Hard Protein Corona Research Articles

A lysozyme corona complex for the controlled pharmacokinetic release of probucol from mesoporous silica particles.

Mesoporous silica particles (MSPs) enhance the release kinetics of poorly soluble compound probucol (PB) under the influence of a pore-blocking protein corona, prepared with lysozyme protein adsorption. In vivo oral administration experiments show a prolongation in the time to reach maximum systemic concentration and half-life of PB released from the lysozyme-MSP complex in comparison to the MSP alone. Specific hard protein corona complexes can act as functional diffusion barriers for the controlled release of drugs from MSP based formulations.

The interaction between nanoparticles-protein corona complex and cells and its toxic effect on cells.

Once nanoparticles (NPs) contact with the biological fluids, the proteins immediately adsorb onto their surface, forming a layer called protein corona (PC), which bestows the biological identity on NPs. Importantly, the NPs-PC complex is the true identity of NPs in physiological environment. Based on the affinity and the binding and dissociation rate, PC is classified into soft protein corona, hard protein corona, and interfacial protein corona. Especially, the hard PC, a protein layer relatively stable and closer to their surface, plays particularly important role in the biological effects of the complex. However, the abundant corona proteins rarely correspond to the most abundant proteins found in biological fluids. The composition profile, formation and conformational change of PC can be affected by many factors. Here, the influence factors, not only the nature of NPs, but also surface chemistry and biological medium, are discussed. Likewise, the formed PC influences the interaction between NPs and cells, and the associated subsequent cellular uptake and cytotoxicity. The uncontrolled PC formation may induce undesirable and sometimes opposite results: increasing or inhibiting cellular uptake, hindering active targeting or contributing to passive targeting, mitigating or aggravating cytotoxicity, and stimulating or mitigating the immune response. In the present review, we discuss these aspects and hope to provide a valuable reference for controlling protein adsorption, predicting their behavior invivo experiments and designing lower toxicity and enhanced targeting nanomedical materials for nanomedicine.

Mass spectrometric approach for the analysis of the hard protein corona of nanoparticles in living cells

The diagnostic and therapeutic application of nanoparticles requires comprehensive knowledge of their interaction with the biomolecular surroundings. The formation of the protein corona on nanoparticles that were internalized by living cells is yet to be understood. In this study, we present a robust approach for the electrophoretic and mass spectrometric analysis of the hard protein corona composition formed in living cells on ~30 nm citrate-stabilized gold nanoparticles, i.e., the proteins with the highest affinity towards the gold nanoparticle surface. The gold nanoparticles were internalized by MCF-7 cells for 24 h followed by the extraction of the hard protein corona‑gold nanoparticle bioconjugates from living cell cultures. The extracted proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by ESI-Q-TOF-MS, which allowed to identify 108 hard corona proteins. The experiments were repeated with J774 macrophage cells with incubation times of 1.5 h, 3 h, 6 h, and 24 h, and the results showed that the hard protein corona remained unchanged over time. Therefore, the proposed experimental approach proved to be a valuable tool for identifying hard corona proteins of nanoparticles internalized by living cells.

Influence of a Protein Corona on the Oral Pharmacokinetics of Testosterone Released from Mesoporous Silica

AbstractThe formation of a protein corona adsorbed on mesoporous silica particles with 2D and 3D pore structures after incubation in fetal bovine serum is investigated. The compositional properties of the resulting protein–particle complex is determined by using mass spectrometry as a function of serum incubation time. The effect of the adsorbed proteins on the drug release properties of the mesoporous particles when these are used to deliver the hormone TST is examined in dissolution media. Changes to the pharmacokinetic parameters are validated in vivo in a rat model. Small changes to the maximum plasma concentration, terminal half‐life, and area under the plasma concentration time curve are observed with pre‐incubation of a hard protein corona around the particles. This strategy is used to slow down the release of pharmaceutical drugs loaded onto mesoporous silica, and acts as a formulation strategy for future nanomedical applications involving advanced drug delivery.

Possibilities and Limitations of Different Separation Techniques for the Analysis of the Protein Corona.

One of the biggest challenges in the field of nanomedicine is the adsorption of biomolecules on the nanomaterial upon contact with a biological medium. The interactions of the resulting protein corona are essential for their behavior in a biological system. Thus, it is now commonly accepted that understanding the formation and consequently understanding the influence of the protein corona on the biological response is crucial. However, the outcome of the protein corona characterization cannot easily be compared between different studies and techniques, since many different sample preparation procedures exist that are suitable for different materials or methods. Depending on the applied procedure, the nanomaterial-protein system will be altered in a certain way, so that it is necessary to consider the individual influence on the protein corona. Accordingly, the aim of this Minireview is to give an overview of the applied sample preparation methods for the analysis of the protein corona and to evaluate their influence on the outcome of the results especially with regard to the introduced terms "soft" and "hard protein corona". Special focus will be placed on the comparison of the most commonly used techniques such as centrifugation, magnetic, and chromatographic separation.

Zwitterionic polymer ligands: an ideal surface coating to totally suppress protein-nanoparticle corona formation?

In the last few years, zwitterionic polymers have been developed as antifouling surface coatings. However, their ability to completely suppress protein adsorption at the surface of nanoparticles in complex biological media remains undemonstrated. Here we investigate the formation of hard (irreversible) and soft (reversible) protein corona around model nanoparticles (NPs) coated with sulfobetaine (SB), phosphorylcholine (PC) and carboxybetaine (CB) polymer ligands in model albumin solutions and in whole serum. We show for the first time a complete absence of protein corona around SB-coated NPs, while PC- and CB-coated NPs undergo reversible adsorption or partial aggregation. These dramatic differences cannot be described by naïve hard/soft acid/base electrostatic interactions. Single NP tracking in the cytoplasm of live cells corroborate these in vitro observations. Finally, while modification of SB polymers with additional charged groups lead to consequent protein adsorption, addition of small neutral targeting moieties preserves antifouling and enable efficient intracellular targeting.

The hard protein corona of stealth liposomes is sparse

The protein corona is widely recognized as a key concept in contemporary nanomedicine. In recent years, the interest in the protein corona has reached new heights as a number of reports have suggested that a comprehensive protein corona can form around nanomaterials that previously were thought to be resistant to protein binding. For example, PEGylated stealth liposomes were long thought to be protein repellent, but a number of recent studies have found that a significant protein corona forms around such liposomes in the bloodstream. Prompted by these surprising recent findings, we here present an extensive quantitative study of the binding of blood proteins to standard PEGylated stealth liposomes. To make the study relevant for targeted as well as non-targeted drug delivery systems, the liposomes were prepared both with and without a targeting antibody conjugated to their surface. The prepared liposomes were either incubated in vitro in fetal bovine serum or administered in vivo into the bloodstream of mice. Subsequently, the liposomes were recovered and analyzed using a variety of techniques. There was very little protein binding to the liposomes recovered after in vitro incubation. In contrast, there was more protein binding to the liposomes recovered after in vivo circulation, but a deeper analysis estimated that the bound proteins still only covered a very low fraction of the liposomal surface area. Both the liposomes recovered after in vitro incubation and the liposomes recovered after in vivo circulation completely retained their size and receptor targeting characteristics. Taken collectively, our results thus demonstrate that the hard protein corona of both non-targeted and antibody-targeted stealth liposomes is sparse and does not affect the structure and function of the liposomes.

Interaction of functionalized nanoparticles with serum proteins and its impact on colloidal stability and cargo leaching.

The majority of effort in the area of polymeric nanocarriers is aimed at providing controlled drug delivery in vivo. Therefore, it is essential to understand the delicate interplay of polymeric NPs with serum proteins in order to forecast their performance in a biological system. In this study, the interaction of serum proteins with functionalized polymeric colloids as a function of particle charge and hydrophobicity was investigated. Moreover, impact on NP stability and cargo leaching was assessed. The hard protein corona of polymeric NPs with either uncharged methoxy groups (methoxy-NPs), positively charged amine groups (amine-NPs), negatively charged carboxylic acid groups (carboxyl-NPs) or zwitterionic NPs decorated with amine and carboxylic acid groups (zwitterion-NPs) was quantitatively and qualitatively analyzed and correlated with the respective colloidal stability using fluorescence resonance energy transfer. Positively charged amine-NPs displayed an enhanced interaction with serum proteins via electrostatic interactions resulting in a hard corona consisting of diverse protein components. As revealed by FRET and agarose gel electrophoresis, the enhanced adsorption of proteins onto the colloidal surface significantly altered the NP identity and severely impaired the colloidal integrity as the lipophilic cargo was continuously leached out of the hydrophobic NP core. These results highlight the importance of generating a profound knowledge of the bio-nano interface as adherence of biomolecules can severely compromise the performance of a colloidal drug delivery system by changing its identity and integrity.

Targeted Nanoparticle Binding to Hydroxyapatite in a High Serum Environment for Early Detection of Heart Disease

The impact of the protein-rich in vivo environment on targeted binding of functionalized nanoparticles has been an active field of research over the past several years. Current research aims at better understanding the nature of the protein corona and how it may be possible for targeted binding to occur even in the presence of serum. Much of the current research focuses on nanoparticles targeted to particular cell receptors or features with the aim of cellular uptake. However, similar research has not been performed on nanoparticles that are targeted to non-protein disease features, such as hydroxyapatite (HA). HA is a crystalline calcium-phosphate mineral that is present in large quantities in bone, and in smaller quantities in diseased cardiovascular tissue in cases of atherosclerosis or various stenoses. Our work aims to gain a better understanding of the behavior of PEGylated, peptide-coated superparamagnetic iron oxide nanoparticles (SPIONs) in a biologically-relevant high-protein environment (50% serum). We first determined that specific binding to HA occurs at significantly higher rates than non-specific binding in the absence of serum protein. We then examined nanoparticle interactions with serum proteins, including determination of the relative quantities of protein in the hard vs. soft protein corona. Finally, we examined specific and non-specific binding of targeted SPIONs in 50% serum, and determined that targeted binding may still occur with significant (p < 0.05) selectivity. We hypothesize that this may be because the nature of the binding interactions between the peptides and the HA are, by definition, less specific than the protein-protein interactions required for nanoparticles to bind to specific cells or cell features. These results suggest that these targeted SPIONs may be further developed for use in early detection of heart diseases such as atherosclerosis and aortic stenosis.

Preservation of the soft protein corona in distinct flow allows identification of weakly bound proteins

Nanocarriers that are used for targeted drug delivery come in contact with biological liquids and subsequently proteins will adsorb to the nanocarriers’ surface to form the so called ‘protein corona’. The protein corona defines the biological identity and determines the biological response towards the nanocarriers in the body. To make nanomedicine safe and reliable it is required to get a better insight into this protein corona and, therefore, the adsorbed proteins have to be characterized. Currently, centrifugation is the common method to isolate the protein corona for further investigations. However, with this method it is only possible to investigate the strongly bound proteins, also referred to as ‘hard protein corona’. Therefore, we want to introduce a new separation technique to separate nanoparticles including the soft protein corona containing also loosely bound proteins for further characterization. The used separation technique is the asymmetric flow field-flow fractionation (AF4). We were able to separate the nanoparticles with proteins forming the soft protein corona and were able to show that in our system only the hard protein corona directly influenced the cell uptake behavior. Statement of SignificanceCurrently, there is an ongoing debate whether only strongly bound proteins (hard corona) or also loosely bound proteins (soft corona) contribute to the biological identity of nanocarriers, because up to now isolation of the soft corona was not possible. Here, asymmetric flow field-flow fractionation was used to isolate nanoparticles with a preserved soft corona from the biological medium. This enabled the characterization of the soft corona composition and to evaluate its influence on cellular uptake. For our system we found that only the strongly bound proteins (hard corona) determined cell internalization. This method can now be used to evaluate the impact of the soft corona further and to characterize nanomaterials that cannot be separated from blood plasma by other means.

Analysis of hard protein corona composition on selective iron oxide nanoparticles by MALDI-TOF mass spectrometry: identification and amplification of a hidden mastitis biomarker in milk proteome.

Surface active maghemite nanoparticles (SAMNs) are able to recognize and bind selected proteins in complex biological systems, forming a hard protein corona. Upon a 5-min incubation in bovine whey from mastitis-affected cows, a significant enrichment of a single peptide characterized by a molecular weight at 4338Da originated from the proteolysis of aS1-casein was observed. Notably, among the large number of macromolecules in bovine milk, the detection of this specific peptide can hardly be accomplished by conventional analytical techniques. The selective formation of a stable binding between the peptide and SAMNs is due to the stability gained by adsorption-induced surface restructuration of the nanomaterial. We attributed the surface recognition properties of SAMNs to the chelation of iron(III) sites on their surface by sterically compatible carboxylic groups of the peptide. The specific peptide recognition by SAMNs allows its easy determination by MALDI-TOF mass spectrometry, and a threshold value of its normalized peak intensity was identified by a logistic regression approach and suggested for the rapid diagnosis of the pathology. Thus, the present report proposes the analysis of hard protein corona on nanomaterials as a perspective for developing fast analytical procedures for the diagnosis of mastitis in cows. Moreover, the huge simplification of proteome complexity by exploiting the selectivity derived by the peculiar SAMN surface topography, due to the iron(III) distribution pattern, could be of general interest, leading to competitive applications in food science and in biomedicine, allowing the rapid determination of hidden biomarkers by a cutting edge diagnostic strategy. Graphical abstract The topography of iron(III) sites on surface active maghemite nanoparticles (SAMNs) allows the recognition of sterically compatible carboxylic groups on proteins and peptides in complex biological matrixes. The analysis of hard protein corona on SAMNs led to the determination of a biomarker for cow mastitis in milk by MALDI-TOF mass spectrometry.

The Nature of a Hard Protein Corona Forming on Quantum Dots Exposed to Human Blood Serum.

Biological responses of cells and organisms to nanoparticle exposure crucially depend on the properties of the protein adsorption layer ("protein corona") forming on nanoparticle surfaces and their characterization is a crucial step toward a deep, mechanistic understanding of their build-up. Previously, adsorption of one type of model protein on nanoparticles was systematically studied in situ by using fluorescence correlation spectroscopy. Here, the first such study of interactions is presented between water-solubilized CdSe/ZnS quantum dots (QDs) and a complex biofluid, human blood serum. Despite the large number of proteins in serum, a protein layer of well-defined (average) thickness forming on QD surfaces is observed. Both the thickness and the apparent binding affinity depend on the type of QD surface ligand. Kinetic experiments reveal that the protein corona formed from serum is irreversibly bound, whereas the one formed from human serum albumin was earlier observed to be reversible. By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry, the most abundant serum proteins contributing to the formation of a hard corona on the QDs are identified.

Interactions of cationic polystyrene nanoparticles with marine bivalve hemocytes in a physiological environment: Role of soluble hemolymph proteins

The bivalve Mytilus galloprovincialis has proven as a suitable model invertebrate for evaluating the potential impact of nanoparticles (NPs) in the marine environment. In particular, in mussels, the immune system represents a sensitive target for different types of NPs. In environmental conditions, both NP intrinsic properties and those of the receiving medium will affect particle behavior and consequent bioavailability/uptake/toxicity. However, the evaluation of the biological effects of NPs requires additional understanding of how, once within the organism, NPs interact at the molecular level with cells in a physiological environment. In mammalian systems, different NPs associate with serum soluble components, organized into a “protein corona”, which affects particle interactions with target cells. However, no information is available so far on the interactions of NPs with biological fluids of aquatic organisms.In this work, the influence of hemolymph serum (HS) on the in vitro effects of amino modified polystyrene NPs (PS-NH2) on Mytilus hemocytes was investigated. Hemocytes were incubated with PS-NH2 suspensions in HS (1, 5 and 50µg/mL) and the results were compared with those obtained in ASW medium. Cell functional parameters (lysosomal membrane stability, oxyradical production, phagocytosis) were evaluated, and morphological changes were investigated by TEM. The activation state of the signalling components involved in Mytilus immune response (p38 MAPK and PKC) was determined. The results show that in the presence of HS, PS-NH2 increased cellular damage and ROS production with respect to ASW medium. The effects were apparently mediated by disregulation of p38 MAPK signalling. The formation of a PS-NH2-protein corona in HS was investigated by centrifugation, and 1D- gel electrophoresis and nano-HPLC-ESI-MS/MS. The results identified the Putative C1q domain containing protein (MgC1q6) as the only component of the PS-NH2 hard protein corona in Mytilus hemolymph. These data represent the first evidence for the formation of a NP bio-corona in aquatic organisms and underline the importance of the recognizable biological identity of NPs in physiological exposure medium when testing their potential impact environmental model organisms. Although the results obtained in vitro do not entirely reflect a realistic exposure scenario and the more complex formation of a bio-corona that is likely to occur in vivo, these data will contribute to a better understanding of the effects of NPs in marine invertebrates.

Interaction of functionalized PEG-PLA/PLGA nanoparticles with serum proteins and impact on nanoparticle stability and cell toxicity

Event Abstract Back to Event Interaction of functionalized PEG-PLA/PLGA nanoparticles with serum proteins and impact on nanoparticle stability and cell toxicity Kathrin Abstiens1 and Achim M. Goepferich1* 1 University of Regensburg, Department of Pharmaceutical Technology, Germany Introduction: Formation of a nanoparticle (NP) protein corona upon administration into the bloodstream plays a pivotal role concerning safety and efficacy of nanoparticle formulations. As a result of the complex interplay between chemical and physical factors, it is particularly challenging to forecast the extent and composition of protein adsorption onto different nanomaterials. Therefore, the aim of this study was to characterize the interaction of serum proteins with poly(lactic-co-glycolic acid) blended poly(ethylene glycol)-polylactid acid (PEG-PLA/PLGA) NPs depending on particle charge and net charge density. Moreover, impact on NP stability and cell toxicity in vitro was assessed. For this purpose methoxy- (m), carboxy- (C) and amino-functionalized (N) PEG-PLA copolymers were used to prepare NPs with a negative (NP510C), positive (NP510N) or uncharged (NP510m) surface as well as dual charged particles (NP510CN). Materials and Methods: PEG-PLA copolymers were synthesized by ring-opening polymerization and NPs were prepared by nanoprecipitation as previously described [1]. Size and zeta potential was measured on a Zetasizer Nano ZS. Serum protein adsorption was evaluated by incubation of functionalized nanoparticles with fetal calf serum (FCS) at 37 °C. Composition of the hard protein corona was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and total protein amount was determined using Micro BCA Assay. The involvement of bovine serum albumin (BSA) in the soft and hard protein corona was assessed by DLS upon incubation with NP at 37 °C. NP stability was evaluated with a fluorescence resonance energy transfer (FRET) experiment as previously described [2]. Influence on cell toxicity was investigated by MTT assay. Results and Discussion: NP surface charge has been shown to influence protein corona formation [3]. As depicted in Fig. 1 A, positively charged NP adsorbed significantly more serum proteins than negatively charged. Furthermore, interaction with serum proteins was even more decreased when particle surface was dual charged (NP510CN) compared to uncharged nanoparticles (NP510m). A FRET experiment revealed that NP stability in serum is directly correlated with the extent of protein adsorption. This may be due to the fact that formation of a protein corona has a strong impact on NP integrity. As a consequence thereof an escape of the lipophilic cargo was facilitated which led to a decrease of the FRET ratio over time [Fig. 1 B]. As interactions with serum proteins are likely to influence cell-NP interactions, cytotoxicity of NPs was assessed by MTT assay. It could be observed that cytotoxicity of NPs was significantly decreased when cell media was supplemented with FCS which indicates that unspecific cellular uptake was interfered by formation of a protein corona. Figure 1: A) Quantification of the hard protein corona of different NP formulations determined by BCA assay B) FRET stability of NP formulations in PBS pH 7.4 and FCS as a function of time Conclusion: The electrostatic interaction of polymeric NP with serum proteins can be regulated by combining differently functionalized PEG-PLA copolymers. Such tailored NPs can be used to control the extent of protein adsorption onto NP surface and thus NP stability and toxicity in vitro.

Complementary analysis of the hard and soft protein corona: sample preparation critically effects corona composition.

Here we demonstrate how a complementary analysis of nanocapsule-protein interactions with and without application media allows gaining insights into the so called hard and soft protein corona. We have investigated how both human plasma and individual proteins (human serum albumin (HSA), apolipoprotein A-I (ApoA-I)) adsorb and interact with hydroxyethyl starch (HES) nanocapsules possessing different functionalities. To analyse the hard protein corona we used sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and a protein quantitation assay. No significant differences were observed with regards to the hard protein corona. For analysis of the soft protein corona we characterized the nanocapsule-protein interaction with isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). DLS and ITC measurements revealed that a high amount of plasma proteins were adsorbed onto the capsules' surface. Although HSA was not detected in the hard protein corona, ITC measurements indicated the adsorption of an HSA amount similar to plasma with a low binding affinity and reaction heat. In contrast, only small amounts of ApoA-I protein adsorb to the capsules with high binding affinities. Through a comparison of these methods we have identified ApoA-I to be a component of the hard protein corona and HSA as a component of the soft corona. We demonstrate a pronounced difference in the protein corona observed depending on the type of characterization technique applied. As the biological identity of a particle is given by the protein corona it is crucial to use complementary characterization techniques to analyse different aspects of the protein corona.

Surface group mediates the adsorption of in situ generated thrombin and its interaction with anti-thrombin in the protein corona of SBA-15

The surface groups of mesoporous silica (MPS) play an important role in mediating the hard protein corona (HPC) formation on its surface, leading to bio-identities with completely different procoagulant activities.

Variation of Protein Corona Composition of Gold Nanoparticles Following Plasmonic Heating

It is well recognized that the primary interaction of most biological environments with nanoparticles (NPs) is strongly influenced by a long-lived ("hard") protein corona that surrounds the NP and remains strongly adsorbed to its surface. The amount and composition of associated proteins in the corona adsorbed onto the NPs is related to several important factors, including the physicochemical properties of the NPs and the composition of the protein solution. Here, for the first time, it is shown that plasmonic heat induction (by laser activation) leads to significant changes in the composition of the hard protein corona adsorbed on low aspect ratio gold nanorods. Using mass spectrometry, several proteins in the corona were identified whose concentrations change most substantially as a result of photoinduced (plasmonic) heating versus simple thermal heating. Molecular modeling suggests that the origin of these changes in protein adsorption may be the result of protein conformational changes in response to much higher local temperatures that occur near the gold nanorods during photoinduced, plasmonic heating. These results may define new applications in vivo for NPs with hyperthermia capability and better define the likely interactions of cells with NPs after plasmonic heating. Potential changes in the protein corona following hyperthermia treatment may influence the final biological fate of plasmonic NPs in clinical applications and help elucidate safety considerations for hyperthermia applications.

Protein corona on magnetite nanoparticles and internalization of nanoparticle–protein complexes into healthy and cancer cells

Superparamagnetic magnetite nanoparticles (MNPs) of different surface properties are incubated in complicated living fluid, including fetal bovine serum solution, cell complete culture medium and cell culture system with/without serum, to investigate the alteration of protein corona and its impact on cell internalization. The MNPs prepared by co-precipitation method are functionalized with L-Lysine (Lys), Glucosamic acid (GA) to obtain amine, carboxyl and hydroxyl groups, separately. All the particles adsorb serum proteins to form MNPs-protein complexes with the surface charge changing into negative. 1D SDS/PAGE gel images analysis indicates that the composition and content of hard protein corona on the surface of NPs are related to their functional groups and agglomeration, and the total amount of protein in the medium. In cell culture system, particles not only adsorb serum proteins, but also associate with cytosolic proteins arising from HepG2 and L02 cells. GA modified MNPs (MNPs-GA) exhibit bovine serum albumin anti-adsorption capability because of the terminal hydroxyl and carboxyl groups. MNPs-GA also shows the highest cellular uptake and label efficiency compared with uncoated MNPs and Lys modified MNPs, due to larger aggregates formation and specific protein corona composition, rather than commonly approved electrostatic interaction between particles and cells. For the first time, our results provide visualized reports on previously neglected, but indispensable protein corona of the MNPs after interaction with both healthy and cancer cells, suggesting that cytosolic protein corona from cells and aggregation of particles are important factors needed to be account for on studying the nano-bio interface.

Formation and cell translocation of carbon nanotube-fibrinogen protein corona

The binding of plasma fibrinogen with both single-walled and multi-walled carbon nanotubes (SWNTs and MWNTs) has been examined. Specifically, our absorbance study indicated that MWNTs were coated with multi-layers of fibrinogen to render a "hard protein corona," while SWNTs were adsorbed with thin layers of the protein to precipitate out of the aqueous phase. In addition, static quenching as a result of energy transfer from fluorescently labeled fibrinogen to their nanotube substrates was revealed by Stern-Volmer analysis. When exposed to HT-29 cells, the nanotubes and fibrinogen could readily dissociate, possibly stemming from their differential affinities for the amphiphilic membrane bilayer.

Hardening of the Nanoparticle–Protein Corona in Metal (Au, Ag) and Oxide (Fe3O4, CoO, and CeO2) Nanoparticles

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.