Category Archives: News

Nano State

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The NanoCenter researchers , Phil Demokritou, Joseph Brain and Georgios Pyrgiotakis were featured in a four page special story at the Harvard School of Public Health magazine. They discussed the impact of nano in the society and the importance of the center research. Further more they talked about the Engineered Water Nanostructures a novel, chemical free method developed in the Center that is promising for the air inactivation of pathogenes.

You can read the whole story here.

Harvard School of Public Health researchers develop technique to measure the quantity of engineered nanomaterials delivered to cells

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Press Release in pdf

Boston, MA— Thousands of consumer products containing engineered nanoparticles — microscopic particles found in everyday items from cosmetics and clothing to building materials — enter the market every year. Concerns about possible environmental health and safety issues of these nano-enabled products continue to grow with scientists struggling to come up with fast, cheap, and easy-to-use cellular screening systems to determine possible hazards of vast libraries of engineered nanomaterials. However, determining how much exposure to engineered nanoparticles could be unsafe for humans requires precise knowledge of the amount (dose) of nanomaterials interacting with cells and tissues such as lungs and skin.

With chemicals, this is easy to do but when it comes to nanoparticles suspended in physiological media, this is not trivial. Engineered nanoparticles in biological media interact with serum proteins and form larger agglomerates which alter both their so called effective density and active surface area and ultimately define their delivery to cell dose and bio-interactions. This behavior has tremendous implications not only in measuring the exact amount of nanomaterials interacting with cells and tissue but also in defining hazard rankings of various engineered nanomaterials (ENMs). As a result, thousands of published cellular screening assays are difficult to interpret and use for risk assessment purposes.

Scientists at the Center for Nanotechnology and Nanotoxicology at Harvard School of Public Health (HSPH) have discovered a fast, simple, and inexpensive method to measure the effective density of engineered nanoparticles in physiological fluids, thereby making it possible to accurately determine the amount of nanomaterials that come into contact with cells and tissue in culture.

The method, referred to as the Volumetric Centrifugation Method (VCM), will be published in the March 28, 2014 Nature Communications.

The new discovery will have a major impact on the hazard assessment of engineered nanoparticles, enabling risk assessors to perform accurate hazard rankings of nanomaterials using cellular systems. Furthermore, by measuring the composition of nanomaterial agglomerates in physiologic fluids, it will allow scientists to design more effective nano-based drug delivery systems for nanomedicine applications.

“The biggest challenge we have in assessing possible health effects associated with nano exposures is deciding when something is hazardous and when it is not, based on the dose level. At low levels, the risks are probably miniscule,” said senior author Philip Demokritou, associate professor of aerosol physics in the Department of Environmental Health at HSPH. “The question is: At what dose level does nano-exposure become problematic? The same question applies to nano-based drugs when we test their efficiency using cellular systems. How much of the administered nano-drug will come in contact with cells and tissue? This will determine the effective dose needed for a given cellular response,” Demokritou said.

Federal regulatory agencies do not require manufacturers to test engineered nanoparticles, if the original form of the bulk material has already been shown to be safe. However, there is evidence that some of these materials could be more harmful in the nanoscale — a scale at which materials may penetrate cells and bypass biological barriers more easily and exhibit unique physical, chemical, and biological properties compared to larger size particles. Nanotoxicologists are struggling to develop fast and cheap toxicological screening cellular assays to cope with the influx of vast forms of engineered nanomaterials and avoid laborious and expensive animal testing. However, this effort has been held back due to the lack of a simple-to-use, fast, method to measure the dose-response relationships and possible toxicological implications. While biological responses are fairly easy to measure, scientists are struggling to develop a fast method to assess the exact amount or dose of nanomaterials coming in contact with cells in biological media.

“Dosimetric considerations are too complicated to consider in nano-bio assessments, but too important to ignore,” Demokritou said. “Comparisons of biological responses to nano-exposures usually rely on guesstimates based on properties measured in the dry powder form (e.g., mass, surface area, and density), without taking into account particle-particle and particle-fluid interactions in biological media. When suspended in fluids, nanoparticles typically form agglomerates that include large amounts of the suspending fluid, and that therefore have effective densities much lower than that of dry material. This greatly influences the particle delivery to cells, and reduces the surface area available for interactions with cells,” said Glen DeLoid, research associate in the Department of Environmental Health, one of the two lead authors of the study. “The VCM method will help nanobiologists and regulators to resolve conflicting in vitro cellular toxicity data that have been reported in the literature for various nanomaterials. These disparities likely result from lack of or inaccurate dosimetric considerations in nano-bio interactions in a cellular screening system,” said Joel Cohen, doctoral student at HSPH and one of the two lead authors of the study.

Wolfgang Kreyling, a nanotoxicologist at the German Research Center for Environmental Health who was not involved in the study, says this method should help toxicologists to understand the nano-bio interactions and address possible nano hazards for the vast libraries of engineered nanoparticles (ENPs) currently in use.

“The paper by DeLoid et al in Nature Communications is a major achievement which offers a solution to solve the pending issues of the apparent ENM density and an easy way to determine the latter by the application of the volumetric centrifugation method. Hence, this paper provides a versatile concept easy to achieve which allows for a rather precise estimate of ENM dosimetry to in vitro cell cultures which hopefully will improve the power of toxicological studies using in vitro cell cultures when comparing to in vivo studies. In this case this would be a major contribution in aiming to reduce in vivo experimental animal work,” Kreyling said.

Other authors of the study include Georgios Pyrgiotakis, research fellow at HSPH, Liying Rojanasakul, and Raymond Derk from the National Institute for Occupational Safety and Health, and Wendel Wohlleben from BASF, Germany.

This research project was supported by NIEHS grant (ES-000002), NSF grant (ID 1235806) and the Center for Nanotechnology and Nanotoxicology at HSPH. This work was performed in part at the Harvard Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Infrastructure Network (NNIN), which is supported by the National Science Foundation under NSF award number ECS-0335765.

“Estimating the effective density of engineered nanomaterials for in vitro dosimetry,” Glen DeLoid, Joel M. Cohen, Tom Darrah, Raymond Derk, Liying Rojanasakul, Georgios Pyrgiotakis, Wendel Wohlleben, and Philip Demokritou, Nature Communications, online March 28, 2014.

First High Throughput Genotox Assay

Protocol for the high throughput Comet Assay. (A) Assembly of macrowell comet array. Agarose gel with microwells is sandwiched between a glass substrate and a bottomless 96-well plate and sealed with mechanical force. Approximately 300 arrayed microwells comprise the bottom of each macrowell. (B) Preparation of the nanoparticle suspension according to the protocol by Cohen at al. (C) Protocol for exposing the cells to the nanoparticles. (D) Loading of the exposed cell samples in the macrowells and running the microwell assay.

Protocol for the high throughput Comet Assay. (A) Assembly of macrowell comet array. Agarose gel with microwells is sandwiched between a glass substrate and a bottomless 96-well plate and sealed with mechanical force. Approximately 300 arrayed microwells comprise the bottom of each macrowell. (B) Preparation of the nanoparticle suspension according to the protocol by Cohen at al. (C) Protocol for exposing the cells to the nanoparticles. (D) Loading of the exposed cell samples in the macrowells and running the microwell assay.

Nanomaterials are part of daily life. Although there is a wide range of methods to evaluate their potential toxic effects, there is no way to evaluate gene damage.

Scientists at Harvard University in the School of Public Health in collaboration with the research Group of Bevin P. Engelward at MIT, have developed a screening assay to detect the genotoxic potential of nanomaterials. Metal oxide nanoparticles in biological systems can generate reactive oxygen species, which can overwhelm innate antioxidant defenses and cause oxidative stress. Oxidative stress, among other factors, has been associated with DNA damage and mutations, precursors to cancer. As more and more commercial products contain nanomaterials consisting of metal oxides such as titanium dioxide and zinc oxide, screening assays such as these are crucial to reducing potential health hazards. Christa Watson, postdoctoral research fellow at HSPH, suggests that accurate toxicity assessments of nanomaterials before they are incorporated into consumer products can help us prevent similar consequences that we are currently facing from asbestos exposures such as mesothelioma. Current efforts are ongoing to understand the novel toxicities nanomaterials may pose on public safety. This research was recently published in ACS Nano February 14, 2014 DOI: 10.1021/nn404871p

Research at the HSPH NanoCenter revolutionize sunscreens!

ZnO is a widely used material in cosmetics and food applications. The ions however, that are leaching have been linked to potential adverse effects. In our center we developed a safer formulation concept to mitigate toxicity by encapsulating the materials in a thin layer of Silica.

ZnO is a widely used material in cosmetics and food applications. The ions however, that are leaching have been linked to potential adverse effects. In our center we developed a safer formulation concept to mitigate toxicity by encapsulating the materials in a thin layer of Silica.

One of the main constituents of sunscreen is the ZnO particles. ZnO nanoparticles are sought out for UV-filter applications thanks to their inherent optoelectronic properties and are, therefore, broadly used today in cosmetics and polymers. Preliminary toxicological data, however, point out that they can induce significant DNA damage and genotoxicity due to their Zn2+ ion leaching. It has become important for the nanotechnology industry, to devise scalable, safer-by-design approaches to minimize the ZnO nanoparticle dissolution and toxicity without altering their desired optoelectronic properties.

G. Sotiriou the lead author of the paper.

In their work, the researchers demonstrated a safer-by-design approach for ZnO nanorods using a scalable flame aerosol process. This technology allows for controlled synthesis of high-purity ZnO nanorods with highly crystalline core and a nanothin amorphous silica shell that improves their biocompatibility. The as-prepared nanorods exhibit high transparency in the visible range, but strong absorption in the UV rendering them suitable for use in sunscreens and polymers. Furthermore, it is demonstrated that the hermetic silica coating does not alter the desired optoelectronic properties of the core ZnO nanorods while their DNA damage potential has been 3-fold decreased.

You can read more at the Chemistry World news article or, directly the paper.

New England Nanotechnology Association Breakfast

NEMA

Please join us for our next meeting of the Massachusetts chapter of the New England Nanotechnology Association (NENA) to hear about some new developments in nanotechnology and nanotoxicology.

We’ll enjoy a continental breakfast, spend a little time networking, and hear about these new developments and other topics of interest. There is no cost to attend, so please RSVP today. Thank you and we look forward to seeing you soon.

Agenda:
8:30 – 9:00AM:  Networking and refreshments

9:00 – 9:10AM:  Welcome and introductions - William S. Rogers, Jr., Esq., Partner, Prince Lobel Tye LLP, Boston, MA

9:10-9:20AM: Speaker - Eric S. Howard, Corporate and Outreach Manager, NSF Center for High-rate Nanomanufacturing, Northeastern University, Boston, MA

9:20-9:35AM:  Speaker - Philip Demokritou, Ph. D., Assoc. Professor and Director, Center for Nanotechnology and Nanotoxicology, Harvard School of Public Health, Cambridge, MA
9:35-9:45AM: Speaker - Joseph Brain, Ph.D., Cecil K. and Philip Drinker Professor of Environmental Physiology, Department of Environmental Health, Harvard School of Public Health, Cambridge, MA
9:45-10:00: Networking and adjourn

Click here to RSVP – deadline is 2/21/14

Can’t make the event? Join our LinkedIn Discussion Page.

We must have a complete list of attendees two days prior to the event in order to arrange visitor passes. No one can get in without a visitor’s pass.

If you have any questions, please don’t hesitate to contact me.

Sincerely,
William S. Rogers, Jr.

Prince Lobel Tye LLP

wsrogers@princelobel.com

617-456-8112

Panasonic continues its support to our Nanocenter

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During their visit a delegation from Panasonic headed by Mr. Oketa continued their financial support to the Center of Nanotechnology and Nanotoxicology through a generous contribution to the Environmental Nanotechnology  postdoctoral Fellowship.

The panasonic delegates that visited our center were Takemi Oketa, Mitsuhiro Sano and Yosuke Mizuyama.

In the image above Prof. Demokritou receives the gift from Mr. Oketa in front of Dr. Mizuyama, Pyrgiotakis and Mr. Sano.

Researchers from our Center made it to the cover of the Enviromental Science: Nano

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Phil Demokritou et al. published recently a high impact paper at the Enviromental Science: Nano a new Journal from the Royal Society of Chemistry. Environmental Science: Nano covers the benefits and implications of nano-science and nanotechnology on environmental health and safety, and the sustainable design, development and use of nanotechnologies. This includes design, applications, life cycle implications, characterization in biological and environmental media, environmental and biological interactions and fate, transformations, transport, reactivity, biological uptake and ecotoxicity, and other areas of sustainable nanotechnology, such as interactions with pollutants and remediation of environmental contaminants by nanomaterials.

Our publication fits the objective of the journal and it was selected by the editors as the most innovative research article to decorate the cover of the first issue of the journal. You can find here the abstract of the paper and the link to the publisher. If you have access to the RSC you will be able to access the publications.

Screen Shot 2014-01-27 at 4.31.48 PMAbstract: Airborne pathogens are associated with the spread of infectious diseases and increased morbidity and mortality. Herein we present an emerging chemical free, nanotechnology-based method for airborne pathogen inactivation. This technique is based on transforming atmospheric water vapor into Engineered Water Nano-Structures (EWNS) via electrospray. The generated EWNS possess a unique set of physical, chemical, morphological and biological properties. Their average size is 25 nm and they contain reactive oxygen species (ROS) such as hydroxyl and superoxide radicals. In addition, EWNS are highly electrically charged (10 electrons per particle on average). A link between their electric charge and the reduction of their evaporation rate was illustrated resulting in an extended lifetime (over an hour) at room conditions. Furthermore, it was clearly demonstrated that the EWNS have the ability to interact with and inactivate airborne bacteria. Finally, inhaled EWNS were found to have minimal toxicological effects, as illustrated in an acute in-vivo inhalation study using a mouse model. In conclusion, this novel, chemical free, nanotechnology-based method has the potential to be used in the battle against airborne infectious diseases.

Joel Cohen Defended his Doctoral Thesis

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On September 23, Joel Cohen defended  his doctoral thesis    on Nanotoxicology. Joel over the course of his studies published several peer reviewed papers, one book chapter and awarded one patent (with his coworkers).

Join us in congratulating Joel Cohen for successfully defending his ScD thesis and obtaining his doctoral degree!

Thesis Abstract: There is a great need for screening tools capable of rapidly and accurately assessing engineered nanomaterial (ENM) toxicity. One impediment to the development of reliable in vitro screening methods is the need for accurate and relevant dosimetry. In a typical in vitro cytotoxicity study ENM powders are suspended in liquid media for application to cells. ENMs in liquid suspension can form large fractal agglomerates thereby altering (1) the total number of free particles, (2) the total surface area available for biointeractions, and (3) the effective size and density of the particles, two properties that influence their fate and transport and determine the effective dose actually delivered to cells in culture over the duration of exposure. I present here a methodology for in vitro nanotoxicology that takes into consideration particokinetics and enables accurate determination and reporting of effective dosimetry. This methodology is based upon (1) standardization of ENM liquid suspension preparation; (2) careful characterization of critical ENM transformations in exposure media including agglomerate effective density; and (3) numeric calculation of the delivered to cell dose as a function of exposure time.

This methodology is then employed to investigate ENM translocation across cellular monolayers in vitro. Relatively little is known about the fate of industrially relevant engineered nanomaterials (ENMs) in the lungs. These interactions are important when considering inhalation exposure and subsequent translocation of ENMs across the thin epithelial lining layer of the lung. I present a novel method for tracking well-characterized industrially relevant metal oxide ENMs made radioactive in vitro. Nano-sized CeO2 of various primary particle diameter (27 and 119nm), ZnO, SiO2-coated-CeO2 and SiO2-coated-ZnO particles generated by flame spray pyrolysis were neutron activated in a nuclear reactor, forming the gamma emitting isotopes 141Ce and 65Zn respectively. To investigate ENM translocation using an in vitro model for the alveolar epithelium, we cultured Calu-3 lung epithelial cells cultured to confluency on transwell inserts with 3μm pores and exposed them to neutron activated ENM dispersions below the pre-determined toxic dose. The effects of ENM exposure on monolayer barrier integrity and tight junctions were evaluated, and ENM translocation across the cellular monolayer was assessed following 2, 4 and 24 hours of exposure by gamma spectrometry. My results demonstrate that industrially relevant ENM agglomerates translocate predominantly via a transcellular pathway without compromising monolayer integrity or disrupting tight junctions. In order from greatest to least translocation the ENMs investigated rank as follows: ZnO> SiO2 coated ZnO > SiO2 coated CeO2 > CeO2 large > CeO2 small. I also demonstrate the effects of particle transport translocation across the alveolar epithelium, emphasizing the importance of accurate dosimetry when comparing ENM-cellular interactions for large panels of materials.

New Publication by Georgios Pyrgiotakis

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Georgios Pyrgiotakis et al. published one of the first attempts to quantify the effect of the protein corona in the nano-bio interactions. The work was carried out in collaboration with the Particle Technology Laboratory at the Swiss Federal Institute of Technology (ETH). Here is the abstract of the publication and the link to access it at Langmuir.

Abstract

Particle–particle interactions in physiological media are important determinants for nanoparticle fate and transport. Herein, such interactions are assessed by a novel atomic force microscopy (AFM)-based platform. Industry-relevant CeO2, Fe2O3, and SiO2nanoparticles of various diameters were made by the flame spray pyrolysis (FSP)-based Harvard Versatile Engineering Nanomaterials Generation System (Harvard VENGES). The nanoparticles were fully characterized structurally and morphologically, and their properties in water and biological media were also assessed. The nanoparticles were attached on AFM tips and deposited on Si substrates to measure particle–particle interactions. The corresponding force was measured in air, water, and biological media that are widely used in toxicological studies. The presented AFM-based approach can be used to assess the agglomeration potential of nanoparticles in physiological fluids. The agglomeration potential of CeO2 nanoparticles in water and RPMI 1640 (Roswell Park Memorial Institute formulation 1640) was inversely proportional to their primary particle (PP) diameter, but for Fe2O3nanoparticles, that potential is independent of PP diameter in these media. Moreover, in RPMI+10% Fetal Bovine Serum (FBS), the corona thickness and dispersibility of the CeO2 are independent of PP diameter, while for Fe2O3, the corona thickness and dispersibility were inversely proportional to PP diameter. The present method can be combined with dynamic light scattering (DLS), proteomics, and computer simulations to understand the nanobio interactions, with emphasis on the agglomeration potential of nanoparticles and their transport in physiological media.

Just Announced: CytoViva Visit

The NanoCenter is very happy to host Cytoviva as they present their latest microscope (Cytoviva) that uses the so-called “hyperspectral imaging”, i.e. measuring the scattering profile of samples, and being able to distinguish cells, and different types of materials. Especially for studies of inorganic particles with cells, it offers several advantages because it is a “label-free” detection method. It is not limited to inorganic particles, however, they claim that also lysosomes and other organic particles can be easily detected. It also has the possibility to use standard fluorescent dyes if we install the appropriate filters.

Title: Nano-scale Hyperspectral Microscopy

Speaker: Byron J. Cheatham, Senior VP, CytoViva, Inc.

Date: Monday July 22

Time: 10:00 am

Place: Room 1302

Abstract: CytoViva, Inc. provides a patented (US patents No. 7,542,203, 7,564,623) nanoscale optical microscope capability integrated with proprietary hyperspectral imaging. This integrated technology was specifically designed for optical observation, spectral characterization and mapping of nano-materials as they interact with biologicals and composite materials. The patented illumination optics of the microscope system utilizes structured oblique-angle illumination to produce a very high signal-to-noise image. Scatter from nano-scale materials imaged with CytoViva’s structured oblique-angle illumination optics can produce as much as seven times more signal intensity when compared to standard darkfield microscope optics.

Integrated hyperspectral imaging on the microscope enables capture of the unique VNIR reflectance spectra (400nm-1,000nm) of nano-scale materials within a wide range of biological and composite environments at a spectral resolution of 2.5nm. The system creates a hyperspectral image of these samples, enabling the nano-materials to be spectrally characterized and mapped throughout the entire sample.

Today over 250 nano-focused laboratories utilize CytoViva technology for nano-drug delivery, nano-toxicology and nano-materials related research initiatives. Additionally the technology is utilized in certain pathogen related studies.

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