Particulate Air Pollution Research meets Nanomaterial Toxicology: Evidence for beyond the lung


140402 #Dia  Flemming Cassee ambasadeur innovatie RIVM Foto Tjitske SluisSpeaker: Flemming R. Cassee, PhD

Senior Inhalation Toxicologist,

National Institute for Public Health and the Environment (RIVM), Netherlands

Institute for Risk Assessment Sciences, Utrecht University, Utrecht, Netherlands

Date: March 20, 2015

Time: 9:30-10:30pm

Place: 665 Huntington Ave,

Bldg. 1, Room 1302,

Boston, MA 02115


Abstract: Ambient particulate matter (PM) has been extensively studied in the past two decades because of the association of the level of PM and several markers for morbidity as well as premature death in the general population. Much has been done to clean up the air that we breathe and in most places standards for PM are met. There are still hot spots, such as busy traffic locations for which abatement measure need to be taken to further reduce the concentrations. Since the effects are still noted below current air quality standards, and the cost of reducing PM further are getting higher and higher, targeted research is needed to identify the most harmful constituents and the related sources of emissions. Of special interest are the ultrafine or nano-scale particles as they seem to contribute to the adverse health effects and are not well captured by current standards. The effects of nanoparticles may very well be observed in organs other than those that correspond to the port of entry – for example, the central nervous system. In light of different distribution patterns on inhalation and the likelihood that nanoparticles can escape natural defence mechanisms, such as phagocytosis, it is likely that this size fraction will also be linked to biological pathways and responses that differ from larger sized particles. Whether or not nanoparticles translocate is dependent on several factors including size and solubility. Small particles (<10nm) translocate with greater efficiency than large particles (<100nm). Soluble particles can directly diffuse across the lung membrane. While small soluble particles have been shown to translocate into the circulatory system, carbon core nanoparticles do not diffuse across the lung membranes to the same extent, although soluble coatings from them can. Examples for translocation to and adverse effects on the cardiovascular and central nervous system after inhalation exposure to nanoparticles will be presented.

Biographical Sketch: Flemming R. Cassee, PhD, is senior inhalation toxicologist at RIVM and is involved in the research on the adverse health effects of air pollution. He has written a number of scientific reports and articles in international journals and has presented several papers at international conferences. In addition, he serves in a number of international advisory committees. He is senior-scientific advisor on Inhalation Toxicology and leader of several projects on Ambient Particulate Matter and Health. Dr. Cassee is member of WHO and EU committees and invited reviewer of the US EPA and Health Effects Institute. He has a position as professor in inhalation toxicology at the Institute of Risk Assessment Sciences (IRAS) at the University of Utrecht, the Netherlands. He has been active in toxicology for more than 20 years with a prime interest in the adverse health effects of air pollutants and inhaled nanomaterials.

Nanotechnology to the Rescue: Using Engineered Water Nanostructures (EWNS) to inactivate Foodborne Microorganisms

Screen Shot 2015-02-25 at 3.53.15 PM

The burden of foodborne diseases worldwide is huge, with serious economic and public health consequences. The U.S. Department of Agriculture’s (USDA’s) Economic Research Service reported in 2014 that foodborne illnesses are costing the economy more than $15.6 billion and about 53,245 Americans visit the hospital annually due to foodborne illnesses. The food industry is in search of effective intervention methods that can be applied form “farm to fork” to ensure the safety of the food chain and be consumer and environment friendly at the same time.

Researchers at the Center for Nanotechnology and Nanotoxicology of the Harvard T. Chan School of Public Health are currently exploring the effectiveness of a nanotechnology based, chemical free, intervention method for the inactivation of foodborne and spoilage microorganisms on fresh produce and on food production surfaces. This method utilizes Engineered Water Nanostructures (EWNS) generated by electrospraying of water. EWNS possess unique properties; they are 25 nm in diameter, remain airborne in indoor conditions for hours, contain Reactive Oxygen Species (ROS), have very strong surface charge (on average 10e/structure) and have the ability to interact and inactivate pathogens by destroying their membrane.

In a study funded by the USDA and just published this week in the premier Environmental Science and Technology journal, the efficacy of these tiny water nanodroplets, in inactivating representative foodborne pathogens such as Escherichia coli, Salmonella enterica and Listeria innocua, on stainless steel surfaces and on tomatoes, was assessed showing significant log reductions in inactivation of select food pathogens. These promising results could open up the gateway for further exploration into the dynamics of this method in the battle against foodborne disease. More importantly this novel, chemical-free, cost effective and environmentally friendly intervention method holds great potential for development and application in the food industry, as a ‘green’ alternative to existing inactivation methods.

Dr. Philip Demokritou, Associate professor at Department of Environmental Health and lead author of the study mentioned, “Nanotechnology can bring on the table new useful intervention approaches that can be used in the battle against food borne diseases. Using water in its engineered nanoscale form can be a ‘game changer’, cost effective approach that can be easily deployed at various intervention points across the ‘farm to fork’ line”.

Drs. Georgios Pyrgiotakis and Pallavi Vedantam, post-doctoral research fellows in the Department of Environmental Health who are currently further exploring the prospects of this novel high throughput system believe that this technology could not only significantly decrease the microbial load on the fresh produce but also extend shelf life of produce and reduce the number of cases of foodborne illnesses on consumption. The Center for Disease Control (CDC) has estimated 48 million such cases each year in the United States alone, which include 3,000 deaths. Hence, this method could potentially landscape the preventable public health challenge of foodborne infections and craft best ways forward. Prof. Mitchell and his team at SEAS also collaborated in this study.

EST publication can be accessed here. For more information on nano related research at Harvard T.H. Chan School of Public Health please visit our website at


Printer Emitted Particles: Are they safe?

Engineered nanomaterials (ENMs) incorporated into toner formulations of toner used in every day laser printers. During the print jobs it is likely that the particles can be released in the air.

Recently, our group developed a lab based Printer Exposure Generation System that allows the generation and sampling of airborne PEPs for subsequent physicochemical, morphological and toxicological analyses. This platform was used to evaluate PM emission profiles from 11 laser printers currently on the market. It was shown that the particle number concentration of PEPs varied across different printer models/manufacturers and reached as high as 1.3 million particles/cm3 and with modal diameters <200 nm. The detailed assessment of both toners and PEPs confirmed not only the presence of nanoscale materials in the airborne state but also their complex chemistries, which include elemental and organic carbon and inorganic compounds such as metals and metal oxides. It has been now confirmed that toners are classified as nano-enabled products. Additionally, we have performed in-depth toxicological analysis evaluating the effects of exposure to PEPs using physiologically relevant cell lines in both mono- and co-culture experimental systems. We have shown that PEPs caused significant cytotoxicity, membrane integrity damage, reactive oxygen species (ROS) production, pro-inflammatory cytokine release, angiogenesis, actin remodeling, gap cell junctions and epigenetic changes in cells at doses comparable to those from real world exposure scenarios representative of inhalation exposures in the range of 1-200 hours. We may conclude that laser printer-emitted engineered nanoparticles can be deleterious to lung cells and may cause persistent genetic modifications that could translate to pulmonary disorders.


  1. Pirela SV, Pyrgiotakis G, Bello D, Thomas T, Castranova V, Demokritou P. 2014a. Development and characterization of an exposure platform suitable for physico-chemical, morphological and toxicological characterization of printer-emitted particles (peps). Inhal Toxicol 26:400-408.
  2. Pirela SV, Sotiriou GA, Bello D, Shafer M, Bunker KL, Castranova V, et al. 2014b. Consumer exposures to laser printer-emitted engineered nanoparticles: A case study of life-cycle implications from nano-enabled products. Nanotoxicology:1-9.
  3. Pirela SV, Miousse IR, Lu X, Castranova V, Thomas T, Qian Y, et al. 2015. Laser printer-emitted engineered nanoparticles lead to cytotoxicity, inflammation and changes in dna methylation in human cells. Environmental health perspectives.
  4. Sisler JD, Pirela SV, Friend S, Farcas M, Schwegler-Berry D, Shvedova A, et al. 2014. Small airway epithelial cells exposure to printer-emitted engineered nanoparticles induces cellular effects on human microvascular endothelial cells in an alveolar-capillary co-culture model. Nanotoxicology:1-11.

News Release: A new bug killer


The Center Director Dr. Phil Demokritou and the Research Fellow Georgios Pyrgiotakis are featured in the prestigious magazine “The Economist” (issue July 7) that is focus on the upcoming “Big things in Nanotechnology”. They discuss the applications of their research on the Engineered Water Nanostructures  (EWNS) in aspects of daily life. This feature is one more on the long list of recognition the research on the topic that the center has earned. You can read the entire story of on the online version of The Economist here.

The research has been featured in numerous print and on line publications:

  • Our paper has been Featured by RSC’s journal Chemistry World (link)
  • It was also featured in the German NPR radio (link)
  • It was featured on the cover of the Enviromental Science: Nano

Nano State

Screen Shot 2014-05-15 at 5.15.00 PM

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

Screen Shot 2014-04-04 at 4.26.03 PM

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.

Predicting the Impact of Engineered Nanomaterials on Lung Diseases

Title: Predicting the Impact of Engineered Nanomaterials on Lung Diseases


Speaker: Dr. James C. Bonner

Associate Professor

Department of Environmental & Molecular Toxicology,

North Carolina State University, Raleigh, NC


Date: February  27, 2014
Time: 12:30-1:30 pm
Place: 665 Huntington Ave, Building 1, Room 1302, Boston, MA 02115

Abstract:  The nanotechnology revolution offers enormous societal and economic benefits for innovation in the fields of engineering, electronics, and medicine. Nevertheless, evidence from rodent inhalation studies show that biopersistent engineered nanomaterials, including carbon nanotubes and metal nanoparticles, have the potential to stimulate immune, inflammatory, or fibroproliferative responses in the lung and pleura. These data suggest possible risks for pulmonary fibrosis or the development of pleural disease as a consequence of occupational or consumer exposure. Some engineered nanomaterials also exacerbate pre-existing allergen-induced inflammation by altering the balance of distinct T-helper cell phenotypes, suggesting that they could serve as sensitizers or adjuvants to alter the innate immune response.  These findings suggest that individuals with asthma or other pre-existing respiratory diseases would be particularly susceptible to the adverse health effects of nanomaterials. Due to their nanoscale dimensions and increased surface area per unit mass, engineered nanomaterials have a much greater potential to reach the distal regions of the lung, generate reactive oxygen species, and alter cell signaling pathways linked to disease pathogenesis. The goal of this presentation will be to discuss mechanisms through which engineered nanomaterials cause lung, airway, and pleural disease, especially in the context of susceptible individuals with pre-existing disease. Functionalization of nanomaterials through processes such as atomic layer deposition will also be discussed as a means of altering the pathogenicity of nanomaterials.