Detecting Cryptic Epitopes Created by Nanoparticles

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Science's STKE  21 Mar 2006:
Vol. 2006, Issue 327, pp. pe14
DOI: 10.1126/stke.3272006pe14


As potential applications of nanotechnology and nanoparticles increase, so too does the likelihood of human exposure to nanoparticles. Because of their small size, nanoparticles are easily taken up into cells (by receptor-mediated endocytosis), whereupon they have essentially free access to all cellular compartments. Similarly to macroscopic biomaterial surfaces (that is, implants), nanoparticles become coated with a layer of adsorbed proteins immediately upon contact with physiological solutions (unless special efforts are taken to prevent this). The process of adsorption often results in conformational changes of the adsorbed protein, which may be affected by the larger curvature of nanoparticles compared with implant surfaces. Protein adsorption may result in the exposure at the surface of amino acid residues that are normally buried in the core of the native protein, which are recognized by the cells as "cryptic epitopes." These cryptic epitopes may trigger inappropriate cellular signaling events (as opposed to being rejected by the cells as foreign bodies). However, identification of such surface-exposed epitopes is nontrivial, and the molecular nature of the adsorbed proteins should be investigated using biological and physical science methods in parallel with systems biology studies of the induced alterations in cell signaling.

The living world uses a system of compartmentalization, through which cells control which reactants colocalize where and when, to build numerous coupled signaling processes that allow for communication and regulation. The outcome is an intricate network that processes complex molecular information (1, 2). As just one example, spatial and temporal control of calcium signaling is responsible for regulation of atrial cardiomyocytes during excitation-contraction coupling (3). Nature has built-in mechanisms to sense and respond to physiochemical and mechanical stimuli of various kinds. Foreign materials (macroscopic surfaces or nanoparticles) may harness these mechanisms and may elicit unexpected biological effects. For example, integrins are a widely expressed family of heterodimeric proteins that act as molecular bridges between surface-adsorbed extracellular matrix proteins and interacting cells. Integrin-mediated adhesion is associated with a complex cascade of biochemical and biomechanical events, including activation of numerous intracellular signaling enzymes, such as focal adhesion kinase and extracellular signal–regulated kinase (46). The presence of the foreign surface may alter gene expression leading to phenotypic variation (7). The overall biological outcome of perturbation by a foreign body is determined by the signaling pathways that have evolved naturally and that are triggered in normally functioning cells. In the future, a major effort combining approaches from biological (2, 6, 8) and physical (9) sciences will enable determination of the "principal" pathways of the signal processing network that are activated by given classes of perturbation.

The nature of biological responses to macroscopic foreign materials is a well-studied subject (10, 11), and the medical-device industry in particular has investigated the nature of the in vivo response to implants. The overall outcome for tissue surrounding foreign materials is often acute inflammation, chronic inflammation (involving monocytes and lymphocytes), the formation of granulation tissue (involving fibroblast proliferation and migration and new capillary formation), and, finally, formation of fibrous capsules (10). This so-called foreign body reaction is a normal physiological response to the implanted biomaterial. Once a steady state is obtained (with encapsulation of the material), an implant is considered biocompatible and suitable for human use. However, dramatic and persistent inflammatory responses are observed for some materials, leading to cell injury and fibrosis and, in some more extreme cases, uncontrolled proliferation and phenotype shift (as, for example, in restenosis). Other phenomena are observed for particles that are known to be "toxic," including asbestos, quartz, and others. Exposure to some of these toxic particles can lead to serious and incurable diseases (1214).

An emerging branch of cell-biomaterial interactions relates to the fate of nanoparticles in vivo. Nanoparticles have potential for use in many technical and medical applications, including in diagnostics and therapeutics (1517), and may offer advantages such as controlled or targeted release during drug or gene delivery. Novel cancer therapies that show promise include the use of ceramic-based nanoparticles or dendritic polymer nanoparticles (18, 19). Polymeric nanoparticles are being developed as contrast agents for magnetic resonance imaging (MRI) and echography, offering improved noninvasive diagnostic capabilities (20, 21). Nanoparticles can be made from a vast range of materials, such as metals (gold, silver), metal oxides (TiO2, SiO2), inorganic materials (carbon nanotubes, quantum dots), polymeric materials, and lipids. These particles can range from several to several hundreds of nanometers in diameter. Nanoparticles have access to various intra- and extracellular spaces and may thereby provoke a large range of responses in biological systems. In physiological environments, foreign materials are typically coated with proteins, and, at the surface, the conformation of these proteins may be disrupted or the proteins may aggregate in various manners, which can trigger unexpected cellular responses. The small size of nanoparticles means that a much larger (and often poorly understood) surface can be presented to the biological fluid. As much as 1 m2 of foreign surface is introduced per milliliter by 60-nm particles present at 1% volume fraction.

Nanoparticles may affect signaling both from outside the cell (when they adhere to the cell surface) and upon entering cells. The primary means by which particles enter cells (apart from phagocytes that have specific internalizing machinery) is through receptor-mediated endocytosis. Receptor-mediated endocytosis is controlled by clathrin, a triskeletal peripheral membrane protein that, when activated, induces the formation of cagelike capsules that engulf the particle (or other foreign body) and draw it into the cell. The particle first enters the primary endosomal compartment and then transits through the secondary endosomal compartment to different locations depending on aspects, as yet poorly understood, of the particle and its surface (22). The molecular structure of clathrin is such that there is a natural size limit (about 120 nm) for these cages and, thereby, a size cutoff above which particles may not use this endocytotic route. Particles smaller than 70 nm are efficient in harnessing the endocytotic machinery of the cell. An optimal particle radius of around 25 nm has been suggested on the basis of experimental data (23) and calculations of membrane wrapping around particles (22). The relation between nanoparticle size and uptake into cells, as well as that between nanoparticle size (curvature) and protein adsorption, is shown schematically in Fig. 1.

Fig. 1.

Cellular interaction with nanoparticles of various sizes. (A) Upon encountering biological solutions, nanoparticles interact with proteins, causing conformational changes in the protein's structure, which may expose cryptic epitopes. The smaller nanoparticles (with the greatest curvature) may cause the greatest disruption to protein conformation. (B) Nanoparticles of 15 nm (yellow) or 70 nm (green) can be endocytosed by receptor-mediated endocytosis. Larger nanoparticles (for example, 250 nm) (purple) remain outside cells.

There are growing concerns that interactions of nanoparticles with cells may lead to profound and as yet unknown consequences (24, 25). Transcriptomics may be one approach that can be used to connect intracellular regulatory processes to surfaces of different types (7). With the use of oligonucleotide microarrays, the presence of asbestos fibers in cells was found to result in increased expression of genes associated with cancer and metastasis. In particular, expression of the early response proto-oncogene fra-1, which encodes a transcription factor, was increased in pulmonary mesothelioma cells that were exposed to asbestos (26). Silencing of fra-1 resulted in greatly diminished expression of cd44, which encodes a cell surface glycoprotein, and c-met, which encodes a receptor tyrosine kinase, thereby making the connection to genes governing motility and invasion processes during metastasis in mesothelioma (26). The biological response to implanted materials or nanoparticles largely depends on the proteins that adsorb to the surface of the foreign body. When implant materials or nanoparticles enter a biological fluid, the surface-adsorbed proteins will be disrupted to some degree, unless concerted efforts are made to prevent this interference (27). The conformational change of these proteins may determine the early (and possibly longer term) biological responses. In aqueous solution, native globular proteins are folded with their hydrophobic groups clustered in the core, away from solvent. Billions of years of evolution of the amino acid sequence has selected hydrophobic residues with shape complementarity to allow for close packing of the cores (2830). Proteins are nevertheless marginally stable, because the beneficial interactions that govern the native structure are counterbalanced by a large entropy loss associated with going from a large ensemble of states to a more restricted set of conformations, as well as by repulsive electrostatic interactions present in the native state (31).

Conformational changes at the time of surface adsorption of proteins vary greatly. Some proteins are bound in nativelike fashion; some experience increased domain motions; others undergo denaturation of the tertiary structure; and, in some cases, even the secondary structure is lost (32, 33). The protein may be bound in a preferred orientation (34, 35), and the degree of denaturation depends on the foreign surface composition (7), as well as its curvature (36, 37). Nanoparticles may induce structural effects quite different from those of "flat" surfaces (33). Furthermore, mutagenesis studies show that small variations in sequence can greatly change the binding affinity of a protein for a surface and the structural consequences of adsorption. Altered functional properties of the adsorbed protein (ligand binding, catalytic and other functions) may also interfere with extra-, inter-, or intracellular signaling pathways.

Clearly some proteins are more apt to bind to foreign surfaces than others (38). The time course for the conformational change upon adsorption may range from seconds to days (39). The rates of protein adsorption, conformational change, and possibly exchange with other proteins will be critical parameters that influence the cellular signaling effects of a foreign surface (40). Many biological processes are under kinetic rather than thermodynamic control, and the time order at which the surface "sees" different biological environments may determine its fate in terms of which proteins are eventually presented to the cells and which cellular responses are invoked. Nature has evolved safety mechanisms to clear the system of age-modified and denatured proteins (41), whereas partial unfolding may provoke adverse signaling, such as overexpression of inflammatory factors [for example, cytokines (42, 43)] or reactive oxygen species (44), or may lead to uncontrolled aggregation or amyloidosis diseases (45). Protein adsorption on implanted surfaces may result in surface exposure of residues that are normally buried in the protein core, here termed "cryptic epitopes." These surface-adsorbed proteins with their disrupted conformation are similar enough to the native protein that the cells do not recognize them as denatured, but are different enough from the native protein that they trigger inappropriate cellular processes.

Thus, one of the most pressing issues that need to be addressed is the identification of these surface-exposed cryptic epitopes that result from proteins interacting with engineered surfaces (46). This is not a trivial task, and much of what we know about adsorbed proteins derives from rather low resolution methods (Table 1). Detailed structural studies of adsorbed proteins are challenging, because the main method used for atomic-level description of proteins in solution [nuclear magnetic resonance (NMR) spectroscopy] is not applicable to extended surfaces or slowly tumbling particles. Techniques like neutron reflectivity and scattering, as well as solid-state NMR, offer the potential for detailed structural studies of adsorbed proteins and are beginning to be applied to these questions (47, 48). It is necessary to understand both the molecular nature of the cryptic epitopes and the biological consequences of them. Does partial protein denaturation and the exposure of cryptic epitopes cause larger effects on signaling than those associated with complete protein denaturation? Complete protein denaturation may trigger the body’s defenses to recognize it as a foreign body, whereas the cryptic epitopes do not. Thus, it is clear that the issues of protein adsorption on flat surfaces or nanoparticles, adsorbed protein conformation, and induced cellular signaling pathways are deeply entwined and need to be studied in parallel. However, the solution properties of nanoparticles (their aggregation behavior in physiological environments), the type and quantity of adsorbed proteins, and the signaling pathways that they may influence are all critically dependent on many biological parameters. These are attributes such as pH, temperature, redox potential, and the concentrations of low-molecular-weight ligands and cofactors, including the presence or absence of metal ions that are critical to the structure and function of ion-binding proteins and signaling cascades. Therefore, in order to be meaningful, any studies of these complex systems need to be conducted under well-controlled solution conditions, with the use of extremely well characterized and purified surfaces or nanoparticles (to ensure that no impurities are bound to the proteins), and by using precisely defined buffer systems and protein preparations. The last-named is more complicated than previously understood, as it has recently been shown that, within humans, there are many variants of the common serum proteins and that large percentages of the population may have a variant, as opposed to what is currently considered the wild-type protein (49). Once the molecular and biological responses to nanoparticles have been better understood, nanoparticles are predicted to become versatile tools in medicine and research, offering new therapeutic and diagnostic solutions. These nanoparticle-based systems will be less invasive; could induce fewer side effects as a result of improved targeting; and may be multifunctional, offering simultaneous diagnosis and treatment.

Table 1. A review of the techniques (physical and biological) that have been applied to understanding the issues of protein adsorption at surfaces and conformational changes upon adsorption, as well as to understanding the effects of surfaces on cellular signaling processes.


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