Nanosafety: a Perspective on Nano-Bio Interactions.

Engineered nanomaterials offer numerous benefits to society ranging from environmental remediation to biomedical applications such as drug or vaccine delivery as well as clean and cost-effective energy production and storage, and the promise of a more sustainable way of life. However, as nanomaterials of increasing sophistication enter the market, close attention to potential adverse effects on human health and the environment is needed. Here a critical perspective on nanotoxicological research is provided; the authors argue that it is time to leverage the knowledge regarding the biological interactions of nanomaterials to achieve a more comprehensive understanding of the human health and environmental impacts of these materials. Moreover, it is posited that nanomaterials behave like biological entities and that they should be regulated as such.

Exploiting the ferroaddiction of pancreatic cancer cells using Fe-doped nanoparticles.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor survival rates. Here, we evaluated iron-doped hydroxyapatite (FeHA) as a potential nanomedicine-based approach to combat PDAC. FeHA, in combination with a sublethal dose of the glutathione peroxidase 4 (GPX4) inhibitor RSL3, was found to trigger ferroptosis in KRAS mutant PANC-1 cells, but not in BxPC3 cells, while sparing normal human cells (fibroblasts and peripheral blood mononuclear cells). These findings were recapitulated in 3D spheroids generated using PDAC cells harboring wild-type versus mutant KRAS. Moreover, ferroptosis induction by FeHA plus RSL3 was reversed by the knockdown of STEAP3, a metalloreductase responsible for converting Fe3+ to Fe2+. Taken together, our data show that FeHA is capable of triggering cancer cell death in a KRAS-selective, STEAP3-dependent manner in PDAC cells.

Nanomaterial characterization: Understanding nano-bio interactions.

Nanomaterials are marvelously small, yet they may be harnessed to great effect. Much has been learned with respect to the biological interactions and effects of nanomaterials during the past decade. However, characterization of nanomaterials is typically performed on as-synthesized materials. We posit that nanomaterials are dynamic entities and should be studied and regulated as such. Hence, characterization of nanomaterials should take into account their biotransformation. However, in situ characterization of nanomaterials as they undergo dynamic changes (coronation, dissolution, degradation) in a living system remains a formidable challenge in nanosafety. Material scientists and toxicologists need to join forces to address this issue.

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.

Copper oxide nanoparticles trigger macrophage cell death with misfolding of Cu/Zn superoxide dismutase 1 (SOD1).

BACKGROUND: Copper oxide (CuO) nanoparticles (NPs) are known to trigger cytotoxicity in a variety of cell models, but the mechanism of cell death remains unknown. Here we addressed the mechanism of cytotoxicity in macrophages exposed to CuO NPs versus copper chloride (CuCl2). METHODS: The mouse macrophage cell line RAW264.7 was used as an in vitro model. Particle uptake and the cellular dose of Cu were investigated by transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS), respectively. The deposition of Cu in lysosomes isolated from macrophages was also determined by ICP-MS. Cell viability (metabolic activity) was assessed using the Alamar Blue assay, and oxidative stress was monitored by a variety of methods including a luminescence-based assay for cellular glutathione (GSH), and flow cytometry-based detection of mitochondrial superoxide and mitochondrial membrane potential. Protein aggregation was determined by confocal microscopy using an aggresome-specific dye and protein misfolding was determined by circular dichroism (CD) spectroscopy. Lastly, proteasome activity was investigated using a fluorometric assay. RESULTS: We observed rapid cellular uptake of CuO NPs in macrophages with deposition in lysosomes. CuO NP-elicited cell death was characterized by mitochondrial swelling with signs of oxidative stress including the production of mitochondrial superoxide and cellular depletion of GSH. We also observed a dose-dependent accumulation of polyubiquitinated proteins and loss of proteasomal function in CuO NP-exposed cells, and we could demonstrate misfolding and mitochondrial translocation of superoxide dismutase 1 (SOD1), a Cu/Zn-dependent enzyme that plays a pivotal role in the defense against oxidative stress. The chelation of copper ions using tetrathiomolybdate (TTM) prevented cell death whereas inhibition of the cellular SOD1 chaperone aggravated toxicity. Moreover, CuO NP-triggered cell death was insensitive to the pan-caspase inhibitor, zVAD-fmk, and to wortmannin, an inhibitor of autophagy, implying that this was a non-apoptotic cell death. ZnO NPs, on the other hand, triggered autophagic cell death. CONCLUSIONS: CuO NPs undergo dissolution in lysosomes leading to copper-dependent macrophage cell death characterized by protein misfolding and proteasomal insufficiency. Specifically, we present novel evidence for Cu-induced SOD1 misfolding which accords with the pronounced oxidative stress observed in CuO NP-exposed macrophages. These results are relevant for our understanding of the consequences of inadvertent human exposure to CuO NPs.

Severe congenital neutropenia-associated JAGN1 mutations unleash a calpain-dependent cell death programme in myeloid cells.

Severe congenital neutropenia (SCN) of autosomal recessive inheritance, also known as Kostmann disease, is characterised by a lack of neutrophils and a propensity for life-threatening infections. Using whole-exome sequencing, we identified homozygous JAGN1 mutations (p.Gly14Ser and p.Glu21Asp) in three patients with Kostmann-like SCN, thus confirming the recent attribution of JAGN1 mutations to SCN. Using the human promyelocytic cell line HL-60 as a model, we found that overexpression of patient-derived JAGN1 mutants, but not silencing of JAGN1, augmented cell death in response to the pro-apoptotic stimuli, etoposide, staurosporine, and thapsigargin. Furthermore, cells expressing mutant JAGN1 were remarkably susceptible to agonists that normally trigger degranulation and succumbed to a calcium-dependent cell death programme. This mode of cell death was completely prevented by pharmacological inhibition of calpain but unaffected by caspase inhibition. In conclusion, our results confirmed the association between JAGN1 mutations and SCN and showed that SCN-associated JAGN1 mutations unleash a calcium- and calpain-dependent cell death in myeloid cells.

Structure and function analysis of the C. elegans aminophospholipid translocase TAT-1.

The Caenorhabditis elegans aminophospholipid translocase TAT-1 maintains phosphatidylserine (PS) asymmetry in the plasma membrane and regulates endocytic transport. Despite these important functions, the structure-function relationship of this protein is poorly understood. Taking advantage of the tat-1 mutations identified by the C. elegans million mutation project, we investigated the effects of 16 single amino acid substitutions on the two functions of the TAT-1 protein. Two substitutions that alter a highly conserved PISL motif in the fourth transmembrane domain and a highly conserved DKTGT phosphorylation motif, respectively, disrupt both functions of TAT-1, leading to a vesicular gut defect and ectopic PS exposure on the cell surface, whereas most other substitutions across the TAT-1 protein, often predicted to be deleterious by bioinformatics programs, do not affect the functions of TAT-1. These results provide in vivo evidence for the importance of the PISL and DKTGT motifs in P4-type ATPases and improve our understanding of the structure-function relationship of TAT-1. Our study also provides an example of how the C. elegans million mutation project helps decipher the structure, functions, and mechanisms of action of important genes.

Cobalt nanoparticles trigger ferroptosis-like cell death (oxytosis) in neuronal cells: Potential implications for neurodegenerative disease.

The neurotoxicity of hard metal-based nanoparticles (NPs) remains poorly understood. Here, we deployed the human neuroblastoma cell line SH-SY5Y differentiated or not into dopaminergic- and cholinergic-like neurons to study the impact of tungsten carbide (WC) NPs, WC NPs sintered with cobalt (Co), or Co NPs versus soluble CoCl2 . Co NPs and Co salt triggered a dose-dependent cytotoxicity with an increase in cytosolic calcium, lipid peroxidation, and depletion of glutathione (GSH). Co NPs and Co salt also suppressed glutathione peroxidase 4 (GPX4) mRNA and protein expression. Co-exposed cells were rescued by N-acetylcysteine (NAC), a precursor of GSH, and partially by liproxstatin-1, an inhibitor of lipid peroxidation. Furthermore, in silico analyses predicted a significant correlation, based on similarities in gene expression profiles, between Co-containing NPs and Parkinson's disease, and changes in the expression of selected genes were validated by RT-PCR. Finally, experiments using primary human dopaminergic neurons demonstrated cytotoxicity and GSH depletion in response to Co NPs and CoCl2 with loss of axonal integrity. Overall, these data point to a marked neurotoxic potential of Co-based but not WC NPs and show that neuronal cell death may occur through a ferroptosis-like mechanism.

Intra- and Extracellular Degradation of Neutrophil Extracellular Traps by Macrophages and Dendritic Cells.

Neutrophil extracellular traps (NETs) composed of nuclear DNA associated with histones and granule proteins are involved in the extracellular killing of pathogens. Excessive NET formation has been implicated in several noninfectious pathological conditions. The disposal of NETs is, therefore, important to prevent inadvertent effects resulting from the continued presence of NETs in the extracellular environment. In this study, we investigated the interaction of NETs released by freshly isolated, PMA-stimulated primary human neutrophils with primary human monocyte-derived macrophages or dendritic cells (DCs). NETs were internalized by macrophages, and removal of the protein component prevented engulfment of NETs, whereas complexation with LL-37 restored the uptake of "naked" (protein-free) NETs. NETs were also found to dampen the bacterial LPS-induced maturation of DCs. Cytokine profiling was conducted by using a multiplex array following the interaction of NETs with macrophages or DCs, and NETs alone were found to be noninflammatory, whereas immunomodulatory effects were noted in the presence of LPS with significant upregulation of IL-1ß secretion, and a marked suppression of other LPS-induced factors including vascular endothelial growth factor (VEGF) in both cell types. Moreover, macrophage digestion of NETs was dependent on TREX1 (also known as DNaseIII), but not DNaseII, whereas extracellular DNase1L3-mediated degradation of NETs was observed for DCs. Collectively, these findings shed light on the interactions between NETs and phagocytic cells and provide new insights regarding the clearance of NETs, double-edged swords of innate immunity.

Multi-walled carbon nanotubes elicit concordant changes in DNA methylation and gene expression following long-term pulmonary exposure in mice.

Pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) causes inflammation and fibrosis. Our previous work has shown that industrially produced MWCNTs trigger specific changes in gene expression in the lungs of exposed animals. To elucidate whether epigenetic effects play a role for these gene expression changes, we performed whole genome bisulphite sequencing to assess DNA methylation patterns in the lungs 56 days after exposure to MWCNTs. Lung tissues were also evaluated with respect to histopathological changes and cytokine profiling of bronchoalveolar lavage (BAL) fluid was conducted using a multi-plex array. Integrated analysis of transcriptomics data and DNA methylation data revealed concordant changes in gene expression. Functional analysis showed that the muscle contraction, immune system/inflammation, and extracellular matrix pathways were the most affected pathways. Taken together, the present study revealed that MWCNTs exert epigenetic effects in the lungs of exposed animals, potentially driving the subsequent gene expression changes.

Nanoparticles and innate immunity: new perspectives on host defence.

The innate immune system provides the first line of defence against foreign microbes and particulate materials. Engineered nanoparticles can interact with the immune system in many different ways. Nanoparticles may thus elicit inflammation with engagement of neutrophils, macrophages and other effector cells; however, it is important to distinguish between acute and chronic inflammation in order to identify the potential hazards of nanoparticles for human health. Nanoparticles may also interact with and become internalised by dendritic cells, key antigen-presenting cells of the immune system, where a better understanding of these processes could pave the way for improved vaccination strategies. Nanoparticle characteristics such as size, shape and deformability also influence nanoparticle uptake by a plethora of immune cells and subsequent immune responses. Furthermore, the corona of adsorbed biomolecules on nanoparticle surfaces should not be neglected. Complement activation represents a special case of regulated and dynamic corona formation on nanoparticles with important implications in clearance and safety. Additionally, the inadvertent binding of bacterial lipopolysaccharide to nanoparticles is important to consider as this may skew the outcome and interpretation of immunotoxicological studies. Here, we discuss nanoparticle interactions with different cell types and soluble mediators belonging to the innate immune system.

Kostmann disease and other forms of severe congenital neutropenia.

Congenital neutropenia with autosomal recessive inheritance was first described by the Swedish paediatrician Rolf Kostmann who coined the term 'infantile genetic agranulocytosis'. The condition is now commonly referred to as Kostmann disease. These patients display a maturation arrest of the myelopoiesis in the bone marrow and reduced neutrophil numbers and suffer from recurrent, often life-threatening infections. The molecular mechanism underlying congenital neutropenia has been intensively investigated, and mutations in genes that impinge on programmed cell death have been identified. The present review provides an overview of these studies.

Brave new world revisited: Focus on nanomedicine.

Nanomedicine is at a crossroads: with relatively few success stories in terms of clinical translation despite more and more research on increasingly sophisticated nanomaterials, it is important to consider whether we are on the right track. Indeed, it is crucial that we address the fact that while considerable efforts are being made to overcome barriers to translation from the bench to the clinic, scientists are still struggling to decipher fundamental aspects of nanomaterial interactions with biological systems. We believe that a key to the successful adoption of nanomedicines in oncology and beyond lies in a deeper understanding of underlying biological processes and in decoding interactions between engineered nanomaterials and biological systems. Here we provide an overview of progress in nanomedicine during the past 5 years.

Next-Generation Sequencing Reveals Differential Responses to Acute versus Long-Term Exposures to Graphene Oxide in Human Lung Cells.

Numerous studies have addressed the biological impact of graphene-based materials including graphene oxide (GO), yet few have focused on long-term effects. Here, RNA sequencing is utilized to unearth responses of human lung cells to GO. To this end, the BEAS-2B cell line derived from normal human bronchial epithelium is subjected to repeated, low-dose exposures of GO (1 or 5 µg mL-1 ) for 28 days or to the equivalent, cumulative amount of GO for 48 h. Then, samples are analyzed by using the NovaSeq 6000 sequencing system followed by pathway analysis and gene ontology enrichment analysis of the differentially expressed genes. Significant differences are seen between the low-dose, long-term exposures and the high-dose, short-term exposures. Hence, exposure to GO for 48 h results in mitochondrial dysfunction. In contrast, exposure to GO for 28 days is characterized by engagement of apoptosis pathways with downregulation of genes belonging to the inhibitor of apoptosis protein (IAP) family. Validation experiments confirm that long-term exposure to GO affects the apoptosis threshold in lung cells, accompanied by a loss of IAPs. These studies reveal the sensitivity of RNA-sequencing approaches and show that acute exposure to GO is not a good predictor of the long-term effects of GO.

Humanin is a novel regulator of Hedgehog signaling and prevents glucocorticoid-induced bone growth impairment.

Glucocorticoids (GCs) are frequently used to treat chronic disorders in children, including inflammation and cancer. Prolonged treatment with GCs is well known to impair bone growth, an effect linked to increased apoptosis and suppressed proliferation in growth plate chondrocytes. We hypothesized that the endogenous antiapoptotic protein humanin (HN) may prevent these effects. Interestingly, GC-induced bone growth impairment and chondrocyte apoptosis was prevented in HN overexpressing mice, HN-treated wild-type mice, and in HN-treated cultured rat metatarsal bones. GC-induced suppression of chondrocyte proliferation was also prevented by HN. Furthermore, GC treatment reduced Indian Hedgehog expression in growth plates of wild-type mice but not in HN overexpressing mice or HN-treated wild-type animals. A Hedgehog (Hh) antagonist, vismodegib, was found to suppress the growth of cultured rat metatarsal bones, and this effect was also prevented by HN. Importantly, HN did not interfere with the desired anti-inflammatory effects of GCs. We conclude that HN is a novel regulator of Hh signaling preventing GC-induced bone growth impairment without interfering with desired effects of GCs. Our data may open for clinical studies exploring a new possible strategy to prevent GC-induced bone growth impairment by cotreating with HN.-Zaman, F., Zhao, Y., Celvin, B., Mehta, H. H., Wan, J., Chrysis, D., Ohlsson, C., Fadeel, B., Cohen, P., Sävendahl, L. Humanin is a novel regulator of Hedgehog signaling and prevents glucocorticoid-induced bone growth impairment.

Tumor selective uptake of drug-nanodiamond complexes improves therapeutic outcome in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths and novel treatment approaches are urgently needed. Here we show that poly(ethylene glycol)-functionalized nanodiamonds loaded with doxorubicin (ND-PEG-DOX) afforded a considerable improvement over free drug in an orthotopic pancreatic xenograft model. ND-PEG-DOX complexes were also superior to free DOX in 3-dimensional (3D) tumor spheroids of PDAC. ND-PEG showed no cytotoxicity towards macrophages, and histopathological analysis showed no abnormalities of major organs upon in vivo administration of ND-PEG-DOX. These results provide evidence that ND-mediated drug delivery may serve as a means of improving the therapeutic outcome in PDAC.

Calcium-dependent cyto- and genotoxicity of nickel metal and nickel oxide nanoparticles in human lung cells.

BACKGROUND: Genotoxicity is an important toxicological endpoint due to the link to diseases such as cancer. Therefore, an increased understanding regarding genotoxicity and underlying mechanisms is needed for assessing the risk with exposure to nanoparticles (NPs). The aim of this study was to perform an in-depth investigation regarding the genotoxicity of well-characterized Ni and NiO NPs in human bronchial epithelial BEAS-2B cells and to discern possible mechanisms. Comparisons were made with NiCl2 in order to elucidate effects of ionic Ni. METHODS: BEAS-2B cells were exposed to Ni and NiO NPs, as well as NiCl2, and uptake and cellular dose were investigated by transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS). The NPs were characterized in terms of surface composition (X-ray photoelectron spectroscopy), agglomeration (photon cross correlation spectroscopy) and nickel release in cell medium (ICP-MS). Cell death (necrosis/apoptosis) was investigated by Annexin V-FITC/PI staining and genotoxicity by cytokinesis-block micronucleus (cytome) assay (OECD 487), chromosomal aberration (OECD 473) and comet assay. The involvement of intracellular reactive oxygen species (ROS) and calcium was explored using the fluorescent probes, DCFH-DA and Fluo-4. RESULTS: NPs were efficiently taken up by the BEAS-2B cells. In contrast, no or minor uptake was observed for ionic Ni from NiCl2. Despite differences in uptake, all exposures (NiO, Ni NPs and NiCl2) caused chromosomal damage. Furthermore, NiO NPs were most potent in causing DNA strand breaks and generating intracellular ROS. An increase in intracellular calcium was observed and modulation of intracellular calcium by using inhibitors and chelators clearly prevented the chromosomal damage. Chelation of iron also protected against induced damage, particularly for NiO and NiCl2. CONCLUSIONS: This study has revealed chromosomal damage by Ni and NiO NPs as well as Ni ionic species and provides novel evidence for a calcium-dependent mechanism of cyto- and genotoxicity.

Transcriptional profiling reveals gene expression changes associated with inflammation and cell proliferation following short-term inhalation exposure to copper oxide nanoparticles.

Our recent studies revealed a dose-dependent proinflammatory response to copper oxide nanoparticles (CuO NPs) in rats following short-term inhalation exposure for five consecutive days. Here transcriptomics approaches were applied using the same model to assess global gene expression in lung tissues obtained 1 day post-exposure and after a recovery period of 22 days from rats exposed to clean air or 6 hour equivalent doses of 3.3 mg m-3 (low dose) and 13.2 mg m-3 (high dose). Microarray analyses yielded about 1000 differentially expressed genes in the high-dose group and 200 in low-dose compared to the clean air control group, and less than 20 after the recovery period. Pathway analysis indicated cell proliferation/survival and inflammation as the main processes triggered by exposure to CuO NPs. We did not find significant perturbations of pathways related to oxidative stress. Upregulation of epithelial cell transforming protein 2 (Ect2), a known oncogene, was noted and ECT2 protein was upregulated in the lungs of exposed animals. Proliferation of alveolar epithelial cells was demonstrated based on Ki67 expression. The gene encoding monocyte chemoattractant protein 1 (or CCL2) was also upregulated and this was confirmed by immunohistochemistry. However, no aberrant DNA methylation of inflammation-associated genes was observed. In conclusion, we have found that inhalation of CuO NPs in rats causes upregulation of the oncoprotein ECT2 and the chemokine CCL2 and other proinflammatory markers as well as proliferation in bronchoalveolar epithelium after a short-term inhalation exposure. Thus, pathways known to be associated with neoplastic processes and inflammation were affected in this model.

Degradation of Single-Layer and Few-Layer Graphene by Neutrophil Myeloperoxidase.

Biodegradability of graphene is one of the fundamental parameters determining the fate of this material in vivo. Two types of aqueous dispersible graphene, corresponding to single-layer (SLG) and few-layer graphene (FLG), devoid of either chemical functionalization or stabilizing surfactants, were subjected to biodegradation by human myeloperoxidase (hMPO) mediated catalysis. Graphene biodegradation was also studied in the presence of activated, degranulating human neutrophils. The degradation of both FLG and SLG sheets was confirmed by Raman spectroscopy and electron microscopy analyses, leading to the conclusion that highly dispersed pristine graphene is not biopersistent.

Cytotoxic and Proinflammatory Effects of Metal-Based Nanoparticles on THP-1 Monocytes Characterized by Combined Proteomics Approaches.

Thorough characterization of toxic effects of nanoparticles (NP) is desirable due to the increasing risk of potential environmental contamination by NP. In the current study, we combined three recently developed proteomics approaches to assess the effects of Au, CuO, and CdTe NP on the innate immune system. The human monocyte cell line THP-1 was employed as a model. The anticancer drugs camptothecin and doxorubicin were used as positive controls for cell death, and lipopolysaccharide was chosen as a positive control for proinflammatory activation. Despite equivalent overall toxicity effect (50 ± 10% dead cells), the three NP induced distinctly different proteomics signatures, with the strongest effect being induced by CdTe NP, followed by CuO and gold NP. The CdTe toxicity mechanism involves down-regulation of topoisomerases. The effect of CuO NP is most reminiscent of oxidative stress and involves up-regulation of proteins involved in heat response. The gold NP induced up-regulation of the inflammatory mediator, NF-κB, and its inhibitor TIPE2 was identified as a direct target of gold NP. Furthermore, gold NP triggered activation of NF-κB as evidenced by phosphorylation of the p65 subunit. Overall, the combined proteomics approach described here can be used to characterize the effects of NP on immune cells.