in vivo 33: 1011-1026 (2019)

doi:10.21873/invivo.11570

Review

Plasma Medicine: A Field of Applied Redox Biology

THOMAS VON WOEDTKE1,2, ANKE SCHMIDT1, SANDER BEKESCHUS3,
KRISTIAN WENDE3 and KLAUS-DIETER WELTMANN1

1Leibniz Institute for Plasma Science and Technology, INP Greifswald, Greifswald, Germany;
2Greifswald University Medicine, Greifswald, Germany;
3ZIK plasmatis at INP Greifswald, Greifswald, Germany

Abstract. Plasma medicine comprises the application of therapeutic effects based on direct interaction of plasma with
physical plasma directly on or in the human body for living tissue. Plasma application for the treatment of medical
therapeutic purposes. Three most important basic plasma materials or devices is an important subject of research and
effects are relevant for medical applications: i) inactivation has been utilized for several years now (1-8). However, the
of a broad spectrum of microorganisms, including multidrug- core area of plasma medicine – as a new field of research –
resistant pathogens, ii) stimulation of cell proliferation and focuses on the use of plasma technology in direct treatment
angiogenesis with lower plasma treatment intensity, and iii) of living cells and tissues. The aim of applied plasma
inactivation of cells by initialization of cell death with higher medicine is to exploit a differentiated interaction of specific
plasma treatment intensity, above all in cancer cells. Based plasma components with specific structural, as well as
on own published results as well as on monitoring of functional elements or functionalities of living cells to
relevant literature the aim of this topical review is to control, and ideally, normalize therapeutic effects. Besides
summarize the state of the art in plasma medicine and its antimicrobial activity, exposure of mammalian cells to
connect it to redox biology. One of the most important results physical plasma can lead either to stimulation or inhibition
of basic research in plasma medicine is the insight that of cellular function (9). Consequently, most research
biological plasma effects are mainly mediated via reactive and primary medical application of physical plasma is
oxygen and nitrogen species influencing cellular redox- concentrated on wound healing and cancer treatment. During
regulated processes. Plasma medicine can be considered a recent years, a broad spectrum of different plasma sources
field of applied redox biology. (called by many different names and abbreviations) has been
designed and dedicated for biomedical applications (9-12).
Plasma medicine is a new field of research combining
plasma physics, life science and clinical medicine. Basically, Plasma Generation and Plasma Sources
medical application of physical plasma comprises two for Biomedical Applications
principal approaches: i) use of plasma-based or plasma-
supplemented techniques to treat surfaces, materials or Physical plasma is a special excited gas state, sometimes
devices to realize specific qualities for subsequent special named “the fourth state of matter” following solid, liquid, and
medical applications, and ii) direct application of physical gaseous states. It can be generated by a continuous supply of
plasma on or in the human (or animal) body to apply energy to the atoms or molecules of a neutral gas until an
excited state is achieved. The energy required may be
provided separately by thermal, chemical, electrical and
radiative resources or a combination of all. However, the
This article is freely accessible online. predominant ionizing mechanism is the collision process that
involves inelastic collision, electron impact, radiative
Correspondence to: Thomas von Woedtke, Leibniz Institute for interactions and charge exchange. As the typical life span of
Plasma Science and Technology (INP Greifswald), Felix-Hausdorff-
excited states is about 10 ns stopping the energy supply starts
Str. 2, 17489 Greifswald, Germany. Tel: +49 3834554445, e-mail:
woedtke@inp-greifswald.de a depletion process rapidly quenching the plasma. The
electron impact ionization is the most robust procedure
Key Words: Plasma medicine, redox biology, cold atmospheric generating a plasma for biomedical purposes. The energy is
plasma (CAP), review. transferred by inelastic and elastic collisions of high-energy

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electrons generated by a strong electric field with the atoms tissue with possible biological consequences (20). Also, the
or molecules in the gas resulting in its partial ionization. The emitted electromagnetic radiation, above all the ultraviolet
temperature of such partially ionized gas is always (UV) light, has the potential to elicit biological effects (21,
substantially lower than the characteristic ionization 22). However, according to the current state of knowledge,
temperature. In a well-designed plasma source, ambient free electrons, high energy states of atoms and molecules
temperature of the plasma can be achieved. The along with ions and radicals in the plasma and those
physicochemical characteristics of plasma can be complex generated in secondary reactions are the main components
and they depend on a multitude of parameters, including the of the chemical reactivity and biological activity of a plasma
type and composition of the gas or gas mixture used for (23). The sum of the CAP derived chemical entities is often
plasma generation, the applied energy and electrode circumscribed as reactive species.
configuration, the pressure, and the environment. A large number of plasma sources that are potentially
Consequentially, a broad range of parameters can be useful for medical applications are described in the literature.
controlled by the plasma source design. With regard to its They differ in their plasma generation mechanism, source
application, especially in the medical context, useful geometry, working gases, and, consequently, vary in their
classifications are thermal versus non-thermal plasmas and application characteristics (9, 12, 24-28). During recent
low pressure versus atmospheric pressure plasmas (13-15). years, mainly two fundamental concepts of CAP devices
For a direct application on living tissue as the main aim of have been tested and are partially applied for medical
plasma medicine, only plasma generated under atmospheric purposes: i) dielectric barrier discharges (DBD) and ii)
conditions should be used. Medical treatment techniques plasma jets (9, 29-32).
using such plasmas have been firmly established for a long In Figure 1, three technical principles of plasma sources
time in the field of electro surgery, even if they were not intended for biomedical applications are depicted (11, 30).
explicitly referred to as plasma medicine at the time. Such The volume DBD (Figure 1A) is characterized by plasma
techniques, like argon plasma coagulation (APC), rely on ignition in a gap between an isolated high voltage electrode
precisely targeted thermal necrotization of tissue to achieve and the target to be treated. Consequently, cultured cells or
hemostasis (cauterization), or to cut or remove tissue (16, 17). living tissues in biomedical application are part of the
Furthermore, several plasma-based devices in cosmetics, e.g. discharge electrode configuration. Plasma has a direct
for wrinkle removal and skin regeneration, also rely on contact with the target to be treated and the target is directly
thermal plasma effects (18, 19). Since the 1990s, technologies exposed to the electrical field that is necessary for plasma
for stable and reproducible plasma generation at low generation (9, 31). In the surface DBD (Figure 1B), plasma
temperature under atmospheric conditions are available on a is ignited around an individually designed electrode structure
larger scale, facilitating the generation of so-called cold (e.g. circular or grid-like), which is isolated from a counter
atmospheric plasmas (CAP). This has led to considerable electrode. There is no direct contact of the active plasma
intensification of research in the field of medical applications with the target to be treated, instead impact is achieved by
of physical plasma at tissue-compatible temperatures. In transport processes bringing the reactive species to the living
terms of medical application, “cold” means temperatures tissue. With both DBD configurations, atmospheric air
lower than 40˚C at the target site during plasma treatment (9). usually serves as the working gas for plasma generation.
Simplifying, generation of CAP and its components can Both volume and surface DBD devices are suitable to
be summarized with the following three steps (9, 11, 14): generate plasmas over larger areas (9, 31). In a plasma jet
i) Ionization and excitation of atoms or molecules of a device (Figure 1C), the electrode setup for plasma generation
neutral gas (argon, helium, oxygen, nitrogen, air, or mixtures is located in or around a tube-like arrangement, in most cases
thereof) via electron impact by supplying electrical energy; inside a pen-like device. Diverse electrode configurations
ii) Interaction of electrons and high energy states of atoms can be used, e.g. pin electrodes, ring electrodes, plate
or molecules with reaction partners in the plasma phase and electrodes etc. The plasma is ignited inside the device using
its vicinity (ambient air, liquids, surfaces), generating a working gas that is flowing through the tube. The so-called
secondary and tertiary reactive species; plasma effluent (or afterglow) is carried out along the gas
iii) Emission of electromagnetic radiation (UV, visible flow and can be brought into direct contact with the target
light, IR/heat, electric fields) formed by excitation and to be treated. In order to maintain a low temperature and to
depletion processes or charge transport. achieve excellent controllability of the discharge, most
It is important to note that the plasma state is maintained plasma jet devices are using noble gases (helium or argon)
as long as the energy supply exists, i.e. it is not possible to as working gas, often doped with small amounts of
store a plasma like a gas. molecular gases (nitrogen, oxygen). The target to be treated
Because plasma contains highly motile electrons it is is not part of the electrode configuration. However, because
conductive and can transfer electrical current to cells and of the conductivity of the plasma and its afterglow, small

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Figure 1. Example photographs of the most common cold atmospheric plasma source principles for biomedical applications: volume DBD (A),
surface DBD (B), and plasma jet (C).

electrical currents may pass to the target. By choosing an occurring is so far not fully understood. Current knowledge
appropriate design of electrode and high voltage waveform assumes that at the interface between gas phase and liquid
these currents can be easily controlled (9, 29, 32). (or solid) target as well as in the target bulk a considerable
One of the best-investigated plasma sources for biomedical rearrangement of the ROS/RNS pattern occurs (Figure 2)
application is the argon-driven cold atmospheric pressure (32, 35-38). Beside an interaction of the plasma derived
plasma jet, kINPen (Figure 1C) (32-34). A needle electrode species among themselves, the interaction with target
inside a dielectric capillary is powered with a sinusoidal high biomolecules results in the formation of diverse chemical
voltage (2-6 kVpp) with a frequency of 1.0-1.1 MHz (power structures acting as a messenger or a beacon.
<3.5W in the hand-held unit). Argon gas with a flow rate of Because all plasma sources for biomedical applications are
3-5 standard liters per minute (slm) is used as the working working under atmospheric air conditions or use ambient air
gas. The plasma is generated at the tip of the needle and is as working gas, the generation of ROS and RNS from air-
subsequently released with the feed gas flow into the based oxygen and nitrogen is a corresponding feature of all
atmospheric environment, thereby generating a typical plasma these plasma sources. However, the composition and
effluent with a length of 9-12 mm and with 1 mm in diameter. quantity of plasma-generated ROS and RNS, as well as UV
Under these conditions, the electron density in the core irradiance, electrical field and other characteristics, are
plasma region near the high voltage electrode tip is in the strongly dependent on specific plasma sources and device
order of 1012 cm–1 and one order of magnitude lower in the parameters as working gas composition, power input and
visible effluent zone. However, electron density depends on temperature (39).
several parameters and can be varied by admixture of
molecular gases, such as oxygen and nitrogen (32). Biological Plasma Effects and its Medical Use:
The reactive species generated inside the plasma or as a Focus on Wound Healing
result of plasma interactions with the surrounding media are
considered the most important components responsible for Among the vast number of experimental reports on biological
biological plasma effects. In the kINPen, the argon-based plasma effects (using different plasma sources and devices
plasma effluent is exposed to atmospheric air containing under varying conditions), three effects most important for a
predominantly oxygen, nitrogen, and water. Traces of these, medical application are consistently reported (30):
especially the water, are contained in the working gas in low i) Effective inactivation of a broad spectrum of
ppm-amounts, too. These atmospheric air compounds are the microorganisms including multidrug-resistant pathogens;
precursors for secondarily generated non-radical and radical ii) Stimulation of cell proliferation and angiogenesis with
reactive oxygen and nitrogen species (ROS, RNS). lower plasma treatment intensity and time;
Generation of ROS and RNS can also be modulated by iii) Initialization of (programmed) cell death with higher
controlled admixture of oxygen, nitrogen, water or air to the plasma treatment intensity and time, primarily in cancer
argon working gas flow, or by gas shielding and modification cells.
of the atmosphere around the plasma effluent (32). When the With the improved availability of CAP technology in the
effluent containing the ROS, RNS, and residual high energy 1990s, its antimicrobial activity was in the early focus of
states targets a liquid (a tissue), a number of transport research with regard to microbial decontamination or
processes and tertiary reactions with target molecules sterilization of materials and devices (40, 41). Extensive

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programmed cell death in bacteria is being discussed as a

potential mechanism (49). Other physical mechanisms are
currently under investigation, for instance electrostatic
disruption by plasma-derived charged particles, or
electroporation caused by plasma-related electric fields (49, 50).
Regardless of the specific mode of action, early
experimental evidence on microbicidal plasma effects on
heat or radiation-sensitive material surfaces spurred further
efforts to investigate plasma effects on contaminated or
infected tissue. Several in vitro studies on effective
inactivation of clinically relevant microorganisms and
viruses have produced promising results (51-58). The first
clinical investigations on antiseptic plasma effects on tissue
and wounds that followed have shown rather modest
microbial reduction rates (59-63). However, improvement of
wound healing in general has been partially seen during
these clinical investigations (61, 64). Some additional case
reports and clinical investigations with different plasma
devices support these findings (65, 66).
Starting in 2013, the first cold atmospheric pressure
plasma devices received CE certification as medical devices
for the purpose of treating chronic wounds as well as
pathogen-based skin diseases in Germany and Europe. These
include the argon-driven, jet-like CAP devices kINPen MED
(neoplas tools GmbH Greifswald, Germany) and SteriPlas
(ADTEC, Hunslow, UK) as well as the DBD-based devices
PlasmaDerm (CINOGY GmbH Duderstadt, Germany) and
plasma care (terraplasma medical GmbH Garching,
Germany), the latter two using atmospheric air as working
Figure 2. Schematic of interactions between the kINPen plasma effluent gas. Certification of all these devices was based on
with aqueous liquid. There are four reaction regions: (A) core plasma comprehensive physical and biological characterization of
region, (B) effluent and plasma-ambient air interaction zone, (C) the respective plasma source accompanied with clinical
plasma/gas/liquid interface, and (D) bulk liquid. Seven processes are
investigations (9, 10, 25, 34, 53, 55, 59-64). However, even
dominant for the generation of ROS and RNS, and their transfer: (1),
(2) mass transfer from plasma to liquid based on gas/plasma component if randomized controlled clinical trials are not available as
dissolution and gas and liquid flow, respectively, (3) photolysis based yet (as unfortunately is the case for many wound therapies
on the plasma UV radiation, (4); positive ions and clusters, which due to a lack of standardization in wound scoring), positive
induce sputtering processes releasing water, gases, or electrons from experiences are consistently reported from the medical
the liquid, (5) negative ions, clusters, and cluster transport, (6) water
practice. Particularly in chronic wounds, e.g. venous leg
evaporation, and (7) electron impact or transport. (Adapted from 32).
ulcers where any conventional therapeutic options have been
exhausted, a clear benefit of CAP treatment has been found.
Some practitioners have reported a re-start or acceleration of
wound healing process in more than 80% of cases as a
research has been done on the mechanisms of low-pressure cold preliminary result, particularly following the use of the
plasma interaction with microorganisms. This was mainly kINPen MED (11, 30). Now, the most important aim is to
attributed to UV-based DNA damage in combination with validate these results by systematic clinical data.
erosion of microorganism structures by UV-based These clinical experiences confirm a very early hypothesis
photodesorption and etching processes by reactive plasma in plasma medicine research, that plasma effects on wound
species (42-45). However, the knowledge on mechanisms of healing may be a result of a two-step activity: antiseptics on
inactivation of microorganisms by CAP is limited. Microbicidal wound surface in combination with stimulation of tissue
CAP effects are mainly attributed to the activity of ROS and regeneration (67). Several experimental in vitro studies could
RNS leading to oxidative damage and modification of demonstrate a direct impact of CAP on cell proliferation and
cytoplasmatic membrane, proteins, and DNA (46-48). migration as well as on angiogenesis (68-76). The
Furthermore, induction of apoptosis-like processes of stimulating effect on skin tissue regeneration was confirmed

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in several in vivo animal experiments (69, 74-90) and in 37). Moreover, it has been shown several times that liquids,
human volunteers or patients with reasonably defined such as water, physiological saline, or cell culture media
wounds (91-93). It has to be pointed out that these last- become biologically effective following plasma treatment
mentioned wound healing effects in vivo were demonstrated (100-105). This underlines a key role of liquid phase
in acute wounds without any interfering microbial composition for biological plasma effects. ROS and RNS
contamination. With plasma treatment, the spontaneous like superoxide (O2–•), hydrogen peroxide (H2O2), hydroxyl
wound healing process was not impeded, and there was an radical (•OH), singlet oxygen (1O2), ozone (O3), and RNS,
acceleration in the early stage of wound healing. With this such as nitric oxide (•NO), nitrogen dioxide (•NO2) and
direct proof of stimulation of wound healing by plasma peroxynitrite (ONOO-), are transferred from plasma into the
treatment, it seems that the antiseptic plasma effect can be liquid environment of cells and tissue, or they are generated
partially pushed into the background because it may turn out by a very complex network of secondary liquid reactions
that it is not the dominating process as it was assumed for (Figure 2) (23, 106-111).
several years. Consequently, as a next step in clinical This insight of the central role of ROS and RNS has
research, it should be investigated, if this early stimulation opened up the door to the field of redox biology to explain
of wound healing may have any beneficial effects also in and interpret biological effects caused by CAP. Redox
acute wounds, e.g. with regard to scar formation or biology can be taken as the interface between the more or
prevention of complications in wound healing. Possible less unspecific impact of external factors and the specific
fields of application of CAP in acute wound healing could response and adaptation of a cell or an organism via its
be in patients with co-morbidities leading to a high risk of metabolic and macromolecular structures (112). Meanwhile,
disturbed wound healing and subsequent chronification, in it is well known that ROS and RNS are not solely harmful
the case of large-area burns or in the treatment of skin graft in cells, but also serve as signaling molecules via reversible
donor and acceptor sites (92, 94). oxidations and reductions of specific protein structures with
Nevertheless, there should be no doubt that plasma-induced cysteine as a major reaction target (113). In a
antiseptics have an important additional effect in the case of comprehensive in vitro study using different jet-based
contaminated wounds. Here, a very important question is why plasma devices with different working gas mixtures, the
plasma is destructive or inactivating for microorganisms while cysteine-oriented plasma chemistry could be proven.
stimulating repair mechanisms on mammalian cells. There is Furthermore, it could be demonstrated that cysteine is a
some evidence that the ROS-RNS composition resulting from useful and sensitive tracer compound to discriminate
plasma generation under atmospheric air conditions is more between the chemical potential of different plasma sources
toxic for microorganisms because both oxygen- and nitrogen- or gas mixtures, respectively (114).
containing reactive species are required for antimicrobial One of the most important players for ROS and RNS-
effects, whereas mammalian cell toxicity is mostly dependent based regulation in cell physiology is the mammalian Kelch-
on oxygen-based reactive species (95). Another very like ECH-associated protein 1 (Keap1)-nuclear factor
interesting insight is that, because of the role of ROS erythroid 2-related factor (Nrf2) pathway. In regular cell
generated by immune cells to fight wound infection, wounded physiology, it uses cysteine oxidation to respond to increased
tissue takes cytoprotective measures to promote some tissue ROS levels. By redox modification of cysteine-residues of
“resilience” for protection against ROS caused damage (96). Keap1, Nrf2 is released from its complex and the E3
This physiological mechanism may also be protective against ubiquitin ligase cullin 3 (CUL3). Subsequently, Nrf2
plasma-generated ROS and RNS. translocates from the cytosol to the nucleus where it binds
to antioxidant responsive elements (ARE) on DNA,
Redox Biology as the Scientific promoting the upregulation of antioxidant genes (113, 115).
Grounding of Plasma Medicine It has been demonstrated that CAP treatment of human
keratinocytes in vitro leads to the stimulation of this Nrf2
The fact that the biological effects of CAPs are mainly based pathway, resulting in the translocation of Nrf2 into the
on ROS and RNS, was primarily reasoned from experimental nucleus and the subsequent activation of Nrf2-ARE-targets,
observations in vitro. Plasma effects on mammalian cells such as glutathione (GSH), glutathione reductase (GSR),
were found to be dependent on cell culture media glutathione S-transferase (GST), superoxide dismutase
composition, each exhibiting a different antioxidative (SOD), heme oxygenase 1 (HMOX-1), and NADPH quinine
potential. Additionally, biological plasma effects could be oxidoreductase 1 (NQO1) (90, 116). Moreover, in an acute
extinguished when antioxidants like N-Acetylcystein (NAC) wound healing study in mice, an early activation of the Nrf2
were added (97-99). A multitude of investigations on plasma- pathway has also been demonstrated in vivo and ex vivo on
liquid interactions has demonstrated the occurrence of ROS skin tissue, dermal fibroblasts and epidermal keratinocytes
and RNS in liquid phases following plasma treatment (36, (Figure 3) (76, 117).

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Figure 3. CAP treatment induces an early translocation of Nrf2 from the cytoplasm to the nucleus (lower images) in skin tissues (A, 3 s CAP
treatment), dermal fibroblasts (B, 60 s CAP treatment) and in epidermal keratinocytes (C, 60 s CAP treatment) in contrast to untreated controls
(upper images). Immunofluorescent staining of nuclei (blue: DAPI - 4’,6-Diamidino-2-phenylindole), Nrf2 (green in A, red in B, C), and F-actin
cytoskeleton (green in B, C: Phalloidin-FITC). Scale bars=100 μm (See 117 for experimental details).

This Nrf2 pathway stimulation by CAP treatment seems to act as sensing element in the redox stress reaction of the Nrf2
be one of the most important mechanisms to protect pathway, CAP stabilizes the architecture of F-actin
mammalian cells from genotoxic plasma effects. Indeed, a cytoskeleton and focal adhesions, and increases granulation
huge number of in vitro studies report on potential genotoxic tissue formation and matrix deposition (117, 132). As a key
CAP effects on isolated, naked, or cellular DNA (118). regulator in macrophages, Nrf2 mediates the infiltration of
However, several investigations of potential genotoxic effects macrophages and neutrophils and the upregulation and
of CAP by in vitro standard procedures for mutagenicity secretion of pro- and anti-inflammatory ligands (e.g. TNFα,
testing of chemical substances has demonstrated no extended TGFβ, IL-1β etc.), which activate signaling and intracellular
mutation rate of plasma treated cells (119-123). The main generation of reactive oxygen and nitrogen species (117).
conclusion is that CAP treatment causes no enhanced Furthermore, CAP supports angiogenesis by recruiting
genotoxic risk. This has also been confirmed in an animal endothelial cells, stimulates growth factor expression like
study using hairless immunocompetent mice (124), in CAP- keratinocyte growth factor (KGF), epidermal growth factor
treated skin biopsies (125-127), and in clinical follow-up (EGF), or vascular epidermal growth factor (VEGF), and
investigations of plasma-treated wounds (128, 129). activates protein kinase B (Akt), which induces Nrf2
Meanwhile, it is well known that ROS and RNS also play expression (70, 71, 138, 139). Nrf2-regulated inflammation
an important role as secondary messengers in the and angiogenesis is also shown in numerous studies (133-
orchestration of wound healing processes (130, 131). This 137). Appropriate effects of CAP are also attributed to the
idea of redox-based repair of destroyed tissue becomes regulation of Nrf2 (90, 116, 117).
important in connection with acute and chronic wound Moreover, the activity of the transcription factor p53
healing supported by CAP. There is some evidence that the depends on the stage of wound healing and reactive species
Nrf2 pathway does not only function in cellular defense concentration (90, 117). The tumor suppressor protein p53
against increased ROS levels but it also has central influences cell proliferation and apoptosis and has a central
regulatory effects in wound healing (Figure 4) (117, 132). role in angiogenesis and cell cycle regulation and DNA repair.
The schematic overview in Figure 4 summarizes the state When the expression of p53 is relatively low, p53 enhances
of knowledge on molecular patterns in wound healing in the protein level of Nrf2 and its target genes promote cell
response to CAP treatment and aligns these results with survival depending on the cyclin-dependent kinase inhibitor 1
insights from redox biology and research on molecular (p21). When p53 expression is high, the Nrf2-mediated
biology of healing processes in acute wounds (90, 117, 124, survival response is inhibited by p53 (142, 143).
132). Briefly, it is assumed that CAP treatment leads to a Taken together, CAP treatment leads to an accelerated
transient and reversible modification of proteins and the lipid repair in acute wounds. All these findings are mainly based
bilayer, which contribute to normal or pathologic stages of on numerous in vitro and in vivo studies using the argon-
wound healing (117, 140, 141). This is crucially mediated by driven cold atmospheric plasma jet kINPen (32, 34).
the Nrf2 pathway (90, 116, 117). Its key role in up-regulation The long-term and systematic investigation of molecular
of detoxifying and antioxidant genes is mentioned above (113, biological processes of plasma-supplemented wound healing
115). Moreover, via activation of Keap1, which does not only processes has opened several interconnections of plasma

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Figure 4. CAP treatment improves wound healing processes via redox-regulated pathways. Enhanced wound healing by CAP involves a distinct
temporal pattern of inflammation, a promoting of pro-angiogenetic factors, and a balance of cellular proliferation and apoptotic events. The cellular
redox homeostasis is maintained and cells are defended from damage by a strong modulation of the nuclear E2-related factor (Nrf2) pathway.

medicine and redox biology. In wound healing, insights from As it was mentioned above, depending on plasma treatment
redox biology, indicating that redox-sensitive processes are intensity and time, it is possible to inactivate mammalian cells
driving factors in tissue repair (130, 131), can serve as a by initializing programmed cell death in them. This is true
sound scientific basis to confirm CAP applications in this particularly for cancer cells. After several reports on apoptosis
field. There is no doubt that this may be true also for other induction in cancer cells in vitro (152-155), animal studies on
fields of biomedical application of cold atmospheric plasma. transcutaneous plasma treatment of subcutaneously induced
solid tumors could prove the general concept of plasma-
Medical Application of Physical Plasma – supported tumor treatment (156, 157). However, there are
Present and Future several open questions about the mechanisms of plasma attack
on cancer cells, a possible selectivity with regard to healthy
Besides wound healing, several other indications for plasma tissue or on possible secondary effects distant from the region
application in dermatology are being taken into of local plasma treatment. Most current hypotheses are based
consideration, mainly in the treatment of pathogen-based on a predominant role of plasma-generated redox active species
and/or inflammatory skin irritations and diseases (128, 144- (158). Briefly, it is assumed that CAP treatment causes
146). Also, anti-infective plasma applications have been apoptosis of cancer cells through a selective rise of intracellular
tested in ophthalmology (147-149), while plasma in dentistry ROS and corresponding ROS-based death pathways. In that
is under research for several years, too. Possible dental regard, enhanced sensitivity of cancer cells may be caused by
applications include antimicrobial plasma activity, enhanced ROS levels in the cancer resulting from its unique
inactivation and removal of biofilm on teeth and on dental metabolic activities (113, 159). Other hypotheses attribute
implants, disinfection of tooth root canal, plasma-assisted differences in cell sensitivity to significant variations of
cleaning and optimization of tooth and implant surfaces to aquaporins (AQPs) among different cell lines. Enhanced
improve bone integration. Additionally, in-growth or bonding generation of long-lived species, such as H2O2 via extracellular
of dental fillings and prostheses, decontamination and superoxide dismutase (Ex-SOD, SOD3) on the cytoplasmatic
coating of dental prosthesis, antimicrobial treatment of the membrane of cancer cells and a subsequent trigger of immune
oral mucosa, oral wound healing and tooth whitening are attack on tumoral tissues via H2O2-mediated (second
under investigation (150). For more details on promising messenger) lymphocyte activation is also discussed (160, 161).
clinical applications of CAP, see a recent review by Another interesting hypothesis is based on the specific action
Metelmann et al. (151). of CAP via singlet oxygen (1O2) generation and the subsequent

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Figure 5. Different aspects of CAP-triggered oxidative stress, its control, adaptive response and physiological consequences. (Adapted from176).

induction of intercellular ROS-RNS-dependent apoptosis- biochemical and molecular biological pathways to transfer
inducing signaling (162). Finally, a plasma-induced stimulation their energy into chemical energy to realize metabolic and
of immunogenic cell death via damage associated molecular signaling processes. From this point of view, redox gradients
patterns (DAMPs) is under discussion (163, 164). are eventually the driving forces of life (171). Because the
There are several promising experimental results leading to primary process of generation of cold atmospheric plasma is
the situation that plasma application in cancer therapy is now the acceleration of electrons by electrical fields, plasma
one of the most attractive research fields in plasma medicine application may be considered as an initial part of a complex
(155). Based on the experimental proof of inactivation of single electron-transfer process transferring electrical energy via
layers of cancer cells by local plasma treatment (165), first of complex chemical reaction cascades into biological effects.
all a supportive plasma application in combination with One of the main advantages of biomedical application of
surgical tumor resections in cases where large-scale tumor CAP is that the active components, such as ROS and RNS,
removal is impossible, seems to be realistic (166, 167). are generated locally and only for the required duration of
Moreover, first CAP applications in palliative care in patients the application primarily by a physical process. By means of
with advanced squamous cell carcinoma of head and neck have variations of several plasma parameters, the essential ROS
not only resulted in the intended reduction of microbial load and RNS-based energy transfer from plasma to cells and
and resulting reduction of typical fetid odor, but in some tissue can be easily controlled. This is the main reason why
particular cases also in transient tumor remission (168-170). one can describe the biomedical plasma application as a field
Further research will tell us if any direct plasma application is of applied redox biology.
useful for the reduction or complete removal of solid tumors The insight that CAP impact may result either in cell and
and will lead to a “paradigm shift in cancer therapy” as it was tissue stimulation or in cell death, depending on exposure
predicted some years ago (156). conditions (e.g. treatment time), fits very well with the
theory of oxidative stress mainly introduced and developed
Conclusion and Outlook by Sies (172-176). According to that, oxidative stress can be
differentiated between oxidative eustress and oxidative
It is a very interesting perspective to attribute vital processes distress depending on low or high oxidant exposure. These
to electron flow, i.e. energetic electrons are processed by processes are strongly controlled by adaptive cellular

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responses, including the Nrf2 pathway that plays a central Acknowledgements

role above all in cell defence (115, 176, 177).
While CAP serves as a controllable source of redox- The authors acknowledge stimulating discussions on redox biology
active species, the cellular environment and its and their significance for plasma medicine with David Graves, UC
antioxidative capacity may act as a “ROS-RNS sink”. The Berkeley, USA, and Georg Bauer, Medical Center – University of
Freiburg, Germany. The German Federal Ministry of Education and
interaction of both components will define the degree of
Research financially supports basic research on plasma medicine at
exposure of the living system to redox active species. INP Greifswald (grant numbers 03Z22DN11&12). Plasma medicine
Depending on the cellular ability of adaptive response as research at INP Greifswald is part of the Research Alliance “Leibniz
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10.1016/j.redox.2016.12.035 Accepted April 24, 2019

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