LUNDQVIST ET AL. VOL. 5 ’ NO. 9 ’ 7503–7509 ’ 2011 www.acsnano.org 7503 August 23, 2011 C 2011 American Chemical Society The Evolution of the Protein Corona around Nanoparticles: A Test Study Martin Lundqvist, †,‡, * Johannes Stigler, † Tommy Cedervall, ‡ Tord Bergga ˚ rd, § Michelle B. Flanagan, ^ Iseult Lynch, † Giuliano Elia, z and Kenneth Dawson †, * † Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Dublin, Ireland, ‡ Center for Molecular Protein Science, Biochemistry, Lund University, Lund, Sweden, § Alligator Bioscience AB, Lund, Sweden, ^ Conway Institute, University College Dublin, Dublin, Ireland, and z Mass Spectrometry Resource, Conway Institute, University College Dublin, Dublin, Ireland N anoparticles may potentially enter our body via several different routes, for example, by inhalation, ingestion, or uptake through the skin. How- ever, regardless of the method of entry, biological fluid will surround nanoparticles once they have entered a biological envir- onment. When nanoparticles come into contact with a biological fluid their surface will be covered with a “corona” of biological macromolecules. The composition of the corona depends on the nanoparticle size and surface characteristics, 1,2 which deter- mine protein binding specificities and affi- nities. Thus, some particles will have a stable hard core of biological macromolecules (that we name the hard corona) that interact strongly with the surface as well as a more loosely bound outer layer of biological macromolecules that associate less strongly both to the particle surface and to the strongly associated biological macromole- cules. Some particles will only have a “weak” corona, meaning that most of the biological macromolecules will have a weak associa- tion to the surface, for example, pegylated particles. Assuming a sufficiently long resi- dence time, the biological macromolecules that surround a nanoparticle will determine its biological fate, as it is this corona of biomolecules that cells “see” and interact with. The majority of the identified biologi- cal macromolecules surrounding nanopar- ticles are proteins, though recently we have also reported the presence of some lipids. 3 We have reported detailed pictures for the “hard” corona formed around nanoparticles of different materials, including copolymer and polystyrene nanoparticles, of di fferent sizes and with different surface properties. 1,4,5 The importance of the protein corona for determining any possible toxicity from differ- ent nanoparticles has been reported. 6,7 Xia et al. have a recent publication in which they mapped the adsorption of a set of small- molecule probes to different nanoparticles and transformed the results into a biological surface-adsorption index. 8 The hard corona around NIPAM:BAM co- polymer particles is quite specific, with a small number of proteins contributing to the main part of the corona. 4,5 The interac- tions between the proteins that build up the corona and the copolymer particles have been characterized. 5,9 These data were used to generate a theoretical model for the formation of the corona around the copo- lymer particles over time. 9 The model shows that immediately after being introduced into the blood the particle will be sur- rounded by serum albumin, but with time the serum albumin will be replaced with less abundant proteins that have a higher asso- ciation rate constant and lower dissociation rate constant. 9 Casals et al. have also shown the transition from a loosely attached pro- tein corona from media containing 10% of fetal bovine serum around gold nanoparti- cles that over time, evolves toward an irre- versible attached protein corona. 10 * Address correspondence to Martin.Lundqvist@biochemistry.lu.se. Received for review July 3, 2011 and accepted August 23, 2011. Published online 10.1021/nn202458g ABSTRACT The importance of the protein corona formed around nanoparticles upon entering a biological fluid has recently been highlighted. This corona is, when sufficiently long-lived, thought to govern the particles' biological fate. However, even this long-lived “hard” corona evolves and re- equilibrates as particles pass from one biological fluid to another, and may be an important feature for long-term fate. Here we show the evolution of the protein corona as a result of transfer of nanoparticles from one biological fluid (plasma) into another (cytosolic fluid), a simple illustrative model for the uptake of nanoparticles into cells. While no direct comparison can be made to what would happen in, for example, the uptake pathway, the results confirm that significant evolution of the corona occurs in the second biological solution, but that the final corona contains a “fingerprint” of its history. This could be evolved to map the transport pathways utilized by nanoparticles, and eventually to predict nanoparticle fate and behavior. KEYWORDS: protein corona . biological fluid . cytosolic fluid . nanoparticles . proteomics ARTICLE