Margriet V. D. Z. Park

THE EFFECT OF PARTICLE SIZE ON THE CYTOTOXICITY, INFLAMMATION, DEVELOPMENTAL TOXICITY AND GENOTOXICITY OF SILVER NANOPARTICLES

Silver nanoparticles are of interest to be used as antimicrobial agents in wound dressings and coatings in medical devices, but potential adverse effects have been reported in the literature. The most pronounced effect of silver nanoparticles and the role of particle size in determining these effects, also in comparison to silver ions, are largely unknown. Effects of silver nanoparticles of different sizes (20, 80, 113 nm) were compared in in vitro assays for cytotoxicity, inflammation, genotoxicity and developmental toxicity. Silver nanoparticles induced effects in all endpoints studied, but effects on cellular metabolic activity and membrane damage were most pronounced. In all toxicity endpoints studied, silver nanoparticles of 20 nm were more toxic than the larger nanoparticles. In L929 fibroblasts, but not in RAW 264.7 macrophages, 20 nm silver nanoparticles were more cytotoxic than silver ions. Collectively, these results indicate that effects of silver nanoparticles on different toxic endpoints may be the consequence of their ability to inflict cell damage. In addition, the potency of silver in the form of nanoparticles to induce cell damage compared to silver ions is cell type and size-dependent.

Physicochemical characteristics of nanomaterials that affect pulmonary inflammation

The increasing manufacture and use of products based on nanotechnology raises concerns for both workers and consumers. Various studies report induction of pulmonary inflammation after inhalation exposure to nanoparticles, which can vary in aspects such as size, shape, charge, crystallinity, chemical composition, and dissolution rate. Each of these aspects can affect their toxicity, although it is largely unknown to what extent. The aim of the current review is to analyse published data on inhalation of nanoparticles to identify and evaluate the contribution of their physicochemical characteristics to the onset and development of pulmonary inflammation. Many physicochemical characteristics of nanoparticles affect their lung deposition, clearance, and pulmonary response that, in combination, ultimately determine whether pulmonary inflammation will occur and to what extent. Lung deposition is mainly determined by the physical properties of the aerosol (size, density, shape, hygroscopicity) in relation to airflow and the anatomy of the respiratory system, whereas clearance and translocation of nanoparticles are mainly determined by their geometry and surface characteristics. Besides size and chemical composition, other physicochemical characteristics influence the induction of pulmonary inflammation after inhalation. As some nanoparticles dissolve, they can release toxic ions that can damage the lung tissue, making dissolution rate an important characteristic that affects lung inflammation. Fibre-shaped materials are more toxic to the lungs compared to spherical shaped nanoparticles of the same chemical composition. In general, cationic nanoparticles are more cytotoxic than neutral or anionic nanoparticles. Finally, surface reactivity correlates well with observed pulmonary inflammation. With all these characteristics affecting different stages of the events leading to pulmonary inflammation, no unifying dose metric could be identified to describe pulmonary inflammation for all nanomaterials, although surface reactivity might be a useful measure. To determine the extent to which the various characteristics influence the induction of pulmonary inflammation, the effect of these characteristics on lung deposition, clearance, and pulmonary response should be systematically evaluated. The results can then be used to facilitate risk assessment by categorizing nanoparticles according to their characteristics.

Systemic and immunotoxicity of silver nanoparticles in an intravenous 28 days repeated dose toxicity study in rats.

Because of its antibacterial activity nanosilver is one of the most commonly used nanomaterials. It is increasingly used in a variety of both medical and consumer products resulting in an increase in human exposure. However, the knowledge on the systemic toxicity of nanosilver is relatively limited. To determine the potential systemic toxicity of silver nanoparticles (Ag-NP) with different sizes (20 nm and 100 nm) a 28-days repeated dose toxicity study was performed in rats using intravenous administration. The toxic effect of the 20 nm Ag-NP was performed using the bench mark dose (BMD) approach. Treatment with a maximum dose of 6 mg/kg body weight was well tolerated by the animals. However, both for 20 nm and 100 nm Ag-NP growth retardation was observed during the treatment. A severe increase in spleen size and weight was present which was due to an increased cell number. Both T and B cell populations showed an increase in absolute cell number, whereas the relative cell numbers remained constant. At histopathological evaluation brown and black pigment indicating Ag-NP accumulation was noted in spleen, liver, and lymph nodes. Ag-NP was also detected incidentally in other organs. Clinical chemistry indicated liver damage (increased alkaline phosphatase, alanine transaminase, and aspartate transaminase) that could not be confirmed by histopathology. Hematology showed a decrease in several red blood cell parameters. The most striking toxic effect was the almost complete suppression of the natural killer (NK) cell activity in the spleen at high doses. Other immune parameters affected were: decreased interferon-γ and interleukin (IL)-10 production by concanavalin-A stimulated spleen cells, increased IL-1β and decreased IL-6, IL-10 and TNF-α production by lipopolysaccharide stimulated spleen cells, increase in serum IgM and IgE, and increase in blood neutrophilic granulocytes. For the spleen weight a critical effect dose of 0.37 mg/kg body weight (b.w.) could be established. The lowest critical effect dose (CED) for a 5% change compared to control animals was observed for thymus weight (CED05 0.01 mg/kg b.w.) and for functional immune parameters, i.e. decrease in NK cell activity (CED05 0.06 mg/kg b.w.) and LPS stimulation of spleen cells (CED05 0.04 mg/kg b.w.). These results show that for nanosilver the most sensitive parameters for potential adverse responses were effects on the immune system.

In vitro developmental toxicity test detects inhibition of stem cell differentiation by silica nanoparticles.

While research into the potential toxic properties of nanomaterials is now increasing, the area of developmental toxicity has remained relatively uninvestigated. The embryonic stem cell test is an in vitro screening assay used to investigate the embryotoxic potential of chemicals by determining their ability to inhibit differentiation of embryonic stem cells into spontaneously contracting cardiomyocytes. Four well characterized silica nanoparticles of various sizes were used to investigate whether nanomaterials are capable of inhibition of differentiation in the embryonic stem cell test. Nanoparticle size distributions and dispersion characteristics were determined before and during incubation in the stem cell culture medium by means of transmission electron microscopy (TEM) and dynamic light scattering. Mouse embryonic stem cells were exposed to silica nanoparticles at concentrations ranging from 1 to 100 microg/ml. The embryonic stem cell test detected a concentration dependent inhibition of differentiation of stem cells into contracting cardiomyocytes by two silica nanoparticles of primary size 10 (TEM 11) and 30 (TEM 34) nm while two other particles of primary size 80 (TEM 34) and 400 (TEM 248) nm had no effect up to the highest concentration tested. Inhibition of differentiation of stem cells occurred below cytotoxic concentrations, indicating a specific effect of the particles on the differentiation of the embryonic stem cells. The impaired differentiation of stem cells by such widely used particles warrants further investigation into the potential of these nanoparticles to migrate into the uterus, placenta and embryo and their possible effects on embryogenesis.

The status of in vitro toxicity studies in the risk assessment of nanomaterials

Nanotechnology applications already on the market or in development promise great benefits for humans as well as the environment. Simultaneously, the pressure to advance the development of fast methods for evaluating the potential risks of increased human exposure to nanomaterials is augmented. One way forward would be to enhance the role of in vitro toxicity studies in risk assessment procedures of nanomaterials. However, to maximize the use of in vitro assays for this purpose, their values and limitations need to be revealed. Even in risk assessment frameworks for regular chemicals, in vitro studies play a minor role. A comparative analysis of published in vitro data with nanomaterials demonstrates that there are a number of issues that need resolving before in vitro studies can play a role in the risk assessment of nanomaterials.

Particle size dependent deposition and pulmonary inflammation after short-term inhalation of silver nanoparticles

BackgroundAlthough silver nanoparticles are currently used in more than 400 consumer products, it is not clear to what extent they induce adverse effects after inhalation during production and use. In this study, we determined the lung burden, tissue distribution, and the induction and recovery of adverse effects after short-term inhalation exposure to 15 nm and 410 nm silver nanoparticles.MethodsRats were nose-only exposed to clean air, 15 nm silver nanoparticles (179 μg/m3) or 410 nm silver particles (167 μg/m3) 6 hours per day, for four consecutive days. Tissue distribution and the induction of pulmonary toxicity were determined at 24 hours and 7 days after exposure and compared with the internal alveolar dose. Presence of silver nanoparticles in lung cells was visualized by transmission electron microscopy (TEM).ResultsExposure to 15 nm silver nanoparticles induced moderate pulmonary toxicity compared to the controls, indicated by a 175-fold increased influx of neutrophils in the lungs, a doubling of cellular damage markers in the lungs, a 5-fold increase in pro-inflammatory cytokines, and a 1.5-fold increase in total glutathione at 24 hours after exposure. All the observed effects disappeared at 7 days after exposure. No effects were observed after exposure to 410 nm silver particles. The internal alveolar mass dose of the 15 nm nanoparticles was 3.5 times higher compared to the 410 nm particles, which equals to a 66,000 times higher particle number. TEM analysis revealed 15 nm nanoparticles in vesicles and nuclei of lung cells, which were decreased in size to <5 nm at 24 hours after exposure. This demonstrates substantial dissolution of the silver nanoparticles.ConclusionThe results show a clear size-dependent effect after inhalation of similar mass concentrations of 15 nm and 410 nm silver (nano)particles. This can be partially explained by the difference in the internal alveolar dose between the 15 nm and 410 nm silver (nano)particles as well as by a difference in the release rate of silver ions.

Genotoxicity evaluation of amorphous silica nanoparticles of different sizes using the micronucleus and the plasmid lacZ gene mutation assay

Abstract We investigated the potential of four well-characterized amorphous silica nanoparticles to induce chromosomal aberrations and gene mutations using two in vitro genotoxicity assays. Transmission electron microscopy (TEM) was used to verify the manufacturers nominal size of 10, 30, 80 and 400 nm which showed actual sizes of 11, 34, 34 and 248 nm, respectively. The 80 (34) nm silica nanoparticles induced chromosomal aberrations in the micronucleus assay using 3T3-L1 mouse fibroblasts and the 30 (34) and 80 (34) nm silica nanoparticles induced gene mutations in mouse embryonic fibroblasts carrying the lacZ reporter gene. TEM imaging demonstrated that the majority of nanoparticles were localized in vacuoles and not in the nucleus of 3T3-L1 cells, indicating that the observed DNA damage was most likely a result of indirect mechanisms. Further studies are needed to reveal these mechanisms and to determine the biological relevance of the effects of these particular silica nanoparticles in vivo.

A perspective on the developmental toxicity of inhaled nanoparticles

This paper aimed to clarify whether maternal inhalation of engineered nanoparticles (NP) may constitute a hazard to pregnancy and fetal development, primarily based on experimental animal studies of NP and air pollution particles. Overall, it is plausible that NP may translocate from the respiratory tract to the placenta and fetus, but also that adverse effects may occur secondarily to maternal inflammatory responses. The limited database describes several organ systems in the offspring to be potentially sensitive to maternal inhalation of particles, but large uncertainties exist about the implications for embryo-fetal development and health later in life. Clearly, the potential for hazard remains to be characterized. Considering the increased production and application of nanomaterials and related consumer products a testing strategy for NP should be established. Due to large gaps in data, significant amounts of groundwork are warranted for a testing strategy to be established on a sound scientific basis.

Progress and future of in vitro models to study translocation of nanoparticles

AbstractThe increasing use of nanoparticles in products likely results in increased exposure of both workers and consumers. Because of their small size, there are concerns that nanoparticles unintentionally cross the barriers of the human body. Several in vivo rodent studies show that, dependent on the exposure route, time, and concentration, and their characteristics, nanoparticles can cross the lung, gut, skin, and placental barrier. This review aims to evaluate the performance of in vitro models that mimic the barriers of the human body, with a focus on the lung, gut, skin, and placental barrier. For these barriers, in vitro models of varying complexity are available, ranging from single-cell-type monolayer to multi-cell (3D) models. Only a few studies are available that allow comparison of the in vitro translocation to in vivo data. This situation could change since the availability of analytical detection techniques is no longer a limiting factor for this comparison. We conclude that to further develop in vitro models to be used in risk assessment, the current strategy to improve the models to more closely mimic the human situation by using co-cultures of different cell types and microfluidic approaches to better control the tissue microenvironments are essential. At the current state of the art, the in vitro models do not yet allow prediction of absolute transfer rates but they do support the definition of relative transfer rates and can thus help to reduce animal testing by setting priorities for subsequent in vivo testing.

Horizon scan of nanomedicinal products

AIM A horizon scan of nanomedicinal product on the market or undergoing clinical investigation by analyzing the current nanomedicinal landscape. MATERIALS & METHODS The horizon scan includes a search of literature, clinical trial registries and the internet. RESULTS This horizon scan yielded 175 nanomedicinal products. Most products were intended for cancer treatment, followed by infectious diseases. Polymer conjugates, liposomes and protein nanoparticles were the most used structures for nanomedicinal products. CONCLUSIONS This paper provides an overview of nanomedicinal products on the market or undergoing clinical investigation, their application areas and specific properties.