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
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.
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.
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.
William S. Rogers, Jr.
Prince Lobel Tye LLP
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.
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.
Abstract: 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.
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.
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.
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.
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.
Joseph D. Brain is the Cecil K. and Philip Drinker Professor of Environmental Physiology and former chair of the Department of Environmental Health at the Harvard School of Public Health. Dr. Brain joined the Department in 1962 as a young graduate student researching the body’s response to inhaled gases, particulates, and microbes and has remained at the School since that time—teaching and mentoring countless students, engaging in ground-breaking research, and leading his colleagues with fervor and foresight.
To honor Dr. Brain for his significant contributions to the Department of Environmental Health and the field of environmental physiology, the school is in the process of establishing the Joseph D. Brain Fellowship Fund in Environmental Health. The Fund will provide much-needed resources for environmental health scholars to pursue doctoral and post-doctoral study at the Harvard School of Public Health. Support provided by the fund will enable talented and dedicated students who otherwise would be unable to afford tuition and expenses to pursue their educational goals. It also allows the work started by Dr. Brain to be carried out and expanded by promising scholars who are preparing to begin their careers in the field.
Our new website is launching soon. A portal to all things nano. Explore the projects, the researcher and the contact the investigators via the highly interactive website. Use the website to explore our labs and learn more about the upcoming events. Launching day July 1st.