Molecular Mechanisms in Metal Oxide Nanoparticle-Tryptophan Interactions.

One of the crucial metabolic processes for both plant and animal kingdoms is the oxidation of the amino acid tryptophan (TRP) that regulates plant growth and controls hunger and sleeping patterns in animals. Here, we report revolutionary insights into how this process can be crucially affected by interactions with metal oxide nanoparticles (NPs), creating a toolbox for a plethora of important biomedical and agricultural applications. Molecular mechanisms in TRP-NP interactions were revealed by NMR and optical spectroscopy for ceria and titania and by X-ray single-crystal study and a computational study of model TRP-polyoxometalate complexes, which permitted the visualization of the oxidation mechanism at an atomic level. Nanozyme activity, involving concerted proton and electron transfer to the NP surface for oxides with a high oxidative potential, like CeO2 or WO3, converted TRP in the first step into a tricyclic organic acid belonging to the family of natural plant hormones, auxins. TiO2, a much poorer oxidant, was strongly binding TRP without concurrent oxidation in the dark but oxidized it nonspecifically via the release of reactive oxygen species (ROS) in daylight.

Microfluidic Cell Microarray Platform for High Throughput Analysis of Particle-Cell Interactions.

With advances in nanotechnology, particles with various size, shape, surface chemistry, and composition can be easily produced. Nano- and microparticles have been extensively explored in many industrial and clinical applications. Ensuring that the particles themselves are not possessing toxic effects to the biological system is of paramount importance. This paper describes a proof of concept method, in which a microfluidic system is used in conjunction with a cell microarray technique aiming to streamline the analysis of particle-cell interaction in a high throughput manner. Polymeric microparticles, with different particle surface functionalities, were first used to investigate the efficiency of particle-cell adhesion under dynamic flow. Silver nanoparticles (AgNPs, 10 nm in diameter) perfused at different concentrations (0 to 20 µg/mL) in parallel streams over the cell microarray exhibited a higher toxicity compared to the static culture in the 96-well-plate format. This developed microfluidic system can be easily scaled up to accommodate a larger number of microchannels for high throughput analysis of the potential toxicity of a wide range of particles in a single experiment.

Single Cell Level Quantification of Nanoparticle-Cell Interactions Using Mass Cytometry.

Quantification of cell-associated nanoparticles (NPs) is a paramount question in both nanomedicine and nanotoxicology. Inductively coupled plasma mass spectrometry is a well-established method to resolve cell-associated (metal) NPs in bulk cell populations, however, such analysis at single cell level remains a challenge. Here we used mass cytometry, a technique that combines single cell analysis and time-of-flight mass spectrometry, to quantitatively analyze extra- and intracellular silver (Ag) in individual Ag NP exposed human T-lymphocytes. The results revealed significant population heterogeneity: for example, in lymphocytes exposed to 3 µg of 30 nm branched polyethylene imine coated Ag NPs/mL the extracellularly bound Ag varied from 79 to 560 fg and cellular uptake from 17 to 121 fg. Similar amplitude of heterogeneity was observed in cells exposed to various doses of Ag NPs with other sizes and surface coatings, demonstrating the importance of single cell analysis when studying NP-cell interactions. Although mass cytometry has some shortcomings such as inability to analyze potential transformation or dissolution of NPs in cells, we consider this method as the most promising for quantitative assessment of cell-NP interaction at single cell level.

Dual-Action Cancer Therapy with Targeted Porous Silicon Nanovectors.

There is a pressing need to develop more effective therapeutics to fight cancer. An idyllic chemotherapeutic is expected to overcome drug resistance of tumors and minimize harmful side effects to healthy tissues. Antibody-functionalized porous silicon nanoparticles loaded with a combination of chemotherapy drug and gold nanoclusters (AuNCs) are developed. These nanocarriers are observed to selectively deliver both payloads, the chemotherapy drug and AuNCs, to human B cells. The accumulation of AuNCs to target cells and subsequent exposure to an external electromagnetic field in the microwave region render them more susceptible to the codelivered drug. This approach represents a targeted two-stage delivery nanocarrier that benefits from a dual therapeutic action that results in enhanced cytotoxicity.

Pan-European inter-laboratory studies on a panel of in vitro cytotoxicity and pro-inflammation assays for nanoparticles.

The rapid development of nanotechnologies and increased production and use of nanomaterials raise concerns about their potential toxic effects for human health and environment. To evaluate the biological effects of nanomaterials, a set of reliable and reproducible methods and development of standard operating procedures (SOPs) is required. In the framework of the European FP7 NanoValid project, three different cell viability assays (MTS, ATP content, and caspase-3/7 activity) with different readouts (absorbance, luminescence and fluorescence) and two immune assays (ELISA of pro-inflammatory cytokines IL1-ß and TNF-α) were evaluated by inter-laboratory comparison. The aim was to determine the suitability and reliability of these assays for nanosafety assessment. Studies on silver and copper oxide nanoparticles (NPs) were performed, and SOPs for particle handling, cell culture, and in vitro assays were established or adapted. These SOPs give precise descriptions of assay procedures, cell culture/seeding conditions, NPs/positive control preparation and dilutions, experimental well plate preparation, and evaluation of NPs interference. The following conclusions can be highlighted from the pan-European inter-laboratory studies: Testing of NPs interference with the toxicity assays should always be conducted. Interference tests should be designed as close as possible to the cell exposure conditions. ATP and MTS assays gave consistent toxicity results with low inter-laboratory variability using Ag and CuO NPs and different cell lines and therefore, could be recommended for further validation and standardization. High inter-laboratory variability was observed for Caspase 3/7 assay and ELISA for IL1-ß and TNF-α measurements.

DNA melting and genotoxicity induced by silver nanoparticles and graphene.

We have revealed a connection between DNA-nanoparticle (NP) binding and in vitro DNA damage induced by citrate- and branched polyethylenimine-coated silver nanoparticles (c-AgNPs and b-AgNPs) as well as graphene oxide (GO) nanosheets. All three types of nanostructures triggered an early onset of DNA melting, where the extent of the melting point shift depends upon both the type and concentration of the NPs. Specifically, at a DNA/NP weight ratio of 1.1/1, the melting temperature of lambda DNA dropped from 94 °C down to 76 °C, 60 °C, and room temperature for GO, c-AgNPs and b-AgNPs, respectively. Consistently, dynamic light scattering revealed that the largest changes in DNA hydrodynamic size were also associated with the binding of b-AgNPs. Upon introduction to cells, b-AgNPs also exhibited the highest cytotoxicity, at the half-maximal inhibitory (IC50) concentrations of 3.2, 2.9, and 5.2 mg/L for B and T-lymphocyte cell lines and primary lymphocytes, compared to the values of 13.4, 12.2, and 12.5 mg/L for c-AgNPs and 331, 251, and 120 mg/L for GO nanosheets, respectively. At cytotoxic concentrations, all NPs elicited elevated genotoxicities via the increased number of micronuclei in the lymphocyte cells. However, b-AgNPs also induced micronuclei at subtoxic concentrations starting from 0.1 mg/L, likely due to their stronger cellular adhesion and internalization, as well as their subsequent interference with normal DNA synthesis or chromosome segregation during the cell cycle. This study facilitates our understanding of the effects of NP chemical composition, surface charge, and morphology on DNA stability and genotoxicity, with implications ranging from nanotoxicology to nanobiotechnology and nanomedicine.

Toxicity of metal oxide nanoparticles in Escherichia coli correlates with conduction band and hydration energies.

Metal oxide nanoparticles (MOx NPs) are used for a host of applications, such as electronics, cosmetics, construction, and medicine, and as a result, the safety of these materials to humans and the environment is of considerable interest. A prior study of 24 MOx NPs in mammalian cells revealed that some of these materials show hazard potential. Here, we report the growth inhibitory effects of the same series of MOx NPs in the bacterium Escherichia coli and show that toxicity trends observed in E. coli parallel those seen previously in mammalian cells. Of the 24 materials studied, only ZnO, CuO, CoO, Mn2O3, Co3O4, Ni2O3, and Cr2O3 were found to exert significant growth inhibitory effects; these effects were found to relate to membrane damage and oxidative stress responses in minimal trophic media. A correlation of the toxicological data with physicochemical parameters of MOx NPs revealed that the probability of a MOx NP being toxic increases as the hydration enthalpy becomes less negative and as the conduction band energy approaches those of biological molecules. These observations are consistent with prior results observed in mammalian cells, revealing that mechanisms of toxicity of MOx NPs are consistent across two very different taxa. These results suggest that studying nanotoxicity in E. coli may help to predict toxicity patterns in higher organisms.

Mapping the dawn of nanoecotoxicological research.

Some researchers consider nanotechnology the next industrial revolution, and consumer products and a variety of industries increasingly use synthetic nanoparticles. In this Account, we review the initial accomplishments of nanoecotoxicology, a discipline that is just a decade old. This new subdiscipline of ecotoxicology faces two important and challenging problems: the analysis of the safety of nanotechnologies in the natural environment and the promotion of sustainable development while mitigating the potential pitfalls of innovative nanotechnologies. In this Account, we provide a snapshot of the publicly available scientific information regarding the ecotoxicity of engineered nanoparticles. We pay special attention to information relevant to aquatic freshwater species commonly used for risk assessment and regulation. Just as the development of ecotoxicology has lagged behind that of toxicology, nanoecotoxicological research has developed much more slowly than nanotoxicology. Although the first nanotoxicolology papers were published in 1990s, the first nanoecotoxicology papers came out in 2006. A meta-analysis of scientific publications covering different environmental impacts of nanomaterials showed that the importance of research into the environmental impact of nanotechnology has gradually increased since 2005. Now the most frequently cited papers in the environmental disciplines are often those that focus on synthetic nanoparticles. The first nanoecotoxicology studies focused on adverse effects of nanoparticles on fish, algae and daphnids, which are ecotoxicological model organisms for classification and labeling of chemicals (these model organisms are also used in the EU chemical safety policy adopted in 2007: Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH)). Based on our experience, we propose a multitrophic battery of nanoecotoxicological testing that includes particle-feeding and a priori particle-"proof" prokaryotic and eukaryotic organisms at different food-chain levels. Using this battery of selected test organisms, we demonstrated that TiO2 nanoparticles were toxic to algae and that ZnO and CuO nanoparticles were toxic to several aquatic invertebrate test species. Thus, one single biotest cannot predict the ecotoxicological effects of chemicals/nanoparticles, and researchers should use several tests instead. Moreover, produced nanoparticles usually vary in features such as size, shape, and coating; therefore, a single nanoparticle species may actually include many entities with different physicochemical properties. An ecotoxicity analysis of all these variants would require a huge number of laboratory tests. To address these issues, high throughput bioassays and computational (QSAR) models that serve as powerful alternatives to conventional (eco)toxicity testing must be implemented to handle both the diversity of nanomaterials and the complexity of ecosystems.

Antibacterial activity of solid surfaces is critically dependent on relative humidity, inoculum volume, and organic soiling.

Antimicrobial surface materials potentially prevent pathogen transfer from contaminated surfaces. Efficacy of such surfaces is assessed by standard methods using wet exposure conditions known to overestimate antimicrobial activity compared to dry exposure. Some dry test formats have been proposed but semi-dry exposure scenarios e.g. oral spray or water droplets exposed to ambient environment, are less studied. We aimed to determine the impact of environmental test conditions on antibacterial activity against the model species Escherichia coli and Staphylococcus aureus. Surfaces based on copper, silver, and quaternary ammonium with known or claimed antimicrobial properties were tested in conditions mimicking microdroplet spray or larger water droplets exposed to variable relative air humidity in the presence or absence of organic soiling. All the environmental parameters critically affected antibacterial activity of the tested surfaces from no effect in high-organic dry conditions to higher effect in low-organic humid conditions but not reaching the effect size demonstrated in the ISO 22169 wet format. Copper was the most efficient antibacterial surface followed by silver and quaternary ammonium based coating. Antimicrobial testing of surfaces using small droplet contamination in application-relevant conditions could therefore be considered as one of the worst-case exposure scenarios relevant to dry use surfaces.

Synthesis and Antibacterial Properties of Novel Quaternary Ammonium Lignins.

The ongoing demand for effective antimicrobial materials persists, and lignin emerges as a promising natural antibacterial material with renewable properties. The adaptability of lignin to various chemical modifications offers avenues to enhance its antimicrobial activity. Here, we employed chloromethylation and subsequent functionalization with variable tertiary N-alkyl dimethyl amines to produce C6-C18 quaternary ammonium lignins (QALs) from hardwood (aspen), softwood (pine), and grass (barley straw). Successful synthesis of QALs was confirmed through NMR and FTIR analysis results along with an increase in the surface ζ-potential. Antibacterial activity of QALs against clinical strains of Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus was assessed using minimal bactericidal concentration (MBC) assay and agar growth inhibition zone (ZOI) test. The antibacterial activity of QALs was found to be higher than that of the unmodified lignins. QALs with longer alkyl chains demonstrated an MBC of 0.012 mg/L against K. pneumoniae already after 1 h of exposure with similar effect size reached after 24 h for S. aureus. For all the lignins, an increase in alkyl chain length resulted in an increase in their bactericidal activity. MBC values of C14-C18 QALs were consistently lower than the MBC values of QALs with shorter alkyl chains. Besides the alkyl chain length, MBC values of barley and pine QALs were negatively correlated with the surface ζ-potential. While alkyl chain length was one of the key properties affecting the MBC values in a liquid-based test, the agar-based ZOI test demonstrated an antibacterial optimum of QALs at C12-C14, likely due to limited diffusion of QALs with longer alkyl chains in a semisolid medium.