Biomolecular Corona Research Articles

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials.

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as "self". For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis - mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography - mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 µL of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (<20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Implications of Biomolecular Corona for Molecular Imaging.

The development of nanoparticle probes has opened up new possibilities for molecular imaging in the era of precision medicine. There are a wide range of nanoprobes that are being used for various modalities that have demonstrated promising potential in early detection, disease monitoring, and theranostics. However, the rate ofsuccessfulclinical translation of the nanoprobes is very low and is affected by the lack of our understanding about nanoparticle interaction with biological fluids after systemic administration, thus representing an unmet clinical need. One of the poorly understood issues relates to the formation of biomolecular corona, a layer of biomolecules formed on the surface of nanoscale materials during their interactions with biological fluids. The biomolecular corona has several significant effects on the biodistribution of nanoprobes and their imaging ability by (i) reducing their targeting efficacy and (ii) affecting the intrinsic imaging properties (e.g., contrast capacity of magnetic nanoprobes). This review provides insights on the importance of considering biomolecular corona in the development of nanoprobes, which may enable their more efficient utilization for molecular imaging applications.

Denatured corona proteins mediate the intracellular bioactivities of nanoparticles via the unfolded protein response

Biomolecular corona formed on nanoparticles (NPs) influences the latter's in vivo biological effects. Nanomaterials with different physicochemical properties exert similar adverse effects, such as cytotoxicity, suggesting the existence of ubiquitous signals during various corona formations that mediate common and fundamental cellular events. Here, we discover the involvement of the unfolded protein response (UPR) and recruited chaperones in the corona. Specially, heat shock protein 90 kDa α class B member 1 (Hsp90ab1) is abundantly enriched in the corona, accompanied by substantial aggregation of misfolded protein on particles intracellularly. Further analysis reveals the particulate matter 2.5 (PM2.5) and metal-containing particles are more capable of denaturing proteins. The recruited Hsp90ab1 activates diverse NPs' pathological behaviour by heat stress response (HSR), which were significantly reversed by geldanamycin (GA), the inhibitor of Hsp90ab1. Murine lung inflammation induced by PM2.5 and iron oxide NPs (Fe3O4NPs) is suppressed by GA, highlighting that Hsp90ab1-mediated UPR is a potential target for the treatment of environmental pollution-related illnesses. Based on our findings, the UPR and Hsp90ab1 presented in the corona of particles initiate fundamental intracellular reactions that lead to common pathological outcomes, which may provide new insights for understanding nanotoxicity and designing therapeutic approaches for diseases associated with environmental pollution.

Lipid and protein corona of food-grade TiO2 nanoparticles in simulated gastrointestinal digestion

In the presence of biological matrices, engineered nanomaterials, such as TiO2, develop a biomolecular corona composed of lipids, proteins, etc. In this study, we analyzed the biocorona formed on the food grade TiO2 (E171) going through an in vitro simulated gastrointestinal digestion system in either a fasting food model (FFM), a standardized food model (SFM), or a high fat food model (HFFM). Lipids and proteins were extracted from the biocorona and underwent untargeted lipidomic and label-free shotgun proteomic analyses. Our results showed that the biocorona composition was different before and after food digestion. After digestion, more diverse lipids were adsorbed compared to proteins, most of which were the enzymes added to the simulated digestion system. The corona lipid profile was distinct from the digested food model they presented in, although similarity in the lipid profiles between the corona and the food matrix increased with the fat content in the food model. The corona formed in the two low-fat environments of FFM and SFM shared a higher degree of similarity while very different from their corresponding matrix, with some lipid species adsorbed with high enrichment factors, indicating specific interaction with the TiO2 surface outperforming lipid matrix concentration in determination of corona formation. Formation of the biocorona may have contributed to the reduced oxidative stress as well as toxicological impacts observed in cellular studies. The present work is the first to confirm persistent adsorption of biomolecules could occur on ingested nanomaterials in food digestae. More future studies are needed to study the in vivo impacts of the biocorona, and shed light on how the biocorona affects the biotransformations and fate of the ingested nanomaterials, which may impose impacts on human health.

Curcumin and isonicotinic acid hydrazide functionalized gold nanoparticles for selective anticancer action

Besides physicochemical properties, careful design of biomolecular surface corona can dictate therapeutic potential of nanoparticles. Therefore, we demonstrate a new approach to improve the anticancer potential of gold nanoparticles (AuNPs) by developing a strong corona of curcumin and isonicotinic acid hydrazide (INH) around them. Here, curcumin reduced AuNPsCur are surface-functionalized with biologically active INH (AuNPsCur@INH). Our investigations on human lung squamous carcinoma (LK-2) and human lung fibroblast (TIG-120) cells indicate selective toxicity of functionalized nanoparticles. Conjugation of curcumin and INH on the surface of AuNPs induced anticancer action through reactive oxygen species (ROS) generation. The further extension of this study revealed apoptosis and morphological changes in LK-2 and TIG-120 cells, respectively. Moreover, the anticancer potential of these nanoparticles is compared with standard cisplatin. The outcomes of the work determine that the facile functional surfaces on nanoparticles can be important in controlling the biological activities of nanoparticles.

The Biomolecular Corona of Lipid Nanoparticles for Gene Therapy.

Gene therapy holds great potential for treating almost any disease by gene silencing, protein expression, or gene correction. To efficiently deliver the nucleic acid payload to its target tissue, the genetic material needs to be combined with a delivery platform. Lipid nanoparticles (LNPs) have proven to be excellent delivery vectors for gene therapy and are increasingly entering into routine clinical practice. Over the past two decades, the optimization of LNP formulations for nucleic acid delivery has led to a well-established body of knowledge culminating in the first-ever RNA interference therapeutic using LNP technology, i.e., Onpattro, and many more in clinical development to deliver various nucleic acid payloads. Screening a lipid library in vivo for optimal gene silencing potency in hepatocytes resulted in the identification of the Onpattro formulation. Subsequent studies discovered that the key to Onpattro's liver tropism is its ability to form a specific "biomolecular corona". In fact, apolipoprotein E (ApoE), among other proteins, adsorbed to the LNP surface enables specific hepatocyte targeting. This proof-of-principle example demonstrates the use of the biomolecular corona for targeting specific receptors and cells, thereby opening up the road to rationally designing LNPs. To date, however, only a few studies have explored in detail the corona of LNPs, and how to efficiently modulate the corona remains poorly understood. In this review, we summarize recent discoveries about the biomolecular corona, expanding the knowledge gained with other nanoparticles to LNPs for nucleic acid delivery. In particular, we address how particle stability, biodistribution, and targeting of LNPs can be influenced by the biological environment. Onpattro is used as a case study to describe both the successful development of an LNP formulation for gene therapy and the key influence of the biological environment. Moreover, we outline the techniques available to isolate and analyze the corona of LNPs, and we highlight their advantages and drawbacks. Finally, we discuss possible implications of the biomolecular corona for LNP delivery and we examine the potential of exploiting the corona as a targeting strategy beyond the liver to develop next-generation gene therapies.

Tracing the In Vivo Fate of Nanoparticles with a “Non-Self” Biological Identity

Nanoparticles can acquire a biomolecular corona with a species-specific biological identity. However, "non-self" incompatibility of recipient biological systems is often not considered, for example, when rodents are used as a model organism for preclinical studies of biomolecule-inspired nanomedicines. Using zebrafish embryos as an emerging model for nanobioimaging, here we unravel the in vivo fate of intravenously injected 70 nm SiO2 nanoparticles with a protein corona preformed from fetal bovine serum (FBS), representing a non-self biological identity. Strikingly rapid sequestration and endolysosomal acidification of nanoparticles with the preformed FBS corona were observed in scavenger endothelial cells within minutes after injection. This led to loss of blood vessel integrity and to inflammatory activation of macrophages over the course of several hours. As unmodified nanoparticles or the equivalent dose of FBS proteins alone failed to induce the observed pathophysiology, this signifies how the corona enriched with a differential repertoire of proteins can determine the fate of the nanoparticles in vivo. Our findings thus reveal the adverse outcome triggered by incompatible protein coronas and indicate a potential pitfall in the use of mismatched species combinations during nanomedicine development.

Role of Physicochemical Properties of Protein in Modulating the Nanoparticle-Bio Interface.

Nanoparticles, on exposure to the biological milieu, tend to interact with macromolecules to form a biomolecular corona. The biomolecular corona confers a unique biological identity to nanoparticles, and its protein composition plays a deterministic role in the biological fate of nanoparticles. The physiological behavior of proteins stems from their physicochemical properties, including surface charge, hydrophobicity, and structural stability. However, there is insufficient understanding about the role of physicochemical properties of proteins in biomolecular corona formation. We hypothesized that the physicochemical properties of proteins would influence their interaction with nanoparticles and have a deterministic effect on nanoparticle-cell interactions. To test our hypothesis, we used model proteins from different structural classes to understand the effect of secondary structure elements of proteins on the nanoparticle-protein interface. Further, we modified the surface of proteins to study the role of protein surface characteristics in governing the nanoparticle-protein interface. For this study, we used mesoporous silica nanoparticles as a model nanoparticle system. We observed that the surface charge of proteins governs the nature of the primary interaction and the extent of subsequent secondary interactions causing structural rearrangements of the protein. We also observed that the secondary structural contents of proteins significantly affected both the extent of secondary interactions at the nanoparticle-protein interface and the dispersion state of the nanoparticle-protein complex. Further, we studied the interactions of different protein-coated nanoparticles with different cells (fibroblast, carcinoma, and macrophage). We observed that different cells internalized the nanoparticle-protein complex as a function of secondary structural components of the protein. The type of model protein had a significant effect on their internalization by macrophages. Overall, we observed that the physicochemical characteristics of proteins had a significant role in modulating the nanoparticle-bio-interface at the level of both biomolecular corona formation and nanoparticle internalization by cells.

Behavior and Bio-Interactions of Anthropogenic Particles in Marine Environment for a More Realistic Ecological Risk Assessment

Due to production, usage and disposal of nano-enabled products as well as fragmentation of bulk materials, anthropogenic nanoscale particles (NPs) can enter the natural environment and through different compartments (air, soil and water) end up into the sea. With the continuous increase of production and associated emissions and discharges, they can reach concentrations able to exceed toxicity-thresholds for living species inhabiting marine coastal areas. Behavior and fate of NPs in marine waters are driven by transformation processes occurring as a function of NP intrinsic and extrinsic properties in the receiving seawaters. All those aspects have been overlooked in ecological risk assessment. This review critically reports ecotoxicity studies in which size distribution, surface charges and bio-nanointeractions have been considered for a more realistic risk assessment of NPs in marine environment. Two emerging and relevant NPs, the metal-based titanium dioxide (TiO2), and the polystyrene (PS), a proxy for nanoplastics, are reviewed and their impact on marine biota (from planktonic species to invertebrates and fish) discussed as a function of particle size and surface charges (negative vs positive) which affect their behavior and interaction with the biological material. Uptake of NPs is related to their nanoscale size, however in vivo studies clearly demonstrated that transformation (agglomerates/aggregates) occurring in both artificial and natural seawater drive to different exposure routes and biological responses at cellular and organism level. Adsorption of single particles or agglomerates onto the body surface or their internalization in feces can impair motility and affect sinking or floating behavior with consequences on population and ecological function. Particle complex dynamics in natural seawater is almost unknown although it determines the effective exposure scenarios. Based on the latest predicted environmental concentrations for TiO2 and PS NPs in the marine environment, current knowledge gaps and future research challenges encompass the comprehensive study of bio-nano interactions. As such, the analysis of NP biomolecular coronas can enable a better assessment of particle uptake and related cellular pathways leading to toxic effects. Moreover, the formation of an environmentally-derived corona (i.e. eco-corona) in seawater accounts for NP physical-chemical alterations, rebounding on interaction with living organisms and toxicity.

Sub-10 nm Radiolabeled Barium Sulfate Nanoparticles as Carriers for Theranostic Applications and Targeted Alpha Therapy.

The treatment of cancer patients with α‐particle‐emitting therapeutics continues to gain in importance and relevance. The range of radiopharmaceutically relevant α‐emitters is limited to a few radionuclides, as stable chelators or carrier systems for safe transport of the radioactive cargo are often lacking. Encapsulation of α‐emitters into solid inorganic systems can help to diversify the portfolio of candidate radionuclides, provided, that these nanomaterials effectively retain both the parent and the recoil daughters. We therefore focus on designing stable and defined nanocarrier‐based systems for various clinically relevant radionuclides, including the promising α‐emitting radionuclide 224Ra. Hence, sub‐10 nm barium sulfate nanocontainers were prepared and different radiometals like 89Zr, 111In, 131Ba, 177Lu or 224Ra were incorporated. Our system shows stabilities of >90 % regarding the radiometal release from the BaSO4 matrix. Furthermore, we confirm the presence of surface‐exposed amine functionalities as well as the formation of a biomolecular corona.

Surface Modification of Spider Silk Particles to Direct Biomolecular Corona Formation.

In recent years, spider silk-based materials have attracted attention because of their biocompatibility, processability, and biodegradability. For their potential use in biomaterial applications, i.e., as drug delivery systems and implant coatings for tissue regeneration, it is vital to understand the interactions between the silk biomaterial surface and the biological environment. Like most polymeric carrier systems, spider silk material surfaces can adsorb proteins when in contact with blood, resulting in the formation of a biomolecular corona. Here, we assessed the effect of surface net charge of materials made of recombinant spider silk on the biomolecular corona composition. In-depth proteomic analysis of the biomolecular corona revealed that positively charged spider silk materials surfaces interacted predominantly with fibrinogen-based proteins. This fibrinogen enrichment correlated with blood clotting observed for both positively charged spider silk films and particles. In contrast, negative surface charges prevented blood clotting. Genetic engineering allows the fine-tuning of surface properties of the spider silk particles providing a whole set of recombinant spider silk proteins with different charges or peptide tags to be used for, for example, drug delivery or cell docking, and several of these were analyzed concerning the composition of their biomolecular corona. Taken together this study demonstrates how the surface net charge of recombinant spider silk surfaces affects the composition of the biomolecular corona, which in turn affects macroscopic effects such as fibrin formation and blood clotting.

On‐Chip Electrical Monitoring of Real‐Time “Soft” and “Hard” Protein Corona Formation on Carbon Nanoparticles

AbstractWhen 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.

Surface Coating Structure and Its Interaction with Cytochrome c in EG6-Coated Nanoparticles Varies with Surface Curvature.

The composition, orientation, and conformation of proteins in biomolecular coronas acquired by nanoparticles in biological media contribute to how they are identified by a cell. While numerous studies have investigated protein composition in biomolecular coronas, relatively little detail is known about how the nanoparticle surface influences the orientation and conformation of the proteins associated with them. We previously showed that the peripheral membrane protein cytochrome c adopts preferred poses relative to negatively charged 3-mercaptopropionic acid (MPA)-gold nanoparticles (AuNPs). Here, we employ molecular dynamics simulations and complementary experiments to establish that cytochrome c also assumes preferred poses upon association with nanoparticles functionalized with an uncharged ligand, specifically ω-(1-mercaptounde-11-cyl)hexa(ethylene glycol) (EG6). We find that the display of the EG6 ligands is sensitive to the curvature of the surface-and, consequently, the effective diameter of the nearly spherical nanoparticle core-which in turn affects the preferred poses of cytochrome c.

Probing protein adsorption onto polymer-stabilized silver nanocolloids towards a better understanding on the evolution and consequences of biomolecular coronas.

The use of noble metal nanoparticles in biomedical and biotechnological applications is nowadays well established. Particularly, silver nanoparticles (AgNPs) were proven to be effective for instance as a biocide agent. They also find applications in tumor therapies and sensing applications being encouraging tools for in-vivo imaging. In this framework, whenever they are in contact with living systems, they are rapidly coated by a protein corona thereby influencing a variety of biological events including cellular uptake, blood circulation lifetime, cytotoxicity and, ultimately, the therapeutic effect. Taking these considerations into account, we have explored the behavior of polymer-coated AgNPs in model protein environments focusing on the self-development of protein coronas. The polymers polyethyleneimine (PEI), polyvinylpyrrolidone (PVP) and poly(2-vinyl pyridine)-b-poly(ethylene oxide) (PEO-b-P2VP) were used as stabilizing agents. The chemical nature of the polymer capping remarkably influences the behavior of the hybrid nanomaterials in protein environments. The PEO-b-P2VP and PVP-stabilized AgNPs are essentially inert to the model proteins adsorption. On the other hand, the PEI-stabilized AgNPs interact strongly with bovine serum albumin (BSA). Nevertheless, the same silver colloids were evidenced to be stable in IgG and lysozyme environments. The BSA adsorption into the PEI-stabilized AgNPs is most probably driven by hydrogen bonding and van der Waals interactions as suggested by isothermal titration calorimetry data. The development of protein coronas around the AgNPs may have relevant implications in a variety of biological events. Therefore, further investigations are currently underway to evaluate the influence of its presence on the cytotoxicity, hemolytic effects and biocide properties of the produced hybrid nanomaterials.

The Biomolecular Corona in 2D and Reverse: Patterning Metal–Phenolic Networks on Proteins, Lipids, Nucleic Acids, Polysaccharides, and Fingerprints

AbstractThe adsorption of biomolecules onto nanomaterials can alter the performance of the nanomaterials in vitro and in vivo. Recent studies have primarily focused on the protein “corona”, formed upon adsorption of proteins onto nanoparticles in biological fluids, which can change the biological fate of the nanoparticles. Conversely, interactions between nanomaterials and other classes of biomolecules namely, lipids, nucleic acids, and polysaccharides have received less attention despite their important roles in biology. A possible reason is the challenge associated with investigating biomolecule interactions with nanomaterials using current technologies. Herein, a protocol is developed for studying bio–nano interactions by depositing four classes of biomolecules (proteins, lipids, nucleic acids, and polysaccharides) and complex biological media (blood) onto planar substrates, followed by exposure to metal–phenolic network (MPN) complexes. The MPNs preferentially interact with the biomolecule over the inorganic substrate (glass), highlighting that patterned biomolecules can be used to engineer patterned MPNs. Subsequent formation of silver nanoparticles on the MPN films maintains the patterns and endows the films with unique reflectance and fluorescence properties, enabling visualization of latent fingerprints (i.e., invisible residual biomolecule patterns). This study demonstrates the potential complexity of the biomolecule corona as all classes of biomolecules can adsorb onto MPN‐based nanomaterials.

A comprehensive analysis of liposomal biomolecular corona upon human plasma incubation: The evolution towards the lipid corona

When drug nanocarriers enter a physiological environment, their surface gets coated by a dynamic biomolecular corona (BMC) mainly constituted by proteins. Although a deep investigation has been performed on the composition of BMC in terms of proteins, scarce attention has been posed to low molecular weight metabolites present in human plasma. In this work, for the first time, the investigation of the BMC of liposomal nanoparticles (NPs) constituted by 1,2-dioleoyl-3-trimethylammonium-propane polar lipid has been carried out by an ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry based untargeted metabolomics approach. Compounds were tentatively identified based on matches with online databases and comparison of MS/MS spectra with available spectral libraries. Moreover, a comparison of three metabolite extraction strategies, including an ultrafiltration membrane based protocol, a methanol extraction based protocol, and Wessel & Flügge protocol, was performed. Methanol extraction procedure resulted in the widest metabolic coverage of liposomal NP BMC. A total of 193 metabolites has been tentatively identified, 166 of which belonged to the class of lipids including phospholipids, steroids, carnitines, fatty alcohols, diglycerides and fatty acids. The high abundance of lipids in the BMC can be explained by the adsorption of plasma lipoproteins onto liposome surface, confirming previous works on other kinds of NPs. Lipids are important bioactive molecules, which could impact NP circulation and uptake by cells. Extending the investigation of BMC beyond the protein corona and towards the “lipid corona” may be the keystone of a better understanding and control of NP fate in human body.

Nanozyme antioxidants as emerging alternatives for natural antioxidants: Achievements and challenges in perspective

Abstract Nanozymes are nanoparticulated structures with the inherent enzymatic activities, which their advances in bio/nanomedicine field are progressively growing regarding their exceptional properties, such as nano‐size, irregular shape, rich surface chemistry, low cost production, etc. These features enable nanostructures to mimic the cellular antioxidant enzymes as novel candidates for treatment of oxidative stress-induced disorders. Oxidative stress reflects principally an imbalance between generation of reactive oxygen species (ROS), such as hydroxyl and superoxide radicals, and the level of antioxidant defense system, so that this imbalance can lead to harsh pathological conditions. Here we present prevalent nanozyme antioxidants as well as their therapeutic concerns from diverse points of view including bio/cytocompatibility and cellular uptake, biomolecular corona, effect of pH and temperature on enzymatic function, and in vivo analysis, which are of paramount importance for reducing the bench-to-clinic gap.

Timing of Heparin Addition to the Biomolecular Corona Influences the Cellular Uptake of Nanocarriers.

Few studies have considered the interaction of nanocarriers with drugs and the implications for their individual efficiency. Here, we demonstrate that heparin, a common anticoagulant, interacts with nanocarriers. Hence, nanocarriers, precoated with heparin and plasma in different conditions, were incubated with cancer cells, as well as primary cells from human blood. The relation between the timing of the heparin's addition to the nanocarrier and the cellular uptake extent was assessed by flow cytometry. Through proteomics the effect of heparin on the biomolecular corona composition was determined. We found that HeLa cells, monocytes and macrophages reacted differently to the presence of heparin: the uptake of the precoated nanocarriers decreased for HeLa and primary monocytes, while it increased for macrophages. Heparin induced no obvious change in the protein corona composition; thus, we suggest that heparin itself, through its adsorption on the nanocarrier, was responsible for the change of uptake.

Virus-Sized Gold Nanorods: Plasmonic Particles for Biology.

Plasmons, collective oscillations of conduction-band electrons in nanoscale metals, are well-known phenomena in colloidal gold and silver nanocrystals that produce brilliant visible colors in these materials that depend on the nanocrystal size and shape. Under illumination at or near the plasmon bands, gold and silver nanocrystals exhibit properties that enable fascinating biological applications: (i) the nanocrystals elastically scatter light, providing a straightforward way to image them in complex aqueous environments; (ii) the nanocrystals produce local electric fields that enable various surface-enhanced spectroscopies for sensing, molecular diagnostics, and boosting of bound fluorophore performance; (iii) the nanocrystals produce heat, which can lead to chemical transformations at or near the nanocrystal surface and can photothermally destroy nearby cells. While all the above-mentioned applications have already been well-demonstrated in the literature, this Account focuses on several other aspects of these nanomaterials, in particular gold nanorods that are approximately the size of viruses (diameters of ∼10 nm, lengths up to 100 nm). Absolute extinction, scattering, and absorption properties are compared for gold nanorods of various absolute dimensions, and references for how to synthesize gold nanorods with four different absolute dimensions are provided. Surface chemistry strategies for coating nanocrystals with smooth or rough shells are detailed; specific examples include mesoporous silica and metal-organic framework shells for porous (rough) coatings and polyelectrolyte layer-by-layer wrapping for "smooth" shells. For self-assembled-monolayer molecular coating ligands, the smoothest shells of all, a wide range of ligand densities have been reported from many experiments, yielding values from less than 1 to nearly 10 molecules/nm2 depending on the nanocrystal size and the nature of the ligand. Systematic studies of ligand density for one particular ligand with a bulky headgroup are highlighted, showing that the highest ligand density occurs for the smallest nanocrystals, even though these ligand headgroups are the most mobile as judged by NMR relaxation studies. Biomolecular coronas form around spherical and rod-shaped nanocrystals upon immersion into biological fluids; these proteins and lipids can be quantified, and their degree of adsorption depends on the nanocrystal surface chemistry as well as the biophysical characteristics of the adsorbing biomolecule. Photothermal adsorption and desorption of proteins on nanocrystals depend on the enthalpy of protein-nanocrystal surface interactions, leading to light-triggered alteration in protein concentrations near the nanocrystals. At the cellular scale, gold nanocrystals exert genetic changes at the mRNA level, with a variety of likely mechanisms that include alteration of local biomolecular concentration gradients, changes in mechanical properties of the extracellular matrix, and physical interruption of key cellular processes-even without plasmonic effects. Microbiomes, both organismal and environmental, are the likely first point of contact of nanomaterials with natural living systems; we see a major scientific frontier in understanding, predicting, and controlling microbe-nanocrystal interactions, which may be augmented by plasmonic effects.

Analysis of complexes formed by small gold nanoparticles in low concentration in cell culture media.

New nanomaterials are constantly developed with applications in everything from cosmetics to high tech electronics. Assessing their biological impact has been done by analysis of their adsorbed protein corona, in vitro cell assays, and larger scale ecotoxicological studies. This has proved to be a huge challenge due to the wide range of available nanomaterials and their unpredictable behaviour in different environments. Furthermore, the enormous number of experimental variables make comparisons difficult. Concentration is one of these variables and can vary greatly depending on the aim of the study. When analysing the protein corona, concentrations are often higher than in cell assays. Using a combination of complementary techniques, we have characterised 20 nm gold nanoparticles in a concentration level commonly used in cell studies. We compare their behaviour in a commonly used, protein rich medium and one protein poor medium over 24 hours. Under these conditions, the NPs were stable in protein rich environment but underwent gradual aggregation in protein poor medium. We characterise the biomolecular corona in both media. In protein poor medium, we can describe the often overlooked aggregation. The aggregates’ morphology is confirmed by cryo-TEM. Finally, in the protein poor medium, by infrared spectroscopy, we have identified the amino acid arginine in the biomolecular corona which drives the aggregation.