The effectiveness of electrochemotherapy (ECT) in local eradication of tumours in human and veterinary medicine has been proven. ECT consists of increasing the uptake of cytotoxic drugs by means of pulsed electric fields (PEFs) that transiently permeabilise the cell membrane. Still, this tumour treatment includes some drawbacks that are linked to the characteristics of the intense electric pulses (EPs) used. Meanwhile, the emerging field of cancer therapies that are based on the application of non-thermal plasmas (NTP) has recently garnered interest because of their potentialities as rich sources of reactive species. In this work, we investigated the potential capabilities of the combined application of indirect NTP treatment and microsecond PEFs (µsPEFs) to outperform in vitro cell electropermeabilisation, the basis of ECT. Thus, phosphate-buffered saline (PBS) was plasma-treated (pPBS) and used afterwards to explore the effects of its combination with µsPEFs. Analysis of two different cell lines (DC-3F Chinese hamster lung fibroblasts and malignant B16-F10 murine melanoma cells), by flow cytometry, revealed that this combination resulted in significant increases of the level of cell membrane electropermeabilisation, even at very low electric field amplitude. The B16-F10 cells were more sensitive to the combined treatment than DC-3F cells. Importantly, the percentage of permeabilised cells reached values similar to those of cells exposed to classical electroporation field amplitude (1100 V/cm) when the cells were treated with pPBS before and after being exposed only to very low PEF amplitude (600 V/cm). Although the level of permeabilisation of the cells that are treated by the pPBS and the PEFs at 600 V/cm is lower than the level reached after the exposure to µsPEFs alone at 1100 V/cm, the combined treatment opens the possibility to reduce the amplitude of the EPs used in ECT, potentially allowing for a novel ECT with reduced side-effects.

Non-thermal plasma (NTP) medical applications are now well established but the translation from in vitro plasma effects to in vivo effects remains far from being intuitive. Among various possible reasons, the translation may be disturbed by a loose control over the electrical characteristics of the different targets (e.g. cell culture dish, animal models) met during the development process, a parameter generally neglected and often unmentioned in papers. The aim of this study is to raise consciousness on how target electrical parameters (e.g. conductivity, electric potential...) play a major role in determining plasma treatment conditions. This effect is of particular relevance in plasma medicine where we move from treating small in vitro samples to humans, passing by animal models. The plasma conditions on targets with different electrical characteristics are compared by means of electrical measurements, optical emission spectroscopy and basic liquid analysis. Commonly tested in vitro targets induce the generation of a plasma significantly different from that produced in contact with a human body. We demonstrate how by means of a basic and easy to implement electrical circuit it is possible to “compensate” the electrical differences between in vitro models, mice and human body and in such a way to reach more reproducible treatment conditions between in vitro and in vivo targets. The proposed method for the control of the target electrical parameters could greatly favor the transition from in vitro and in vivo models to patients for many plasma devices developed for biomedical applications.

Amélioration de l’efficacité de l’électrochimiothérapie par association avec des liquides traités par des plasmas froids : mise au point des protocoles expérimentaux et preuve de concept: L'électrochimiothérapie (ECT) - une méthode établie pour l'éradication locale des tumeurs cutanées basée sur la technologie d'électroporation - est largement utilisée en Europe en médecine humaine et vétérinaire avec très peu d'effets secondaires systémiques. Les plasmas froids (NTP) ont récemment pris de l'ampleur pour leur application en cancérologie grâce à leur riche source en espèces réactives à courte durée de vie ainsi qu'en espèces réactives de l'oxygène et de l'azote à longue durée de vie (RONS) qui peuvent être transférées aux liquides traités par du NTP. Nous cherchions à combiner ces technologies afin d'améliorer les capacités de l’ECT et de mieux comprendre la contribution des RONS dans ces stratégies anticancéreuses. En tant qu'étude multidisciplinaire utilisant à la fois de la physique, de la chimie, des biotechnologies et de la médecine, cette thèse visait à mettre en évidence les effets bénéfiques de la combinaison de l’ECT et du plasma froid. Une nouvelle source de plasma d'hélium multi-jet (PMJ) a été utilisée pour des études in vitro sur trois lignées cellulaires différentes (les cellules DC-3F, B16-F10 et LPB), et pour des études in vivo sur les souris nude et C57Bl/6 porteuses de tumeurs sous-cutanées. Nous avons démontré que le pH et la température de stockage du PBS traité par le PMJ (pPBS), ayant une influence importante sur la stabilité des trois principaux RONS (H₂O₂, NO₂- et NO₃-), étaient cruciaux pour conserver la capacité anticancéreuse du pPBS. Le traitement combinant du pPBS et une ECT à une amplitude de champs électrique plus faible que celle utilisée habituellement a permis de restaurer l’efficacité de l’ECT, ralentissant la croissance des tumeurs chez la souris et, en conséquence, améliorant le taux de survie des animaux traités.