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Characterization of the bionano interface and mapping extrinsic interactions of the corona of nanomaterials
Abstract
Nanoparticles in physiological environments are known to selectively adsorb proteins and other biomolecules forming a tightly bound biomolecular 'corona' on their surface. Where the exchange times of the proteins are sufficiently long, it is believed that the protein corona constitutes the particle identity in biological milieu. Here we show that proteins in the corona retain their functional characteristics and can specifically bind to cognate proteins on arrays of thousands of immobilised human proteins. The biological identity of the nanomaterial is seen to be specific to the blood plasma concentration in which they are exposed. We show that the resulting in situ nanoparticle interactome is dependent on the protein concentration in plasma, with the emergence of a small number of dominant protein-protein interactions. These interactions are those driven by proteins that are adsorbed onto the particle surface and whose binding epitopes are subsequently expressed or presented suitably on the particle surface. We suggest that, since specific tailored protein arrays for target systems and organs can be designed, their use may be an important element in an overall study of the biomolecular corona.
... The principle of the method was based on the evaluation of the adsorption of individual proteins and the establishment of a map of surface adsorption/exclusion assumed to reflect the surface coverage property of the tested NM. The choice of proteins to probe surface properties of NM was also consistent with their key role in mechanisms controlling the in vivo activity of these compounds [37,40,41]. The proposed method was designed to provide a simple model evaluating NM surface functionality that can be implemented in routine to be used in quality control of NMs. ...
... The profile of adsorbed proteins deduced from this analysis determines the so called "biological identity" of the nanomaterial [42]. It is very sensitive to the surface coverage properties of the nanomaterial and its determination raised interests to develop predictive models for the in vivo behavior of NMs including their cellular uptake, stability and biodistribution [41,[43][44][45]. The method would provide a screenshot of what happens after NM's injection into the blood [41,42,[46][47][48]. ...
... It is very sensitive to the surface coverage properties of the nanomaterial and its determination raised interests to develop predictive models for the in vivo behavior of NMs including their cellular uptake, stability and biodistribution [41,[43][44][45]. The method would provide a screenshot of what happens after NM's injection into the blood [41,42,[46][47][48]. However, currently, methods allowing the establishment of the protein corona are based on the most advanced techniques applied in proteomic analysis using last generation of liquid chromatography -mass spectrometry (LC-MS) instruments. ...
A faithful characterization of nanomedicine (NM) is needed for a better understanding of their in vivo outcomes. Size and surface charge are studied with well-established methods. However, other relevant parameters for the understanding of NM behavior in vivo remain largely inaccessible. For instance, the reactive surface of nanomedicines, which are often grafted with macromolecules to decrease their recognition by the immune system, is excluded from a systematic characterization. Yet, it is known that a subtle modification of NM's surface characteristics (grafting density, molecular architecture and conformation of macromolecules) is at the root of major changes in the presence of biological components. In this work, a method that investigates the steric hindrance properties of the NMs' surface coverage based on its capacity to exclude or allow adsorption of well-defined proteins was developed based on capillary electrophoresis. A series of proteins with different molecular weights (MW) were used as molecular probes to screen their adsorption behavior on nanoparticles bearing different molecular architectures at their surface. This novel strategy evaluating to some degree a functionality of NMs can bring additional information about their shell property and might allow for a better perception of their behavior in the presence of biological components. The developed method could discriminate NM with a high surface coverage excluding high MW proteins from NM with a low surface coverage that allowed high MW proteins to adsorb on their surface. The method has the potential for further standardization and automation for a routine use. It can be applied in quality control of NMs and to investigate interactions between proteins and NM in different situations.
... The so-called "protein corona" was also shown to influence nanomedicines' targeting properties and affect particle distribution inside the cells (Bertoli et al., 2016;Mahon, Salvati, Baldelli Bombelli, Lynch, & Dawson, 2012;Wang et al., 2013). The majority of studies investigating the relationship between nanoparticles, protein corona, and particle distribution to and inside of the cells used "hard" nanomaterials, such as colloidal metals, metal oxides, and silica (Bertoli et al., 2014(Bertoli et al., , 2016Deville et al., 2016;Dobrovolskaia et al., 2014;Gibbons et al., 2015;Kelly et al., 2015;Maiolo et al., 2014;O'Connell et al., 2015;Wan et al., 2015;Wang et al., 2013). Studies of "soft" materials such as liposomes and dendrimers are limited to total protein binding due to the nuances in methodologies currently available for separating nanoparticle-bound proteins from the bulk plasma (Ilinskaya & Dobrovolskaia, 2016). ...
... Bertoli et al., 2014;Bertoli, Garry, Monopoli, Salvati, & Dawson, 2016;Deville et al., 2016;Gibbons et al., 2015;Kelly et al., 2015;Maiolo, Bergese, Mahon, Dawson, & Monopoli, 2014;O'Connell et al., 2015;Wan et al., 2015; ...
Gene therapeutics including siRNAs, anti‐sense oligos, messenger RNAs, and CRISPR ribonucleoprotein complexes offer unmet potential to treat over 7,000 known genetic diseases, as well as cancer, through targeted in vivo modulation of aberrant gene expression and immune cell activation. Compared with viral vectors, nonviral delivery vectors offer controlled immunogenicity and low manufacturing cost, yet suffer from limitations in toxicity, targeting, and transduction efficiency. Structured DNA assemblies fabricated using the principle of scaffolded DNA origami offer a new nonviral delivery vector with intrinsic, yet controllable immunostimulatory properties and virus‐like spatial presentation of ligands and immunogens for cell‐specific targeting, activation, and control over intracellular trafficking, in addition to low manufacturing cost. However, the relative utilities and limitations of these vectors must clearly be demonstrated in preclinical studies for their clinical potential to be realized. Here, we review the major capabilities, opportunities, and challenges we foresee in translating these next‐generation delivery and vaccine vectors to the clinic.
This article is categorized under:
Therapeutic Approaches and Drug Discovery > Emerging Technologies
Biology‐Inspired Nanomaterials > Nucleic Acid‐Based Structures
Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease
Abstract
Virus‐like nucleic acid nanoparticles can be programmed to deliver and release CRISPR RNPs, HDR templates, and siRNAs either alone, or in combination, for next‐generation in vivo gene therapeutic applications.
... Thus, while it is clear that nanomaterials may adsorb proteins when incubated in FBS-containing cell culture medium 49 , and while this layer of proteins may impact on cell uptake or cytotoxicity, this does not mean that the material itself is unavailable for interactions with cell surface receptors. Recent work has shown that the protein corona composition on the surface of nanoparticles changes with increasing plasma concentrations 50,51 , and it was suggested that while the surface is "relatively well covered" when nanoparticles are immersed in 10% plasma, the so-called 'hard' corona continues to evolve at increasing concentrations of plasma 50 . In addition, in a previous study of dextran-coated superparamagnetic iron oxide, Simberg et al. 52 reported that both the dextran coating and the iron oxide core remained accessible to specific probes after incubation in plasma, suggesting that the nanoparticle surface could be available for recognition by macrophages, regardless of protein coating. ...
... Thus, while it is clear that nanomaterials may adsorb proteins when incubated in FBS-containing cell culture medium 49 , and while this layer of proteins may impact on cell uptake or cytotoxicity, this does not mean that the material itself is unavailable for interactions with cell surface receptors. Recent work has shown that the protein corona composition on the surface of nanoparticles changes with increasing plasma concentrations 50,51 , and it was suggested that while the surface is "relatively well covered" when nanoparticles are immersed in 10% plasma, the so-called 'hard' corona continues to evolve at increasing concentrations of plasma 50 . In addition, in a previous study of dextran-coated superparamagnetic iron oxide, Simberg et al. 52 reported that both the dextran coating and the iron oxide core remained accessible to specific probes after incubation in plasma, suggesting that the nanoparticle surface could be available for recognition by macrophages, regardless of protein coating. ...
Carbon-based nanomaterials including carbon nanotubes (CNTs) have been shown to trigger inflammation. However, how these materials are 'sensed' by immune cells is not known. Here we compared the effects of two carbon-based nanomaterials, single-walled CNTs (SWCNTs) and graphene oxide (GO), on primary human monocyte-derived macrophages. Genome-wide transcriptomics assessment was performed at sub-cytotoxic doses. Pathway analysis of the microarray data revealed pronounced effects on chemokine-encoding genes in macrophages exposed to SWCNTs, but not in response to GO, and these results were validated by multiplex array-based cytokine and chemokine profiling. Conditioned medium from SWCNT-exposed cells acted as a chemoattractant for dendritic cells. Chemokine secretion was reduced upon inhibition of NF-κB, as predicted by upstream regulator analysis of the transcriptomics data, and Toll-like receptors (TLRs) and their adaptor molecule, MyD88 were shown to be important for CCL5 secretion. Moreover, a specific role for TLR2/4 was confirmed by using reporter cell lines. Computational studies to elucidate how SWCNTs may interact with TLR4 in the absence of a protein corona suggested that binding is guided mainly by hydrophobic interactions. Taken together, these results imply that CNTs may be 'sensed' as pathogens by immune cells.
... These biological materials include proteins, peptides, lipids, nucleic acids, metabolites, and others. The association of these biomolecules is dependent on a number of factors including the physicochemical properties of the nanoparticle, the physiological environment, and the duration of incubation [1,2]. This collection of biomolecules on the nanoparticle's surface is collectively referred to as the biocorona. ...
Formation of the biocorona on the surface of nanoparticles is a significant obstacle for the development of safe and effective nanotechnologies, especially for nanoparticles with biomedical applications. Following introduction into a biological environment nanoparticles are rapidly coated with biomolecules resulting in formation of the nanoparticle-biocorona. The addition of these biomolecules alters the nanoparticle’s physicochemical characteristics, functionality, biodistribution, and toxicity. To synthesize effective nanotherapeutics and to more fully understand possible toxicity following human exposures it is necessary to elucidate these interactions between the nanoparticle and the biological media resulting in biocorona formation. A thorough understanding of the mechanisms by which the addition of the biocorona governs nanoparticle-cell interactions is also required. Through elucidating the formation and the biological impact of the biocorona, the field of nanotechnology can reach its full potential. This understanding of the biocorona will ultimately allow for more effective laboratory screening of nanoparticles and enhanced biomedical applications. The importance of the nanoparticle-biocorona has been appreciated for a decade however there remain numerous future directions for research which are necessary for study. This perspectives article will summarize the unique challenges presented by the nanoparticle-biocorona and avenues of future needed investigation.
... In this context, numerous studies have been carried out to investigate the composition of the corona formed around many types of NPs. [5][6][7][8][9][10][11][12][13][14][15][16][17][18] In most of these studies, tandem mass spectrometry and shotgun proteomics has proved to be a powerful and indispensable tool to obtain large datasets. ...
In order to direct nanocarriers to their targets efficiently, we have to understand the interactions occuring at the nano-bio interface between nanocarriers and human proteins, which forms the layer called the corona. However, experiments aiming to identify and quantify the proteins in the corona, especially critical steps in the separation of nanoparticles from biological media may affect the corona composition. Here, we used nano-LC MS/MS to compare the protein corona contents obtained after using two different separation methods. We showed that applying centrifugation versus magnetization to isolate nanoparticles surrounded by a corona resulted in protein loss and a reshuffling of their respective abundances.
... Similarly, in another work, QCM was also used to screen for nanoparticle binding to cells directly grown on the sensor surface (Gianneli et al., 2017). Another interesting approach to identify all epitopes exposed on the corona was reported by O'Connell et al. (2015), who FIGURE 4 | Mapping of receptor binding motifs on the biomolecular corona and identification of receptors mediating uptake by cells. Schematic illustration of epitope mapping of ApoB-100 on the biomolecular corona of SiO 2 nanoparticles by 5 nm immunogold nanoparticles conjugated with antibody anti-ApoB100 (A). ...
Nanoparticles are promising tools for nanomedicine in a wide array of therapeutic and diagnostic applications. Yet, despite the advances in the biomedical applications of nanomaterials, relatively few nanomedicines made it to the clinics. The formation of the biomolecular corona on the surface of nanoparticles has been known as one of the challenges toward successful targeting of nanomedicines. This adsorbed protein layer can mask targeting moieties and creates a new biological identity that critically affects the subsequent biological interactions of nanomedicines with cells. Extensive studies have been directed toward understanding the characteristics of this layer of biomolecules and its implications for nanomedicine outcomes at cell and organism levels, yet several aspects are still poorly understood. One aspect that still requires further insights is how the biomolecular corona interacts with and is “read” by the cellular machinery. Within this context, this review is focused on the current understanding of the interactions of the biomolecular corona with cell receptors. First, we address the importance and the role of receptors in the uptake of nanoparticles. Second, we discuss the recent advances and techniques in characterizing and identifying biomolecular corona-receptor interactions. Additionally, we present how we can exploit the knowledge of corona-cell receptor interactions to discover novel receptors for targeting of nanocarriers. Finally, we conclude this review with an outlook on possible future perspectives in the field. A better understanding of the first interactions of nanomaterials with cells, and -in particular -the receptors interacting with the biomolecular corona and involved in nanoparticle uptake, will help for the successful design of nanomedicines for targeted delivery.
... In designing NPs to interact with proteins, it is important to consider how NP size, surface chemistry, and shape impact the protein's ability to remain folded and its likelihood to bind to protein regions distant from the active site. Many investigations have been conducted with the aim of learning how to manipulate the ability of NPs to change protein structure and flexibility [13][14][15][16][17][18] . For instance, Jonsson and his colleagues 19 found that smaller nanoparticles (NPs) have weaker ability to denature proteins than larger ones. ...
The ability of nanoparticles to alter protein structure and dynamics plays an important role in their medical and biological applications. We investigate allosteric effects of gold nanoparticles on human serum albumin protein using molecular simulations. The extent to which bound nanoparticles influence the structure and dynamics of residues distant from the binding site is analyzed. The root mean square deviation, root mean square fluctuation and variation in the secondary structure of individual residues on a human serum albumin protein are calculated for four protein-gold nanoparticle binding complexes. The complexes are identified in a brute-force search process using an implicit-solvent coarse-grained model for proteins and nanoparticles. They are then converted to atomic resolution and their structural and dynamic properties are investigated using explicit-solvent atomistic molecular dynamics simulations. The results show that even though the albumin protein remains in a folded structure, the presence of a gold nanoparticle can cause more than 50% of the residues to decrease their flexibility significantly, and approximately 10% of the residues to change their secondary structure. These affected residues are distributed on the whole protein, even on regions that are distant from the nanoparticle. We analyze the changes in structure and flexibility of amino acid residues on a variety of binding sites on albumin and confirm that nanoparticles could allosterically affect the ability of albumin to bind fatty acids, thyroxin and metals. Our simulations suggest that allosteric effects must be considered when designing and deploying nanoparticles in medical and biological applications that depend on protein-nanoparticle interactions.
... Our suggestion is supported by recent literature showing that LDL-enriched NPs present functional motifs that allow recognition by LDL receptor 38 . Our future biological understanding of how NPs interact with cells will confirm these conclusions more in depth by benefiting from a detailed knowledge of the arrangements of functional motifs of BC components at the nanoscale [39][40][41] . Figure 4 summarizes our present understanding of the role played by BC on the MC liposome association with HeLa cells. ...
Following exposure to biological milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomolecular corona (BC) that defines many of their biological outcomes, such as the elicited immune response, biodistribution, and targeting capabilities. In spite of this, the BC role in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulation, biological fluid, and target cell (respectively). By confocal fluorescence microscopy experiments and image correlation analyses, we quantitatively demonstrate that the BC promotes a neat switch of cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano liquid chromatography tandem mass spectrometry identifies Apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in clathrin-enriched membrane domains. Our results highlight the crucial role of BC as an intrinsic trigger of specific NP-cell interactions and biological responses and set the basis for a rational exploitation of the BC for targeted delivery.
... Recently, O'Connell et al. have shown the importance of using protein interaction databases in the investigation of the corona around polystyrene nanoparticles by using a protein microarray. 51 Thus, in the current study, we infer that NP-protein and protein-protein interactions are concomitant phenomena, driven by both physicochemical and biological properties. The emerging science of networks now allows both types of influences to be taken into account. ...
Magnetic mesoporous silica nanoparticles (M-MSNs) represent promising targeting tools for theranostics. Engineering the interaction of nanoparticles (NPs) with biological systems requires an understanding of protein corona formation around the nanoparticles as this drives the biological fate of nanocarriers. We investigated the behavior of proteins in contact with M-MSNs by high-throughput comparative proteomics, using human and bovine sera as biological fluids, in order to assess the adsorption dynamics of proteins in these media. Using systems biology tools, and especially protein–protein interaction databases, we demonstrated how the protein network builds up within the corona over the course of the experiment. Based on these results, we introduce and discuss the role of the “corona interactome” as an important factor influencing protein corona evolution. The concept of the "corona interactome" is an original methodology which could be generalized to all NPs candidates. Based on this, pre-coating nanocarriers with specific proteins presenting minimal interactions with opsonins might provide them properties such as stealth.
... In order to make a durable immobilized FR coating on fabric, we exploited the ability of aminoacidic biomacromolecules to irreversibly coat nanoparticles (NPs) according to the protein corona theory (Lynch and Dawson 2008;O'Connell et al. 2015), as well as the affinity of metal oxide NPs towards naturally hydrophilic fibers, as demonstrated by ceramization processes reported in our previous works (Ortelli et al. 2014(Ortelli et al. , 2015. The design strategy adopted in this work is schematically shown in Fig. 1. ...
A novel durable intumescent flame retardant coating, based on metal oxide nanoparticles (NPs) and biomacromolecules, was designed and applied on cotton fabrics. Specifically, different TiO2 NPs/proteins systems were deposited by dip-pad-dry-cure process and the morphology of the resulting coating were assessed by SEM analysis. Enhancement of durability (i.e. resistance to washing treatments) was verified by release tests carried out in static and dynamic conditions. Flammability and cone calorimetry tests were performed for evaluating the fire behavior of the treated fabrics. More specifically, in horizontal flame spread tests, the different nanoparticle/protein based coatings provided an increase of the total burning time and a decrease of the burning rate. Furthermore, the residues at the end of the test were significantly higher with respect to untreated cotton fabric. In particular, casein-based systems seemed to be more effective as compared to the whey proteins counterparts. Cone calorimetry tests showed better fire performances for the coatings based on TiO2/caseins with respect to TiO2/whey proteins, which did not seem to be so effective in protecting the underlying fabric from the heat flux. Therefore, due to their high char-forming character, casein-based coatings may represent an effective and durable fire-resistant finishing alternative to standard flame retardant treatments for cotton.
... For this purpose, various analytical methods have been developed to characterize the protein corona [36][37][38]. Using these methods, the absorbed protein profiles of nanoparticles with different characteristics have been compared, revealing that certain particle properties result in unique patterns of protein binding [39][40][41]. These and other studies have demonstrated that nanoparticle characteristics have a profound impact on the composition of the protein corona [30], which has spurred the notion that nanoparticles could be engineered to favor binding of specific biomolecules. ...
Introduction: The ultimate goal in the field of drug delivery is to exclusively direct therapeutic agents to pathological tissues in order to increase therapeutic efficacy and eliminate side effects. This goal is challenging due to multiple transport obstacles in the body. Strategies that improve drug transport exploit differences in the characteristics of normal and pathological tissues. Within the field of oncology, these concepts have laid the groundwork for a new discipline termed transport oncophysics.
Areas covered: Efforts to improve drug biodistribution have mainly focused on nanocarriers that enable preferential accumulation of drugs in diseased tissues. A less common approach to enhance drug transport involves priming strategies that modulate the biological environment in ways that favor localized drug delivery. This review discusses a variety of priming and nanoparticle design strategies that have been used for drug delivery.
Expert opinion: Combinations of priming agents and nanocarriers are likely to yield optimal drug distribution profiles. Although priming strategies have yet to be widely implemented, they represent promising solutions for overcoming biological transport barriers. In fact, such strategies are not restricted to priming the tumor microenvironment but can also be directed toward healthy tissue in order to reduce nanoparticle uptake.
... In the past decades, nanotechnology has shown great potentials in the biomedical fields [9][10][11]. Recently, more attention has been paid to nano-bio interactions, such as nano-protein and nano-cell interactions [12][13][14][15][16][17]. These interactions are of noncovalent bindings, including the electrostatic force, the hydrophobic interaction and the hydrogen bonding [18,19], and have been shown to induce protein adsorption and cytotoxicity [20][21][22]. ...
Aim:
Recently, nano-bio interactions and their biomedical impacts have drawn much attention, but nano-bacteria interaction and its function are unknown. Herein, we aim to synthesize drug-free and cationic nanoparticles (CNPs) and investigate CNP-bacteria interaction and its antibiofilm effect.
Materials & methods:
The bioactivity of CNPs against Streptococcus mutans was examined by colony-forming units counting and scanning electron microscopy. CNP-bacteria interaction force was measured by atomic force microscopy.
Results:
CNPs (217.7 nm, 14.7 mv) showed a concentration-dependent activity against bacteria. Particularly, CNPs at 200 μg/ml completely inhibited planktonic bacterial growth and biofilm formation, and disrupted ∼70% mature biofilm. CNP-bacteria interaction force was up to 184 nN.
Conclusion:
CNPs have great potentials for convenient local use for prevention and treatment of bacteria-related oral diseases.
... In fact, by increasing their pharmaceutical bioavailability and targeting, NPs avoid systemic toxicity while retaining pharmacological activity. 1 After intravenous injection, NPs adsorb a multitude of biological molecules (proteins, lipids, glycans, and metabolites) from plasma on their surface. These biomole cules continuously recycle 2,3 on the NP surface, thereby forming a dynamic shell known as a "biomolecular corona." 4 Most research studying the biomolecular corona has focused on protein composition characterization: 5,6 therefore, the biomolecular corona is generally called the "protein corona" (PC). 7,8 This natural coating provides NPs with a new biological identity and significantly affects their fate, pharmacokinetics, and cellular interactions. ...
A thorough understanding of interactions occurring at the interface between nanocarriers and biological systems is crucial to predict and interpret their biodistribution, targeting, and efficacy, and thus design more effective drug delivery systems. Upon intravenous injection, nanoparticles are coated by a protein corona (PC). This confers a new biological identity on the particles that largely determines their biological fate. Liposomes have great pharmaceutical versatility, so, as proof of concept, their PC has recently been implicated in the mechanism and efficiency of their internalization into the cell. In an attempt to better understand the interactions between nanocarriers and biological systems, we analyzed the plasma proteins adsorbed on the surface of multicomponent liposomes. Specifically, we analyzed the physical properties and ultrastructure of liposome/PC complexes and the aggregation process that occurs when liposomes are dispersed in plasma. The results of combined confocal microscopy and flow cytometry experiments demonstrated that the PC favors liposome internalization by both macrophages and tumor cells. This work provides insights into the effects of the PC on liposomes’ physical properties and, consequently, liposome-liposome and liposome-cell interactions.
... Recent reports have shed light on the complexity of these interactions due to non-specic associations of the NP surface with a variety of molecular entities that result in a biomolecular corona. 3,4 Because of these interactions, the control over the colloidal stability is oen compromised that leads to undesirable aggregation and localization of NP. 5 Additionally, the surface of most nanoscale imaging platforms, such as quantum dots, carbon nanotubes and plasmonic contrast agents, are functionalized with antibodies, peptides or aptamers to enable recognition of specic cell surface receptors. 6,7 However, the appearance of the corona alters the physico-chemical properties of the NP adversely inuencing cellular recognition and targeting specicity. ...
Developing effective theranostic nanoplex platforms for personalized disease treatment necessitates an understanding of and the ability to control live cell-nanoparticle interactions. However, aggregation of nanoparticles on the cell surface and their subsequent internalization is sparsely understood and adversely impact cellular recognition and viability. Here we report a facile method of precisely modulating the aggregation and uptake for silver nanoparticles without altering their surface geometry or functionalization. Exploiting the stabilization properties of trehalose, our approach enables uptake of nanoparticles while reducing aggregation on cell surface and maintaining cell viability. Electron microscopy reveals the larger utilization of endosomal structures in the trehalose-rich environment compared to the nanoparticles’ “free” cytosolic diffusion patterns in the control group. Additionally, in the presence of trehalose, plasmon-enhanced Raman spectroscopy confirms the preservation of the protein structure in the vicinity of the nanoparticles reinforcing the promise of the proposed route for label-free, multiplexed intracellular monitoring.
... It should be noted that it may also be the soft corona proteins (not investigated in this study) that lead to the increased cell uptake properties of TMV in cMEM. Future studies could set out to identify the pool of coronal interaction partners (so called interactome), [ 47,48 ] but this is beyond the present work. ...
Biomolecules in bodily fluids such as plasma can adsorb to the surface of nanoparticles and influence their biological properties. This phenomenon, known as the protein corona, is well established in the field of synthetic nanotechnology but has not been described in the context of plant virus nanoparticles (VNPs). The interaction between VNPs derived from Tobacco mosaic virus (TMV) and plasma proteins is investigated, and it is found that the VNP protein corona is significantly less abundant compared to the corona of synthetic particles. The formed corona is dominated by complement proteins and immunoglobulins, the binding of which can be reduced by PEGylating the VNP surface. The impact of the VNP protein corona on molecular recognition and cell targeting in the context of cancer and thrombosis is investigated. A library of functionalized TMV rods with polyethylene glycol (PEG) and peptide ligands targeting integrins or fibrin(ogen) show different dispersion properties, cellular interactions, and in vivo fates depending on the properties of the protein corona, influencing target specificity, and non-specific scavenging by macrophages. Our results provide insight into the in vivo properties of VNPs and suggest that the protein corona effect should be considered during the development of efficacious, targeted VNP formulations.
... Entry of NPs into an environment leads to the absorption of reactive molecules present in that environment onto the NP surface. If NPs enter a biological system, the adsorbed molecules form a bio-corona, which is a dynamic system that can include proteins, peptides, lipids, nucleic acids, and different metabolites [46]. The composition of the bio-corona is primarily dependent on the physicochemical properties of the NPs, the chemical composition of the nano-biointerface, and the duration of incubation [47]. ...
Background:
The surface of a nanoparticle adsorbs molecules from its surroundings with a specific affinity determined by the chemical and physical properties of the nanomaterial. When a nanoparticle is exposed to a biological system, the adsorbed molecules form a dynamic and specific surface layer called a bio-corona. The present study aimed to identify the metabolites that form the bio-corona around anatase TiO2 nanoparticles incubated with leaves of the model plant Arabidopsis thaliana.
Results:
We used an untargeted metabolomics approach and compared the metabolites isolated from wild-type plants with plants deficient in a class of polyphenolic compounds called flavonoids.
Conclusions:
These analyses showed that TiO2 nanoparticle coronas are enriched for flavonoids and lipids and that these metabolite classes compete with each other for binding the nanoparticle surface.
... Their findings can be used to describe the biological identity of various nanoparticles at the molecular level. In an in situ study, O'Connell et al. (2015) proposed deployment of protein arrays which are specifically designed to target particular systems and organs in order to study the biomolecular corona. They showed that the protein concentration in plasma and a small quantity of dominant protein-protein interactions are the major factors governing the nanoparticle interactome. ...
Nanoparticles can adsorb diverse type of biomolecules (e.g., proteins) when being in contact with biological fluids. Surface properties of the coated nanoparticles, in terms of type and amount of associated proteins, dictate their interactions with biological systems. In this perspective, we will focus on the recent advances and pitfalls in the field of protein corona.
... A simple method to predict protein-NP interactions before performing more complex in vivo experiments is to expose NPs to biological solutions obtained from blood ex vivo and characterize the consequences in vitro. Previous studies have shown that the concentration of these biological solutions affects protein corona composition, which in turn impacts the NPs' interactions with biomolecules, their cytotoxicity, and cellular uptake (Kim et al., 2014;O'Connell et al., 2015). However, little attention had been paid to whether the identity of the solution used to create the protein corona, namely serum versus plasma, affects the NPs' physical properties and cellular uptake. ...
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 opsinins, such as fibrinogen, on the surfaces of the NPs exposed to plasma, but not serum, because these components trigger NP internalization by immune cells. Because 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.
... Higher Ce content at the root surface has Considering that the NPs can be adsorbed (Cui et al., 2018) and the presence of mucilage on the root epidermis, we can assume that the particles and the sorbed ions are physically bound to the roots mucilage. The organic molecules exudated by roots can adsorb on the CeO 2 NPs surface, forming a bio-corona that may modulate their interaction with the root (O'Connell et al., 2015;Walczyk et al., 2010). The composition of the corona is primarily dependent on the physicochemical properties of NPs and the surrounding interface (Kurepa et al., 2020), and might change the NPs dissolution properties, affecting its stabilization and behavior. ...
This study evaluated the uptake and translocation of cerium nanoparticles (CeO2 NPs) and soluble
Ce(NO3)3 by soybean plants (Glycine max L. Merrill) under the whole plant life-cycle and relevant
environmental concentrations, 0.062 and 0.933 mg kg�1, which represent maximal values for 2017 in
agricultural soils and sludge treated soils, respectively. The experiments were carried out using a nutrient
solution. Cerium was detected in the soybean roots epidermis and cortex, leaves, and grains, but it
neither impaired plant development nor grain yield. The concentration of Ce in the shoot increased as a
function of time for plants treated with Ce(NO3)3, while it remained constant for plants treated with
CeO2 NPs. It means that CeO2 NPs were absorbed in the same rate as biomass production, which suggests
that they are taken up and transported by water mass flow. Single-particle inductively coupled plasma
mass spectrometry revealed clusters of CeO2 NPs in leaves of plants treated with 25 nm CeO2 NPs (ca. 30
e45 nm). The reprecipitation of soluble cerium from Ce(NO3)3 within the plant was not confirmed.
Finally, bioconcentration factors above one were found for the lowest concentrated treatments. Since
soybean is a widespread source of protein for animals, we draw attention to the importance of evaluating
the effects of Ce entrance in the food chain and its possible biomagnification
Silica nanoparticles (SiO2-NPs) has been extensively exploited in biomedical fields and mostly designed to enter the circulatory system, however, few studies focused on the potential adverse effects of SiO2-NPs exposure on the blood-brain barrier (BBB) that serves as a critical barrier between the central nervous system (CNS) and the peripheral circulation. This study attempts to provide an understanding of whether and how SiO2-NPs disrupts the BBB in vitro and in vivo. Through a human BBB model, we found that SiO2-NPs could induce tight junction loss and cytoskeleton arrangement, and increase inflammatory response and the release of vascular endothelial growth factor (VEGF) of brain microvessel endothelial cells (BMECs), which further activates astrocytes to amplify the generation of VEGF and increase the aquaporin-4 expression, and thus causing BBB disruption through a complex immunoregulatory loop between BMECs and astrocytes under SiO2-NPs exposure. Additionally, our data show that inhibition of reactive oxygen species (ROS) and Rho-kinase (ROCK) could effectively protect the SiO2-NPs-induced BBB dysfunction. In vivo studies further confirmed that SiO2-NPs could cause the BBB paracellular opening, oxidative stress and astrocyte activation in brains of Sprague-Dawley (SD) rats. These findings demonstrate that SiO2-NPs could disturb BBB structure and function and induce BBB inflammation, and suggest that these effects may occur through ROS and ROCK-mediated pathways, which not only improve neurotoxicity evaluation for SiO2-NPs but also provide useful information in development of SiO2-NPs in neuro-therapeutics and nanodiagnostics.
Despite colloidal gold nanoparticles (AuNP) being proposed for a multitude of biomedical applications, there is a lack of understanding on how the protein corona (PC) formation over AuNP influences its interaction with blood components. Herein, 40 and 80 nm AuNP with branched polyethylenimine, lipoic acid, and polyethylene glycol surface coatings were exposed to human plasma, and time-dependent evolution of the PC was evaluated using differential centrifugation sedimentation. Further, the impact of PC-AuNP interaction with human blood components was studied by evaluating red blood cell (RBC) aggregation, hemolysis, platelet activation and aggregation, prothrombin time, activated partial thromboplastin time, complement activation and cytokine release. In contrast to bare AuNP, PC-coated AuNP exhibited enhanced compatibility with RBC, platelets, and lymphocytes. More importantly, PC-AuNP did not activate the platelet coagulation cascade or complement system or elicit an immune response up to a relatively higher dose of 100 μg/mL. This study suggests that, irrespective of the physicochemical properties, the adsorption of the PC over AuNP significantly influences its biological impact by alleviating adverse hematotoxicity of bare NP.
Nanoparticles (NPs) are extensively being used in modern nano-based therapies and nanoprotein formualtions.The exposure to these comprehensively used NPs persuade changes in protein structure and functionality, hence raises grave health issues. In this study, we thoroughly investigated the interaction and adsorption of bovine serum albmin (BSA) with CoFe2O4 NPs by Circular dichroism(CD), fourier transform infrared (FTIR), absorption, fluorescence spectroscopic techniques, scanning electron microscopy (SEM), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), thermogravimetric analysis (TGA) and dynamic light scattering (DLS), respectively. The results corroborate, the CoFe2O4 NPs cause fluorescence quenching in BSA by static quenching mechanism. The negative values of van’t Hoff thermodynamic expressions (ΔH, ΔS and ΔG) corroborates the spontaniety and exothermic nature of static quenching. The major contributers of higher binding affinity among CoFe2O4 NPs with BSA were van der Waals forces and hydrogen bonding. Furthermore, BSA protein corona formation on CoFe2O4 NPs was confirmed by SEM, TGA , DLS and zeta potential studies. TGA, DLS and zeta potential results confirmed the formation of thick layer of BSA on CoFe2O4 NPs with negative boost in zeta potential. This coating of BSA over CoFe2O4 NPs leads to decrease the magnetic saturation value from 50.4 to 46.2 emu, hence the magnetic character of CoFe2O4 NPs. The development of protein corona on CoFe2O4 NPs was further estimated by comparing the steady state fluorescnec quenching and theoretical data. In addition, FTIR, UV-CD, UV–vis spectroscopy and three dimensional fluorescence (3D) techniques confirmed that CoFe2O4 NPs binding could induce microenvironment perturbations leading to secondary and tertiary conformation changes in BSA. Furthermore, synchronous fluorescence spectroscopy (SFS) affirmed the significant changes in microenvironment around trytophan (typ) residue by CoFe2O4 NPs. The denaturation of BSA biochemistry by CoFe2O4 NPs was advanced by assaying esterase activity.
Nanotechnology has a potential to transform healthcare by improving the quality of drugs and medical devices. The benefits of using nanomaterials in medical devices include improving device durability, decreasing bacterial adhesion and biofilm formation, and providing slow and controlled release of device-associated drugs. Using nanoparticles as implantable materials and components of medical devices also poses some safety concerns and regulatory challenges. This chapter reviews hematological and immunological toxicities relevant to the nanomaterials used in medical devices. Regulatory challenges, translational considerations, and literature case studies pertinent to the immunological safety of nanotechnology-based devices are also discussed.
Protein coronas incorporate with nanoparticles (NPs) are now becoming a new trend in research and can introduce novel applications in various fields and majorly used in biomedical aspect. The unique features presented by protein coronas can be exploited in the design of the nanomaterial, rather than combat their adsorption. Uncontrolled protein coronation may lead to cytotoxicity, a reduced blood circulation half-life, and nonspecific targeting to a diseased cell. However, a comprehensive understanding and design of suitable nanomaterials with varied functional proteins can allow selective protein coronation, which can help to tailor their therapeutic properties for nano-drug delivery vehicles. Protein corona is a complex structure and its uniqueness varies with different nanomaterials and nanoparticles. Upon contact with biological media, biomolecules adsorb onto a nanoparticle’s surface forming a layer mainly composed of proteins, which is called protein corona. Formation of protein-NP corona is influenced by many parameters such as the nanoparticle’s physicochemical properties and biological environmental factors. This dynamic structure constitutes the new interface with biological systems and consequently has a deep impact on the nanoparticle’s biological fate and response. In summary, it can be stated that novel investigations about protein-NP corona will assist to develop potential therapeutic against life-threatening diseases and other applications.
This book provides a comprehensive overview of the cascade of events activated in the body following the implant of biomaterials and devices. It is one of the first books to shed light on the role of the host immune response on therapeutic efficacy, and reviews the state-of-the-art for both basic science and medical applications. The text examines advantages and disadvantages of the use of synthetic versus natural biomaterials. Particular emphasis is placed on the role of biomimicry in the development of smart strategies able to modulate infiltrating immune cells, thus reducing side effects (such as acute and chronic inflammation, fibrosis and/or implant rejection) and improving the therapeutic outcome (healing, tissue restoration). Current cutting-edge approaches in tissue engineering, regenerative medicine, and nanomedicine offer the latest insights into the role immunomodulation in improving tolerance during tissue transplant in the treatment of orthopaedic, pancreatic, and hepatic diseases. "Immune Response to Implanted Materials and Devices" is intended for an audience of graduate students and professional researchers in both academia and industry interested in the development of smart strategies, which are able to exploit the self-healing properties of the body and achieve functional tissue restoration.
Nanoparticles (NPs) represent one of the most promising tools for drug‐targeting and drug‐delivery. However, a deeper understanding of the complex dynamics that happen after their in vivo administration is required. Particularly, plasma proteins tend to associate to NPs, forming a new surface named the ‘protein corona’ (PC). This surface is the most exposed as the ‘visible side’ of NPs and therefore, can have a strong impact on NP biodistribution, targeting efficacy and also toxicity. The PC consists of two poorly delimited layers, known as ‘hard corona’ (HC) and ‘soft corona’ (SC), that are affected by the complexity of the environment and the formed protein‐surface equilibrium during in vivo blood circulation. The HC corona is formed by proteins strongly associated to the NPs, while the SC is an outer layer consisting of loosely bound proteins. Several studies attempted to investigate the HC, which is easier to be isolated, but yielded poor reproducibility, due to varying experimental conditions. As a consequence, full mapping of the HC for different NPs is still lacking. Moreover, the current knowledge on the SC, which may play a major role in the ‘first’ interaction of NPs once in vivo, is very limited, mainly due to the difficulties in preserving it after purification. Therefore, multi‐disciplinary approaches leading to the obtainment of a major number of information about the PC and its properties is strongly needed to fully understand its impact and to better support a more safety and conscious application of nanotechnology in medicine. WIREs Nanomed Nanobiotechnol 2017, 9:e1467. doi: 10.1002/wnan.1467
This article is categorized under:
• Therapeutic Approaches and Drug Discovery > Emerging Technologies
• Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
Due to its enormous relevance the corona formation of adsorbed proteins around nanoparticles is widely investigated. A comparison of different experimental techniques is given. Direct measurements of proteins, such as typically performed with mass spectrometry, will be compared with indirect analysis, in which instead information about the protein corona is gathered from changes in the properties of the nanoparticles. The type of measurement determines also whether before analysis purification from unbound excess proteins is necessary, which may change the equilibrium, or if measurements can be performed in situ without required purification. Pros and contras of the different methods will be discussed.
Ball-milling utilizes mechanical stress to modify properties of carbon nanotubes (CNTs) including size, capping, and functionalization. Ball-milling, however, may introduce structural defects resulting in altered CNT-biomolecule interactions. Nanomaterial-biomolecule interactions result in the formation of the biocorona (BC), which alters nanomaterial properties, function, and biological responses. The formation of the BC is governed by the nanomaterial physicochemical properties and the physiological environment. Underlying disease states such as cardiovascular disease can alter the biological milieu possibly leading to unique BC identities. In this ex vivo study, we evaluated variations in the formation of the BC on single-walled CNTs (SWCNTs) due to physicochemical alterations in structure resulting from ball-milling and variations in the environment due to the high-cholesterol disease state. Increased ball-milling time of SWCNTs resulted in enhanced structural defects. Following incubation in normal mouse serum, label-free quantitative proteomics identified differences in the biomolecular content of the BC due to the ball-milling process. Further, incubation in cholesterol-rich mouse serum resulted in the formation of unique BCs compared to SWCNTs incubated in normal serum. Our study demonstrates that the BC is modified due to physicochemical modifications such as defects induced by ball-milling and physiological disease conditions, which may result in variable biological responses.
Aim:
A comprehensive understanding of nanoparticle (NP)-protein interaction (protein corona formation) is required. So far, many factors influencing this interaction have been investigated, like size and ζ potential. However, NPs exposure concentration has always been ignored. Herein, we aim to disclose the correlation of NPs exposure concentration with protein adsorption.
Materials & methods:
Four polymeric NPs systems possessing similar sizes (230 ± 20 nm) but varied ζ potentials (-30 ∼ +40 mv) were prepared. Physicochemical properties and protein adsorption upon NP-protein interaction were characterized.
Results:
Protein adsorption capacity and adsorbed protein types were NPs concentration-dependent.
Conclusion:
Considering the critical impacts of protein adsorption on NPs delivery, our work could be an urgent warning about the possible risks of dosage adjustment of nanoformulations.
The interaction of molecules with the surface of nanoparticles is important in many fields of study, including drug delivery and nanoparticle toxicity. Solution-state NMR has the potential to provide structural as well as dynamic information regarding molecules adsorbed to the nanoparticle surface. Here, we use Saturation-Transfer Difference NMR (STD-NMR) to examine small molecules binding to the surface of polystyrene nanoparticles. Binding constants for this nonspecific adsorption are determined from the initial slope of the STD buildup curve at several ligand concentrations. We also use the STD-NMR technique to quantify the association of solvent water molecules with the nanoparticle surface. The results presented here will be useful to future studies involving peptides and proteins adsorbed on nanoparticle surfaces.
The recent discovery of the biological corona is revolutionising our understanding of the in vivo behaviour of nanomaterials. Accurate analysis of corona bioactivity is essential for predicting the fate of nanomaterials and thereby improving nanomedicine design. Nevertheless, current biotechniques for protein analysis are not readily adaptable for analysing corona proteins, given that their conformation, activity, and interaction may largely differ from those of the native proteins. Here, we introduce and propose tailor-made modifications to five types of mainstream bioanalytical methodologies. We specifically illustrate how these modifications can translate existing techniques for protein analysis into competent tools for dissecting the composition, bioactivity, and interaction (with both nanomaterials and the tissue) of corona formed on specific nanomaterial surfaces.
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.
Les nanomédecines injectées par voie intraveineuse interagissent avec les éléments biologiques qui les entourent dans le compartiment sanguin. Parmi ces interactions, celles avec les protéines sanguines se révèlent être très importantes dans le devenir de ces nanovecteurs, leur conférant une identité biologique influençant leur chemin jusqu’au tissu et aux cellules cibles. La compréhension et le contrôle de ces phénomènes reste un enjeu crucial dans le développement des nanomédecines. Des méthodes permettant une étude facilitée de ces interactions sont nécessaires à cet égard. Les travaux de cette thèse ont eu pour but de développer des méthodes, utilisables en routine, permettant une caractérisation fine des nanomédecines et de leurs interactions avec les protéines plasmatiques, applicables dans un contexte clinique. Ils s’inscrivent dans un projet intitulé « Nano Innovation for CancEr » (NICE, BPI France) regroupant un consortium de partenaires industriels en développement clinique de nanomédecines.Dans un premier temps, un travail bibliographique sur les méthodes actuellement mises en œuvre pour une telle caractérisation ont pu mettre en avant deux limitations majeures. (i) D’une part, la complexité des méthodes actuelles disponibles pour lesquelles la spécificité des équipements et l’expertise requise limitent une utilisation à large échelle. (ii) D’autre part, les propriétés aujourd’hui caractérisées en routine (taille, morphologie globale, charge) ne sont que grossières comparées à la finesse des processus biologiques qui interagissent et « analysent » les nanovecteurs une fois introduits dans le milieu biologique. Ces deux aspects limitent aujourd’hui un développement plus sûr des nanomédecines pour une bonne reproductibilité en clinique et garantir des essais de contrôle qualité fiables.Au cours de nos travaux, nous avons développé des méthodes permettant de répondre en partie à la problématique posée par la caractérisation des nanomédecines. Une méthode d’analyse à haut débit de l’activation du système du complément par immunoélectrophorèse en deux dimensions a été développée et validée. Elle permet l’analyse reproductible de l’activation de la protéine C3. Elle est applicable à l’étude de l’effet de la présence de nanoparticules dans le sérum humain et leur degré d’action sur la cascade du complément. Cette méthode a été utilisée pour mener une étude plus fondamentale du mécanisme de l’activation du système du complément en regard de l’architecture de la surface de nanoparticules.Une deuxième méthode d’étude de l’activation du complément produit par des nanomédecine a été proposée sur la base de la résonnance plasmonique de surface (SPR). Une puce permettant un screening automatisé de l’activation du complément a été développée. L’application de cette méthode comparée à d’autres méthodes d’études de l’activation du système du complément (Immunoélectrophorèse 2D, ELISA) a permis d’identifier des biais lors de leur application à l’évaluation des nanomédecines.Enfin, une approche originale de caractérisation de la surface de nanoparticules a été proposée utilisant des protéines pour sonder la capacité de la surface des nanoparticules à adsorber ou repousser ces dernières. Dans cette méthode, l’électrophorèse capillaire est utilisée comme outil analytique permettant une analyse directe de l’échantillon sans séparation préalable des nanomédecines.Les méthodes développées au cours de ces travaux peuvent être appliquées à la caractérisation de nanomédecines et proposées comme des méthodes de contrôle en routine de façon plus générale. Un développement de la caractérisation dans ce sens constitue l’un des leviers pour une translation plus fructueuse des nanomédecines entrant en phase clinique.
The design of nanoparticles is critical for their efficient use in many applications ranging from biomedicine to sensing and energy. While shape and size are responsible for the properties of the inorganic nanoparticle core, the choice of ligands is of utmost importance for the colloidal stability and function of the nanoparticles. Moreover, the selection of ligands employed in nanoparticle synthesis can determine their final size and shape. Ligands added after nanoparticle synthesis infer both new properties as well as provide enhanced colloidal stability. In this article, we provide a comprehensive review on the role of the ligands with respect to the nanoparticle morphology, stability, and function. We analyze the interaction of nanoparticle surface and ligands with different chemical groups, the types of bonding, the final dispersibility of ligand-coated nanoparticles in complex media, their reactivity, and their performance in biomedicine, photodetectors, photovoltaic devices, light-emitting devices, sensors, memory devices, thermoelectric applications, and catalysis.
This review gives an overview of protein adsorption at solid/liquid interface. Compared to the other ones, we have focus on three main questions with the point of view of the protein. The first question is related to the kinetic and especially the using of Langmuir model to describe the protein adsorption. The second question is about the concept of hard and soft protein. In this part, we report the protein structural modification induced by adsorption regarding their intrinsic structure. This allows formulating of a new concept to classify the protein to predict their behavior at solid/liquid interface. The last question is related to the protein corona. We give an overview about the soft/hard corona and attempt to make correlation with the concept of hard/soft protein
Over the last decade nanomaterials have had a major impact on human health for the early detection and treatment of many diseases. The future success of clinically translatable nanomaterials lies in the combination of several functionalities to realize a personalized medical experience for patients. To maintain promises, concerns arising from toxic potential and off‐target accumulation of nanomaterials must be addressed first. Upon introduction to a complex biological system (e.g., following systemic administration), nanomaterials interact with all the encountered biomolecules and form the protein corona, a complex coating of plasma proteins that provides them with a totally new biological identity. As the protein corona controls the nanomaterial behavior in vivo, a precise knowledge of the relationship between biological identity and physiological response is needed but not yet achieved. Based on impressive progress made thus far, this review critically discusses how the protein corona activates immune response and influences the targeted delivery of nanomaterials. Furthermore, we comment on emerging strategies to manipulate protein binding in order to promote formation of designer artificial coronas and achieve a desired therapeutic outcome. We conclude by debating challenges that must be overcome to obtain widespread clinical adoption of nanomaterials.
This article is categorized under:
• Nanotechnology Approaches to Biology > Cells at the Nanoscale
• Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials
• Therapeutic Approaches and Drug Discovery > Emerging Technologies
When nanoparticles encounter a biological fluid, proteins, lipids, and other biomolecules adsorb on the nanoscale surface consequently leading to the evolution of a protein shell or “corona.” The corona formed is dynamic in nature and depends on the “synthetic identity” of the NPs, ultimately affecting their biological response. In this paper, an integrated microfluidic platform coupled with an electrical resistance measurement setup is developed to monitor and investigate the real‐time formation of a biomolecular corona of carbon nanoparticles. “Soft” and “hard” corona formation stages are effectively discriminated based on their nanoscale surface chemistries when combined with a time‐frequency tool known as wavelet transform and machine‐learning techniques. Additionally, the corona and its composition are studied using different techniques such as dynamic light scattering, nanoparticle tracking analysis, zeta potential, excitation–emission profiles, 1D sodium dodecyl polyacrylamide gel electrophoresis and subsequently, liquid chromatography‐mass spectrometry analysis. The dynamic setup can eventually be used as a valuable tool for screening of any nanoparticles formulations with distinct surface chemistries for the purpose of reduced protein adsorption/weaker protein corona formation and consequently enhance the success of targeted drug delivery. An integrated microfluidic platform coupled with electrical resistance measurement setup aided with wavelet transform and machine learning algorithms is developed to monitor the real‐time formation of protein corona and discrimination of their fouling behavior based on nanoscale surface chemistries.
One of the main diseases contributing to human death are malignant tumors. Phototherapy is a promising approach for cancer therapy, and functional nanoparticles with targeting ligands are commonly used to improve the therapeutic efficiency. However, recent studies have shown that nanoparticles in contact with a biological fluid can rapidly form a “protein corona” on their surface, which will remarkably decrease the targeting ability. Here, we describe the preparation of hybrid nanomaterials with Bi2S3 nanorods as the core, and fluorescein-isothiocyanate and folic acid-modified human serum albumin (HSA-FITC-FA) as the shell. By using fluorescent binding label (FITC) and imaging techniques, we discovered the image of the cell lysosomes, indicating that the photothermal therapy agent was predominantly targeted to and accumulated in lysosomes. Combined with photothermal therapy agent (Bi2S3 nanorods) and targeting ligand (FA), the obtained product shows enhanced photothermal therapy under near-infrared region laser irradiation. Additionally, SDS-PAGE shows that the modified HSA shell could remarkably reduce the reabsorption of protein corona from blood serum, minimized the adverse effect of protein corona on targetability. Taken together, the results indicate that our strategy has the potential for preparing efficient photothermal nanomaterials with image-guided subcellular organelle-targeting cancer cell ablation ability.
Nanomedicine aims to find novel solutions for urgent biomedical needs. Despite this, one of the most challenging hurdles that nanomedicine faces is to successfully target therapeutic nanoparticles to cells of interest in vivo. As for any biomaterials, once in vivo, nanoparticles can interact with plasma biomolecules, forming new entities for which the name protein coronas (PCs) have been coined. The PC can influence the in vivo biological fate of a nanoparticle. Thus for guaranteeing the desired function of an engineered nanomaterial in vivo, it is crucial to dissect its PC in terms of formation and evolution within the body. In this contribution we will review the 'good' and 'bad' sides of the PC, starting from the scientific aspects to the technological applications.
It is now well established that the surface of nanoparticles (NPs) in a biological environment is immediately modified by the adsorption of biomolecules with the formation of a protein corona and it is also accepted that the protein corona, rather than the original nanoparticle surface, defines a new biological identity. Consequently, a methodology to effectively study the interaction between nanomaterials and the biological corona encountered within an organism is a key objective in nanoscience for understanding the impact of the nanoparticle-protein interactions on the biological response in vitro and in vivo. Here, we outline an integrated methodology to address the different aspects governing the formation and the function of the protein corona of polystyrene nanoparticles coated with Transferrin by different strategies. Protein-NP complexes are studied both in situ (in human plasma, full corona FC) and after washing (hard corona, HC) in terms of structural properties, composition and second-order interactions with protein microarrays. Human protein microarrays are used to effectively study NP-corona/proteins interactions addressing the growing demand to advance investigations of the extrinsic function of corona complexes. Our data highlight the importance of this methodology as an analysis to be used in advance of the application of engineered NPs in biological environments.
Nanomaterials hold promise as multifunctional diagnostic and therapeutic agents. However, the effective application of nanomaterials is hampered by limited understanding and control over their interactions with complex biological systems. When a nanomaterial enters a physiological environment, it rapidly adsorbs proteins forming what is known as the protein 'corona'. The protein corona alters the size and interfacial composition of a nanomaterial, giving it a biological identity that is distinct from its synthetic identity. The biological identity determines the physiological response including signalling, kinetics, transport, accumulation, and toxicity. The structure and composition of the protein corona depends on the synthetic identity of the nanomaterial (size, shape, and composition), the nature of the physiological environment (blood, interstitial fluid, cell cytoplasm, etc.), and the duration of exposure. In this critical review, we discuss the formation of the protein corona, its structure and composition, and its influence on the physiological response. We also present an 'adsorbome' of 125 plasma proteins that are known to associate with nanomaterials. We further describe how the protein corona is related to the synthetic identity of a nanomaterial, and highlight efforts to control protein-nanomaterial interactions. We conclude by discussing gaps in the understanding of protein-nanomaterial interactions along with strategies to fill them (167 references).
Gold nanorods (GNRs) stabilized with cetyltrimethylammonium bromide (CTAB) and GNR functionalized via a ligand exchange method with either thiolated polyethylene glycol (PEG(5000)) or mercaptohexadecanoic acid (MHDA) were investigated for their stability in biological media and subsequent toxicological effects to HaCaT cells. GNR-PEG and GNR-MHDA exhibited minimal effects on cell proliferation, whereas GNR-CTAB reduced cell proliferation significantly due to the inherent toxicity of the cationic surfactant to cells. Cell uptake studies indicated relatively low uptake for GNR-PEG and high uptake for GNR-MHDA. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that GNR-PEG induced less significant and unique changes in the transcription levels of 84 genes related to stress and toxicity compared to GNR-MHDA. The results demonstrate that, although cell proliferation was not affected by both particles, there is a significant difference in gene expression in GNR-MHDA exposed cells, suggesting long-term implications for chronic exposure.
It is now clearly emerging that besides size and shape, the other primary defining element of nanoscale objects in biological media is their long-lived protein ("hard") corona. This corona may be expressed as a durable, stabilizing coating of the bare surface of nanoparticle (NP) monomers, or it may be reflected in different subpopulations of particle assemblies, each presenting a durable protein coating. Using the approach and concepts of physical chemistry, we relate studies on the composition of the protein corona at different plasma concentrations with structural data on the complexes both in situ and free from excess plasma. This enables a high degree of confidence in the meaning of the hard protein corona in a biological context. Here, we present the protein adsorption for two compositionally different NPs, namely sulfonated polystyrene and silica NPs. NP-protein complexes are characterized by differential centrifugal sedimentation, dynamic light scattering, and zeta-potential both in situ and once isolated from plasma as a function of the protein/NP surface area ratio. We then introduce a semiquantitative determination of their hard corona composition using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray liquid chromatography mass spectrometry, which allows us to follow the total binding isotherms for the particles, identifying simultaneously the nature and amount of the most relevant proteins as a function of the plasma concentration. We find that the hard corona can evolve quite significantly as one passes from protein concentrations appropriate to in vitro cell studies to those present in in vivo studies, which has deep implications for in vitro-in vivo extrapolations and will require some consideration in the future.
The chemical composition, size, shape and surface characteristics of nanoparticles affect the way proteins bind to these particles, and this in turn influences the way in which nanoparticles interact with cells and tissues. Nanomaterials bound with proteins can result in physiological and pathological changes, including macrophage uptake, blood coagulation, protein aggregation and complement activation, but the mechanisms that lead to these changes remain poorly understood. Here, we show that negatively charged poly(acrylic acid)-conjugated gold nanoparticles bind to and induce unfolding of fibrinogen, which promotes interaction with the integrin receptor, Mac-1. Activation of this receptor increases the NF-κB signalling pathway, resulting in the release of inflammatory cytokines. However, not all nanoparticles that bind to fibrinogen demonstrated this effect. Our results show that the binding of certain nanoparticles to fibrinogen in plasma offers an alternative mechanism to the more commonly described role of oxidative stress in the inflammatory response to nanomaterials.
In a physiological environment, nanoparticles selectively absorb proteins to form 'nanoparticle-protein coronas', a process governed by molecular interactions between chemical groups on the nanoparticle surfaces and the amino-acid residues of the proteins. Here, we propose a biological surface adsorption index to characterize these interactions by quantifying the competitive adsorption of a set of small molecule probes onto the nanoparticles. The adsorption properties of nanomaterials are assumed to be governed by Coulomb forces, London dispersion, hydrogen-bond acidity and basicity, polarizability and lone-pair electrons. Adsorption coefficients of the probe compounds were measured and used to create a set of nanodescriptors representing the contributions and relative strengths of each molecular interaction. The method successfully predicted the adsorption of various small molecules onto carbon nanotubes, and the nanodescriptors were also measured for 12 other nanomaterials. The biological surface adsorption index nanodescriptors can be used to develop pharmacokinetic and safety assessment models for nanomaterials.
MicroRNAs (miRNAs) are single-stranded regulatory RNAs, frequently expressed as clusters. Previous studies have demonstrated that the six-miRNA cluster miR-17 approximately 92 has important roles in tissue development and cancers. However, the precise role of each miRNA in the cluster is unknown. Here we show that overexpression of miR-17 results in decreased cell adhesion, migration and proliferation. Transgenic mice overexpressing miR-17 showed overall growth retardation, smaller organs and greatly reduced haematopoietic cell lineages. We found that fibronectin and the fibronectin type-III domain containing 3A (FNDC3A) are two targets that have their expression repressed by miR-17, both in vitro and in transgenic mice. Several lines of evidence support the notion that miR-17 causes cellular defects through its repression of fibronectin expression. Our single miRNA expression assay may be evolved to allow the manipulation of individual miRNA functions in vitro and in vivo. We anticipate that this could serve as a model for studying gene regulation by miRNAs in the development of gene therapy.
Rapid growth in nanotechnology is increasing the likelihood of engineered nanomaterials coming into contact with humans and the environment. Nanoparticles interacting with proteins, membranes, cells, DNA and organelles establish a series of nanoparticle/biological interfaces that depend on colloidal forces as well as dynamic biophysicochemical interactions. These interactions lead to the formation of protein coronas, particle wrapping, intracellular uptake and biocatalytic processes that could have biocompatible or bioadverse outcomes. For their part, the biomolecules may induce phase transformations, free energy releases, restructuring and dissolution at the nanomaterial surface. Probing these various interfaces allows the development of predictive relationships between structure and activity that are determined by nanomaterial properties such as size, shape, surface chemistry, roughness and surface coatings. This knowledge is important from the perspective of safe use of nanomaterials.
Plasma contains thousands of proteins, but a small number of these proteins comprise the majority of protein molecules and mass.
We surveyed proteomic studies to identify candidates for high-abundance polypeptide chains. We searched the literature for information on the plasma concentrations of the most abundant components in healthy adults and for the molecular mass of the mature polypeptide chains in plasma. Because proteomic studies usually dissociate proteins into polypeptide chains or detect short peptide segments of proteins, we summarized data on individual peptide chains for proteins containing multiple subunits or polypeptides. We collected data on about 150 of the most abundant polypeptides in plasma. The abundant polypeptides span approximately the top 4 logs of concentration in plasma, from 650 to 0.06 micromol/L on a molar basis or from about 50,000 to 1 mg/L mass abundance.
Data on the concentrations of the high-abundance peptide chains in plasma assist in understanding the composition of plasma and potential approaches for clinical laboratory or proteomic analysis of plasma proteins. Development of more extensive databases regarding the plasma concentrations of proteins in health and diseases would promote diagnostic and proteomic advances.
The human plasma proteome holds the promise of a revolution in disease diagnosis and therapeutic monitoring provided that major challenges in proteomics and related disciplines can be addressed. Plasma is not only the primary clinical specimen but also represents the largest and deepest version of the human proteome present in any sample: in addition to the classical "plasma proteins," it contains all tissue proteins (as leakage markers) plus very numerous distinct immunoglobulin sequences, and it has an extraordinary dynamic range in that more than 10 orders of magnitude in concentration separate albumin and the rarest proteins now measured clinically. Although the restricted dynamic range of conventional proteomic technology (two-dimensional gels and mass spectrometry) has limited its contribution to the list of 289 proteins (tabulated here) that have been reported in plasma to date, very recent advances in multidimensional survey techniques promise at least double this number in the near future. Abundant scientific evidence, from proteomics and other disciplines, suggests that among these are proteins whose abundances and structures change in ways indicative of many, if not most, human diseases. Nevertheless, only a handful of proteins are currently used in routine clinical diagnosis, and the rate of introduction of new protein tests approved by the United States Food and Drug Administration (FDA) has paradoxically declined over the last decade to less than one new protein diagnostic marker per year. We speculate on the reasons behind this large discrepancy between the expectations arising from proteomics and the realities of clinical diagnostics and suggest approaches by which protein-disease associations may be more effectively translated into diagnostic tools in the future.
Symplastic spermatids (sys) male mice are sterile due to a recessive mutation that causes defective adhesion between spermatids and Sertoli cells within the seminiferous epithelium. We show that the mutation in sys mice involves a deletion of 1.24 Mb of chromosome 14. Comparative genomic analysis suggests that this region contains only one gene, Fndc3a. A genetic complementation analysis using mice with a specific mutation within Fndc3a verifies that mutation of Fndc3a is the cause of male sterility in sys mice. Fndc3a is a member of a three-gene family in mice. Fndc3a, which is expressed in several tissues including testis, encodes a novel protein composed of a proline-rich amino-terminus, nine fibronectin type-III domains, and a hydrophobic carboxy-terminus. The proline-rich region of each family member contains conserved amino acids that include a PPGY consensus binding site for type I WW domain containing proteins. The hydrophobic carboxy-terminus is similar to that found in 'tail-anchored' proteins, integral membrane proteins that are localized to the cytosolic face of the endoplasmic reticulum. Immunohistochemical staining indicated that FNDC3A localizes to the acrosome of spermatids, as well as to Leydig cells in the mouse testis. Acrosomal localization of FNDC3A is observed in spermatids between step 2 and step 10 inclusive. In step 12 spermatids, FNDC3A is largely absent from the acrosomal region with immunostaining being localized to vesicular structures located within the cytoplasm of elongate spermatids. Models are presented for the function of FNDC3A in mediating spermatid-Sertoli adhesion during mouse spermatogenesis.
Due to their small size, nanoparticles have distinct properties compared with the bulk form of the same materials. These properties are rapidly revolutionizing many areas of medicine and technology. Despite the remarkable speed of development of nanoscience, relatively little is known about the interaction of nanoscale objects with living systems. In a biological fluid, proteins associate with nanoparticles, and the amount and presentation of the proteins on the surface of the particles leads to an in vivo response. Proteins compete for the nanoparticle "surface," leading to a protein "corona" that largely defines the biological identity of the particle. Thus, knowledge of rates, affinities, and stoichiometries of protein association with, and dissociation from, nanoparticles is important for understanding the nature of the particle surface seen by the functional machinery of cells. Here we develop approaches to study these parameters and apply them to plasma and simple model systems, albumin and fibrinogen. A series of copolymer nanoparticles are used with variation of size and composition (hydrophobicity). We show that isothermal titration calorimetry is suitable for studying the affinity and stoichiometry of protein binding to nanoparticles. We determine the rates of protein association and dissociation using surface plasmon resonance technology with nanoparticles that are thiol-linked to gold, and through size exclusion chromatography of protein-nanoparticle mixtures. This method is less perturbing than centrifugation, and is developed into a systematic methodology to isolate nanoparticle-associated proteins. The kinetic and equilibrium binding properties depend on protein identity as well as particle surface characteristics and size.
Nanoparticles present enormous surface areas and are found to enhance the rate of protein fibrillation by decreasing the lag time for nucleation. Protein fibrillation is involved in many human diseases, including Alzheimer's, Creutzfeld-Jacob disease, and dialysis-related amyloidosis. Fibril formation occurs by nucleation-dependent kinetics, wherein formation of a critical nucleus is the key rate-determining step, after which fibrillation proceeds rapidly. We show that nanoparticles (copolymer particles, cerium oxide particles, quantum dots, and carbon nanotubes) enhance the probability of appearance of a critical nucleus for nucleation of protein fibrils from human β2-microglobulin. The observed shorter lag (nucleation) phase depends on the amount and nature of particle surface. There is an exchange of protein between solution and nanoparticle surface, and β2-microglobulin forms multiple layers on the particle surface, providing a locally increased protein concentration promoting oligomer formation. This and the shortened lag phase suggest a mechanism involving surface-assisted nucleation that may increase the risk for toxic cluster and amyloid formation. It also opens the door to new routes for the controlled self-assembly of proteins and peptides into novel nanomaterials.
• amyloid
• nanotoxicology
• surface-assisted nucleation
Nanoparticles in a biological milieu are known to form a sufficiently long-lived and well-organized 'corona' of biomolecules to confer a biological identity to the particle. Because this nanoparticle-biomolecule complex interacts with cells and biological barriers, potentially engaging with different biological pathways, it is important to clarify the presentation of functional biomolecular motifs at its interface. Here, we demonstrate that by using antibody-labelled gold nanoparticles, differential centrifugal sedimentation and various imaging techniques it is possible to identify the spatial location of proteins, their functional motifs and their binding sites. We show that for transferrin-coated polystyrene nanoparticles only a minority of adsorbed proteins exhibit functional motifs and the spatial organization appears random, which is consistent, overall, with a stochastic and irreversible adsorption process. Our methods are applicable to a wide array of nanoparticles and can offer a microscopic molecular description of the biological identity of nanoparticles.
The significance of a protein corona on nanoparticles in modulating particle properties and their biological interactions has been widely acknowledged. The protein corona is derived from proteins in biological fluids, many of which are glycosylated. To date, the glycans on the proteins have been largely overlooked in studies of nanoparticle-cell interactions. In this study, we demonstrate that glycosylation of the protein corona plays an important role in maintaining the colloidal stability of nanoparticles and influences nanoparticle-cell interactions. The removal of glycans from the protein corona enhances cell membrane adhesion and cell uptake of nanoparticles in comparison with the fully glycosylated form, resulting in the generation of a pro-inflammatory milieu by macrophages. This study highlights that the post-translational modification of proteins can significantly impact nanoparticle-cell interactions by modulating the protein corona properties.
Nanoparticles (NPs) when exposed to biological fluids are coated by specific proteins which form the so called protein corona. While some adsorbing proteins exchange with the surroundings on a short time scale, described as a “dynamic” corona, others with higher affinity and long lived interaction with the NP surface form a “hard” corona, which is believed to mediate NP interaction with cellular machineries. In depth NP protein corona characterization is therefore a necessary step in understanding the relationship between surface layer structure and biological outcomes. In the present work we evaluate the protein composition and stability over time and we systematically challenge the formed complexes with surfactants. Each challenge is characterized through different physicochemical measurements (DLS, zeta potential and DCS) alongside proteomic evaluation in titration type experiments (surfactant titration). 100 nm silicon oxide (Si) and 100 nm carboxylated polystyrene (PS-COOH) NPs cloaked by human plasma HC were titrated with CHAPS (zwitterionic), TritonX-100 (non-ionic), SDS (anionic) and DTAB (cationic) surfactants. Composition and density of HC together with size and -potential of NP-HC complexes were tracked at each step after surfactant titration. Results on Si NP-HC complexes showed that SDS removes most of the HC, while DTAB induces NPs agglomeration. Analogous results were obtained for PS NP-HC complexes. Interestingly, CHAPS and TritonX-100, thanks to similar surface binding preferences, enable selective extraction of Apolipoprotein-AI (ApoAI) from Si NP hard coronas, leaving unaltered the dispersion physicochemical properties. These findings indicate that surfactant titration can enable the study of NP-HC stability through surfactant variation and also selective separation of certain proteins from the HC. This approach thus has an immediate analytical value as well as potential applications in HC engineering.
Nanoparticles in contact with cells and living organisms generate quite novel interactions at the interface between the nanoparticle surface and the surrounding biological environment. However, a detailed time resolved molecular level description of the evolving interactions as nanoparticles are internalized and trafficked within the cellular environment is still missing and will certainly be required for the emerging arena of nanoparticle-cell interactions to mature. In this paper promising methodologies to map out the time resolved nanoparticle-cell interactome for nanoparticle uptake are discussed. Thus silica coated magnetite nanoparticles are presented to cells and their magnetic properties used to isolate, in a time resolved manner, the organelles containing the nanoparticles. Characterization of the recovered fractions shows that different cell compartments are isolated at different times, in agreement with imaging results on nanoparticle intracellular location. Subsequently the internalized nanoparticles can be further isolated from the recovered organelles, allowing the study of the most tightly nanoparticle-bound biomolecules, analogous to the ‘hard corona’ that so far has mostly been characterized in extracellular environments. Preliminary data on the recovered nanoparticles suggest that significant portion of the original corona (derived from the serum in which particles are presented to the cells) is preserved as nanoparticles are trafficked through the cells.
In biological fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiological systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biological relevancy. Here we show that label-free snapshot proteomics can be used to obtain quantitative time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (<0.5 minutes) and, over time, to change significantly in terms of the amount of bound protein, but not in composition. Rapid corona formation is found to affect haemolysis, thrombocyte activation, nanoparticle uptake and endothelial cell death at an early exposure time.
Over the last decade the existence of "the corona," a natural interface between nanomaterials and living matter in biological milieu, evolved from a vague concept into broadly recognized fact. This robust shell arises (to some extent) on the surface of all nanoparticles (NPs), even the ones designed to avoid its formation upon contact with biological fluids and confers a biological identity to the nanomaterials such that they can engage with cellular machinery. The NP corona consists of those proteins (and other biomolecules such as lipids and sugars) residing on the NP surface for a sufficient timescale to influence the NP's properties and interactions with living systems. This chapter aims to provide simple protocols, as well as notes on potential pitfalls, to help researchers to perform basic experiments in this field as the basis for a more mechanistic approach to study and understand NP-protein corona complexes. This work has been supported by INSPIRE (Integrated NanoScience Platform for Ireland) funded by the Irish Government's Programme for Research in Third Level Institutions, Cycle 4, National Development Plan 2007-2013, and 3MICRON (NMP-2009-LA-245572), NAMDIATREAM (NMP4-LA-2010-246479) and QualityNano (INFRA-2010-262163) funded by the European Commission 7th Framework Programme.
Nanoparticles have been proposed as carriers for drugs, genes and therapies to treat various diseases. Many strategies have been developed to target nanomaterials to specific or over-expressed receptors in diseased cells, and these typically involve functionalizing the surface of nanoparticles with proteins, antibodies or other biomolecules. Here, we show that the targeting ability of such functionalized nanoparticles may disappear when they are placed in a biological environment. Using transferrin-conjugated nanoparticles, we found that proteins in the media can shield transferrin from binding to both its targeted receptors on cells and soluble transferrin receptors. Although nanoparticles continue to enter cells, the targeting specificity of transferrin is lost. Our results suggest that when nanoparticles are placed in a complex biological environment, interaction with other proteins in the medium and the formation of a protein corona can 'screen' the targeting molecules on the surface of nanoparticles and cause loss of specificity in targeting.
The innovative use of engineered nanomaterials in medicine, be it in therapy or diagnosis, is growing dramatically. This is motivated by the current extraordinary control over the synthesis of complex nanomaterials with a variety of biological functions (e.g. contrast agents, drug-delivery systems, transducers, amplifiers, etc.). Engineered nanomaterials are found in the bio-context with a variety of applications in fields such as sensing, imaging, therapy or diagnosis. As the degree of control to fabricate customized novel and/or enhanced nanomaterials evolves, often new applications, devices with enhanced performance or unprecedented sensing limits can be achieved. Of course, interfacing any novel material with biological systems has to be critically analyzed as many undesirable adverse effects can be triggered (e.g. toxicity, allergy, genotoxicity, etc.) and/or the performance of the nanomaterial can be compromised due to the unexpected phenomena in physiological environments (e.g. corrosion, aggregation, unspecific absorption of biomolecules, etc.). Despite the need for standard protocols for assessing the toxicity and bio-performance of each new functional nanomaterial, these are still scarce or currently under development. Nonetheless, nanotoxicology and relating adverse effects to the physico-chemical properties of nanomaterials are emerging areas of the utmost importance which have to be continuously revisited as any new material emerges. This review highlights recent progress concerning the interaction of nanomaterials with biological systems and following adverse effects.
Protein adsorption to nanoparticles (NPs) is a key prerequisite to understand NP-cell interactions. While the layer thickness of the protein corona has been well characterized in many cases, the absolute number of bound proteins and their exchange dynamics in body fluids is difficult to assess. Here we measure the number of molecules adsorbed to sulfonate (PSOSO(3)H) and carboxyl-(PSCOOH) polystyrene NPs using fluorescence correlation spectroscopy. We find that the fraction of molecules bound to NPs falls onto a single, universal adsorption curve, if plotted as a function of molar protein-to-NP ratio. The adsorption curve shows the build-up of a strongly bound monolayer up to the point of monolayer saturation (at a geometrically defined protein-to-NP ratio), beyond which a secondary, weakly bound layer is formed. While the first layer is irreversibly bound (hard corona), the secondary layer (soft corona) exhibits dynamic exchange, if competing unlabeled is added. In the presence of plasma proteins, the hard corona is stable, while the soft corona is almost completely removed. The existence of two distinct time scales in the protein off-kinetics, for both NP types studied here, indicates the possibility of an exposure memory effect in the NP corona.
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.
Simple, stable, and specific methods for immobilizing proteins on gold surfaces are needed for the development of applications that rely on the oriented attachment of proteins to gold surfaces. We report a direct, stable, genetically encodable method for the oriented chemisorption of proteins to gold nanoparticles (Au NPs) through the tetracysteine motif (C-C-P-G-C-C) while simultaneously suppressing protein physisorption. Mutants of ubiquitin (Ub) and enhanced green fluorescent protein (eGFP) containing the tetracysteine motif were produced and displayed stronger adsorption to the NPs than did native proteins. An eGFP mutant with a dicysteine motif (G-C-C) did not show a significant improvement in binding to Au NPs compared to that of the wild-type protein. The binding of the proteins to Au NPs of various sizes (14, 18, 28, and 39 nm) was explored. The small Ub tetracysteine mutant stabilized several sizes of Au NPs, and the eGFP tetracysteine mutant clearly had the strongest chemisorption to the 18 nm NPs. The control of binding orientation for proteins bearing a tetracysteine motif was demonstrated through the enhanced specific binding of protein-NP conjugates to immobilized targets.
The development of appropriate in vitro protocols to assess the potential toxicity of the ever expanding range of nanoparticles represents a challenging issue, because of the rapid changes of their intrinsic physicochemical properties (size, shape, reactivity, surface area, etc.) upon dispersion in biological fluids. Dynamic formation of protein coating around nanoparticles is a key molecular event, which may strongly impact the biological response in nanotoxicological tests. In this work, by using citrate-capped gold nanoparticles (AuNPs) of different sizes as a model, we show, by several spectroscopic techniques (dynamic light scattering, UV-visible, plasmon resonance light scattering), that proteins-NP interactions are differently mediated by two widely used cellular media (i.e., Dulbecco Modified Eagle's medium (DMEM) and Roswell Park Memorial Institute medium (RPMI), supplemented with fetal bovine serum). We found that, while DMEM elicits the formation of a large time-dependent protein corona, RPMI shows different dynamics with reduced protein coating. Characterization of these nanobioentities was also performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectroscopy, revealing that the average composition of protein corona does not reflect the relative abundance of serum proteins. To evaluate the biological impact of such hybrid bionanostructures, several comparative viability assays onto two cell lines (HeLa and U937) were carried out in the two media, in the presence of 15 nm AuNPs. We observed that proteins/NP complexes formed in RPMI are more abundantly internalized in cells as compared to DMEM, overall exerting higher cytotoxic effects. These results show that, beyond an in-depth NPs characterization before cellular experiments, a detailed understanding of the effects elicited by cell culture media on NPs is crucial for standardized nanotoxicology tests.
Nanoparticles are of an appropriate size to interact with cells, and are likely to use a range of cellular machinery for internalisation and trafficking to various sub-cellular compartments. It is now understood that once in contact with biological fluids, the nanoparticle surface gets covered by a highly specific layer of proteins, forming the nanoparticle protein corona. This protein layer is stable for times longer than the typical time scale of nanoparticle import, and thus can impact on particle uptake and trafficking inside the cells. In this work, the effect of the corona composition on nanoparticle uptake has been investigated, by studying the impact of serum heat inactivation and complement depletion on the load of nanoparticles accumulated inside the cell. For the same material and nanoparticle size, cellular uptake was found to be significantly different when the nanoparticles were dispersed in medium where the serum was heat inactivated or not heat inactivated, even for non-specialized cells, suggesting that different sera can lead to different nanoparticle doses. The fact that uptake was correlated with the amount of protein bound into the nanoparticle corona suggests the need for commonly agreed dispersion protocols for in vitro nanoparticle-cell studies.
Nanotoxicology is still a new discipline. In this Perspective, both its origins and its future trends are discussed. In particular, we note several issues we consider important for publications in this field.
Cellular binding of cationic nanoparticles in the presence of serum proteins was probed with two-colour fluorescence microscopy. Cationic nanoparticles associate with serum proteins in solution and bind to the cell surface as a single anionic complex. Displacement of serum proteins from the nanoparticles was found to be protein dependent.
In this work, we explore the formation of the protein corona after exposure of metallic Au nanoparticles (NPs), with sizes ranging from 4 to 40 nm, to cell culture media containing 10% of fetal bovine serum. Under in vitro cell culture conditions, zeta potential measurements, UV-vis spectroscopy, dynamic light scattering and transmission electron microscope analysis were used to monitor the time evolution of the inorganic NP-protein corona formation and to characterize the stability of the NPs and their surface state at every stage of the experiment. As expected, the red-shift of the surface plasmon resonance peak, as well as the drop of surface charge and the increase of the hydrodynamic diameter indicated the conjugation of proteins to NPs. Remarkably, an evolution from a loosely attached toward an irreversible attached protein corona over time was observed. Mass spectrometry of the digested protein corona revealed albumin as the most abundant component which suggests an improved biocompatibility.
It is now known that nanoparticles, when exposed to biological fluid, become coated with proteins and other biomolecules to form a 'protein corona'. Recent systematic studies have identified various proteins that can make up this corona, but these nanoparticle-protein interactions are still poorly understood, and quantitative studies to characterize them are few in number. Here, we have quantitatively analysed the adsorption of human serum albumin onto small (10-20 nm in diameter) polymer-coated FePt and CdSe/ZnS nanoparticles by using fluorescence correlation spectroscopy. The protein corona forms a monolayer with a thickness of 3.3 nm. Proteins bind to the negatively charged nanoparticles with micromolar affinity, and time-resolved fluorescence quenching experiments show that they reside on the particle for approximately 100 s. These new findings deepen our quantitative understanding of the protein corona, which is of utmost importance in the safe application of nanoscale objects in living organisms.
Fluorescence correlation spectroscopy is used as a quantitative method to understand the binding and exchange behaviour of proteins on the surfaces of nanoparticles.
In a biological environment, nanoparticles immediately become covered by an evolving corona of biomolecules, which gives a biological identity to the nanoparticle and determines its biological impact and fate. Previous efforts at describing the corona have concerned only its protein content. Here, for the first time, we show, using size exclusion chromatography, NMR, and pull-down experiments, that copolymer nanoparticles bind cholesterol, triglycerides and phospholipids from human plasma, and that the binding reaches saturation. The lipid and protein binding patterns correspond closely with the composition of high-density lipoprotein (HDL). By using fractionated lipoproteins, we show that HDL binds to copolymer nanoparticles with much higher specificity than other lipoproteins, probably mediated by apolipoprotein A-I. Together with the previously identified protein binding patterns in the corona, our results imply that copolymer nanoparticles bind complete HDL complexes, and may be recognized by living systems as HDL complexes, opening up these transport pathways to nanoparticles. Apolipoproteins have been identified as binding to many other nanoparticles, suggesting that lipid and lipoprotein binding is a general feature of nanoparticles under physiological conditions.
Nanoparticle size and plasma binding profile contribute to a particle's longevity in the bloodstream, which can have important consequences for therapeutic efficacy. In this study an approximate doubling in nanoparticle hydrodynamic size was observed upon in vitro incubation of 30- and 50-nm colloidal gold in human plasma. Plasma proteins that bind the surface of citrate-stabilized gold colloids have been identified. Effects of protein binding on the nanoparticle hydrodynamic size, elements of coagulation, and the complement system have been investigated. The difference in size measurements obtained from dynamic light scattering, electron microscopy, and scanning probe microscopy are also discussed.
Fibronectin type III modules are versatile components of many proteins. Recent structures of module pairs show how these modules are joined together.
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