3479 © 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com small 2011, 7, No. 24, 3479–3486 1. Introduction Study of the molecular characteristics of inorganic nano- particles (NPs) is an expanding field of research that focuses on the use of different NP conjugates for diagnosis and therapy in medicine. [1] NPs dispersed in biological fluids are sensitive to such an environment and suffer modifications. [2] One of the most significant alterations is the formation of the NP–protein corona as a result of the adsorption of proteins onto the inorganic surface. Currently, protein corona forma- Hardening of the Nanoparticle–Protein Corona in Metal (Au, Ag) and Oxide (Fe 3 O 4 , CoO, and CeO 2 ) Nanoparticles Eudald Casals, Tobias Pfaller, Albert Duschl, Gertie J. Oostingh, and Víctor F. Puntes* tion is gaining attention in the field of inorganic NPs, [3] since this spontaneous coating provides the biological identity to the NP–protein corona composite and determines the inter- actions between the NPs and the host in living systems. Regarding this interaction, what has been observed is a fast conjugation dynamics where within minutes the NP is coated by proteins. However, this coating is not permanent but tran- sient and evolves towards a more stable configuration. [3b,4] In fact, the interfacial chemistry between blood serum pro- teins and inorganic surfaces is known to be governed by the Vroman effect. [5] In 1962, Leo Vroman reported how the exposure of hydrophobic inorganic powders to blood plasma results in the removal of coagulation factors, and the inor- ganic surface becomes more hydrophilic. This effect has a competitive adsorption hierarchy: the highest mobility pro- teins arrive first and are later replaced by less motile proteins that have a higher affinity for the surface. This may take up to a few hours. However, with longer residence times (days), we have observed that this equilibrium further slows down towards a permanent protein corona [3a] that we dubbed the hard protein corona, which remains associated with the NP even in protein-free media, where the maintenance of the chemical equilibrium would imply the partial desorption of the NP-associated proteins. We attribute this to protein crowding effects at the NP surface. [3c,6] Thus, the strength of DOI: 10.1002/smll.201101511 E. Casals, Prof. V. F. Puntes CIN2(ICN-CSIC) Catalan Institute of Nanotechnology and Universitat Autònoma de Barcelona (UAB) Campus de la UAB, Edifici Q, 08193 Bellaterra, Barcelona, Spain E-mail: victor.puntes@uab.es Dr. T. Pfaller, Dr. A. Duschl, Dr. G. J. Oostingh Department of Molecular Biology University of Salzburg Salzburg, Austria Prof. V. F. Puntes Institut Català de Recerca i Estudis Avançats (ICREA) Barcelona, Spain The surface modifications of metal and metal oxide nanoparticles with sizes ranging from 7 to 20 nm dispersed in commonly used cell culture medium supplemented with serum are investigated. All the tested nanoparticles adsorb proteins onto their surface, thereby forming a protein corona through a dynamic process evolving towards an irreversible coating (hard protein corona). Despite the fact that the studied nanomaterials have similar characteristics of hydrophobicity and surface charge, different temporal patterns of the protein corona formation are observed that can be considered a fingerprint for nanoparticle identification. Some of the biological and toxicological implications of the formation of the nanoparticle–protein corona are studied using the human monocytic cell line THP-1 exposed to cobalt oxide nanoparticles. Results show that production of reactive oxygen species is decreased if the nanoparticles are preincubated for 48 h with serum. Nanoparticle–Protein Corona