Center for Nanotechnology and Nanotoxicology

Boston, MA, United States

Center for Nanotechnology and Nanotoxicology

Boston, MA, United States
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Deloid G.M.,Center for Nanotechnology and Nanotoxicology | Cohen J.M.,Center for Nanotechnology and Nanotoxicology | Pyrgiotakis G.,Center for Nanotechnology and Nanotoxicology | Demokritou P.,Center for Nanotechnology and Nanotoxicology
Nature Protocols | Year: 2017

Evidence continues to grow of the importance of in vitro and in vivo dosimetry in the hazard assessment and ranking of engineered nanomaterials (ENMs). Accurate dose metrics are particularly important for in vitro cellular screening to assess the potential health risks or bioactivity of ENMs. To ensure meaningful and reproducible quantification of in vitro dose, with consistent measurement and reporting between laboratories, it is necessary to adopt standardized and integrated methodologies for (i) generation of stable ENM suspensions in cell culture media; (ii) colloidal characterization of suspended ENMs, particularly of properties that determine particle kinetics in an in vitro system (size distribution and formed agglomerate effective density); and (iii) robust numerical fate and transport modeling for accurate determination of the ENM dose delivered to cells over the course of the in vitro exposure. Here we present an integrated comprehensive protocol based on such a methodology for in vitro dosimetry, including detailed standardized procedures for each of these three critical aims. The entire protocol requires ∼6-12 h to complete.

Pal A.K.,Center for Nanotechnology and Nanotoxicology | Watson C.Y.,Center for Nanotechnology and Nanotoxicology | Pirela S.V.,Center for Nanotechnology and Nanotoxicology | Singh D.,Center for Nanotechnology and Nanotoxicology | And 3 more authors.
Toxicological Sciences | Year: 2015

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.

Deloid G.,Center for Nanotechnology and Nanotoxicology | Cohen J.M.,Center for Nanotechnology and Nanotoxicology | Darrah T.,Duke University | Derk R.,U.S. National Institute for Occupational Safety and Health | And 4 more authors.
Nature Communications | Year: 2014

The need for accurate in vitro dosimetry remains a major obstacle to the development of cost-effective toxicological screening methods for engineered nanomaterials. An important key to accurate in vitro dosimetry is the characterization of sedimentation and diffusion rates of nanoparticles suspended in culture media, which largely depend upon the effective density and diameter of formed agglomerates in suspension. Here we present a rapid and inexpensive method for accurately measuring the effective density of nano-agglomerates in suspension. This novel method is based on the volume of the pellet obtained by benchtop centrifugation of nanomaterial suspensions in a packed cell volume tube, and is validated against gold-standard analytical ultracentrifugation data. This simple and cost-effective method allows nanotoxicologists to correctly model nanoparticle transport, and thus attain accurate dosimetry in cell culture systems, which will greatly advance the development of reliable and efficient methods for toxicological testing and investigation of nano-bio interactions in vitro. © 2014 Macmillan Publishers Limited. All rights reserved.

Cohen J.M.,Center for Nanotechnology and Nanotoxicology | Teeguarden J.G.,Pacific Northwest National Laboratory | Demokritou P.,Center for Nanotechnology and Nanotoxicology
Particle and Fibre Toxicology | Year: 2014

Background: There is a great need for screening tools capable of rapidly assessing nanomaterial toxicity. One impediment to the development of reliable in vitro screening methods is the need for accurate measures of cellular dose. We present here a methodology that enables accurate determination of delivered to cell dose metrics. This methodology includes (1) standardization of engineered nanomaterial (ENM) suspension preparation; (2) measurement of ENM characteristics controlling delivery to cells in culture; and (3) calculation of delivered dose as a function of exposure time using the ISDD model. The approach is validated against experimentally measured doses, and simplified analytical expressions for the delivered dose (Relevant In Vitro Dose (RID)f function) are derived for 20 ENMs. These functions can be used by nanotoxicologists to accurately calculate the total mass (RIDM), surface area (RIDSA), or particle number (RIDN) delivered to cells as a function of exposure time.Results: The proposed methodology was used to derive the effective density, agglomerate diameter and RID functions for 17 industrially-relevant metal and metal oxide ENMs, two carbonaceous nanoparticles, and non-agglomerating gold nanospheres, for two well plate configurations (96 and 384 well plates). For agglomerating ENMs, the measured effective density was on average 60% below the material density. We report great variability in delivered dose metrics, with some materials depositing within 24 hours while others require over 100 hours for delivery to cells. A neutron-activated tracer particle system was employed to validate the proposed in vitro dosimetry methodology for a number of ENMs (measured delivered to cell dose within 9% of estimated).Conclusions: Our findings confirm and extend experimental and computational evidence that agglomerate characteristics affect the dose delivered to cells. Therefore measurement of these characteristics is critical for effective use of in vitro systems for nanotoxicology. The mixed experimental/computational approach to cellular dosimetry proposed and validated here can be used by nanotoxicologists to accurately calculate the delivered to cell dose metrics for various ENMs and in vitro conditions as a function of exposure time. The RID functions and characterization data for widely used ENMs presented here can together be used by experimentalists to design and interpret toxicity studies. © 2014 Cohen et al.; licensee BioMed Central Ltd.

Pal A.K.,University of Massachusetts Lowell | Bello D.,University of Massachusetts Lowell | Cohen J.,Center for Nanotechnology and Nanotoxicology | Demokritou P.,Center for Nanotechnology and Nanotoxicology
Nanotoxicology | Year: 2015

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 (R2 = 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. © 2015 Informa UK Ltd. All rights reserved: reproduction in whole or part not permitted

Cohen J.M.,Center for Nanotechnology and Nanotoxicology | Derk R.,U.S. National Institute for Occupational Safety and Health | Wang L.,U.S. National Institute for Occupational Safety and Health | Godleski J.,Center for Nanotechnology and Nanotoxicology | And 3 more authors.
Nanotoxicology | Year: 2014

Relatively little is known about the fate of industrially relevant engineered nanomaterials (ENMs) in the lungs that can be used to convert administered doses to delivered doses. Inhalation exposure and subsequent translocation of ENMs across the epithelial lining layer of the lung might contribute to clearance, toxic effects or both. To allow precise quantitation of translocation across lung epithelial cells, we developed a method for tracking industrially relevant metal oxide ENMs in vitro using neutron activation. The versatility and sensitivity of the proposed in vitro epithelial translocation (INVET) system was demonstrated using a variety of industry relevant ENMs including CeO2 of various primary particle diameter, ZnO, and SiO2-coated CeO2 and ZnO particles. ENMs were neutron activated, forming gamma emitting isotopes 141Ce and 65Zn, respectively. Calu-3 lung epithelial cells cultured to confluency on transwell inserts were exposed to neutron-activated ENM dispersions at sub-lethal doses to investigate the link between ENM properties and translocation potential. The effects of ENM exposure on monolayer integrity was monitored by various methods. ENM translocation across the cellular monolayer was assessed by gamma spectrometry following 2, 4 and 24 h of exposure. Our results demonstrate that ENMs translocated in small amounts (e.g. <0.01% of the delivered dose at 24 h), predominantly via transcellular pathways without compromising monolayer integrity or disrupting tight junctions. It was also demonstrated that the delivery of particles in suspension to cells in culture is proportional to translocation, emphasizing the importance of accurate dosimetry when comparing ENM-cellular interactions for large panels of materials. The reported INVET system for tracking industrially relevant ENMs while accounting for dosimetry can be a valuable tool for investigating nano-bio interactions in the future. © 2014 Informa UK Ltd. All rights reserved.

Fusco S.,ETH Zurich | Sakar M.S.,ETH Zurich | Kennedy S.,Wyss Institute for Biologically Inspired Engineering | Peters C.,ETH Zurich | And 8 more authors.
Advanced Materials | Year: 2014

The presented microrobotic platform combines together the advantages of self-folding NIR light sensitive polymer bilayers, magnetic alginate microbeads, and a 3D manipulation system, to propose a solution for targeted, on-demand drug and cell delivery. First feasibility studies are presented together with the potential of the full design. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Watson C.,Center for Nanotechnology and Nanotoxicology | Ge J.,Massachusetts Institute of Technology | Cohen J.,Center for Nanotechnology and Nanotoxicology | Pyrgiotakis G.,Center for Nanotechnology and Nanotoxicology | And 2 more authors.
ACS Nano | Year: 2014

The likelihood of intentional and unintentional engineered nanoparticle (ENP) exposure has dramatically increased due to the use of nanoenabled products. Indeed, ENPs have been incorporated in many useful products and have enhanced our way of life. However, there are many unanswered questions about the consequences of nanoparticle exposures, in particular, with regard to their potential to damage the genome and thus potentially promote cancer. In this study, we present a high-throughput screening assay based upon the recently developed CometChip technology, which enables evaluation of single-stranded DNA breaks, abasic sites, and alkali-sensitive sites in cells exposed to ENPs. The strategic microfabricated, 96-well design and automated processing improves efficiency, reduces processing time, and suppresses user bias in comparison to the standard comet assay. We evaluated the versatility of this assay by screening five industrially relevant ENP exposures (SiO2, ZnO, Fe2O3, Ag, and CeO2) on both suspension human lymphoblastoid (TK6) and adherent Chinese hamster ovary (H9T3) cell lines. MTT and CyQuant NF assays were employed to assess cellular viability and proliferation after ENP exposure. Exposure to ENPs at a dose range of 5, 10, and 20 μg/mL induced dose-dependent increases in DNA damage and cytotoxicity. Genotoxicity profiles of ZnO > Ag > Fe2O3 > CeO2 > SiO2 in TK6 cells at 4 h and Ag > Fe 2O3 > ZnO > CeO2 > SiO2 in H9T3 cells at 24 h were observed. The presented CometChip platform enabled efficient and reliable measurement of ENP-mediated DNA damage, therefore demonstrating the efficacy of this powerful tool in nanogenotoxicity studies. © 2014 American Chemical Society.

Pyrgiotakis G.,Center for Nanotechnology and Nanotoxicology | McDevitt J.,Center for Nanotechnology and Nanotoxicology | Yamauchi T.,Panasonic | Demokritou P.,Center for Nanotechnology and Nanotoxicology
Journal of Nanoparticle Research | Year: 2012

This is a study focusing on the potential to deactivate biological agents (bacteria and endospores) using engineered water nanostructures (EWNS). The EWNS were generated using an electrospray device that collects water by condensing atmospheric water vapor on a Peltier-cooled electrode. A high voltage is applied between the collection electrode and a grounded electrode resulting in aerosolization of the condensed water and a constant generation of EWNS. Gram-negative Serratia marcescens, gram-positive Staphylococcus aureus, and Bacillus atrophaeus endospores were placed on stainless steel coupons and exposed to generated EWNS at multiple time intervals. Upon exposures, the bacteria were recovered and placed on nutrient agar to grow, and the colony forming units were counted. Ozone levels as well as air temperature and relative humidity were monitored during the experiments. Qualitative confirmation of bacterial destruction was also obtained by transmission electron microscopy. In addition, important EWNS aerosol properties such as particle number concentration as a function of size as well as the average surface charge of the generated EWNS were measured using real-time instrumentation. It was shown that the novel electrospray method can generate over time a constant flux of EWNS. EWNS have a peak number concentration of ~8,000 particles per cubic centimeter with a modal peak size around 20 nm. The average surface charge of the generated EWNS was found to be 10 ± 2 electrons per particle. In addition, it was shown that the EWNS have the potential to deactivate both bacteria types from surfaces. At the same administrate dose, however, the endospores were not inactivated. This novel method and the unique properties of the generated EWNS could potentially be used to develop an effective, environmentally friendly, and inexpensive method for bacteria inactivation. © 2012 Springer Science+Business Media B.V.

Cohen J.,Center for Nanotechnology and Nanotoxicology | Deloid G.,Center for Nanotechnology and Nanotoxicology | Pyrgiotakis G.,Center for Nanotechnology and Nanotoxicology | Demokritou P.,Center for Nanotechnology and Nanotoxicology
Nanotoxicology | Year: 2013

In vitro toxicity assays are efficient and inexpensive tools for screening the increasing number of engineered nanomaterials (ENMs) entering the consumer market. However, the data produced by in vitro studies often vary substantially among different studies and from in vivo data. In part, these discrepancies may be attributable to lack of standardisation in dispersion protocols and inadequate characterisation of particle-media interactions which may affect the particle kinetics and the dose delivered to cells. In this study, a novel approach for preparation of monodisperse, stabilised liquid suspensions is presented and coupled with a numerical model which estimates delivered dose values. Empirically derived material-and media-specific functions are presented for each media-ENM system that can be used to convert administered doses to delivered doses. The interactions of ENMs with a variety of physiologic media were investigated and the importance of this approach was demonstrated by in vitro cytotoxicity assays using THP-1 macrophages. © 2013 Informa UK, Ltd.

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