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International audience; Non-thermal plasmas, produced from dielectric barrier discharges in air or from rare gas plasma jet devices, generate at room temperature, long and short lifetime reactive species (RS) in gaseous phase or in the liquid solution on which they impinge, ionic species, UV photons and intense transient electric fields (EF). In the last decade, the main focus was drawn on the RS to understand and optimize their action in the frame of the broad topic of “Plasma medicine”. More recently, the characterization and implication of EF in biomedical applications is studied, considering the generation of ns to μs duration, kV/cm amplitude features of plasma EF. Plasma have been shown to be a potential alternative to more “conventional technologies” for gene transfer, cell permeabilization, drug delivery in various applications including for cancer therapy. Quite recently, the concepts of reversible electroporation, especially the models of so called pore formation in cell membranes were broaden and detailed involving not only the key role of EF but also the oxidation of cell membrane associated with RS. Thus it appears we attend to a unique opportunity to reach more insights and achieve cell manipulation process optimization with considering the potential complementary or synergistic delivery of pulsed electric fields and plasma for in vitro and in vivo protocols.Besides a quick overview of some significant works focused on the use of non-thermal plasmas in interaction with cell membranes, the lecture will document the results obtained so far with the Plasma Gun developed at GREMI for first, cancer cell permeabilization with various plasma delivery protocols, and second on the first demonstrations of the positive combined action of pulsed electric fields (PEF) and plasma for antitumor action. Cell permeabilization has been performed in vitro for different adherent cell lines, exposed to plasma jet delivered at various pulse repetition rates for short duration and followed with cell incubation in plasma treated solution. The combination of plasma and PEF is studied in vitro and with murine models considering the delivery of pulsed voltage and current pulses (electroporation) together with plasma treated solutions in contact with biological targets (suspended cancer cells in electroporation cuvettes or tumors) at different delays with respect to the electroporation step.AcknowledgmentsWork supported by PLASCANCER project INCa-PlanCancer-n°17CP087-00 and GDR CNRS 2025 HAPPYBIO

International audience; Biological applications of Low Temperature Non Thermal Plasmas (LTNTP) have underwent a very rapid development over the recent years and have, even more recently, attracted a lot of attention in the field of agriculture and related domains. Their ability to fight harmful microorganisms and to promote decontamination makes them particularly attractive in that they do not generate chemical residues that must be reprocessed. They are also very attractive to generate interesting reactive species in liquid media that can then be sprayed or used for watering or for soil fertilization. The quality of seeds not only includes their capacity of rapid germination and fruitful growth, but also their protection against, at least, major well know diseases that can threaten their development (for example diseases provoked by Tilletia caries or Ustilago tritici (smut) in the case of wheat). Taking into account the fact that, in the coming years, the new regulations will lead to the impossibility to use the up to now frequently used chemicals, seed producers will have to find new physical treatments if they don’t want to face problems when putting their production on the market. They are then facing another problem: physical treatment must cure seed from pathogens, but, at the same time, they must be totally safe for seed germination and grow. Also, the longer term effects on plants and potential gene modifications should be considered. Due to seed characteristics, especially design to survive in aggressive environments during the latency period, this represents a huge challenge not easy to overcome. LTNTP appear as a very interesting potential treatment in that domain, especially for very high added value seeds and slip, and can open new routes in agriculture processing.On one hand, due the complex nature of plasmas produced at atmospheric pressure in air, experiments must be conducted to allow determination of plasma components (at least RONS and specific species like CO, UV and Electric field) main action trends. On another hand, due to the increasing use of plasma treated water, it is suitable to find new ways of producing different cocktails of usable reactive species of agricultural interest. Experiments, dedicated to that, have been conducted on various seeds and discharge systems (including jet, multijets, volumic plasmas, low pressure plasmas, bubbling plasma treated water) and will be presented.

International audience; The last decade has seen an impressive increase of the research dedicated to the biomedical applications of low temperature plasmas, especially with plasma sources working at atmospheric pressure. In this new trend, beside decontamination/sterilization and surface treatment that have already a quite long story through low-pressure plasma research and developments, medical applications are tacking an increasing place underlined by the actual numerous clinical trials. Medical applications of low temperature plasmas now concern a very wide range of domains, including primary haemostasis and blood coagulation, dental care, skin decontamination and hygiene, wound and ulcer treatment, dermatology, cancer treatment. Biological applications are also now extended to agriculture and, more recently, to cosmetic. Despite the huge number of in vitro and in vivo experiments there are still numerous challenges to overcome linked to the nature of the encountered target (biological tissues and materials, organs and their direct environment, liquids) that have a direct effect on the produced plasma itself and on the generated species. It is clear that the extremely strong coupling between the characteristics of the plasma and those of the target, as already shown, will play a very important role in the results observed during the treatments. A variation in the chemical or physical characteristics of the target will involve significant differences in the gas flow, the local temperature, or the induced electric field, resulting de facto in variations in the production of the reactive species. It also concerns the transposition of the results between the in vitro and the in vivo experiments that are carried out under extremely different conditions, especially concerning the equivalent electric circuit of the reactor / plasma / biological target assembly. These problems directly affect the identification of the processes involved and currently limit the possibility of a definition of a "dose" in plasma treatments. Recently, study reported led to reflections on non-sustainable tumor response. The loss of effectiveness under long-term plasma treatment of cancer tissue opens questions about plasma application and protocol. It must be considered that the treated area is morphologically and chemically changing over the time, from activated surrounding to more normal tissues that are less humid and bacteriologically cleaner. This aspect is particularly important for the development of efficient systems and protocols in plasma cancer treatment but also for any other plasma therapeutically approaches. It induce a in real-time in situ control of plasma production and at longer term a protocol adaptation taking into account the biological target evolution. Some progress are already done in that domain

International audience; Atmospheric pressure plasma jets have been extensively studied for several years as theyshowed very promising results in numerous fields such as material processing or plasma medicine.What makes cold plasma interesting is the presence of electrons, ions, reactive oxygen andnitrogen species, visible and UV light and high transient electric fields (EF). Even if the latter playa key role, from the production and propagation of the plasma to the efficiency of applications,they are far from being fully understood. This work focuses on the comparison of measurementsof EF produced by a Plasma Gun (PG) discharge with an electro-optic probe based on Pockelseffect [1] with 2D simulations of atmospheric pressure discharges propagating in a geometry closeto the Plasma Gun’s one. Plasma Gun consists in a vertically downward oriented capillary with aninner high-voltage electrode and an outer grounded one. Plasma is powered with μs-durationvoltage pulses from single shot to 4kHz.In [2] the electric field is evaluated by the simulations inside the capillary, in the glass andoutside. The aim of this work is to compare the radial profile of the electric field outside thecapillary between experiments and simulations to get information on the plasma inside it. Theelectro-optic probe was placed at different levels alongside the capillary and was getting far fromit. Measurements were executed every 5mm over 5cm. At the middle of the capillary a decreaseinversely proportional to the distance has been found. It reminds the shape of an EF produced byan infinite uniformly charged cylinder. 2D simulations were in a good agreement finding also thesame slope for EF. But simulations permit also to get information on the distribution of chargedensity and especially to know if the main contribution is from volume or surface charge density.Results show that the radial profile of EF outside the tube is mostly determined by the volumecharge density in positive polarity while in negative polarity the surface charge density isdominant. Simulations have provided values of average volume charge density in the order of 7.5nC/cm3 in positive polarity and of average surface charge density around 1nC/cm² in negativepolarity. Near the electrodes, a decrease inversely proportional to the square of the distance hasbeen found, showing that there is an axial position dependence. A time dependence investigationwill also be presented.X.D. acknowledges his grant funding Thermofisher Scientific INEL/Région Centre Val de Loire.References[1] G. Gaborit et al., IEEE Trans. Plasma Sci., 42 (2014).[2] P. Viegas, F. Pechereau and A. Bourdon, Plasma Source Science and Technology, in press(2018)

International audience; Compelling evidence suggests that Cold Atmospheric Pressure Plasma (CAPPs) has potential as a new cancer therapy. However, knowledge about cellular signaling events and toxicity subsequent to plasma treatment of is still poorly documented. The aim of this study was to focus on the interaction between 3 different types of plasma (He, He-O2, He-N2) and human epithelial cell lines to gain better insight into plasma-cell interaction. We choose epithelial cell lines (human keratinocytes, human fibroblasts, human colorectal carcinoma and skin melanoma) to gain insight into plasma-cell interaction and to determine which cellular pathways are induced by CAPP treatment in normal and malignant cells. We provide evidence that the proteasome, a major intracellular proteolytic system which is important for tumor cell growth and survival, is a target for (He or He-N2) CAPP. This is accompanied by apoptosis, DNA damage, mitochondrial dysfunction and mild oxidative protein damage. By differential label-free quantitative proteomic analysis we found that CAPP triggers antioxidant and cellular defense but also seen cancerous processes in keratinocytes. Moreover we found that malignant cells are more resistant to CAPP treatment than normal cells. However, reactiveoxygen and nitrogen species (RONS) are not the only actors involved in cell death; electric field and charged particles could play a significant role especially for He-O2 CAPP. Particular attention was paid to the contribution of the electric field (EF) and RONS in cellular response to plasma treatment. Taken together, our findings provide insight into potential mechanisms of CAPP-induced cell death.

International audience; The quality of seeds not only includes their capacity of rapid germination and fruitful growth, but also their protection against, at least, major well know diseases that can threaten their development (for example diseases provoked by Tilletia caries or Ustilago tritici (smut) in the case of wheat). Taking into account the fact that, in the coming years, the new regulations will lead to the impossibility to use the up to now frequently used chemicals, seed producers will have to find new physical treatments if they don’t want to face problems when putting their production on the market. They are then facing another problem: physical treatment must cure seed from pathogens, but, at the same time, they must be totally safe for seed germination and grow. Also, the longer term effects on plants and potential gene modifications should be considered. Due to seed characteristics, especially design to survive in aggressive environments during the latency period, this represents a huge challenge not easy to overcome. Low Temperature Non Thermal Plasmas (LTNTP) appear as a very interesting potential treatment in that domain, especially for very high added value seeds, and can open new routes in the seed processing.Due the complex nature of plasmas produced at atmospheric pressure in air, preliminary experiments must be conducted to allow determination of plasma components (at least RONS, UV and Electric field) main action trends. Experiments, dedicated to that, have been conducted on various seeds and discharge systems (including jet, multijets, volumic plasmas, low pressure plasmas) and will be presented with emphasis on growth regulation and potential toxicity.

International audience; The quality of seeds not only includes their capacity of rapid germination and fruitful growth, but also their protection against, at least, major well know diseases that can threaten their development (for example diseases provoked by Tilletia caries or Ustilago tritici (smut) in the case of wheat). Taking into account the fact that, in the coming years, the new regulations will lead to the impossibility to use the up to now frequently used chemicals, seed producers will have to find new physical treatments if they don’t want to face problems when putting their production on the market. They are then facing another problem: physical treatment must cure seed from pathogens, but, at the same time, they must be totally safe for seed germination and grow. Also, the longer term effects on plants and potential gene modifications should be considered. Due to seed characteristics, especially design to survive in aggressive environments during the latency period, this represents a huge challenge not easy to overcome. Low Temperature Non Thermal Plasmas (LTNTP) appear as a very interesting potential treatment in that domain, especially for very high added value seeds, and can open new routes in the seed processing.Due the complex nature of plasmas produced at atmospheric pressure in air, preliminary experiments must be conducted to allow determination of plasma components (at least RONS, UV and Electric field) main action trends. Experiments, dedicated to that, have been conducted on various seeds and discharge systems (including jet, multijets, volumic plasmas, low pressure plasmas) and will be presented with emphasis on growth regulation and potential toxicity.

International audience; The antitumor action of Low Temperature Non Thermal Plasma (LTNTP) has been now widely demonstrated both in vitro and in vivo (on animal models and more recently on human [1]) on a large variety of cancer cell lines and tumor types. Nevertheless, due to the great variety of plasma components able to play a role, the precise understanding of the process chain leading to the observed effects is far to be reached. It is not easy to isolate or to measure the plasma component individual action or, in contrast, to evaluate the potential benefit or synergy of combined ones. Up to now, many experiments in vitro have been realized with that intention, but the translation to in vivo results is rather complicated, even, in many cases, impossible due to the complex environment of the cells in vivo, including vascularization (then blood flow and oxygenation/physioxia) and the body response (signaling, recruitment, immune system). Generally, the first step of dedicated studies concerns in vitro cell viability or toxicity in culture media with the analysis of both the induced changes in the cells and in the medium. It must be stressed that the used experimental conditions together with the fact to treat cells in multiwell plates outside their incubation chamber may generate cell stress that can affect their behavior and then leading to biased results. This presentation will show some of the issues and challenges associated with in vitro cancerous cells treatments by a plasma jet (here a Plasma Gun), also in relation with in vivo conditions associated with the hypoxic/physioxic environment.We will present results on direct effect of plasma treatment on both the culture medium and cells, including liquid evaporation, oxygen deprivation, extra and intracellular RONS generation, membrane poration, cell depolarization, with the potential role of electric field in the treated and adjacent wells [2]. We will particularly emphasize the importance of cell oxygenation level which is a key factor in the proliferation/regression of tumors in hypoxic environment. It will be shown that an uncontrolled plasma jet in vitro treatments can lead to drastic changes in the oxygen pressure (and temperature) in cell culture during the experiment with sequences of maxima (leading to normoxia) and minima (potentially leading to strong hypoxia). The latter contrasts with the plasma oxygen pressure and blood flow increases measured in the in vivo conditions [3]. This also means that a great attention must paid in the modeling of interactions of plasma jets with physiological liquid media since the initial concentration of oxygen can be greatly affected. AcknowledgmentsThis work is founded by ARD 2020 Cosmetoscience PLASMACOSM project. X.D. is supported by INEL/Region Centre-Val de Loire fellowship.References[1] Metelmann et al, Clinical Plasma Medicine, 3 17-23 (2015).[2] E. Robert, T. Darny, S. Dozias, S. Iseni, and J. M. Pouvesle, Phys. Plasmas 22, 122007 (2015).[3] G. Collet, E. Robert, A. Lenoir, M. Vandamme, T. Darny, S. Dozias, C. Kieda, and J. M. Pouvesle, Plasma Sources Sci. Technol.23, 012005 (2014).

International audience; Any medical treatments with plasma jets implies plasma biological target interactions. In vivo, in most cases, the targeted tissue or organ is electrically conductive. Many studies have been devoted to the characterization of free jets (i.e without target in front of) but only a few concern plasma jets touching conductive targets. However, it has been shown that this contact leads to a strong increase of reactive species production [1] and helium metastable production in helium plasma jet [2]. Recently, numerical model evidence target effect depending of target nature ( dielectric or metallic) [3]. We studied the influence of conductive target on plasma properties in µs helium Plasma Gun discharge by a cross correlation of two diagnostics. Helium metastable concentration measurements by laser absorption spectroscopy (optical bench LPGP [4]) were correlated with two orthogonal components of electric field ( longitudinal and radial) measurements realized with electro-optic probe [5]. These two diagnostics are both spatially and temporally resolved. Comparing with free jet, it first appears that the helium metastable production is very dependent on target distance from capillary end, not only in the plasma plume, but also all along the capillary. With 1 cm target gap, the helium metastable concentration strongly increases by more than one order of magnitude close to target surface (1,8.1012 cm-3) compare to free jet plasma plume at same position (1,4.1011 cm-3). In the capillary the concentration follows a quasi linear increase of almost factor 3 between the beginning and the capillary end, whereas no increase occurs in free jet. With the correlation of metastable fast production and electric field components time evolutions, we have identified a mechanism triggered by the ionization front contact on the conductive target. This contact is immediately followed by a very fast counter propagation of a secondary front from the target surface to the high voltage electrode through the remnant plasma tail of the primary front. As observed, this lead to a greatly enhanced production of helium metastable inside the capillary and inside plasma plume. AcknowledgmentsThis work is supported by ANR BLAN 093003 PAMPA, APR PLASMEDNORM, TD is supported by MENSRReferences[1]S. Yonemori and R. Ono, J. Phys. D. Appl. Phys., vol. 47, no. 12, p. 125401, 2014.[2]K. Urabe, T. Morita, K. Tachibana, and B. N. Ganguly, J. Phys. D. Appl. Phys., vol. 43, p. 095201, 2010.[3]S. A. Norberg, E. Johnsen, and M. J. Kushner, J. Appl. Phys., vol. 118, no. 1, p. 013301, 2015.[4]G. Cadot, C. Douat, V. Puech, and N. Sadeghi, IEEE Trans. Plas. Sci., vol.42,10, p.2446 (2014)[5]E. Robert, T. Darny, S. Dozias, S. Iseni, and J. M. Pouvesle, “New insights on the propagation of pulsed atmospheric plasma streams: From single jet to multi jet arrays,” Phys. Plasmas, vol. 22, no. 12, p. 122007, 2015.

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International audience; The present work deals with plasma polymerisation of TetraMethylDiSilOxanewith a Dielectric Barier Discharge plasma jet at atmospheric pressure. Depending on parameters likevoltage, frequency, carrier gas and monomer injection, the deposited polymer looks like gelfilm or transparent film with fringes. Deposits are characterized by Fourier Transformed IRspectroscopy and contact angle measurements.Keywords: Atmospheric pressure DBD plasma jet, Low temperature, Plasmapolymerisation, TetraMethylDiSilOxane.

International audience; Many recent experiments or treatments related to biological applications of cold plasmas have been recently performed using plasma jets. It concerns either applications in vitro or in vivo. The observed effect are most of the time attributed to the very rich chemistry generated by the interaction of the rare gas plasma plume with the surrounding environment constituted either from the ambient air or this last one in complex interaction with liquids at the interface with the target (nutritious medium for in vitro experiments, organ liquids for in vivo applications). Recent experiments performed on tissue oxygenation1 or breast cancer treatments on immunocompetent mice2 lead to the conclusion that probably the involved chemistry couldn’t, alone, completely allow to describe the observed phenomena. This, especially under very soft treatment conditions, was suggesting possible triggering of some immune system chain processes, but also possible modifications in the microenvironment of tissue and tumors. In this context, there is still an unknown role of the electric field associated with the ionization front or generated in the environment of the plasma plume tip.In this work, we measured the electric field in real conditions of Plasma Gun (GREMI plasma jet) applications on various biological samples and targets (tissues and liquids), also in free jet conditions, using a new device (bi-component EOP Kapteos probe) that allows time (ns) and space (1 mm3) resolved measurements in a two radial and longitudinal directions.We will present data obtained along the discharge capillary together with measurements along and in the plasma plume and at different level in biological samples. Both in the capillary and the plume, high longitudinal induced electric fields have been measured just ahead of the apparition of a strong radial electric field in good agreement with Kushner model results. More interesting, is the measurement of a significant electric field presence rather deep in tissue of euthanatized animals (down to two millimeters) at levels that may affect cell permeability or behavior. 1. G. Collet, E. Robert, A. Lenoir, M. Vandamme, T. Darny, S. Dozias, C. Kieda, , J.M. Pouvesle, “Plasma jet-induced tissue oxygenation: potentialities for new therapeutic strategies” Plasma Sources Sci. Technol. 23 (2014) 0120052. G. Collet, L. Ridou, E. Robert, M. Vandamme, T. Darny, S. Dozias, C. Kieda, , J.M. Pouvesle “NTP antitumor soft treatment: evidence of a triggering effect? ”, ICPM5, May 18-23, 2014, Nara (Japan)______________________* Work supported by Région Centre through APR PLASMEDNORM and MENRES through ANR PAMPA

International audience; The antitumor effect of Non Thermal Plasmas (NTP) has been clearly shown in vivo on murine models with various cancer types (bladder, colon, glioblastoma, melanoma, ovary, pancreas). Although the mechanism is far from being fully understood, the therapeutic effect is now totally admitted. To date, the literature reports mainly induction of apoptosis, effect which is not restricted to cancer cells and probably mask other aspects of the NTP for cancer therapy. In case of plasma jet experiments, the observed effect are most of the time attributed to the very rich chemistry generated by the interaction of the rare gas plasma plume with the surrounding environment constituted either from the ambient air, or this last one in complex interaction with liquids at the interface with the targeted organ. Our recent experiments performed on tissue oxygenation1 or breast cancer treatments on immunocompetent mice2 lead to the conclusion that probably the involved chemistry couldn’t, alone, completely allow to describe the observed phenomena. This, especially under very soft treatment conditions, is suggesting possible triggering of some immune system chain processes, but also possible modifications in the microenvironment of tissue and tumors. In this context, there is still an unknown role of the electric field associated with the ionization front or generated in the environment of the plasma plume tip. Taking into consideration the recent vessel normalization based-cancer treatment, the NTP effect should be further investigated in view of blood vessels structure and function (blood flow) as well as tumor hypoxia compensation to confirm a possible NTP-based adjuvant approach for cancer treatments. These results suggest new ways, especially combined therapy, to consider the plasma and its therapeutic delivery in NTP-based tumor therapy. All these aspects will be discussed during the presentation, including recent results on electric field measurements in plasma biological application conditions. This work is supported by the APR Region Centre PLASMEDNORM.References[1] G. Collet, E. Robert, A. Lenoir, M. Vandamme, T. Darny, S. Dozias, C. Kieda, , J.M. Pouvesle, “Plasma jet-induced tissue oxygenation: potentialities for new therapeutic strategies” Plasma Sources Sci. Technol. 23 (2014) 012005[2] G. Collet, L. Ridou, E. Robert, M. Vandamme, T. Darny, S. Dozias, C. Kieda, , J.M. Pouvesle “NTP antitumor soft treatment: evidence of a triggering effect? ”, ICPM5, May 18-23, 2014, Nara (Japan)

International audience; Non thermal plasma jets delivered at atmospheric pressure have been recently successfully used in an impressive number of experimental works including plasma diagnostics, biomedical treatments and material processing. The generation of plasma occurs in a discharge reactor and is then delivered either as a plasma effluent or propagates towards the target through fast ionization wave or streamer mechanisms. While the plasma source setups are very simple, it has been evidenced that many parameters may significantly influence the plasma characteristics offering at the same time a large versatility for plasma delivery but also requiring a special attention to match and characterize the plasma features for any specific application.In this work, emphasis will be given on two critical topics necessarily involved in any plasma jet biomedical applications. The first consists in the major influence of the target over which plasma jet impingement occurs. It has been shown that depending on the conductivity of the target, secondary plasma generation occurs from the target, leading to a critical modification of the reactive species (RS) generation. The second main issue concerns the strong interplay between the rare gas flow and the plasma species generated during plasma jet ionization wave propagation. Drastic modification of the rare gas flow features have been recently evidenced and characterized through Schlieren visualization and ICCD imaging [1].This work is supported by the APR Region Centre PLASMEDNORM and ANR 2010 BLAN 093001 PAMPA.References[1] E. Robert, V. Sarron, T Darny, D. Riès, S. Dozias, J Fontane, L. Joly and J.M. Pouvesle, Rare gas flow structuration in plasma jet experiment, Plasma Sources Sci. Technol. 23 0120003 (2014).

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