Biocompatibility of Water-Dispersible Pristine Graphene and Graphene Oxide Using a Close-to-Human Animal Model: A Pilot Study on Swine.

Graphene-based materials (GBMs) are of considerable interest for biomedical applications, and the pilot study on the toxicological and immunological impact of pristine graphene (GR) and graphene oxide (GO) using swine as a close-to-human provides valuable insights. First, ex vivo experiments are conducted on swine blood cells, then GBMs are injected intraperitoneally (i.p.) into swine. Hematological and biochemical analyses at various intervals indicate that neither GO nor GR cause systemic inflammation, pro-coagulant responses, or renal or hepatic dysfunction. Importantly, no systemic toxicity is observed. Analysis of a panel of 84 immune-related genes shows minimal impact of GO and GR. The animals are sacrificed 21 days post-injection, and transient absorption imaging and Raman mapping show the presence of GO and GR in the mesentery only. Histological evaluation reveals no signs of alterations in other organs. Thus, clusters of both materials are detected in the mesentery, and GO aggregates are surrounded only by macrophages with the formation of granulomas. In contrast, modest local reactions are observed around the GR clusters. Overall, these results reveal that i.p. injection of GBMs resulted in a modest local tissue reaction without systemic toxicity. This study, performed in swine, provides essential guidance for future biomedical applications of graphene.

Hazard assessment of hexagonal boron nitride and hexagonal boron nitride reinforced thermoplastic polyurethane composites using human skin and lung cells.

Hexagonal boron nitride (hBN) is an emerging two-dimensional material attracting considerable attention in the industrial sector given its innovative physicochemical properties. Potential risks are associated mainly with occupational exposure where inhalation and skin contact are the most relevant exposure routes for workers. Here we aimed at characterizing the effects induced by composites of thermoplastic polyurethane (TPU) and hBN, using immortalized HaCaT skin keratinocytes and BEAS-2B bronchial epithelial cells. The composite was abraded using a Taber® rotary abraser and abraded TPU and TPU-hBN were also subjected to photo-Fenton-mediated degradation mimicking potential weathering across the product life cycle. Cells were exposed to the materials for 24 h (acute exposure) or twice per week for 4 weeks (chronic exposure) and evaluated with respect to material internalization, cytotoxicity, and proinflammatory cytokine secretion. Additionally, comprehensive mass spectrometry-based proteomics and metabolomics (secretomics) analyses were performed. Overall, despite evidence of cellular uptake of the material, no significant cellular and/or protein expression profiles alterations were observed after acute or chronic exposure of HaCaT or BEAS-2B cells, identifying only few pro-inflammatory proteins. Similar results were obtained for the degraded materials. These results support the determination of hazard profiles associated with cutaneous and pulmonary hBN-reinforced polymer composites exposure.

Remediation of Metal Oxide Nanotoxicity with a Functional Amyloid.

Understanding the environmental health and safety of nanomaterials (NanoEHS) is essential for the sustained development of nanotechnology. Although extensive research over the past two decades has elucidated the phenomena, mechanisms, and implications of nanomaterials in cellular and organismal models, the active remediation of the adverse biological and environmental effects of nanomaterials remains largely unexplored. Inspired by recent developments in functional amyloids for biomedical and environmental engineering, this work shows their new utility as metallothionein mimics in the strategically important area of NanoEHS. Specifically, metal ions released from CuO and ZnO nanoparticles are sequestered through cysteine coordination and electrostatic interactions with beta-lactoglobulin (bLg) amyloid, as revealed by inductively coupled plasma mass spectrometry and molecular dynamics simulations. The toxicity of the metal oxide nanoparticles is subsequently mitigated by functional amyloids, as validated by cell viability and apoptosis assays in vitro and murine survival and biomarker assays in vivo. As bLg amyloid fibrils can be readily produced from whey in large quantities at a low cost, the study offers a crucial strategy for remediating the biological and environmental footprints of transition metal oxide nanomaterials.

Two-dimensional molybdenum disulfide nanosheets evoke nitric oxide-dependent antibacterial effects.

Nanomaterials are currently being explored as novel antimicrobial agents. In this study, we first investigated the ability of two-dimensional (2D) molybdenum disulfide (MoS2) nanosheets to trigger neutrophil extracellular traps (NETs) using neutrophil-differentiated HL-60 cells as well as primary human peripheral blood neutrophils. We then addressed whether the MoS2 nanosheets themselves function as antibacterial agents. We found that MoS2 and Na2MoO4 both triggered NETs, as evidenced by the quantification of neutrophil elastase (NE) activity and immunofluorescence staining of extracellular NE, as well as scanning electron microscopy. The release of NETs was found to be nitric oxide (NO)-dependent. We also found that the MoS2 nanosheets but not the soluble salt prompted acellular NO production in the presence of NaNO2. The acellular generation of NO, suggestive of nanozyme properties of the MoS2 nanosheets, was demonstrated by electron paramagnetic resonance analysis. Electrochemical analysis using cyclic voltammetry confirmed the redox transition of the MoS2 nanosheets. Finally, MoS2 nanosheets inhibited the growth of Escherichia coli in the presence of sodium nitrate. Taken together, MoS2 nanosheets triggered cellular effects as well as acellular antibacterial effects, and we provided evidence for nitrite reductase-like properties of MoS2.

Spike Protein Fragments Promote Alzheimer's Amyloidogenesis.

Alzheimer's disease (AD) is a major cause of dementia inducing memory loss, cognitive decline, and mortality among the aging population. While the amyloid aggregation of peptide Aß has long been implicated in neurodegeneration in AD, primarily through the production of toxic polymorphic aggregates and reactive oxygen species, viral infection has a less explicit role in the etiology of the brain disease. On the other hand, while the COVID-19 pandemic is known to harm human organs and function, its adverse effects on AD pathobiology and other human conditions remain unclear. Here we first identified the amyloidogenic potential of 1058HGVVFLHVTYV1068, a short fragment of the spike protein of SARS-CoV-2 coronavirus. The peptide fragment was found to be toxic and displayed a high binding propensity for the amyloidogenic segments of Aß, thereby promoting the aggregation and toxicity of the peptide in vitro and in silico, while retarding the hatching and survival of zebrafish embryos upon exposure. Our study implicated SARS-CoV-2 viral infection as a potential contributor to AD pathogenesis, a little explored area in our quest for understanding and overcoming Long Covid.

Graphene oxide elicits microbiome-dependent type 2 immune responses via the aryl hydrocarbon receptor.

The gut microbiome produces metabolites that interact with the aryl hydrocarbon receptor (AhR), a key regulator of immune homoeostasis in the gut1,2. Here we show that oral exposure to graphene oxide (GO) modulates the composition of the gut microbiome in adult zebrafish, with significant differences in wild-type versus ahr2-deficient animals. Furthermore, GO was found to elicit AhR-dependent induction of cyp1a and homing of lck+ cells to the gut in germ-free zebrafish larvae when combined with the short-chain fatty acid butyrate. To obtain further insights into the immune responses to GO, we used single-cell RNA sequencing to profile cells from whole germ-free embryos as well as cells enriched for lck. These studies provided evidence for the existence of innate lymphoid cell (ILC)-like cells3 in germ-free zebrafish. Moreover, GO endowed with a 'corona' of microbial butyrate triggered the induction of ILC2-like cells with attributes of regulatory cells. Taken together, this study shows that a nanomaterial can influence the crosstalk between the microbiome and immune system in an AhR-dependent manner.

Understanding the bidirectional interactions between two-dimensional materials, microorganisms, and the immune system.

Two-dimensional (2D) materials such as the graphene-based materials, transition metal dichalcogenides, transition metal carbides and nitrides (MXenes), black phosphorus, hexagonal boron nitride, and others have attracted considerable attention due to their unique physicochemical properties. This is true not least in the field of medicine. Understanding the interactions between 2D materials and the immune system is therefore of paramount importance. Furthermore, emerging evidence suggests that 2D materials may interact with microorganisms - pathogens as well as commensal bacteria that dwell in and on our body. We discuss the interplay between 2D materials, the immune system, and the microbial world in order to bring a systems perspective to bear on the biological interactions of 2D materials. The use of 2D materials as vectors for drug delivery and as immune adjuvants in tumor vaccines, and 2D materials to counteract inflammation and promote tissue regeneration, are explored. The bio-corona formation on and biodegradation of 2D materials, and the reciprocal interactions between 2D materials and microorganisms, are also highlighted. Finally, we consider the future challenges pertaining to the biomedical applications of various classes of 2D materials.

Two-Dimensional Transition Metal Dichalcogenides Trigger Trained Immunity in Human Macrophages through Epigenetic and Metabolic Pathways.

Trained immunity is a recently described phenomenon whereby cells of the innate immune system undergo long-term epigenetic and/or metabolic reprogramming following a short-term interaction with microbes or microbial products. Here, it is shown that 2D transition metal dichalcogenides (TMDs) trigger trained immunity in primary human monocyte-derived macrophages. First, aqueous dispersions of 2D crystal formulations of MoS2 and WS2 are tested, and no cytotoxicity is found despite avid uptake of these materials by macrophages. However, when macrophages are pre-exposed to TMDs, followed by a resting period, this causes a marked modulation of immune-specific gene expression upon subsequent challenge with a microbial agent (i.e., bacterial lipopolysaccharides). Specifically, MoS2 triggers trained immunity through an epigenetic pathway insofar as the histone methyltransferase inhibitor methylthioadenosine reverses these effects. Furthermore, MoS2 triggers an elevation of cyclic adenosine monophosphate (cAMP) levels in macrophages and increased glycolysis is also evidenced in cells subjected to MoS2 training, pointing toward a metabolic rewiring of the cells. Importantly, it is observed that MoS2 triggers the upregulation of Mo-dependent enzymes in macrophages, thus confirming that Mo is bioavailable in these cells. In conclusion, MoS2 is identified as a novel inducer of trained immunity. Thus, TMDs could potentially be harnessed as immunomodulatory agents.

Biodegradation of graphdiyne oxide in classically activated (M1) macrophages modulates cytokine production.

Graphdiyne oxide (GDYO) is a carbon-based nanomaterial possessing sp2 and sp-hybridized carbon atoms with many promising applications. However, its biocompatibility and potential biodegradability remain poorly understood. Using human primary monocyte-derived macrophages as a model we show here that GDYO elicited little or no cytotoxicity toward classically activated (M1) and alternatively activated (M2) macrophages. Moreover, GDYO reprogrammed M2 macrophages towards M1 macrophages, as evidenced by the elevation of specific cell surface markers and cytokines and the induction of NOS2 expression. We could also show inducible nitric oxide synthase (iNOS)-dependent biodegradation of GDYO in M1 macrophages, and this was corroborated in an acellular system using the peroxynitrite donor, SIN-1. Furthermore, GDYO elicited the production of pro-inflammatory cytokines in a biodegradation-dependent manner. Our findings shed new light on the reciprocal interactions between GDYO and human macrophages. This is relevant for biomedical applications of GDYO such as the re-education of tumor-associated macrophages or TAMs.

Nitric oxide-dependent biodegradation of graphene oxide reduces inflammation in the gastrointestinal tract.

Understanding the biological fate of graphene-based materials such as graphene oxide (GO) is crucial to assess adverse effects following intentional or inadvertent exposure. Here we provide first evidence of biodegradation of GO in the gastrointestinal tract using zebrafish as a model. Raman mapping was deployed to assess biodegradation. The degradation was blocked upon knockdown of nos2a encoding the inducible nitric oxide synthase (iNOS) or by pharmacological inhibition of NOS using l-NAME, demonstrating that the process was nitric oxide (NO)-dependent. NO-dependent degradation of GO was further confirmed in vitro by combining a superoxide-generating system, xanthine/xanthine oxidase (X/XO), with an NO donor (PAPA NONOate), or by simultaneously producing superoxide and NO by decomposition of SIN-1. Finally, by using the transgenic strain Tg(mpx:eGFP) to visualize the movement of neutrophils, we could show that inhibition of the degradation of GO resulted in increased neutrophil infiltration into the gastrointestinal tract, indicative of inflammation.

Graphene and other 2D materials: a multidisciplinary analysis to uncover the hidden potential as cancer theranostics.

Cancer represents one of the main causes of death in the world; hence the development of more specific approaches for its diagnosis and treatment is urgently needed in clinical practice. Here we aim at providing a comprehensive review on the use of 2-dimensional materials (2DMs) in cancer theranostics. In particular, we focus on graphene-related materials (GRMs), graphene hybrids, and graphdiyne (GDY), as well as other emerging 2DMs, such as MXene, tungsten disulfide (WS2), molybdenum disulfide (MoS2), hexagonal boron nitride (h-BN), black phosphorus (BP), silicene, antimonene (AM), germanene, biotite (black mica), metal organic frameworks (MOFs), and others. The results reported in the scientific literature in the last ten years (>200 papers) are dissected here with respect to the wide variety of combinations of imaging methodologies and therapeutic approaches, including drug/gene delivery, photothermal/photodynamic therapy, sonodynamic therapy, and immunotherapy. We provide a unique multidisciplinary approach in discussing the literature, which also includes a detailed section on the characterization methods used to analyze the material properties, highlighting the merits and limitations of the different approaches. The aim of this review is to show the strong potential of 2DMs for use as cancer theranostics, as well as to highlight issues that prevent the clinical translation of these materials. Overall, we hope to shed light on the hidden potential of the vast panorama of new and emerging 2DMs as clinical cancer theranostics.

Environmental Hazard Potential of Nano-Photocatalysts Determined by Nano-Bio Interactions and Exposure Conditions.

Nano-photocatalysts are known for their ability to degrade pollutants or perform water splitting catalyzed by light. Being the key functional ingredients of current and future products, the potential of nano-photocatalysts releasing into the environment and causing unintended harm to living organisms warrants investigation. Risk assessment of these materials serves as an important step to allow safe implementation and to avoid irrational fear. Using TiO2 and g-C3 N4 as representative nano-photocatalysts, this study evaluates their hazard potential in zebrafish. Under simulated solar light, nano-photocatalysts up to 100 mg L-1 show no acute toxicity to zebrafish embryos due to the protection of chorions. The short-lived reactive oxygen species generated by nano-photocatalysts only exert injury to the hatched larvae at and above 50 mg L-1 . The input of solar energy, determined by the depth of water, irradiation time, and light intensity, greatly influences the toxicity outcome. Increasing concentrations of natural organic matters contribute positively to the hazard potential at 0-10 mg L-1 while gradually diminishing the hazardous effect above 10 mg L-1 . This study demonstrates the importance of nano-bio interactions and environmental exposure conditions in determining the safety profile of nano-photocatalysts.

Redox Activity and Nano-Bio Interactions Determine the Skin Injury Potential of Co3O4-Based Metal Oxide Nanoparticles toward Zebrafish.

Redox-active metal oxide nanoparticles show varying oxidizing capacities and injury potentials toward biological systems. Here, two metal oxide libraries including transition-metal-doped Co3O4 and PdO-Co3O4 with strong chemical contacts were design-synthesized and used to investigate their biological injury potential and mechanisms using zebrafish as a model organism. Among different dopants, Cu significantly increased the oxidizing capacity of Co3O4. An increased amount of PdO resulted in higher density of heterojunctions, which also led to higher oxidizing capacity. The oxidizing capacity of these nanoparticles was positively correlated with higher mortality of dechorionated embryos and severe larval skin injury upon exposure. Using transgenic zebrafish Tg(LysC:eGFP), we show in real time that the redox-active nanoparticles induced skin injury and activated the infiltration of immune cells. Such inflammatory response was confirmed by the increased mRNA expression level of Nrf2a, HO-1, IL-1ß, and IL-6 genes. Although the exposure to the nanoparticles alone was not lethal, the skin injury did lower the tolerance level against other environmental contaminants. More importantly, after withdrawing from the nanoparticle exposure, larvae with skin injury could recover within 24 h in uncontaminated medium, indicating such injury was transient and recoverable.

Reduced graphene oxide composites and its real-life application potential for in-situ crude oil removal.

Crude oil pollution can cause severe and long-term ecological damage and oil cleanup has become a worldwide challenge. Conventional treatment strategies like in-situ burning, manual skimmer and bioremediation were labor-intensive and time-consuming. The high viscosity of crude oil also posed difficulty for traditional absorbents. Herein, to address these limitations, we designed and fabricated a floating absorbent that was comprised of reduced graphene oxide (RGO), melamine sponge (MS), and a 3D-printed mounting platform. Through a facile one-pot hydrothermal method, graphene oxide (GO) was simultaneously reduced to RGO and loaded in MS (RGO-MS). The resulted RGO-MS composites possess desirable hydrophobicity/oleophilicity for oil absorption with a water contact angle of 122°. The effective light-to-heat conversion allowed the RGO-MS composite to absorb approximately 95 times its own weight of crude oil within 12 min under light irradiation. A 3D-printed mounting platform for RGO-MS composites was further fabricated to improve its applicability and allow easy retrieval. Taking advantages of the RGO's hydrophobicity/oleophilicity and photothermal property, the floating ability of MS, this study demonstrated the real-life applicability of RGO-MS composites for in-situ crude oil cleanup.

Mitigation of Amyloidosis with Nanomaterials.

Amyloidosis is a biophysical phenomenon of protein aggregation with biological and pathogenic implications. Among the various strategies developed to date, nanomaterials and multifunctional nanocomposites possessing certain structural and physicochemical traits are promising candidates for mitigating amyloidosis in vitro and in vivo. The mechanisms underpinning protein aggregation and toxicity are introduced, and opportunities in materials science to drive this interdisciplinary field forward are highlighted. Advancement of this emerging frontier hinges on exploitation of protein self-assembly and interactions of amyloid proteins with nanoparticles, intracellular and extracellular proteins, chaperones, membranes, organelles, and biometals.

Inhibition of amyloid beta toxicity in zebrafish with a chaperone-gold nanoparticle dual strategy.

Alzheimer's disease (AD) is the most prevalent form of neurodegenerative disorders, yet no major breakthroughs have been made in AD human trials and the disease remains a paramount challenge and a stigma in medicine. Here we eliminate the toxicity of amyloid beta (Aß) in a facile, high-throughput zebrafish (Danio rerio) model using casein coated-gold nanoparticles (ßCas AuNPs). ßCas AuNPs in systemic circulation translocate across the blood brain barrier of zebrafish larvae and sequester intracerebral Aß42 and its elicited toxicity in a nonspecific, chaperone-like manner. This is evidenced by behavioral pathology, reactive oxygen species and neuronal dysfunction biomarkers assays, complemented by brain histology and inductively coupled plasma-mass spectroscopy. We further demonstrate the capacity of ßCas AuNPs in recovering the mobility and cognitive function of adult zebrafish exposed to Aß. This potent, safe-to-use, and easy-to-apply nanomedicine may find broad use for eradicating toxic amyloid proteins implicated in a range of human diseases.

Mitigating Human IAPP Amyloidogenesis In Vivo with Chiral Silica Nanoribbons.

Amyloid fibrils generally display chirality, a feature which has rarely been exploited in the development of therapeutics against amyloid diseases. This study reports, for the first time, the use of mesoscopic chiral silica nanoribbons against the in vivo amyloidogenesis of human islet amyloid polypeptide (IAPP), the peptide whose aggregation is implicated in type 2 diabetes. The thioflavin T assay and transmission electron microscopy show accelerated IAPP fibrillization through elimination of the nucleation phase and shortening of the elongation phase by the nanostructures. Coarse-grained simulations offer complementary molecular insights into the acceleration of amyloid aggregation through their nonspecific binding and directional seeding with the nanostructures. This accelerated IAPP fibrillization translates to reduced toxicity, especially for the right-handed silica nanoribbons, as revealed by cell viability, helium ion microscopy, as well as zebrafish embryo survival, developmental, and behavioral assays. This study has implicated the potential of employing chiral nanotechnologies against the mesoscopic enantioselectivity of amyloid proteins and their associated diseases.

Graphene quantum dots against human IAPP aggregation and toxicity in vivo.

The development of biocompatible nanomaterials has become a new frontier in the detection, treatment and prevention of human amyloid diseases. Here we demonstrated the use of graphene quantum dots (GQDs) as a potent inhibitor against the in vivo aggregation and toxicity of human islet amyloid polypeptide (IAPP), a hallmark of type 2 diabetes. GQDs initiated contact with IAPP through electrostatic and hydrophobic interactions as well as hydrogen bonding, which subsequently drove the peptide fibrillization off-pathway to eliminate the toxic intermediates. Such interactions, probed in vitro by a thioflavin T kinetic assay, fluorescence quenching, circular dichroism spectroscopy, a cell viability assay and in silico by discrete molecular dynamics simulations, translated to a significant recovery of embryonic zebrafish from the damage elicited by IAPP in vivo, as indicated by improved hatching as well as alleviated reactive oxygen species production, abnormality and mortality of the organism. This study points to the potential of using zero-dimensional nanomaterials for in vivo mitigation of a range of amyloidosis.

In Vivo Mitigation of Amyloidogenesis through Functional-Pathogenic Double-Protein Coronae.

Amyloid diseases are global epidemics with no cure available. Herein, we report a first demonstration of in vivo mitigation of amyloidogenesis using biomimetic nanotechnology. Specifically, the amyloid fragments (ba) of ß-lactoglobulin, a whey protein, were deposited onto the surfaces of carbon nanotubes (baCNT), which subsequently sequestered human islet amyloid polypeptide (IAPP) through functional-pathogenic double-protein coronae. Conformational changes at the ba-IAPP interface were studied by Fourier transform infrared, circular dichroism, and X-ray scattering spectroscopies. baCNT eliminated the toxic IAPP species from zebrafish embryos, as evidenced by the assays of embryonic development, cell morphology, hatching, and survival as well as suppression of oxidative stress. In addition to IAPP, baCNT also displayed high potency against the toxicity of amyloid-ß, thereby demonstrating the broad applicability of this biomimetic nanotechnology and the use of an embryonic zebrafish model for the high-throughput screening of a range of amyloidogenesis and their inhibitors in vivo.

Photocatalytic Degradation of 4-Nitrophenol by C, N-TiO2: Degradation Efficiency vs. Embryonic Toxicity of the Resulting Compounds.

The photocatalytic activity of TiO2 based photocatalysts can be improved by structural modification and elemental doping. In this study, through rational design, one type of carbon and nitrogen co-doped TiO2 (C, N-TiO2) photocatalyst with mesoporous structure was synthesized with improved photocatalytic activity in degrading 4-nitrophenol under simulated sunlight irradiation. The photocatalytic degradation efficiency of the C, N-TiO2 was much higher than the anatase TiO2 (A-TiO2) based on absorbance and HPLC analyses. Moreover, using zebrafish embryos, we showed that the intermediate degradation compounds generated by photocatalytic degradation of 4-nitrophenol had higher toxicity than the parent compound. A repeated degradation process was necessary to render complete degradation and non-toxicity to the zebrafish embryos. Our results demonstrated the importance of evaluating the photocatalytic degradation efficiency in conjunction with the toxicity assessment of the degradation compounds.