Dynamic changes of protein corona compositions on the surface of zinc oxide nanoparticle in cell culture media

  • Vo-Van Giau
  • Yoon-Hee Park
  • Kyu-Hwan Shim
  • Sang-Wook SonEmail author
  • Seong-Soo A. AnEmail author
Research Article


The potential applications of nanomaterials used in nanomedicine as ingredients in drug delivery systems and in other products continue to expand. When nanomaterials are introduced into physiological environments and driven by energetics, they readily associate proteins forming a protein corona (PC) on their surface. This PC could result in an alteration of the nanomaterial’s surface characteristics, affecting their interaction with cells due to conformational changes in adsorbed protein molecules. However, our current understanding of nanobiological interactions is still very limited. Utilizing a liquid chromatography–mass spectroscopy/mass spectroscopy technology and a Cytoscape plugin (ClueGO) approach, we examined the composition of the PC for a set of zinc oxide nanoparticles (ZnONP) from cell culture media typically and further analyzed the biological interaction of identified proteins, respectively. In total, 36 and 33 common proteins were investigated as being bound to ZnONP at 5 min and 60 min, respectively. These proteins were further analyzed with ClueGO, a Cytoscape plugin, which provided gene ontology and the biological interaction processes of identified proteins. Proteins bound to the surface of nanoparticles that may modify the structure, therefore the function of the adsorbed protein could be consequently affect the complicated biological processes.


ZnONPs nanoparticles protein corona ClueGO 


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This research was supported by a National Research Foundation of Korea (NRF) awarded by the Korean government (NRF- 2017R1A2B4012636).


  1. 1.
    Blunk T, Hochstrasser D F, Sanchez J C, Muller B W, Muller R H. Colloidal carriers for intravenous drug targeting: Plasma protein adsorption patterns on surface-modified latex particles evaluated by two-dimensional polyacrylamide gel electrophoresis. Electrophoresis, 1993, 14(12): 1382–1387CrossRefGoogle Scholar
  2. 2.
    Ehrenberg M S, Friedman A E, Finkelstein J N, Oberdorster G, McGrath J L. The influence of protein adsorption on nanoparticle association with cultured endothelial cells. Biomaterials, 2009, 30 (4): 603–610CrossRefGoogle Scholar
  3. 3.
    Lundqvist M, Augustsson C, Lilja M, Lundkvist K, Dahlbäck B, Linse S, Cedervall T. The nanoparticle protein corona formed in human blood or human blood fractions. PLoS One, 2017, 12(4): e0175871CrossRefGoogle Scholar
  4. 4.
    Beduneau A, Ma Z, Grotepas C B, Kabanov A, Rabinow B E, Gong N, Mosley R L, Dou H, Boska M D, Gendelman H E. Facilitated monocyte-macrophage uptake and tissue distribution of superparmagnetic iron-oxide nanoparticles. PLoS One, 2009, 4(2): e4343CrossRefGoogle Scholar
  5. 5.
    Clift M J, Bhattacharjee S, Brown D M, Stone V. The effects of serum on the toxicity of manufactured nanoparticles. Toxicology Letters, 2010, 198(3): 358–365CrossRefGoogle Scholar
  6. 6.
    Khanna P, Ong C, Bay B H, Baeg G H. Nanotoxicity: An interplay of oxidative stress, inflammation and cell death. Nanomaterials (Basel, Switzerland), 2015, 5(3): 1163–1180Google Scholar
  7. 7.
    Lartigue L,Wilhelm C, Servais J, Factor C, Dencausse A, Bacri J C, Luciani N, Gazeau F. Nanomagnetic sensing of blood plasma protein interactions with iron oxide nanoparticles: Impact on macrophage uptake. ACS Nano, 2012, 6(3): 2665–2678CrossRefGoogle Scholar
  8. 8.
    Lunov O, Syrovets T, Loos C, Beil J, Delacher M, Tron K, Nienhaus G U, Musyanovych A, Mailänder V, Landfester K, et al. Differential uptake of functionalized polystyrene nanoparticles by human macrophages and a monocytic cell line. ACS Nano, 2011, 5(3): 1657–1669CrossRefGoogle Scholar
  9. 9.
    Walkey C D, Olsen J B, Song F, Liu R, Liu R, Guo H, Olsen D W, Cohen Y, Emili A, Chan WC. Protein corona fingerprinting predicts the cellular interaction of gold and silver nanoparticles. ACS Nano, 2014, 8(3): 2439–2455CrossRefGoogle Scholar
  10. 10.
    Gu Z, Yang Z, Chong Y, Ge C, Weber J K, Bell D R, Zhou R. Surface curvature relation to protein adsorption for carbon-based nanomaterials. Scientific Reports, 2015, 5(1): 10886CrossRefGoogle Scholar
  11. 11.
    Marucco A, Gazzano E, Ghigo D, Enrico E, Fenoglio I. Fibrinogen enhances the inflammatory response of alveolar macrophages to TiO2, SiO2 and carbon nanomaterials. Nanotoxicology, 2016, 10(1): 1–9Google Scholar
  12. 12.
    Ge C, Du J, Zhao L,Wang L, Liu Y, Li D, Yang Y, Zhou R, Zhao Y, Chai Z, Chen C. Binding of blood proteins to carbon nanotubes reduces cytotoxicity. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(41): 16968–16973CrossRefGoogle Scholar
  13. 13.
    Hajipour M J, Raheb J, Akhavan O, Arjmand S, Mashinchian O, Rahman M, Abdolahad M, Serpooshan V, Laurentj L, Mahmoudi M. Personalized disease-specific protein corona influences the therapeutic impact of graphene oxide. Nanoscale, 2015, 7(19): 8978–8994CrossRefGoogle Scholar
  14. 14.
    Salvati A, Pitek A S, Monopoli M P, Prapainop K, Bombelli F B, Hristov D R, Kelly P M, Åberg C, Mahon E, Dawson K A. Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface. Nature Nanotechnology, 2013, 8(2): 137–143CrossRefGoogle Scholar
  15. 15.
    Deng Z J, Liang M, Monteiro M, Toth I, Minchin R F. Nanoparticleinduced unfolding of fibrinogen promotes Mac-1 receptor activation and inflammation. Nature Nanotechnology, 2011, 6(1): 39–44CrossRefGoogle Scholar
  16. 16.
    Saikia J, Yazdimamaghani M, Hadipour M. S P, Ghandehari H. Differential protein adsorption and cellular uptake of silica nanoparticles based on size and porosity. ACS Applied Materials & Interfaces, 2016, 8(50): 34820–34832Google Scholar
  17. 17.
    Anders C B, Eixenberger J E, Franco N A, Hermann R J, Rainey K D, Chess J J, Punnooseb A, Wingett D G. ZnO nanoparticle preparation route influences surface reactivity, dissolution and cytotoxicity. Environmental Science. Nano, 2018, 5(2): 572–588CrossRefGoogle Scholar
  18. 18.
    Rasmussen J W, Martinez E, Louka P, Denise G,Wingett D G. Zinc oxide nanoparticles for selective destruction of tumor cells and potential for drug delivery applications. Expert Opinion on Drug Delivery, 2010, 7(9): 1063–1077CrossRefGoogle Scholar
  19. 19.
    Scherzad A, Meyer T, Kleinsasser N, Hackenberg S. Molecular mechanisms of zinc oxide nanoparticle-induced genotoxicity short running title: Genotoxicity of ZnO NPs. Materials (Basel), 2017, 10 (12): e1427Google Scholar
  20. 20.
    Singh N, Manshian B, Jenkins G J, Griffiths S M, Williams P M, Maffeis T G, Wright C J, Doak S H. Nanogenotoxicology: The DNA damaging potential of engineered nanomaterials. Biomaterials, 2009, 30(23–24): 3891–3914CrossRefGoogle Scholar
  21. 21.
    Rajput V D, Minkina T M, Behal A, Sushkova S N, Mandzhieva S, Singh R, Gorovtsov A, Tsitsuashvili V S, Purvis W O, Ghazaryan K A, et al. Effects of zinc-oxide nanoparticles on soil, plants, animals and soil organisms: A review. Environmental Nanotechnology, Monitoring & Management, 2018, 9: 76–84CrossRefGoogle Scholar
  22. 22.
    Wahab R, Siddiqui M A, Saquib Q, Dwivedi S, Ahmad J, Musarrat J, Al-Khedhairy A A, Shin H S. ZnO nanoparticles induced oxidative stress and apoptosis in HepG2 and MCF-7 cancer cells and their antibacterial activity. Colloids and Surfaces. B, Biointerfaces, 2014, 117(1): 267–276CrossRefGoogle Scholar
  23. 23.
    Chevallet M, Gallet B, Fuchs A, Jouneau P H, Um K, Mintz E, Michaud-Soret I. Metal homeostasis disruption and mitochondrial dysfunction in hepatocytes exposed to sub-toxic doses of zinc oxide nanoparticles. Nanoscale, 2016, 8(43): 18495–18506CrossRefGoogle Scholar
  24. 24.
    Zhang W, Zhao Y, Li F, Li L, Feng Y, Min L, Ma D, Yu S, Liu J, Zhang H, et al. Zinc oxide nanoparticle caused plasma metabolomic perturbations correlate with hepatic steatosis. Frontiers in Pharmacology, 2018, 9: 57CrossRefGoogle Scholar
  25. 25.
    Leite-Silva V R, Liu D C, Sanchez W Y, Studier H, Mohammed Y H, Holmes A, Becker W, Grice J E, Benson H A, Roberts M S. Effect of flexing and massage on in vivo human skin penetration and toxicity of zinc oxide nanoparticles. Nanomedicine (London), 2016, 11(10): 1193–1205CrossRefGoogle Scholar
  26. 26.
    Sayes C M, Reed K L, Warheit D B. Assessing toxicity of fine and nanoparticles: Comparing in vitro measurements to in vivo pulmonary toxicity profiles. Toxicological Sciences: An Official Journal of the Society of Toxicology, 2007, 97(1): 163–180CrossRefGoogle Scholar
  27. 27.
    Xia T, Zhao Y, Sager T, George S, Pokhrel S, Li N, Schoenfeld D, Meng H, Lin S,Wang X, et al. Decreased dissolution of ZnO by iron doping yields nanoparticles with reduced toxicity in the rodent lung and zebrafish embryos. ACS Nano, 2011, 5(2): 1223–1235CrossRefGoogle Scholar
  28. 28.
    Warheit D B, Sayes C M, Reed K L. Nanoscale and fine zinc oxide particles: Can in vitro assays accurately forecast lung hazards following inhalation exposures? Environmental Science & Technology, 2009, 43(20): 7939–7945CrossRefGoogle Scholar
  29. 29.
    Giau V V, An S S. Emergence of exosomal miRNAs as a diagnostic biomarker for Alzheimer’s disease. Journal of the Neurological Sciences, 2016, 15(360): 141–152CrossRefGoogle Scholar
  30. 30.
    Mana M N, Fougères P A, Leung Y H, Liu F, Djurišić A B, Giesy J P, Leung K M Y. Physicochemical characteristics and toxicity of surface-modified zinc oxide nanoparticles to freshwater and marine microalgae. Scientific Reports, 2017, 7(1): 15909CrossRefGoogle Scholar
  31. 31.
    Milani S, Bombelli F B, Pitek A S, Dawson K A, Rädler J. Reversible versus irreversible binding of transferrin to polystyrene nanoparticles: Soft and hard corona. ACS Nano, 2012, 6(3): 2532–2541CrossRefGoogle Scholar
  32. 32.
    Pederzoli F, Tosi G, Vandelli M A, Belletti D, Forni F, Ruozi B. Protein corona and nanoparticles: How can we investigate on? Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology, 2017, 9(6): e1467Google Scholar
  33. 33.
    Rao A, Long H, Harley-Trochimczyk A, Pham T, Zettl A, Carraro C, Maboudian R. In situ localized growth of ordered metal oxide hollow sphere array on microheater platform for sensitive, ultra-fast gas sensing. ACS Applied Materials & Interfaces, 2017, 9(3): 2634–2641CrossRefGoogle Scholar
  34. 34.
    Schöttler S, Landfester K, Mailänder V. Controlling the stealth effect of nanocarriers through understanding the protein corona. Angewandte Chemie International Edition in English, 2016, 55(31): 8806–8815CrossRefGoogle Scholar
  35. 35.
    Tenzer S, Docter D, Kuharev J, Musyanovych A, Fetz V, Hecht R, Schlenk F, Fischer D, Kiouptsi K, Reinhardt C, et al. Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. Nature Nanotechnology, 2013, 8(10): 772–781CrossRefGoogle Scholar
  36. 36.
    Saha K, Rahimi M, Yazdani M, Kim S T, Moyano D F, Hou S, Das R, Mout R, Rezaee F, Mahmoudi M, et al. Regulation of macrophage recognition through the interplay of nanoparticle surface functionality and protein corona. ACS Nano, 2016, 10(4): 4421–4430CrossRefGoogle Scholar
  37. 37.
    Shannahan J H, Brown J M, Chen R, Ke P C, Lai X, Mitra S, Witzmann F A. Comparison of nanotube-protein corona composition in cell culture media. Small, 2013, 9(12): 2171–2181CrossRefGoogle Scholar
  38. 38.
    Shannahan J H, Lai X, Ke P C, Podila R, Brown J M, Witzmann F A. Silver nanoparticle protein corona composition in cell culture media. PLoS One, 2013, 8(9): e74001CrossRefGoogle Scholar
  39. 39.
    Kim K M, Choi M H, Lee J K, Jeong J, Kim Y R, Kim M K, Paek S M, Oh J M. Physicochemical properties of surface charge-modified ZnO nanoparticles with different particle sizes. International Journal of Nanomedicine, 2014, 9(Suppl 2): 41–56Google Scholar
  40. 40.
    Shim K H, Hulme J, Maeng E H, Kim M K. An S S A. Analysis of zinc oxide nanoparticles binding proteins in rat blood and brain homogenate. International Journal of Nanomedicine, 2014, 9(Suppl 2): 217–224Google Scholar
  41. 41.
    Cedervall T, Lynch I, Foy M, Berggård T, Donnelly S C, Cagney G, Linse S, Dawson K A. Detailed identification of plasma proteins adsorbed on copolymer nanoparticles. Angewandte Chemie International Edition in English, 2007, 46(30): 5754–5756CrossRefGoogle Scholar
  42. 42.
    Lara S, Alnasser F, Polo E, Garry D, Giudice M C L, Hristov D R, Rocks L, Salvati A, Yan Y, Dawso N K A. Identification of receptor binding to the biomolecular corona of nanoparticles. ACS Nano, 2017, 11(2): 1884–1893CrossRefGoogle Scholar
  43. 43.
    Kreuter J, Shamenkov D, Petrov V, Ramge P, Cychutek K, Koch- Brandt C, Alyautdin R. Apolipoprotein-mediated transport of nanoparticle-bond drugs across the blood-brain barrier. Journal of Drug Targeting, 2002, 10(4): 317–325CrossRefGoogle Scholar
  44. 44.
    Palchetti S, Colapicchioni V, Digiacomo L, Caracciolo G, Pozzi D, Capriotti A L, La Barbera G, Laganà A. The protein corona of circulating PEGylated liposomes. Biochimica et Biophysica Acta, 2016, 1858(2): 189–196CrossRefGoogle Scholar
  45. 45.
    Ritz S, Schöttler S, Kotman N, Baier G, Musyanovych A, Kuharev J, Landfester K, Schild H, Jahn O, Tenzer S, et al. Protein corona of nanoparticles: Distinct proteins regulate the cellular uptake. Biomacromolecules, 2015, 16(4): 1311–1321CrossRefGoogle Scholar
  46. 46.
    Lundqvist M, Stigler J, Elia G, Lynch I, Cedervall T, Dawson K A. Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(38): 14265–14270CrossRefGoogle Scholar
  47. 47.
    Townson J L, Lin Y S, Agola J O, Carnes E C, Leong H S, Lewis J D, Haynes C L, Brinker C J. Differing in vivo characteristics of sizeand charge-matched mesoporous silica nanoparticles. Journal of the American Chemical Society, 2013, 135(43): 16030–16033CrossRefGoogle Scholar
  48. 48.
    Röcker C, Pötzl M, Zhang F, Parak W J, Nienhaus G U. A quantitative fluorescence study of protein monolayer formation on colloidal nanoparticles. Nature Nanotechnology, 2009, 4(9): 577–580CrossRefGoogle Scholar
  49. 49.
    Deng Z J, Mortimer G, Schiller T, Musumeci A, Martin D, Minchin R F. Differential plasma protein binding to metal oxide nanoparticles. Nanotechnology, 2009, 20(45): 455101CrossRefGoogle Scholar
  50. 50.
    Lesniak A, Campbell A, Monopoli M P, Lynch I, Salvati A, Dawson K A. Serum heat inactivation affects protein corona composition and nanoparticle uptake. Biomaterials, 2010, 31(36): 9511–5918CrossRefGoogle Scholar
  51. 51.
    Lesniak A, Fenaroli F, Monopoli M P, Åberg C, Dawson K A, Salvati A. Effects of the presence or absence of a protein corona on silica nanoparticle uptake and impact on cells. ACS Nano, 2012, 6 (7): 5845–5857CrossRefGoogle Scholar
  52. 52.
    Lesniak A, Salvati A, Santos-Martinez M J, Radomski M W, Dawson K A, Åberg C. Nanoparticle adhesion to the cell membrane and its effect on nanoparticle uptake efficiency. Journal of the American Chemical Society, 2013, 135(4): 1438–1444CrossRefGoogle Scholar
  53. 53.
    Murdock R C, Braydich-Stolle L, Schrand A M, Schlager J J, Hussain S M. Characterization of nanomaterial dispersion in solution prior to in vitro exposure using dynamic light scattering technique. Toxicological Sciences: An Official Journal of the Society of Toxicology, 2008, 101(2): 239–253CrossRefGoogle Scholar
  54. 54.
    Maiorano G, Sabella S, Sorce B, Brunetti V, Malvindi M A, Cingolani R, Pompa P P. Effects of cell culture media on the dynamic formation of protein-nanoparticle complexes and influence on the cellular response. ACS Nano, 2010, 4(12): 7481–7491CrossRefGoogle Scholar
  55. 55.
    Wang J, Jensen U B, Jensen G V, Shipovskov S, Balakrishnan V S, Otzen D, Pedersen J S, Besenbacher F, Sutherland D S. Soft interactions at nanoparticles alter protein function and conformation in a size dependent manner. Nano Letters, 2011, 11(11): 4985–4991CrossRefGoogle Scholar
  56. 56.
    Zhou Y, Fang X, Gong Y, Xiao A, Xie Y, Liu L, Cao Y. The interactions between ZnO nanoparticles (NPs) and α-linolenic acid (LNA) complexed to BSA did not influence the toxicity of ZnO NPs on HepG2 cells. Journal of the American Chemical Society, 2017, 135(4): 1438–1444Google Scholar
  57. 57.
    Go M R, Yu J, Bae S H, Kim H J, Choi S J. Effects of interactions between ZnO nanoparticles and saccharides on biological responses. International Journal of Molecular Sciences, 2018, 19(2): 486CrossRefGoogle Scholar

Copyright information

© Higher Education Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Bionanotechnology and Gachon Medical Research InstituteGachon University and Gachon Medical Research InstituteSeongnamKorea
  2. 2.Laboratory of Cell Signaling and Nanomedicine, Department of Dermatology and Division of Brain Korea 21 Project for Biomedical ScienceKorea University College of MedicineSeoulKorea

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