Updates from Medicine

Cold atmospheric pressure (physical) plasma in dermatology:

where are we today?
Peter C. Friedman, MD, PhD

The Skin Center Dermatology Group, New Abstract

City, NY, USA Cold atmospheric pressure plasma is physical plasma (essentially ionized gas) created at
room temperature and atmospheric pressure, and it has complex effects on cells, tissues,
Correspondence
Peter C. Friedman, MD, PHD
and living organisms. These effects are studied extensively for medical and dermatological
The Skin Center Dermatology Group use. This article reviews current achievements and new trends in clinical dermatological
200 E Eckerson Rd cold plasma research, discusses the basics of plasma physics and plasma engineering,
New City and describes the most important areas of laboratory plasma research to provide a well-
NY 10956
rounded understanding of the nature, present applications, and future promise of this
USA
E-mail: [email protected]
exciting, emerging technology.

Conflict of interest: None.

Funding source: None.

doi: 10.1111/ijd.15110

“regular” gas, where entire molecules or atoms move freely,

Introduction
plasma is in a higher energy state where atoms lose their elec-
Over the course of the past several years, clinical trials and trons – become ionized – and the resulting electrons and ions
small case series appeared in the dermatological literature move freely and independently from each other. If all particles
describing the use of cold plasma in skin conditions, such as (electrons, ions) are in energy equilibrium, meaning that they
chronic wounds, onychomycoses, actinic keratoses, and have the same very high temperature, we get hot plasma: the
warts.1-4 If someone decides to look deeper, it is easy to find corona of the sun, the plasma in a fusion reactor, or a dis-
the vast body of literature describing the effects of cold plasma charge arc such as in welding or arc lamps. There are exam-
on living tissues from laboratories all over the world. Countless ples of hot plasma use in medicine, mostly as a surgical tool,
publications exist showing the ability of cold plasma to kill bac- but this review is limited to cold plasmas. If only the electrons
teria, selectively inhibit the growth of cancer cells, and induce are hot, but the rest of the particles, representing the main
stem cell differentiation, just to name the most important ones. mass of the gas, are at a lower temperature – the plasma is in
Compared to the large amount of accumulated in vitro and energy non-equilibrium – we get cold plasma, which can be cool
animal data, clinical studies may seem to be just trickling drops, enough to be safe on the skin surface. Of cold plasmas, those
but the opening of the flood gates is inevitable: we are likely to created under atmospheric pressure conditions (i.e. not in a
witness a cold plasma boom in medicine and especially in der- vacuum) are the most feasible for medical applications. Various
matology very soon. shorter or longer designations have been used to identify this
In this article, we will review the highlights of cold plasma type of plasma based on the linguistic tastes of different
physics and experimental data with a focus on the most recent research groups, but lately consensus seems to be forming
clinical developments relevant to dermatology. around the designation “Cold Atmospheric Plasma” or CAP.
CAP is a complex entity, and its composition is not uniform: it
depends on the gas in which it was created and on the way it
Physics of cold physical plasma
was created. The main active components of plasma are reac-
In medicine, plasma means blood plasma, but there is another tive oxygen and nitrogen species, but other reactive species,
use of the term in physics, where the entity “plasma” is referred charged particles, its electric field, and partly even the UV pho-
to as the fourth state of matter or as an ionized gas. This is tons and ozone generated during plasma production may play a
what can be distinguished as physical plasma. Compared to role in its complex effects on living tissues. 1

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2 Updates from Medicine Cold plasma in dermatology Friedman

Just as plasma composition is complex, so are its interac- the apoptosis-inducing effect of CAP-treated medium on mela-
tions with biological targets. For example, plasma’s inhibitory noma cells, the results depended on the exposure length.22
effect on bacteria is believed to be related both to direct oxida- Exposure time is only useful as a valid measure of plasma dose
tive stress caused by reactive species, and damage to the cell when used in the context of a single plasma source but not
wall and cell membrane caused by charged particles contained when comparing different plasma devices as the composition of
in plasma.5 Some plasma-induced changes in eukaryotic cells the plasma generated by them can differ greatly. For example,
are attributed to reactive oxygen or nitrogen species acting using the very same jet plasma device but different feeding
directly as signaling molecules6 or via inducing oxidative stress- gases changes the biological effect exerted on bacterial
related triggering of various pathways,7,8 or by other mecha- growth.23 A detailed study using jet plasma and a 3D skin
nisms, such as changing intracellular ion concentrations.9,10 model showed that interleukin expression induced by CAP can
Likely it is the differences between different cell lines in their be modified by changing not only the length of exposure but
status of cell proliferation and cell death-related pathways that also energy input, carrier gas.24 One possible approach is to
can explain plasma’s intriguing selectivity when it comes to characterize every system for every application individually. The
malignant and nonmalignant cells.11 The effect of plasma as an more comprehensive way would be to develop core markers of
electric entity, unrelated to reactive species, is also a considera- plasma effect to enable comparison between different devices.
tion. Studies using not plasma but nanosecond electric fields on One such attempt was based on the significance of reactive
cell cultures demonstrated increased intracellular calcium influx, species in CAP effect: in a complex study the utility of total oxi-
and effects on intracellular signaling just as it is seen with CAP dation potential as a plasma dose measure was proposed.25
application.12,13 On the other hand, plasma effects are observed The authors suggest that total oxidation potential quantifies the
even when using experimental designs that eliminate the pres- effects of generated reactive species, the main driver of – for
ence of an electric field, underscoring the complex nature of example – CAP-induced decontamination, while also acknowl-
these interactions.14,15 It seems that not just one but multiple edging that other factors that have no influence on total oxida-
plasma components together shape the effect of CAP, and that tion potential may also be important in different CAP functions.
effect also varies based on the targeted biological structures. Dose measurement and dose limit setting ties into plasma
Another important question is the depth of penetration of safety as well. From that perspective, another approach is
plasma. It seems it can be answered only in terms of penetra- under development to use biological assays to measure the
tion depth of specific components of CAP as it greatly differs. A effects of CAP devices, which ultimately may be the most
study measuring CAP penetration through tissue using slices of meaningful way to assess not just safety limits but also effective
pig muscle of various thicknesses found that while only 5% of doses.26
reactive oxygen species penetrated 0.5 mm of tissue, the pene- CAP parameters, concentrations of species, and their ener-
tration rate of reactive nitrogen species was 80%. The authors gies are very much controllable, which permits for any specific
also detected some reactive species penetration through 1.25 treatment to find a niche where efficacy of treatment can be
mm tissue slice.16 In animal experiments, plasma was shown to achieved without short and long-term skin damage.
induce apoptosis throughout a 2.8 mm thick tumor in a mouse.
The same study also demonstrated penetration of plasma-in-
Plasma device designs
duced reactive species through a 1 mm thick layer of pig skin
and multiple millimeters of agarose gel and noted differences in CAP is primarily created by high frequency electric discharge,
the penetration depth of various species and with different using a pulse generator. Settings of the pulse generator, such
plasma settings.17 Plasma penetration depth was also exam- as frequency, power, wave form, and also the composition of
ined in human mucosa: a Raman microspectroscopy study gas in which the plasma is generated and the length of applica-
detected CAP tissue penetration to about 270 lm of the human tion, have a significant impact on the biological effects of CAP.
cervix mucosa.18 Biological liquids present on the target sur- Clinical trials have used three main types of CAP device
faces also influence not only the depth of plasma and reactive designs: jet, dielectric barrier discharge (DBD), and surface
species penetration but also the composition of plasma compo- microdischarge plasmas. These devices have very important
nents reaching deeper layers of the targeted tissue.19,20 The practical differences, which determine how and for what pur-
concepts and challenges of tracking penetration of plasma and pose they can be used best. The physics, engineering, and
reactive species into tissues, the various surface and deep characteristics of the different CAP devices have been
interactions, and the challenges represented by half life differ- described in great detail.27-34 The following is a concise sum-
ences between different components of plasma were reviewed mary of their main features. The plasma jet has an electrode
and discussed in detail by Szili et al.21 away from the skin, and the plasma generated around the elec-
In any medical treatment, the amount of “medicine” given is trode is literally blown onto the skin using gas flow, typically a
of great importance. Just as plasma penetration is complex, so noble gas, such as argon or helium (Fig. 1). This design
is the dosing of plasma. For example, in experiments showing reduces potential issues related to electric contact with the skin,

International Journal of Dermatology 2020 ª 2020 the International Society of Dermatology

Friedman Cold plasma in dermatology Updates from Medicine 3

Figure 1 Helium plasma jet. Handpiece of a plasma jet device with Figure 2 Floating-electrode DBD plasma. Top panel: pulse
outflow of helium plasma showing the characteristic “plasma glow”. generator and hand-held electrode. Bottom panel: Plasma is being
In this particular device, plasma is generated by a rectangular generated using the electrode tip. The electrode connected to the
electrode inside the hand piece. Most of the plasma develops at the pulse generator must be kept near the treated surface. Either the
corners of the electrode, resulting in the brighter, more “plasma- tip or the side of the electrode can be used, but plasma will only
rich” streams in the plasma flow. The device pictured is a form where the gap between the electrode and the target surface
Piezobrush, manufactured by Relyon Plasma GmbH (formerly does not exceed 1 mm. The device pictured is an experimental
Reinhausen Plasma GmbH) prototype described previously by Friedman et al.3

device contamination problems, and need for sterilizing elec- this issue. Alternatively, moving an electrode over the surface
trodes. It also facilitates the treatment of uneven, incongruous can also result in sufficient cumulative plasma delivery over
surfaces but is only applicable for larger target areas when time even for a larger target area. Because of the close proxim-
wider, multi-jet torches are used, or by moving the jet or torch ity to the skin, contact is practically unavoidable. This necessi-
over the treated surface. The plasma energy delivered to the tates electrode sterilization or single use parts to avoid
surface is limited, and most jet plasmas need a continuous contamination. The safety of electricity affecting the target sur-
source of gas requiring considerable installation reducing, but face must also be addressed. A variation of DBD principles
not entirely eliminating, portability. DBD plasma devices with a called surface microdischarge uses a powered plane electrode
so-called floating electrode create CAP directly on the surface and a grounded wire-mesh electrode near the skin. This setup
of the skin by generating electric discharges between an elec- eliminates the electric current going through the skin and may
trode connected to the pulse generator and the skin, which cover a larger area, helping to overcome some of the limitations
serves as the other electrode (Fig. 2). This plasma can have of floating-electrode DBD plasma designs, while other issues,
substantially higher energy compared to jet plasmas. Because such as contact contamination concerns, remain.
of the need for the electrode to cover the target area with a very A fundamentally different type of plasma delivery yet to be
narrow but steady gap, uneven, large, or nonflat surfaces repre- seen in published clinical trials in dermatology but used exten-
sent a challenge for floating-electrode DBD plasma. Engineering sively in cell culture based plasma experiments is indirect
variously shaped, larger, possibly flexible electrodes may solve plasma: application of plasma to a liquid, such as cell culture

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4 Updates from Medicine Cold plasma in dermatology Friedman

medium or saline solution, which is then used to create the interesting from a dermatological perspective, while others will
effects of plasma treatment on the desired target without the be referenced as needed when reviewing corresponding clinical
presence of the original plasma source. It works because at plasma applications.
least some of the components of plasma that are responsible For general reference purposes, one of the earliest most
for its effect on living tissues, such as free oxygen and nitrogen comprehensive experimental studies was published by Dobrynin
radicals, can survive in solutions and may have the same effect et al.40 The authors presented extensive experimental findings
as applying plasma itself. Indirect plasma, however, is more describing the biological effects of plasma on different types of
complex than mixing plasma and a liquid. Plasma generation in living cells, analyzing and discussing in detail the physics of the
the vicinity of a liquid alters the physics of the process. Plasma- interaction and the molecular and biological changes in the tar-
liquid interactions can be created using different settings, such get cells. The expansion of plasma science resulted in many
as direct discharge in liquid, gas phase discharges, and multi- reviews of either the entirety of in vitro plasma research,41,42 or
phase discharges. The generated reactive species differ in the specific subtopics, such as cancer CAP studies,43,44 antimicro-
liquid and gas phase and vary according to the creation bial effects,45,46 and biological safety,47 for example.
method, liquid, and plasma parameters used.35,36 Chemical and
biochemical reactions induced by CAP are also important parts Selective cell death of malignancies
of the equation.37 Indirect plasma can be an appealing CAP A literature review from 2018 found 190 original research arti-
delivery method for a number of fields in medicine.38 Adjusting cles published up to and including 2017 describing CAP use in
the plasma “dose” to obtain a predetermined composition of oncology, which included in vitro experiments, in vivo animal
reactive species based on the needs of a particular application studies, and clinical trials (of which they have found only
requires the careful selection of plasma sources, delivery three).43 There was an exponential increase of the number of
parameters, and treated liquid.39 publications annually during the period of 2005 and 2017.
If in fact proven to be equivalent to actual plasma delivery, Eighty-seven percent of these works studied human cancer cell
indirect plasma could eliminate many practical issues of plasma lines, and 11% of those were melanomas, but murine mela-
treatments, as the treated intermediary liquid can be stored, noma cells (9% of the total publications) were the most studied
shipped, and may also be used over very large areas of skin. of all the murine cell lines at 58.6%. The three identified clinical
As indirect plasma removes plasma generation from the trea- trials described plasma use in nondermatological cancer treat-
ted area, some plasma components are not transferred. Two of ments, showing encouraging results applying CAP as a part of
these are UV photons and ozone. Eliminating UV and ozone a combination treatment for malignant pleural mesotheliomas48
exposure of plasma-treated skin can easily be seen as a bene- and advanced head and neck cancers,49,50 although in the latter
fit, as it voids patient safety concerns related to these factors. case, it is possible that the clinical improvement had to do with
Table 1 summarizes the most important features of various the antibacterial and wound healing effect of plasma, which we
plasma device designs. will review under clinical applications.
In vitro studies show that CAP can selectively induce cell
death in a variety of malignant cell lines. As early as 2007, Frid-
In vitro plasma research
man et al.22 showed that low dose plasma treatment induces
CAP has numerous, well-investigated effects on biological tar- apoptosis in cultured melanoma cells using a floating-electrode
gets, but their detailed analysis is beyond the scope of this DBD plasma device comparing treated and nontreated cells.
review. Here we will only discuss the two research areas most Another group went one step further by comparing CAP effect

Table 1 Plasma device types

High-energy Easy to use on Easy to use on Independent use by

plasma UV exposure large areas uneven surfaces Fully portable patients is feasible

Plasma Jet No Yes Somewhat Yes Somewhat No

FE-DBDa Yes Yes No No Yes No
SMDb No Yes Somewhat Somewhat Yes Somewhat
Indirect plasma No No Yes Yes Yes Yes

The table shows the typical features of various plasma generation technologies, based only on devices that have been used in published
research. Other device designs may have different characteristics. These features are important when evaluating the best target diseases for
each device type.
a
Floating-electrode dielectric barrier discharge plasma.
b
Surface microdischarge plasma.

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Friedman Cold plasma in dermatology Updates from Medicine 5

on melanoma cells and primary human keratinocytes. After attributed to increased Wnt signaling caused by CAP-generated
using helium jet CAP treatment, they found a substantially reactive oxygen species.60 Taken together with another study,
higher percentage of cell death in melanoma cells, and the dif- which (although without examining stem cells specifically) showed
ference was shown to be caused by CAP-induced apoptosis, as that CAP treatment stimulated angiogenesis in an ex vivo experi-
demonstrated using TUNEL assay.51 This selectivity is likely not mental setting,61 it seems that CAP may be a very promising tool
absolute: adjustments of plasma characteristics, such as to heal wounds and to assist in tissue engineering.
plasma emission intensity and power, could be used to optimize Neural cell differentiation, for example, is also influenced by
the selective ablation and migration inhibition effect of a helium plasma. A study found that CAPinduced neural differentiation in
jet plasma on cultured human metastatic MDA-MB-231 breast cultured mouse neuroblastoma cells. The authors demonstrated
cancer cells, using bone marrow-derived human mesenchymal that this was a result of a complex cascade leading to the acti-
stem cells as controls.52 In another study, comparison of CAP vation of the Trk/Ras/ERK signaling pathway by CAP-derived
effect on four different head and neck squamous cell carcinoma cytosolic and mitochondrial reactive species.62 Another study
cell lines and two normal oral cavity epithelial cell lines in vitro using a culture of immortalized murine neural stem cell line
showed significant but varying inhibitory effect on the malignant C17.2 also found increased neural differentiation when treated
cell lines and only minimal deleterious effect on the nonmalig- with CAP. While no specific signaling pathway was identified,
nant cells.53 A comparison of A431 squamous cell carcinoma the authors concluded that CAP achieved its effect via reactive
cells and HaCaT keratinocytes treated with indirect plasma (cell nitrogen species.63 These in vitro findings raise the hope of
culture medium and phosphate buffered saline previously using CAP as a potential treatment for neurodegenerative dis-
exposed to CAP) showed a dose-dependent increase in apopto- eases, although given the complexity of the neural tissue, a lot
sis of the squamous cell carcinoma cells, which was attributed of work still needs to be done using what one author described
to the presence of reactive oxygen species.54 These findings as “more realistic models of neurological disease”.64
suggest that because of the different plasma sensitivities of var- Keratinocytes: an ex vivo study of CAP-treated skin biopsy
ious cancer (and nonmalignant) cell lines, adjustment and opti- specimens maintained in organ culture found no change in
mization may be needed not only when targeting malignant select differentiation markers but showed increased basal ker-
cells of different tissue origins but even those of the same cell atinocyte proliferation and Ki67 expression. This effect was only
type. Demonstrating the complexity of CAP treatment, Biscop observed when a specific treatment time was used: longer or
et al.55 evaluated the influence of cell type, cancer type, and shorter CAP exposure failed to achieve the same results.65 A
cell culture medium composition on the efficacy of direct and complex and detailed study using both HaCaT human ker-
indirect CAP treatment and found that the selectivity of in vitro atinocyte culture and an in vivo mouse model examined the
CAP effect is indeed influenced by those variables, especially effect of CAP on cell proliferation in the context of wound heal-
when indirect plasma treatment is used. Changes in treatment ing. They found induction of epidermal cell proliferation and
parameters may influence not just what plasma does but also increased skin remodeling when compared to untreated con-
the way it does it: it was shown using surface microdischarge trols.66 The authors also showed CAP-mediated b-catenin acti-
plasma on cultured melanoma cells, that while higher doses of vation and translocation to the nucleus in both in vitro and
plasma cause apoptosis, shorter exposure time leads to non- in vivo models, which seem to support the above discussed
apoptotic cell inactivation via induction of cell senescence.56 A implication of Wnt signaling (which is profoundly linked to b-
follow-up study using a similar experimental setup demonstrated catenin activation) being the effector of CAP treatment in mouse
that the melanoma cell senescence effect of in vitro CAP treat- limb bud growth experiments. Another study using argon jet
ment was triggered by plasma-induced intracellular Calcium plasma on in vitro cell culture studies with human fibroblasts
influx.9 The in vivo effects are perhaps even more complex: and an in vivo mouse skin wound healing model found that
while they are possibly simply the result of CAP-induced apop- plasma treatment induced the expression of IL-6, IL-8, MCP-1,
tosis or cell senescence, it was also suggested that CAP can TGF-ß1, and TGF-ß2, and promoted the production of collagen
restore the immunogenicity of malignant cells,57 or it can cause type I and alpha-SMA, which play a role in wound healing.67
58
direct immune stimulation to induce cancer ablation. Angiogenesis and tissue macrophage activation induced by
CAP are also likely to improve wound healing. Miller et al.
Stem cell differentiation and cell proliferation and wound demonstrated that microsecond pulsed DBD plasma treatment
healing stimulates the production of vascular endothelial growth factor,
Perhaps the most exciting CAP research topic is the induction of matrix metalloproteinase-9, and CXCL 1 that in turn induces
stem cell differentiation and cell proliferation using plasma. One of angiogenesis in mouse aortic rings in vitro.61 A host of proan-
the earliest studies showed enhanced differentiation of chondro- giogenesis factors, including growth factors, cytokines are mod-
cytes and osteoblasts after CAP treatment, first in cell culture,59 ulated by CAP in an autocrine and paracrine way, providing
and later in an organ culture system where plasma-treated mouse further insight into at least one of the ways CAP enhances
limb buds showed superior growth and survival, which the authors wound healing.68 Other mechanisms, such as influencing redox-

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6 Updates from Medicine Cold plasma in dermatology Friedman

mediated tissue response, activation of the nuclear E2-related bacteria and induction of wound healing, while lowering treatment
factor signaling, and stabilization of the scaffolding function and frequency to create an effective and practical solution.
actin network in dermal fibroblasts, have also been studied.69 A case report of CAP plasma treating chronic postoperative
The role of CAP modulation of the nuclear E2-related factor auditory canal and nasopharynx bacterial infection following
pathway (along with plasma-induced p53 inhibition) was con- tympanoplasty, incus resection, and reconstructive surgery of
firmed by studying plasma effects in a dermal, full-thickness the auditory canal highlights that plasma can be used for differ-
wound model in mice. In the same study, the authors described ent kinds of chronic infectious and nonhealing wounds.77 Bacte-
a complex CAP treatment-related change in the inflammatory rial load reduction is only part of the way CAP improves
response leading to a conclusion that singling out one pathway healing: a randomized, placebo-controlled clinical trial of 37
as the main driver of changes in wound healing was not appro- patients with herpes zoster showed that CAP treatment was
priate as effects of CAP as well as their consequences are superior to placebo in improving herpes zoster-related pain.78 A
complex and many.70 Another in vitro study using mouse fibrob- single case report found CAP beneficial even in a case of Hai-
lasts and human keratinocytes detected elevated expression of ley-Hailey disease, where the primary etiology of chronic ero-
genes that the authors considered important for maintaining sions is a genetic mutation, not infection.79
skin function (such as Type I collagen, fibronectin, and VEGF)
leading to the suggestion of CAP being potentially beneficial for Onychomycosis
skin rejuvenation.71 Argon jet plasma was used successfully to inactivate fungal prolif-
eration of Trichophyton interdigitale, Trichophyton rubrum,
Microsporum canis, and Candida albicans isolates ex vivo, with dif-
Clinical cold plasma applications in
ferent species responding differently: Candida albicans was the
dermatology
most and Microsporum canis the least susceptible to CAP.80 An
Wound healing in vitro study using surface microdischarge plasma achieved fungal
The cold plasma application most extensively studied in clinical tri- growth inhibition in Trichophyton rubrum and Microsporum canis
als is the treatment of wounds. While the pathogenesis of chronic but only when daily 10-minute long treatments were used for
wounds is complex, the presence of bacteria does play a role. 9 days.81 Another ex vivo nail infection model based study used
Plasma has a well-documented inhibitory effect on bacteria,72,73 helium jet plasma to successfully inhibit Trichophyton rubrum
and this is what initially led to the idea of using cold plasma to adhesion, germination, and growth in vitro.82 The first clinical pilot
reduce bacterial load of wounds and thus facilitate healing.2 The used DBD plasma on toenails. The authors achieved over 50%
first published studies employed jet-type plasmas using argon as clinical and about 15% mycological cure in 13 patients who have
carrier gas.74 The daily treatments may not be practical for all completed the study.83 Given the recalcitrant nature of onychomy-
patients, and the onerous treatment schedule was in fact an identi- cosis and the limited options to treat it, a nail treatment CAP device
fied reason for patient dropout. Another group used a floating-elec- could be a useful addition to our tool box.
trode DBD plasma device for chronic wound treatment with three
applications a week for 8 weeks, followed by an observation period Actinic keratosis treatment
for 4 weeks. The DBD plasma treatment – similarly to jet plasma – A recent, landmark, large scale, multicenter study comparing
resulted in a reduction of bacterial load, and it also provided some the four most common field-directed treatments of actinic ker-
advantage to wound healing as assessed by ulcer size.1 A more atosis (AK) found that only 29–75% of patients achieved at least
recent trial – also using argon jet plasma – had a once a week treat- 75% lesion count reduction 12 months after the end of treat-
ment schedule. Not only the authors achieved significant decrease ment, with an adverse effect percentage in the 90s,84 indicating
of bacterial load in plasma-treated patients, they also used Pres- that there is certainly room for improvement, and there is an
sure Ulcer Scale for Healing scores to demonstrate the superiority unmet need for highly effective and well-tolerated AK treat-
of CAP treatment of chronic wounds compared to wound care ments. The extensive in vitro research showing the ability of
alone.75 Reducing bacterial load is not the only beneficial effect CAP to induce selective cell death in various cancer cell lines
plasma has on wounds. As we have reviewed above, CAP has paired with evidence of its safety and great tolerability laid the
been shown to induce stem cell differentiation and cell proliferation foundation for clinical use in AK, which may also serve as a
in various cell types. A study investigating the effects of argon jet clinical surrogate for (skin) cancer, the ultimate target of many
plasma on skin graft donor sites in a non-bacteria-colonized envi- plasma researchers. The first reported trial – by our group –
ronment demonstrated a positive impact on wound healing, com- used a floating-electrode DBD plasma device for lesion-directed
pared to untreated controls in a split-site experimental design.76 treatment of AKs. Seventeen lesions were treated (in five
Advances in engineering play an important role in the future of patients) only one time and evaluated one month later. Nine
plasma wound treatment: the development of hand-held, portable lesions resolved fully, and three improved significantly with a
devices increases access to treatment. Fine tuning plasma settings single treatment, which corresponds to a 53% clearance rate
may also help to optimize delivery to reach maximum inhibition of and a 70% rate of at least significant improvement or clearance.

International Journal of Dermatology 2020 ª 2020 the International Society of Dermatology

Friedman Cold plasma in dermatology Updates from Medicine 7

double-stranded DNA viruses just like the human papillo-

mavirus. It has been long established that plasma induces intra-
cellular calcium influx.40 There is evidence that the well-
established anti-wart agent, imiquimod, may act at least in part
by inducing intracellular calcium influx.89 Heat therapy, which
was shown to be effective at treating warts,90 also possibly
exerts its effect by triggering calcium influx, based on studies
examining the effect of hyperthermia on epithelial tumor cell
Figure 3 Field-directed actinic keratosis treatment using floating-
electrode DBD plasma. Left panel: chest with multiple actinic lines.91 These findings make it feasible that either by inducing
keratoses before being treated using a 10-cm long quartz covered intracellular calcium influx, or possibly by prohibiting viral repli-
electrode with multiple passes of slow sideways motion utilizing the cation,or both, CAP may be an effective treatment against
side of the electrode to evenly cover the entire target area. warts. Our group has published the first case series showing
Treatment settings were based on our previously reported lesion
the efficacy of CAP in adult patients with warts.4 Not only was
directed AK treatment study.3 Middle panel: significant decrease of
AK lesion number one month after a single treatment. The patient CAP able to clear warts effectively and without recurrence for at
received four additional treatments at 1-month intervals. Right least 5 months, the treatments were entirely painless and there
panel: Maintained improvement five months after completing the was no discomfort or blistering following the procedure. This tol-
last treatment of the series. (Khan A, Fridman G, Fridman A, erability is crucial when treating larger warts and special popula-
Friedman PC, unpublished data. The photographs were taken as
tions, such as children (Fig. 4). A follow-up study also
part of an IRB controlled clinical trial: Western Institutional Review
Board, date: 11/10/2016, protocol number: 20130084, ClinicalTria demonstrated the efficacy and exceptional tolerability of CAP
ls.gov, registration number: NCT02759900) when used to treat warts in children.92 Both of these proof-of-
concept trials were limited by small patient number, and while
they are promising, further research must confirm their findings
The procedure was extremely tolerable with no discomfort dur- on substantially larger number of patients. Additional studies
ing or after treatment.3 Later, another group used jet plasma for should also investigate the optimal settings of plasma delivery
a series of field-directed AK treatments of seven patients. Tar- for different types of warts.
get areas were treated twice a week, seven times total. The
authors presented before and after total lesions counts and Treatment of hair loss
counts of improved lesions according to the Olsen clinical AK As discussed above, CAP has been shown to induce stem cell
severity scale. They found that the total lesion count decreased differentiation in various tissues. Minoxidil, one of the most
by 23%, and 53% of lesions were given a lower Olsen grading, established treatment modalities for androgenetic alopecia
marking clinical improvement. Patients tolerated the treatments (AGA), may induce hair follicle stem cell differentiation and hair
well.85 While it is encouraging that two different types of plasma growth via triggering cellular calcium influx.93 As CAP has also
devices, using different protocols, were shown to be effective been shown to induce cellular calcium influx in vitro,40 it can be
and tolerable for AK treatment, larger trials are needed to con- hypothesized that CAP may be able to improve AGA by induc-
firm these findings and to fine tune our plasma delivery tech- ing hair follicle stem cell differentiation based on its similar
niques to achieve the best possible outcome. Although the DBD effect on other stem cell populations and specifically via
plasma study produced higher clearance and improvement
rates with a single application than the jet plasma trial with
seven applications, the comparison is not entirely valid because
one trial was lesion directed, the other field directed. Modifying
electrode design and delivery technique may allow to keep the
higher efficacy of DBD plasma even when used for larger target
areas and field-directed treatment of AKs (Khan A, Fridman G,
Fridman A, Friedman PC, unpublished data) (Fig. 3). It also
needs to be investigated what effect, if any, CAP field treatment
has on long-term keratinocyte cancer development, as the
Figure 4 Extensive plantar warts treated with floating-electrode
transformation rate of AK into skin cancer may not be directly DBD plasma. Left panel: plantar warts prior to plasma treatment.
linked to the clinical severity of AK lesions.86 Right panel: fully resolved warts 3 months after a single treatment
with a floating-electrode DBD plasma device using previously
reported treatment settings.4 (Friedman PC, Fridman G, Fridman A,
Treatment of viral warts
unpublished data. The photographs were taken as part of an IRB
A recent disease target of CAP in clinical trials is viral warts.
controlled clinical trial: Western Institutional Review Board, date: 11/
The rationale stems from multiple sources: plasma has been 10/2016, protocol number: 20130084, ClinicalTrials.gov, registration
shown to destabilize adenoviruses,87,88 which are nonenveloped number: NCT02759900)

ª 2020 the International Society of Dermatology International Journal of Dermatology 2020

8 Updates from Medicine Cold plasma in dermatology Friedman

triggering cellular calcium influx. A recent rat model based study safe and tolerable, at least within the given exposure parame-
showed that CAP treatment using a nitrogen-CAP jet-type ters. Yet another study examining a jet-type plasma device also
device increased hair follicle diameter.94 Using CAP on the concluded that the application was safe but noted that changing
human scalp presents challenges, as the large surface area parameters, like extending exposure time, may increase risks.99
would require long treatment times using currently available The presence of reactive species raises a concern of muta-
devices and the presence of hair may impede plasma delivery. genicity for CAP. Jet-type and DBD CAP devices were exam-
Another concern is the questionable feasibility of frequent, ined for mutagenicity, and they were found safe within the
ongoing, office-based treatments for a chronic condition. As dis- operating parameters studied.100,101 Another approach, study of
cussed above, a plasma-treated liquid medium can exert the skin barrier function and skin moisture, also found CAP tolera-
effects of plasma and, in this case, it would allow for easy treat- ble and safe.102 As mentioned in the context of plasma dosing,
ment of the entire scalp and the treatment could be self-admin- another approach is to examine the effects of CAP on the
istered by the patients at home. Our group has begun a clinical actual biological targets, in our case on the human skin. This
trial based on the above principles (clinicaltrials.gov: approach uses measurements to assess changes in the struc-
NCT04379752). Preliminary assessment showed that ongoing ture and function of the human skin after being treated with
indirect plasma treatment for the scalp is well tolerated and pre- plasma. One such proposed model uses dermatoscopy, confo-
ferred by patients (Khan A, Fridman G, Fridman A, Friedman cal laser scanning microscopy, hyperspectral imaging and also
PC, submitted for publication). Large, controlled clinical trials quality-of-life questions and visual assessment to determine the
and examination of the actual effects of direct and indirect CAP safety and tolerability of CAP.26 All studies addressing safety
on human hair follicles are needed to explore this potential highlight the device and treatment setting specific nature of their
application. results. Even though CAP seems to be generally safe, likely
every single device needs to be tested. To ensure that CAP
can be used with confidence, there is a need for a comprehen-
Clinical safety of plasma
sive approach. International efforts on developing plasma safety
Plasma is a very complex physical entity, and all plasma com- standards are coordinated by IOPMS (International Organization
ponents may raise safety concerns. For some aspects of on Plasma Medical Device Standardization), which – at the time
plasma such as UV radiation, ozone, nitrogen oxide, and electri- of writing this article – is co-chaired by Dr. Eun Ha Choi
cal current, there are already existing safety standards issued (Kwangwoon University, Seoul, Korea) and Dr. Kai Masur (Leib-
by national regulatory authorities. Assuring compliance with niz-Institute for Plasma Science and Technology, Greifswald,
such standards is a matter of simply measuring the values for Germany), with Dr. Alexander Fridman (C. & J. Nyheim Plasma
every CAP device. Other secondary aspects may require more Institute, Drexel University, Philadelphia, PA, USA) representing
CAP-specific analysis and perhaps the development of new the United States on its board. IOPMS is considering device
safety standards. For example, chromosome damage of cul- safety under the major aspects: (i) biomedical safety, related to
tured brain cancer cells caused by DBD plasma was measured optimization of protocols, suppression of side effects, detailed
using a micronucleus assay, which can be used as a tool to analysis of clinical studies; (ii) physical, technological, and elec-
measure possible genotoxicity in other cells and tissues as tric safety, related to limitations of generation of ozone, UV radi-
well.95 A more recent study using such assay, along with apop- ation, leak currents, electromagnetic interaction with other
tosis assay and hypoxanthine phosphoribosyl transferase devices in medical environment; (iii) safety aspects related to
(HPRT) gene mutation assay on plasma-treated liver cells, device (and treatment protocol), sensitivity to device positioning,
found time-dependent formation of micronucleus formation after dosimetry, environmental conditions. The diversity of different
CAP exposure, but no delayed genomic instability like delayed types of plasma devices represents a substantial challenge for
reproductive cell death and micronucleus formation was found IOPMS. The goal is to address all these concerns in a way that
in the progeny cells, nor was there an increased HPRT mutation is applicable to all medical plasma technologies and to develop
frequency either in the target cells or their progeny.96 One study safety standards and parameters that can be adopted by regu-
concluded that when using a jet-type plasma, UV radiation did latory bodies worldwide.
not penetrate beyond the stratum corneum and the exposure
was one order of magnitude lower than that of sun exposure.97
Future trends
Another study presented an ex vivo model to examine the
safety of any CAP devices. The authors analyzed skin speci- As much as it can be predicted, the near future of plasma der-
mens treated with a surface microdischarge plasma using matology will likely be determined by two main factors, both
microscopy, electron microscopy, and a DNA double-strand related to the beginning of commercialization: expansion of clini-
break assay. They found no signs of structural changes, but cal use areas driven by commercial competition and by the clini-
there was an increase of double-strand DNS breaks at some cal availability of this technology, and intensified basic research
settings.98 Overall, the authors found the tested CAP device because of increased interest and funding stemming from

International Journal of Dermatology 2020 ª 2020 the International Society of Dermatology

Friedman Cold plasma in dermatology Updates from Medicine 9

mainstream acceptance of CAP technology. Besides different proliferative, or – in contrast – degenerative skin conditions can
plasma devices now sold for wound treatment in the European be evaluated for plasma treatment, not to mention using plasma
Union and the Canady HeliosTM Cold Plasma Therapy helium to reverse skin aging by inducing stem cell differentiation.
plasma jet currently in clinical trial to augment cancer surgery Another field not discussed here in detail is transcutaneous drug
(www.usmedinnovations.com), there are at least three condi- delivery. Plasma has been shown to induce the penetration of
tions (onychomycosis, actinic keratosis, warts) with successful topically applied substances into the deeper layers of the skin,
proof-of-concept studies awaiting commercialization. As there is opening the door for enhancement of the efficacy of medica-
an overlap between the abilities of various types of devices from tions targeting the skin and also enabling topical administration
different research teams, there will likely be a competition of medications intended for systemic absorption.106-108
between respective device manufacturers to provide the “best” The cost of plasma devices can vary greatly, but if one takes
plasma treatment for the already established target diseases the German hand-held device plasma care as an indicator, or
and also to expand the scope of each device or device family to even the more sophisticated machines intended for research
new skin conditions to capture the largest market share possi- use, the price can be substantially below even that of the
ble. Off-label use is more the rule than the exception in derma- cheapest lasers used so frequently in dermatology offices.
tology. Once cold plasma devices will be available, it will not be Depending on the application, simple, low cost devices may suf-
surprising to see case reports of their innovative use for various fice, such as a battery operated hand-held ‘flashlight’ DBD CAP
skin ailments. Based on the success of actinic keratosis pilot device described in 2012, which had an estimated cost of
studies, skin cancer clinical trials are imminent, especially for around 100 USD.109 In the United States, some treatments,
squamous cell carcinomas, which can be seen as part of a con- such as destruction of warts and actinic keratoses, can feasibly
tinuum that includes actinic keratosis on the other end (Fig. 5). be billed to insurance companies using Current Procedural Ter-
Any condition that can be improved by reducing the number of minology (CPT) codes, while others, such as onychomycosis
microorganisms on the skin is also a fair target. Plasma is treatments, can be offered on a relatively affordable cash-fee
known to disrupt biofilms,72,103 which are shown to play a role basis given the low cost of the devices. Alternatively, a develop-
in itch in patients with atopic dermatitis, for example.104 While ing plasma dermatology community may lobby for its own CPT
the first trial using CAP to reduce pruritus failed to show bene- codes, just as we saw it happen for reflectance confocal micro-
fit,105 further studies in this field are expected using different scopy. The lower price range also means that there will be less
settings and different devices. Skin microbiome is gaining more of a barrier adapting CAP, in fact it is feasible that one derma-
and more recognition, and plasma seems to be well positioned tology practice will own more than one type of CAP device, just
to modify it to our benefit. Based on our constantly evolving like it is the case with lasers today.
understating of CAP causing selective inhibition of malignant As we have seen in all plasma research areas, CAP is a
cell growth and induction of cell proliferation, many malignant, complex entity with even more complex effects on cells and tis-
sues, which we are only beginning to understand. A very excit-
ing and rapidly evolving field is the influence of plasma on
signaling. For example, CAP effect on intracellular calcium
influx is emerging as a putative crucial step in modulating vari-
ous signaling pathways and mitochondrial and endoplasmic
reticulum responses. We hypothesize that it is possibly one of
the main mechanisms of most biological plasma effects shown
in clinical trials. It has been well demonstrated that modifying
delivery parameters can profoundly change the biological
response to CAP. It will require extensive and expensive
Figure 5 Atypical squamous cell proliferation treated with floating- in vitro, in vivo, and clinical research to explore this field. Fine
electrode DBD plasma. Left panel: This extensive, multifocal, tuning delivery settings, protocols, and target selection can
ulcerated eyelid lesion was diagnosed to be within the actinic likely improve efficacy numbers of plasma treatment even for
keratosis spectrum, although in situ squamous cell carcinoma was
conditions where there are successful proof-of-concept clinical
part of the differential diagnosis. The entire lesion was treated one
studies, but it requires large and costly clinical trials. Commer-
time with a floating-electrode DBD plasma device using previously
reported treatment settings.3 Right panel: no sign of recurrent lesion cialization is hoped to increase available funding for plasma
15 months after a single CAP treatment, although a new lesion is research to better understand how plasma does what it does on
noted medially from the treatment area. (Friedman PC, Fridman G, the laboratory and clinical level as well, partly because it is in
Fridman A, unpublished data. The photographs were taken as part the interest of industry to support at least some research to
of an IRB controlled clinical trial: Western Institutional Review
improve their devices and remain competitive, and partly
Board, date: 11/10/2016, protocol number: 20130084, ClinicalTria
ls.gov, registration number: NCT02759900) because government and other funding for academia can also

ª 2020 the International Society of Dermatology International Journal of Dermatology 2020

10 Updates from Medicine Cold plasma in dermatology Friedman

be more accessible in a field that is established as a recognized 4 Which of the following plasma device types are used in cur-
and accepted medical treatment. rent clinical dermatological research?
If further laboratory and clinical research can lead to a better a Interpolation electrode plasma
understanding of how plasma works, and this knowledge is b Surface microdischarge plasma
paired with advances in engineering, it is not impossible to envi- c High-pressure contact plasma
sion plasma technology that, for example, can selectively elimi- d Floating-electrode DBD plasma
nate only certain bacteria from the skin surface to influence
5 Published clinical studies used cold plasma for which of the
diseases via the cutaneous microbiome, or can alter cell fate at
following conditions?
will, down to the level of very small groups of cells.
a Psoriasis
b Bullous pemphigoid
CONCLUSION
c Actinic keratosis
Plasma is a physical entity that has a variety of effects on living d Pruritus
tissues, many of them potentially important for medical applica-
6 All plasma effects result from electric changes to the cell
tions. It is easy and relatively inexpensive to create cold plasma
membrane, without any detectable changes in intracellular
at room temperature and under atmospheric pressure so it can
signaling pathways.
be applied to the surface of the skin, with no discomfort, pain, or
a True
known harmful effects. Plasma medicine is just taking its first
b False
baby steps, but it shows promise for the treatment of various skin
diseases. The way plasma acts on living tissues is very complex, 7 Because of their engineering simplicity, cold plasma devices
but with the expansion of clinical use and research, our under- can easily use safety standards already in place for any other
standing of what plasma does and what it can be used for will electric devices.
deepen. Extensive laboratory research and recent proof-of-con- a True
cept clinical trials raise the hope that plasma may be the answer b False
to some of the most stubborn problems in dermatology by
8 Cold plasma was shown to selectively induce cell death in
destroying hard to treat bacteria or fungi, causing selective cell
malignant cell lines but not in nonmalignant controls.
death in malignancies, and inducing tissue regeneration.
a True
b False
Acknowledgments
9 Cold plasma was shown to induce stem cell differentiation in
The author thanks Dr. Alexander Fridman, his collaborator and various types of stem cells.
friend, for his advice and guidance, especially for sections a a True
describing the basics of plasma physics. b b False

10 The extreme high cost of current cold plasma devices is

Questions (answers provided after predicted to be the most important barrier of the adoption of
references) this technology.
a a True
1 Among others, which components are implicated in the bio- b b False
logical effect of cold plasma?
a Gas pressure of carrier gas
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International Journal of Dermatology 2020 ª 2020 the International Society of Dermatology

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