An examination of the mechanisms driving the therapeutic effects of an AAV expressing a soluble variant of VEGF receptor-1.

In previous animal model studies, we demonstrated the potential of rAAV2-sVEGFRv-1, which encodes a truncated variant of the alternatively spliced soluble version of VEGF receptor-1 (VEGFR1), as a human gene therapy for age-related macular degeneration (AMD) and diabetic retinopathy (DR). Here, we elucidate in vitro some of the mechanisms by which rAAV2-sVEGFRv-1 exerts its therapeutic effects. Human umbilical vein endothelial cells (HUVECs) were infected with rAAV2-sVEGFRv-1 or a control virus vector in the presence of members of the VEGF family to identify potential binding partners via ELISA, which showed that VEGF-A, VEGF-B, and placental growth factor (PlGF) are all ligands of its transgene product. In order to determine the effects of rAAV2-sVEGFRv-1 on cell proliferation and permeability, processes that are important to the progression AMD and DR, HUVECs were infected with the therapeutic virus vector under the stimulation of VEGF-A, the major driver of the neovascularization that characterizes the forms of these conditions most associated with vision loss. rAAV2-sVEGFRv-1 treatment, as a result, markedly reduced the extent to which these processes occurred, with the latter determined by measuring zonula occludens 1 expression. Finally, the human microglial HMC3 cell line was used to show the effects of the therapeutic virus vector upon inflammatory processes, another major contributor to angiogenic eye disease pathophysiology, with rAAV2-sVEGFRv-1 reducing therein the secretion of pro-inflammatory cytokines interleukin (IL)-1ß and IL-6. Combined with our previously published in vivo data, the in vitro activity of the expressed transgene here further demonstrates the great promise of rAAV2-sVEGFRv-1 as a potential human gene therapeutic for addressing angiogenic ocular conditions.

scAAV2-Mediated Expression of Thioredoxin 2 and C3 Transferase Prevents Retinal Ganglion Cell Death and Lowers Intraocular Pressure in a Mouse Model of Glaucoma.

Elevated intraocular pressure (IOP) in glaucoma causes retinal ganglion cell (RGC) loss and damage to the optic nerve. Although IOP is controlled pharmacologically, no treatment is available to restore retinal and optic nerve function. In this paper, we aimed to develop a novel gene therapy for glaucoma using an AAV2-based thioredoxin 2 (Trx2)-exoenzyme C3 transferase (C3) fusion protein expression vector (scAAV2-Trx2-C3). We evaluated the therapeutic effects of this vector in vitro and in vivo using dexamethasone (DEX)-induced glaucoma models. We found that scAAV2-Trx2-C3-treated HeLa cells had significantly reduced GTP-bound active RhoA and increased phosphor-cofilin Ser3 protein expression levels. scAAV2-Trx2-C3 was also shown to inhibit oxidative stress, fibronectin expression, and alpha-SMA expression in DEX-treated HeLa cells. NeuN immunostaining and TUNEL assay in mouse retinal tissues was performed to evaluate its neuroprotective effect upon RGCs, whereas changes in mouse IOP were monitored via rebound tonometer. The present study showed that scAAV2-Trx2-C3 can protect RGCs from degeneration and reduce IOP in a DEX-induced mouse model of glaucoma, while immunohistochemistry revealed that the expression of fibronectin and alpha-SMA was decreased after the transduction of scAAV2-Trx2-C3 in murine eye tissues. Our results suggest that AAV2-Trx2-C3 modulates the outflow resistance of the trabecular meshwork, protects retinal and other ocular tissues from oxidative damage, and may lead to the development of a gene therapeutic for glaucoma.

Biokinetics of subacutely co-inhaled same size gold and silver nanoparticles.

BACKGROUND: Toxicokinetics of nanomaterials, including studies on the absorption, distribution, metabolism, and elimination of nanomaterials, are essential in assessing their potential health effects. The fate of nanomaterials after inhalation exposure to multiple nanomaterials is not clearly understood. METHODS: Male Sprague-Dawley rats were exposed to similar sizes of silver nanoparticles (AgNPs, 10.86 nm) and gold nanoparticles (AuNPs, 10.82 nm) for 28 days (6-h/day, 5-days/week for four weeks) either with separate NP inhalation exposures or with combined co-exposure in a nose-only inhalation system. Mass concentrations sampled from the breathing zone were AuNP 19.34 ± 2.55 µg/m3 and AgNP 17.38 ± 1.88 µg/m3 for separate exposure and AuNP 8.20 µg/m3 and AgNP 8.99 µg/m3 for co-exposure. Lung retention and clearance were previously determined on day 1 (6-h) of exposure (E-1) and on post-exposure days 1, 7, and 28 (PEO-1, PEO-7, and PEO-28, respectively). In addition, the fate of nanoparticles, including translocation and elimination from the lung to the major organs, were determined during the post-exposure observation period. RESULTS: AuNP was translocated to the extrapulmonary organs, including the liver, kidney, spleen, testis, epididymis, olfactory bulb, hilar and brachial lymph nodes, and brain after subacute inhalation and showed biopersistence regardless of AuNP single exposure or AuNP + AgNP co-exposure, showing similar elimination half-time. In contrast, Ag was translocated to the tissues and rapidly eliminated from the tissues regardless of AuNP co-exposure. Ag was continually accumulated in the olfactory bulb and brain and persistent until PEO-28. CONCLUSION: Our co-exposure study of AuNP and AgNP indicated that soluble AgNP and insoluble AuNP translocated differently, showing soluble AgNP could be dissolved into Ag ion to translocate to the extrapulmonary organs and rapidly removed from most organs except the brain and olfactory bulb. Insoluble AuNPs were continually translocated to the extrapulmonary organs, and they were not eliminated rapidly.

The Acute and Short-Term Inhalation of Carbon Nanofiber in Sprague-Dawley Rats.

The inhalation toxicity of carbon nanofibers (CNFs) is not clearly known due to relatively few related studies reported. An acute inhalation study and short-term inhalation study (5 days) were therefore conducted using Sprague-Dawley rats. In the acute inhalation study, the rats were grouped and exposed to a fresh air control or to low (0.238 ± 0.197), moderate (1.935 ± 0.159), or high (24.696 ± 6.336 mg/m3) CNF concentrations for 6 h and thereafter sacrificed at 14 days. For the short-term inhalation study, the rats were grouped and exposed to a fresh air control or low (0.593 ± 0.019), moderate (2.487 ± 0.213), or high (10.345 ± 0.541 mg/m3) CNF concentrations for 6 h/day for 5 days and sacrificed at 1, 3, and 21 days post-exposure. No mortality was observed in the acute inhalation study. Thus, the CNF LC50 was higher than 25 mg/m3. No significant body or organ weight changes were noted during the 5 days short-term inhalation study or during the post-exposure period. No significant effects of toxicological importance were observed in the hematological, blood biochemical, and coagulation tests. In addition, the bronchoalveolar lavage (BAL) fluid cell differential counts and BAL inflammatory markers showed no CNF-exposure-relevant changes. The histopathological examination also found no CNF-exposure-relevant histopathological lesions. Thus, neither acute nor 5 days inhalation exposure to CNFs induced any noticeable toxicological responses.

Lung retention and particokinetics of silver and gold nanoparticles in rats following subacute inhalation co-exposure.

BACKGROUND: Inhalation exposure to nanomaterials in workplaces can include a mixture of multiple nanoparticles. Such ambient nanoparticles can be of high dissolution or low dissolution in vivo and we wished to determine whether co-exposure to particles with different dissolution rates affects their biokinetics. METHODS AND RESULTS: Rats were exposed to biosoluble silver nanoparticles (AgNPs, 10.86 nm) and to biopersistent gold nanoparticles (AuNPs, 10.82 nm) for 28 days (6-h/day, 5-days/week for 4 weeks) either with separate NP inhalation exposures or with combined co-exposure. The separate NPs mass concentrations estimated by the differential mobility analyzer system (DMAS) were determined to be 17.68 ± 1.69 µg/m3 for AuNP and 10.12 ± 0.71 µg/m3 for AgNP. In addition, mass concentrations analyzed by atomic absorption spectrometer (AAS) via filter sampling were for AuNP 19.34 ± 2.55 µg/m3 and AgNP 17.38 ± 1.88 µg/m3 for separate exposure and AuNP 8.20 ± 1.05 µg/m3 and AgNP 8.99 ± 1.77 µg/m3 for co-exposure. Lung retention and clearance were determined on day 1 (6-h) of exposure (E-1) and on post-exposure days 1, 7, and 28 (PEO-1, PEO-7, and PEO-28, respectively). While the AgNP and AuNP deposition rates were determined to be similar due to the similarity of NP size of both aerosols, the retention half-times and clearance rates differed due to the difference in dissolution rates. Thus, when comparing the lung burdens following separate exposures, the AgNP retention was 10 times less than the AuNP retention at 6-h (E-1), and 69, 89, and 121 times lower less than the AuNP retention at PEO-1, PEO-7, and PEO-28, respectively. In the case of AuNP+AgNP co-exposure, the retained AgNP lung burden was 14 times less than the retained AuNP lung burden at E-1, and 26, 43, and 55 times less than the retained AuNP lung burden at PEO-1, PEO-7, and PEO-28, respectively. The retention of AuNP was not affected by the presence of AgNP, but AgNP retention was influenced in the presence of AuNP starting at 24 h after the first day of post day of exposure. The clearance of AgNPs of the separate exposure showed 2 phases; fast (T1/2 3.1 days) and slow (T1/2 48.5 days), while the clearance of AuNPs only showed one phase (T1/2 .81.5 days). For the co-exposure of AuNPs+AgNPs, the clearance of AgNPs also showed 2 phases; fast (T1/2 2.2 days) and slow (T1/2 28.4 days), while the clearance of AuNPs consistently showed one phase (T1/2 54.2 days). The percentage of Ag lung burden in the fast and slow clearing lung compartment was different between separate and combined exposure. For the combined exposure, the slow and fast compartments were each 50% of the lung burden. For the single exposure, 1/3 of the lung burden was cleared by the fast rate and 2/3 of the lung burden by the slow rate. CONCLUSIONS: The clearance of AgNPs follows a two- phase model of fast and slow dissolution rates while the clearance of AuNPs could be described by a one- phase model with a longer half-time. The co-exposure of AuNPs+AgNPs showed that the clearance of AgNPs was altered by the presence of AuNPs perhaps due to some interaction between AgNP and AuNP affecting dissolution and/or mechanical clearance of AgNP in vivo.

Even lobar deposition of poorly soluble gold nanoparticles (AuNPs) is similar to that of soluble silver nanoparticles (AgNPs).

BACKGROUND: Information on particle deposition, retention, and clearance is important when evaluating the risk of inhaled nanomaterials to human health. The revised Organization Economic Cooperation and Development (OECD) inhalation toxicity test guidelines now require lung burden measurements of nanomaterials after rodent subacute and sub-chronic inhalation exposure (OECD 412, OECD 413) to inform on lung clearance behavior and translocation after exposure and during post-exposure observation (PEO). Lung burden measurements are particularly relevant when the testing chemical is a solid poorly soluble nanomaterial. Previously, the current authors showed that total retained lung burden of inhaled soluble silver nanoparticles (AgNPs) could be effectively measured using any individual lung lobe. METHODS AND RESULTS: Accordingly, the current study investigated the evenness of deposition/retention of poorly soluble gold nanoparticles (AuNPs) after 1 and 5 days of inhalation exposure. Rats were exposed nose-only for 1 or 5 days (6 h/day) to an aerosol of 11 nm well-dispersed AuNPs. Thereafter, the five lung lobes were separated and the gold concentrations measured using an inductively coupled plasma-mass spectrophotometer (ICP-MS). The results showed no statistically significant difference in the AuNP deposition/retention among the different lung lobes in terms of the gold mass per gram of lung tissue. CONCLUSIONS: Thus, it would seem that any rat lung lobe can be used for the lung burden analysis after short or long-term NP inhalation, while the other lobes can be used for collecting and analyzing the bronchoalveolar lavage fluid (BALF) and for the histopathological analysis. Therefore, combining the lung burden measurement, histopathological tissue preparation, and BALF assay from one rat can minimize the number of animals used and maximize the number of endpoints measured.

Mode of silver clearance following 28-day inhalation exposure to silver nanoparticles determined from lung burden assessment including post-exposure observation periods.

Recently revised OECD inhalation toxicity testing guidelines require measurements of lung burden immediately after and for periods following exposure for nanomaterials. Lung burden is a function of pulmonary deposition and retention of nanoparticles. Using lung burden studies as per OECD guidelines, it may be possible to assess clearance mechanisms of nanoparticles. In this study, male rats were exposed to silver nanoparticle (AgNP) aerosols (18.1-19.6 nm) generated from a spark generator. Exposure groups consisted of (1) control (fresh air), (2) low (31.2 ± 8.5 µg/m3), (3) moderate (81.8 ± 11.4 µg/m3), and (4) high concentrations (115.6 ± 30.5 µg/m3). Rats were exposed for 6-h/day, 5-days/week for 4 weeks (28-days) based on the revised OECD test guideline 412. Bronchoalveolar lavage (BAL) fluids were collected on post-exposure observation (PEO)-1 and PEO-7 days and analyzed for inflammatory cells and inflammatory biomarkers. The lung burdens of Ag from AgNPs were measured on PEO-1, PEO-7, and PEO-28 days to obtain quantitative mass concentrations per lung. Differential counting of blood cells and inflammatory biomarkers in BAL fluid and histopathological evaluation of lung tissue indicated that exposure to the high concentrations of AgNP aerosol induced inflammation at PEO-1, slowly resolved at PEO-7 and completely resolved at PEO-28 days. Lung burden measurement suggested that Ag from AgNPs was cleared through two different modes; fast and slow clearance. The fast clearance component was concentration-dependent with half-times ranging from two to four days and clearance rates of 0.35-0.17/day-1 from low to high concentrations. The slow clearance had half-times of 100, 57, and 76 days and clearance rates of 0.009, 0.012, and 0.007/day-1 for the high, moderate and low concentration exposure. The exact mechanism of clearance is not known currently. The fast clearance component which was concentration-dependent could be dependent on the dissolution of AgNPs and the slow clearance would be due to slow clearance of the low dissolution AgNPs secondary particles originating from silver ions reacting with biogenic anions. These secondary AgNPs might be cleared by mechanisms other than dissolution such as mucociliary escalation, translocation to the lymphatic system or other organs.

28-Day inhalation toxicity study with evaluation of lung deposition and retention of tangled multi-walled carbon nanotubes.

Lung deposition and retention measurements are now required by the newly revised OECD inhalation toxicity testing guidelines 412 and 413 when evaluating the clearance and biopersistence of poorly soluble nanomaterials, such as multi-walled carbon nanotubes (MWCNTs). However, evaluating the lung deposition concentration is challenging with certain nanomaterials, such as carbon-based and iron-based nanomaterials, as it is difficult to differentiate them from endogenous elements. Therefore, the current 28-day inhalation toxicity study investigated the lung retention kinetics of tangled MWCNTs. Male Sprague Dawley rats were exposed to MWCNTs at 0, 0.257, 1.439, and 4.253 mg/m3 for 28 days (6 h/day, 5 days/week, 4 weeks). Thereafter, the rats were sacrificed at day 1, 7, and 28 post-exposure and the pulmonary inflammatory response evaluated by analyzing the bronchoalveolar lavage fluid. Plus, the blood biochemistry, hematology, and histopathology of the lungs were also examined. The lung deposition and retention of MWCNTs were determined based on the elemental carbon content in the lungs after tissue digestion. The number of polymorphonuclear cells and LDH concentration were both found to be significantly higher with the medium and high concentrations (1.439 and 4.253 mg/m3) and dose dependent. The estimated retention half-life for the high concentration (4.253 mg/m3) was about 35 days. The results of this study indicate that tangled MWCNTs seem to have a relatively shorter retention half-life when compared to previous reports on rigid MWCNTs, and the no-observed adverse effect level (NOAEL) for the tested tangled MWCNTs was 0.257 mg/m3 in a previous rat 28-day subacute inhalation toxicity study.

Derivation of occupational exposure limits for multi-walled carbon nanotubes and graphene using subchronic inhalation toxicity data and a multi-path particle dosimetry model.

In this study, we aimed to provide the recommended occupational exposure limits (OELs) for multi-walled carbon nanotubes (MWCNTs) and graphene nanomaterials based on data from a subchronic inhalation toxicity study using a lung dosimetry model. We used a no observed adverse effect level (NOAEL) of 0.98 mg m-3 and 3.02 mg m-3 in rats for MWCNTs and graphene, respectively. The NOAELs were obtained from a 13-week inhalation study in rats. The deposition fractions of MWCNTs and graphene in the respiratory tract of rats and humans were calculated by using the multi-path particle dosimetry model (MPPD model, v3.04). The deposition fraction in the alveolar region was 0.0527 and 0.0984 for MWCNTs and 0.0569 and 0.1043 for graphene in rats and human lungs, respectively. Then, the human equivalent exposure concentrations (HECs) of MWCNTs and graphene were calculated according to the method by the National Institute for Occupational Safety and Health (NIOSH). The HEC was estimated to be 0.17 mg m-3 for MWCNTs and to be 0.54 mg m-3 for graphene, which was relevant to the rat NOAEL of 0.98 mg m-3 and 3.02 mg m-3 for MWCNTs and graphene, respectively. Finally, we estimated the recommended OELs by applying uncertainty factors (UFs) to the HEC as follows: an UF of 3 for species differences (rats to humans), 2 for an experimental duration (subchronic to chronic), and 5 for inter-individual variations among workers. Thus, the OEL was estimated to be 6 µg m-3 for MWCNTs and 18 µg m-3 for graphene. These values could be useful in preventing the adverse health effects of nanoparticles in workers.

Lobar evenness of deposition/retention in rat lungs of inhaled silver nanoparticles: an approach for reducing animal use while maximizing endpoints.

BACKGROUND: Information on particle deposition, retention and clearance are important for the evaluation of the risk of inhaled nanomaterials to human health. Recent revised OECD inhalation toxicity test guidelines require to evaluate the lung burden of nanomaterials after rodent subacute and subchronic inhalation exposure (OECD 412, OECD 413). These revised test guidelines require additional post-exposure observation (PEO) periods that include lung burden measurements that can inform on lung clearance behavior and translocation. The latter being particularly relevant when the testing chemical is a solid poorly soluble nanomaterial. Therefore, in the spirit of 3 R's, we investigated whether measurement of retained lung burden of inhaled nanoparticles (NPs) in individual lung lobes is sufficient to determine retained lung burden in the total lung. If it is possible to use only one lobe, it will reduce animal use and maximize the number of endpoints evaluated. RESULTS: To achieve these goals, rats were exposed nose-only for 1 or 5 days (6 h/day) to an aerosol of 20 nm well-dispersed silver nanoparticles (AgNPs), which is the desired particle diameter resulting in maximum deposition in the pulmonary region when inhaled as singlets. After exposure, the five lung lobes were separated and silver concentration was measured using inductively coupled plasma-mass spectrophotometer (ICP-MS). The results showed that the retention of deposited silver nanoparticle in the different lung lobes did not show any statistically significant difference among lung lobes in terms of silver mass per gram lung lobe. This novel finding of evenness of retention/deposition of inhaled 20 nm NPs in rats for all five lobes in terms of mass per unit tissue weight contrasts with earlier studies reporting greater apical lobe deposition of inhaled micro-particles in rodents. The difference is most likely due to preferred and efficient deposition of inhaled NPs by diffusion vs. additional deposition by sedimentation and impaction for micron-sized particles. CONCLUSION: AgNPs following acute inhalation by rats are evenly retained in each lung lobe in terms of mass per unit lung tissue weight. Accordingly, we suggest sampling any of the rat lung lobes for lung burden analysis can be used to determine deposited or retained total lung burden after short-term inhalation of NPs and using the other lobes for collecting and analyzing bronchoalveolar lavage fluid (BALF) and for histopathological analysis. Therefore, by combining lung burden measurement, histopathological tissue preparation, and BALF assay in the same rat will reduce the number of animals used and maximize the number of endpoints measured.

Short-term inhalation study of graphene oxide nanoplates.

Graphene oxides possess unique physicochemical properties with important potential applications in electronics, pharmaceuticals, and medicine. However, the toxicity following inhalation exposure to graphene oxide has not yet been clarified. Therefore, this study conducted a short-term graphene oxide inhalation toxicity analysis using a nose-only inhalation exposure system and male Sprague-Dawley rats. A total of four groups (15 rats per group) were exposed: (1) control (fresh air), (2) low concentration (0.76 ± 0.16 mg/m3), (3) moderate concentration (2.60 ± 0.19 mg/m3), and (4) high concentration (9.78 ± 0.29 mg/m3). The rats were exposed to graphene oxide for 6 h/day for 5 days, followed by recovery for 1, 3, and 21 days. No significant body or organ weight changes were noted after the short-term exposure or during the recovery period. Similarly, no significant systemic effects of toxicological importance were noted in the hematological assays, bronchoalveolar lavage fluid (BAL) inflammatory markers, BAL fluid cytokines, or blood biochemical assays following the graphene oxide exposure or during the post-exposure observation period. Moreover, no significant differences were observed in the BAL cell differentials, such as lymphocytes, macrophages, or polymorphonuclear cells. Graphene oxide-ingested alveolar macrophages as a spontaneous clearance reaction were observed in the lungs of all the concentration groups from post 1 day to post 21 days. Histopathological examination of the liver and kidneys did not reveal any significant test-article-relevant histopathological lesions. Importantly, similar to previously reported graphene inhalation data, this short-term nose-only inhalation study found only minimal or unnoticeable graphene oxide toxicity in the lungs and other organs.

Subacute inhalation toxicity study of synthetic amorphous silica nanoparticles in Sprague-Dawley rats.

Synthetic amorphous silica nanoparticles (SiNPs) are one of the most applied nanomaterials and are widely used in a broad variety of industrial and biomedical fields. However, no recent long-term inhalation studies evaluating the toxicity of SiNPs are available and results of acute studies are limited. Thus, we conducted a subacute inhalation toxicity study of SiNPs in Sprague-Dawley rats using a nose-only inhalation system. Rats were separated into four groups and target concentrations selected in this study were as follows: control (fresh air), low- (0.407 ± 0.066 mg/m3), middle- (1.439 ± 0.177 mg/m3) and high-concentration group (5.386 ± 0.729 mg/m3), respectively. The rats were exposed to SiNPs for four consecutive weeks (6 hr/day, 5 days/week) except for control group of rats which received filtered fresh air. After 28-days of inhalation exposure to SiNPs, rats were sacrificed after recovery periods of one, seven and 28 days. Although there were minimal toxic changes such as temporary decrease of body weight after exposure, increased levels of red blood cells (RBCs) and hemoglobin (Hb) concentration, the lung histopathological findings and inflammatory markers in bronchoalveolar lavage (BAL) fluid including polymorphonuclear (PMN) leukocyte, lactate dehydrogenase (LDH), albumin and protein did not show significant changes at any recovery period. The results of this study suggest that the subacute inhalation of SiNPs had no toxic effects on the lung of rats at the concentrations and selected time points used in this study.

Down-regulation of MicroRNA-126 in Glioblastoma and its Correlation with Patient Prognosis: A Pilot Study.

Glioblastoma is the most common primary malignant tumor of the adult human brain. Although microRNA-126 (miR-126) has been reported to exhibit expression abnormalities in various types of cancer, to date very few studies have examined changes in miR-126 level in glioblastoma. In this pilot study, we investigated the changes in miR-126 expression in newly-dissected primary glioblastoma to explore possible roles of miR-126 in patient prognosis. Total RNA was extracted from tumoral and adjacent non-cancerous tissues from 14 patients' paired frozen specimens. Using an established quantitative reverse transcriptase-PCR protocol, the levels of miR-126 in glioblastoma and adjacent non-tumor brain tissues were compared against small nucleolar RNA U48 (RNU48) as a reference gene. The expression of miR-126 in glioblastoma samples was significantly lower than in paired non-tumoral controls (p<0.05). Importantly, age-adjusted analyses suggest that glioblastoma patients with higher relative intratumoral miR-126 expression (i.e. 53-79% relative to that of the control tissue; n=7) had significantly improved survival duration than patients whose miR-126 levels were lower (i.e. 12-48%, n=7; stratified log-rank analysis p=0.011 when the dividing threshold was set at ≥51%; total: n=14, male: 8; female: 6). Thus, intraglioblastoma miR-126 may be down-regulated relative to normal tissue and patients with less down-regulation of intratumoral miR-126 expression could have improved postsurgical prognosis. Future clinical studies with larger sample sizes should be performed to validate this observation.

Exposure monitoring of graphene nanoplatelets manufacturing workplaces.

Graphenes have emerged as a highly promising, two-dimensional engineered nanomaterial that can possibly substitute carbon nanotubes. They are being explored in numerous R&D and industrial applications in laboratories across the globe, leading to possible human and environmental exposures to them. Yet, there are no published data on graphene exposures in occupational settings and no readily available methods for their detection and quantitation exist. This study investigates for the first time the potential exposure of workers and research personnel to graphenes in two research facilities and evaluates the status of the control measures. One facility manufactures graphene using graphite exfoliation and chemical vapor deposition (CVD), while the other facility grows graphene on a copper plate using CVD, which is then transferred to a polyethylene terephthalate (PET) sheet. Graphene exposures and process emissions were investigated for three tasks - CVD growth, exfoliation, and transfer - using a multi-metric approach, which utilizes several direct reading instruments, integrated sampling, and chemical and morphological analysis. Real-time instruments included a dust monitor, condensation particle counter (CPC), nanoparticle surface area monitor, scanning mobility particle sizer, and an aethalometer. Morphologically, graphenes and other nanostructures released from the work process were investigated using a transmission electron microscope (TEM). Graphenes were quantified in airborne respirable samples as elemental carbon via thermo-optical analysis. The mass concentrations of total suspended particulate at Workplaces A and B were very low, and elemental carbon concentrations were mostly below the detection limit, indicating very low exposure to graphene or any other particles. The real-time monitoring, especially the aethalometer, showed a good response to the released black carbon, providing a signature of the graphene released during the opening of the CVD reactor at Workplace A. The TEM observation of the samples obtained from Workplaces A and B showed graphene-like structures and aggregated/agglomerated carbon structures. Taken together, the current findings on common scenarios (exfoliation, CVD growth, and transfer), while not inclusive of all graphene manufacturing processes, indicate very minimal graphene or particle exposure at facilities manufacturing graphenes with good manufacturing practices.

Case study on risk evaluation of printed electronics using nanosilver ink.

BACKGROUND: With the ever-increasing development of nanotechnology, our society is being surrounded by possible risks related to exposure to manufactured nanomaterials. The consumer market already includes many products that contain silver nanoparticles (AgNPs), including various household products, such as yoga mats, cutting boards, running shirts, and socks. There is a growing concern over the release of AgNPs in workplaces related to the manufacture and application of nanomaterials. OBJECTIVE: This study investigated the release of AgNPs during the operation of a printed electronics printer. METHODS: Using an exposure simulation chamber, a nanoparticle collector, scanning mobility particle sizer (SMPS), condensation particle counter (CPC), dust monitor, and mixed cellulose ester (MCE) filters are all connected to measure the AgNP exposure levels when operating a printed electronics printer. RESULTS: A very small amount of AgNPs was released during the operation of the printed electronics printer, and the number of AgNPs inside the exposure simulation chamber was lower than that outside background. In addition, when evaluating the potential risks for consumers and workers using a margin of exposure (MOE) approach and target MOE of 1000, the operational results far exceeded the target MOE in this simulation study and in a previous workplace exposure study. CONCLUSION: The overall results indicate a no-risk concern level in the case of printed electronics using nanosilver ink.

28-Day inhalation toxicity of graphene nanoplatelets in Sprague-Dawley rats.

Graphene, a two-dimensional engineered nanomaterial, is now being used in many applications, such as electronics, biological engineering, filtration, lightweight and strong nanocomposite materials, and energy storage. However, there is a lack of information on the potential health effects of graphene in humans based on inhalation, the primary engineered nanomaterial exposure pathway in workplaces. Thus, an inhalation toxicology study of graphene was conducted using a nose-only inhalation system for 28 days (6 h/day and 5 days/week) with male Sprague-Dawley rats that were then allowed to recover for 1-, 28-, and 90-day post-exposure period. Animals were separated into 4 groups (control, low, moderate, and high) with 15 male rats (5 rats per time point) in each group. The measured mass concentrations for the low, moderate, and high exposure groups were 0.12, 0.47, and 1.88 mg/m(3), respectively, very close to target concentrations of 0.125, 0.5, and 2 mg/m(3). Airborne graphene exposure was monitored using several real-time instrumentation over 10 nm to 20 µm for size distribution and number concentration. The total and respirable elemental carbon concentrations were also measured using filter sampling. Graphene in the air and biological media was traced using transmission electron microscopy. In addition to mortality and clinical observations, the body weights and food consumption were recorded weekly. At the end of the study, the rats were subjected to a full necropsy, blood samples were collected for blood biochemical tests, and the organ weights were measured. No dose-dependent effects were recorded for the body weights, organ weights, bronchoalveolar lavage fluid inflammatory markers, and blood biochemical parameters at 1-day post-exposure and 28-day post-exposure. The inhaled graphenes were mostly ingested by macrophages. No distinct lung pathology was observed at the 1-, 28- and 90-day post-exposure. The inhaled graphene was translocated to lung lymph nodes. The results of this 28-day graphene inhalation study suggest low toxicity and a NOAEL of no less than 1.88 mg/m(3).

Pulmonary Responses of Sprague-Dawley Rats in Single Inhalation Exposure to Graphene Oxide Nanomaterials.

Graphene is receiving increased attention due to its potential widespread applications in future. However, the health effects of graphene have not yet been well studied. Therefore, this study examined the pulmonary effects of graphene oxide using male Sprague-Dawley rats and a single 6-hour nose-only inhalation technique. Following the exposure, the rats were allowed to recover for 1 day, 7 days, or 14 days. A total of three groups were compared: control (fresh air), low concentration (0.46 ± 0.06 mg/m(3)), and high concentration (3.76 ± 0.24 mg/m(3)). The exposure to graphene oxide did not induce significant changes in the body weights, organ weights, and food consumption during the 14 days of recovery time. The microalbumin and lactate dehydrogenase levels in the bronchoalveolar lavage (BAL) fluid were not significantly changed due to the exposure. Similarly, total cell count, macrophages, polymorphonuclear leukocytes, and lymphocytes were not significantly altered in the BAL fluid. Plus, the histopathological examination of the rat lungs only showed an uptake of graphene oxide in the alveolar macrophages of the high-concentration group. Therefore, these results demonstrate that the single inhalation exposure to graphene oxide induce minimal toxic responses in rat lungs at the concentrations and time points used in the present study.

Aquatic Toxicity Comparison of Silver Nanoparticles and Silver Nanowires.

To better understand the potential ecotoxicological impact of silver nanoparticles (AgNPs) and silver nanowires (AgNWs) released into freshwater environments, the toxicities of these nanomaterials were assessed and compared using Organization for Economic Cooperation and Development (OECD) test guidelines, including a "Daphnia sp., acute immobilization test," "Fish, acute toxicity test," and "freshwater alga and cyanobacteria, growth inhibition test." Based on the estimated median lethal/effective concentrations of AgNPs and AgNWs, the susceptibility to the nanomaterials was different among test organisms (daphnia > algae > fish), suggesting that the AgNPs are classified as "category acute 1" for Daphnia magna, "category acute 2" for Oryzias latipes, and "category acute 1" for Raphidocelis subcapitata, while the AgNWs are classified as "category acute 1" for Daphnia magna, "category acute 2" for Oryzias latipes, and "category acute 2" for Raphidocelis subcapitata, according to the GHS (Globally Harmonized System of Classification and Labelling of Chemicals). In conclusion, the present results suggest that more attention should be paid to prevent the accidental or intentional release of silver nanomaterials into freshwater aquatic environments.

5-Day repeated inhalation and 28-day post-exposure study of graphene.

Graphene has recently been attracting increasing attention due to its unique electronic and chemical properties and many potential applications in such fields as semiconductors, energy storage, flexible electronics, biosensors and medical imaging. However, the toxicity of graphene in the case of human exposure has not yet been clarified. Thus, a 5-day repeated inhalation toxicity study of graphene was conducted using a nose-only inhalation system for male Sprague-Dawley rats. A total of three groups (20 rats per group) were compared: (1) control (ambient air), (2) low concentration (0.68 ± 0.14 mg/m(3) graphene) and (3) high concentration (3.86 ± 0.94 mg/m(3) graphene). The rats were exposed to graphene for 6 h/day for 5 days, followed by recovery for 1, 3, 7 or 28 days. The bioaccumulation and macrophage ingestion of the graphene were evaluated in the rat lungs. The exposure to graphene did not change the body weights or organ weights of the rats after the 5-day exposure and during the recovery period. No statistically significant difference was observed in the levels of lactate dehydrogenase, protein and albumin between the exposed and control groups. However, graphene ingestion by alveolar macrophages was observed in the exposed groups. Therefore, these results suggest that the 5-day repeated exposure to graphene only had a minimal toxic effect at the concentrations and time points used in this study.

Acute toxicity comparison of single-walled carbon nanotubes in various freshwater organisms.

While the commercialization of single-walled carbon nanotubes (SWCNTs) is rapidly expanding, the environmental impact of this nanomaterial is not well understood. Therefore, the present study evaluates the acute aquatic toxicity of SWCNTs towards two freshwater microalgae (Raphidocelis subcapitata and Chlorella vulgaris), a microcrustacean (Daphnia magna), and a fish (Oryzias latipes) based on OECD test guidelines (201, 202, and 203). According to the results, the SWCNTs inhibited the growth of the algae R. subcapitata and C. vulgaris with a median effective concentration (EC50) of 29.99 and 30.96 mg/L, respectively, representing "acute category 3" in the Globally Harmonized System (GHS) of classification and labeling of chemicals. Meanwhile, the acute toxicity test using O. latipes and D. magna did not show any mortality/immobilizing effects up to a concentration of 100.00 mg/L SWCNTs, indicating no hazard category in the GHS classification. In conclusion, SWCNTs were found to induce acute ecotoxicity in freshwater microalgae, yet not in D. magna and medaka fish.