Search

International audience; The last decade has seen an impressive increase of the research dedicated to the biological applications of low temperature atmospheric pressure plasmas. Medical appli-cations are tacking an increasing place underlined by many clinical trials. They now concern numerous domains, including 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. That must therefore be taken into account in the applied treatments and complicates the definition of a "plasma dose" expected by many.

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; 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; Molecular delivery in cells combining low cytotoxicity and high efficiency using physical techniques remains to be optimized In this work, we report on the use of a cold atmospheric pressure plasma jet device called Plasma Gun as an innovative strategy for cell membrane permeabilization In our previous study[ 1 we focused on evaluating key parameters needed to achieve an efficient molecular delivery We showed that a low number of pulses (between 1 000 and 100 000 and a 30 minute incubation at 37 C of the treated cells were enhancing molecular uptake in a ring shape pattern with no significant toxicity In this report, we investigated cell permeabilization kinetics and surprisingly, best permeabilization levels were reached when molecular injections in the cell medium and plasma treatments were not simultaneous Indeed, best efficiency was measured when the injection was performed a few minutes after plasma This transient permeabilization was also measured for longer delays of molecular injection which confirms the non toxic nature of our plasma treatment conditions

International audience; Drug delivery in cancer cells combining low cytotoxicity and high efficiency remains to be optimized with physical techniques. Several studies have shown that cold atmospheric plasmas (CAPs) have demonstrated their relevance in biomedical applications and are prone to be quite useful tools in medicine. Plasma could be used to deliver chemotherapy [1] or nucleic acids [2] via enhancement of cell permeabilization. Parameters needed for helium plasma to induce cell membrane permeabilization on cancer cells were investigated (Fig.1). A frequency of 100 Hz for 100 s, corresponding to 10,000 pulses, with an applied voltage of 14 kV was used. Both plasma electric field and endocytosis involvement in molecular uptake were studied [3]. Kinetics of plasma-induced permeabilization were also studied. Highest permeabilization efficiency was observed when propidium iodide was added after treatment (up to 40% of propidium iodide positive cells in both HeLa and 4T1). No significant toxicity was observed in these conditions as the viability in HeLa cells was measured to be of 80%. A treatment combining doxorubicin and plasma also showed improvement of cell membrane permeabilization. Figure 1: Round-shaped permeabilization spot of propidium iodide in 4T1 cells after plasma jet treatmentReferences [1] W. Zhu, S.-J. Lee, N. J. Castro, D. Yan, M. Keidar, and L. G. Zhang, Scientific Report, 6, (2016).[2] M. Jinno, Y. Ikeda, H. Motomura, Y. Kido and S. Satoh. Archives Biochemistry Biophysics, 605, 59-66 (2016) [3] V. Vijayarangan, A. Delalande, S. Dozias, J.-M. Pouvesle, C. Pichon, and E. Robert, IEEE Transactions on Radiation and Plasma Medical Sciences, 2, 1-7 (2017).

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 which 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 hemostasis 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. In this talk, after a presentation of the context, the plasma devices, and the main applications, we will focus on the different problems linked to the plasma/target interaction, including treatments of tissues and liquids. Beside the induced changes in gas flow, the radical production and the potential role of the strong electric field generated around the plasma plume of atmospheric plasma jet systems, we will discuss possible changes induced in microenvironment of living targets. Throughout this presentation, we will emphasize on the fact that plasma diagnostics must be performed in real treatment conditions. We will also tackle the main issues, challenges and opportunities linked to the control of these multimodal action of non-equilibrium cold plasmas on living organisms.

International audience; Cold atmospheric plasmas (CAPs) have demonstrated to be quite useful tools in biomedicalapplications and their relevance in medicine is getting more and more recognized. Drug deliveryin cancer cells combining low cytotoxicity and high efficiency remains to be optimized withphysical techniques. Several studies have shown that plasma could be used to deliverchemotherapy [1] or nucleic acids [2] via enhancement of cell permeabilization. We investigatedthe parameters needed for helium plasma to induce cell membrane permeabilization on cancercells for drug delivery purposes. 10,000 plasma pulses were used at a frequency of 100 Hz for100 s with an applied voltage of 14 kV. Both plasma electric field and endocytosis involvementin molecular uptake were also studied [3]. Kinetics of plasma-induced permeabilization werestudied. Highest permeabilization efficiency was observed when propidium iodide was addedafter treatment (up to 40% of propidium iodide positive cells in both HeLa and 4T1). Nosignificant toxicity was observed in these conditions as the viability in HeLa cells was measuredto be of 80%. Doxorubicin was used as an anticancer drug. A treatment combining doxorubicinand plasma decreased the concentration of doxorubicin needed to achieve 50% of cytotoxicity byenhancing doxorubicin uptake in cancer cells.References[1] W. Zhu, S.-J. Lee, N. J. Castro, D. Yan, M. Keidar, and L. G. Zhang, Scientific Report, 6,(2016).[2] M. Jinno, Y. Ikeda, H. Motomura, Y. Kido and S. Satoh. Archives BiochemistryBiophysics, 605, 59-66 (2016)[3] V. Vijayarangan, A. Delalande, S. Dozias, J.-M. Pouvesle, C. Pichon, and E.

International audience; Plasma discharges have been known for quite a long time as a method to allow for cell permeabilization and likely to involve electric fields, UV photons and charged or excited particles, e.g.[1]. Comparison of different plasma devices, including so called “cold atmospheric pressure plasma” based on dielectric barrier discharge, but also combined role of electrical and chemical factors was more recently investigated with its potential interest as a new method for cell transfection [2]. In a very recent paper [3] not only radical produced by the plasma source but also intense electric field, as high as 150 kV/cm delivered on adherent cells are reported to induce transient increase in intracellular calcium concentrations, thereby playing a key role in dell response to plasma stimulus [3].In this work, we report on the use of plasma jet, so called Plasma Gun developed at GREMI, as an innovative drug delivery technique for adherent, HeLa and 4T1 cells. The role of different plasma source operation parameters but also plasma delivery protocols has been studied. Propidium iodide and FITC-Dextran were used as permeabilization markers. Cellular uptake was assessed by fluorescence microscopy and flow cytometry. Cell viability and reactive oxygen and nitrogen species produced were measured. Percentage of propidium iodide positive cells was the highest for 1,000, 10,000,and 100,000 pulses corresponding to 100 s-long treatments [4]. We found that cellular uptake was more efficient after a 30-minute incubation time at 37°C. Drug uptake kinetics was also investigated showing that surprisingly best permeabilization levels were achieved when plasma treatment is processed a few minutes before and not simultaneously with the drug injection in the culture medium. Transient permeabilization following plasma treatment was measured for longer delays for drug injection also confirming the non-toxic nature of plasmas when a limited number of pulses are delivered. The specific role of plasma induced transient electric fields is under investigation to try to elucidate not only the plasma mode of action but the ring shape permeabilization patterns, already previously reported by other groups [5], as well

National audience

National audience

International audience; The parameters needed for helium plasma jet to induce a cell membrane permeabilization on cancerous cells for drug delivery purposes have been investigated. Using our optimal settings, different fluorescent molecular markers were used in order to determine pore size, opening kinetics and the importance of endocytosis in the process. It is shown that plasma induced chemistry but also electric field play a role in drug uptake and that endocytosis is one of the mode of membrane permeabilzation.

International audience; Atmospheric pressure plasma jets have demonstrated their ability in biomedical applications thanks to their low gas temperature and their capacity to produce radicals, ions, electrons, UV radiation and electric fields. However the understanding of the interactions between the plasma and living cells and tissues is still far from being completely understood. Recently, Robert et al characterized two components of the electric field from a plasma jet and showed that the latter can propagate deeply in tissues on several mm [1]. In this work, we focus on the study of the electric field induced by the plasma and its influence on the cell membrane. Propidium iodide, dextran sulfate and plasmid DNA are used to measure the permeability of the membrane, while an electro-optic probe is used to measure the longitudinal and the radial components of the electric field. The two components are both spatially and temporally resolved. To investigate the contribution of the electric field on the cell membrane, a dielectric barrier is used between the plasma and the biological target. A comparison with and without the barrier will be presented for both biological and agriculture applications. [1] E. Robert et al, 2015, Phys. Plasmas 22 122007

International audience; Cold atmospheric pressure plasmas (CAPs) are promising therapeutic strategies in numerous biomedical fields such as, oncology and dermatology (wound healing, disinfection of tissues). CAPS are constituted by different components that may have effects on cells (e.g. photons, charged particles, reactive oxygen and nitrogen species, electric field, etc.). Among them, reactive oxygen and nitrogen species (RONS) are often considered as main actors responsible for the effects of plasma treatment on treated cells [1]. Depending on their gaseous composition, plasmas generated with similar electrical conditions could act in a very different way on cells and so, potentially, have completely different applications.The exact effects of plasma treatment on living cells are not very well characterized. This study aimed to investigate those effects in both human keratinocytes and HeLa cancer cell line. Particular attention was paid to the contribution of the electric field (EF) and RONS in cellular response to plasma treatment. Indeed, if the cell membrane gets charged, an electric stress could cause its rupture leading to cell death [2]. It is also well known that the electric field alone is able to cause either a reversible or irreversible depolarization of cell membrane, favorable to the increase of membrane permeability to certain species [3].In this work, plasmas were generated using a Plasma Gun feeded with helium or He-0.5% O2 mixtures. An electro optic electric field sensor was used to measure the electric field exposure of cell and culture media. It was measured that pure He plasma was more toxic than He-0.5% O2 plasma. H2O2 and NO2- were the main species detected in treated PBS post treatment, in higher concentrations following He than He-O2 plasmas exposure. Electric field alone treatment, achieved either with inserting a dielectric plate between the plasma jet and the well plates, or by irradiating the well plates from their bottom, lead to much less but still significant cell death.Regarding He-0.5% O2, EF measured was lower but plasmic membrane depolarization observed was much more important than that obtained with He plasma. Following the exposure to only a few thousands of plasma shots, He and He-O2 plasmas were also shown to induce potent permeabilization of HeLa plasma membrane, revealed through 4 kDa Dextran fluorescence detected in the cell nucleus. He-O2 plasma was the more efficient, leading to up to a 30% uptake for this dextran small molecule. Plasma or electric field alone exposure during a few thousands pulses was finally shown to allow for very limited but unambiguous penetration of much larger molecules as plasmid DNA in the HeLa cells.This work supports that strong cell depolarization with He-O2 plasmas can be obtained, same as plasma electric field which permeabilization effect does favor the penetration of chemical species such as RONS or larger molecules, but surprisingly leads to a much lower cell death.References[1] M. Ishaq, M. Evans, K. Ostrikov, International Journal of Cancer.,134, 1517 (2014). [2] N. Babaeva, N. Ning, D. Graves, M. Kushner, Journal of Physics, 45 11 115203 (2012)[3] Leduc M, Guay D, Leask RL, Coulombe S. New Journal of Physics, 11 115021 (2009)

International audience

National audience