Silver nanoparticle interactions with glycated and non-glycated human serum albumin mediate toxicity.

Introduction: Biomolecules bind to and transform nanoparticles, mediating their fate in biological systems. Despite over a decade of research into the protein corona, the role of protein modifications in mediating their interaction with nanomaterials remains poorly understood. In this study, we evaluated how glycation of the most abundant blood protein, human serum albumin (HSA), influences the formation of the protein corona on 40 nm silver nanoparticles (AgNPs) and the toxicity of AgNPs to the HepG2 human liver cell line. Methods: The effects of glycation on AgNP-HSA interactions were quantified using circular dichroism spectroscopy to monitor protein structural changes, dynamic light scattering to assess AgNP colloidal stability, zeta potential measurements to measure AgNP surface charge, and UV-vis spectroscopy and capillary electrophoresis (CE) to evaluate protein binding affinity and kinetics. The effect of the protein corona and HSA glycation on the toxicity of AgNPs to HepG2 cells was measured using the WST cell viability assay and AgNP dissolution was measured using linear sweep stripping voltammetry. Results and Discussion: Results from UV-vis and CE analyses suggest that glycation of HSA had little impact on the formation of the AgNP protein corona with protein-AgNP association constants of ≈2x107 M-1 for both HSA and glycated HSA (gHSA). The formation of the protein corona itself (regardless of whether it was formed from HSA or glycated HSA) caused an approximate 2-fold decrease in cell viability compared to the no protein AgNP control. While the toxicity of AgNPs to cells is often attributed to dissolved Ag(I), dissolution studies showed that the protein coated AgNPs underwent less dissolution than the no protein control, suggesting that the protein corona facilitated a nanoparticle-specific mechanism of toxicity. Overall, this study highlights the importance of protein coronas in mediating AgNP interactions with HepG2 cells and the need for future work to discern how protein coronas and protein modifications (like glycation) may alter AgNP reactivity to cellular organisms.

Silver Nanoparticle Surface Chemistry Determines Interactions with Human Serum Albumin and Cytotoxic Responses in Human Liver Cells.

Engineered nanomaterials (ENMs) are synthesized with a diversity of surface chemistries that mediate biochemical interactions and physiological response to the particles. In this work, silver engineered nanomaterials (AgENMs) are used to evaluate the role of surface charge in protein interactions and cellular cytotoxicity. The most abundant protein in blood, human serum albumin (HSA), was interacted with 40 nm AgENMs with a range of surface-charged coatings: positively charged branched polyethyleneimine (bPEI), negatively charged citrate (CIT), and circumneutral poly(ethylene glycol) (PEG). HSA adsorption to AgENMs was monitored by UV-vis spectroscopy and dynamic light scattering, while changes to the protein structure were evaluated with circular dichroism spectroscopy. Binding affinity for citrate-coated AgENMs and HSA is largest among the three AgENM coatings; yet, HSA lost the most secondary structure upon interaction with bPEI-coated AgENMs compared to the other two coatings. HSA increased AgENM oxidative dissolution across all particle types, with the greatest dissolution for citrate-coated AgENMs. Results indicate that surface coating is an important consideration in transformation of both the particle and protein upon interaction. To connect results to cellular outcomes, we also performed cytotoxicity experiments with HepG2 cells across all three AgENM types with and without HSA. Results show that bPEI-coated AgENMs cause the greatest loss of cell viability, both with and without inclusion of HSA with the AgENMs. Thus, surface coatings on AgENMs alter both biophysical interactions with proteins and particle cytotoxicity. Within this study set, positively charged bPEI-coated AgENMs cause the greatest disruption to HSA structure and cell viability.

Isolation Methods Influence the Protein Corona Composition on Gold-Coated Iron Oxide Nanoparticles.

Upon exposure to a biological environment, nanoparticles (NPs) acquire biomolecular coatings, the most studied of which is the protein corona. This protein corona gives NPs a new biological identity that will determine various biological responses including cellular uptake, biodistribution, and toxicity. The standard method to isolate NPs from a biological matrix in order to study their coronas is centrifugation, but more gentle means of retrieval may enable deeper understanding of both irreversibly bound hard coronas and more loosely bound soft coronas. In this study, magnetic gold-coated iron oxide NPs were incubated with rainbow trout gill cell total protein extracts and mass spectrometric proteomic analysis was conducted to determine the composition of the protein coronas isolated by either centrifugation or magnetic retrieval. The number of washes were varied to strip away the soft coronas and isolate the hard corona. Hundreds of proteins were adsorbed to the NPs. Some proteins were common to all isolation methods and many others were particular to the isolation method. Some qualitative trends in protein character were discerned from quantitative proteomic analyses, but more importantly, a new kind of protein corona was identified, mixed corona, in which the labile or inert nature of the protein-NP interaction is dependent upon sample history.

Hypothalamic kinin B1 receptor mediates orexin system hyperactivity in neurogenic hypertension.

Brain orexin system hyperactivity contributes to neurogenic hypertension. We previously reported upregulated neuronal kinin B1 receptor (B1R) expression in hypertension. However, the role of central B1R activation on the orexin system in neurogenic hypertension has not been examined. We hypothesized that kinin B1R contributes to hypertension via upregulation of brain orexin-arginine vasopressin signaling. We utilized deoxycorticosterone acetate (DOCA)-salt hypertension model in wild-type (WT) and B1R knockout (B1RKO) mice. In WT mice, DOCA-salt-treatment increased gene and protein expression of orexin A, orexin receptor 1, and orexin receptor 2 in the hypothalamic paraventricular nucleus and these effects were attenuated in B1RKO mice. Furthermore, DOCA-salt- treatment increased plasma arginine vasopressin levels in WT mice, but not in B1RKO mice. Cultured primary hypothalamic neurons expressed orexin A and orexin receptor 1. B1R specific agonist (LDABK) stimulation of primary neurons increased B1R protein expression, which was abrogated by B1R selective antagonist R715 but not by the dual orexin receptor antagonist, ACT 462206, suggesting that B1R is upstream of the orexin system. These data provide novel evidence that B1R blockade blunts orexin hyperactivity and constitutes a potential therapeutic target for the treatment of salt-sensitive hypertension.

Environmental dimensions of the protein corona.

The adsorption of biomolecules to the surface of engineered nanomaterials, known as corona formation, defines their biological identity by altering their surface properties and transforming the physical, chemical and biological characteristics of the particles. In the first decade since the term protein corona was coined, studies have focused primarily on biomedical applications and human toxicity. The relevance of the environmental dimensions of the protein corona is still emerging. Often referred to as the eco-corona, a biomolecular coating forms upon nanomaterials as they enter the environment and may include proteins, as well as a diverse array of other biomolecules such as metabolites from cellular activity and/or natural organic matter. Proteins remain central in studies of eco-coronas because of the ease of monitoring and structurally characterizing proteins, as well as their crucial role in receptor engagement and signalling. The proteins within the eco-corona are optimal targets to establish the biophysicochemical principles of corona formation and transformation, as well as downstream impacts on nanomaterial uptake, distribution and impacts on the environment. Moreover, proteins appear to impart a biological identity, leading to cellular or organismal recognition of nanomaterials, a unique characteristic compared with natural organic matter. We contrast insights into protein corona formation from clinical samples with those in environmentally relevant systems. Principles specific to the environment are also explored to gain insights into the dynamics of interaction with or replacement by other biomolecules, including changes during trophic transfer and ecotoxicity. With many challenges remaining, we also highlight key opportunities for method development and impactful systems on which to focus the next phase of eco-corona studies. By interrogating these environmental dimensions of the protein corona, we offer a perspective on how mechanistic insights into protein coronas in the environment can lead to more sustainable, environmentally safe nanomaterials, as well as enhancing the efficacy of nanomaterials used in remediation and in the agri-food sector.

The Primarily Undergraduate Nanomaterials Cooperative: A New Model for Supporting Collaborative Research at Small Institutions on a National Scale.

The Primarily Undergraduate Nanomaterials Cooperative (PUNC) is an organization for research-active faculty studying nanomaterials at Primarily Undergraduate Institutions (PUIs), where undergraduate teaching and research go hand-in-hand. In this perspective, we outline the differences in maintaining an active research group at a PUI compared to an R1 institution. We also discuss the work of PUNC, which focuses on community building, instrument sharing, and facilitating new collaborations. Currently consisting of 37 members from across the United States, PUNC has created an online community consisting of its Web site (nanocooperative.org), a weekly online summer group meeting program for faculty and students, and a Discord server for informal conversations. Additionally, in-person symposia at ACS conferences and PUNC-specific conferences are planned for the future. It is our hope that in the years to come PUNC will be seen as a model organization for community building and research support at primarily undergraduate institutions.

The role of human serum and solution chemistry in fibrinogen peptide-nanoparticle interactions.

In living systems, the biomolecules that coat nanoparticles (NPs) alter the NP biological identity and response. Although some biomolecules are more effective in mediating NP stability or biological fate, it is difficult to monitor an individual biomolecule within the complexity of the biota. To understand the dependence of protein-NP interactions on common variations in blood, we have evaluated binding between silica NPs and a model gamma-fibrinogen (GF) peptide. Fibrinogen is commonly identified within the protein corona fingerprint of human serum, but its abundance on the NP varies. To assess the relative importance of human serum and solution conditions, GF peptide and silica NP interactions were evaluated with and without serum across pH, NaCl concentrations, and glucose concentrations. Initial evaluation of the GF peptide and silica NP complexes using circular dichroism and dynamic light scattering show little change in the secondary structure of the peptide and no significant agglomeration of NPs, suggesting peptide-NP complexes are stable across study conditions. Fluorescence anisotropy was used to monitor GF peptide-NP binding. Both with and without serum, binding constants for the gamma-fibrinogen peptide vary significantly upon addition of diluted HS (1:500) and 29 mM sodium chloride. Yet, results indicated that gamma-fibrinogen binding interactions with silica NPs are comparatively insensitive to physiologically relevant pH changes and dramatic increases in glucose concentrations. Results highlight the importance of blood chemistries, which vary across individuals and disease states, in mediating protein corona formation.

Low-Dose Silver Nanoparticle Surface Chemistry and Temporal Effects on Gene Expression in Human Liver Cells.

Silver nanoparticles (AgNPs) are widely incorporated into consumer and biomedical products for their antimicrobial and plasmonic properties with limited risk assessment of low-dose cumulative exposure in humans. To evaluate cellular responses to low-dose AgNP exposures across time, human liver cells (HepG2) are exposed to AgNPs with three different surface charges (1.2 µg mL-1 ) and complete gene expression is monitored across a 24 h period. Time and AgNP surface chemistry mediate gene expression. In addition, since cells are fed, time has marked effects on gene expression that should be considered. Surface chemistry of AgNPs alters gene transcription in a time-dependent manner, with the most dramatic effects in cationic AgNPs. Universal to all surface coatings, AgNP-treated cells responded by inactivating proliferation and enabling cell cycle checkpoints. Further analysis of these universal features of AgNP cellular response, as well as more detailed analysis of specific AgNP treatments, time points, or specific genes, is facilitated with an accompanying application. Taken together, these results provide a foundation for understanding hepatic response to low-dose AgNPs for future risk assessment.

Estrogen-dependent hypersensitivity to diabetes-evoked cardiac autonomic dysregulation: Role of hypothalamic neuroinflammation.

AIMS: To investigate if autonomic dysregulation is exacerbated in female rats, subjected to diabetes mellitus (DM), via a paradoxical estrogen (E2)-evoked provocation of neuroinflammation/injury of the hypothalamic paraventricular nucleus (PVN). MAIN METHODS: We measured cardiac autonomic function and conducted subsequent PVN neurochemical studies, in DM rats, and their respective controls, divided as follows: male, sham operated (SO), ovariectomized (OVX), and OVX with E2 supplementation (OVX/E2). KEY FINDINGS: Autonomic dysregulation, expressed as sympathetic dominance (higher low frequency, LF, band), only occurred in DM E2-replete (SO and OVX/E2) rats, and was associated with higher neuronal activity (c-Fos) and higher levels of TNFα and phosphorylated death associated protein kinase-3 (p-DAPK3) in the PVN. These proinflammatory molecules likely contributed to the heightened PVN oxidative stress, injury and apoptosis. The PVN of these E2-replete DM rats also exhibited upregulations of estrogen receptors, ERα and ERß, and proinflammatory adenosine A1 and A2a receptors. SIGNIFICANCE: The E2-dependent autonomic dysregulation likely predisposes DM female rats and women to hypersensitivity to cardiac dysfunction. Further, upregulations of proinflammatory mediators including adenosine A1 and A2 receptors, TNFα and DAPK3, conceivably explain the paradoxical hypersensitivity of DM females to PVN inflammation/injury and the subsequent autonomic dysregulation in the presence of E2.

Restoration of Adiponectin-Connexin43 Signaling Mitigates Myocardial Inflammation and Dysfunction in Diabetic Female Rats.

ur preclinical findings replicated women's hypersensitivity to type-2 diabetes mellitus (T2DM)-evoked cardiac dysfunction along with demonstrating estrogen (E2)-dependent disruption of the cardiac adiponectin (APN)-connexin43 (Cx43) signaling. Whether the latter molecular anomaly underlies this women's cardiovascular health problem remains unknown. We hypothesized that restoration of the disrupted APN-Cx43 signaling alleviates this sex/E2-dependent cardiac dysfunction in diabetic female rats. To test this hypothesis, we administered the adiponectin receptor 1 (AdipoR1) agonist AdipoRon (30 mg/kg/d for 10 days) to female sham operated (SO) and ovariectomized (OVX) rats, which exhibited and lacked the T2DM left ventricular (LV) dysfunction, respectively, when fed high-fat diet and received low dose streptozotocin regimen; nondiabetic control SO and OVX rats received control diet and vehicle for streptozotocin. In T2DM SO rats, LV dysfunction, AdipoRon mitigated: (1) LV hypertrophy, (2) reductions in fractional shortening, LV developed pressure, dP/dtmax, dP/dtmin, and Tau. In LV tissues of the same rats, AdipoRon reversed reduction in Cx43 and elevations in TNFα, heme-oxygenase 1 (HO-1), and circulating cardiovascular risk factor asymmetric dimethylarginine. The findings also revealed ovarian hormones independent effects of AdipoRon, which included dampening of the pro-oxidant enzyme HO-1. These novel findings yield new insight into a causal role for compromised APN-Cx43 signaling in the E2-dependent hypersensitivity to T2DM-evoked cardiac inflammation and dysfunction. Equally important, the findings identify restoration of Cx43 signaling as a viable therapeutic modality for alleviating this women's cardiovascular health-related problem.

Emerging investigator series: characterization of silver and silver nanoparticle interactions with zinc finger peptides.

In biological systems, chemical and physical transformations of engineered silver nanomaterials (AgENMs) are mediated, in part, by proteins and other biomolecules. Metalloprotein interactions with AgENMs are also central in understanding toxicity and antimicrobial and resistance mechanisms. Despite their readily available thiolate and amine ligands, zinc finger (ZF) peptides have thus far escaped study in reaction with AgENMs and their Ag(I) oxidative dissolution product. We report spectroscopic studies that characterize AgENM and Ag(I) interactions with two ZF peptides that differ in sequence, but not in metal binding ligands: the ZF consensus peptide CP-CCHC and the C-terminal zinc finger domain of HIV-1 nucleocapsid protein p7 (NCp7_C). Both ZF peptides catalyze AgENM (10 and 40 nm, citrate coated) dissolution and agglomeration, two important AgENM transformations that impact bioreactivity. AgENMs and their oxidative dissolution product, Ag(I)(aq), mediate changes to ZF peptide structure and metalation as well. Spectroscopic titrations of Ag(I) into apo-ZF peptides show an Ag(I)-thiolate charge transfer band, indicative of Ag(I)-ZF binding. Fluorescence studies of the Zn(II)-NCp_7 complex indicate that the Ag(I) also effectively competes with the Zn(II) to drive Zn(II) displacement from the ZFs. Upon interaction with AgENMs, Zn(II) bound ZF peptides show a secondary structural change in circular dichroism spectroscopy toward an apo-like structure. The results suggest that Ag(I) and AgENMs may alter ZF protein function within the cell.

Publisher Correction: On the issue of transparency and reproducibility in nanomedicine.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: On the issue of transparency and reproducibility in nanomedicine.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Best practice in reporting corona studies: Minimum information about Nanomaterial Biocorona Experiments (MINBE).

Characterisation of the adsorption of biomolecules, or a biocorona, on nanomaterials has proliferated in the past 10 years, as protein corona studies provide molecular level insight into mechanisms of cellular recognition, uptake, and toxicity of nanomaterials. At the crossroads of two rapidly evolving orthogonal fields, nanoscience and proteomics, the interdisciplinarity of protein corona studies creates challenges for experimental design and reporting. Here we propose a flexible checklist for experimental design and reporting guidelines to outline Minimum Information about Nanomaterial Biocorona Experiments (MINBE). The checklist for experimental design, compiled after review of reporting within the protein corona literature, provides researchers with prompts to ensure best practice experimental approaches for each stage of the workflow, collated from the nanoscience, proteomics, and bioinformatics fields. Reporting guidelines are also assembled from established sources, integrated to span the entire workflow and extended and modified to aid interdisciplinary researchers in the most challenging stages of the workflow. Where appropriate, de novo guidelines to address areas specific to protein corona studies, including exposure conditions and isolation of adsorbed proteins, were written. The MINBE guidelines provide protein corona researchers with a conduit between materials science techniques and proteomics. Implementation of these guidelines is anticipated to catalyse enhanced quality, impact, and extent of data mining and computational modelling of protein corona composition and its role in nanosafety and nanomedicine. Furthermore, high quality experimental design and reporting in the bio-nanosciences will enhance the next phase of targeted nanomedicines and sustainable nanotechnologies.

Estrogen-Dependent Disruption of Adiponectin-Connexin43 Signaling Underlies Exacerbated Myocardial Dysfunction in Diabetic Female Rats.

The reasons for the higher severity of type 2 diabetes (T2DM)-associated cardiomyopathy in women, despite their inherent estrogen (E2)-dependent cardioprotection, remain unknown. We hypothesized that the reliance of the healthy females' hearts on augmented adiponectin (APN)-connexin 43 (Cx43) signaling becomes paradoxically detrimental when disrupted by T2DM in an E2-dependent manner. We tested this hypothesis in high-fat, low- dose streptozotocin diabetic rats and their controls with the following designations: 1) sham-operated (SO), 2) ovariectomized (OVX), 3) ovariectomized with E2 supplementation (OVX + E2), and 4) male. E2-replete (SO or OVX + E2) diabetic rats exhibited higher mortality and greater increases in left ventricular (LV) mass and reduced LV developed pressure, LV contractility, and fractional shortening but preserved ejection fraction. Further, compared with respective nondiabetic counterparts, the hearts of these E2-replete diabetic rats exhibited greater upregulation of cardiac estrogen receptor α and reductions in Cx43 expression and in the phosphorylation levels of the survival molecules extracellular regulating kinases 1/2 and phosphorylated AKT (pAKT). Whereas serum APN was reduced, independent of sex and ovarian hormone status in all DM rats, cardiac APN was most drastically reduced in DM SO rats. The present translational findings are the first to implicate ovarian hormones/E2 in the exacerbated myocardial dysfunction in female diabetic subjects and to suggest a pivotal role for malfunctioning cardiac APN-Cx43 signaling in this sex/E2-specific clinical problem.

Machine learning provides predictive analysis into silver nanoparticle protein corona formation from physicochemical properties.

Proteins encountered in biological and environmental systems bind to engineered nanomaterials (ENMs) to form a protein corona (PC) that alters the surface chemistry, reactivity, and fate of the ENMs. Complexities such as the diversity of the PC and variation with ENM properties and reaction conditions make the PC population difficult to predict. Here, we support the development of predictive models for PC populations by relating biophysicochemical characteristics of proteins, ENMs, and solution conditions to PC formation using random forest classification. The resulting model offers a predictive analysis into the population of PC proteins in Ag ENM systems of various ENM size and surface coatings. With an area under the receiver operating characteristic curve of 0.83 and F1-score of 0.81, a model with strong performance has been constructed based upon experimental data. The weighted contribution of each variable provides recommendations for mechanistic models based upon protein enrichment classification results. Protein biophysical properties such as pI and weight are weighted heavily. Yet, ENM size, surface charge, and solution ionic strength also proved essential to an accurate model. The model can be readily modified and applied to other ENM PC populations. The model presented here represents the first step toward robust predictions of PC fingerprints.

Graphene Oxide Sheets Combine into Conductive Coatings by Direct Oxidative Electropolymerization.

New coatings are obtained when graphene oxide is further oxidized at moderate anodic potentials (≤~1.3 V vs. Ag/AgCl). Based on a variety of spectroscopic and electrochemical observations, the coatings are attributed to the direct electropolymerization of graphene oxide sheets via oxidation of the phenol edge groups on graphene. Depending on the applied potential, ether or carboxylic groups are formed. The coatings obtained via further oxidation are characterized by a lower O/C ratio due to decarboxylation and a higher content of C=C bonds. These bonds extend aromatic conjugation into the combined graphene oxide sheets and are responsible for the highly conductive nature of these coatings.

Beyond the passive interactions at the nano-bio interface: evidence of Cu metalloprotein-driven oxidative dissolution of silver nanoparticles.

BACKGROUND: In a biological system, an engineered nanomaterial (ENM) surface is altered by adsorbed proteins that modify ENM fate and toxicity. Thus far, protein corona characterizations have focused on protein adsorption, interaction strength, and downstream impacts on cell interactions. Given previous reports of Ag ENM disruption of Cu trafficking, this study focuses on Ag ENM interactions with a model Cu metalloprotein, Cu(II) azurin. The study provides evidence of otherwise overlooked ENM-protein chemical reactivity within the corona: redox activity. RESULTS: Citrate-coated Ag ENMs of various sizes (10-40 nm) reacted with Cu(II) azurin resulted in an order of magnitude more dissolved ionic silver (Ag(I)(aq)) than samples of Ag ENMs only, ENMs mixed Cu(II) ions, or control proteins such as cytochrome c and horse radish peroxidase. This dramatic increase in ENM oxidative dissolution was observed even when Cu(II) azurin was combined with a diverse mixture of Escherchia coli proteins to mimic the complexity of the cellular conona. SDS PAGE results confirm that the multiprotein ENM corona includes azurin. A Cu(I)(aq) colorimetric indicator confirms Cu(II) azurin reduction upon interaction with Ag ENMs, but not with the addition of ionic silver, Ag(I)(aq). CONCLUSIONS: Cu(II) azurin and 10-40 nm Ag ENMs react to catalyze Ag ENM oxidative dissolution and reduction of the model Cu metalloprotein. Results push the current evaluation of protein-ENM characterization beyond passive binding interactions and enable the proposal of a mechanism for reactivity between a model Cu metalloprotein and Ag ENMs.

Spectroscopic characterization of copper(I) binding to apo and metal-reconstituted zinc finger peptides.

Cu(I) exhibits high affinity for thiolate ligands, suggesting that thiol-rich zinc or iron binding sites may be subject to disruption during copper stress conditions. Zinc fingers constitute a large class of metalloproteins that use a combination of cysteine and histidine residues that bind Zn(II) as a structural element. Despite the shared preference of both copper and zinc for thiolate and amine coordination, the susceptibility of zinc finger domains toward copper substitution is not well studied. We report spectroscopic studies that characterize the Cu(I) binding properties of the zinc finger consensus peptides CP-CCHH, CP-CCHC, and CP-CCCC and the C-terminal zinc finger domain of HIV-1 nucleocapsid protein p7 (NCp7_C). Cu(I) binds to both the apopeptides and the Co(II)-substituted peptides, and the stoichiometry of Cu(I) binding is dependent on the number of cysteine thiols at the metal binding site. Fluorescence studies of the Zn(II)-NCp7_C complex indicate that Cu(I) also effectively competes with Zn(II) at the metal binding site, despite the high affinity of Zn(II) for the CCHC binding motif. Circular dichroism studies on both CP-CCHC and NCp7_C show that the conformations of the Cu(I)-bound complexes differ substantially from those of the Zn(II) species, implying that Cu(I) substitution is likely to impact zinc finger function. These results show that for the peptides studied here, Cu(I) is the thermodynamically favored metal despite the known high Zn(II) affinity of zinc finger domains, suggesting that Cu(I)-substituted zinc finger domains might be relevant in the context of both copper toxicity mechanisms and copper-responsive transcription factors.