By Dr. Kim Van Vliet, Dr. Megha Agrawal, Dr.

Shyamasri Biswas, Contributing Editors

Thin Films of Powerful Viruses

Could Change the World

ome biological molecules, such as viruses, naturally form

well-defined structures that can be exploited for a variety
of uses, such as the development of homogeneous thin
films based devices for advanced biomedical applications. The
protein shell of viruses, called capsids have several properties
that make them advantageous. Their particles are small in size,
have precise dimensions, and many of these viral capsids have
been characterized at atomic resolution. High vacuum and ultra-high vacuum technology based state-of-the-art microscopic
techniques have been applied to characterize such protein shell
thin films. Vacuum technologists together with thin films experts
and biotechnologists have come together to study and exploit viruses for new applications in medical technologies.
Virus capsids come in a variety of shapes, either icosahedral
(spherical) or helical shaped filaments. Each virus generally produces viral capsids that are identical in size, shape and composition with high fidelity. Viral capsid proteins also can be easily
modified to add functional groups that confer specific biochemical properties. Some viruses have capsids that self-assemble
by supplying a limited number of components in vitro; thereby
eliminating the need for living cells. Hence, virus capsids can
be easily mass produced with high fidelity. Capsids also possess
many protein-protein interactions which provide stability. This
is advantageous during purification, as well as for storage over
time which may be required for use in a wide variety of applications. In addition to external modifications of the virus capsid,
the internal cavities of these protein capsids also provide space
for small cargo for drug delivery applications.
For virus particles that are able to self-assemble and exhibit
crystalline ordering, genetically engineered viral material can be
produced and then utilized to generate thin films comprised of

multiple layers of virus particles arranged on a conductive gold

surface. For example, virus capsids of a genetically engineered
M13 bacteriophage, were recently used to produce thin films that
are capable of generating electricity based on the piezoelectric
effect, where a charge accumulates based on some sort of stress,
such as motion or vibration. Then if the piezoelectric effect is
large enough, it can be used to transform mechanical energy into
electricity (1). Seung-Wuk Lee, and coworkers recently produced
a device that contains a large number of these films and by using
their thumb to squeeze the device, they were able to generate 6
nA of current at 400 mV, allowing them to power a liquid crystal
display. The fabrication process to make the M13 capsid-based
thin films is simple and more cost effective compared to conventional piezoelectric materials, such as quartz, and some types of
ceramics and polymers which often require complex fabrication
processes to render them useful for practical applications. The
M13 bacteriophage is considered relatively safe to humans because it only infects bacteria. Upon infecting bacteria, the phage
can use the bacterial host to synthesize and secrete millions of
new phage particles with ease. The filamentous bacteriophage
M13 has a long rod-like shape and is about 6.5 nanometers in
diameter and 880 nanometers in length. (Figure 1a) Each M13
phage capsid is comprised of about 2700 copies of a major coat
protein, pVIII and 5 copies of minor coat proteins, pIII and pIX.
Structurally, pVIII protein is -helical and these are aligned at
a 20 angle with respect to the axis of the virus (dashed line),
shown in Figure 1a. The protein has a dipole moment directed
from the amino terminus to the carboxy terminus, shown as positive (blue) and negative (red) ends, and this leads to the piezoelectric effects in the M13 bacteriophage thin films, although
the origin of the piezoelectric effect is unclear (Figure 1b). One

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June 2014 Vacuum Technology & Coating

Figure 1. Baceteriophage M13 biofilm. (a) Bacteriophage M13 structure. (b) One explanation for the origin of the piezoelectric effect. (c) Alternate explanation for the piezoelectric effect. [Credit: Yu, S.M. 2012 (2)].

explanation is that compressing the virus from above (blue arrow) causes neighboring protein helices to rub against each other
(purple arrows), resulting in deformation of the helical structure
(green arrows) and the development of new electric dipole moments, as shown in Figure 1b. Another explanation is shown in
Figure 1c, where although the virus is symmetrical in the radial
direction, compressive pressure could break the symmetry and
produce new charges on the top and bottom surfaces of the virus.
The piezoelectric effect of bactetiophage M13 has been further
enhanced by genetically engineering the viral capsid protein to
include four negatively charged glutamate residues on the end of
the protein which results in an even more negative charge on one
end of the protein. This increases the charge difference between
the positive and negative ends of the protein, increasing the voltage of the M13 capsid. Stacking about 20 single layer M13 biofilms on top of each other in the device also enhanced the system.
Such a multilayer thin film protein structure is advantageous as it
exhibits enhanced functionality.
Although the electricity generated in this first device is only
about one-fourth the energy generated from a AAA battery, these
M13 thin films open up the possibility that in the future LED
screens could be powered by trillions of tiny viruses that can
convert vibrations into electricity. This first piezoelectric device
holds promise for viral electronics, including consumer electronics or sensors that can convert vibrations or pressure to electricity to charge themselves. In the future, this technology could be
incorporated in shoes, doors, and even carpets to generate small
amounts of electricity.

Development of M13 bacteriophage for useful applications

is not limited to the generation of electricity. These viruses are
also being modified for use in cancer diagnostics and molecular
imaging Genetically engineered M13 bacteriophage are promis-

Figure 2. Genetically engineered viral capsid protein for magnetic resonance imaging. Thin films of M13 phage (yellow), SPARC binding
peptide (pink), Triglutamate motif allows for the assembly iron oxide
nanocrystals (black) along the surface of the viral coat. [Credit: Ghosh,
D. et al 2012 (3)].

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ing contrast agents and the development of novel particles with

higher signal-to-noise ratios will result in improved imaging
techniques. Rather than utilizing the capsid protein to generate
electricity, Ghoush, et al (3) have engineered the M13 phage to
display a tumor targeting peptide. This tumor targeting peptide
binds SPARC glycoprotein which is highly overexpressed in
various cancers. In addition to incorporating this tumor-targeting
peptide, they have also modified the M13 phage capsid to display a peptide motif for thin film assembly of iron oxide particles
(NPs)to allow for magnetic resonance imaging for specific tumor
detection. In mouse models of prostate cancer, they have shown
that the engineered M13 phage achieves targeting and delivers a
larger payload of (NPs)per SPARC bound compared to conventional methods. The use of virus-based technologies for imaging
allows for specificity in the process and improved methods of
cancer detection in addition to the ability to provide treatments
directly to the effected regions. In addition to targeting peptides,
and peptide motifs for thin film assembly of NPs, it may also be
possible to incorporate chemotherapeutic agents for drug delivery and therapy. These genetically engineered M13 viral particles
are promising tools for tumor detection and improved therapies.
This is not the first time that thin film bacteriophage have been
used for biotechnology applications. One of the earliest uses of
biotechnology in humans is the use of bacteriophage to treat bacterial infections. A conservative estimate of the number of bacteriophages on Earth is in excess of 1031 making them one of
the most abundant life forms on the planet. (4). Bacteriophage
can be divided into two groups, the lysogenic phage or the lytic
phage. The lytic phage infect bacteria, lyse the cells, and release
progeny phage that can go and infect more bacterial cells. Bacteriophage with a broad host range, those that can infect several
bacterial strains are most useful as therapeutic agents. Prior to the
discovery of antibiotics, bacteriophage were used to treat diseases such as typhoid and dysentery. Upon treatment in the presence
of susceptible bacteria, the level of phage increase and remain
high for a period of time which enables the phage to control the
infection. After the discovery of antibiotics, phage therapy was
largely discontinued in the western world; however, it is still in
use in parts of Europe even today. Due to the increased use of
antibiotics, some bacteria have become resistant to treatment
with antimicrobials. This has led to renewed interest in the use
of phage therapy worldwide for the control of resistant bacteria. In a recent study by Marinelli, et al (5), they investigated the
bacteria that infect sebaceous glands, Propionibacterium acnes,
to identify bacteriophage capable of infecting this clinically relevant bacteria which are the causative agent of acne. The isolated
P. acnes phages were shown to infect a broad range of P. acnes
clinical bacterial subtypes. These studies support further investigation of bacteriophage P. acnes for the development of a targeted approach to acne treatment. The resurgence of phage therapy
for use in the clinic provides exciting alternative treatment strategies for antibiotic resistant bacteria which are now posing health
threats worldwide.
Similar to bacteriophage lysing their host cells, there are other
viruses that have the ability to lyse cancer cells. These viruses
have been placed into a group now known as oncolytic virus-

es. These viruses infect and multiply within malignant cells and
then lyse the cancer cells without affecting normal cells. This
property of oncolytic viruses is either inherent or genetically engineered. Several types of oncolytic viruses have been developed
as therapeutic agents. One such virus is adenovirus, which is approximately 70 to 90 nanometers in size. Several modifications
of this virus have been tested in clinical trials for head and neck
cancers. One modified adenovirus based product, H101, has already been approved for use in China. Another oncolytic virus,
coxsackie virus, is currently in clinical trials for the treatment
of malignant melanomas. Engineered versions of poliovirus are
also being tested for oncolytic potential against glioblastoma
multiforme, which are the most aggressive malignant brain tumors in humans. One of the more promising oncolytic viruses
is reovirus, which is a virus with no known associated disease in
humans and is named reo which is short for respiratory enteric
orphan virus. Exposure to reovirus is common in humans and
most humans have been infected with this virus at some point
during their lives. Reovirus particles are 60 to 90 nanometers in
diameter and the virus replicates in cells that have an activated
Ras pathway, which is a common mutation in cancer cells, while
leaving normal cells unaffected. Approximately 1/3 of human
cancers have mutations in the Ras gene and approximately 2/3
of cancers may have an activated Ras signaling pathway due to
other activating mutations in the signaling pathway. When reovirus was tested in combination with chemotherapies, there were
enhanced effects, possibly due to enhanced ability of the virus to
spread from cell to cell due to the chemotherapeutic agent used.
Reovirus has shown promising results in Phase I and Phase II
clinical trials and provides hope for patients with aggressive untreatable cancers.
Cowpea mosaic virus (CPMV) has also been used to detect invasive forms of cancer. In one such report, Cowpea mosaic virus
nanoparticles in homogeneous thin film form have been shown
to interact with vimetin, a type III intermediate filament which
is overexpressed in a number of cancers like breast, prostrate,
and colon cancer, as well as osteosarcomas (6). Vimetin plays a
major role in epithelial mesenchymal transitions which is a sign
of tumor invasiveness and aggression. In tumor metastasis vimetin plays a key role in cell adhesion in migrating cells. Apart
from being cytosolic, vimetin has been found to be exposed on
the surface or secreted outside of activated macrophages. CPMV
particles can be attached with fluorescent labels and can be given
to patients in the form of an intravenous injection. These particles
can then bind to vimetin on the surface of tumor cells. One advantage of using plant CPMV particles is that they can be cleared
rapidly from the circulation without any toxicity or pathological
effects. It has been shown that fluorescently labelled CPMV particles can be internalized by endothelial cells in vivo, a process
which is mediated by vimentin that is expressed on the surface of
cancer cells (6). This leads to state-of-the-art vacuum technology
based high resolution imaging of the vasculature, which is especially useful in the case of tumors.
In addition to developing CPMV for imaging vasculature and
cancers, this virus is also being developed as a viral protein expression system to allow for high levels of protein expression in

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June 2014 Vacuum Technology & Coating

plants in a short period of time. In doing this, CPMV provides

a platform to display peptides for use as a vaccine. The cowpea
plant that the virus infects is an edible host, which is ideal for
an oral vaccine, particularly a vaccine that may have widespread
utility in third world countries. Rae, C. et al reported that the use
of CPMV nanoparticles is a feasible strategy for oral delivery
of therapeutics after evaluating the vector in mouse studies and
demonstrating that the CPMV particle was stable under natural
conditions of ingestion (7). Plants not only serve as the vaccine
delivery mechanism but also function to protect the vaccine antigen from being degraded as it encounters the low pH environment of the stomach. Edible vaccines will be relatively inexpensive to produce and administer, particularly to the approximately
20 percent of the worlds children that currently do not have
access to vaccines that could protect them from preventable infectious diseases. Edible vaccines will also be ideal as a low cost
method to immunize livestock against pathogens (8).
CPMV isnt the only virus that is being utilized to express
proteins for therapeutic purposes. Another promising biomedicine is based on a small virus called adeno-associated virus or
AAV. The virus capsids are about 25 nanometers in diameter and
in nature the virus requires a helper virus such as adenovirus to
replicate. As a part of the virus replication cycle, the virus produces intact empty capsids and then uses a specific set of proteins to insert the viral genome into these preformed capsids.
Researchers have learned how to trick the virus into inserting
therapeutic genes in the capsid instead of the viral genome. Because the virus does not replicate on its own, it has an excellent
safety profile and has been successfully tested in clinical trials.
An advantage of this system over protein therapies is that a corrected gene for the defective protein is used, and as a result there
is long-term expression of the therapeutic protein. Recently, in
Europe, a medical product based on this virus, Glybera, was approved for use in the clinic to treat lipoprotein lipase deficiency.
AAV is currently being developed to express foreign proteins to
stimulate an immune response for vaccine purposes, as well as
treating genetic diseases and provides the potential for new therapies where none currently exist.
In conclusion plenty of opportunities exist for biotechnologists, thin film and vacuum technologists and physicists and
chemists to collaborate for the research and development of virus
particle based technology. It provides hope for viral electronics,
as well as new and improved therapeutics. The development of
these viral particles will be useful for practical applications such
as generation of electricity, as well as in healthcare applications,
such as the development of novel diagnostics, vacuum technology based higher resolution imaging and new treatment options,
particularly for a variety of cancers, which have a better prognosis with early detection. A renewed interest in bacteriophage
therapies also provides another weapon against bacteria that have
developed antibiotic resistance and therefore are no longer treatable with current antibiotic regimens (9). Virus-based technologies also open up the possibility for edible vaccines in the future.
This may provide low cost access to vaccinations and eliminate
preventable diseases worldwide. The applications of the powerful viruses describe here have the potential to change the world.

References
1. Virus-based piezoelectric energy generation. Lee BY, Zhang J, Zueger C, Chung W-J, You SY, Wang E, Meyer J, Ramesh R, and Lee
S-W. Nature Nanotechnology (2012), 7: 351-356.
2. Piezoelectric devices: Squeezed virus produces electricity. Yu, S.M.
Nature Nanotechnology (2012), 7: 343-344.
3. M13-templated magnetic nanoparticles for targeted in vivo imaging
of prostate cancer. Ghosh D, Lee Y, Thomas S, Kohli AG, Yun DS,
Belcher AM, Kelly KA. Nature nanotechnology (2012), 7, 677-682.
4. Global phage diversity. Rohwer F. Cell (2003), 113(2):141.
5. Propionibacterium acnes bacteriophages display limited genetic
diversity and broad killing activity against bacterial skin isolates.
Marinelli LJ, Fitz-Gibbon S, Hayes C, Bowman C, Inkeles M, Loncaric A, Russell DA, Jacobs-Sera D, Cokus S, Pellegrini M, Kim J,
Miller JF, Hatfull GF, Modlin RL. mBio (2012), 3(5):e00279-12.
doi:10.1128/mBio.00279-12.
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M. Nanomedicine (2011), 6(2), 351-364.
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9. 
Phage and Phage Therapy website: <http://www.evergreen.edu/
phage/>

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