Toxicity assessment of CeO2 and CuO nanoparticles at the air-liquid interface using bioinspired condensational particle growth.

CeO2 and CuO nanoparticles (NPs) are used as additives in petrodiesel to enhance engine performance leading to reduced diesel combustion emissions. Despite their benefits, the additive application poses human health concerns by releasing inhalable NPs into the ambient air. In this study, a bioinspired lung cell exposure system, Dosimetric Aerosol in Vitro Inhalation Device (DAVID), was employed for evaluating the toxicity of aerosolized CeO2 and CuO NPs with a short duration of exposure (≤10 min vs. hours in other systems) and without exerting toxicity from non-NP factors. Human epithelial A549 lung cells were cultured and maintained within DAVID at the air-liquid interface (ALI), onto which aerosolized NPs were deposited, and experiments in submerged cells were used for comparison. Exposure of the cells to the CeO2 NPs did not result in detectable IL-8 release, nor did it produce a significant reduction in cell viability based on lactate dehydrogenase (LDH) assay, with a marginal decrease (10%) at the dose of 388 µg/cm2 (273 cm2/cm2). In contrast, exposure to CuO NPs resulted in a concentration dependent reduction in LDH release based on LDH leakage, with 38% reduction in viability at the highest dose of 52 µg/cm2 (28.3 cm2/cm2). Cells exposed to CuO NPs resulted in a dose dependent cellular membrane toxicity and expressed IL-8 secretion at a global dose five times lower than cells exposed under submerged conditions. However, when comparing the ALI results at the local cellular dose of CuO NPs to the submerged results, the IL-8 secretion was similar. In this study, we demonstrated DAVID as a new exposure tool that helps evaluate aerosol toxicity in simulated lung environment. Our results also highlight the necessity in choosing the right assay endpoints for the given exposure scenario, e.g., LDH for ALI and Deep Blue for submerged conditions for cell viability.

Examining cellular responses to reconstituted antibody protein liquids.

Protein ionic liquids (PIL) are a new class of biologic stabilizers designed to protect the functionality and extend the shelf-life of biotechnological and therapeutic agents making them more readily available, and resistant to austere environments. Protein biorecognition elements such as monoclonal antibodies are commonly utilized therapeutics that require the robust stabilization offered by PILs, but biocompatibility remains an important issue. This study has focused on characterizing the biocompatibility of an antibody based PIL by exposing multiple cells types to a cationized immunoglobulin suspended in an anionic liquid (IgG-IL). The IgG-IL caused no significant alterations in cellular health for all three cell types with treatments < 12.5 µg/mL. Concentrations ≥ 12.5 µg/mL resulted in significant necrotic cell death in A549 and HaCaT cells, and caspase associated cell death in HepG2 cells. In addition, all cells displayed evidence of oxidative stress and IL-8 induction in response to IgG-IL exposures. Therapeutic Ig can be utilized with a wide dose range that extends into concentrations we have found to exhibit cytotoxicity raising a toxicity concern and a need for more extensive understanding of the biocompatibility of IgG-ILs.

Engineered aluminum nanoparticle induces mitochondrial deformation and is predicated on cell phenotype.

The main role of mitochondria is to generate the energy necessary for the cell to survive and adapt to different environmental stresses. Energy demand varies depending on the phenotype of the cell. To efficiently meet metabolic demands, mitochondria require a specific proton homeostasis and defined membrane structures to facilitate adenosine triphosphate production. This homeostatic environment is constantly challenged as mitochondria are a major target for damage after exposure to environmental contaminants. Here we report changes in mitochondrial structure profiles in different cell types using electron microscopy in response to particle stress exposure in three different representative lung cell types. Endpoint analyses include nanoparticle intracellular uptake; quantitation of mitochondrial size, shape, and ultrastructure; and confirmation of autophagosome formation. Results show that low-dose aluminum nanoparticles exposure (1 ppm; 1 µg/mL; 1.6 × 1 0-7 µg/cell)) to primary and asthma cells incurred significant mitochondrial deformation and increases in mitophagy, while cancer cells exhibited only slight changes in mitochondrial morphology and an increase in lipid body formation. These results show low-dose aluminum nanoparticle exposure induces subtle changes in the mitochondria of specific lung cells that can be quantified with microscopy techniques. Furthermore, within the lung, cell type by the nature of origin (i.e. primary vs. cancer vs. asthma) dictates mitochondrial morphology, metabolic health, and the metabolic stress response of the cell.

Biofabrication of endothelial cell, dermal fibroblast, and multilayered keratinocyte layers for skin tissue engineering.

The skin serves a substantial number of physiological purposes and is exposed to numerous biological and chemical agents owing to its large surface area and accessibility. Yet, current skin models are limited in emulating the multifaceted functions of skin tissues due to a lack of effort on the optimization of biomaterials and techniques at different skin layers for building skin frameworks. Here, we use biomaterial-based approaches and bioengineered techniques to develop a 3D skin model with layers of endothelial cell networks, dermal fibroblasts, and multilayered keratinocytes. Analysis of mechanical properties of gelatin methacryloyl (GelMA)-based bioinks mixed with different portions of alginate revealed bioprinted endothelium could be better modeled to optimize endothelial cell viability with a mixture of 7.5% GelMA and 2% alginate. Matrix stiffness plays a crucial role in modulating produced levels of Pro-Collagen I alpha-1 and matrix metalloproteinase-1 in human dermal fibroblasts and affecting their viability, proliferation, and spreading. Moreover, seeding human keratinocytes with gelatin-coating multiple times proved to be helpful in reducing culture time to create multiple layers of keratinocytes while maintaining their viability. The ability to fabricate selected biomaterials for each layer of skin tissues has implications in the biofabrication of skin systems for regenerative medicine and disease modeling.

Human Nontumorigenic Microglia Synthesize Strongly Fluorescent Au/Fe Nanoclusters, Retaining Bioavailability.

The ability for cells to self-synthesize metal-core nanoclusters (mcNCs) offers increased imaging and identification opportunities. To date, much work has been done illustrating the ability for human tumorigenic cell lines to synthesize mcNCs; however, this has not been illustrated for nontumorigenic cell lines. Here, we present the ability for human nontumorigenic microglial cells, which are the major immune cells in the central nervous system, to self-synthesize gold (Au) and iron (Fe) core nanoclusters, following exposures to metallic salts. We also show the ability for cells to internalize presynthesized Au and Fe mcNCs. Cellular fluorescence increased in most exposures and in a dose dependent manner in the case of Au salt. Scanning transmission electron microscopic imaging confirmed the presence of the metal within cells, while transmission electron microscopy images confirmed nanocluster structures and self-synthesis. Interestingly, self-synthesized nanoclusters were of similar size and internal structure as presynthesized mcNCs. Toxicity assessment of both salts and presynthesized NCs illustrated a lack of toxicity from Au salt and presynthesized NCs. However, Fe salt was generally more toxic and stressful to cells at similar concentrations.

In Vitro Aerosol Exposure to Nanomaterials: From Laboratory to Environmental Field Toxicity Testing.

Exposure to nanomaterials (NMs) is inevitable, requiring robust toxicological assessment to understand potential environmental and human health effects. NMs are favored in many applications because of their small size; however, this allows them to easily aerosolize and, subsequently, expose humans via inhalation. Toxicological assessment of NMs by conventional methods in submerged cell culture is not a relevant way to assess inhalation toxicity of NMs because of particle interference with bioassays and changes in particokinetics when dispersed in medium. Therefore, an in vitro aerosol exposure chamber (AEC) was custom designed and used for direct deposition of NMs from aerosols in the environment to the air-liquid interface of lung cells. Human epithelial lung cell line, A549, was used to assess the toxicity of copper, nickel, and zinc oxide nanopowders aerosolized by acoustic agitation in laboratory study. Post optimization, the AEC was used in the field to expose the A549 cells to NM aerosols generated from firing a hand gun and rifle. Toxicity was assessed using nondestructive assays for cell viability and inflammatory response, comparing the biologic effect to the delivered mass dose measured by inductively coupled plasma-mass spectrometry. The nanopowder exposure to submerged and ALI cells resulted in dose-dependent toxicity. In the field, weapon exhaust from the M4 reduced cell viability greater than the M9, while the M9 stimulated inflammatory cytokine release of IL-8. This study highlights the use of a portable chamber with the capability to assess toxicity of NM aerosols exposed to air-liquid interface in vitro lung cell culture.

Depletion of phosphatidylinositol 4-phosphate at the Golgi translocates K-Ras to mitochondria.

Ras proteins are small GTPases localized to the plasma membrane (PM), which regulate cellular proliferation, apoptosis and differentiation. After a series of post-translational modifications, H-Ras and N-Ras traffic to the PM from the Golgi via the classical exocytic pathway, but the exact mechanism of K-Ras trafficking to the PM from the ER is not fully characterized. ATP5G1 (also known as ATP5MC1) is one of the three proteins that comprise subunit c of the F0 complex of the mitochondrial ATP synthase. In this study, we show that overexpression of the mitochondrial targeting sequence of ATP5G1 perturbs glucose metabolism, inhibits oncogenic K-Ras signaling, and redistributes phosphatidylserine (PtdSer) to mitochondria and other endomembranes, resulting in K-Ras translocation to mitochondria. Also, it depletes phosphatidylinositol 4-phosphate (PI4P) at the Golgi. Glucose supplementation restores PtdSer and K-Ras PM localization and PI4P at the Golgi. We further show that inhibition of the Golgi-localized PI4-kinases (PI4Ks) translocates K-Ras, and PtdSer to mitochondria and endomembranes, respectively. We conclude that PI4P at the Golgi regulates the PM localization of PtdSer and K-Ras.This article has an associated First Person interview with the first author of the paper.

Achievements and Challenges for Real-Time Sensing of Analytes in Sweat within Wearable Platforms.

Physiological sensors in a wearable form have rapidly emerged on the market due to technological breakthroughs and have become nearly ubiquitous with the Apple Watch, FitBit, and other wearable devices. While these wearables mostly monitor simple biometric signatures, new devices that can report on the human readiness level through sensing molecular biomarkers are critical to optimizing the human factor in both commercial sectors and the Department of Defense. The military is particularly interested in real-time, wearable, minimally invasive monitoring of fatigue and human performance to improve the readiness and performance of the war fighter. However, very few devices have ventured into the realm of reporting directly on biomarkers of interest. Primarily this is because of the difficulties of sampling biological fluids in real-time and providing accurate readouts using highly selective and sensitive sensors. When additional restrictions to only use sweat, an excretory fluid, are enforced to minimize invasiveness, the demands on sensors becomes even greater due to the dilution of the biomarkers of interest, as well as variability in salinity, pH, and other physicochemical variables which directly impact the read-out of real-time biosensors. This Account will provide a synopsis not only on exemplary demonstrations and technological achievements toward implementation of real-time, wearable sweat sensors but also on defining problems that still remain toward implementation in wearable devices that can detect molecular biomarkers for real world applications. First, the authors describe the composition of minimally invasive biofluids and then identify what biomarkers are of interest as biophysical indicators. This Account then reviews demonstrated techniques for extracting biofluids from the site of generation and transport to the sensor developed by the authors. Included in this discussion is a detailed description on biosensing recognition elements and transducers developed by the authors to enable generation of selective electrochemical sensing platforms. The authors also discuss ongoing efforts to identify biorecognition elements and the chemistries necessary to enable high affinity, selective biorecognition elements. Finally, this Account presents the requirements for wearable, real-time sensors to be (1) highly stable, (2) portable, (3) reagentless, (4) continuous, and (5) responsive in real-time, before delving into specific methodologies to sense classes of biomarkers that have been explored by academia, government laboratories, and industry. Each platform has its areas of greatest utility, but also come with corresponding weaknesses: (1) ion selective electrodes are robust and have been demonstrated in wearables but are limited to detection of ions, (2) enzymatic sensors enable indirect detection of metabolites and have been demonstrated in wearables, but the compounds that can be detected are limited to a subset of small molecules and the sensors are sensitive to flow, (3) impedance-based sensors can detect a wide range of compounds but require further research and development for deployment in wearables. In conclusion, while substantial progress has been made toward wearable molecular biosensors, substantial barriers remain and need to be solved to enable deployment of minimally invasive, wearable biomarker monitoring devices that can accurately report on psychophysiological status.

Condensational particle growth device for reliable cell exposure at the air-liquid interface to nanoparticles.

A first-of-its-kind aerosol exposure device for toxicity testing, referred to as the Dosimetric Aerosol in Vitro Inhalation Device (DAVID), was evaluated for its ability to deliver airborne nanoparticles to lung cells grown as air-liquid interface (ALI) cultures. For inhalation studies, ALI lung cell cultures exposed to airborne nanoparticles have more relevancy than the same cells exposed in submerged culture because ALI culture better represents the respiratory physiology and consequently more closely reflect cellular response to aerosol exposure. In DAVID, water condensation grows particles as small as 5 nm to droplets sized > 5 µm for inertial deposition at low flow rates. The application of DAVID for nanotoxicity analysis was evaluated by measuring the amount and variability in the deposition of uranine nanoparticles and then assessing the viability of ALI cell cultures exposed to clean-air under the same operational conditions. The results showed a low coefficient of variation, < 0.25, at most conditions, and low variability in deposition between the exposure wells, trials, and operational flow rates. At an operational flow rate of 4 LPM, no significant changes in cell viability were observed, and minimal effects observed at 6 LPM. The reliable and gentle deposition mechanism of DAVID makes it advantageous for nanoparticle exposure.

Development of reference metal and metal oxide engineered nanomaterials for nanotoxicology research using high throughput and precision flame spray synthesis approaches.

There is a growing need to develop and characterize reference metal and metal oxide engineered nanomaterials (ENMs) of high purity and tunable intrinsic properties suitable for nanotoxicology research. Here a high throughput (volume) and precision flame spray pyrolysis (FSP) approach coupled with state-of-the-art characterization techniques are utilized to generate such reference ENMs. The lab-based and industrially relevant FSP system, termed as Versatile Engineered Nanomaterials Generation System (VENGES), synthesizes the metals and metal oxides, at high throughput manner with controlled properties, such as primary particle size, aggregate diameter, shape, crystallinity, stoichiometry and surface chemistry. A nanopanel of nine reference ENMs (silica, silver, silver supported on silica, alumina, ceria and iron oxide) was synthesized and characterized using combined electron microscopy, advanced spectroscopic techniques and physical analyses (e.g., BET, XRD, TEM, pycnometry, XPS, ICP-MS and FTIR). ENMs show a high degree of chemical purity and stoichiometry, and low content of carbon residuals, and are sterile and free of bacteria and endotoxins. Further, their colloidal properties and their implication in in-vitro dosimetry have been also investigated in both environmental and test biological media. The suitability of reference ENMs and protocols developed in this study brings forth new arenas to generate reliable and reproducible toxicological data in an effort to reduce conflicting and contradicting inter-laboratory data on relative toxic effects of ENMs.

Implementation of physiological fluids to provide insight into the characterization, fate, and biological interactions of silver nanoparticles.

Silver nanoparticles (AgNPs) are being increasingly utilized in consumer and medical applications. However, there remains conflicting reports on their safety, which are evaluated through a combination of in vitro and in vivo exposure models. These discrepancies may arise, in part, due to the inherent differences between cell-based and animal systems. It is well established that nanotoxicological effects are highly dependent on the unique physicochemical properties and behavior of the particle set, including size, surface chemistry, agglomeration, and ionic dissolution. However, recent studies have identified that these properties vary as a function of exposure environment; providing a rationale for the contradictory results between in vitro and in vivo assessments. Artificial physiological fluids are emerging as a powerful tool as they allow for the characterization of NPs in an environment which they would likely encounter in vivo, in addition to having the experimental advantages of flexibility and consistency. Here, we demonstrated that the utilization of artificial fluids provided a mechanism to assess AgNP behavior and induced bioresponses in environments that they would likely encounter in vivo. AgNPs were introduced within an alveolar-based exposure model, which included alveolar epithelial (A549) cells incubated within artificial alveolar fluid (AF). Additionally, the particles underwent extensive characterization within both AF and lysosomal fluid, which the AgNPs would encounter following cellular internalization. Following incubation in physiological environments AgNP properties were significantly modified versus a traditional media environment, including alterations to both extent of agglomeration and rate of ionic dissolution. Moreover, when A549s were exposed to AgNPs in AF, the cells displayed lower cytotoxicity and stress rates, corresponding to a fluid-dependent drop in silver ion production. This work highlights the need for enhanced in vitro models that more closely mimic in vivo exposure environments in order to capture true NP behaviors and cellular interactions.

Characterization of exposure to byproducts from firing lead-free frangible ammunition in an enclosed, ventilated firing range.

U.S. Air Force small arms firing ranges began using copper-based, lead-free frangible ammunition in the early 2000s due to environmental and health concerns related to the use of lead-based ammunition. Exposure assessments at these firing ranges have routinely detected chemicals and metals in amounts much lower than their mass-based occupational exposure limits, yet, instructors report work-related health concerns including respiratory distress, nausea, and headache. The objective of this study at one firing range was to characterize the aerosol emissions produced by weapons during firing events and evaluate the ventilation system's effectiveness in controlling instructor exposure to these emissions. The ventilation system was assessed by measuring the range static air pressure differential and the air velocity at the firing line. Air flow patterns were near the firing line. Instructor exposure was sampled using a filter-based air sampling method for metals and a wearable, real-time ultrafine particle counter. Area air sampling was simultaneously performed to characterize the particle size distribution, morphology, and composition. In the instructor's breathing zone, the airborne mass concentration of copper was low (range = <1 µg/m3 to 16 µg/m3), yet the ultrafine (nanoscale) particle number concentration increased substantially during each firing event. Ultrafine particles contained some copper and were complex in morphology and composition. The ventilation assessment found that the average velocity across all shooting lanes was acceptable compared to the recommended guideline (20% of the ideal 0.38 m/s (75 ft/min). However, uniform, downrange airflow pattern requirements were not met. These results suggest that the mass-based occupational exposure limits, as applied to this environment, may not be protective enough to eliminate health complaints reported by instructors whose full-time job involves training personnel on weapons that fire lead-free frangible ammunition. Using an ultrafine particle counter appears to be an alternative method of assessing ventilation effectiveness in removing ultrafine particulate produced during firing events.

In situ Synthesis of Fluorescent Gold Nanoclusters by Nontumorigenic Microglial Cells.

To date, the directed in situ synthesis of fluorescent gold nanoclusters (AuNCs) has only been demonstrated in cancerous cells, with the theorized synthesis mechanism prohibiting AuNC formation in nontumorigenic cell lines. This limitation hinders potential biostabilized AuNC-based technology in healthy cells involving both chemical and mechanical analysis, such as the direct sensing of protein function and the elucidation of local mechanical environments. Thus, new synthesis strategies are required to expand the application space of AuNCs beyond cancer-focused cellular studies. In this contribution, we have developed the methodology and demonstrated the direct in situ synthesis of AuNCs in the nontumorigenic neuronal microglial line, C8B4. The as-synthesized AuNCs form in situ and are stabilized by cellular proteins. The clusters exhibit bright green fluorescence and demonstrate low (<10%) toxicity. Interestingly, elevated ROS levels were not required for the in situ formation of AuNCs, although intracellular reductants such as glutamate were required for the synthesis of AuNCs in C8B4 cells. To our knowledge, this is the first-ever demonstration of AuNC synthesis in nontumorigenic cells and, as such, it considerably expands the application space of biostabilized fluorescent AuNCs.

Modification of the protein corona-nanoparticle complex by physiological factors.

Nanoparticle (NP) effects in a biological system are driven through the formation and structure of the protein corona-NP complex, which is dynamic by nature and dependent upon factors from both the local environment and NP physicochemical parameters. To date, considerable data has been gathered regarding the structure and behavior of the protein corona in blood, plasma, and traditional cell culture medium. However, there exists a knowledge gap pertaining to the protein corona in additional biological fluids and following incubation in a dynamic environment. Using 13nm gold NPs (AuNPs), functionalized with either polyethylene glycol or tannic acid, we demonstrated that both particle characteristics and the associated protein corona were altered when exposed to artificial physiological fluids and under dynamic flow. Furthermore, the magnitude of observed behavioral shifts were dependent upon AuNP surface chemistry. Lastly, we revealed that exposure to interstitial fluid produced protein corona modifications, reshaping of the nano-cellular interface, modified AuNP dosimetry, and induction of previously unseen cytotoxicity. This study highlights the need to elucidate both NP and protein corona behavior in biologically representative environments in an effort to increase accurate interpretation of data and transfer of this knowledge to efficacy, behavior, and safety of nano-based applications.

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

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.

Modulation of miRNA-155 alters manganese nanoparticle-induced inflammatory response.

Regulation of gene expression by non-coding RNAs, such as microRNAs (miRNAs), is increasingly being examined in a variety of disciplines. Here we evaluated changes in miRNA expression following metallic nanoparticle (NP) exposure in a mouse neuronal co-culture model. Exposure to manganese (Mn) NPs resulted in oxidative stress, inflammation, and toxicity. Next-generation sequencing (NGS) following an 8 h exposure to Mn NPs (low and high doses) revealed several miRNA candidates that modulate NP induced responses. The lead candidate identified was miR-155, which showed a dose dependent decrease in expression upon Mn exposure. Introduction of a miR-155 mimic into the co-culture to restore miR-155 expression completely abrogated the Mn NP-induced gene and protein expression of inflammatory markers TNF-α and IL-6. Taken together, this study is the first report where global NP-induced miRNA expression changes were used to identify and then modulate negative impacts of metallic NP exposure in a neuronal model. These findings demonstrate that unique miRNA expression profiles provide novel targets for manipulating gene and protein expression, and therefore provide the potential of modifying cellular responses to NP exposure.

Distribution and accumulation of 10 nm silver nanoparticles in maternal tissues and visceral yolk sac of pregnant mice, and a potential effect on embryo growth.

We examined the distribution of silver in pregnant mice and embryos/fetuses following intravenous injections of 10 nm silver nanoparticles (AgNPs) or soluble silver nitrate (AgNO3) at dose levels of 0 (citrate buffer control) or 66 µg Ag/mouse to pregnant mice on gestation days (GDs) 7, 8 and 9. Selected maternal tissues and all embryos/fetuses from control, AgNP- and AgNO3-treated groups on GD10 and control and AgNP-treated groups on GD16 were processed for the measurement of silver concentrations, intracellular AgNP localization, histopathology and gross examination of tissue morphology. Inductively-coupled plasma mass spectrometry revealed silver in all examined tissues following either AgNP or AgNO3 treatment, with highest concentrations of silver in maternal liver, spleen and visceral yolk sac (VYS), and lowest concentrations in embryos/fetuses. For VYS, mean silver concentration following AgNO3 treatment (4.87 ng Ag/mg tissue) was approximately two-fold that following AgNP treatment (2.31 ng Ag/mg tissue); for all other tissues examined, mean silver concentrations following either AgNP or AgNO3 treatment were not significantly different from each other (e.g. 2.57 or 2.84 ng Ag/mg tissue in maternal liver and 1.61 or 2.50 ng Ag/mg tissue in maternal spleen following AgNP or AgNO3 treatment, respectively). Hyperspectral imaging revealed AgNP aggregates in maternal liver, kidney, spleen and VYS from AgNP-treated mice, but not AgNO3-treated mice. Additionally, one or more embryos collected on GD10 from eight of ten AgNP-treated mice appeared small for their age (i.e. Theiler stage 13 [GD8.5] or younger). In the control group (N = 11), this effect was seen in embryos from only one mouse. In conclusion, intravenous injection of 10 nm AgNPs to pregnant mice resulted in notable silver accumulation in maternal liver, spleen and VYS, and may have affected embryonic growth. Silver accumulation in embryos/fetuses was negligible.

The role of biological fluid and dynamic flow in the behavior and cellular interactions of gold nanoparticles.

BACKGROUND: Due to their distinctive physicochemical properties, nanoparticles (NPs) have proven to be extremely advantageous for product and application development, but are also capable of inducing detrimental outcomes in biological systems. Standard in vitro methodologies are currently the primary means for evaluating NP safety, as vast quantities of particles exist that require appraisal. However, cell-based models are plagued by the fact that they are not representative of complex physiological systems. The need for a more accurate exposure model is highlighted by the fact that NP behavior and subsequent bioresponses are highly dependent upon their surroundings. Therefore, standard in vitro models will likely produce inaccurate NP behavioral analyses and erroneous safety results. As such, the goal of this study was to develop an enhanced in vitro model for NP evaluation that retained the advantages of cell culture, but implemented the key physiological variables of accurate biological fluid and dynamic flow. RESULTS: In this study, a cellular microenvironment was modeled and created after an inhalation exposure scenario. This system comprised of A549 lung epithelial cells, artificial alveolar fluid (AAF), and biologically accurate dynamic flow. Under the influence of microenvironment variables, tannic acid coated gold NPs (AuNPs) displayed modulated physicochemical characteristics, including increased agglomeration, disruption of the spectral signature, and decreased rate of ionic dissolution. Furthermore, AuNP deposition efficiency, internalization patterns, and the nano-cellular interface varied as a function of fluid composition and flow condition. AAF incubation simultaneously influenced both AuNPs and cellular behavior, through excessive NP agglomeration and alteration to A549 morphology. Dynamic flow targeted the nano-cellular interface, with differential responses including modified deposition, internalization patterns, and cellular elongation. Lastly, the biocompatibility of the system was verified to ensure cellular health following AAF exposure and fluid dynamics. CONCLUSIONS: This study confirmed the feasibility of improving standard in vitro models through the incorporation of physiological variables. Utilization of this enhanced system demonstrated that to elucidate true NP behavior and accurately gauge their cellular interactions, assessments should be carried out in a more complex and relevant biological exposure model.

Assessment of Carbon- and Metal-Based Nanoparticle DNA Damage with Microfluidic Electrophoretic Separation Technology.

In this study, we examined the feasibility of extracting DNA from whole cell lysates exposed to nanoparticles using two different methodologies for evaluation of fragmentation with microfluidic electrophoretic separation. Human lung macrophages were exposed to five different carbon- and metal-based nanoparticles at two different time points (2 h, 24 h) and two different doses (5 µg/ml, 100 µg/ml). The primary difference in the banding patterns after 2 h of nanoparticle exposure is more DNA fragmentation at the higher NP concentration when examining cells exposed to nanoparticles of the same composition. However, higher doses of carbon and silver nanoparticles at both short and long dosing periods can contribute to erroneous or incomplete data with this technique. Also comparing DNA isolation methodologies, we recommend the centrifugation extraction technique, which provides more consistent banding patterns in the control samples compared to the spooling technique. Here we demonstrate that multi-walled carbon nanotubes, 15 nm silver nanoparticles and the positive control cadmium oxide cause similar DNA fragmentation at the short time point of 2 h with the centrifugation extraction technique. Therefore, the results of these studies contribute to elucidating the relationship between nanoparticle physicochemical properties and DNA fragmentation results while providing the pros and cons of altering the DNA isolation methodology. Overall, this technique provides a high throughput way to analyze subcellular alterations in DNA profiles of cells exposed to nanomaterials to aid in understanding the consequences of exposure and mechanistic effects. Future studies in microfluidic electrophoretic separation technologies should be investigated to determine the utility of protein or other assays applicable to cellular systems exposed to nanoparticles.

At the Crossroads of Nanotoxicology in vitro: Past Achievements and Current Challenges.

The exponential growth in the employment of nanomaterials (NMs) has given rise to the field of nanotoxicology; which evaluates the safety of engineered NMs. Initial nanotoxicological studies were limited by a lack of both available materials and accurate biodispersion characterization tools. However, the years that followed were marked by the development of enhanced synthesis techniques and characterization technologies; which are now standard practice for nanotoxicological evaluation. Paralleling advances in characterization, significant progress was made in correlating specific physical parameters, such as size, morphology, or coating, to resultant physiological responses. Although great strides have been made to advance the field, nanotoxicology is currently at a crossroads and faces a number of obstacles and technical limitations not associated with traditional toxicology. Some of the most pressing and influential challenges include establishing full characterization requirements, standardization of dosimetry, evaluating kinetic rates of ionic dissolution, improving in vitro to in vivo predictive efficiencies, and establishing safety exposure limits. This Review will discuss both the progress and future directions of nanotoxicology: highlighting key previous research successes and exploring challenges plaguing the field today.