Synthesis, Characterization, and Toxicity Assessment of Zinc Oxide-Doped Manganese Oxide Nanoparticles in a Macrophage Model.

The doping of engineered nanomaterials (ENMs) is a key tool for manipulating the properties of ENMs (e.g., electromagnetic, optical, etc.) for different therapeutic applications. However, adverse health outcomes and the cellular biointeraction of doped ENMs, compared to undoped counterparts, are not fully understood. Previously, we have shown that doping manganese oxide nanoparticles with ZnO (ZnO-MnO2 NPs) improved their catalytic properties. In this study, we assessed the toxicity of ZnO-MnO2 NPs in Raw 264.7 cells. NPs were prepared via an eco-friendly, co-precipitation method and characterized by several techniques, including transmission and scanning electron microscopy, X-ray diffraction, and Fourier transform infrared. The physicochemical properties of ZnO-MnO2 NPs, including size, morphology, and crystalline structure, were almost identical to MnO2 NPs. However, ZnO-MnO2 NPs showed slightly larger particle aggregates and negative charge in cell culture media. Exposure to ZnO-MnO2 NPs resulted in lower toxicity based on the cell viability and functional assay (phagocytosis) data. Exposure to both NPs resulted in the activation of the cell inflammatory response and the generation of reactive oxygen species (ROS). Despite this, exposure to ZnO-MnO2 NPs was associated with a lower toxicity profile, and it resulted in a higher ROS burst and the activation of the cell antioxidant system, hence indicating that MnO2 NP-induced toxicity is potentially mediated via other ROS-independent pathways. Furthermore, the cellular internalization of ZnO-MnO2 NPs was lower compared to MnO2 NPs, and this could explain the lower extent of toxicity of ZnO-MnO2 NPs and suggests Zn-driven ROS generation. Together, the findings of this report suggest that ZnO (1%) doping impacts cellular biointeraction and the consequent toxicological outcomes of MnO2 NPs in Raw 264.7 cells.

Electronic cigarette vapor disrupts key metabolic pathways in human lung epithelial cells.

The steady increase in the use of electronic cigarettes (ECs) has reached an epidemic level, increasing mortality and morbidity, mainly due to pulmonary toxicity. Several mechanisms are involved in EC-induced toxicity, including oxidative stress and increased inflammation. Concurrently, the integrity of cellular metabolism is essential for cellular homeostasis and mitigation of toxic insults. However, the effects of EC on cellular metabolism remain largely unknown. In this study, we investigated the metabolic changes induced by EC in human lung epithelial cells (A549) using an untargeted metabolomics approach. A549 cells were exposed to increasing EC vapor extract concentrations, and cell viability, oxidative stress, and metabolomic changes were assessed. Our findings show that ECs induce cell death and increase oxidative stress in a concentration-dependent manner. Metabolomic studies demonstrated that ECs induce unique metabolic changes in key cellular metabolic pathways. Our results revealed that exposure to ECs induced clear segregation in metabolic responses which is driven significantly by number of essential metabolites such as aminoacids, fatty acids, glutathione, and pyruvate. Interstingly, our metabolomics results showed that each concentration of ECs induced unqiues pattern of metabolic changes, suggesting the complexity of ECs induced cytotoxcity. Disrupted metabolites were linked to essential cellular pathways, such as fatty acid biosynthesis, as well as glutathione, pyruvate, nicotinate and nicotinamide, and amino acid metabolisms. These results highlight the potential adverse effects of ECs on cellular metabolism and emphasize the need for further research to fully understand the long-term consequences of EC use. Overall, this study demonstrates that ECs not only induce cell death and oxidative stress but also disrupt cellular metabolism in A549 lung epithelial cells.

Topical Sustained-Release Dexamethasone-Loaded Chitosan Nanoparticles: Assessment of Drug Delivery Efficiency in a Rabbit Model of Endotoxin-Induced Uveitis.

Uveitis is an ocular illness that if not treated properly can lead to a total loss of vision. In this study, we evaluated the utility of HA-coated Dexamethasone-sodium-phosphate (DEX)-chitosan nanoparticles (CSNPs) coated with hyaluronic acid (HA) as a sustained ocular delivery vehicle for the treatment of endotoxin-induced-uveitis (EIU) in rabbits. The CSNPs were characterized for particle size, zeta potential, polydispersity, surface morphology, and physicochemical properties. Drug encapsulation, in vitro drug release, and transcorneal permeation were also evaluated. Finally, eye irritation, ocular pharmacokinetics, and pharmacodynamics were in vivo. The CSNPs ranged from 310.4 nm and 379.3 nm pre-(uncoated) and post-lyophilization (with HA-coated), respectively. The zeta potentials were +32 mV (uncoated) and -5 mV (HA-uncoated), while polydispersity was 0.178-0.427. Drug encapsulation and loading in the CSNPs were 73.56% and 6.94% (uncoated) and 71.07% and 5.54% (HA-coated), respectively. The in vitro DEX release over 12 h was 77.1% from the HA-coated and 74.2% from the uncoated NPs. The physicochemical properties of the CSNPs were stable over a 3-month period when stored at 25 °C. Around a 10-fold increased transcorneal-flux and permeability of DEX was found with HA-CSNPs compared to the DEX-aqueous solution (DEX-AqS), and the eye-irritation experiment indicated its ocular safety. After the ocular application of the CSNPs, DEX was detected in the aqueous humor (AH) till 24 h. The area under the concentrations curve (AUC0-24h) for DEX from the CSNPs was 1.87-fold (uncoated) and 2.36-fold (HA-coated) higher than DEX-AqS. The half-life (t1/2) of DEX from the uncoated and HA-coated NPs was 2.49-and 3.36-fold higher, and the ocular MRT0-inf was 2.47- and 3.15-fold greater, than that of DEX-AqS, respectively. The EIU rabbit model showed increased levels of MPO, TNF-α, and IL-6 in AH. Topical DEX-loaded CSNPs reduced MPO, TNF-α, and IL-6 levels as well as inhibited NF-κB expression. Our findings demonstrate that the DEX-CSNPs platform has improved the delivery properties and, hence, the promising anti-inflammatory effects on EIU in rabbits.

Adverse Responses following Exposure to Subtoxic Concentrations of Zinc Oxide and Nickle Oxide Nanoparticles in the Raw 264.7 Cells.

The incorporation of engineered nanomaterials (ENMs) in biomedical and consumer products has been growing, leading to increased human exposure. Previous research was largely focused on studying direct ENM toxicity in unrealistic high-exposure settings. This could result in overlooking potential adverse responses at low and subtoxic exposure levels. This study investigated adverse cellular outcomes to subtoxic concentrations of zinc oxide (ZnONPs) or nickel oxide (NiONPs) nanoparticles in the Raw 264.7 cells, a macrophage-like cell model. Exposure to both nanoparticles resulted in a concentration-dependent reduction of cell viability. A subtoxic concentration of 6.25 µg/mL (i.e., no observed adverse effect level) was used in subsequent experiments. Exposure to both nanoparticles at subtoxic levels induced reactive oxygen species generation. Cellular internalization data demonstrated significant uptake of NiONPs, while there was minimal uptake of ZnONPs, suggesting a membrane-driven interaction. Although subtoxic exposure to both nanoparticles was not associated with cell activation (based on the expression of MHC-II and CD86 surface markers), it resulted in the modulation of the lipopolysaccharide-induced inflammatory response (TNFα and IL6), and cells exposed to ZnONPs had reduced cell phagocytic capacity. Furthermore, subtoxic exposure to the nanoparticles distinctly altered the levels of several cellular metabolites involved in cell bioenergetics. These findings suggest that exposure to ENMs at subtoxic levels may not be devoid of adverse health outcomes. This emphasizes the importance of establishing sensitive endpoints of exposure and toxicity beyond conventional toxicological testing.

In Vitro Safety Assessment of In-House Synthesized Titanium Dioxide Nanoparticles: Impact of Washing and Temperature Conditions.

Titanium dioxide nanoparticles (TiO2 NPs) have been widely used in food, cosmetics, and biomedical research. However, human safety following exposure to TiO2 NPs remains to be fully understood. The aim of this study was to evaluate the in vitro safety and toxicity of TiO2 NPs synthesized via the Stöber method under different washing and temperature conditions. TiO2 NPs were characterized by their size, shape, surface charge, surface area, crystalline pattern, and band gap. Biological studies were conducted on phagocytic (RAW 264.7) and non-phagocytic (HEK-239) cells. Results showed that washing amorphous as-prepared TiO2 NPs (T1) with ethanol while applying heat at 550 °C (T2) resulted in a reduction in the surface area and charge compared to washing with water (T3) or a higher temperature (800 °C) (T4) and influenced the formation of crystalline structures with the anatase phase in T2 and T3 and rutile/anatase mixture in T4. Biological and toxicological responses varied among TiO2 NPs. T1 was associated with significant cellular internalization and toxicity in both cell types compared to other TiO2 NPs. Furthermore, the formation of the crystalline structure induced toxicity independent of other physicochemical properties. Compared with anatase, the rutile phase (T4) reduced cellular internalization and toxicity. However, comparable levels of reactive oxygen species were generated following exposure to the different types of TiO2, indicating that toxicity is partially driven via non-oxidative pathways. TiO2 NPs were able to trigger an inflammatory response, with varying trends among the two tested cell types. Together, the findings emphasize the importance of standardizing engineered nanomaterial synthesis conditions and evaluating the associated biological and toxicological consequences arising from changes in synthesis conditions.

Development of Imeglimin Electrospun Nanofibers as a Potential Buccal Antidiabetic Therapeutic Approach.

The prevalence of type 2 diabetes (T2D) has been growing worldwide; hence, safe and effective antidiabetics are critically warranted. Recently, imeglimin, a novel tetrahydrotriazene compound, has been approved for use in T2D patients in Japan. It has shown promising glucose-lowering properties by improving pancreatic beta-cell function and peripheral insulin sensitivity. Nevertheless, it has several drawbacks, including suboptimal oral absorption and gastrointestinal (GI) discomfort. Therefore, this study aimed to fabricate a novel formulation of imeglimin loaded into electrospun nanofibers to be delivered through the buccal cavity to overcome the current GI-related adverse events and to provide a convenient route of administration. The fabricated nanofibers were characterized for diameter, drug-loading (DL), disintegration, and drug release profiles. The data demonstrated that the imeglimin nanofibers had a diameter of 361 ± 54 nm and DL of 23.5 ± 0.2 µg/mg of fibers. The X-ray diffraction (XRD) data confirmed the solid dispersion of imeglimin, favoring drug solubility, and release with improved bioavailability. The rate of drug-loaded nanofibers disintegration was recorded at 2 ± 1 s, indicating the rapid disintegration ability of this dosage form and its suitability for buccal delivery, with a complete drug release after 30 min. The findings of this study suggest that the developed imeglimin nanofibers have the potential to be given via the buccal route, thereby achieving optimal therapeutic outcomes and improving patient compliance.

Topical Application of Linezolid-Loaded Chitosan Nanoparticles for the Treatment of Eye Infections.

Linezolid (LZ) loaded chitosan-nanoparticles (CSNPs) was developed by the ionic-gelation method using Tripolyphosphate-sodium as a crosslinker for topical application for the treatment of bacterial eye infections. Particles were characterized by Zeta-Sizer (Malvern Nano-series). TEM was used for structural morphology. Encapsulation and drug loading were estimated by measuring the unencapsulated drug. In-vitro drug release in STF (pH 7) was performed through a dialysis membrane. Storage stability of LZ-CSNPs was checked at 25 °C and 40 °C for six months. The antimicrobial potency of NPs was evaluated on different Gram-positive strains. Ocular irritation and pharmacokinetic studies were completed in rabbits. Ex-vivo transcorneal permeation of the drug was determined through the rabbit cornea. Ionic interaction among the oppositely charged functional groups of CS and TPP generated the CSNPs. The weight ratio at 3:1, wt/wt (CS/TPP) with 21.7 mg of LZ produced optimal NPs (213.7 nm with 0.387 of PDI and +23.1 mV of ZP) with 71% and 11.2% encapsulation and drug loading, respectively. Around 76.7% of LZ was released from LZ-AqS within 1 h, while 79.8% of LZ was released from CSNPs at 12 h and 90% at 24 h. The sustained drug release property of CSNPS was evaluated by applying kinetic models. The linearity in the release profile suggested that the release of LZ from CSNPs followed the Higuchi-Matrix model. LZ-CSNPs have shown 1.4 to 1.6-times improved antibacterial activity against the used bacterial strains. The LZ-CSNPs were "minimally-irritating" to rabbit eyes and exhibited 4.4-times increased transcorneal permeation of LZ than from LZ-AqS. Around 3-, 1.2- and 3.1-times improved Tmax, Cmax, and AUC0-24 h, respectively were found for LZ-CSNPs during the ocular pharmacokinetic study. AqS has shown 3.1-times faster clearance of LZ. Conclusively, LZ-CSNPs could offer a better alternative for the prolonged delivery of LZ for the treatment of bacterial infections in the eyes.

Identification and Characterization of Plasmids and Genes from Carbapenemase-Producing Klebsiella pneumoniae in Makkah Province, Saudi Arabia.

Klebsiella pneumoniae (K. pneumoniae) is involved in several hospital and community-acquired infections. The prevalence of K. pneumoniae-producing-carbapenemase (KPC) resistance genes rapidly increases and threatens public health worldwide. This study aimed to assess the antibiotic resistance level of K. pneumoniae isolates from Makkah Province, Saudi Arabia, during the Islamic 'Umrah' ritual and to identify the plasmid types, presence of genes associated with carbapenem hydrolyzing enzymes, and virulence factors. The phenotypic and genotypic analyses based on the minimum inhibitory concentration (MIC), biofilm formation, PCR, and characterization of KPC-encoding plasmids based on the replicon typing technique (PBRT) were explored. The results showed that most isolates were resistant to carbapenem antibiotics and other antibiotics classes. This study identified sixteen different replicons of plasmids in the isolates and multiple genes encoding carbapenem factors, with blaVIM and blaOXA-48 being the most prevalent genes identified in the isolates. However, none of the isolates exhibited positivity for the KPC production activity. In addition, this study also identified six virulence-related genes, including kfu, wabG, uge, rmpA, fimH, and a capsular polysaccharide (CPS). Together, the data reported in this study indicate that the isolated K. pneumoniae during the pilgrimage in Makkah were all resistant to carbapenem antibiotics. Although the isolates lacked KPC production activity, they carried multiple carbapenem-resistant genes and virulence factors, which could drive their resistant phenotype. The need for specialized methods for KPC detection, monitoring the possibility of nosocomial transmission, and diverse therapeutic alternatives are necessary for controlling the spreading of KPC. This study can serve as a reference for clinicians and researchers on types of K. pneumoniae commonly found during religious gathering seasons in Saudi Arabia.

Recent nanotechnology advancements to treat multidrug-resistance pancreatic cancer: Pre-clinical and clinical overview.

Pancreatic cancer (PC) remains one of the most lethal and incurable forms of cancer and has a poor prognosis. One of the significant therapeutic challenges in PC is multidrug resistance (MDR), a phenomenon in which cancer cells develop resistance toward administered therapy. Development of novel therapeutic platforms that could overcome MDR in PC is crucial for improving therapeutic outcomes. Nanotechnology is emerging as a promising tool to enhance drug efficacy and minimize off-target responses via passive and/or active targeting mechanisms. Over the past decade, tremendous efforts have been made to utilize nanocarriers capable of targeting PC cells while minimizing off-target effects. In this review article, we first give an overview of PC and the major molecular mechanisms of MDR, and then we discuss recent advancements in the development of nanocarriers used to overcome PC drug resistance. In doing so, we explore the developmental stages of this research in both pre-clinical and clinical settings. Lastly, we discuss current challenges and gaps in the literature as well as potential future directions in the field.

Silver Nanoparticle-Based Combinations with Antimicrobial Agents against Antimicrobial-Resistant Clinical Isolates.

The increasing prevalence of antimicrobial-resistant (AMR) bacteria along with the limited development of antimicrobials warrant investigating novel antimicrobial modalities. Emerging inorganic engineered nanomaterials (ENMs), most notably silver nanoparticles (AgNPs), have demonstrated superior antimicrobial properties. However, AgNPs, particularly those of small size, could exert overt toxicity to mammalian cells. This study investigated whether combining AgNPs and conventional antimicrobials would produce a synergistic response and determined the optimal and safe minimum inhibitory concentration (MIC) range against several wild-type Gram-positive and -negative strains and three different clinical isolates of AMR Klebsiella pneumoniae. Furthermore, the cytotoxicity of the synergistic combinations was assessed in a human hepatocyte model. The results showed that the AgNPs (15-25 nm) were effective against Gram-negative bacteria (MIC of 16-128 µg/mL) but not Gram-positive strains (MIC of 256 µg/mL). Both wild-type and AMR K. pneumoniae had similar MIC values following exposure to AgNPs. Importantly, co-exposure to combinations of AgNPs and antimicrobial agents, including kanamycin, colistin, rifampicin, and vancomycin, displayed synergy against both wild-type and AMR K. pneumoniae isolates (except for vancomycin against AMR strain I). Notably, the tested combinations demonstrated no to minimal toxicity against hepatocytes. Altogether, this study indicates the potential of combining AgNPs with conventional antimicrobials to overcome AMR bacteria.

Ceramide expression in relation to breast cancer molecular subtypes in Saudi women.

BACKGROUND/INTRODUCTION: Despite advances in the diagnosis and management of breast cancer (BC), it is still associated with high mortality rates. New biomarkers are being developed for the diagnosis, treatment, and prediction of responses of BC. Ceramide (CER), a bioactive sphingolipid, has emerged recently as a useful diagnostic tool in several types of tumors. In this study, we evaluated CER expression in invasive BC and assessed its relation to the molecular subtypes of BC. MATERIALS AND METHODS: The clinical data and histopathological slides of 50 patients with invasive ductal carcinoma were retrieved and reviewed. The cases were then stained with a mouse monoclonal anti-ceramide antibody. Pearson correlation was used to assess the correlation between CER percentage and intensity and other clinical and pathological variables. RESULTS: CER expression showed a direct relationship with estrogen and progesterone receptors Allred scores. However, it showed an inverse relation with tumor grade, HER2/neu status and Ki-67 index. CONCLUSIONS: CER expression is likely to be associated with luminal BC molecular subtypes. However, more research is needed to confirm these results and to explore its relation to the different clinical outcomes, including response to treatment and prognosis.

Adverse cardiovascular responses of engineered nanomaterials: Current understanding of molecular mechanisms and future challenges.

Nanotechnology is spanning multiple fields of study from materials science to computer engineering and drug discovery. Since the early 21st century, nanotechnology and nano-enabled research have received great attention and governmental funding accompanied with interest to ensure human and environmental safety of engineered nanomaterials (ENMs). Optimal functioning of the cardiovascular (CV) system is of utmost importance for the overall health of the body. Following exposure, ENMs essentially end up in the circulation (at least partially) and hence it is key to assess any associated adverse CV consequences. Accumulating research suggests that exposure to ENMs (different compositions and physicochemical properties) has the capacity to directly and indirectly interact with CV components resulting in adverse events and worsening of CV complications. However, the underlying molecular mechanisms driving these events remain to be elucidated. In this article, we review state-of-art literature on ENM-associated adverse CV responses and discuss the potential underlying molecular mechanisms.

Aggravation of autism-like behavior in BTBR T+tf/J mice by environmental pollutant, di-(2-ethylhexyl) phthalate: Role of nuclear factor erythroid 2-related factor 2 and oxidative enzymes in innate immune cells and cerebellum.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder which manifests itself in early childhood and is distinguished by recurring behavioral patterns, and dysfunction in social/communication skills. Ubiquitous environmental pollutant, di-2-ethylhexyl phthalate (DEHP) is one of the most frequently used plasticizers in various industrial products, e.g. vinyl flooring, plastic toys, and medical appliances. DEHP gets easily released into the environment and leads to human exposure through various routes. DEHP has been described to be linked with oxidative stress in various organs in animal/human studies. Increased concentration of DEHP has also been detected in ASD children which indicates an association between phthalates exposure and ASD. However, effect of DEHP on autism-like behavior has not been investigated previously. Therefore, this study probed the effect of DEHP on autism-like behavior (marble burying, self-grooming and sociability) and innate immune cells (dendritic cells/neutrophils)/cerebellar oxidant-antioxidant balance (NFkB, iNOS, NADPH oxidase, nitrotyrosine, lipid peroxides, Nrf2, SOD, GPx) in BTBR and C57 mice. Our data show that DEHP treatment causes worsening of autism-like behavior in BTBR mice which is associated with enhancement of oxidative stress in innate immune cells and cerebellum with concomitant lack of antioxidant protection. DEHP also causes oxidative stress in C57 mice in both innate immune cells and cerebellar compartment, however there is Nrf2-mediated induction of enzymatic antioxidants which protects them from upregulated oxidative stress. This proposes the notion that ubiquitous environmental pollutants such as DEHP may be involved in the pathogenesis/progression of ASD through dysregulation of antioxidant-antioxidant balance in innate immune cells and cerebellum.

Engineered Nanomaterials and Type I Allergic Hypersensitivity Reactions.

Type I allergic hypersensitivity disorders (atopy) including asthma, atopic dermatitis, allergic rhinitis, and food allergy are on the rise in developed and developing countries. Engineered nanomaterials (ENMs) span a large spectrum of material compositions including carbonic, metals, polymers, lipid-based, proteins, and peptides and are being utilized in a wide range of industries including healthcare and pharmaceuticals, electronics, construction, and food industry, and yet, regulations for the use of ENMs in consumer products are largely lacking. Prior evidence has demonstrated the potential of ENMs to induce and/or aggravate type I allergic hypersensitivity responses. Furthermore, previous studies have shown that ENMs could directly interact with and activate key T-helper 2 (Th2) effector cell types (such as mast cells) and the complement system, which could result in pseudoallergic (non-IgE-mediated) hypersensitivity reactions. Nevertheless, the underlying molecular mechanisms of ENM-mediated induction and/or exacerbation of type I immune responses are poorly understood. In this review, we first highlight key examples of studies that have demonstrated inherent immunomodulatory properties of ENMs in the context of type I allergic hypersensitivity reactions, and most importantly, we attempt to put together the potential molecular mechanisms that could drive ENM-mediated stimulation and/or aggravation of type I allergic hypersensitivity responses.

5-aminoisoquinolinone attenuates social behavior deficits and immune abnormalities in the BTBR T+ Itpr3tf/J mouse model for autism.

Autism spectrum disorder (ASD) is diagnosed by core symptoms including impaired social communication and the presence of repetitive and stereotypical behaviors. There is also evidence for immune dysfunction in individuals with ASD, but it is a disease that is still insufficiently controlled by current treatment strategies. The use of 5-aminoisoquinolinone (5-AIQ) ameliorates several immune-mediated symptoms including rheumatoid arthritis and colitis, and has neuroprotective properties; however, its role in ASD is not yet characterized. In this study, we investigated the effect of 5-AIQ on sociability tests, self-grooming, marble burying, and locomotor activities in BTBR T+ Itpr3tf/J (BTBR) mice, which serve as an ASD animal model. We further investigated the possible molecular mechanism of 5-AIQ administration on CXCR4-, CXCR6-, IFN-γ-, IL-22-, NOS2-, STAT1-, T-bet-, and RORγT-producing CD3+ T cells isolated from the spleens of treated mice. We also explored its effects on mRNA expression in brain tissue. Our results showed that in BTBR mice, 5-AIQ treatment significantly prevented self-grooming and marble burying behaviors and enhanced social interactions without any adverse effects on locomotor activity/anxiety level. Additionally, 5-AIQ treatment substantially decreased CXCR4-, CXCR6-, IFN-γ-, IL-22-, NOS2-, STAT1-, T-bet-, and RORγT-producing CD3+ T cells in the spleen. Furthermore, 5-AIQ treatment decreased CXCR4, IFN-γ, IL-22, STAT1, and RORγT mRNA expression levels in brain tissue. Our findings demonstrated that 5-AIQ improved behavioral and immune abnormalities associated with ASD, which supports the hypothesis that 5-AIQ has important therapeutic potential for the treatment of behavioral and neuroimmune dysfunctions in ASD.

Defect-induced electronic states amplify the cellular toxicity of ZnO nanoparticles.

Zinc oxide nanoparticles (ZnO NPs) are used in numerous applications, including sunscreens, cosmetics, textiles, and electrical devices. Increased consumer and occupational exposure to ZnO NPs potentially poses a risk for toxicity. While many studies have examined the toxicity of ZnO NPs, little is known regarding the toxicological impact of inherent defects arising from batch-to-batch variations. It was hypothesized that the presence of varying chemical defects in ZnO NPs will contribute to cellular toxicity in rat aortic endothelial cells (RAECs). Pristine and defected ZnO NPs (oxidized, reduced, and annealed) were prepared and assessed three major cellular outcomes; cytotoxicity/apoptosis, reactive oxygen species production and oxidative stress, and endoplasmic reticulum (ER) stress. ZnO NPs chemical defects were confirmed by X-ray photoelectron spectroscopy and photoluminescence. Increased toxicity was observed in defected ZnO NPs compared to the pristine NPs as measured by cell viability, ER stress, and glutathione redox potential. It was determined that ZnO NPs induced ER stress through the PERK pathway. Taken together, these results demonstrate a previously unrecognized contribution of chemical defects to the toxicity of ZnO NPs, which should be considered in the risk assessment of engineered nanomaterials.

Exposure to silver nanoparticles primes mast cells for enhanced activation through the high-affinity IgE receptor.

Mast cells are a key effector cell in type I allergic reactions. It has been shown that environmental exposures such as diesel exhaust and heavy metals exacerbate mast cell degranulation and activation. Today, the use of engineered nanomaterials (ENMs) is rapidly expanding and silver nanoparticles (AgNP) are one of the mostly widely utilized ENMs, primarily for their antimicrobial properties, and are being incorporated into many consumer and biomedical products. We assessed whether pre-exposure of bone marrow-derived mast cells (BMMCs) to 20 nm AgNPs enhanced degranulation and activation to an allergen (dinitrophenol-conjugated human serum albumin) by measuring ß-hexosaminidase release, LTB4 and IL-6 production. In addition, we assessed reactive oxygen species (ROS) generation, cell oxidative stress and toxicity as well as total and individual protein tyrosine phosphorylation (p-Tyr). We found that pre-exposure of BMMCs to AgNPs results in exacerbated allergen-mediated mast cell degranulation, LTB4 production and IL-6 release. Exposure of BMMCs to AgNPs exacerbated allergen-induced ROS generation, however, this was not associated with oxidative stress nor cell death. Finally, pre-exposure to AgNPs enhanced allergen-mediated global p-Tyr as well as individual proteins including Syk, PLCγ and LAT. Our findings indicate that pre-exposure to AgNPs exacerbates mast cell allergen-mediated phosphorylation of FcεR1-linked tyrosine kinases and ROS generation resulting in amplified early and late-phase responses. These findings suggest that exposure to AgNPs has the potential to prime mast cells to allergic immune responses, which could be of particular concern to atopic populations as the use of AgNPs in consumer and biomedical products rapidly increases.

Silver nanoparticle immunomodulatory potential in absence of direct cytotoxicity in RAW 264.7 macrophages and MPRO 2.1 neutrophils.

Engineered nanomaterials (ENM) are being used in a wide range of consumer products and pharmaceuticals; hence, there is an increasing risk for human exposure and potential adverse outcomes. The immune system, vital in host defense and protection against environmental agents, is typically initiated and executed by innate effector immune cells including macrophages and neutrophils. Previous literature has reported the immune system as a major target of ENM toxicity; however, there is inconsistency regarding the immunotoxicity of ENM. This could be attributed to differences in ENM physicochemical properties, cellular models examined, biocorona formation, etc. Thus, the current study examined the toxicity and immunomodulatory effects of silver nanoparticles (AgNP), one of the most utilized ENM in consumer and medical products, in two key innate immune cell models, e.g. RAW 264.7 cells (macrophages) and differentiated MPRO 2.1 cells (promyelocytes/neutrophils). The results showed that despite a generation of reactive oxygen species, exposure to 20 nm citrate-coated AgNP was not associated with major oxidative damage, inflammatory responses, nor cytotoxicity. Nevertheless, and most importantly, pre-exposure to the AgNP for 24 h enhanced RAW 264.7 cell phagocytic ability as well as the release of inflammatory cytokine interleukin-6 in response to lipopolysaccharide (LPS). In MPRO 2.1 cells, AgNP pre-exposure also resulted in enhanced phagocytic ability; however, these cells manifest reduced cell degranulation (elastase release) and oxidative burst in response to phorbol myristate acetate (PMA). Taken together, these findings indicated to us that exposure to AgNP, despite not being directly (cyto)toxic to these cells, had the potential to alter immune cell responses. The findings underscore the import of assessing immune cell function post-exposure to ENM beyond the standard endpoints such as oxidative stress and cytotoxicity. In addition, these findings further illustrate the importance of understanding the underlying molecular mechanisms of ENM-cellular interactions, particularly in the immune system.

Methods for Assessing Mast Cell Responses to Engineered Nanomaterial Exposure.

Mast cells are key effector cells in inflammatory and allergic immune responses such as asthma, rhinitis, and atopic dermatitis. Activation of mast cells leads to immediate release of preformed mediators such as histamine and proteases, which can regulate vascular permeability and the function of a number of immune and nonimmune cells. Engineered nanomaterials (ENM) have been utilized for a wide range of applications and introduced into a number of consumer products; yet the consequent increase in human exposure and any potential adverse effects have not been fully evaluated. Modulation of the immune system function has been shown to be a major toxicological consequence of ENM exposure. The implication of mast cells in ENM-mediated toxicity, including the most widely utilized carbon and metal-based ENMs, has been previously demonstrated; and therefore, understanding direct ENM interaction with mast cells at the cellular and molecular level is of critical importance for the safe implementation of ENMs into consumer products.

Biocorona formation contributes to silver nanoparticle induced endoplasmic reticulum stress.

Prior research has demonstrated cells exposed to silver nanoparticles (AgNPs) undergo endoplasmic reticulum (ER) stress leading to cellular apoptosis and toxicity, however, the fundamental mechanism underlying AgNP-induced ER stress is unknown. We hypothesize the biophysical interactions between AgNPs and adsorbed proteins lead to misfolded proteins to elicit an ER stress response. Our investigation examined rat aortic endothelial cells (RAEC) exposed to 20 or 100 nm AgNPs with or without a biocorona (BC) consisting of bovine serum albumin (BSA), high density lipoprotein (HDL) or fetal bovine serum (FBS) to form a complex BC. The presence of a BC consisting of BSA or FBS proteins significantly reduced uptake of 20 nm and 100 nm AgNPs in RAEC. Western blot analysis indicated robust activation of the IREα and PERK pathways in RAEC exposed to 20 nm despite the reduction in uptake by the presence of a BC. This was not observed for the 100 nm AgNPs. Hyperspectral darkfield microscopy qualitatively confirmed that the preformed BC was maintained following uptake by RAEC. Transmission electron microscopy demonstrated a size dependent effect on the sub-cellular localization of AgNPs. Overall, these results suggest that AgNP size, surface area and BC formation governs the induction of ER stress and alterations in intracellular trafficking.