22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Helium-generated cold plasma in controlling infection and healing
P. Brun1, V. Russo1, P. Brun2, E. Tarricone2, S. Corrao2, V. Deligianni3, A. Leonardi4, R. Cavazzana5, M. Zuin5 and
E. Martines5
1
Department of Molecular Medicine, University of Padua, Unit of Microbiology, Padua, Italy
2
Department of Molecular Medicine, University of Padua, Unit of Histology, Padua, Italy
3
Department of Ophthalmology, S. Antonio Hospital, Padua, Italy
4
Department of Neuroscience, University of Padua, Unit of Ophthalmology, Padua, Italy
5
Consorzio RFX, Padua, Italy
Abstract: Cold atmospheric pressure plasma has been proposed for the treatment of
infectious keratitis, one of the leading causes of monocular blindness worldwide. Our
recent studies revealed that plasma-generated intracellular reactive species are mandatory
for the antimicrobial effects and also regulate signalling pathways in eukaryotic cells.
Indeed, plasma treatment induces gene and protein expression involved in healing of the
wound in a dose-dependent fashion.
Keywords: reactive oxygen species, eukaryotic cells, infection, bacteria
1. Introduction
Infectious keratitis is a common, highly contagious
disease caused by adhesion, penetration, and
proliferation
of
pathogenic
or
commensal
microorganisms in the cornea, the outermost part of the
eye.
Indeed, the ocular surface is continuously
exposed to microbes (bacteria, fungi, protozoa, and
viruses) usually restrained by the host immune system.
However traumas, as may occur with poorly fitting
contact lenses, injuries, surgical procedures, or
concomitant infectious diseases such as herpetic cold
sores on the lip or upper respiratory infections expose
the outer layer of the cornea and the deeper tissues to a
high microbial load. Upon the infection, inflammatory
reactions take place and alter the architecture of the
cornea leading to scratched surface, shingles,
ulcerations, and destruction of corneal tissue. Actually,
infectious keratitis must be promptly and adequately
treated to prevent corneal perforation and subsequent
dissemination of the infection to underlying ocular
tissues, a complication eventually leading to permanent
impairment of the eyesight [1].
Microbiological assays performed on human corneal
samples revealed a wide range of infectious agents in
keratitis.
Thus, Staphylococcus epidermidis,
Streptococcus
pneumoniae
and
Pseudomonas
aeruginosa are reported to be the most common
pathogens associated with bacterial keratitis.
Filamentous fungi such as Fusarium species and
Aspergillus species as well unicellular fungi, such as
yeasts of the Saccharomycetaceae and Tremellaceae
families are common in mycotic keratitis. Amoebic
keratitis, such as ones caused by Acanthamoeba, is
closely associated with contact lens wear, whereas viral
keratitis, mainly caused by Herpes Simplex Virus
Type 1 (HSV-1), Varicella Zoster virus or Adenovirus
accounts for the most part of infectious keratitis in
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developed countries [2]. Since infectious keratitis
caused by different microbial agents lacks of
pathognomonic clinical appearance, the initial medical
treatments include the topical or systemic
administration of broad-spectrum antimicrobial drugs.
However, the increasing outbreak of multi- or pan-drug
resistant microbial strains and the anatomy of the
cornea characterized by no blood vessels compromise
the efficacy of therapies, extend the treatment period,
adversely affect the wound healing process and worsen
the prognosis of the disease [3].
For all these reasons novel therapeutic strategies
would be a great breakthrough in the treatment of
corneal infection. Low-temperature (cold) atmospheric
pressure plasmas (CAP) have been successfully used to
inactivate various microbes on surfaces and in solution
and to foster healing of skin lesions. Moreover since
antimicrobial effects of CAP are mainly ascribed to a
space-localized burst of reactive species, plasmas are
emerging as promising tools for the treatment of
localized infection in living tissues, such as microbial
keratitis.
2. Methods
2.1. Helium generated CAP
The plasma was produced by applying a
radiofrequency (RF) electric field to a flow of helium
at atmospheric pressure. This plasma source has been
described elsewhere [4] and is schematically outlined
in Fig. 1. As indicated, the source consists of two
coaxial tubes, each closed at one end by a double brass
grid. The outer tube is made out of copper and
electrically grounded while the inner one is made out
of insulating material. Plasma was generated between
two grids acting as electrodes. The two parallel grids
are positioned 1 mm away from one other. The electric
field is formed in the space between the two grids by
1
applying a RF voltage difference supplied by a RF
generator coupled to the source by a matching network.
The matching network raises the voltage to the value
needed for helium ionization of about 1000 V peak-topeak. Despite the high voltage value, the current
flowing in the plasma is so low that the dissipated
power is below 1 W.
The chosen operational
frequency is 4.8 MHz.
The gas flow rate is
1.5 litres/min.
Fig. 1. Schematic representation of helium generated
plasma source.
2.2. Treatment of microbes and eukaryotic cells
Microbes were isolated from clinical specimens,
characterized and cultured following standardized
protocols. For the plasma treatment, bacteria and fungi
were diluted to 1x104 colony forming unit (CFU)/mL.
Eukaryotic cells were growth and cultured in DMEM
containing 10% fetal bovine serum, 2 mM
L-glutamine, 100 U/ml penicillin, and 100 mg/ml
streptomycin, (all provided by Gibco, Italy). One day
before plasma treatment, cells were seeded in 24-well
tissue culture plates at 3x105 cells/well. Microbes and
cells were then exposed for 2 - 5 minutes to the plasma
afterglow in open air, as means the chemical-enriched
helium flow probably accounting for the generation of
reactive species.
2.3. Determination of CAP effects on microbes and
eukaryotic cells
The inactivation of bacteria and fungi by CAP
treatment was assessed by colony enumeration [5].
Thus, microbes were aseptically diluted in warm
medium and cultured for 24 - 72 hours on solid
medium under standard microbiological protocols.
The effects of CAP treatment on eukaryotic cells
were determined by evaluating the generation of
reactive species, expression of genes, intracellular
signalling pathways, and cell proliferation. Methods
were extensively described elsewhere [6]. Briefly,
ROS
generation
was
assessed
using
2’,7’-dichlorodihydrofluorescein
diacetate
(H 2 DCFDA; Molecular Probes), probe specific for the
detection of reactive oxygen species. Gene expression
2
and signalling pathways were investigated by
quantitative PCR analysis and Western Blot
techniques, respectively. Cellular proliferation was
evaluated using flow cytometer by the partitioning
among the daughter cells of fluorescent dye
carboxyfluorescein diacetate succinimidyl ester (CFSE,
Molecular Probe).
3. Results
3.1. CAP treatment selectively inactivated microbes
As reported in Fig. 2, two minutes of CAP treatment
substantially inactivated bacteria and fungi. However,
our studies revealed that helium-generated plasma
highly inactivated Gram-negative bacteria. Thus, the
calculated percentage in growth reduction of
Escherichia coli, Proteus mirabilis, and Pseudomonas
aeruginosa ranged between 97 and 61. The growth of
Gram-positive bacteria (Staphylococcus epidermidis,
Staphylococcus
pyogens,
and
Streptococcus
agalactiae) and fungi (Trichophytum rubrum,
Malassenzia furfur, and Candida albicans) instead
decreased only by 30%. The diverse effects of CAP
treatment are ascribed to different cell wall structures
of bacteria. Indeed, the outer layers of Gram-negative
bacteria are particularly enriched in lipids whereas a
thick wall of proteins and sugars protects Grampositive bacteria and fungi. These considerations
strengthen the role of reactive species in mediating the
antimicrobial effects of CAP. Thus, 1 minute after
CAP treatment ROS increased in bacterial cells and
amplified over time directly inducing bacterial cells
death.
Fig. 2. Effects of CAP treatment on microbes.
3.2. CAP-generated ROS promoted wound-healing in
primary cultured cells
Akin to bacteria, primary fibroblast cells cultured
from healthy subjects reported increased intracellular
ROS levels following two as well five minutes CAP
treatment. In eukaryotic cells however plasma-induced
intracellular ROS were characterized by lower
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concentrations and shorter half-lives as compared to
ROS generated by incubation with hydrogen peroxide
(H 2 O 2 ), a well known pro-oxidant agent (Fig. 3).
Moreover, 5 minutes of CAP treatment induced higher
ROS levels than cells treated for only two minutes.
follows different biological effects [7]. Thus, two
minutes of CAP treatment induced cell proliferation,
migration, and healing of wound after 24 hours
whereas these effects took more time in fibroblasts
exposed to CAP for five minutes.
Fig. 3. ROS generation in fibroblast cells measured
5 minutes after CAP exposure.
4. Conclusions
The selectively effect of helium-generated plasma to
mainly inactivate facultative anaerobic Gram-negative
bacteria without damaging host cells represents a great
advantage in the treatment of polymicrobial infection.
Indeed, antimicrobial agents kill or slow the growth of
both commensal and pathogenic microbes leading to
microbial dysbiosis. Moreover, drugs usually impair
host tissue integrity thus worsening the prognosis of
the infection [3]. It is now well accepted that the
biological effects of CAP are largely mediated by the
generation of intracellular ROS. However, while
cellular compartments of organelles protect eukaryotic
cells from reactive specie burst, bacteria are vulnerable
to low levels of ROS. This effect is however dosedependent [8]. Thus, increased concentration of ROS
leads to different cellular phenotype and function.
These considerations pave the way to a number of
different application of plasma in disinfection and
antisepsis of human tissues. Thus, fine modulation of
plasma power might result in increasing antibacterial
effects, broadening the spectrum of susceptible
microbial species or targeting specific microbial
species. At the same, CAP-generated ROS in host cells
might represent an additional source of reactive
species, helpful in controlling intracellular pathogens
or addressing specific cellular functions.
Actually, primary cells exposed to CAP for two
minutes did not experience membrane injury, DNA
damage, or necrosis/apoptosis. On the contrary, cells
exposed to CAP for 5 minutes reported increased
expression of Annexin V, a membrane protein
associated to inflammation and cellular stress.
The different levels of CAP-generated intracellular
ROS lead to diverse cellular responses. Thus, two
minutes of treatment resulted in transient protein
phosphorilation and increased nuclear translocation of
NF-κB, a transcriptional factor involved in cellular
responses to stimuli such as stress and free radicals.
Following five minutes of CAP exposure instead
NF-κB activation lasted for longer time.
Fig. 4. Western Blot analysis revealing activation of
NFκB protein after 4 - 24 hours in fibroblasts treated
with CAP for 2 or 5 minutes.
Actually, NF-κB phosphorilation works as checkpoint in the cellular fate and the extent of activation
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