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 &
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
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
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.
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
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
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
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 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
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
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
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
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
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.
ENGINEERED WATER NANOSTRUCTURES (EWNS) AND USES THEREOF
[ 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
NANOCARRIERS FOR THE DELIVERY OF ACTIVE INGREDIENTS
[ 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.