Internalisation of engineered nanoparticles into mammalian cells in vitro: influence of cell type and particle properties


Cellular internalisation of industrial engineered nanoparticles is undesired and a reason for concern. Here we investigated and compared the ability of seven different mammalian cell cultures in vitro to incorporate six kinds of engineered nanoparticles, focussing on the role of cell type and particle properties in particle uptake. Uptake was examined using light and electron microscopy coupled with energy dispersive X-ray spectroscopy (EDX) for particle element identification. Flow cytometry was applied for semi-quantitative analyses of particle uptake and for exploring the influence on uptake by the phagocytosis inhibitor Cytochalasin D (CytoD). All particles studied were found to enter each kind of cultured cells. Yet, particles were never found within cell nuclei. The presence of the respective particles within the cells was confirmed by EDX. Live-cell imaging revealed the time-dependent process of internalisation of technical nanoparticles, which was exemplified by tungsten carbide particle uptake into the human skin cells, HaCaT. Particles were found to co-localise with lysosomal structures within the cells. The incorporated nanoparticles changed the cellular granularity, as measured by flow cytometry, already after 3 h of exposure in a particle specific manner. By correlating particle properties with flow cytometry data, only the primary particle size was found to be a weakly influential property for particle uptake. CytoD, an inhibitor of actin filaments and therewith of phagocytosis, significantly inhibited the internalisation of particle uptake in only two of the seven investigated cell cultures. Our study, therefore, supports the notion that nanoparticles can enter mammalian cells quickly and easily, irrespective of the phagocytic ability of the cells.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 99

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9


  1. Ashwood P, Thompson RP, Powell JJ (2007) Fine particles that adsorb lipopolysaccharide via bridging calcium cations may mimic bacterial pathogenicity towards cells. Exp Biol Med 232:107–117

  2. Bao L, Chen S, Wu L, Hei TK, Wu Y, Yu Z, Xu A (2007) Mutagenicity of diesel exhaust particles mediated by cell–particle interaction in mammalian cells. Toxicology 229:91–100

  3. Bastian S, Busch W, Kühnel D, Springer A, Meißner T, Holke R, Scholz S, Iwe M, Pompe W, Gelinsky M, Potthoff A, Richter V, Ikonomidou C, Schirmer K (2009) Toxicity of tungsten carbide and cobalt-doped tungsten carbide nanoparticles in mammalian cells in vitro. Environ Health Perspect 117:530–536

  4. Baun A, Sorensen SN, Rasmussen RF, Hartmann NB, Koch CB (2008) Toxicity and bioaccumulation of xenobiotic organic compounds in the presence of aqueous suspensions of aggregates of nano-C(60). Aquat Toxicol 86:379–387

  5. Bhattacharya K, Davoren M, Boertz J, Schins R, Hoffmann E, Dopp E (2009) Titanium dioxide nanoparticles induce oxidative stress and DNA-adduct formation but not DNA-breakage in human lung cells. Part Fibre Toxicol 6:17

  6. Busch W, Kühnel D, Schirmer K, Scholz S (2010) Tungsten carbide cobalt nanoparticles exert hypoxia-like effects on the gene expression level in human keratinocytes. BMC Genomics 11:65

  7. Cedervall T, Lynch I, Lindman S, Berggard T, Thulin E, Nilsson H, Dawson KA, Linse S (2007) Understanding the nanoparticle–protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Proc Natl Acad Sci USA 104:2050–2055

  8. Chang GH, Barbaro NM, Pieper RO (2000) Phosphatidylserine-dependent phagocytosis of apoptotic glioma cells by normal human microglia, astrocytes, and glioma cells. Neuro Oncol 2:174–183

  9. Chen M, von Mikecz A (2005) Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO2 nanoparticles. Exp Cell Res 305:51–62

  10. Chithrani BD, Ghazani AA, Chan WC (2006) Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett 6:662–668

  11. Deguchi S, Yamazaki T, Mukai SA, Usami R, Horikoshi K (2007) Stabilization of C60 nanoparticles by protein adsorption and its implications for toxicity studies. Chem Res Toxicol 20:854–858

  12. Geiser M, Rothen-Rutishauser B, Kapp N, Schurch S, Kreyling W, Schulz H, Semmler M, Im Hof V, Heyder J, Gehr P (2005) Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells. Environ Health Perspect 113:1555–1560

  13. Gojova A, Guo B, Kota RS, Rutledge JC, Kennedy IM, Barakat AI (2007) Induction of inflammation in vascular endothelial cells by metal oxide nanoparticles: effect of particle composition. Environ Health Perspect 115:403–409

  14. Gratton SE, Ropp PA, Pohlhaus PD, Luft JC, Madden VJ, Napier ME, DeSimone JM (2008) The effect of particle design on cellular internalization pathways. Proc Natl Acad Sci USA 105:11613–11618

  15. Haberzettl P, Duffin R, Kramer U, Hohr D, Schins RP, Borm PJ, Albrecht C (2007) Actin plays a crucial role in the phagocytosis and biological response to respirable quartz particles in macrophages. Arch Toxicol 81:459–470

  16. Harush-Frenkel O, Rozentur E, Benita S, Altschuler Y (2008) Surface charge of nanoparticles determines their endocytic and transcytotic pathway in polarized MDCK cells. Biomacromolecules 9:435–443

  17. Hildebrand H, Kühnel D, Potthoff A, Mackenzie K, Springer A, Schirmer K (2009) Evaluating the cytotoxicity of palladium/magnetite nano-catalysts intended for wastewater treatment. Environ Pollut 158:65–73

  18. Johnsen S, Widder EA (1999) The physical basis of transparency in biological tissue: ultrastructure and the minimization of light scattering. J Theor Biol 199:181–198

  19. Kaksonen M, Toret CP, Drubin DG (2006) Harnessing actin dynamics for clathrin-mediated endocytosis. Nat Rev Mol Cell Biol 7:404–414

  20. Kanno S, Furuyama A, Hirano S (2007) A murine scavenger receptor MARCO recognizes polystyrene nanoparticles. Toxicol Sci 97:398–406

  21. Kreuter J, Shamenkov D, Petrov V, Ramge P, Cychutek K, Koch-Brandt C, Alyautdin R (2002) Apolipoprotein-mediated transport of nanoparticle-bound drugs across the blood–brain barrier. J Drug Target 10:317–325

  22. Kühnel D, Busch W, Meißner T, Springer A, Potthoff A, Richter V, Gelinsky M, Scholz S, Schirmer K (2009) Agglomeration of tungsten carbide nanoparticles in exposure medium does not prevent uptake and toxicity toward a rainbow trout gill cell line. Aquat Toxicol 93:91–99

  23. 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 39:9370–9376

  24. Long TC, Saleh N, Tilton RD, Lowry GV, Veronesi B (2006) Titanium dioxide (P25) produces reactive oxygen species in immortalized brain microglia (BV2): implications for nanoparticle neurotoxicity. Environ Sci Technol 40:4346–4352

  25. Lundqvist M, Stigler J, Elia G, Lynch I, Cedervall T, Dawson KA (2008) Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc Natl Acad Sci USA 105:14265–14270

  26. Maynard AD, Kuempel ED (2005) Airborne nanostructured particles and occupational health. J Nanopart Res 7:587–614

  27. Meißner T, Potthoff A, Richter V (2009) Physico-chemical characterization in the light of toxicological effects. Inhal Toxicol 21(Suppl 1):35–39

  28. Meißner T, Kühnel D, Busch W, Oswald S, Richter V, Michaelis A, Schirmer K, Potthoff A (2010) Physical–chemical characterization of tungsten carbide nanoparticles as a basis for toxicological investigations. Nanotoxicology 4:196–206

  29. Naß R, Albayrak S, Aslan M, Schmidt H (1994) Colloidal processing and sintering of nano-scale TiN. In: 5th international conference in ceramic processing, science and technology. Friedrichshafen, Germany

  30. Oh JM, Choi SJ, Lee GE, Kim JE, Choy JH (2009) Inorganic metal hydroxide nanoparticles for targeted cellular uptake through clathrin-mediated endocytosis. Chem Asian J 4:67–73

  31. Papis E, Rossi F, Raspanti M, Dalle-Donne I, Colombo G, Milzani A, Bernardini G, Gornati R (2009) Engineered cobalt oxide nanoparticles readily enter cells. Toxicol Lett 189:253–259

  32. Potthoff A, Meißner T, Richter V, Busch W, Kühnel D, Bastian S, Iwe M, Springer A (2009) Evaluation of health risks of nanoparticles—a contribution to a sustainable development of nanotechnology. Solid State Phenom 151:183–189

  33. Razani B, Woodman SE, Lisanti MP (2002) Caveolae: from cell biology to animal physiology. Pharmacol Rev 54:431–467

  34. Rejman J, Oberle V, Zuhorn IS, Hoekstra D (2004) Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J 377:159–169

  35. Reyes L, Davidson MK, Thomas LC, Davis JK (1999) Effects of Mycoplasma fermentans incognitus on differentiation of THP-1 cells. Infect Immun 67:3188–3192

  36. Roldan A, Gogg S, Ferrini M, Schillaci R, De Nicola AF (1997) Glucocorticoid regulation of in vitro astrocyte phagocytosis. Biocell 21:83–89

  37. Schubert W, Frank PG, Razani B, Park DS, Chow CW, Lisanti MP (2001) Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo. J Biol Chem 276:48619–48622

  38. Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43

  39. Stearns R, Paulauskis J, Godleski J (2001) Endocytosis of ultrafine particles by A549 cells. Am J Respir Cell Mol Biol 24:108–115

  40. Stoeger T, Takenaka S, Frankenberger B, Ritter B, Karg E, Maier K, Schulz H, Schmid O (2009) Deducing in vivo toxicity of combustion-derived nanoparticles from a cell-free oxidative potency assay and metabolic activation of organic compounds. Environ Health Perspect 117:54–60

  41. Stringer B, Imrich A, Kobzik L (1995) Flow cytometric assay of lung macrophage uptake of environmental particulates. Cytometry 20:23–32

  42. Thomas EW (1992) Brain macrophages: evaluation of microglia and their functions. Brain Res Rev 17:61–74

  43. Xia T, Kovochich M, Brant J, Hotze M, Sempf J, Oberley T, Sioutas C, Yeh JI, Wiesner MR, Nel AE (2006) Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm. Nano Lett 6:1794–1807

  44. Yumoto R, Nishikawa H, Okamoto M, Katayama H, Nagai J, Takano M (2006) Clathrin-mediated endocytosis of FITC-albumin in alveolar type II epithelial cell line RLE-6TN. Am J Physiol Lung Cell Mol Physiol 290:L946–L955

  45. Zhang LW, Monteiro-Riviere NA (2009) Mechanisms of quantum dot nanoparticle cellular uptake. Toxicol Sci 110:138–155

Download references


This research was supported by the German Federal Ministry for Education and Research (BMBF) within the INOS project (Identification and Evaluation of Health and Environmental Effects of Technical Particles at the Nanoscale; Grant #03X0013C), WB was additionally supported by the Max Buchner Forschungsstiftung and by the Helmholtz Impulse and Networking Fund through Helmholtz Interdisciplinary Graduate School for Environmental Research (HIGRADE). Stefan Scholz (Department Bioanalytical Ecotoxicology, UFZ) is acknowledged for constructive discussions.

Author information

Correspondence to Wibke Busch.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Movie of HaCaT cells incorporating WCL particles. Photographs of HaCaT cells, exposed to 30 μg/mL WCL nanoparticles, were taken every 10 min during a time period of 2 days using an inverse microscope (Leica DMI 4000B, magnification 200×). The pictures are shown as a time flow movie in the Additional file 2. (MPEG 21380 kb)

Cell culture protocols. The cell lines and isolated cells used in the study and their standard treatments are explained in the Additional file 1. A table shows the numbers of seeded cells for the different experiments. (DOC 39 kb)

Movie of HaCaT cells incorporating WCL particles. Photographs of HaCaT cells, exposed to 30 μg/mL WCL nanoparticles, were taken every 10 min during a time period of 2 days using an inverse microscope (Leica DMI 4000B, magnification 200×). The pictures are shown as a time flow movie in the Additional file 2. (MPEG 21380 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Busch, W., Bastian, S., Trahorsch, U. et al. Internalisation of engineered nanoparticles into mammalian cells in vitro: influence of cell type and particle properties. J Nanopart Res 13, 293–310 (2011).

Download citation


  • Engineered nanoparticles
  • Uptake
  • Mammalian cells
  • Flow cytometry
  • Microscopy
  • Cytochalasin D
  • Health and safety