Lack of Detectable Direct Effects of Silver and Silver Nanoparticles on Mitochondria in Mouse Hepatocytes.

Silver nanoparticles (AgNPs) are well-proven antimicrobial nanomaterials, yet little is elucidated regarding the mechanism underlying cytotoxicity induced by these nanoparticles. Here, we tested the hypothesis that mitochondria are primary intracellular targets of two AgNPs and silver ions in mouse hepatocytes (AML12) cultured in glucose- and galactose-based media. AML12 cells were more sensitive to mitochondrial uncoupling when grown with galactose rather than glucose. However, 24 h treatments with 15 nm AgNPs and 6 nm GA-AgNPs (5 and 10 µg/mL) and AgNO3 (1 and 3 µg/mL), concentrations that resulted in either 10 or 30% cytotoxicity, failed to cause more toxicity to AML12 cells grown on galactose than glucose. Furthermore, colocalization analysis and subcellular Ag quantification did not show any enrichment of silver content in mitochondria in either medium. Finally, the effects of the same exposures on mitochondrial respiration were mild or undetectable, a result inconsistent with mitochondrial toxicity causing cell death. Our results suggest that neither ionic Ag nor the AgNPs that we tested specifically target mitochondria and are inconsistent with mitochondrial dysfunction being the primary cause of cell death after Ag exposure under these conditions.

Differential Reactivity of Copper- and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm.

Reliable predictions of the environmental fate and risk of engineered nanomaterials (ENMs) require a better understanding of ENM reactivity in complex, biologically active systems for chronic low-concentration exposure scenarios. Here, simulated freshwater wetland mesocosms were dosed with ENMs to assess how their reactivity and seasonal changes in environmental parameters influence ENM fate in aquatic systems. Copper-based ENMs (Kocide), known to dissolve in water, and gold nanoparticles (AuNPs), stable against dissolution in the absence of specific ligands, were added weekly to mesocosm waters for 9 months. Metal accumulation and speciation changes in the different environmental compartments were assessed over time. Copper from Kocide rapidly dissolved likely associating with organic matter in the water column, transported to terrestrial soils and deeper sediment where it became associated with organic or sulfide phases. In contrast, Au accumulated on/in the macrophytes where it oxidized and transferred over time to surficial sediment. A dynamic seasonal accumulation and metal redox cycling were found between the macrophyte and the surficial sediment for AuNPs. These results demonstrate the need for experimental quantification of how the biological and chemical complexity of the environment, combined with their seasonal variations, drive the fate of metastable ENMs.

Formulation and Validation of a Functional Assay-Driven Model of Nanoparticle Aquatic Transport.

Here, we present a model for the prediction of nanoparticle fate in aquatic environments, parametrized using functional assays that take into account conditions of the environmental media and nanoparticle properties. The model was used to explore scenarios for five nanomaterials in a freshwater wetland setting and compared with experimental results obtained in mesocosm studies. Material characteristics used in the model were size, density, dissolution rate constants, and surface attachment efficiencies. Model predictions and experimentally measured removal rate constants from the water column were strongly correlated, with Pearson correlation coefficient 0.993. Further, the model predicted removal rate constants quantitively very close to measured rates. Of particular importance for accurate predictions were two key processes beyond the usual heteroaggregation with suspended solids. These were homoaggregation of nanomaterials and nanomaterial attachment to aquatic plant surfaces. These results highlight the importance of including all relevant aggregation and deposition processes over short time scales for nanoparticle transport, while demonstrating the utility of functional assays for surface attachment as model inputs.

Tailoring the Core-Satellite Nanoassembly Architectures by Tuning Internanoparticle Electrostatic Interactions.

The use of plasmonic nanoplatforms has received increasing interest in a wide variety of fields ranging from theranostics to environmental sensing to plant biology. In particular, the development of plasmonic nanoparticles into ordered nanoclusters has been of special interest due to the new chemical functionalities and optical responses that they can introduce. However, achieving predetermined nanocluster architectures from bottom-up approaches in the colloidal solution state still remains a great challenge. Herein, we report a one-pot assembly approach that provides flexibility in precise control of core-satellite nanocluster architectures in the colloidal solution state. We found that the pH of the assembly medium plays a vital role in the hierarchy of the nanoclusters. The architecture along with the size of the satellite gold nanoparticles determines the optical responses of nanoclusters. Using electron microscopy and optical spectroscopy, we introduce a set of design rules for the synthesis of distinct architectures of silica-core gold satellites nanoclusters in the colloidal solution state. Our findings provide insight into advancing the colloidal solution state nanoclusters formation with predictable architectures and optical properties.

Engineered nanoparticles interact with nutrients to intensify eutrophication in a wetland ecosystem experiment.

Despite the rapid rise in diversity and quantities of engineered nanomaterials produced, the impacts of these emerging contaminants on the structure and function of ecosystems have received little attention from ecologists. Moreover, little is known about how manufactured nanomaterials may interact with nutrient pollution in altering ecosystem productivity, despite the recognition that eutrophication is the primary water quality issue in freshwater ecosystems worldwide. In this study, we asked two main questions: (1) To what extent do manufactured nanoparticles affect the biomass and productivity of primary producers in wetland ecosystems? (2) How are these impacts mediated by nutrient pollution? To address these questions, we examined the impacts of a citrate-coated gold nanoparticle (AuNPs) and of a commercial pesticide containing Cu(OH)2 nanoparticles (CuNPs) on aquatic primary producers under both ambient and enriched nutrient conditions. Wetland mesocosms were exposed repeatedly with low concentrations of nanoparticles and nutrients over the course of a 9-month experiment in an effort to replicate realistic field exposure scenarios. In the absence of nutrient enrichment, there were no persistent effects of AuNPs or CuNPs on primary producers or ecosystem productivity. However, when combined with nutrient enrichment, both NPs intensified eutrophication. When either of these NPs were added in combination with nutrients, algal blooms persisted for >50 d longer than in the nutrient-only treatment. In the AuNP treatment, this shift from clear waters to turbid waters led to large declines in both macrophyte growth and rates of ecosystem gross primary productivity (average reduction of 52% ± 6% and 92% ± 5%, respectively) during the summer. Our results suggest that nutrient status greatly influences the ecosystem-scale impact of two emerging contaminants and that synthetic chemicals may be playing an under-appreciated role in the global trends of increasing eutrophication. We provide evidence here that chronic exposure to Au and Cu(OH)2 nanoparticles at low concentrations can intensify eutrophication of wetlands and promote the occurrence of algal blooms.

Comparative Persistence of Engineered Nanoparticles in a Complex Aquatic Ecosystem.

During nanoparticle environmental exposure, presence in the water column is expected to dominate long distance transport as well as initial aquatic organism exposure. Much work has been done to understand potential ecological and toxicological effects of these particles. However, little has been done to date to understand the comparative persistence of engineered particles in realistic environmental systems. Presented here is a study of the water column lifetimes of 3 different classes of nanoparticles prepared with a combination of surface chemistries in wetland mesocosms. We find that, when introduced as a single pulse, all tested nanoparticles persist in the water column for periods ranging from 36 h to 10 days. Specifically, we found a range of nanoparticle residence times in the order Ag > TiO2 > SWCNT > CeO2. We further explored the hypothesis that heteroaggregation was the primary driving factor for nanoparticle removal from the water column in all but one case, and that values of surface affinity (α) measured in the laboratory appear to predict relative removal rates when heteroaggregation dominates. Though persistence in the water column was relatively short in all cases, differences in persistence may play a role in determining nanoparticle fate and impacts and were poorly predicted by currently prevailing benchmarks such as particle surface preparation.

Size-Based Differential Transport, Uptake, and Mass Distribution of Ceria (CeO2) Nanoparticles in Wetland Mesocosms.

Trace metals associated with nanoparticles are known to possess reactivities that are different from their larger-size counterparts. However, the relative importance of small relative to large particles for the overall distribution and biouptake of these metals is not as well studied in complex environmental systems. Here, we have examined differences in the long term fate and transport of ceria (CeO2) nanoparticles of two different sizes (3.8 vs 185 nm), dosed weekly to freshwater wetland mesocosms over 9 months. While the majority of CeO2 particles were detected in soils and sediments at the end of nine months, there were significant differences observed in fate, distribution, and transport mechanisms between the two materials. Small nanoparticles were removed from the water column primarily through heteroaggregation with suspended solids and plants, while large nanoparticles were removed primarily by sedimentation. A greater fraction of small particles remained in the upper floc layers of sediment relative to the large particles (31% vs 7%). Cerium from the small particles were also significantly more bioavailable to aquatic plants (2% vs 0.5%), snails (44 vs 2.6 ng), and insects (8 vs 0.07 µg). Small CeO2 particles were also significantly reduced from Ce(IV) to Ce(III), while aquatic sediments were a sink for untransformed large nanoparticles. These results demonstrate that trace metals originating from nanoscale materials have much greater potential than their larger counterparts to distribute throughout multiple compartments of a complex aquatic ecosystem and contribute to the overall bioavailable pool of the metal for biouptake and trophic transfer.

Mechanistic Insights from Discrete Molecular Dynamics Simulations of Pesticide-Nanoparticle Interactions.

Nanoscale particles have the potential to modulate the transport, lifetimes, and ultimate uptake of pesticides that may otherwise be bound to agricultural soils. Engineered nanoparticles provide a unique platform for studying these interactions. In this study, we utilized discrete molecular dynamics (DMD) as a screening tool for examining nanoparticle-pesticide adsorptive interactions. As a proof-of-concept, we selected a library of 15 pesticides common in the United States and 4 nanomaterials with likely natural or incidental sources, and simulated all possible nanoparticle-pesticide pairs. The resulting adsorption coefficients derived from DMD simulations ranged over several orders of magnitude, and in many cases were significantly stronger than pesticide adsorption on clay surfaces, highlighting the significance of specific nanoscale phases as a preferential media with which pesticides may associate. Binding was found to be significantly enhanced by the capacity to form hydrogen bonds with slightly hydroxylated fullerols, highlighting the importance of considering the precise nature of weathered nanomaterials as opposed to pristine precursors. Results were compared to experimental adsorption studies using selected pesticides, with a Pearson correlation coefficient of 0.97.

Measuring Nanoparticle Attachment Efficiency in Complex Systems.

As process-based environmental fate and transport models for engineered nanoparticles are developed, there is a need for relevant and reliable measures of nanoparticle behavior. The affinity of nanoparticles for various surfaces (α) is one such measure. Measurements of the affinity of nanoparticles obtained by flowing particles through a porous medium are constrained by the types of materials or exposure scenarios that can be configured into such column studies. Utilizing glass beads and kaolinite as model collector surfaces, we evaluate a previously developed mixing method for measuring nanoparticle attachment to environmental surfaces, and validate this method with an equivalent static column system over a range of organic matter concentrations and ionic strengths. We found that, while both impacted heteroaggregation rates in a predictable manner when varied individually, neither dominated when both parameters were varied. The theory behind observed nanoparticle heteroaggregation rates (αßB) to background particles in mixed systems is also experimentally validated, demonstrating both collision frequency (ß) and background particle concentration (B) to be independent for use in fate modeling. We further examined the effects of collector particle composition (kaolinite vs glass beads) and nanoparticle surface chemistry (PVP, citrate, or humic acid) on α, and found a strong dependence on both.

Nanoparticle Surface Affinity as a Predictor of Trophic Transfer.

Nanoscale materials, whether natural, engineered, or incidental, are increasingly acknowledged as important components in large, environmental systems with potential implications for environmental impact and human health. Mathematical models are a useful tool for handling the rapidly increasing complexity and diversity of these materials and their exposure routes. Presented here is a mathematical model of trophic transfer driven by nanomaterial surface affinity for environmental and biological surfaces, developed in tandem with an experimental functional assay for determining these surface affinities. We found that nanoparticle surface affinity is a strong predictor of uptake through predation in a simple food web consisting of the algae Chlorella vulgaris and daphnid Daphnia magna. The mass of nanoparticles internalized by D. magna through consuming nanomaterial-contaminated algae varied linearly with surface-attachment efficiency. Internalized quantities of gold nanoparticles in D. magna ranged from 8.3 to 23.6 ng/mg for nanoparticle preparations with surface-attachment efficiencies ranging from 0.07 to 1. This model, coupled with the functional-assay approach, may provide a useful screening tool for existing materials as well as a predictive model for their development.

PAMAM dendrimers and graphene: materials for removing aromatic contaminants from water.

We present results from experiments and atomistic molecular dynamics simulations on the remediation of naphthalene by polyamidoamine (PAMAM) dendrimers and graphene oxide (GrO). Specifically, we investigate 3rd-6th generation (G3-G6) PAMAM dendrimers and GrO with different levels of oxidation. The work is motivated by the potential applications of these emerging nanomaterials in removing polycyclic aromatic hydrocarbon contaminants from water. Our experimental results indicate that GrO outperforms dendrimers in removing naphthalene from water. Molecular dynamics simulations suggest that the prominent factors driving naphthalene association to these seemingly disparate materials are similar. Interestingly, we find that cooperative interactions between the naphthalene molecules play a significant role in enhancing their association to the dendrimers and GrO. Our findings highlight that while selection of appropriate materials is important, the interactions between the contaminants themselves can also be important in governing the effectiveness of a given material. The combined use of experiments and molecular dynamics simulations allows us to comment on the possible factors resulting in better performance of GrO in removing polyaromatic contaminants from water.

Structure-function relationship of PAMAM dendrimers as robust oil dispersants.

PAMAM dendrimers have recently been investigated as efficient and biocompatible oil dispersants utilizing their encapsulation capacity; however, their high cationic charge density has been shown to be cytotoxic. It is therefore imperative to mitigate cationic charge-induced toxicity and understand the effects of such changes. Presented here is a synergistic experimental and computational approach to examine the effects of varying terminal surface charge on the capacity of dendrimers to disperse model liner, polycyclic aromatic, and hybrid hydrocarbons. Uncharged dendrimers collapse by forming intramolecular hydrogen bonds, which reduce the hosting capability. On the other hand, changing the surface charges from positive to negative greatly shifts the pKa of tertiary amines of the PAMAM dendrimer interior. As a result, the negatively charged dendrimers have a significant percentage of tertiary amines protonated, ∼30%. This unexpected change in the interior protonation state causes electrostatic interactions with the anionic terminal groups, leading to contraction and a marked decrease in hydrocarbon hosting capacity. The present work highlights the robust nature of dendrimer oil dispersion and also illuminates potentially unintended or unanticipated effects of varying dendrimer surface chemistry on their encapsulation or hosting efficacy, which is important for their environmental, industrial, and biomedical applications.

Exploiting the physicochemical properties of dendritic polymers for environmental and biological applications.

In this perspective we first examine the rich physicochemical properties of dendritic polymers for hosting cations, anions, and polyaromatic hydrocarbons. We then extrapolate these conceptual discussions to the use of dendritic polymers in humic acid antifouling, oil dispersion, copper sensing, and fullerenol remediation. In addition, we review the state-of-the-art of dendrimer research and elaborate on its implications for water purification, environmental remediation, nanomedicine, and energy harvesting.

Delivery, Fate, and Mobility of Silver Nanoparticles in Citrus Trees.

Crop disease control is crucial for the sustainable development of agriculture, with recent advances in nanotechnology offering a promising solution to this pressing problem. However, the efficacy of nanoparticle (NP) delivery methods has not been fully explored, and knowledge regarding the fate and mobility of NPs within trees is still largely unknown. In this study, we evaluate the efficiency of NP delivery methods and investigate the mobility and distribution of NPs with different surface coatings (citrate (Ct), polyvinylpyrrolidone (PVP), and gum Arabic (GA)) within Mexican lime citrus trees. In contrast to the limited delivery efficiency reported for foliar and root delivery methods, petiole feeding and trunk injection are able to deliver a large amount of NPs into trees, although petiole feeding takes much longer time than trunk injection (7 days vs 2 h in citrus trees). Once NPs enter plants, steric repulsive interactions between NPs and conducting tube surfaces are predicted to facilitate NP transport throughout the plant. Compared to PVP and Ct, GA is highly effective in inhibiting the aggregation of NPs in synthetic sap and enhancing the mobility of NPs in trees. Over a 7 day experimental period, the majority of the Ag recovered from trees (10 mL, 10 ppm GA-AgNP suspension) remain throughout the trunk (81.0% on average), with a considerable amount in the roots (11.7% on average), some in branches (4.4% on average), and a limited amount in leaves (2.9% on average). Furthermore, NP concentrations during injection and tree incubation time postinjection are found to impact the distribution of Ag in tree. We also present evidence for a transport pathway that allows NPs to move from the xylem to the phloem, which disperses the NPs throughout the plant architecture, including to the roots.

Caveats to the use of MTT, neutral red, Hoechst and Resazurin to measure silver nanoparticle cytotoxicity.

The extensive use of silver nanoparticles (AgNPs) in manufactured products will inevitably increase environmental exposure, highlighting the importance of accurate toxicity assessments. A frequent strategy to estimate AgNP cytotoxicity is to use absorbance or fluorescent-based assays. In this study we report that AgNPs - with or without surface functionalizations (polyvinyl pyrrolidone or gum arabic), and of different sizes (2-15 nm) - can interfere with the spectrometric quantification of different dyes commonly used in cytotoxicity assays, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red (NR), Hoechst, and Resazurin. Some AgNP types caused more interference than others, which was dependent on the assay. Overall most AgNPs caused the direct reduction of MTT, as well as Hoechst and NR fluorescence quenching, and absorbed light at the same wavelength as NR. None of the AgNPs tested caused the direct reduction of Resazurin; however, depending on AgNP characteristics and concentration, they may still promote fluorescence quenching of this dye. Our results show that AgNPs with different size and coatings can interfere with spectroscopy-based assays to different degrees, suggesting that their cytotoxicity may be underestimated or overestimated. We suggest that when using any spectroscopy-based assay it is essential that each individual nanoparticle formulation be tested first for potential interferences at all intended concentrations.

Gold nanoparticle biodissolution by a freshwater macrophyte and its associated microbiome.

Predicting nanoparticle fate in aquatic environments requires mimicking of ecosystem complexity to observe the geochemical processes affecting their behaviour. Here, 12 nm Au nanoparticles were added weekly to large-scale freshwater wetland mesocosms. After six months, ~70% of Au was associated with the macrophyte Egeria densa, where, despite the thermodynamic stability of Au0 in water, the pristine Au0 nanoparticles were fully oxidized and complexed to cyanide, hydroxyls or thiol ligands. Extracted biofilms growing on E. densa leaves were shown to dissolve Au nanoparticles within days. The Au biodissolution rate was highest for the biofilm with the lowest prevalence of metal-resistant taxa but the highest ability to release cyanide, known to promote Au0 oxidation and complexation. Macrophytes and the associated microbiome thus form a biologically active system that can be a major sink for nanoparticle accumulation and transformations. Nanoparticle biotransformation in these compartments should not be ignored, even for nanoparticles commonly considered to be stable in the environment.

Reducing Intestinal Digestion and Absorption of Fat Using a Nature-Derived Biopolymer: Interference of Triglyceride Hydrolysis by Nanocellulose.

Engineered nanomaterials are increasingly added to foods to improve quality, safety, or nutrition. Here we report the ability of ingested nanocellulose (NC) materials to reduce digestion and absorption of ingested fat. In the small intestinal phase of an acellular simulated gastrointestinal tract, the hydrolysis of free fatty acids (FFA) from triglycerides (TG) in a high-fat food model was reduced by 48.4% when NC was added at 0.75% w/w to the food, as quantified by pH stat titration, and by 40.1% as assessed by fluorometric FFA assay. Furthermore, translocation of TG and FFA across an in vitro cellular model of the intestinal epithelium was significantly reduced by the presence of 0.75% w/w NC in the food (TG by 52% and FFA by 32%). Finally, in in vivo experiments, the postprandial rise in serum TG 1 h after gavage with the high fat food model was reduced by 36% when 1.0% w/w NC was administered with the food. Scanning electron microscopy and molecular dynamics studies suggest two primary mechanisms for this effect: (1) coalescence of fat droplets on fibrillar NC (CNF) fibers, resulting in a reduction of available surface area for lipase binding and (2) sequestration of bile salts, causing impaired interfacial displacement of proteins at the lipid droplet surface and impaired solubilization of lipid digestion products. Together these findings suggest a potential use for NC, as a food additive or supplement, to reduce absorption of ingested fat and thereby assist in weight loss and the management of obesity.

Direct observation of a single nanoparticle-ubiquitin corona formation.

The advancement of nanomedicine and the increasing applications of nanoparticles in consumer products have led to administered biological exposure and unintentional environmental accumulation of nanoparticles, causing concerns over the biocompatibility and sustainability of nanotechnology. Upon entering physiological environments, nanoparticles readily assume the form of a nanoparticle-protein corona that dictates their biological identity. Consequently, understanding the structure and dynamics of a nanoparticle-protein corona is essential for predicting the fate, transport, and toxicity of nanomaterials in living systems and for enabling the vast applications of nanomedicine. Here we combined multiscale molecular dynamics simulations and complementary experiments to characterize the silver nanoparticle-ubiquitin corona formation. Notably, ubiquitins competed with citrates for the nanoparticle surface, governed by specific electrostatic interactions. Under a high protein/nanoparticle stoichiometry, ubiquitins formed a multi-layer corona on the particle surface. The binding exhibited an unusual stretched-exponential behavior, suggesting a rich binding kinetics. Furthermore, the binding destabilized the α-helices while increasing the ß-sheet content of the proteins. This study revealed the atomic and molecular details of the structural and dynamic characteristics of nanoparticle-protein corona formation.

Competitive binding of natural amphiphiles with graphene derivatives.

Understanding the transformation of graphene derivatives by natural amphiphiles is essential for elucidating the biological and environmental implications of this emerging class of engineered nanomaterials. Using rapid discrete-molecular-dynamics simulations, we examined the binding of graphene and graphene oxide with peptides, fatty acids, and cellulose, and complemented our simulations by experimental studies of Raman spectroscopy, FTIR, and UV-Vis spectrophotometry. Specifically, we established a connection between the differential binding and the conformational flexibility, molecular geometry, and hydrocarbon content of the amphiphiles. Importantly, our dynamics simulations revealed a Vroman-like competitive binding of the amphiphiles for the graphene oxide substrate. This study provides a mechanistic basis for addressing the transformation, evolution, transport, biocompatibility, and toxicity of graphene derivatives in living systems and the natural environment.