Effect of Particle Size of Zinc Oxides on Cytotoxicity and Cell Permeability in Caco-2 Cells

  • Received : 2011.02.21
  • Accepted : 2011.03.30
  • Published : 2011.06.30


The cell permeability and cytotoxic effects of different-sized zinc oxide (ZnO) particles were investigated using a human colorectal adenocarcinoma cell line called Caco-2. Morphological observation by scanning electron microscopy revealed that three zinc oxides with different mean particle sizes (ZnO-1, 20 nm; ZnO-2, 90~200 nm; ZnO-3, $1\sim5\;{\mu}m$) tended to aggregate, particularly in the case of ZnO-1. When cytotoxicities of all three sizes of zinc oxide particles were measured at concentration ranges of $1\sim1000\;{\mu}g$/mL, significant decreases in cell viability were observed at concentrations of $50\;{\mu}g$/mL and higher. Among the three zinc oxides, ZnO-1 showed the lowest viability at $50\;{\mu}g$/mL in Caco-2 cells, followed by ZnO-2 and ZnO-3. The permeate concentration of ZnO-1 from the apical to the basolateral side in the Caco-2 model system after four hours was about three-fold higher than that of either ZnO-2 or ZnO-3. These results demonstrated that ZnO-1, with a 20 nm mean particle size, had poorer viability and better permeability in Caco-2 cells than ZnO-2 and ZnO-3.


  1. Tarver T. 2006. Food nanotechnology. Food Technol 60: 22-26.
  2. Park B. 2009. Nanotechnology for food safety. Cereal Foods World 54: 158-162.
  3. Bouwmeester H, Dekkers S, Noordam MY, Hagens WI, Bulder AS, de Heer C, ten Voorde SE, Wijnhoven SW, Marvin HJ, Sips AJ. 2009. Review of health safety aspects of nanotechnologies in food production. Regul Toxicol Pharmacol 53: 52-62.
  4. Oberdörster G, Oberdörster E, Oberdörster J. 2005. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 113: 823-839.
  5. Hoet P, Bruske-Hohlfeld I, Salata O. 2004. Nanoparticlesknown and unknown health risks. J Nanobiotechnol 2: 12-26.
  6. Powers KW, Palazuelos M, Moudgil BM, Roberts SM. 2007. Characterization of the size, shape, and state of dispersion of nanoparticles for toxicological studies. Nanotoxicology 1: 42-51.
  7. Stefanidou M, Maravelias C, Dona A, Spiliopolou C. 2006. Zinc: a multipurpose trace element. Arch Toxicol 80: 1-9.
  8. Rosado JL. 2003. Zinc and copper: proposed fortification levels and recommended zinc compounds. J Nutr 133: 2985S-2989S.
  9. Friends of the Earth, Australia, Europe and U.S.A. 2008. Out of the laboratory and on to our plates, nanotechnology in food & agriculture. A report. p 1-63.
  10. Shah P, Jogani V, Bagchi T, Misra A. 2006. Role of Caco-2 cell monolayers in prediction of intestinal drug absorption. Biotech Prog 22: 186-198.
  11. Dobrovolskaia M. 2007. Immunological properties of engineered nanomaterials. Nature Nanotechnol 2: 469-478.
  12. Khan R, Kaushik A, Solanki RR, Ansari AA, Pandey MM, Malhotra BD. 2008. Zinc oxide nanoparticles-chitosan composite film for cholesterol biosensor. Anal Chim Acta 616: 207-213.
  13. Opanasopit P, Aumklad P, Kowapradit J, Ngawhiranpat T, Apirakaramwong A, Rojanarata T. 2007. Effect of salt form and molecular weight of chitosan on in vitro permeability enhancement in intestinal epithelial cells (Caco-2). Pharmaceut Develop Technol 12: 447-455.
  14. Rekha MR, Sharma CP. 2009. Synthesis and evaluation of lauryl succinyl chitosan particles towards oral insulin delivery and absorption. J Control Release 135: 144-151.
  15. Chen FZ, Zhang R, Yuan F, Qin X, Wang M, Huang Y. 2008. In vitro and in vivo study of N-trimethyl chitosan nanoparticles for oral protein delivery. Int J Pharm 49: 226-233.
  16. Lin YH, Chung CK, Chen CT, Liang HF, Chen SC, Sung HW. 2005. Preparation of nanoparticles composed of chitosan/poly-$\gamma$-glutamic acid and evaluation of their permeability through Caco-2 cells. Biomacromolecules 6: 1104-1112.
  17. Markowska M, Oberle R, Juzwin S, Hsu CP, Gryszkiewicz M, Streeter AJ. 2001. Optimizing Caco-2 cell monolayers to increase throughput in drug intestinal absorption analysis. J Pharmacol Toxicol Methods 46: 51-55.
  18. McCall KA, Fierke CA. 2000. Colorimetric and fluorimetric assays to quantitate micromolar concentrations of transition metals. Anal Biochem 284: 307-315.
  19. Tantra R, Tompkins J, Quincey P. 2010. Characterisation of the de-agglomeration effects of bovine serum albumin on nanoparticles in aqueous suspension. Colloids Surf B: Biointerfaces 75: 275-281.
  20. Kato H, Suzuki M, Fujita K, Horie M, Endoh S, Yoshida Y, Iwahashi H, Takahashi K, Nakamura A, Kinugasa S. 2009. Reliable size determination of nanoparticles using dynamic light scattering method for in vitro toxicology assessment. Toxicol in Vitro 23: 927-934.
  21. Zhang Y, Chen Y, Westerhoff P, Hristovski K, Crittenden JC. 2008. Stability of commercial metal oxide nanoparticles in water. Water Res 42: 2204-2212.
  22. Murdock RC, Braydich-Stolle L, Schrand AM, Schlager JJ, Hussain SM. 2008. Characterization of nanomaterial dispersion in solution prior to in vitro exposure using dynamic light scattering technique. Toxicol Sci 101: 239-253.
  23. Yang H, Liu C, Yang D, Zhang H, Xi Z. 2008. Comparative study of cytotoxicity, oxidative stress and genotoxicity induced by four typical nanomaterials: the role of particle size, shape and composition. J Appl Toxicol 29: 69-78.
  24. Heng BC, Zhao X, Xiong S, Ng KW, Boey FYC, Loo JSC. 2010. Toxicity of zinc oxide (ZnO) nanoparticles on human bronchial epithelial cells (BEAS-2B) is accentuated by oxidative stress. Food Chem Toxicol 48: 1762-1766.
  25. Hackenberg S, Scherzed A, Technau A, Kessler M, Froelich K, Ginzkey C, Koehler C, Burghartz M, Hagen R, Kleinsasser N. 2011. Cytotoxic, genotoxic and pro-inflammatory effects of zinc oxide nanoparticles in human nasal mucosa cells in vitro. Toxicol in Vitro 25: 657-663.
  26. Nair S, Sasidharan A, Rani VVD, Menon D, Nair S, Manzoor K, Raina S. 2009. Role of size scale of ZnO nanoparticles and microparticles on toxicity toward bacteria and osteoblast cancer cells. J Mater Sci Mater Med 20: S235-241.
  27. Colon G, Ward BC, Webster TJ. 2006. Increased osteoblast and decreased Staphylococcus epidermidis functions on nanophase ZnO and $TiO_2$. J Biomed Mater Res 78A: 595-604.
  28. Yamashita S, Furubayashi T, Kataoka M, Sakane T, Sezaki H, Tokuda H. 2000. Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells. Eur J Pharm Sci 10: 195-204.
  29. Jia L, Wong H, Cerna C, Weitman SD. 2002. Effect of nanonization on absorption of 301029: ex vivo and in vivo pharmacokinetic correlations determined by liquid chromatography/mass spectrometry. Pharm Res 19: 1091-1096.