Characterizing Nanoparticles in Biological Matrices: Tipping Points in Agglomeration State and Cellular Delivery In Vitro.

Understanding the delivered cellular dose of nanoparticles is imperative in nanomedicine and nanosafety, yet is known to be extremely complex because of multiple interactions between nanoparticles, their environment, and the cells. Here, we use 3-D reconstruction of agglomerates preserved by cryogenic snapshot sampling and imaged by electron microscopy to quantify the "bioavailable dose" that is presented at the cell surface and formed by the process of individual nanoparticle sequestration into agglomerates in the exposure media. Critically, using 20 and 40 nm carboxylated polystyrene-latex and 16 and 85 nm silicon dioxide nanoparticles, we show that abrupt, dose-dependent "tipping points" in agglomeration state can arise, subsequently affecting cellular delivery and increasing toxicity. These changes are triggered by shifts in the ratio of the total nanoparticle surface area to biomolecule abundance, with the switch to a highly agglomerated state effectively changing the test article midassay, challenging the dose-response paradigm for nanosafety experiments. By characterizing nanoparticle numbers per agglomerate, we show these tipping points can lead to the formation of extreme agglomeration states whereby 90% of an administered dose is contained and delivered to the cells by just the top 2% of the largest agglomerates. We thus demonstrate precise definition, description, and comparison of the nanoparticle dose formed in different experimental environments and show that this description is critical to understanding cellular delivery and toxicity. We further empirically "stress-test" the commonly used dynamic light scattering approach, establishing its limitations to present an analysis strategy that significantly improves the usefulness of this popular nanoparticle characterization technique.

Effects of a novel pesticide-particle conjugate on viability and reactive oxygen species generation in neuronal (PC12) cells.

Development of new methods and compounds to eradicate insect vectors are desperately needed. To that end, our team has previously described the synthesis and characterization of a conjugate comprised of a silver nanoparticle core encapsulated by the pyrethroid pesticide, deltamethrin (pesticide encapsulated silver nanoparticle termed "PENS"). For this current work, the PENS conjugate was tested in neuronal cultured cells to compare the cytotoxic responses to the unconjugated pesticide deltamethrin - a known neurotoxic agent and pristine silver nanoparticles. The PC12 (pheochromocytoma of the rat adrenal medulla) cell line was chosen as a model neuronal culture system. Cells were exposed to known concentrations of PENS, deltamethrin or silver nanoparticle suspensions to assess the degree of toxicity in vitro. After 24 hours of incubation, cell viability and intracellular reactive oxygen species (ROS) were measured. Bright field images of high dose exposures to dosing solutions were also acquired to evaluate cell morphology. Exposure to PENS resulted in a 17% decline in viability at the highest concentration of 45 µM while exposure to deltamethrin caused a 47% decrease. These results suggest that cellular viability was less adversely affected by PENS than by the deltamethrin. Also, ROS production following PENS exposure indicated that the newly developed conjugate was responding in a similar manner as that of cells treated with deltamethrin only.

Processing and characterization of stable, pH-sensitive layer-by-layer modified colloidal quantum dots.

Quantum Dots (QDs) stabilized with dihydrolipoic acid (DHLA) were used as a template for layer-by-layer (LbL) modification to study the effect on the QD optical properties. We studied several different polyelectrolytes to determine that large quantities of monodisperse DHLA-QDs could only be obtained with the weak polyelectrolyte pair of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA). The key to this success was the development of a two-step method to split the LbL process into adsorption and centrifugation phases, which require different pH solutions for optimum success. Solution pH is highlighted as an important factor to achieve sufficient QD surface coverage and QD recovery during wash cycles. We optimized the process to scale up synthesis by introducing a solvent precipitation step before ultracentrifugation that, when coupled with the correct pH conditions, results in a mean QD recovery of 86-90% after three wash cycles. We found that adsorption of PAH had a negligible effect on the quantum yield and lifetime but an additional layer of PAA resulted in a substantial decrease in both quantum yield and lifetime that could not be recovered by the addition of more layers. The PAH coating provides a protective coating that extends DHLA-QDs stability, prevents photo-oxidation mediated aggregation, alleviates concerns over batch variability, and results in pH-dependent emission.

A physiologically relevant approach to characterize the microbial response to colloidal particles in food matrices within a simulated gastrointestinal tract.

Colloids on the nanometer size scale are beginning to find increased applications in drinks, foods, food-contact surfaces, and food packaging. While these particles add intrinsic value to the food industry, their potential toxicities warrant careful studies. The physicochemical changes and possible perturbations to microbial communities within the gastrointestinal tract have not been adequately studied. The purpose of this study was to design and perform a simulated digestion protocol to evaluate the effect of colloidal silver in an orange juice suspension when exposed to planktonic bacterial cultures and biofilms. The model system includes four precursor steps in which the silver is exposed to varying pH conditions and incubation times. The gastrointestinally "digested" samples were then incubated with Escherichia coli strains for up to 4h, the average residence time of foods in the GI tract. The physicochemical changes of the colloids and their corresponding biological effects were characterized at every step. The results showed differences between (1) bacterial cultures versus bacterial biofilms, (2) "digested" versus "undigested" silver on bacteria, and (3) differences between "digested" silver nitrate versus silver colloids on bacteria. We conclude that simulated digestion, as well as manner in which bacterial cells are grown, influences the results of toxicity.

Distinct immunomodulatory effects of a panel of nanomaterials in human dermal fibroblasts.

There are many efforts in understanding the effects of nanoparticles on cell viability and metabolism, however, not much is known regarding the distinct molecular mechanisms of inflammation and cellular stress using low dosing concentrations. To address this gap in the literature, we utilized a novel experimental design that specifically probes the effects of a panel of commonly studied engineered nanomaterials along immunomodulatory pathways, including NF-κB. The panel of particles selected for this study included quantum dot nanocrystals, titanium dioxide, hydroxylated fullerenes, and silver nanoparticles. Cell viability, antioxidant activity, select messenger RNA, and protein modulation were studied in primary human dermal fibroblasts (HDF) and NF-κB knockdown HDF cells. Inflammatory and non-inflammatory immune responses were measured using protein and real-time PCR array analysis from HDF cells exposed to sub-lethal concentrations of nanoparticles. Differences in cellular response to nanoparticles in protein and antioxidant experiments were evident in NF-κB knockdown cells. The methods used in the study, along with the resultant data sets, serve as a potential model for studying the complex pathway-specific biochemical responses in cell and tissue systems associated with nanoparticle exposures.

Surface functionalization of silver nanoparticles: novel applications for insect vector control.

Every day, people and animals contract debilitating and life threatening diseases due to bites from infected flies, ticks, and mosquitoes. The current methods utilized to fight against these diseases are only partially effective or safe for humans and animals. When it comes to insect vector control, a conceptual paradigm shift is urgently needed. This work proposes a novel synthetic scheme to produce a nanoparticle-pesticide core-shell conjugate to be used as an active agent against arthropod vectors, such as mosquitoes. As a proof of concept, we conjugated nanosilver to the pyrethroid pesticide deltamethrin. First, electron microscopy and Fourier transform infrared spectroscopy verified the presence of a 15 nm nanosilver core surrounded by deltamethrin. Second, when the conjugate was exposed to mosquitoes for a 24 h bioassay, mortality was observed at 9 × 10(-4) M. Silver was detected in the hemolymph of mosquitoes exposed to the conjugate. We concluded that the newly developed nanoconjugate did not inactivate the primary function of the pesticide and was effective in killing mosquitoes at low concentrations. These results demonstrate the potential to use nanoparticle surfaces to kill insects, specifically vectors of human pathogens.