Marco D. Torelli

Surface chemistry, charge and ligand type impact the toxicity of gold nanoparticles to Daphnia magna

Nanoparticles (NPs) are the basis of a range of emerging technologies used for a variety of industrial, biomedical, and environmental applications. As manufactured NP production increases, so too does the concern about their release into the environment and potentially harmful effects. Creating nanomaterials that have minimal negative environmental impact will heavily influence the sustainability of nanomaterials as a technology. In order to create such NPs, the mechanisms that govern NP toxicity need to be better elucidated. One aspect of NP structure that may influence toxicity is the identity and charge of ligand molecules used to functionalize the NP surface. These surface chemistries have the potential to increase or decrease negative biological impacts, yet their impacts are poorly understood. In this study, the toxicity of three types of functionalized ~4–5 nm gold NPs (AuNPs), polyallylamine hydrochloride (PAH–AuNPs), citrate (Cit–AuNPs) and mercaptopropionic acid (MPA–AuNPs) as well as cetyltrimethylammonium bromide-functionalized gold nanorods (CTAB–AuNRs) were evaluated in the toxicological model species, Daphnia magna. In order to get the most detailed information on NP toxicity in D. magna, both acute and chronic toxicity assays were performed. Acute exposure toxicity assays show that overall the negatively-charged AuNPs tested are orders of magnitude less toxic than the positively-charged AuNPs. However, chronic exposure assays show that both positively and negatively-charged particles impact reproduction but potentially through different mechanisms and dependent upon functional group. In addition, while select ligands used in NP functionalization (such as CTAB) that are toxic on their own can contribute to observed NP toxicity, our acute toxicity assays indicate that minimally toxic ligands (such as PAH) can also cause significant toxicity when conjugated to NPs. This research demonstrates that surface chemistry plays a pivotal role in NP toxicity and that surface chemistry has the potential to affect the sustainability of these materials.

A Citric Acid-Derived Ligand for Modular Functionalization of Metal Oxide Surfaces via “Click” Chemistry

Citric acid is a widely used surface-modifying ligand for growth and processing of a variety of nanoparticles; however, the inability to easily prepare derivatives of this molecule has restricted the development of versatile chemistries for nanoparticle surface functionalization. Here, we report the design and synthesis of a citric acid derivative bearing an alkyne group and demonstrate that this molecule provides the ability to achieve stable, multidentate carboxylate binding to metal oxide nanoparticles, while also enabling subsequent multistep chemistry via the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The broad utility of this strategy for the modular functionalization of metal oxide surfaces was demonstrated by its application in the CuAAC modification of ZnO, Fe(2)O(3), TiO(2), and WO(3) nanoparticles.

Quantitative Determination of Ligand Densities on Nanomaterials by X-ray Photoelectron Spectroscopy

X-ray photoelectron spectroscopy (XPS) is a nearly universal method for quantitative characterization of both organic and inorganic layers on surfaces. When applied to nanoparticles, the analysis is complicated by the strong curvature of the surface and by the fact that the electron attenuation length can be comparable to the diameter of the nanoparticles, making it necessary to explicitly include the shape of the nanoparticle to achieve quantitative analysis. We describe a combined experimental and computational analysis of XPS data for molecular ligands on gold nanoparticles. The analysis includes scattering in both Au core and organic shells and is valid even for nanoparticles having diameters comparable to the electron attenuation length (EAL). To test this model, we show experimentally how varying particle diameter from 1.3 to 6.3 nm leads to a change in the measured AC/AAu peak area ratio, changing by a factor of 15. By analyzing the data in a simple computational model, we demonstrate that ligand densities can be obtained, and, moreover, that the actual ligand densities for these nanoparticles are a constant value of 3.9 ± 0.2 molecules nm(-2). This model can be easily extended to a wide range of core-shell nanoparticles, providing a simple pathway to extend XPS quantitative analysis to a broader range of nanomaterials.

Effects of charge and surface ligand properties of nanoparticles on oxidative stress and gene expression within the gut of Daphnia magna

Concern has been raised regarding the current and future release of engineered nanomaterials into aquatic environments from industry and other sources. However, not all nanomaterials may cause an environmental impact and identifying which nanomaterials may be of greatest concern has been difficult. It is thought that the surface groups of a functionalized nanoparticles (NPs) may play a significant role in determining their interactions with aquatic organisms, but the way in which surface properties of NPs impact their toxicity in whole organisms has been minimally explored. A major point of interaction of NPs with aquatic organisms is in the gastrointestinal tract as they ingest particulates from the water column or from the sediment. The main goal of this study was to use model gold NP (AuNPs) to evaluate the potential effects of the different surfaces groups on NPs on the gut of an aquatic model organism, Daphnia magna. In this study, we exposed daphnids to a range of AuNPs concentrations and assessed the impact of AuNP exposure in the daphnid gut by measuring reactive oxygen species (ROS) production and expression of genes associated with oxidative stress and general cellular stress: glutathione S-transferase (gst), catalase (cat), heat shock protein 70 (hsp70), and metallothionein1 (mt1). We found ROS formation and gene expression were impacted by both charge and the specific surface ligand used. We detected some degree of ROS production in all NP exposures, but positively charged AuNPs induced a greater ROS response. Similarly, we observed that, compared to controls, both positively charged AuNPs and only one negatively AuNP impacted expression of genes associated with cellular stress. Finally, ligand-AuNP exposures showed a different toxicity and gene expression profile than the ligand alone, indicating a NP specific effect.

Quantification of Free Polyelectrolytes Present in Colloidal Suspension, Revealing a Source of Toxic Responses for Polyelectrolyte-Wrapped Gold Nanoparticles

Polyelectrolyte (PE) wrapping of colloidal nanoparticles (NPs) is a standard method to control NP surface chemistry and charge. Because excess polyelectrolytes are usually employed in the surface modification process, it is critical to evaluate different purification strategies to obtain a clean final product and thus avoid ambiguities in the source of effects on biological systems. In this work, 4 nm diameter gold nanoparticles (AuNPs) were wrapped with 15 kDa poly(allylamine hydrochloride) (PAH), and three purification strategies were applied: (a) diafiltration or either (b) one round or (c) two rounds of centrifugation. The bacterial toxicity of each of these three PAH-AuNP samples was evaluated for the bacterium Shewanella oneidensis MR-1 and is quantitatively correlated with the amount of unbound PAH molecules in the AuNP suspensions, as judged by X-ray photoelectron spectroscopy, nuclear magnetic resonance experiments and quantification using fluorescent assay. Dialysis experiments show that, for a 15 kDa polyelectrolyte, a 50 kDa dialysis membrane is not sufficient to remove all PAH polymers. Together, these data showcase the importance of choosing a proper postsynthesis purification method for polyelectrolyte-wrapped NPs and reveal that apparent toxicity results may be due to unintended free wrapping agents such as polyelectrolytes.

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.

Commercial quantities of ultrasmall fluorescent nanodiamonds containing color centers

Optically active nanodiamond particles remain one of the most popular research topics due to the photoluminescent properties of crystallographic defects in the diamond lattice, referred to as color centers. A number of groups are currently undertaking efforts to commercialize this material. Recently, our group succeeded in large-scale production of fluorescent diamond particles containing nitrogen-vacancy (NV) color centers in hundred-gram per batch scales using irradiation with 2-3 MeV electrons. Production of ND-NV fractions with median sizes ranging between 10 nm and 100 nm was achieved. While 100 nm fluorescent nanodiamonds (FNDs) are ~10x brighter than a conventional dye (Atto 532), the brightness of FNDs drops with decreasing particle size. Because of this, significant efforts must be undertaken to elucidate the size/brightness compromise and identify relevant application niches for FND in bioimaging and biolabeling. In order for a new material to be considered for applications in the overcrowded optical reagent market, the reagent must be convenient to use by an end user from the biomedical community, be validated both in vitro and in vivo, and offer measurable and significant (rather than incremental) benefit to end users in specific applications. This paper reports on the characteristics of the ultrasmall (10-40nm) and larger fluorescent nanodiamonds as well as our efforts toward their adaptation for use in the biological science community.

Fluorescent single-digit detonation nanodiamond for biomedical applications

Detonation nanodiamonds (DNDs) have emerged as promising candidates for a variety of biomedical applications, thanks to different physicochemical and biological properties, such as small size and reactive surfaces. In this study, we propose carbon dot decorated single digit (4-5 nm diameter) primary particles of detonation nanodiamond as promising fluorescent probes. Due to their intrinsic fluorescence originating from tiny (1-2 atomic layer thickness) carbonaceous structures on their surfaces, they exhibit brightness suitable for in vitro imaging. Moreover, this material offers a unique, cost effective alternative to sub-10 nm nanodiamonds containing fluorescent nitrogen-vacancy color centers, which have not yet been produced at large scale. In this paper, carbon dot decorated nanodiamonds are characterized by several analytical techniques. In addition, the efficient cellular uptake and fluorescence of these particles are observed in vitro on MDA-MD-231 breast cancer cells by means of confocal imaging. Finally, the in vivo biocompatibility of carbon dot decorated nanodiamonds is demonstrated in zebrafish during the development. Our results indicate the potential of single-digit detonation nanodiamonds as biocompatible fluorescent probes. This unique material will find application in correlative light and electron microscopy, where small sized NDs can be attached to antibodies to act as a suitable dual marker for intracellular correlative microscopy of biomolecules.

Size dependent oxidative stress response of the gut of Daphnia magna to functionalized nanodiamond particles

ABSTRACT Nanodiamonds are a type of engineered nanomaterial with high surface area that is highly tunable and are being proposed for use as a material for medical imaging or drug delivery to composites. With their potential for widespread use they may potentially be released into the aquatic environment as are many chemicals used for these purposes. It is generally thought that nanodiamonds are innocuous, but toxicity may occur due to surface functionalization. This study investigated the potential oxidative stress and antioxidant response of enterocytes in a freshwater invertebrate, Daphnia magna, a common aquatic invertebrate for ecotoxicological studies, in response to two types of functionalized nanodiamonds (polyallylamine and oxidized). We also examined how the size of the nanomaterial may influence toxicity by testing two different sizes (5 nm and 15 nm) of nanodiamonds with the same functionalization. Adults of Daphnia magna were exposed to three concentrations of each of the nanodiamonds for 24 h. We found that both 5 and 15 nm polyallylamine nanodiamond and oxidized nanodiamond induced the production of reactive oxygen species in tissues. The smaller 5 nm nanodiamond induced a significant change in the expression of heat shock protein 70 and glutathione‐S‐transferase. This may suggest that daphnids mounted an antioxidant response to the oxidative effects of 5 nm nanodiamonds but not the comparative 15 nm nanodiamonds with either surface chemistry. Outcomes of this study reveal that functionalized nanodiamond may cause oxidative stress and may potentially initiate lipid peroxidation of enterocyte cell membranes in freshwater organisms, but the impact of the exposure depends on the particle size. HighlightsWe examined the effects of 2 sizes and 2 types of functionalized diamond nanoparticles.Our results suggest a correlation between size and ROS production.5 nm DNPs instigate expression of hsp70 and oxidized DNPs cause greater stress than PAH‐ND particles.