Detection of Beta-Glucan Contamination in Nanoparticle Formulations.

Beta-glucans with diverse chemical structures are produced by a variety of microorganisms and are commonly found in microbial cell walls. ß-(1,3)-D-glucans are present in yeast and fungi, and, for this reason, their traces are commonly used as a sign of yeast or fungal infection or contamination. Despite being less immunologically active than endotoxins, beta-glucans are pro-inflammatory and can activate cytokines and other immunological responses via their cognate pattern recognition receptors. Unlike endotoxins, there is no established threshold pyrogen dose for beta-glucans; as such, their quantity in pharmaceutical products is not regulated. Nevertheless, regulatory agencies recognize the potential contribution of beta-glucans to the immunogenicity of protein-containing drug products and recommend assessing beta-glucans to aid the interpretation of immunotoxicity studies and assess the risk of immunogenicity. The protocol for the detection and quantification of ß-(1,3)-D-glucans in nanoparticle formulations is based on a modified limulus amoebocyte lysate assay. The results of this test are used to inform immunotoxicity studies of nanotechnology-based drug products.

Detection of Intracellular Complement Activation by Nanoparticles in Human T Lymphocytes.

The complement system is complex and includes two main components: the systemic or plasma complement and the so-called intracellular complement or complosome. The complement proteins expressed by the liver and secreted into blood plasma compose the plasma complement system, whereas complement proteins expressed by and functioning inside the cell represent the intracellular complement. The complement system plays an essential role in host defense; however, complement activation may lead to pathologies when uncontrolled. When such undesirable activation of the plasma complement occurs in response to a drug product, it leads to immediate-type hypersensitivity reactions independent of immunoglobulin E. These reactions are often called complement activation-related pseudoallergy (CARPA). In addition to the blood plasma, the complement protein C3 is found in many cells, including lymphocytes, monocytes, endothelial, and even cancer cells. The activation of the intracellular complement generates split products, which are exported from the cell onto the membrane. Since the activation of the intracellular complement in T lymphocytes was found to correlate with autoimmune disorders, and growing evidence is available for the involvement of T lymphocytes in the development of drug-induced hypersensitivity reactions, understanding the ability of nanomaterials to activate intracellular complement may aid in establishing a long-term safety profile for these materials. This chapter describes a flow cytometry-based protocol for detecting intracellular complement activation by engineered nanomaterials.

Evaluation of the Acute Anaphylactoid Reactogenicity of Nanoparticle-Containing Medicines and Vaccines Using the Porcine CARPA Model.

A small fraction, up to 10%, of people treated intravenously with state-of-the-art nanoparticulate drugs or diagnostic agents develop an acute infusion reaction which can be severe or even lethal. Activation of the complement (C) system can play a causal, or contributing role in these atypical, "pseudoallergic" reactions, hence their name, C activation-related pseudoallergy (CARPA). Intravenous (i.v.) administration of the human reaction-triggering (very small) dose of a test sample in pigs triggers a symptom tetrad (characteristic hemodynamic, hematological, skin, and laboratory changes) that correspond to the major human symptoms. Quantitating these changes provides a highly sensitive and reproducible method for assessing the risk of CARPA, enabling the implementation of appropriate preventive measures. Accordingly, the porcine CARPA model has been increasingly used for the safety evaluation of therapeutic and diagnostic nanomedicines and, recently, mRNA-lipid nanoparticle vaccines. This chapter provides details of the experimental procedure followed upon using the model.

Protocol for modeling and simulating lithiation-induced stress in largely deformed spherical nanoparticles using COMSOL.

Here, we present a finite element method-based scheme for solving coupled partial differential equations (PDEs) for the analysis of lithiation-induced stress in largely deformed spherical nanoparticles via the PDE module in COMSOL. We describe steps for software installation and setting PDEs, initial/boundary conditions, and mesh parameters. We then detail procedures for dividing the mesh and analyzing lithium trapping during electrochemical cycling. This protocol can also be extended to analyze a wide range of problems involving diffusion-induced stress. For complete details on the use and execution of this protocol, please refer to Li et al.1.

Comprehensive analysis revealed that titanium dioxide nanoparticles could strengthen the resistance of apple rootstock B9 to saline-alkali stress.

Apple growth and development can be adversely affected by saline-alkali stress, which has become a significant factor restricting the high yield of the apple industry. In recent years, nanomaterials have become a potential source for plant growth and development. Titanium dioxide nanoparticles (TiO2 NPs) play an important role in multiple plant development processes, including mitigating environmental stress. In this study, one-year-old apple rootstock B9 stem cuttings were used as research objects. Different concentrations of TiO2 NPs were applied to the roots before saline-alkali treatment. Principal component analysis showed that 1gkg-1 TiO2 NPs treatment had the best effect in alleviating the stress for B9. It significantly reduced the damage to B9 under salt-alkali stress, increased the content of photosynthetic pigment, enhanced the performance of Photosystem II, and promoted photosynthesis. At the same time, the content of K+ was increased, and the ion toxicity was alleviated. In addition, TiO2 NPs have also been shown to reduce B9 cell damage and lipid peroxidation, increase antioxidant enzyme activity, and regulate the accumulation of solutes. Overall, this study provides a theoretical basis for TiO2 NPs to mitigate the adverse effects of plants under saline-alkali stress and provides useful insights for managing other plants affected by global salinity and alkalinity.

Co-Administration of Nanowired Monoclonal Antibodies to Inducible Nitric Oxide Synthase and Tumor Necrosis Factor Alpha Together with Antioxidant H-290/51 Reduces SiO2 Nanoparticles-Induced Exacerbation of Pathophysiology of Spinal Cord Trauma.

Military personnel are often exposed to silica dust during combat operations across the globe. Exposure to silica dust in US military or service personnel could cause Desert Strom Pneumonitis also referred to as Al Eskan disease causing several organs damage and precipitate autoimmune dysfunction. However, the effects of microfine particles of sand inhalation-induced brain damage on the pathophysiology of traumatic brain or spinal cord injury are not explored. Previously intoxication of silica nanoparticles (50-60 nm size) is shown to exacerbates spinal cord injury induces blood-spinal cord barrier breakdown, edema formation and cellular changes. However, the mechanism of silica nanoparticles-induced cord pathology is still not well known. Spinal cord injury is well known to alter serotonin (5-hydroxytryptamine) metabolism and induce oxidative stress including upregulation of nitric oxide synthase and tumor necrosis factor alpha. This suggests that these agents are involved in the pathophysiology of spinal cord injury. In this review, we examined the effects of combined nanowired delivery of monoclonal antibodies to neuronal nitric oxide synthase (nNOS) together with tumor necrosis factor alpha (TNF-α) antibodies and a potent antioxidant H-290/51 to induce neuroprotection in spinal cord injury associated with silica nanoparticles intoxication. Our results for the first time show that co-administration of nanowired delivery of antibodies to nNOS and TNF-α with H-290/51 significantly attenuated silica nanoparticles-induced exacerbation of spinal cord pathology, not reported earlier.

CsPbBr3 Perovskite Nanoparticles causes Colitis-Like Symptom via Promoting Intestinal Barrier Damage and Gut Microbiota Dysbiosis.

Lead-based perovskite nanoparticles (Pb-PNPs) with superior optoelectronic properties are promising alternatives for the next generation of photovoltaics materials. This raises a great concern about their potential exposure toxicity in biological systems. However, little is known about their adverse effects on the gastrointestinal tract system so far. Here, the aim is to investigate the biodistribution, biotransformation, potential gastrointestinal tract toxicity, and effect on the gut microbiota after oral exposure to the CsPbBr3 perovskite nanoparticles (CPB PNPs). The advanced synchrotron radiation based microscopic X-ray fluorescence scanning and X-ray absorption near-edge spectroscopy demonstrate that high doses of CPB (CPB-H) PNPs can gradually transform into different lead-based compounds, subsequently accumulating in the gastrointestinal tract, especially the colon. Meanwhile, the pathological changes of stomach, small intestine, and colon reveal that CPB-H PNPs have higher gastrointestinal tract toxicity than Pb(Ac)2 , consequently leading to colitis-like symptoms. More importantly, 16S rRNA gene sequencing analysis discloses that CPB-H PNPs cause more significant alterations in the richness and diversity of the gut microbiota related to inflammation, intestinal barrier, and immune function than Pb(Ac)2 . The findings may contribute to shedding light on understanding the adverse effects on gastrointestinal tract and gut microbiota of Pb-PNPs.

[The Effects of Nanoparticles on Nerve Cell Differentiation and Its Mechanisms for Human Health Risk Analysis].

Neurodevelopment is one of the most complex events in human growth and is very sensitive to disruption. Various genetic factors are the main causes of neuronal dysfunction; however, recent epidemiological studies have also revealed relationships between environmental factors and the onset of neurodevelopmental disorders. Humans are regularly exposed to a wide range of environmental factors, among which fine particles have attracted recent interest. In this regards, the development of products containing nanomaterials has expanded substantially in a wide variety of fields including medicine, food, and cosmetics. As the size of the particles in these nanomaterials decreases, their reactivity at the tissue interface and their tissue penetration increases. In addition, the reduction of particle size could alter kinetics and lead to unexpected biological effects compared with those seen with conventional materials. Thus, we need to identify potential sources of unpredictable adverse effects of nanomaterials on neurodevelopment to ensure their safe use. From this perspective, nano-safety science research has been conducted through the collection of toxicity information on nanoparticles based on their physicochemical properties and kinetics via the association analysis of physicochemical properties, kinetics, and toxicity. The results of this nano-safety science research were then used in nano-safety design research to develop safer forms of nanomaterials. In this paper, we introduce findings that demonstrate that nanomaterials translocate into the brain and describe the effects on cranial nerves.

The onset of rare earth metallosis begins with renal gadolinium-rich nanoparticles from magnetic resonance imaging contrast agent exposure.

The leitmotifs of magnetic resonance imaging (MRI) contrast agent-induced complications range from acute kidney injury, symptoms associated with gadolinium exposure (SAGE)/gadolinium deposition disease, potentially fatal gadolinium encephalopathy, and irreversible systemic fibrosis. Gadolinium is the active ingredient of these contrast agents, a non-physiologic lanthanide metal. The mechanisms of MRI contrast agent-induced diseases are unknown. Mice were treated with a MRI contrast agent. Human kidney tissues from contrast-naïve and MRI contrast agent-treated patients were obtained and analyzed. Kidneys (human and mouse) were assessed with transmission electron microscopy and scanning transmission electron microscopy with X-ray energy-dispersive spectroscopy. MRI contrast agent treatment resulted in unilamellar vesicles and mitochondriopathy in renal epithelium. Electron-dense intracellular precipitates and the outer rim of lipid droplets were rich in gadolinium and phosphorus. We conclude that MRI contrast agents are not physiologically inert. The long-term safety of these synthetic metal-ligand complexes, especially with repeated use, should be studied further.

Perspectives on complement and phagocytic cell responses to nanoparticles: From fundamentals to adverse reactions.

The complement system, professional phagocytes and other cells such as Natural killer cells and mast cells are among the important components of the innate arm of the immune system. These constituents provide an orchestrated array of defences and responses against tissue injury and foreign particles, including nanopharmaceuticals. While interception of nanopharmaceuticals by the immune system is beneficial for immunomodulation and treatment of phagocytic cell disorders, it is imperative to understand the multifaceted mechanisms by which nanopharmaceuticals interacts with the immune system and evaluate the subsequent balance of beneficial versus adverse reactions. An example of the latter is adverse infusion reactions to regulatory-approved nanopharmaceuticals seen in human subjects. Here, we discuss collective opinions and findings from our laboratories in mapping nanoparticle-mediated complement and leucocyte/macrophage responses.

Comparative Evaluation of the Effects of Amorphous Silica Nanoparticles on the Erythrocytes of Wistar Normotensive and Spontaneously Hypertensive Rats.

Silica nanoparticles (SiNPs) are one of the most widely used nanomaterials. SiNPs can encounter erythrocytes and hypertension is strongly linked to abnormalities in the functional and structural characteristics of erythrocytes. As little is known about the combinatorial effect of SiNP-hypertension interactions on erythrocytes, the aim of this work was to study the effects triggered by hypertension on SiNPs induced hemolysis and the pathophysiological mechanism underlying it. We compared the interaction of amorphous 50 nm SiNPs at various concentrations (0.2, 1, 5 and 25 µg/mL) with erythrocytes of normotensive (NT) and hypertensive (HT) rats in vitro. Following incubation of the erythrocytes, SiNPs induced significant and dose-dependent increase in hemolysis. Transmission electron microscopy revealed erythrocyte deformity in addition to SiNPs taken up by erythrocytes. The erythrocyte susceptibility to lipid peroxidation was significantly increased. The concentration of reduced glutathione, and activities of superoxide dismutase, and catalase were significantly increased. SiNPs significantly increased intracellular Ca2+. Likewise, the concentration of the cellular protein annexin V and calpain activity was enhanced by SiNPs. Concerningly, all the tested parameters were significantly enhanced in erythrocytes from HT rats compared to NT rats. Our results collectively demonstrate that hypertension can potentially exacerbate the in vitro effect induced by SiNPs.

Ameliorative role of nanocurcumin against the toxicological effects of novel forms of Cuo as nanopesticides: a comparative study.

Copper oxide nanoparticles (CuONPs) have a wide range of uses in agricultural applications. Nanocurcumin (NCur) acts as an antioxidant treatment. The goal of the study is to reduce the toxicity resulting from the use of CuONPs as nanopesticides on living organisms by inducing changes in the morphological shape of CuONPs or treating it with NCur. So, we induced a comparative study between three shapes of CuONPs: CuO nanosphere (CuONSp), CuO nanosheet (CuONS), and CuO nanoflower (CuONF). We characterize each nano-form by using X-ray diffraction (XRD), scanning electron microscope (SEM), transmission electron microscope (HRTEM), and Zetasizer HT device; 36 rats were divided into six groups (n = 6): 1st group was the control group; 2nd group received 50 mg/kg/day of NCur orally for 30 days; 3rd, 4th, and 5th groups received orally 50 mg/kg/day of CuONSp, CuONS, and CuONF, respectively, for 30 days; 6th group received 50 mg/kg/day CuONSp plus 50 mg/kg/day of NCur orally for 30 days. An elevation occurred in malondialdehyde (MDA), liver and kidney functions, tumor necrosis factor-alpha (TNF-α), and B-cell lymphoma 2 (Bcl2) by CuONSp > CuONS > CuONF, respectively. An inhibition occurred in glutathione (GSH), superoxidase (SOD) catalase (CAT), apoptotic Bax gene (Bax), histopathological, and ultrastructural alterations by CuONSp < CuONS < CuONF, respectively. NCur ameliorated these alternations. In conclusion, CuONF is a better form compared to other forms of nanopesticide in agriculture due to its lower toxicity. NCur decreased the biological alternations which induced by CuONSp due to its antioxidant and anti-apoptotic properties.

LncRNA HOTAIRM1 Involved in Nano NiO-Induced Pulmonary Fibrosis via Regulating PRKCB DNA Methylation-Mediated JNK/c-Jun Pathway.

Nickel oxide nanoparticles (Nano NiO) lead to pulmonary fibrosis, and the mechanisms are associated with epigenetics. This study aimed to clarify the regulatory relationship among long noncoding RNA HOXA transcript antisense RNA myeloid-specific 1 (HOTAIRM1), DNA methylation and expression of protein kinase C beta (PRKCB), and JNK/c-Jun pathway in Nano NiO-induced pulmonary fibrosis. Therefore, we constructed the rat pulmonary fibrosis model by intratracheal instillation of Nano NiO twice a week for 9 weeks and established the collagen deposition model by treating BEAS-2B cells with Nano NiO for 24 h. Here, the DNA methylation pattern was analyzed by whole-genome bisulfite sequencing in rat fibrotic lung tissues. Then, we integrated mRNA transcriptome data and found 93 DNA methylation genes with transcriptional significance. Meanwhile, the data showed that Nano NiO caused the down-regulation of lncRNA HOTAIRM1, the hypomethylation, and up-regulation of PRKCB2, JNK/c-Jun pathway activation, and collagen deposition (the up-regulated Col-I and α-SMA) both in vivo and in vitro. DNMTs inhibitor 5-AZDC attenuated Nano NiO-induced PRKCB2 expression, JNK/c-Jun pathway activation, and collagen deposition, but overexpression of PRKCB2 aggravated the changes mentioned indicators in Nano NiO-induced BEAS-2B cells. Furthermore, JNK/c-Jun pathway inhibitor (SP600125) alleviated Nano NiO-induced excessive collagen formation. Additionally, overexpression of HOTAIRM1 restrained the PRKCB hypomethylation, the activation of JNK/c-Jun pathway, and collagen formation induced by Nano NiO in BEAS-2B cells. In conclusion, these findings demonstrated that HOTAIRM1 could arrest Nano NiO-induced pulmonary fibrosis by suppressing the PRKCB DNA methylation-mediated JNK/c-Jun pathway.

Effect of ZnO and CuO nanoparticles on the growth, nutrient absorption, and potential health risk of the seasonal vegetable Medicago polymorpha L.

Background: Medicago polymorpha L., a seasonal vegetable, is commonly grown in China. The increasing use of nanoparticles (NPs) such as ZnO and CuO NPs in agriculture has raised concerns about their potential risks for plant growth and for human consumption. There is a lack of research on the effects of ZnO and CuO NPs on agronomic performance of Medicago polymorpha L. and their potential risks for human health. Methods: In this study, different treatment concentrations of ZnO NPs (25, 50, 100, and 200 mg kg-1) and CuO NPs (10, 25, 50, and 100 mg kg-1) were used to determine their effects on the growth and nutrient absorption of Medicago polymorpha L., as well as their potential risk for human health. Results: The results showed that ZnO and CuO NPs increased the fresh weight of Medicago polymorpha L. by 5.8-11.8 and 3.7-8.1%, respectively. The best performance for ZnO NPs occurred between 25-50 mg kg-1 and the best performance for CuO NPs occurred between 10-25 mg kg-1. Compared with the control, ZnO and CuO NPs improved the macronutrients phosphorus (P), potassium (K), magnesium (Mg), and calcium (Ca). The following micronutrients were also improved: iron (Fe), nickel (Ni), copper (Cu), zinc (Zn), and manganese (Mn), with the exception of nitrogen (N) accumulation. Low treatment concentrations exhibited more efficient nutrient uptake than high treatment concentrations. A comprehensive analysis showed that the optimum concentrations were 25 mg kg-1 for ZnO NPs and 10 mg kg-1 for CuO NPs. The potential non-carcinogenic health risk of Medicago polymorpha L. treated with ZnO and CuO NPs was analyzed according to the estimated daily intake (EDI), the hazard quotient (HQ), and the cumulative hazard quotient (CHQ). Compared with the oral reference dose, the EDI under different ZnO and CuO NPs treatments was lower. The HQ and CHQ under different ZnO and CuO NPs treatments were far below 1. This indicated that Medicago polymorpha L. treated with ZnO and CuO NPs did not pose any non-carcinogenic health risk to the human body. Therefore, ZnO and CuO NPs were considered as a safe nano fertilizer for Medicago polymorpha L. production according to growth analysis and a human health risk assessment.

Safety profile of repeated infusion of platelet-like nanoparticles in healthy dogs.

OBJECTIVE: To assess the safety and efficacy of the platelet-like nanoparticle (PLN), and to assess its safety in repeated administration. ANIMALS: 6 purpose-bred dogs. PROCEDURES: The PLN was administered IV at 3 different doses using a randomized crossover design. Each dog received a full dose of 8 X 1010 particles/10 kg, half dose, and 10 times the dose, with a 14-day washout period between doses. Biochemical, prothrombin time, partial thromboplastin time, and fibrinogen analyses were performed at baseline and 96 hours postinfusion. A CBC, kaolin-activated thromboelastography, platelet function assay closure time, and buccal mucosal bleeding time were performed at baseline and 1, 6, 24, 48, 72, and 96 hours postinfusion. RESULTS: No significant changes were observed over time in the thromboelastography parameters, closure time, and buccal mucosal bleeding time. After the administration of the half dose, hematocrit levels decreased significantly at 1, 6, 24, 48, and 96 hours, with all values within the reference range. The platelet count was decreased significantly at hours 1, 6, 24, 48, and 72 after administration of the half dose, with values less than the reference range at all hours but hour 72. No significant changes in serum biochemistry, coagulation panel, and fibrinogen were observed for all doses. No adverse events were noted during the first infusion. Three dogs experienced transient sedation and nausea after repeat infusion. CLINICAL RELEVANCE: The PLN resulted in a dilution of hematocrit and platelets, and did not significantly alter hemostasis negatively. The safety of repeated doses should be investigated further in dogs.

Where Is Nano Today and Where Is It Headed? A Review of Nanomedicine and the Dilemma of Nanotoxicology.

Worldwide nanotechnology development and application have fueled many scientific advances, but technophilic expectations and technophobic demands must be counterbalanced in parallel. Some of the burning issues today are the following: (1) Where is nano today? (2) How good are the communication and investment networks between academia/research and governments? (3) Is there any spotlight application for nanotechnology? Nanomedicine is a particular arm of nanotechnology within the healthcare landscape, focused on diagnosis, treatment, and monitoring of emerging (such as coronavirus disease 2019, COVID-19) and contemporary (including diabetes, cardiovascular diseases, neurodegenerative disorders, and cancer) diseases. However, it may only represent the bright side of the coin. In fact, in the recent past, the concept of nanotoxicology has emerged to address the dark shadows of nanomedicine. The nanomedicine field requires more nanotoxicological studies to identify undesirable effects and guarantee safety. Here, we provide an overall perspective on nanomedicine and nanotoxicology as central pieces of the giant puzzle of nanotechnology. First, the impact of nanotechnology on education and research is highlighted, followed by market trends and scientific output tendencies. In the next section, the nanomedicine and nanotoxicology dilemma is addressed through the interplay of in silico, in vitro, and in vivo models with the support of omics and microfluidic approaches. Lastly, a reflection on the regulatory issues and clinical trials is provided. Finally, some conclusions and future perspectives are proposed for a clearer and safer translation of nanomedicines from the bench to the bedside.

Better lipids to power next generation of mRNA vaccines.

New delivery systems aim to increase vaccine potency and reduce side effects.

Single-cell suspension preparation from murine organs following in vivo mRNA-LNP exposure.

We describe a protocol to study inflammatory responses triggered by the mRNA-lipid nanoparticle (LNP) vaccine formulations in skin, muscle, and lung and the adaptive immune responses induced in the draining lymph nodes. Here, we will present how to deliver these reagents through intradermal, intramuscular, and intranasal routes, generating single-cell suspensions from the inoculated and target organs for downstream analyses. For complete details on the use and execution of this protocol, please refer to Ndeupen et al. (2021) and (2022).