Dose assessment of SiC nanoparticle dispersions during in vitro assays

  • Jorge Mejia
  • Jean-Pascal Piret
  • Florence Noël
  • Bernard Masereel
  • Olivier Toussaint
  • Stéphane Lucas
Research Paper


Here, we show that key physicochemical parameters of commercial Silicon Carbide nanoparticles, such as the primary particles of about 53 nm in size, the agglomerates size, and the surface composition, are considerably modified with respect to the pristine conditions, during in vitro assessment. The use of sample conditioning stages, such as the pre-dispersion in aqueous media and the subsequent dispersion in a culture medium specific to the in vitro assay, produce modifications as the absorption of N, C, and O, from the culture medium, in the nanoparticles surface. Our results show that the sedimented dose, fraction of sedimented NPs during incubation and consequently in contact with cells seeded at the bottom, of Silicon Carbide nanoparticles can be measured from the particle size distribution obtained using a centrifugal liquid sedimentation technique. It is underlined that the variations observed in the physicochemical properties are related to the in vitro assay conditions. Culture medium and incubation time are found to influence the most the sedimented dose and consequently the cells dose uptake.


SiC nanoparticles Size distribution Surface composition In vitro assays Dose assessment Sedimented dose 



This work is supported by the “Direction Générale des Technologies de la Recherche et de l’Energie” (DGTRE) of the Walloon Region of Belgium (Nanotoxico Project, RW/FUNDP research convention No. 516252). O. Toussaint is a Research Associate of the Belgian FRS/FNRS. The authors acknowledge financial support from the European Union under the Framework 7 program, Qnano (INFRASTRUCTURE-2010-1-262163).


  1. Arora S, Rajwade JM, Paknikar KM (2012) Nanotoxicology and in vitro studies: the need of the hour. YTAAP 258(2):151–165. doi: 10.1016/j.taap.2011.11.010 Google Scholar
  2. ATCC Cultures and Products (2011) Accessed 10 June 2012
  3. Barillet S, Jugan M-L, Simon-Deckers A, Leconte Y, Herlin-Boime N, Mayne-l’Hermite M, Reynaud C, Carriere M (2009) SiC nanoparticles cyto- and genotoxicity to Hep-G2 cells. J Phys Conf Ser 170:012016CrossRefGoogle Scholar
  4. Barillet S, Jugan ML, Laye M, Leconte Y, Herlin-Boime N, Reynaud C, Carrière M (2010a) In vitro evaluation of SiC nanoparticles impact on A549 pulmonary cells: cyto-, genotoxicity and oxidative stress. Toxicol Lett 198(3):324–330CrossRefGoogle Scholar
  5. Barillet S, Simon-Deckers A, Herlin-Boime N, Mayne-L’Hermite M, Reynaud C, Cassio D, Gouget B, Carrière M (2010b) Toxicological consequences of TiO, SiC nanoparticles and multi-walled carbon nanotubes exposure in several mammalian cell types: an in vitro study. J Nanopart Res 12(1):61–73. doi: 10.1007/s11051-009-9694-y CrossRefGoogle Scholar
  6. Bihari P, Vippola M, Schultes S, Praetner M, Khandoga A, Reichel C, Coester C, Tuomi T, Rehberg M, Krombach F (2008) Optimized dispersion of nanoparticles for biological in vitro and in vivo studies. Part Fibre Toxicol 5(1):14CrossRefGoogle Scholar
  7. Bruch J, Rehn B, Song W, Gono E, Malkusch W (1993) Toxicological investigations on silicon carbide. 2. In vitro cell tests and long term injection tests. Br J Ind Med 50(9):807–813Google Scholar
  8. Christian P, Kammer F, Baalousha M, Hofmann T (2008) Nanoparticles: structure, properties, preparation and behaviour in environmental media. Ecotoxicology 17(5):326–343. doi: 10.1007/s10646-008-0213-1 CrossRefGoogle Scholar
  9. Cohen J, DeLoid G, Pyrgiotakis G, Demokritou P (2012) Interactions of engineered nanomaterials in physiological media and implications for in vitro dosimetry. Nanotoxicology 7(4):1–15. doi: 10.3109/17435390.2012.666576 Google Scholar
  10. Dhawan A, Sharma V (2010) Toxicity assessment of nanomaterials: methods and challenges. Anal Bioanal Chem 398(2):589–605CrossRefGoogle Scholar
  11. Dickson MA, Hahn WC, Ino Y, Ronfard V, Wu JY, Weinberg RA, Louis DN, Li FP, Rheinwald JG (2000) Human keratinocytes that express hTERT and also bypass a p16INK4a-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol 20(4):1436–1447. doi: 10.1128/mcb.20.4.1436-1447.2000 CrossRefGoogle Scholar
  12. Fubini B, Ghiazza M, Fenoglio I (2010) Physico-chemical features of engineered nanoparticles relevant to their toxicity. Nanotoxicology 4(4):347–363CrossRefGoogle Scholar
  13. Guevara-Lora I, Czosnek C, Smycz A, Janik J, Kozik A (2009) SiC nanoparticles as potential carriers for biologically active substances. J Phys: Conf Ser 146(1):012022CrossRefGoogle Scholar
  14. Hinderliter P, Minard K, Orr G, Chrisler W, Thrall B, Pounds J, Teeguarden J (2010) ISDD: a computational model of particle sedimentation, diffusion and target cell dosimetry for in vitro toxicity studies. Part Fibre Toxicol 7(1):36CrossRefGoogle Scholar
  15. Iolitec (2007) Silicon(IV)carbide–Technical data sheet. Accessed 5 Aug 2012
  16. Jiang J, Oberdörster G, Biswas P (2009) Characterization of size, surface charge, and agglomeration state of nanoparticle dispersions for toxicological studies. J Nanopart Res 11(1):77–89CrossRefGoogle Scholar
  17. Jones CF, Grainger DW (2009) In vitro assessments of nanomaterial toxicity. Adv Drug Deliv Rev 61(6):438–456CrossRefGoogle Scholar
  18. Kato H, Fujita K, Horie M, Suzuki M, Nakamura A, Endoh S, Yoshida Y, Iwahashi H, Takahashi K, Kinugasa S (2010) Dispersion characteristics of various metal oxide secondary nanoparticles in culture medium for in vitro toxicology assessment. Toxicol In Vitro 24(3):1009–1018CrossRefGoogle Scholar
  19. Laloy J, Robert S, Marbehant C, Mullier F, Mejia J, Piret J-P, Lucas S, Chatelain B, Dogné J-M, Toussaint O, Masereel B, Rolin S (2012) Validation of the calibrated thrombin generation test (cTGT) as the reference assay to evaluate the procoagulant activity of nanomaterials. Nanotoxicology 6(2):213–232. doi: 10.3109/17435390.2011.569096 CrossRefGoogle Scholar
  20. Limbach LK, Li Y, Grass RN, Brunner TJ, Hintermann MA, Muller M, Gunther D, Stark WJ (2005) Oxide nanoparticle uptake in human lung fibroblasts: effects of particle size, agglomeration, and diffusion at low concentrations. Environ Sci Technol 39(23):9370–9376. doi: 10.1021/es051043o CrossRefGoogle Scholar
  21. Lison D, Thomassen LCJ, Rabolli V, Gonzalez L, Napierska D, Seo JW, Kirsch-Volders M, Hoet P, Kirschhock CEA, Martens JA (2008) Nominal and effective dosimetry of silica nanoparticles in cytotoxicity assays. J Toxicol Sci 104(1):155–162. doi: 10.1093/toxsci/kfn072 CrossRefGoogle Scholar
  22. Lozano O, Mejia J, Masereel B, Toussaint O, Lison D, Lucas S (2012a) Development of a PIXE analysis method for the determination of the biopersistence of SiC and TiC nanoparticles in rat lungs. Nanotoxicology 6(3):263–271. doi: 10.3109/17435390.2011.572301 CrossRefGoogle Scholar
  23. Lozano O, Mejia J, Tabarrant T, Masereel B, Dogné J-M, Toussaint O, Lucas S (2012b) Quantification of nanoparticles in aqueous food matrices using particle-induced X-ray emission. Anal Bioanal Chem 403(10):2835–2841. doi: 10.1007/s00216-012-5895-9 CrossRefGoogle Scholar
  24. Maiorano G, Sabella S, Sorce B, Brunetti V, Malvindi MA, Cingolani R, Pompa PP (2010) Effects of cell culture media on the dynamic formation of protein-nanoparticle complexes and influence on the cellular response. ACS Nano 4(12):7481–7491CrossRefGoogle Scholar
  25. Mejia J, Tichelaar F, Saout C, Toussaint O, Bernard M, Mekhalif Z, Lucas S, Delhalle J (2011) Effects of the dispersion methods in Pluronic F108 on the size and the surface composition of MWCNTs and their implications in toxicology assessment. J Nanopart Res 13(2):655–667CrossRefGoogle Scholar
  26. Mejia J, Valembois V, Piret J-P, Tichelaar F, Bernard M, Toussaint O, Delhalle J, Mekhalif Z, Lucas S (2012) Are stirring and sonication pre-dispersion methods equivalent for the in vitro toxicology evaluation of SiC and TiC? J Nanopart Res 14(4):815–833. doi: 10.1007/s11051-012-0815-7 CrossRefGoogle Scholar
  27. Merkus HG (2009) Particle size measurements–fundamentals, practice, quality. Particle technology series, vol 17. Springer Netherlands, Netherlands, pp 329–334. doi: 978-1-4020-9016-5 Google Scholar
  28. Monopoli MP, Walczyk D, Campbell A, Elia G, Lynch I, Baldelli Bombelli F, Dawson KA (2011) Physical-chemical aspects of protein corona: relevance to in vitro and in vivo biological impacts of nanoparticles. J Am Chem Soc 133(8):2525–2534CrossRefGoogle Scholar
  29. Piret JP, Detriche S, Vigneron R, Vankoningsloo S, Rolin S, Mejia Mendoza J, Masereel B, Lucas S, Delhalle J, Luizi F, Saout C, Toussaint O (2010) Dispersion of multi-walled carbon nanotubes in biocompatible dispersants. J Nanopart Res 12(1):75–82CrossRefGoogle Scholar
  30. Pourchez J, Forest V, Boumahdi N, Boudard D, Tomatis M, Fubini B, Herlin-Boime N, Leconte Y, Guilhot B, Cottier M, Grosseau P (2012) In vitro cellular responses to silicon carbide nanoparticles: impact of physico-chemical features on pro-inflammatory and pro-oxidative effects. J Nanopart Res 14(10):1–12. doi: 10.1007/s11051-012-1143-7 CrossRefGoogle Scholar
  31. Singh B, Jena J, Besra L, Bhattacharjee S (2007) Dispersion of nano-silicon carbide (SiC) powder in aqueous suspensions. J Nanopart Res 9(5):797–806CrossRefGoogle Scholar
  32. Stone V, Johnston H, Schins RPF (2009) Development of in vitro systems for nanotoxicology: methodological considerations. Crit Rev Toxicol 39(7):613–626. doi: 10.1080/10408440903120975 CrossRefGoogle Scholar
  33. Svensson I, Artursson E, Leanderson P, Berglind R, Lindgren F (1997) Toxicity in vitro of some silicon carbides and silicon nitrides: whiskers and powders. Am J Ind Med 31(3):335–343. doi: 10.1002/(SICI)1097-0274(199703)31:3<335:AID-AJIM10>3.0.CO;2-1 CrossRefGoogle Scholar
  34. Teeguarden JG, Hinderliter PM, Orr G, Thrall BD, Pounds JG (2007) Particokinetics in vitro: dosimetry considerations for in vitro nanoparticle toxicity assessments. J Toxicol Sci 95(2):300–312. doi: 10.1093/toxsci/kfl165 Google Scholar
  35. Vankoningsloo S, Piret J-P, Saout C, Noel F, Mejia J, Zouboulis CC, Delhalle J, Lucas S, Toussaint O (2010) Cytotoxicity of multi-walled carbon nanotubes in three skin cellular models: effects of sonication, dispersive agents and corneous layer of reconstructed epidermis. Nanotoxicology 4(1):84–97. doi: 10.3109/17435390903428869 CrossRefGoogle Scholar
  36. Vankoningsloo S, Piret J-P, Saout C, Noel F, Mejia J, Coquette A, Zouboulis CC, Delhalle J, Lucas S, Toussaint O (2011) Pro-inflammatory effects of different MWCNTs dispersions in p16INK4A-deficient telomerase-expressing human keratinocytes but not in human SV-40 immortalized sebocytes. Nanotoxicology 6(1):1–17Google Scholar
  37. Wittmaack K (2011) Novel dose metric for apparent cytotoxicity effects generated by in vitro cell exposure to silica nanoparticles. Chem Res Toxicol 24(2):150–158CrossRefGoogle Scholar
  38. Zouboulis CC, Seltmann H, Neitzel H, Orfanos CE (1999) Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95). J Invest Dermatol 113(6):1011–1020CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Jorge Mejia
    • 1
  • Jean-Pascal Piret
    • 2
  • Florence Noël
    • 2
  • Bernard Masereel
    • 3
  • Olivier Toussaint
    • 2
  • Stéphane Lucas
    • 1
  1. 1.Research Centre for the Physics of Matter and Radiation (LARN-PMR) NARILISUniversity of NamurNamurBelgium
  2. 2.Research Unit in Cellular Biology (URBC) NARILISUniversity of NamurNamurBelgium
  3. 3.Department of Pharmacy NAMEDIC, Namur Thrombosis and Homeostasis Center (NTHC) NARILISUniversity of NamurNamurBelgium

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