicology.

[36]  Alexandra Kroll,et al.  Current in vitro methods in nanoparticle risk assessment: limitations and challenges. , 2009, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V.

[37]  David M. Brown,et al.  Interaction between nanoparticles and cytokine proteins: impact on protein and particle functionality , 2010, Nanotechnology.

[38]  Aravind Subramanian,et al.  Perturbational profiling of nanomaterial biologic activity , 2008, Proceedings of the National Academy of Sciences.

[39]  O A Sadik,et al.  Sensors as tools for quantitation, nanotoxicity and nanomonitoring assessment of engineered nanomaterials. , 2009, Journal of environmental monitoring : JEM.

[40]  J. West,et al.  Correlating nanoscale titania structure with toxicity: a cytotoxicity and inflammatory response study with human dermal fibroblasts and human lung epithelial cells. , 2006, Toxicological sciences : an official journal of the Society of Toxicology.

[41]  J. Schnekenburger,et al.  Not ready to use – overcoming pitfalls when dispersing nanoparticles in physiological media , 2008 .

[42]  G Chambers,et al.  Single walled carbon nanotubes induce indirect cytotoxicity by medium depletion in A549 lung cells. , 2008, Toxicology letters.

[43]  Morteza Mahmoudi,et al.  A new approach for the in vitro identification of the cytotoxicity of superparamagnetic iron oxide nanoparticles. , 2010, Colloids and surfaces. B, Biointerfaces.

[44]  V. Prachayasittikul,et al.  Zinc ions bound to chimeric His4/lactate dehydrogenase facilitate decarboxylation of oxaloacetate. , 1993, Protein Engineering.

[45]  R. Hamel,et al.  Carbon black and titanium dioxide nanoparticles induce pro-inflammatory responses in bronchial epithelial cells: Need for multiparametric evaluation due to adsorption artifacts , 2009, Inhalation toxicology.

[46]  M. Lag,et al.  Differential binding of cytokines to environmentally relevant particles: a possible source for misinterpretation of in vitro results? , 2008, Toxicology letters.