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International audience; Due to their unique size-dependent properties, nanoparticles (NPs) have many industrial and biomedical applications. Although NPs are generally characterized based on the size or morphological analysis, the mass of whole particles can be of interest as it represents the total amount of material in the particle regardless of shape, density, or elemental composition. In addition, the shape of nonspherical NPs presents a conceptual challenge, making them difficult to characterize in terms of size or morphological characteristics. Here, we used a novel nano-electro-mechanical sensor mass spectrometry (NEMS-MS) technology to characterize the mass distributions of various NPs. For standard spherical gold NPs, mass distributions covered the range from ∼5 to 250 MDa (8 to ∼415 attograms). Applying the density of gold (19.3 g/cm3) and assuming perfect sphericity, these mass measurements were used to compute the equivalent diameters of the NPs. The sizes determined agreed well with the transmission electron microscopy (TEM) imaging data, with deviations of ∼1.4%. Subsequently, we analyzed the mass distribution of ∼50 nm synthetic silicon dioxide particles, having determined their size by electron microscopy (SEM and TEM). Their estimated density was in line with the literature values derived from differential mobility analyzer and aerosol particle mass analyzer data. Finally, we examined the intact gold nanotetrapods and obtained a mass distribution revealing their controlled polydispersity. The presence of polyethylene glycol coating was also quantified and corroborated nuclear magnetic resonance observations. Our results demonstrate the potential of NEMS-MS-based measurements as an effective means to characterize NPs, whatever their composition, shape or density.

To make surface enhanced Raman scattering (SERS) sensors more practical, we propose nanoporous Ag disks as SERS-active plasmonic structures that can be readily inkjet-printed just before use to avoid degradation of SERS enhancement. Together with the aid of the enhanced plasmonic fields from the nanoporous Ag (confirmed by electromagnetic simulation), we utilize a coffee-ring effect to concentrate target analytes, which is demonstrated by confocal Raman measurements. By using the proposed SERS sensor, Raman signals of TiO2 nanoparticles with a concentration of ppm to sub-ppb have been successfully measured. TiO2 in commercial consumables has been also detected by distinguishing its crystalline phase.

This year, France banned the application of titanium dioxide nanoparticles as a food additive (hereafter, E171) based on the insufficient oral toxicity data. Here, we investigated the subchronic toxic responses of E171 (0, 10, 100, and 1,000 mg/kg) and tried to elucidate the possible toxic mechanism using AGS cells, a human stomach epithelial cell line. There were no dose-related changes in the Organisation for Economic Cooperation and Development test guideline-related endpoints. Meanwhile, E171 deeply penetrated cells lining the stomach tissues of rats, and the IgM and granulocyte-macrophage colony-stimulating factor levels were significantly lower in the blood from rats exposed to E171 compared with the control. The colonic antioxidant protein level decreased with increasing Ti accumulation. Additionally, after 24-h exposure, E171 located in the perinuclear region of AGS cells and affected expression of endoplasmic reticulum stress-related proteins. However, cell death was not observed up to the used maximum concentration. A gene profile analysis also showed that immune response-related microRNAs were most strongly affected by E171 exposure. Collectively, we concluded that the NOAEL of E171 for 90 days repeated oral administration is between 100 and 1,000 mg/kg for both male and female rats. Additionally, further study is needed to clarify the possible carcinogenesis following the chronic accumulation in the colon.

The advancement of analytical methods for nanoparticle measurements is critical both for the growing industrial exploitation of engineered nanoparticles and for developing robust strategies to understand and control the concentration of nanomaterials in humans and the environment. For high value nanoparticles, the measurement of nanoparticle number concentration in a liquid directly impacts the ability to assess the scale and reproducibility of the production process, it allows optimisation of efficiency and supports regulatory compliance. This measurement is also useful to monitor and control the intentional or accidental release of engineered nanoparticles into the environment at the production plant, as well as by end-users., peer-reviewed

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the nanoscale, i.e. 1–100 nm), dynamic nature, and diverse properties. At the same time, reliable and repeatable characterization is paramount to ensure safety and quality in the manufacturing of NMbearing products. There are several methods available to monitor and achieve reliable measurement of nanoscale-related properties, one example of which is UltravioletVisible Spectroscopy (UV-Vis). This is a well-established, simple, and inexpensive technique that provides non-invasive and fast real-time screening evaluation of NM size, concentration, and aggregation state. Such features make UV-Vis an ideal methodology to assess the proficiency testing schemes (PTS) of a validated standard operating procedure (SOP) intended to evaluate the performance and reproducibility of a characterization method. In this paper, the PTS of six partner laboratories from the H2020 project ACEnano were assessed through an interlaboratory comparison (ILC). Standard gold (Au) colloid suspensions of different sizes (ranging 5–100 nm) were characterized by UV-Vis at the different institutions to develop an implementable and robust protocol for NM size characterization., peer-reviewed

Reliable quantification of nanoparticle uptake in mammalian cells is essential to study the effects of nanoparticles in the fields of medicine and environmental science. Most conventional quantification methods, such as electron microscopy or confocal imaging, are laborious and semi-quantitative and therefore not readily applicable to routine analyses. Here, we developed assays to quantify fluorescently labelled nanoparticle uptake in mammalian cells using a flow cytometer. The first approach was to measure the percentage of nanoparticle-containing cells based on a cutoff fluorescence intensity as set from a histogram of control cells, which is a quick and easy way to relatively compare nanoparticle uptake in the same set of experiments. The second approach was to measure the calibrated fluorescence intensity of the nanoparticle-treated cells in molecules of equivalent soluble fluorophore (MESF) values using calibration beads, which allows for comparisons between different sets of experiments. We successfully applied the developed assays to more readily measure fluorescence-labelled silica nanoparticle uptake in A549 lung carcinoma cells in a quantitative rather than semi-quantitative manner. We further tested the assays with nine different types of mammalian cells and investigated the correlation between cell type/size and nanoparticle uptake.

Precise measurement of the size and size distribution of colloidal nanoparticles are an important component of evaluating nanoparticle safety. However, size measurements become increasingly difficult to perform as particle size decreases. The sizes and size distributions of nanoparticles can be determined using a variety of techniques. In this study, we used an electrospray scanning mobility particle sizer (ES-SMPS) to investigate the effect of nanoparticle concentration on the size and size distribution measurements of 20 nm and 50 nm silica nanoparticles. To calculate the size of the water droplets in the electrospray, the droplet size distribution in sucrose solutions was determined at various sucrose concentrations. The average water droplet diameter was ~ 697.5 nm, regardless of sucrose concentration. Based on these results, we estimated the appropriate nanoparticle concentration ranges for the ES-SMPS size determination of 20-nm and 50-nm silica nanoparticles. Reliable measurements of the 20- and 50-nm silica nanoparticles could be performed at concentrations of

Nanomaterials are used in a variety of fields and toxicity assessment is paramount for their development and application. Although most toxicity assessments have been performed in 2D (2-Dimensional) cell culture, the inability to adequately replicate thein vivoenvironment and toxicity is a limitation. To overcome the limitation, a 3D (3-Dimensional) cell culture method has been developed to make an environment closer to anin vivosystem. In this study, 20 nm SiO2nanoparticles were dispersed in serum-containing (SC) and serum-free (SF) media to compare 2D cell culture and 3D cell culture toxicity. The cells were subjected to a 3D cell culture method in which HepG2, a human-derived liver cancer cell line, was mixed on a scaffold. We found that nanoparticles induced toxicity in 2D cell culture, but toxicity was not observed in 3D cell culture similar toin vivoenvironment. However, differences in toxicity were observed between the three types of scaffolds in the absence of serum as the number of cells decreased.

Carbon nanotubes have recently been rated as an effective biomaterial owing to their functionalization ability. However, the safety of multi-walled carbon nanotubes (MWCNTs) has yet to be clearly understood. To investigate how cells differentially react to minor geometric differences, we prepared well-dispersed and stable long and short MWCNTs showing an approximately 100-nm length difference in an in vitro system. Through an optimal combination of bovine serum albumin (BSA) and fetal bovine serum (FBS) biosurfactants and ultrasonication, we first confirmed that the MWCNTs were maintained without aggregation throughout the experiments. Internalized MWCNTs in human coronary artery smooth muscle cells were then quantified in a label-free manner using coherent anti-Stokes Raman scattering, followed by an analysis of their localization via two-photon excitation fluorescence. Intracellular MWCNTs were found to primarily localize in mitochondria with abnormal morphologies. Mitochondrial dysfunction, which was found to result from early stages of oxidative stress that consequently lead to cell death, was then proved via decreasing mitochondrial membrane potentials, with short MWCNTs showing significantly greater cytotoxicity than long MWCNTs. Our results suggest that even small length differences of MWCNTs may lead to differential responses in cells.

Background The International Agency for Research on Cancer classified as a Group B carcinogen inhaled titanium dioxide (TiO 2 ) nanoparticles, and France banned the application of TiO 2 nanoparticles as a food additive based on the insufficient oral toxicity data. However, there still remains controversial due to the insufficient evidence for its safety. Here, we explored the toxicity of food-grade TiO 2 nanoparticles (hereafter, E171) in vivo and in vitro . Methods We investigated the subchronic toxic responses of E171 (0, 10, 100, and 1,000 mg/kg) under the Good Laboratory Practice (GLP) system and tried to elucidate the possible toxic mechanism using AGS cells, a human stomach epithelial cell line. Results There were no dose-related changes in the Organization for Economic Co-operation and Development test guideline-related endpoints. Meanwhile, E171 deeply penetrated cells lining the stomach tissues of rats administered the maximum dose, and blood IgM (male and female) and GM-CSF (female) levels were significantly lower in the E171-treated rats than in the control rats. Colonic SOD-1 (male and female) and SOD-2 (female) protein levels decreased with increasing Ti accumulation. When exposed to AGS cells for 24 h, E171 (40 μg/mL) located in the perinuclear region. The E171 treatment affected expression of ER stress-related proteins but did not induce cell death up to the used maximum concentration (40 μg/mL). A gene profile analysis also showed that immune response-related microRNAs were most strongly affected by E171 exposure. Conclusion Considering the potential toxicity observed in vivo and in vitro test, we concluded that the NOAEL of E171 for 90-days repeated oral administrations is less than 1,000 mg/kg for both male and female rats. Additionally, E171 may attenuate host's defense function against foreign bodies by decreasing antioxidant capacity, thus we propose that chronic toxicity studies on E171 are required to warrant the safety for use in the food industry.

The size of nanoparticles is considered to influence their toxicity, as smaller-sized nanoparticles should more easily penetrate the cell and exert toxic effects. However, conflicting results and unstandardized methodology have resulted in controversy of these size-dependent effects. Here, we introduce a unique approach to study such size-dependent effects of nanoparticles and present evidence that reliably supports this general assumption along with elucidation of the underlying cytotoxic mechanism.We prepared and physically characterized size-controlled (20-50 nm) monodispersed silica nanoparticles (SNPs) in aqueous suspensions. Then, a variety of biochemical assessments are used for evaluating the cytotoxic mechanisms.SNP treatment in three cell lines decreased cell viability and migration ability, while ROS production increased in dose- and size-dependent manners, with SNPs30 nm showing the greatest effects. 30- and 40-nm SNPs were observed similar to these biological activities of 20- and 50-nm, respectively. Under the conventionally used serum-free conditions, both 20-nm and 50-nm SNPs at the ICOur results highlight the need to revise safety guidelines to account for this demonstrated size-dependent cytotoxicity under serum-free conditions, which may be similar to the microenvironment after tissue penetration.

In this work, an extracellular matrix permeability/efficacy assay tip (E-PAT), which can be used in a conventional pipette to simultaneously test drug permeability and efficacy, was developed by growing cells in the extracellular matrix (ECM) layer in a hole at the outlet (bottom) of E-PAT. In the existing permeability/efficacy assay technique conducted using the transwell assays, the Cell/ECM layers were formed on permeable polymer membranes; however, the production of small membranes to realize transwell miniaturization is expensive and difficult. To resolve this issue, in the proposed approach using E-PAT, small Cell/ECM layers were formed in the hole at the outlet (bottom) of the tip without membranes. A pipette was connected to the E-PAT to easily aspirate the Cell/ECM mixture into the hole of the tip, allowing the formation of miniature Cell/ECM layers at the bottom of the tip. Subsequently, the E-PAT was detached from the pipette and dipped into a well filled with media. The cells in the ECM grew three-dimensionally and made Cell/ECM layer in the E-PAT. The permeability/efficacy assays were performed using the Cell/ECM layer in E-PAT. The compounds or nanoparticles supplied through the inlet (hole) at the top of the E-PAT penetrated the Cell/ECM layers and were diluted by the media in the well. By measuring the concentration of the compounds or nanoparticles in the well and the cell viability in the Cell/ECM layer, the permeability and efficacy/cytotoxicity of the compounds in the proposed E-PAT could be concurrently estimated with a high throughput. In particular, E-PAT measured the IC50 (half maximal inhibitory concentration) for 3.383 μM doxorubicin (DOX) and indicated that 73 % 4 μM DOX penetrated the Cell/ECM layers, inducing the death of 57.2 % HepG2 cells. Moreover, the device demonstrated that FITC-SiO2 nanoparticles with a diameter of 25 nm permeated higher into the ECM and killed more HepG2 cells under the serum (fetal bovine serum, FBS) free media conditions than under the serum containing media conditions. The E-PAT could thus measure the compound efficacy as well as the compound permeability. The proposed tip can be a valuable tool to determine whether the efficacy/cytotoxicity of a compound is because of its low permeability.

Background Silica nanoparticles (SiNPs) are widely used for biosensing and diagnostics, and for the targeted delivery of therapeutic agents. Safety concerns about the biomedical and clinical applications of SiNPs have been raised, necessitating analysis of the effects of their intrinsic properties, such as sizes, shapes, and surface physicochemical characteristics, on human health to minimize risk in biomedical applications. In particular, SiNP size-associated toxicological effects, and the underlying molecular mechanisms in the vascular endothelium remain unclear. This study aimed to elucidate the detailed mechanisms underlying the cellular response to exposure to trace amounts of SiNPs and to determine applicable size criteria for biomedical application. Methods To clarify whether these SiNP-mediated cytotoxicity due to induction of apoptosis or necrosis, human ECs were treated with SiNPs of four different non-overlapping sizes under low serum-containing condition, stained with annexin V and propidium iodide (PI), and subjected to flow cytometric analysis (FACS). Two types of cell death mechanisms were assessed in terms of production of reactive oxygen species (ROS), endoplasmic reticulum (ER) stress induction, and autophagy activity. Results Spherical SiNPs had a diameter of 21.8 nm; this was further increased to 31.4, 42.9, and 56.7 nm. Hence, we investigated these effects in human endothelial cells (ECs) treated with these nanoparticles under overlap- or agglomerate-free conditions. The 20-nm SiNPs, but not SiNPs of other sizes, significantly induced apoptosis and necrosis. Surprisingly, the two types of cell death occurred independently and through different mechanisms. Apoptotic cell death resulted from ROS-mediated ER stress. Furthermore, autophagy-mediated necrotic cell death was induced through the PI3K/AKT/eNOS signaling axis. Together, the present results indicate that SiNPs within a diameter of

In this study, we report the first example of a bismuth film electrode plated in situ using activated graphene (AG). The AG was prepared through of process of chemical activation of graphene oxide (GO) with KOH to create pores. The composite electrode was enhanced as an electrochemical sensing platform to determine trace Zn2+, Cd2+, and Pb2+ using differential pulse anodic stripping voltammetry. The surface morphology of the AG-NA/Bi composite film modified electrode was investigated by scanning electron microscopy and transmission electron microscopy. The electrochemical properties of the AG-NA/Bi composite electrode were evaluated to examine the effects of experimental variables, such as deposition potential, deposition time, bismuth concentration, and stirring speed during pre-concentration, on the determination of trace metals in 0.1 M acetate buffer solution (pH 4.5). Linear calibration curves ranged from 5 to 100 μg L−1 for trace heavy metal ions. The detection limits were 0.57 μg L−1 for Zn2+, 0.07 μg L−1 for Cd2+, and 0.05 μg L−1 for Pb2+ (S/N = 3). The limits of detection achieved are much lower than the guideline for drinking water quality provided by the World Health Organization (WHO). The AG-NA/Bi composite film electrodes were successfully applied to analysis trace metals in real samples.

Rattle-structured nanomaterials composed of a gold nanorod in a mesoporous silica nanocapsule (AuNR@mSiO(2)) were prepared by a novel solution-based consecutive process. The drug-loading properties of the nanomaterial and regrowth control of the core nanoparticles were also studied.

Nanoparticles are of great interest due to their wide variety of biomedical and bioengineering applications. However, they affect cellular differentiation and/or intracellular signaling when applied and exposed to target organisms or cells. The brown adipocyte is a cell type important in energy homeostasis and thus closely related to obesity. In this study, we assessed the effects of silica nanoparticles (SNPs) on brown adipocyte differentiation. The results clearly showed that brown adipocyte differentiation was significantly repressed by exposure to SNPs. The brown adipocyte-specific genes as well as mitochondrial content were also markedly reduced. Additionally, SNPs led to suppressed p38 phosphorylation during brown adipocyte differentiation. These effects depend on the size of SNPs. Taken together, these results lead us to suggest that SNP has anti-brown adipogenic effect in a size-dependent manner via regulation of p38 phosphorylation.

We report a facile strategy of graphene/gold nanocomposite based electrochemical biosensor that exhibit good sensitivity and high reproducibility. The graphene/gold nanocomposites were prepared by using sodium hydroxide acting as an accelerator for the reduction of gold ions. The graphene oxide was reduced by chemical and thermal methods after deposition of gold nanoparticles. The composition of the resulting materials was characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, and thermogravimetric analysis (TGA). Also, transmission electron microscope (TEM) was employed to demonstrate the successful deposition of gold nanoparticles on the surface of graphene sheets. The electrochemical behavior of dopamine (DA) on the surface of the graphene/gold nanocomposite modified electrode was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). In comparison to the reduced graphene oxide (rGO) modified electrode, the graphene/gold nanocomposite modified electrode shows good performance for the selective determination of DA. The nanocomposite modified electrode showed a low detection limit (0.095 μM), an enhanced electrochemical oxidation current for DA, and a good separation of potential of DA and ascorbic acid (AA).

Nanotechnology is regarded as the enabling technology of the 21st century. However, only a relatively small number of nano-enabled medical and healthcare products finally made their way to the market. There are several reasons why such innovative approaches fail in translation, with one key factor being the uncertainty surrounding their safety assessment. Although well described, interference reactions of engineered nanomaterials (ENM) with classical cytotoxicity assays remain a major source of uncertainty., Flow cytometry is a powerful, widely used, in vitro technique. Its readout is based on the detection of refracted laser light and fluorescence signals. It is therefore susceptible to ENM interference. Here we investigated possible interferences of ENM in the Annexin V/propidium iodide (PI) assay, which quantifies apoptotic and necrotic cell populations by flow cytometry., Two case studies were conducted using either silica or gold nanoparticles differing in size, specific surface area and surface chemistry. Both ENM types were found to cause distinct interference reactions at realistic concentrations. Silica particles induced false-positive signals; however only in the absence of a protein corona and in conjunction with a particular fluorophore combination (FITC/PI). In contrast, gold particles led to complex quenching effects which were only marginally influenced by the presence of proteins and occurred for both fluorophore combinations analyzed. We present a versatile spike-in approach which is applicable to all ENM and cell types. It further allows for the identification of a broad range of different interference phenomena, thereby increasing the reliability and quality of flow cytometry and ENM hazard assessment.