A health concern regarding the protein corona,aggregation and disaggregation

Nanoparticle (NP)–protein complexes exhibit the “correct identity” of NP in biological media. Therefore, protein–NP interactions should be closely explored to understand and modulate the nature of NPs in medical implementations. This review focuses mainly on the physicochemical parameters such as dimension, surface chemistry, morphology of NPs, and influence of pH on the formation of protein corona and conformational changes of adsorbed proteins by different kinds of techniques. Also, the impact of protein corona on the colloidal stability of NPs is discussed. Uncontrolled protein attachment on NPs may bring unwanted impacts such as protein denaturation and aggregation. In contrast, controlled protein adsorption by optimal concentration, size, pH, and surface modification of NPs may result in potential implementation of NPs as therapeutic agents especially for disaggregation of amyloid fibrils. Also, the effect of NPs-protein corona on reducing the cytotoxicity and clinical implications such as drug delivery, cancer therapy, imaging and diagnosis will be discussed. Validated correlative physicochemical parameters for NP–protein corona formation frequently derived from protein corona fingerprints of NPs which are more valid than the parameters obtained only on the base of NP features. This review may provide useful information regarding the potency as well as the adverse effects of NPs to predict their behavior in vivo.     

Toward a molecular understanding of nanoparticle–protein interactions

Wherever nanoparticles (NPs) come in contact with a living organism, physical and chemical interactions take place between the surfaces of the NPs and biomatter, in particular proteins. When NP are exposed to biological fluids, an adsorption layer of proteins, a “protein corona” forms around the NPs. Consequently, living systems interact with the protein-coated NP rather than with a bare NP. To anticipate biological responses to NPs, we thus require comprehensive knowledge of the interactions at the bio–nano interface. In recent years, a wide variety of biophysical techniques have been employed to elucidate mechanistic aspects of NP–protein interactions. In this brief review, we present the latest findings regarding the composition of the protein corona as it forms on NPs in the blood stream. We also discuss molecular aspects of this adsorption layer and its time evolution. The current state of knowledge is summarized, and issues that still need to be addressed to further advance our understanding of NP–protein interactions are identified.     

In-vitro in-vivo correlation (IVIVC) in nanomedicine: Is protein corona the missing link?

One of the unmet challenges in nanotechnology is to understand and establish the relationship between physicochemical properties of nanoparticles (NPs) and its biological interactions (bio-nano interactions). However, we are still far from assessing the biofate of NPs in a clear and unquestionable manner. Recent developments in the area of bio-nano interface and the understanding of protein corona (PC) has brought new insight in predicting biological interactions of NPs. PC refers to the spontaneous formation of an adsorbed layer of biomolecules on the surface of NPs in a biological environment. PC formation involves the spatiotemporal interplay of an intricate network of biological, environmental and particle characteristics. NPs with its PC can be viewed as a biological entity, which interacts with cells and barriers in a biological system. Recent studies on the bio-nano interface have revealed biological signatures that participate in cellular and physiological bioprocesses and control the biofate and toxicity of NPs. The ability of in-vitro derived parameters to forecast in-vivo consequences by developing a mathematical model forms the basis of in-vitro in-vivo correlation (IVIVC). Understanding the effect of bio-nano interactions on the biological consequences of NPs at the cellular and physiological level can have a direct impact on the translation of future nanomedicines and can lead to the ultimate goal of developing a mathematical IVIVC model. The review summarizes the emerging paradigms in the field of bio-nano-interface which clearly suggests an urgent need to revisit existing protocols in nanotechnology for defining the physicochemical correlates of bio-nano interactions.     

Identifying Metal Nanoparticle Size Effect on Sensing Common Human Plasma Protein by Counting the Sensitivity of Optical Absorption Spectra Damping

Colloidal nanoparticles (NPs) interact with biological fluids such as human plasma to form a protein coating (corona) on the surface of NPs (NP-protein complex). However, the impact of size and type of NPs on binding of the hard corona to the surface of NPs as well as damping of their optical spectra has not been systematically explored. To elucidate the interaction between biological environment (human plasma) and NPs, a photophysical measurement was conducted to quantify the interaction of two different types of NPs (gold (Au) and silver (Ag)) with common human plasma proteins. The colloidal AuNPs and AgNPs were electrostatically stabilized and varied in diameter from 10 to 80 nm in the presence of common human plasma. The sizes of the NPs were determined using transmission electron microscopy (TEM). Optical absorption spectra were obtained for the complexes. Dynamic light scattering (DLS) measurement and zeta potential were used to characterize the sizes, hydrodynamic diameters, and surface charges of the protein-NPs complexes. Protein separation was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to isolate and identify the protein bands. The absorption of proteins to the NPs was found to be strongly dependent on the size and type of NPs. The distance between surface of NPs by absorbed protein bound to the NPs gradually increased with size of NPs, particularly for AgNPs with primary diameter of < 50 nm. The chi-square test proved that AgNPs are a good candidate in sensing the protein complex in human plasma compared with AuNPs mainly for the AgNPs with diameter sized 50 nm.

     

Dispersion Behaviour of Silica Nanoparticles in Biological Media and Its Influence on Cellular Uptake

Given the increasing variety of manufactured nanomaterials, suitable, robust, standardized in vitro screening methods are needed to study the mechanisms by which they can interact with biological systems. The in vitro evaluation of interactions of nanoparticles (NPs) with living cells is challenging due to the complex behaviour of NPs, which may involve dissolution, aggregation, sedimentation and formation of a protein corona. These variable parameters have an influence on the surface properties and the stability of NPs in the biological environment and therefore also on the interaction of NPs with cells. We present here a study using 30 nm and 80 nm fluorescently-labelled silicon dioxide NPs (Rubipy-SiO₂ NPs) to evaluate the NPs dispersion behaviour up to 48 hours in two different cellular media either supplemented with 10% of serum or in serum-free conditions. Size-dependent differences in dispersion behaviour were observed and the influence of the living cells on NPs stability and deposition was determined. Using flow cytometry and fluorescence microscopy techniques we studied the kinetics of the cellular uptake of Rubipy-SiO₂ NPs by A549 and CaCo-2 cells and we found a correlation between the NPs characteristics in cell media and the amount of cellular uptake. Our results emphasize how relevant and important it is to evaluate and to monitor the size and agglomeration state of nanoparticles in the biological medium, in order to interpret correctly the results of the in vitro toxicological assays.     

The importance of selecting a proper biological milieu for protein corona analysis in vitro: Human plasma versus human serum

Nanoparticle (NP) exposure to biological fluids in the body results in protein binding to the NP surface, which forms a protein coating that is called the “protein corona”. To simplify studies of protein–NP interactions and protein corona formation, NPs are incubated with biological solutions, such as human serum or human plasma, and the effects of this exposure are characterized in vitro. Yet, how NP exposure to these two different biological milieus affects protein corona composition and cell response has not been investigated. Here, we explore the differences between the protein coronas that form when NPs are incubated in human serum versus human plasma. NP characterization indicated that NPs that were exposed to human plasma had higher amounts of proteins bound to their surfaces, and were slightly larger in size than those exposed to human serum. In addition, significant differences in corona composition were also detected with gel electrophoresis and liquid chromatography–mass spectrometry/mass spectrometry, where a higher fraction of coagulation proteins and complement factors were found on the plasma-exposed NPs. Flow cytometry and confocal microscopy showed that the uptake of plasma-exposed NPs was higher than that of serum-exposed NPs by RAW 264.7 macrophage immune cells, but not by NIH 3T3 fibroblast cells. This difference is likely due to the elevated amounts of opsonins, such as fibrinogen, on the surfaces of the NPs exposed to plasma, but not serum, because these components trigger NP internalization by immune cells. As the human plasma better mimics the composition of the in vivo environment, namely blood, in vitro protein corona studies should employ human plasma, and not human serum, so the biological phenomena that is observed is more similar to that occurring in vivo.     

Formation of a protein corona influences the biological identity of nanomaterials

The development and testing of nanomaterials is an area of interest due to promising diagnostic and therapeutic applications in the treatment of diseases like cancer or cardiovascular disease. While extensive studies of the physicochemical properties of nanoparticles (NPs) are available, the investigation of the protein corona (PC) that is formed on NPs in biofluids is a relatively new area of research. The fact that few NPs are in clinical use indicates that the biological identity of NPs, which is in large part due to the PC formed in blood or other bodily fluids, may be altered in ways yet to be fully understood. Herein, we review the recent advances in PC research with the intent to highlight the current state of the field. We discuss the dynamic processes that control the formation of the PC on NPs, which involve the transient soft corona and more stable hard corona. Critical factors, like the environment and disease-state that affect the composition and stability of the PC are presented, with the intent of showcasing promising applications for utilizing the PC for disease diagnosis and the identification of disease-related biomarkers. This review summarizes the unique challenges presented by the nanoparticle corona and indicates future directions for investigation.     

An integrative method for evaluating the biological effects of nanoparticle-protein corona

BackgroundNanoplastics in the environment can enter the human body through gastrointestinal intake, dermal contact, and pulmonary inhalation, posing a threat to human health. Protein molecules in body fluids will quickly adsorb on the surfaces of the nanoplastics, forming a protein corona, which has implications for the interaction of the nanoplastics with cells and the metabolic pathways of the nanoplastic within cells. For years, practical tools such as dynamic light scattering, transmission electron microscopy, and liquid chromatography have been developed to understand the protein corona of nanoparticles (NPs), either in vitro or in cellular or molecular level. However, an integrated approach to understand the nanoparticles-protein corona is still lacking.MethodsUsing the most frequently observed environmental nanoplastics, polystyrene nanoplastics (PS), as a standard, we established an integrative structural characterization platform, a biophysical and biochemical evaluation method to investigate the effect of surface charge on protein corona composition. The cellular and molecular mechanisms were also explored through in vitro cellular experiments.ResultsThe first integrative method for characterizing biological properties of NPs-protein corona has been established. This method comprehensively covers the critical aspects to understand NPs-protein corona interactions, from structure to function.ConclusionsThe integrative method for nanoplastics microstructure characterization can be applied to the structural characterization of nanoparticles in nanoscale, which is of universal significance from in vitro characterization to cellular experiments and then to molecular mechanism studies.General significanceThis strategy has high reliability and repeatability and can be applied both in environment and nanomedicine safety assessment.     

Intentional formation of a protein corona on nanoparticles: Serum concentration affects protein corona mass,surface charge,and nanoparticle–cell interaction

The protein corona, which immediately is formed after contact of nanoparticles and biological systems, plays a crucial role for the biological fate of nanoparticles. In the here presented study we describe a strategy to control the amount of corona proteins which bind on particle surface and the impact of such a protein corona on particle-cell interactions. For corona formation, polyethyleneimine (PEI) coated magnetic nanoparticles (MNP) were incubated in a medium consisting of fetal calf serum (FCS) and cell culture medium. To modulate the amount of proteins bind to particles, the composition of the incubation medium was varied with regard to the FCS content. The protein corona mass was estimated and the size distribution of the participating proteins was determined by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Additionally, the zeta potential of incubated particles was measured. Human blood–brain barrier-representing cell line HBMEC was used for in vitro incubation experiments. To investigate the consequences of the FCS dependent protein corona formation on the interaction of MNP and cells flow cytometry and laser scanning microscopy were used. Zeta potential as well as SDS–PAGE clearly reveal an increase in the amount of corona proteins on MNP with increasing amount of FCS in incubation medium. For MNP incubated with lower FCS concentrations especially medium-sized proteins of molecular weights between 30 kDa and 100 kDa could be found within the protein corona, whereas for MNP incubated within higher FCS concentrations the fraction of corona proteins of 30 kDa and less increased. The presence of the protein corona reduces the interaction of PEI-coated MNP with HBMEC cells within a 30 min-incubation.     

Chemical and biological properties of polysaccharide-coated titania nanoparticles: the key role of proteins

In this study, we investigated the physicochemical and biological properties of naked and coated titania nanoparticles. The aim of this study was to verify the effect of the biopolymer coatings (hyaluronic acid and its biphosphonated derivative) and the role of protein adsorption from a cell culture medium on the citotoxicity of nanoparticles. Infrared spectroscopy (FT-IR) was used to investigate the interactions between the nanoparticles and the polysaccharides. The ζ potentials and the average hydrodynamic diameters of naked and coated nanoparticles dispersed in deionized water, medium with and without fetal bovine serum, were measured by means of dynamic light scattering (DLS). FT-IR and DLS measurements indicate that serum proteins are adsorbed on the NPs' surface. The biological tests show that naked and coated TiO(2) NPs do not induce an acute toxic effect on fibroblast cell cultures. This result shows that protein adsorption on NPs is an important factor in explaining the effect of NPs on cellular behavior.     

Personalized liposome–protein corona in the blood of breast,gastric and pancreatic cancer patients

When nanoparticles (NPs) are dispersed in a biofluid, they are covered by a protein corona the composition of which strongly depends on the protein source. Recent studies demonstrated that the type of disease has a crucial role in the protein composition of the NP corona with relevant implications on personalized medicine. Proteomic variations frequently occur in cancer with the consequence that the bio-identity of NPs in the blood of cancer patients may differ from that acquired after administration to healthy volunteers. In this study we investigated the correlation between alterations of plasma proteins in breast, gastric and pancreatic cancer and the biological identity of clinically approved AmBisome-like liposomes as determined by a combination of dynamic light scattering, zeta potential analysis, one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) and semi-quantitative densitometry. While size of liposome–protein complexes was not significantly different between cancer groups, the hard corona from pancreatic cancer patients was significantly less negatively charged. Of note, the hard corona from pancreatic cancer patients was more enriched than those of other cancer types this enrichment being most likely due to IgA and IgG with possible correlations with the autoantibodies productions in cancer. Given the strict relationship between tumor antigen-specific autoantibodies and early cancer detection, our results could be the basis for the development of novel nanoparticle-corona-based screening tests of cancer.     

Do plasma proteins distinguish between liposomes of varying charge density?

Cationic liposomes (CLs) are one of the most employed nonviral nanovector systems in gene therapy. However, their transfection efficiency is strongly affected by interactions with plasma components, that lead to the formation of a “protein corona” onto CL surface. The interactions between nanoparticles entering the body and biomolecules have an essential role for their biodistribution. Because the knowledge of proteins adsorbed onto vector surface could be useful in the screening of new, more efficient and more biocompatible liposomal formulations, the behavior of three CLs with different membrane charge densities was investigated. The proteins of the three coronas were identified by nano-liquid chromatography-tandem mass spectrometry, and quantified with label-free spectral counting strategy. Fibrinogen displayed higher association with CLs with high membrane charge density, while apolipoproteins and C4b-binding protein with CLs with low membrane charge density. These results are discussed in terms of the different lipid compositions of CLs and may have a deep biological impact for in vivo applications. Surface charge of nanoparticles is emerging as a relevant factor determining the corona composition after interaction with plasma proteins. Remarkably, it is also shown that the charge of the protein corona formed around CLs is strongly related to their membrane charge density.     

Effect of protein corona magnetite nanoparticles derived from bread in vitro digestion on Caco-2 cells morphology and uptake

Nanoparticles (NPs) in biological fluids immediately interact with proteins forming a biomolecular corona (PC) that imparts their biological identity. While several studies on the formation of the PC in human plasma have been reported, the PC of orally administrated NPs has been less investigated, mostly in the presence of a food matrix. In fact, food matrixes when digested are subject of several dynamic changes that will certainly affect the PC formed on the NPs. The lack of studies on this topic is clearly related to the difficulty in isolating representative PC NPs from such a complex environment. In this work magnetite NPs were added to in vitro simulated digestion simultaneously with bread and PC NPs were isolated after gastric and duodenal phases by sucrose gradient ultracentrifugation (UC). The PC NPs were characterized in terms of size and protein composition. Translocation studies were then performed on Caco-2 monolayers in a serum free environment and cell morphology was characterized by confocal microscopy. PC NPs isolated from gastric and duodenal phases were different in size, surface charge and protein corona composition. NP cellular uptake was enhanced by the digestive PC inducing morphology changes in the cell monolayer. Overall, in this work we were able to isolate PC NPs from digested fluids in the presence of a food matrix and study their biological response on Caco-2 cells.     

Nanoparticle interactions with blood proteins and what it means: a tutorial review

Nanoparticles find many uses in medicine and biomedical technology. Such applications imply that they must be colloidally stable and do not interact with proteins in the blood or blood serum. A nanoparticle put into the blood will instantaneously be covered by a protein corona that compromises the function of the nanoparticle core, changes the effective size of the nanoparticle, and determines its biological fate. Strategies developed to gain control over nanoparticles in biological fluids, particular in blood, heavily rely on creating a hydrated polymer shell that sterically and osmotically prevents a protein corona from forming. In this tutorial review, we provide an overview of factors that affect the formation of the protein corona in blood and how to prevent it forming. We focus on describing the latest advances in our understanding of how small core-shell nanoparticles (core diameter 4-20 nm in diameter) with a shell of densely grafted polymer chains, a so-called polymer brush, interact with proteins and cells in vitro. Such nanoparticles are among the most well-defined and well-characterized colloids used for biomedical applications, from which an improved understanding of how nanoparticle architecture influences their biological fate can be obtained in detail.     

Modeling of a single nanoparticle interaction with the human blood plasma proteins

When nanoparticles are introduced into a physiological environment, proteins and lipids immediately cover their surface, forming a protein “corona”. It is well recognized that the corona structure influences the biological response of the body. Two deterministic models for corona formation of the human blood serum proteins around a single nanoparticle are presented and studied numerically in this paper. One of them is based on a coupled system of PDEs and involves diffusion of proteins toward the nanoparticle surface. The other one is described by ODEs and is a limit version of the first model as the protein diffusivity tends to infinity. The protein diffusivity influence on the temporal corona structure is studied in detail. Results are presented using figures and discussion.     

A hard-sphere model of protein corona formation on spherical and cylindrical nanoparticles

A nanoparticle (NP) immersed in biological media rapidly forms a corona of adsorbed proteins, which later controls the eventual fate of the particle and the route through which adverse outcomes may occur. The composition and timescale for the formation of this corona are both highly dependent on both the NP and its environment. The deposition of proteins on the surface of the NP can be imitated by a process of random sequential adsorption, and, based on this model, we develop a rate-equation treatment for the formation of a corona represented by hard spheres on spherical and cylindrical NPs. We find that the geometry of the NP significantly alters the composition of the corona through a process independent of the rate constants assumed for adsorption and desorption of proteins, with the radius and shape of the NP both influencing the corona. We further investigate the roles of protein mobility on the surface of the NP and changes in the concentration of proteins.     

A secreted protein microarray platform for extracellular protein interaction discovery

Characterization of the extracellular protein interactome has lagged far behind that of intracellular proteins, where mass spectrometry and yeast two-hybrid technologies have excelled. Improved methods for identifying receptor-ligand and extracellular matrix protein interactions will greatly accelerate biological discovery in cell signaling and cellular communication. These technologies must be able to identify low-affinity binding events that are often observed between membrane-bound coreceptor molecules during cell-cell or cell-extracellular matrix contact. Here we demonstrate that functional protein microarrays are particularly well-suited for high-throughput screening of extracellular protein interactions. To evaluate the performance of the platform, we screened a set of 89 immunoglobulin (Ig)-type receptors against a highly diverse extracellular protein microarray with 686 genes represented. To enhance detection of low-affinity interactions, we developed a rapid method to assemble bait Fc fusion proteins into multivalent complexes using protein A microbeads. Based on these screens, we developed a statistical methodology for hit calling and identification of nonspecific interactions on protein microarrays. We found that the Ig receptor interactions identified using our methodology are highly specific and display minimal off-target binding, resulting in a 70% true-positive to false-positive hit ratio. We anticipate that these methods will be useful for a wide variety of functional protein microarray users.     

Molecular insights on the interference of simplified lung surfactant models by gold nanoparticle pollutants

Inhaled nanoparticles (NPs) are experienced by the first biological barrier inside the alveolus known as lung surfactant (LS), a surface tension reducing agent, consisting of phospholipids and proteins in the form of the monolayer at the air-water interface. The monolayer surface tension is continuously regulated by the alveolus compression and expansion and protects the alveoli from collapsing. Inhaled NPs can reach deep into the lungs and interfere with the biophysical properties of the lung components. The interaction mechanisms of bare gold nanoparticles (AuNPs) with the LS monolayer and the consequences of the interactions on lung function are not well understood. Coarse-grained molecular dynamics simulations were carried out to elucidate the interactions of AuNPs with simplified LS monolayers at the nanoscale. It was observed that the interactions of AuNPs and LS components deform the monolayer structure, change the biophysical properties of LS and create pores in the monolayer, which all interfere with the normal lungs function. The results also indicate that AuNP concentrations >0.1 mol% (of AuNPs/lipids) hinder the lowering of the LS surface tension, a prerequisite of the normal breathing process. Overall, these findings could help to identify the possible consequences of airborne NPs inhalation and their contribution to the potential development of various lung diseases.     

Quantitative proteomics analysis of adsorbed plasma proteins classifies nanoparticles with different surface properties and size

Nanoparticle biological activity, biocompatibility and fate can be directly affected by layers of readily adsorbed host proteins in biofluids. Here, we report a study on the interactions between human blood plasma proteins and nanoparticles with a controlled systematic variation of properties using (18)O-labeling and LC-MS-based quantitative proteomics. We developed a novel protocol to both simplify isolation of nanoparticle bound proteins and improve reproducibility. LC-MS analysis identified and quantified 88 human plasma proteins associated with polystyrene nanoparticles consisting of three different surface chemistries and two sizes, as well as, for four different exposure times (for a total of 24 different samples). Quantitative comparison of relative protein abundances was achieved by spiking an (18)O-labeled "universal" reference into each individually processed unlabeled sample as an internal standard, enabling simultaneous application of both label-free and isotopic labeling quantification across the entire sample set. Clustering analysis of the quantitative proteomics data resulted in distinctive patterns that classified the nanoparticles based on their surface properties and size. In addition, temporal data indicated that the formation of the stable protein corona was at equilibrium within 5 min. The comprehensive quantitative proteomics results obtained in this study provide rich data for computational modeling and have potential implications towards predicting nanoparticle biocompatibility.     

Application of a fluorescent biosensor based‐on magneto‐γ‐Fe2O3–methyldopa nanoparticles for adsorption of human serum albumin

Understanding and controlling the interaction between the polymer methyldopa (2‐amino‐3‐(3,4‐dihydroxyphenyl)‐2‐methyl‐propanoic acid) (PMDP)–γ‐Fe₂O₃ nanoparticles and biological fluids is important if the potential of nanoparticles (NPs) in biomedicine is to be realized. Physicochemical studies on the interactions between proteins and NPs are influenced by the surface properties of the NPs. To identify the effects of the NP surface, interactions between human serum albumin (HSA) and PMDP–γ‐Fe₂O₃ NPs were investigated. Here, the adsorption of HSA onto small (10–30 nm diameter) PMDP–γ‐Fe₂O₃ NPs was quantitatively analyzed using spectroscopic methods. The fluorescence quenching data were checked for the inner‐filter effect, the main confounding factor in the observed quenching. The binding constants, K_a, were calculated at different temperatures, using a nonlinear fit to the experimental data, and the thermodynamic parameters ?H, ?S and ?G were given. The obtained thermodynamic signature suggests that hydrophobic interactions at least are present. This result indicates that the structure of the protein turns from a structureless denatured state at pH 3 into an ordered biologically active native state on addition of PMDP–γ‐Fe₂O₃ NPs. Copyright © 2015 John Wiley & Sons, Ltd.