Plasma extracellular vesicle proteins as promising noninvasive biomarkers for diagnosis of idiopathic pulmonary fibrosis.

High-resolution computed tomography (HRCT) imaging is critical for diagnostic evaluation of Idiopathic Pulmonary Fibrosis (IPF). However, several other interstitial lung diseases (ILDs) often exhibit radiologic pattern similar to IPF on HRCT making the diagnosis of the disease difficult. Therefore, biomarkers that distinguish IPF from other ILDs can be a valuable aid in diagnosis. Using mass spectrometry, we performed proteomic analysis of plasma extracellular vesicles (EVs) in patients diagnosed with IPF, chronic hypersensitivity pneumonitis, nonspecific interstitial pneumonitis, and healthy subjects. A five-protein signature was identified by lasso regression and was validated in an independent cohort using ELISA. The five-protein signature derived from mass spectrometry data showed an area under the receiver operating characteristic curve of 0.915 (95%CI: 0.819-1.011) and 0.958 (95%CI: 0.882-1.034) for differentiating IPF from other ILDs and from healthy subjects, respectively. Stepwise backwards elimination yielded a model with 3 and 2 proteins for discriminating IPF from other ILDs and healthy subjects, respectively, without compromising diagnostic accuracy. In summary, we discovered and validated EV protein biomarkers for differential diagnosis of IPF in independent cohorts. Interestingly, the biomarker panel could also distinguish IPF and healthy subjects with high accuracy. The biomarkers need to be evaluated in large prospective cohorts to establish their clinical utility.

A comparison of clinically relevant sources of mesenchymal stem cell-derived exosomes: Bone marrow and amniotic fluid.

BACKGROUND/PURPOSE: Exosomes may constitute a more practical alternative to live cells in select stem cell-based therapies. We sought to compare exosomes from two mesenchymal stem cell (MSC) sources relevant to perinatal and pediatric diseases. METHODS: Exosomes were isolated by reagent-enhanced centrifugation from cell culture media of banked human bone marrow (bm) and amniotic fluid (af) MSCs after serum starvation. Characterization was by flow exometry for tetraspanin markers CD9, CD63, and CD81, transmission electron microscopy for size and morphology, and tunable resistive pulse sensing for size distribution and concentration. Statistical comparisons of count data were made by Poisson regression modeling and Student's T-test. RESULTS: Exosomes of appropriate size and morphology were isolated with comparable expressions of CD9 (96% vs. 94%), CD63 (88% vs. 66%), and CD81 (71% vs. 63%) for bmMSC and afMSC, respectively. Total exosome yield (particles/mL) adjusted for number of cells was higher from afMSCs than bmMSCs by an estimated 25% (P < 0.001). CONCLUSIONS: While bone marrow and amniotic fluid mesenchymal stem cells are comparable sources of exosomes in size distribution, morphology, and expression of typical surface markers, yield may be higher from amniotic fluid cells. The amniotic fluid appears to be a preferable source of exosomes for clinical applications. LEVEL OF EVIDENCE: N/A (bench laboratory study).

High-Resolution Single Particle Zeta Potential Characterisation of Biological Nanoparticles using Tunable Resistive Pulse Sensing.

Physicochemical properties of nanoparticles, such as size, shape, surface charge, density, and porosity play a central role in biological interactions and hence accurate determination of these characteristics is of utmost importance. Here we propose tunable resistive pulse sensing for simultaneous size and surface charge measurements on a particle-by-particle basis, enabling the analysis of a wide spectrum of nanoparticles and their mixtures. Existing methodologies for measuring zeta potential of nanoparticles using resistive pulse sensing are significantly improved by including convection into the theoretical model. The efficacy of this methodology is demonstrated for a range of biological case studies, including measurements of mixed anionic, cationic liposomes, extracellular vesicles in plasma, and in situ time study of DNA immobilisation on the surface of magnetic nanoparticles. The high-resolution single particle size and zeta potential characterisation will provide a better understanding of nano-bio interactions, positively impacting nanomedicine development and their regulatory approval.

Additive Impairment of Synaptic Signaling in Cultured Cortical Neurons by Exogenously-Applied Oligomerized Amyloid-ß and Airborne Nanoparticles Generated during Photocopying.

Photocopying in offices and printing centers releases nanoparticles that can reach the brain following inhalation. We examined whether subcytotoxic levels of airborne photocopy-emitted nanoparticles could potentiate perturbation of synaptic signaling in cultured neurons following exposure to amyloid-ß (Aß). Signaling was only transiently inhibited by Aß or nanoparticles individually, but remained statistically reduced in cultures receiving both after 24 h. In vitro and in vivo studies with copier emitted nanoparticles have consistently demonstrated inflammation, oxidative stress, and cytotoxicity. Since Aß can accumulate years before cognitive decline, subcytotoxic levels of nanoparticles are one factor that could potentiate Aß-induced impairment of synaptic activity during these early stages.

Linking Exposures of Particles Released From Nano-Enabled Products to Toxicology: An Integrated Methodology for Particle Sampling, Extraction, Dispersion, and Dosing.

Nano-enabled products (NEPs) represent a growing economic global market that integrates nanotechnology into our everyday lives. Increased consumer use and disposal of NEPs at their end of life has led to increased environmental, health and safety (EHS) concerns, due to the potential environmental release of constituent engineered nanomaterials (ENMs) used in the production of NEPs. Although, there is an urgent need to assess particulate matter (PM) release scenarios and potential EHS implications, no current standardized methodologies exist across the exposure-toxicological characterization continuum. Here, an integrated methodology is presented, that can be used to sample, extract, disperse and estimate relevant dose of life cycle-released PM (LCPM), for in vitro and in vivo toxicological studies. The proposed methodology was utilized to evaluate two "real world" LCPM systems simulating consumer use and disposal of NEPs. This multi-step integrated methodology consists of: (1) real-time monitoring and sampling of size fractionated LCPM; (2) efficient extraction of LCPM collected on substrates using aqueous or ethanol extraction protocols to ensure minimal physicochemical alterations; (3) optimized LCPM dispersion preparation and characterization; (4) use of dosimetric techniques for in vitro and in vivo toxicological studies. This comprehensive framework provides a standardized protocol to assess the release and toxicological implications of ENMs released across the life cycle of NEPs and will help in addressing important knowledge gaps in the field of nanotoxicology.

Implications of in vitro dosimetry on toxicological ranking of low aspect ratio engineered nanomaterials.

In vitro high throughput screening platforms based on mechanistic injury pathways are been used for hazard assessment of engineered nanomaterials (ENM). Toxicity screening and other in vitro nanotoxicology assessment efforts in essence compare and rank nanomaterials relative to each other. We hypothesize that this ranking of ENM is susceptible to dispersion and dosimetry protocols, which continue to be poorly standardized. Our objective was to quantitate the impact of dosimetry on toxicity ranking of ENM. A set of eight well-characterized and diverse low aspect ratio ENMs, were utilized. The recently developed in vitro dosimetry platform at Harvard, which includes preparation of fairly monodispersed suspensions, measurement of the effective density of formed agglomerates in culture media and fate and transport modeling was used for calculating the effective dose delivered to cells as a function of time. Changes in the dose-response relationships between the administered and delivered dose were investigated with two representative endpoints, cell viability and IL-8 production, in the human monocytic THP-1 cells. The slopes of administered/delivered dose-response relationships changed 1:4.94 times and were ENM-dependent. The overall relative ranking of ENM intrinsic toxicity also changed considerably, matching notably better the in vivo inflammation data (R(2 )= 0.97 versus 0.64). This standardized dispersion and dosimetry methodology presented here is generalizable to low aspect ratio ENMs. Our findings further reinforce the need to reanalyze and reinterpret in vitro ENM hazard ranking data published in the nanotoxicology literature in the light of dispersion and dosimetry considerations (or lack thereof) and to adopt these protocols in future in vitro nanotoxicology testing.

High resolution characterization of engineered nanomaterial dispersions in complex media using tunable resistive pulse sensing technology.

In vitro toxicity assessment of engineered nanomaterials (ENM), the most common testing platform for ENM, requires prior ENM dispersion, stabilization, and characterization in cell culture media. Dispersion inefficiencies and active aggregation of particles often result in polydisperse and multimodal particle size distributions. Accurate characterization of important properties of such polydisperse distributions (size distribution, effective density, charge, mobility, aggregation kinetics, etc.) is critical for understanding differences in the effective dose delivered to cells as a function of time and dispersion conditions, as well as for nano-bio interactions. Here we have investigated the utility of tunable nanopore resistive pulse sensing (TRPS) technology for characterization of four industry relevant ENMs (oxidized single-walled carbon nanohorns, carbon black, cerium oxide and nickel nanoparticles) in cell culture media containing serum. Harvard dispersion and dosimetry platform was used for preparing ENM dispersions and estimating delivered dose to cells based on dispersion characterization input from dynamic light scattering (DLS) and TRPS. The slopes of cell death vs administered and delivered ENM dose were then derived and compared. We investigated the impact of serum protein content, ENM concentration, and cell medium on the size distributions. The TRPS technology offers higher resolution and sensitivity compared to DLS and unique insights into ENM size distribution and concentration, as well as particle behavior and morphology in complex media. The in vitro dose-response slopes changed significantly for certain nanomaterials when delivered dose to cells was taken into consideration, highlighting the importance of accurate dispersion and dosimetry in in vitro nanotoxicology.

Development of therapeutic polymeric nanoparticles for the resolution of inflammation.

Liver X receptors (LXRs) attenuate inflammation by modulating the expression of key inflammatory genes, making LXRs and their ligands particularly attractive candidates for therapeutic intervention in cardiovascular, metabolic, and/or inflammatory diseases. Herein, enhanced proresolving activity of polymeric nanoparticles (NPs) containing the synthetic LXR agonist GW3965 (LXR-NPs) is demonstrated, developed from a combinatorial library of more than 70 formulations with variations in critical physicochemical parameters. In vitro studies on peritoneal macrophages confirm that LXR-NPs are significantly more effective than the free agonist at downregulating pro-inflammatory mediators (MCP-1 and TNFα), as well as inducing the expression of LXR target genes (ABCA1 and SREBP1c). Through a zymosan-induced acute peritonitis in vivo model, LXR-NPs are found to be more efficient than free GW3965 at limiting the recruitment of polymononuclear neutrophils (50% vs 17%), suppressing the gene expression and secretion of pro-inflammatory factors MCP-1 and TNFα in peritoneal macrophages, and decreasing the resolution interval up to 4 h. Furthermore, LXR-NPs suppress the secretion of MCP-1 and TNFα by monocytes and macrophages more efficiently than the commercial drug dexamethasone. Overall, these findings demonstrate that LXR-NPs are capable of promoting resolution of inflammation and highlight the prospect of LXR-based nanotherapeutics for inflammatory diseases.

Toxicological effects of PM0.25-2.0 particles collected from a photocopy center in three human cell lines.

Printing devices such as photocopiers and printers emit predominantly nanoparticles, which may aggregate with time to form PM0.25-2.0 particles. To date, there are no reports on cytotoxic or genotoxic effects of PM0.25-2.0 particles emitted from photocopiers. To investigate the ability of PM0.25-2 fraction emitted from photocopiers, induce pro-inflammatory cytokines, DNA damage and apoptosis in different human-derived cell lines. Three cell types, i.e. a THP-1 line, primary human nasal and small airway epithelial cells, were used. The airborne PM0.25-2.0 size fraction collected from a photocopy center was characterized for its physicochemical and morphological properties, dispersed in culture media and cells were treated with 30, 100 or 300 µg/ml doses. Levels of 13 cytokines and chemokines in the culture medium harvested at 6 and 24 h of treatment were measured using Luminex cytokine kits. In cells harvested at the same timepoints, DNA damage in cells was studied by a Comet assay, and apoptosis was measured by cytofluorimetry using an Annexin V staining kit. The results indicate that in THP-1 cells, several cytokines (IL-6, IL-8, TNFα and IL-1ß) were significantly elevated. Only IL-8 was significantly elevated in the primary nasal and small airway cells. Cells exposed to PM0.25-2.0 underwent apoptosis in a dose-dependent manner, but no significant differences were found in the extent of DNA damage at either timepoint. Airborne PM0.25-2.0 collected at one photocopier center was capable of inducing several pro-inflammatory cytokines and apoptosis, but no genotoxicity, in all cell lines suggesting a role for PM0.25-2.0 in our previously documented airway inflammation in human volunteers. Further toxicological evaluations of these particles across different toner manufacturers are warranted.

Evaluation of cytotoxic, genotoxic and inflammatory responses of nanoparticles from photocopiers in three human cell lines.

BACKGROUND: Photocopiers emit nanoparticles with complex chemical composition. Short-term exposures to modest nanoparticle concentrations triggered upper airway inflammation and oxidative stress in healthy human volunteers in a recent study. To further understand the toxicological properties of copier-emitted nanoparticles, we studied in-vitro their ability to induce cytotoxicity, pro-inflammatory cytokine release, DNA damage, and apoptosis in relevant human cell lines. METHODS: Three cell types were used: THP-1, primary human nasal- and small airway epithelial cells. Following collection in a large volume photocopy center, nanoparticles were extracted, dispersed and characterized in the cell culture medium. Cells were doped at 30, 100 and 300 µg/mL administered doses for up to 24 hrs. Estimated dose delivered to cells, was ~10% and 22% of the administered dose at 6 and 24 hrs, respectively. Gene expression analysis of key biomarkers was performed using real time quantitative PCR (RT-qPCR) in THP-1 cells at 5 µg nanoparticles/mL for 6-hr exposure for confirmation purposes. RESULTS: Multiple cytokines, GM-CSF, IL-1ß, IL-6, IL-8, IFNγ, MCP-1, TNF-α and VEGF, were significantly elevated in THP-1 cells in a dose-dependent manner. Gene expression analysis confirmed up-regulation of the TNF-α gene in THP-1 cells, consistent with cytokine findings. In both primary epithelial cells, cytokines IL-8, VEGF, EGF, IL-1α, TNF-α, IL-6 and GM-CSF were significantly elevated. Apoptosis was induced in all cell lines in a dose-dependent manner, consistent with the significant up-regulation of key apoptosis-regulating genes P53 and Casp8 in THP-1 cells. No significant DNA damage was found at any concentration with the comet assay. Up-regulation of key DNA damage and repair genes, Ku70 and Rad51, were also observed in THP-1 cells, albeit not statistically significant. Significant up-regulation of the key gene HO1 for oxidative stress, implicates oxidative stress induced by nanoparticles. CONCLUSIONS: Copier-emitted nanoparticles induced the release of pro-inflammatory cytokines, apoptosis and modest cytotoxicity but no DNA damage in all three-human cell lines. Taken together with gene expression data in THP-1 cells, we conclude that these nanoparticles are directly responsible for inflammation observed in human volunteers. Further toxicological evaluations of these nanoparticles, including across different toner formulations, are warranted.

Mapping the biological oxidative damage of engineered nanomaterials.

Novel engineered nanomaterials (ENMs) are being introduced into the market rapidly with little understanding of their potential toxicity. Each ENM is a complex combination of diverse sizes, surface chemistries, crystallinity, and metal impurities. Variability in physicochemical properties is poorly understood but is critically important in revealing adverse effects of ENMs. A need also exists for discovering broad relationships between variations in these physicochemical parameters and toxicological endpoints of interest. Biological oxidative damage (BOD) has been recognized as a key mechanism of nanotoxicity. An assortment of 138 ENMs representing major classes are evaluated for BOD elicited (net decrease in the antioxidant capacity of ENM-exposed human blood serum, as compare to unexposed serum) using the 'Ferric Reducing Ability of Serum' (FRAS) assay. This robust and high-throughput approach has the ability to determine the co-effects which multiple physicochemical characteristics impart on oxidative potential, and subsequently to identify and quantify the influence of individual factors. FRAS BOD approach demonstrated the potential for preliminary evaluation of potential toxicity of ENMs, mapping the within- and between-class variability of ENMs, ranking the potential toxicity by material class, and prioritizing the ENMs for further toxicity evaluation and risk assessment.

Nanoparticles from photocopiers induce oxidative stress and upper respiratory tract inflammation in healthy volunteers.

Photocopiers emit large quantities of nanoparticles (NPs); however, their toxicological properties have not been studied. Here we investigate for the first time early human responses following a day's exposure to NPs from photocopiers. Nine healthy subjects spent 6 h at a busy photocopy centre on 2-3 randomly selected days. Matched nasal lavage and urine samples were collected before and at different time points post-exposure. Nasal lavage samples were analysed for 14 cytokines, inflammatory cells and total protein. Urine samples were analysed for 8-hydroxydeoxyguanosine (8-OH-dG). Exposure assessment was conducted using a suite of instruments. The mean total particle number on exposure days was >5 times higher than background, with size distributions in nanoscale range (peak 30-40 nm). Following exposure, 8-OH-dG and several pro-inflammatory cytokines were elevated 2-10 folds compared with pre-exposure levels and remained elevated for up to 36 h. We conclude that NPs from photocopiers induce upper airway inflammation and oxidative stress.

Screening for Oxidative Stress Elicited by Engineered Nanomaterials: Evaluation of Acellular DCFH Assay.

The DCFH assay is commonly used for measuring free radicals generated by engineered nanomaterials (ENM), a well-established mechanism of ENM toxicity. Concerns exist over susceptibility of the DCFH assay to: assay conditions, adsorption of DCFH onto ENM, fluorescence quenching and light scattering. These effects vary in magnitude depending on ENM physiochemical properties and concentration. A rigorous evaluation of this method is still lacking. The objective was to evaluate performance of the DCFH assay for measuring ENM-induced free radicals. A series of diverse and well-characterized ENM were tested in the acellular DCFH assay. We investigated the effect of sonication conditions, dispersion media, ENM concentration, and the use of horseradish peroxidase (HRP) on the DCFH results. The acellular DCFH assay suffers from high background signals resulting from dye auto-oxidation and lacks sensitivity and robustness. DCFH oxidation is further enhanced by HRP. The number of positive ENM in the assay and their relative ranking changed as a function of experimental conditions. An inverse dose relationship was observed for several Carbon-based ENM. Overall, these findings indicate the importance of having standardized assays for evaluating ENM toxicity and highlights limitations of the DCFH assay for measuring ENM-induced free radicals.

Understanding and correcting for carbon nanotube interferences with a commercial LDH cytotoxicity assay.

The lactate dehydrogenase (LDH) assay accurately quantifies cytotoxicity of chemicals via the measurement of LDH released from damaged cells. In the assay, LDH catalyzes formation of a reporter chromophore that can be quantified spectrophotometrically at its 490 nm peak, a standard assay, and related to the released LDH concentration. However, certain engineered nanomaterials have been reported to produce aberrant values, resulting in inaccurate assessment of toxicity as measured by LDH levels in media. We studied this effect spectroscopically by measuring unexpected changes in the complete visible spectrum of the product chromophore resulting from using either purified LDH or LDH from lysed cells in the presence of varying concentrations of single walled carbon nanotubes (SWCNTs) or carbon nanohorns (SWCNH-oxs). Basically, at constant LDH concentrations, the 490 nm product peak decreased with increasing carbon nanotube concentration, while the 580 nm peak increased to a lesser extent and the maximum absorbing wavelength increased. The product chromophore spectrum was altered in different ways by potential interactions with a number of components in the reaction mixture including: BSA, LDH, SWCNTs, SWCNT-oxs, or various combinations of these species. We propose to improve the accuracy of the LDH assay when evaluated in the presence of varying concentrations of these carbon nanostructures by use of both the 490 and 580 nm peak absorbances combined via regression analysis. Our results indicate that molecular probes of cytotoxicity must be assessed individually for accuracy in the presence of engineered nanomaterials.

Biological oxidative damage by carbon nanotubes: fingerprint or footprint?

Carbon nanotubes (CNTs) have received much attention for performance and toxicity, but vary substantially in terms of impurity type and content, morphology, and surface activity. This study determined the decrease of antioxidant capacity, defined as biological oxidative damage (BOD), of CNTs-exposed serum. The variability in several physicochemical properties of CNTs and their links to BOD elicited in human serum were explored. Tremendous variation in transition metal type and content (104-fold), specific surface area (SSA, nine-fold), and BOD were observed. Mass specific BOD (mBOD) varied from 0.006-0.187 µmol TEU mg(-1), whereas surface area specific BOD (sBOD) varied from 0.068-0.42 µmol TEU m(-2). The sBOD increased in a stepwise fashion from ∼0.1-0.32 µmol TEU m(-2) for tubes with outer diameter less than 10 nm. The mBOD and sBOD may be useful denominators of surface activity and impurity content and assist in designing safer CNTs.