Florian Judée

Les dispositifs plasma à la pression atmosphérique (PA) produisent de nombreuses espèces actives physiques (photons, particules chargées, champ électriques etc.) et chimiques (radicaux libres, espèces réactives de l'oxygène, espèces réactives de l'azote etc.). Ces espèces connues pour leurs effets biologiques directs ou indirects font de l'utilisation des jets de plasmas froids à la PA pour des traitements biomédicaux un sujet de recherche en plein développement. La recrudescence des cas de radiorésistance et chimiorésistance chez les micro-organismes et les cellules cancéreuses impose la recherche de nouveaux modes de traitements innovants. C'est dans ce contexte général que s'inscrit les travaux présentés ici dont l'enjeu majeur est la compréhension des mécanismes d'actions des plasmas froids à la PA sur le cancer colorectal (deuxième cause de mortalité par cancer en France). Le travail de thèse présenté concerne l'utilisation d'un modèle biologique in-vitro (sphéroïde tumoraux multicellulaire du cancer du côlon HCT116) en 3 dimensions qui permet de prendre en compte des paramètres déterminants dans la prolifération tumorale. Ce modèle permet ainsi une meilleure prédiction des résultats in vivo dans l'objectif d'une étude clinique ultérieure. De plus les cinétiques de créations des espèces actives ont été étudiées dans leur globalité depuis le dispositif plasma jusqu'aux interactions intracellulaires à partir d'analyses physiques, biologiques et chimiques. Le traitement indirect par utilisation de liquides activés par jet de plasma d'hélium a été privilégié pour élaborer un traitement endoscopique du cancer colorectal. L'ensemble des travaux menés sur l'observation des interactions des liquides activés par plasma sur les sphéroïdes tumoraux multicellulaires a permis de révéler deux modes d'actions distincts. Le premier étant une génotoxicité du liquide activé induite par la présence de peroxyde d'hydrogène dont l'action induit une cassure double brin de l'ADN intracellulaire conduisant les cellules à la mort par apoptose. Une interaction directe des radicaux libres produits dans le milieu avec les composants de ce dernier (acides aminés, glucose, etc.) ainsi que la présence de nitrites et nitrates induisent également un effet antiprolifératif à long terme du milieu activé par plasma sur les tumeurs HCT116. Différentes méthodes comme la résonance paramagnétique électronique et la spectroscopie d'émission optique ont permis de révéler des voies de création possibles conduisant à la formation de ces espèces actives. Des campagnes d'essais thérapeutiques ont permis d'évaluer la capacité des liquides activés par plasma à cibler davantage les cellules cancéreuses plutôt que les cellules saines ce qui en fait une méthode de traitement sélectif particulièrement prometteuse. Le développement et la caractérisation d'un second jet de plasma d'argon a été réalisé avec l'objectif d'optimiser l'effet antiprolifératif des plasmas sur les tumeurs tout en tenant compte des contraintes imposées par l'utilisation d'un tel dispositif pour le traitement du cancer colorectal.

Two atmospheric pressure plasma jet (APPJ) devices—a plasma gun and a plasma Tesla jet—are compared in terms of safety and therapeutic efficiency to reduce the tumor volume progression of cholangiocarcinoma, i.e. a rare and very aggressive cancer emerging in biliary tree. For this, a three steps methodology is carried out. First, the two APPJ have been benchmarked in regard to their electrical and physico-chemical properties while interacting with material targets: dielectric plate, liquid sample, metal plate and an equivalent electrical circuit of human body. The propagation properties of the ionization wave interacting with these targets are discussed, in particular the profile of the related pulsed atmospheric plasma streams. In a second step, a dermal toxicity survey is performed so as to define an experimental operating window where plasma parameters can be changed without damaging healthy skin of mice during their exposure to plasma and without inducing any electrical hazards (burnings, ventricular fibrillation). Optimal conditions are identified discarding the conditions where slight alterations may be evidenced by histology (e.g. prenecrotic aspect of keratinocytes, alterations in the collagen structure). Hence, for the two APPJ plasma parameters these conditions are as follow: duty cycle = 14%, repetition frequency = 30 kHz, magnitude = 7 kV, gap = 10 mm and exposure time = 1 min. In a third step, the two plasma jets are utilized on cholangiocarcinoma xenograft tumor model developed in immunodeficient mice. The two devices are safe and a significant therapeutic efficiency is demonstrated with the plasma Tesla. In conclusion, we have developed a safe cold atmospheric plasma device with antitumoral properties in preclinical model of cholangiocarcinoma, opening the path for new anticancer treatment opportunities.

Simulations and experimental works have been carried out in a complementary way to engineer a basic material target mimicking the same dielectric properties of the human body. It includes a resistor in parallel with a capacitor, whose values ( R _h = 1500 Ω and C _h = 100 pF) are estimated in regard of parameters commonly utilized upon in vivo campaigns (frequency = 30 kHz, gap = 10 mm, high voltage electrode surface = 12.6 mm ² ). This equivalent electrical human body (EEHB) circuit can be used as a reference and realistic target to calibrate electrical properties of therapeutic plasma sources before their utilization on patients. In this letter, we consider a configuration where this EEHB target interacts with a plasma gun. Plasma power measurements performed in such configuration clearly indicate two operating modes depending on the value of the supplied voltage. Hence, the plasma gun generates pulsed atmospheric plasma streams likely to present therapeutic interest for voltages comprised between 3.0 and 8.5 kV while for higher values, transient arcs of thermal plasma are generated and represent substantial risks for the patient.

Objective: The aim of this work is to investigate the inhibitory effect of Plasma Activated Medium (PAM) on Head and Neck cancerous cells (FaDu). The response of FaDu cells in monolayer cultures and Multi Cellular Tumor Spheroids (MCTS) after treatment with different PAMs will be compared. Background: Head and Neck squamous cell carcinoma is a widespread cancer that responds poorly to anticancer treatments such as chemotherapy and radiotherapy. Nowadays there is a growing interest in cold plasmas and their applications in cancer therapy. Methods: A homemade helium plasma jet is used to produce PAM. The effects of PAM and hydrogen peroxide H2O2 on FaDu 2D cells cultures and MCTS were characterized by evaluating the cell viability with PrestoBlue test and by measuring the size of MCTS. Results: One treatment with PAM induce cell detachment from MCTS since the first day in a PAM exposure dependent manner. This is due to the presence of H2O2 in PAM. However, a rapid spheroids regrowth is observed attributed to a resistance of FaDu cells to H2O2. After multiple treatments of MCTS with PAM we obtained an inhibition of cell growth. MCTS are brought out when comparing PAM effect on 2D versus MCTS. Inversely, PAM induces cell death in the case of 2D cell culture. Conclusion: PAM may be considered as a potentially efficient agent in the therapy of head and neck cancer. We also point out that MCTS is a more valuable model than 2D cell culture for the evaluation of the anti-cancer activity of PAM.

In order to identify aqueous species formed in Plasma activated media (PAM), quantitative investigations of reactive oxygen and nitrogen species (ROS, RNS) were performed and compared to Milli-Q water and culture media without and with Fetal Calf Serum. Electron paramagnetic resonance, fluorometric and colorimetric analysis were used to identify and quantify free radicals generated by helium plasma jet in these liquids. Results clearly show the formation of ROS such as hydroxyl radical, superoxide anion radical and singlet oxygen in order of the micromolar range of concentrations. Nitric oxide, hydrogen peroxide and nitrite-nitrate anions (in range of several hundred micromolars) are the major species observed in PAM. The composition of the medium has a major impact on the pH of the solution during plasma treatment, on the stability of the different RONS that are produced and on their reactivity with biomolecules. To emphasize the interactions of plasma with a complex medium, amino acid degradation by means of mass spectrometry was also investigated using methionine, tyrosine, tryptophan and arginine. All of these components such as long lifetime RONS and oxidized biological compounds may contribute to the cytotoxic effect of PAM. This study provides mechanistic insights into the mechanisms involved in cell death after treatment with PAM.

The aim is the comparison of different plasma parameters of single and double dielectric barrier discharge plasma jet configurations (S-DBD and D-DBD) which are potentially usable in biomedical applications. Both configurations are studied in terms of electric field distribution, electrical discharge characteristics, plasma parameters (estimated by optical emission spectroscopy analysis), and hydrodynamics of the plasma jet for electrical parameters of power supplies corresponding to an applied voltage of 10 kV, pulse duration of 1 μs, frequency of 9.69 kHz, and Ar flow of 2 l/min. We observed that the D-DBD configuration requires half the electrical power one needs to provide in the S-DBD case to generate a plasma jet with similar characteristics: excitation temperature around 4700 K, electron density around 2.5 × 1014 cm−3, gas temperature of about 320 K, a relatively high atomic oxygen concentration reaching up to 1000 ppm, the presence of reactive oxygen and nitrogen species (nitric oxide, hydroxyl radical, and atomic oxygen), and an irradiance in the UV-C range of about 20 μW cm−2. Moreover, it has been observed that D-DBD plasma jet is more sensitive to short pulse durations, probably due to the charge accumulation over the dielectric barrier around the internal electrode. This results in a significantly longer plasma length in the D-DBD configuration than in the S-DBD one up to a critical flow rate (2.25 l/min) before the occurrence of turbulence in the D-DBD case. Conversely, ionization wave velocities are significantly higher in the S-DBD setup (3.35 × 105 m/s against 1.02 × 105 m/s for D-DBD), probably due to the higher electrostatic field close to the high voltage electrode in the S-DBD plasma jet.

This work investigates the regionalized antiproliferative effects of plasma-activated medium (PAM) on colon adenocarcinoma multicellular tumor spheroid (MCTS), a model that mimics 3D organization and regionalization of a microtumor region. PAM was generated by dielectric barrier plasma jet setup crossed by helium carrier gas. MCTS were transferred in PAM at various times after plasma exposure up to 48 hours and effect on MCTS growth and DNA damage were evaluated. We report the impact of plasma exposure duration and delay before transfer on MCTS growth and DNA damage. Local accumulation of DNA damage revealed by histone H2AX phosphorylation is observed on outermost layers and is dependent on plasma exposure. DNA damage is completely reverted by catalase addition indicating that H₂O₂ plays major role in observed genotoxic effect while growth inhibitory effect is maintained suggesting that it is due to others reactive species. SOD and D-mannitol scavengers also reduced DNA damage by 30% indicating that "Equation missing" and OH^* are involved in H₂O₂ formation. Finally, PAM is able to retain its cytotoxic and genotoxic activity upon storage at +4 °C or −80 °C. These results suggest that plasma activated media may be a promising new antitumor strategy for colorectal cancer tumors.

The main bactericidal sources produced by a microwave induced cold argon plasma jet in open air are identified and their relative proportion in the biocide efficiency of the jet is assessed on planktonic Gram-negative bacteria (wild-type strains and deletion mutants of Escherichia coli) diluted in water. In these conditions ultraviolet light (UV) most probably in the UV-C region of the electromagnetic spectrum, is responsible for 86.7 ± 3.2% of the observed bactericidal efficiency of the jet whereas hydrogen peroxide represents 9.9 ± 5.5% of it. The exposition level of the bacteria to UV-C radiations is estimated at 20 mJ cm−2 using a specific photodiode and the influence of the initial bacteria concentration on the apparent antibacterial efficiency of the jet is highlighted.