Philip PYRGIOTAKIS, et al.
Engineered Water Nanostructures ( EWNS )
15 February 2016
Scientific Reports volume 6, Article number: 21073 (2016)

Optimization of a nanotechnology based antimicrobial platform for food safety applications using Engineered Water Nanostructures (EWNS)
Georgios Pyrgiotakis, Pallavi Vedantam, Caroline Cirenza, James McDevitt, Mary Eleftheriadou, Stephen S. Leonard & Philip Demokritou

A chemical free, nanotechnology-based, antimicrobial platform using Engineered Water Nanostructures (EWNS) was recently developed. EWNS have high surface charge, are loaded with reactive oxygen species (ROS) and can interact-with and inactivate an array of microorganisms, including foodborne pathogens. Here, it was demonstrated that their properties during synthesis can be fine tuned and optimized to further enhance their antimicrobial potential. A lab based EWNS platform was developed to enable fine-tuning of EWNS properties by modifying synthesis parameters. Characterization of EWNS properties (charge, size and ROS content) was performed using state-of-the art analytical methods. Further their microbial inactivation potential was evaluated with food related microorganisms such as Escherichia coli, Salmonella enterica, Listeria innocua, Mycobacterium parafortuitum and Saccharomyces cerevisiae inoculated onto the surface of organic grape tomatoes. The results presented here indicate that EWNS properties can be fine-tuned during synthesis resulting in a multifold increase of the inactivation efficacy. More specifically, the surface charge quadrupled and the ROS content increased. Microbial removal rates were microorganism dependent and ranged between 1.0 to 3.8 logs after 45 mins of exposure to an EWNS aerosol dose of 40,000 #/cm3.
Environmental Science: Nano,  Issue 1, 2014

 A chemical free, nanotechnology-based method for airborne bacterial inactivation using engineered water nanostructures
Georgios Pyrgiotakis, et al.

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.

Inactivation of ambient virues using Engineered Water Nanostructures
Demokritou, Philip   
Harvard University, Boston, MA, United States

Despite advances in hygiene, sanitation and the development of vaccines and antibiotics, infectious diseases continue to affect hundreds of millions of people each year with serious health outcomes. Infectious diseases can be transmitted either by air (airborne) or via surfaces (fomites). The toll of infectious disease is further complicated through the evolution of antibiotic-resistant bacteria, while the constant antigenic shift of influenza viruses creates difficulties for vaccine development. Control of these infections remains a challenge and currently relies on interventions that have significant shortcomings, including their own health risks. New, innovative, effective, low cost and most importantly chemical-free, 'green' technologies, possessing fewer drawbacks than the existing ones, are urgently in need in the battle against infections. The investigators have been working on such a novel nanotechnology-based method. It relies on the synthesis of Engineered Water Nanostructures (EWNS) by electrospraying high purity water. Preliminary data indicate that EWNS possess unique physicochemical and biological properties. Most importantly, they are highly mobile and can inactivate bacteria on both surfaces and in the air through damage to their membrane. Here, we plan to assess and optimize EWNS as an alternative, chemical-free method to inactivate viruses in air and on environmental surfaces. The pathogen-EWNS interactions will be investigated using a variety of validated, state-of-the-art analytical methods and biological assays.

The specific aims of this project are:

AIM1 : Development and characterization of a high-throughput EWNS generation platform to study the nano-virus interaction in air and surfaces using relevant bioassay models. The system will be used for the controlled synthesis and property characterization of EWNS. The EWNS generation platform will enable for EWNS property modification (size, surface charge, ROS content, lifetime) and study their effect on the viral inactivation process.

AIM 2 : Inactivation of aerosolized or surface deposited influenza virus (2009 H1N1) following exposure to EWNS will be assessed and optimized using in vitro and in vivo infectivity assays. The role of EWNS properties and electrospray operational parameters on the inactivation potential and mechanisms will be investigated using state of the art analytical methods. The information generated in these studies will lead to the development of applications of this novel, chemical-free approach for the control of virally transmitted infectious diseases such as Influenza. The proposed project spans disciplines in which our investigators have expertise: Nanoparticle synthesis, characterization and environmental nanotechnology (Demokritou), cellular biology, respiratory pathophysiology, aerobiology and infectious diseases (Kobzik, McDevitt). Such a novel chemical free approach, if successful, will reduce risk of infection and have a beneficial economic and public health impact.

Public Health Relevance
Infectious diseases caused from viruses, transmitted via air and surfaces, continue to affect hundreds of millions of people worldwide with serious health outcomes including mortality and morbidity. Current methods of disinfection have major shortcomings such as use of chemicals high-energy consumption and health risks. In this proposal, we examine the ability of a new low cost, low energy, chemical free, intervention method using Engineered Water Nanostructures, to inactivate viruses on surfaces and in the air. Such a novel approach, if successful, will have huge economic and public health impact, and will enhance our arsenal of methods on reducing the risk of infection...
Nanomedicine. 2014 Aug; 10(6): 1175–1183., 2014 Mar 12.
doi: 10.1016/j.nano.2014.02.016

Mycobacteria inactivation using Engineered Water Nanostructures (EWNS)
Georgios Pyrgiotakis, et al.
Airborne transmitted pathogens such as Mycobacterium tuberculosis (Mtb) cause serious, often fatal infectious disease with enormous global health implications. Due to their unique cell wall and slow growth, mycobacteria are among the most resilient microbial forms. Herein we evaluate the ability of an emerging, chemical-free, nanotechnology-based method to inactivate M. parafortuitum (Mtb surrogate). This method is based on the transformation of atmospheric water vapor into engineered water nano-structures (EWNS) via electrospray. We demonstrate that the EWNS can interact with and inactivate airborne mycobacteria, reducing their concentration levels significantly. Additionally, EWNS can inactivate M. parafortuitum on surfaces eight times faster than the control. The mechanism of mycobacteria inactivation was also investigated in this study. It was demonstrated that the EWNS effectively deliver the reactive oxygen species, encapsulated during the electrospray process, to the bacteria oxidizing their cell membrane resulting into inactivation. Overall, this is a method with the potential to become an effective intervention technology in the battle against airborne infections...

EWNS synthesis
The EWNS are synthesized via electrospray, a method used widely to aerosolize particles and fibers from liquid suspensions. Electrospray relies on a strong electric field to aerosolize a liquid, that is typically contained in a fine metal capillary.27 The required strong electric field is generated by the application of a high voltage (most often negative voltage) between the metal capillary and a counter electrode positioned in a fixed distance from the capillary. The strong electric field causes the liquid to break into highly charged droplets.27 This phenomenon, known widely as Rayleigh effect, states that a liquid droplet with high surface charge density is unstable. While the surface charge density increases, the distance among the charges becomes smaller which increases the electrostatic interactions. This results in a non-favorable energy increase of the system forcing the droplet to break into smaller droplets with smaller surface charge density (the overall surface area increases, while the charge remains the same). The only force that counters the breaking is the surface tension. There is a critical diameter, known as Rayleigh diameter, for which the surface tension is not enough to counter the electrostatic interactions and the droplets break into smaller droplets.28

Figures 1, A and B, illustrate the electro-spray module used in this study and the process for the synthesis of EWNS respectively. In brief, a gold plated electrode is cooled down to 6 °C via a Peltier element. The atmospheric water vapor, condensed on the electrode, becomes the source of water for the electrospray. High voltage of approximately 5 kV is applied between the Peltier electrode and a grounded counter electrode causing the water to break into small droplets as it is described above.24,25,29 For this particular experimental setup, the operational environmental conditions were maintained at 20–25 °C and 45–55% Relative Humidity (RH)...

...The results from the lipid peroxidation assay (Figure 4) clearly confirm that the primary inactivation mechanism is the delivery of ROS to bacteria by EWNS nanoaerosol. The EWNS very effectively protect the otherwise short lived ROS and deliver them due to their highly mobile nature to the bacteria, causing significant lipid peroxidation and destruction of the cell membrane. This peroxidation effect disappeared, when the vitamin-C, a well-known antioxidant that prohibits ROS mediated lipid peroxidation,40 is added to the bacteria prior to their exposure. It is important to note that the vitamin-C alone does not seem to have any positive effect to the bacteria since the room air exposed vitamin-C treated bacteria do not show statistically significant peroxidation compared to the control bacteria. These data are in agreement with previous published by the authors work for other bacteria, demonstrating that the presence of EWNS results in the destruction of the bacteria cell membrane.24,25 Collectively, these data conclude that the ROS account for one of the dominant pathways of inactivation of bacteria exposed to EWNS.

The electrospray-generated EWNS were found effective in inactivating M. parafortuitum, an Mtb surrogate. This is a promising finding in the fight against the tuberculosis epidemic, offering a cost effective intervention approach, which can be implemented in a variety of indoor environmental settings thus reducing the risk of transmission. Advantages of the proposed control technology include the ease of use, the low cost, and the low energy consumption. Overall, this is a chemical-free, sustainable, and environmentally friendly technology that has the potential to reduce the risk of transmission of diseases, such as tuberculosis...!divAbstract

A novel method for textile odor removal using engineered water nanostructures
Lisha Zhu, et al.

The malodor attached to textiles not only causes indoor environmental pollution but also endangers people's health even at low concentrations. Existing technologies cannot effectively eliminate the odor. Herein, an effective and environmentally friendly technology was proposed to address this challenging issue. This technology utilizes electrospraying process to produce Engineered Water Nanostructures (EWNS) in a controllable manner. Upon application of a high voltage to the Taylor cone, EWNS can be generated from the condensed vapor water through a Peltier element. Smoking, cooking and perspiration, considered the typical indoor malodorous gases emitted from human activities, were studied in this paper. A headspace SPME method in conjunction with GC-MS was employed for the extraction, detection and quantification of any odor residues. Results indicated that EWNS played a significant role in the deodorization process with removal efficiencies for the three odors were 95.3 ± 0.1%, 100.0 ± 0.0% and 43.7 ± 2.3%, respectively. The Reactive Oxygen Species (ROS) contained in the EWNS, mainly hydroxyl (OH˙) and superoxide radicals Image ID:c9ra01988j-t1.gif are the possible mechanisms for the odor removal. These ROS are strong oxidative and highly reactive and have the ability to convert odorous compounds to non-odorous compounds through various chemical reaction mechanisms. This study showed clearly the potential of the proposed method in the field of odor removal and can be applied in the battle against indoor air pollution...

EWNS synthesis
EWNS were synthesized based on an electrospraying technique,46 which divides liquid into fairly uniform fragments, ranging from few nanometers to hundred microns.47 Herein, water vapor from the air were condensed on an electrode cooled by a Peltier element. Above the electrode, a counter electrode was arranged concentrically. High voltage was applied between the two electrodes (condensing electrode, −5 kV, grounded counter electrodes) to form a Taylor cone due to the electrical shear stress. The capillary is placed at a negative voltage while the counter electrode is connected to a positive voltage. At this point, the liquid jet contained lots of negative ions. Subsequently, since the Coulomb repulsion instability generated by the ions was greater than the surface tension, the liquid jet continued to be dispersed into fine droplets.27,48–50 As the droplets volume decreased, the charge density of the droplets exceeded the limit of surface tension, causing these droplets spontaneously split, eventually reaching a stable radius called Rayleigh critical radius51 and producing EWNS. The generation process of EWNS is illustrated in Fig. 1.

During this electrospray process, some water molecules and oxygen molecules were split or lost electrons under high electric field creating several kinds of ROS, like OH˙ and 52,53 It has been proven that those ROS are encapsulated in EWNS, which prevents them from neutralization by other air molecules and extends significantly their lifetime.54 Detailed information on the EWNS synthesis can be found in the series paper of the Harvard group.27–29,40 Unlike the previous studies by the Harvard group, in this study, a novel EWNS generator was employed with a new type of linear structure electrode developed by the Panasonic Corporation. It can increase significantly the discharging area and produces increased concentrations of ROS per unit of time...
ACS Sustainable Chemistry & Engineering (2019)

Inactivation of Hand Hygiene-Related Pathogens Using Engineered Water Nanostructures
Runze Huang, Nachiket Vaze, Anand Soorneedi, Matthew D. Moore, Yalong Xue, Dhimiter Bello, Philip Demokritou


Hand hygiene is a critical public health issue associated with disease transmission worldwide. Here, a nanotechnology-based approach has been employed to enhance hand hygiene using engineered water nanostructures (EWNS) synthesized by electrospray and ionization of antimicrobial aqueous solutions. The EWNS possess unique properties: have a tunable size in the nanoscale, are electrically charged, which results in a lifespan of hours in room conditions, and can carry both antimicrobial agents and reactive oxygen species (ROS) from ionization of water. More importantly, EWNS are highly mobile, can be directed toward a surface of interest utilizing their electric charge, and can inactivate pathogens by delivering active ingredients (AIs) and ROS. In this study, a variety of AIs commonly used for hand sanitization and food safety, such as hydrogen peroxide, citric acid, lysozyme, and nisin, were utilized to synthesize various EWNS-based nanosanitizers and inactivate hand hygiene-related pathogens. A 0.5 min exposure to various EWNS-based nanosanitizers reduced Escherichia coli, Staphylococcus aureus, and bacteriophage MS2 by ∼3, 1, and 2 log, respectively. More importantly, such an aerosol-based nanocarrier platform, because of its targeted delivery manner, utilizes only nanograms of “nature-inspired” antimicrobials and leaves behind no chemical byproducts, making it an efficient approach for hand sanitization.

Summary of characteristics of EWNS-based nanosanitizers and AI solutions used to generate EWNS-based nanosanitizers

Chemical characterization of the EWNS-based nanosanitizer synthesized with 10% hydrogen peroxide, 1% citric acid, 0.1% lysozyme, and 0.0025% nisin; summary of the antimicrobial efficacy of used AIs using “wet”, suspension tests; time-kill curve of E. coli exposed to the EWNS-based nanosanitizer synthesized with 10% hydrogen peroxide, 1% citric acid, 0.1% lysozyme, and 0.0025% nisin; transmission electron microscopy images of E. coli cells exposed to EWNS-based nanosanitizers; and physicochemical characterizations of EWNS-based nanosanitizers.

Harvard Chan Center for Nanotechnology and Nanotoxicology looks to improve on soap and water

Nanosafety researchers at the Harvard T.H. Chan School of Public Health have developed a new intervention to fight infectious disease by more effectively disinfecting the air around us, our food, our hands, and whatever else harbors the microbes that make us sick. The researchers, from the School’s Center for Nanotechnology and Nanotoxicology, were led by Associate Professor of Aerosol Physics Philip Demokritou, the center’s director, and first author Runze Huang, a postdoctoral fellow there. They used a nano-enabled platform developed at the center to create and deliver tiny, aerosolized water nonodroplets containing non-toxic, nature-inspired disinfectants wherever desired. Demokritou talked to the Gazette about the invention and its application on hand hygiene, which was described recently in the journal ACS Sustainable Chemistry and Engineering.

Q&A with Philip Demokritou

GAZETTE:  Give us a quick overview of the problem you’re trying to solve.
DEMOKRITOU:  If you go back to the ’60s and the invention of many antibiotics, we thought that the chapter on infectious diseases would be closed. Of course, 60 years later, we now know that’s not true. Infectious diseases are still emerging. Microorganisms are smarter than we thought and evolving new strains. It’s a constant battle. And when I talk about infectious diseases, I’m mainly talking about airborne and foodborne diseases: For example, flu and tuberculosis are airborne diseases, respiratory diseases, which cause millions of deaths a year. Foodborne diseases also kill 500,000 people annually and cost our economy billions of dollars.

GAZETTE: Diarrheal diseases are big killers of kids, too.
DEMOKRITOU: It’s a big problem, especially in developing countries with fragmented health care systems.

GAZETTE: What’s wrong with how we sanitize our hands?
DEMOKRITOU: We hear all the time that you have to wash your hands. It’s a primary measure to reduce infectious diseases. More recently, we’re also using antiseptics. Alcohol is OK, but we are also using other chemicals like triclosan and chlorhexadine. There’s research linking these chemicals to the increase in antimicrobial resistance, among other drawbacks. In addition, some people are sensitive to frequent washes and rubbing with chemicals. That’s where new approaches come into play. So, within the last four or five years, we’ve been trying to develop nanotechnology-based interventions to fight infectious diseases.

GAZETTE: So the technology involved here — the engineered water nanostructures — is a couple of years old. What’s new is the application?
DEMOKRITOU: We have the tools to make these engineered nanomaterials and, in this particular case, we can take water and turn it into an engineered water nanoparticle, which carries its deadly payload, primarily nontoxic, nature-inspired antimicrobials, and kills microorganisms on surfaces and in the air.

It is fairly simple, you need 12 volts DC, and we combine that with electrospray and ionization to turn water into a nanoaerosol, in which these engineered nanostructures are suspended in the air. These water nanoparticles have unique properties because of their small size and also contain reactive oxygen species. These are hydroxyl radicals, peroxides, and are similar to what nature uses in cells to kill pathogens. These nanoparticles, by design, also carry an electric charge, which increases surface energy and reduces evaporation. That means these engineered nanostructures can remain suspended in air for hours. When the charge dissipates, they become water vapor and disappear.

Very recently, we started using these structures as a carrier, and we can now incorporate nature-inspired antimicrobials into their chemical structure. These are not super toxic to humans. For instance, my grandmother in Greece used to disinfect her surfaces with lemon juice — citric acid. Or, in milk — and also found in tears — is another highly potent antimicrobial called lysozyme. Nisin is another nature-inspired antimicrobial that bacteria release when they’re competing with other bacteria. Nature provides us with a ton of nontoxic antimicrobials that, if we can find a way to deliver them in a targeted, precise manner, can do the job. No need to invent new and potentially toxic chemicals. Let’s go to nature’s pharmacy and shop.

When we put these nature-inspired antimicrobials into the engineered water nanostructures, their antimicrobial potency increases dramatically. But we do that without using huge quantities of antimicrobials, about 1 percent or 2 percent by volume. Most of the engineered water nanostructure is still water.

At this point, these engineered structures are carrying antimicrobials and are charged, and we can use the charge to direct them to surfaces by applying a weak electric field. You can also release them into the air — they’re highly mobile — and they can move around and inactivate flu virus, for example.

GAZETTE: How would this work with food?
DEMOKRITOU: This nano-enabled platform can be used as an intervention technology for food safety applications as well. When it comes to disinfecting our food, we’re still using archaic approaches developed in the ’50s. For instance, today we put our fresh produce into chlorine-based solutions, which leave residues that can compromise health. It leaves behind byproducts, which are toxic, and you have to find a way to deal with them as well.

Instead, you can use the water nanoaerosols that contain nanogram levels of an active ingredient — nature-inspired and not toxic — and disinfect our food. Currently, this novel invention is being explored for use — from the farm to the fork — to enhance food safety and quality.

GAZETTE: So when you use it on food, you would essentially spray the nanoparticles onto a head of lettuce, for example?
DEMOKRITOU: It depends on the application. You can put this technology in your refrigerator, and it will kill microbes on food surfaces and in the air there and improve food safety. It will also increase shelf life, which is linked to spoilage microorganisms. You can also use this technology for air disinfection. The only thing you need is 12-volt DC, which you can power from your computer USB port. Imagine sitting on a train and you generate an invisible shield of these engineered water nanostructures that protects you and minimizes the risk of getting the flu.

GAZETTE: If you’re on the train with a bunch of sick people?
DEMOKRITOU: Exactly, or on an airplane, anywhere you have microorganisms. Most planes recirculate the air, and all it takes is one sick guy — he doesn’t have to be sitting next to you — to get sick. Unfortunately, that’s a big problem. The newer airplanes have filtration to remove some of these pathogens. But this is a very versatile technology that you can pretty much take with you.

GAZETTE:  Let’s talk about hand hygiene.
DEMOKRITOU:  We know hand hygiene is very important, but in addition to the drawbacks of washing with water or using chemicals, the air dryers commonly used in the bathroom environment can aerosolize microbes and put them back in the air and even back on your hands. So there is room to utilize these engineered water nanostructures and develop an alternative that is airless and waterless — because it uses picogram levels of water, your hands will never get wet.

GAZETTE: So you’re washing your hands, using water. But they don’t get wet?
DEMOKRITOU: Exactly. And it disinfects hands in a matter of 15–20 seconds, as indicated in our recently published study.

GAZETTE: As far as an application goes, do you see something similar to the hand driers we all use at highway rest stops? Only, when you stick your hands in, it doesn’t blow? Do you feel anything at all?
DEMOKRITOU: You don’t feel anything. That’s the problem; this is like magic. You don’t see; you don’t feel; you don’t smell; but your hands are sanitized.

GAZETTE: So how do people know anything’s happened? As humans we want some sort of stimulation.
DEMOKRITOU: We could put a light and music to entertain people, but nobody can see a 25-nanometer particle. We are excited to see that there is interest from industry to pursue commercialization of this technology for hand hygiene. We may soon have an airless, waterless apparatus that can be used across the board, though not necessarily in the bathroom environment. This can be a battery-operated device, it can be placed around airports and other spots where people don’t have time or access to water to wash their hands.

[ PDF ]
Various embodiments of the present invention relate to, among other things, systems for generating engineered water nanostructures (EWNS) comprising reactive oxygen species (ROS) and methods for inactivating at least one of viruses, bacteria, bacterial spores, and fungi in or on a wound of a subject in need thereof or on produce by applying EWNS to the wound or to the produce.

[ PDF ]
Various embodiments of the present invention relate to, among other things, a nano carrier platform for generating enhanced engineered water nanostructures (iEWNS) encapsulating and delivering reactive oxygen species (ROS) and, in some instances, other active ingredients, methods for inactivating at least one of viruses, bacteria, bacterial spores, and fungi on a substrate by applying iEWNS to the substrate.

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