The protein corona spontaneously develops and evolves on the surface of nanoscale materials when they are exposed to biological environments, altering their physiochemical properties and affecting their subsequent interactions with biosystems. In this Review, we provide an overview of the current state of protein corona research in nanomedicine. We next discuss remaining challenges in the research methodology and characterization of the protein corona that slow the development of nanoparticle therapeutics and diagnostics, and we address how artificial intelligence can advance protein corona research as a complement to experimental research efforts. We then review emerging opportunities provided by the protein corona to address major issues in healthcare and environmental sciences. This Review details how mechanistic insights into nanoparticle protein corona formation can broadly address unmet clinical and environmental needs, as well as enhance the safety and efficacy of nanobiotechnology strength also influence surface-binding-induced protein unfolding 45 .For example, adsorption-induced protein conformational changes were quantified by targeting and fluorescently labelling exposed amine groups; these residues, which were originally buried inside the tertiary structures of proteins in native conformations, became exposed because of adsorption-induced conformational disorders 48 . Protein conformational changes can impact protein functionality through the exposure of once-concealed functional epitopes or misfolding that results in complete loss-of-protein function, subsequently impacting 1960s

Effective in silico methods to predict protein corona compositions on engineered nanomaterials (ENMs) could help elucidate the biological outcomes of ENMs in biosystems without the need for conducting lengthy experiments for corona characterization. However, the physicochemical properties of ENMs, used as the descriptors in current modeling methods, are insufficient to represent the complex interactions between ENMs and proteins. Herein, we utilized the fluorescence change (FC) from fluorescamine labeling on a protein, with or without the presence of the ENM, as a novel descriptor of the ENM to build machine learning models for corona formation. FCs were significantly correlated with the abundance of the corresponding proteins in the corona on diverse classes of ENMs, including metal and metal oxides, nanocellulose, and 2D ENMs. Prediction models established by the random forest algorithm using FCs as the ENM descriptors showed better performance than the conventional descriptors, such as ENM size and surface charge, in the prediction of corona formation. Moreover, they were able to predict protein corona formation on ENMs with very heterogeneous properties. We believe this novel descriptor can improve in silico studies of corona formation, leading to a better understanding on the protein adsorption behaviors of diverse ENMs in different biological matrices. Such information is essential for gaining a comprehensive view of how ENMs interact with biological systems in ENM safety and sustainability assessments.

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In the presence of biological matrices, engineered nanomaterials, such as TiO 2 , develop a biomolecular corona composed of lipids, proteins, etc. In this study, we analyzed the biocorona formed on the food grade TiO 2 (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 TiO 2 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 *

Increasing applications of nanomaterials in consumer goods, industrial products, medical practices, etc., calls for the development of tools for rapid separation, quantification, and sizing of nanoparticles to ensure their safe and sustainable employment. While many techniques are available for characterization of pure, homogeneous nanomaterial preparations, particle sizing and counting remains difficult for heterogeneous mixtures that resulted from imperfect synthesis conditions, aggregation from product instability, or degradation during storage. Herein, nanoparticle tracking analysis (NTA) was coupled to asymmetrical flow field flow fraction (AF4) using a splitter manifold to enable online particle separation and counting. The high pressure and flow rate in AF4 were reduced to the levels compatible with NTA by the proper flow splitting design, and a syringe pump was employed to withdraw fluid through the exit port of the NTA and maintain consistent flow rates entering NTA for proper particle sizing. Successful AF4-NTA coupling was demonstrated by analyzing a mixture of polystyrene particles with the average diameters of ∼50, 100, and 200 nm. Good correlation was observed between the amount of each type of particle injected to and measured by the hyphenated system. The particle concentrations acquired using online and offline coupling of AF4-NTA also agreed well with each other. The nonspherical nanoparticles like gold nanorods and hexagonal boron nitride nanosheets were also analyzed to demonstrate the versatile applicability of this system. Our work has proved that AF4-NTA can achieve accurate online particle counting on different populations of the nanomaterials in a mixture, which cannot be done by either AF4 or NTA alone. It will be a valuable tool for rapid characterization of heterogeneous nanomaterial solutions without purification to fulfill the regulation requirement on the nanomaterial-containing products.

The molecular structure of a protein could be altered when attached to nanoparticles (NPs), affecting the performance of NPs present in biological systems. Limited proteolysis coupled with LC-MS/MS could reveal the changes in protein structure when it binds to a variety of entities, including macro-molecules and small drugs, but has not yet been applied to study protein-NP interaction. Herein, adsorption of proteins, transferrin and catalase, on the polystyrene (PS) or iron oxide (IO) NPs was analyzed with this method. Both increased and decreased proteolytic efficiency in certain regions on the proteins were observed. Identification of the peptides affected by protein-NP interaction led to proper prediction of alterations to protein function as well as to colloidal stability of NPs. Overall, the present work has demonstrated the utility of limited proteolysis in helping elucidate the potential biological outcomes of the protein-NP conjugate, obtaining knowledge to guide improvement of the rational design of the protein conjugated NPs for biomedical applications, and to understand the biological behaviors of the engineered NPs. Protein adsorption on the surface of engineered nanoparticles (NPs) unintentionally exposed to biological systems is inevitable. 1-5 On the other hand, intentionally attaching specific proteins (i.e. antibodies) on NPs as the target recognition moieties has also been used to achieve active targeting and assist delivery of NPs for therapeutic purposes. 6-8 While the adsorbed protein corona could be beneficial to the outcomes of the NPs by keeping the NPs from being cleared by the innate immune system; 9-11 structural changes, e.g. unfolding, fibrillation and aggregation, 12-14 could occur to the protein after binding to the NPs, and induce negative impacts on the protein-NP conjugates. 7,15,16 For example, protein unfolding can expose the originally buried cryptic epitopes to induce immune clearance of the NPs and

Despite mounting evidence of micro-nanoplastics (MNPs) in food and drinking water, little is known of the potential health risks of ingested MNPs, and nothing is known of their potential impact on nutrient digestion and absorption. We assessed the effects of environmentally relevant secondary MNPs generated by incineration of polyethylene (PE-I), on digestion and absorption of fat in a high fat food model using a 3-phase in vitro simulated digestion coupled with a tri-culture small intestinal epithelium model. The presence of 400 μg/mL PE-I increased fat digestion by 33% and increased fat absorption by 147 and 145% 1 and 2 h after exposure. Analysis of the PE-I lipid corona during digestion revealed predominantly triacylglycerols with enrichment of fatty acids in the small intestinal phase. Protein corona analysis showed enrichment of triacylglycerol lipase and depletion of β-casein in the small intestinal phase. These findings suggest digestion of triacylglycerol by lipase on the surface of lipid-coated MNPs as a potential mechanism. Further studies are needed to investigate the mechanisms underlying the greater observed increase in fat absorption, to verify these results in an animal model, and to determine the MNP properties governing their effects on lipid digestion and absorption.

Applications of reduced graphene oxide (rGO) in many different areas have been gradually increasing owing to its unique physicochemical characteristics, demanding more understanding of their biological impacts. Herein, we assessed the toxicological effects of rGO in mammary epithelial cells. Because the as-synthesized rGO was dissolved in sodium cholate to maintain a stable aqueous dispersion, we hypothesize that changing the cholate concentration in the dispersion may alter the surface property of rGO and subsequently affect its cellular toxicity. Thus, four types of rGO were prepared and compared: rGO dispersed in 4 and 2 mg/mL sodium cholate, labeled as rGO and concentrated-rGO (c-rGO), respectively, and rGO and c-rGO coated with a protein corona through 1 h incubation in culture media, correspondingly named pro-rGO and pro-c-rGO. Notably, c-rGO and pro-c-rGO exhibited higher toxicity than rGO and pro-rGO and also caused higher reactive oxygen species production, more lipid membrane peroxidation, and more significant disruption of mitochondrial-based ATP synthesis. In all toxicological assessments, pro-c-rGO induced more severe adverse impacts than c-rGO. Further examination of the material surface, protein adsorption, and cellular uptake showed that the surface of c-rGO was coated with a lower content of surfactant and adsorbed more proteins, which may result in the higher cellular uptake observed with pro-c-rGO than pro-rGO. Several proteins involved in cellular redox mediation were also more enriched in pro-c-rGO. These results support the strong correlation between dispersant coating and corona formation and their subsequent cellular impacts. Future studies in this direction could reveal a deeper understanding of the correlation and the specific cellular pathways involved and help gain knowledge on how the toxicity of rGO could be modulated through surface modification, guiding the sustainable applications of rGO.