Elizabeth I. Maurer

Does Shape Matter? Bioeffects of Gold Nanomaterials in a Human Skin Cell Model

Gold nanomaterials (AuNMs) have distinctive electronic and optical properties, making them ideal candidates for biological, medical, and defense applications. Therefore, it is imperative to evaluate the potential biological impact of AuNMs before employing them in any application. This study investigates two AuNMs with different aspect ratios (AR) on mediation of biological responses in the human keratinocyte cell line (HaCaT) to model potential skin exposure to these AuNMs. The cellular responses were evaluated by cell viability, reactive oxygen species (ROS) generation, alteration in gene and protein expression, and inflammatory response. Gold nanospheres, nominally 20 nm in diameter and coated with mercaptopropane sulfonate (AuNS-MPS), formed agglomerates when dispersed in cell culture media, had a large fractal dimension (D(f) = 2.57 ± 0.4) (i.e., tightly bound and densely packed) and were found to be nontoxic even at the highest dose of 100 μg/mL. Highly uniform, 16.7 nm diameter, and 43.8 nm long polyethylene glycol-capped gold nanorods (AuNR-PEG) also formed agglomerates when dispersed into the cell culture media. However, the agglomerates had a smaller fractal dimension (D(f) = 1.28 ± 0.08) (i.e., loosely bound) and were found to be cytotoxic to the HaCaT cells, with a significant decrease in cell viability occurring at 25 μg/mL and higher. Moreover, AuNR-PEG caused significant ROS production and up-regulated several genes involved in cellular stress and toxicity. These results, combined with increased levels of inflammatory and apoptotic proteins, demonstrated that the AuNR-PEG induced apoptosis. Exposure to AuNS-MPS, however, did not show any of the detrimental effects observed from the AuNR-PEG. Therefore, we conclude that shape appears to play a key role in mediating the cellular response to AuNMs.

Slow Release of Ions from Internalized Silver Nanoparticles Modifies the Epidermal Growth Factor Signaling Response

Due to their distinctive physiochemical properties, including a robust antibacterial activity and plasmonic capability, hundreds of consumer and medical products contain colloidal silver nanoparticles (AgNPs). However, even at sub-toxic dosages, AgNPs are able to disrupt cell functionality, through a yet unknown mechanism. Moreover, internalized AgNPs have the potential to prolong this disruption, even after the removal of excess particles. In the present study, we evaluated the impact, mechanism of action, and continual effects of 50 nm AgNP exposure on epidermal growth factor (EGF) signal transduction within a human keratinocyte (HaCaT) cell line. After AgNP expose, EGF signaling was initially obstructed due to the dissolution of particles into silver ions. However, at longer durations, the internalized AgNPs increased EGF signaling activity. This latter behavior correlated to sustained HaCaT stress, believed to be maintained through the continual dissolution of internalized AgNPs. This study raises concerns that even after exposure ceases, the retained nanomaterials are capable of acting as a slow-release mechanism for metallic ions; continually stressing and modifying normal cellular functionality.

Tannic Acid Coated Gold Nanorods Demonstrate a Distinctive Form of Endosomal Uptake and Unique Distribution within Cells

One of the primary challenges associated with nanoparticle-dependent biological applications is that endosomal entrapment in a physiological environment severely limits the desired targeting and functionality of the nanoconstructs. This study sought to overcome that challenge through a systematic approach of gold nanorod (GNR) functionalization: evaluating the influence of both aspect ratio and surface chemistry on targeted cellular internalization rates and preservation of particle integrity. Owing to their unique spectral properties and enhanced surface area, GNRs possess great potential for the advancement of nanobased delivery and imaging applications. However, their ability for efficient intracellular delivery while maintaining their specific physiochemical parameters has yet to be satisfactorily explored. This study identified that longer and positively charged GNRs demonstrated a higher degree of internalization compared to their shorter and negative counterparts. Notably, of the four surface chemistries explored, only tannic acid resulted in retention of GNR integrity following endocytosis into keratinocyte cells, due to the presence of a strong protein corona matrix that served to protect the particles. Taken together, these results identify tannic acid functionalized GNRs as a potential candidate for future development in nanobased biomolecule delivery, bioimaging, and therapeutic applications.

Novel Platform Development Using an Assembly of Carbon Nanotube, Nanogold and Immobilized RNA Capture Element towards Rapid, Selective Sensing of Bacteria

This study examines the creation of a nano-featured biosensor platform designed for the rapid and selective detection of the bacterium Escherichia coli. The foundation of this sensor is carbon nanotubes decorated with gold nanoparticles that are modified with a specific, surface adherent ribonucleiuc acid (RNA) sequence element. The multi-step sensor assembly was accomplished by growing carbon nanotubes on a graphite substrate, the direct synthesis of gold nanoparticles on the nanotube surface, and the attachment of thiolated RNA to the bound nanoparticles. The application of the compounded nano-materials for sensor development has the distinct advantage of retaining the electrical behavior property of carbon nanotubes and, through the gold nanoparticles, incorporating an increased surface area for additional analyte attachment sites, thus increasing sensitivity. We successfully demonstrated that the coating of gold nanoparticles with a selective RNA sequence increased the capture of E. coli by 189% when compared to uncoated particles. The approach to sensor formation detailed in this study illustrates the great potential of unique composite structures in the development of a multi-array, electrochemical sensor for the fast and sensitive detection of pathogens.

The biological impact of concurrent exposure to metallic nanoparticles and a static magnetic field.

The rapid advancement of technology has led to an exponential increase of both nanomaterial and magnetic field utilization in applications spanning a variety of sectors. While extensive work has focused on the impact of these two variables on biological systems independently, the existence of any synergistic effects following concurrent exposure has yet to be investigated. This study sought to ascertain the induced alterations to the stress and proliferation responses of the human adult low calcium, high temperature keratinocyte (HaCaT) cell line by the application of a static magnetic field (approximately 0.5 or 30 mT) in conjunction with either gold or iron oxide nanoparticles for a duration of 24 h. By evaluating targets at a cellular, protein, and genetic level a complete assessment of the HaCaT response was generated. A magnetic field-dependent proliferative effect was found (∼15%), which correlated with a decrease in reactive oxygen species and a simultaneous increase in ki67 expression, all occurring independently of nanoparticle presence. Furthermore, the application of a static magnetic field was able to counteract the cellular stress response induced by nanoparticle exposure through a combination of decreased reactive oxygen species production and modification of gene regulation. Therefore, we conclude that while these variables each introduce the potential to uniquely influence physiological events, no negative synergistic reactions were identified.

Intracellular accumulation and dissolution of silver nanoparticles in L-929 fibroblast cells using live cell time-lapse microscopy

Abstract Cytotoxicity assessments of nanomaterials, such as silver nanoparticles, are challenging due to interferences with test reagents and indicators as well uncertainties in dosing as a result of the complex nature of nanoparticle intracellular accumulation. Furthermore, current theories suggest that silver nanoparticle cytotoxicity is a result of silver nanoparticle dissolution and subsequent ion release. This study introduces a novel technique, nanoparticle associated cytotoxicity microscopy analysis (NACMA), which combines fluorescence microscopy detection using ethidium homodimer-1, a cell permeability marker that binds to DNA after a cell membrane is compromised (a classical dead-cell indicator dye), with live cell time-lapse microscopy and image analysis to simultaneously investigate silver nanoparticle accumulation and cytotoxicity in L-929 fibroblast cells. Results of this method are consistent with traditional methods of assessing cytotoxicity and nanoparticle accumulation. Studies conducted on 10, 50, 100 and 200 nm silver nanoparticles reveal size dependent cytotoxicity with particularly high cytotoxicity from 10 nm particles. In addition, NACMA results, when combined with transmission electron microscopy imaging, reveal direct evidence of intracellular silver ion dissolution and possible nanoparticle reformation within cells for all silver nanoparticle sizes.

The effect of shear flow on nanoparticle agglomeration and deposition in in vitro dynamic flow models

Abstract Traditional in vitro toxicity experiments typically involve exposure of a mono- or co-culture of cells to nanoparticles (NPs) in static conditions with the assumption of 100% deposition (i.e. dose) of well-dispersed particles. However, cellular dose can be affected by agglomeration and the unique transport kinetics of NPs in biological media. We hypothesize that shear flow can address these issues and achieve more predictable dosage. Here, we compare the behavior of gold NPs with diameters of 5, 10 and 30 nm in static and dynamic in vitro models. We also utilize transport modeling to approximate the shear rate experienced by the cells in dynamic conditions to evaluate physiological relevance. The transport kinetics show that NP behavior is governed by both gravity and diffusion forces in static conditions and only diffusion in dynamic conditions. Our results reveal that dynamic systems are capable of producing a more predictable dose compared to static systems, which has strong implications for improving repeatability in nanotoxicity assessments.

Advantages of using imaged-based fluorescent analysis for nanomaterial studies

Currently, there is no standardised method for making nanomaterials or evaluating their toxicity. Studies show great variability and lack of reproducibility in results, with the discrepancies being caused due to assays used to evaluate exposure toxicity. While the traditional biochemical methods for evaluating toxicity have been employed, there is an issue of how the nanomaterials themselves interact with the dyes, which has created some issues. The current methodologies need to be evaluated to determine the toxicity of the nanomaterials and ensure that there is no interaction between the nanomaterials and the reagents used to complete the assays. This study evaluated the mechanisms of nanomaterial toxicity using mitochondrial function, loss of mitochondrial membrane potential and formation of reactive oxygen species as indicators of toxicity, and compared the data obtained on the BD Pathway 435 confocal to data from a plate reader. Furthermore, nanoparticle uptake and localisation were evaluated using th...