Cold atmospheric pressure plasmas (CAPPs) have emerged over the last decade as a new promising therapy to fight cancer. CAPPs’ antitumor activity is primarily due to the delivery of reactive oxygen and nitrogen species (RONS), but the precise determination of the constituents linked to this anticancer process remains to be done. In the present study, using a micro-plasma jet produced in helium (He), we demonstrate that the concentration of H₂O₂, NO₂⁻ and NO₃⁻ can fully account for the majority of RONS produced in plasma-activated buffer. The role of these species on the viability of normal and tumour cell lines was investigated. Although the degree of sensitivity to H₂O₂ is cell-type dependent, we show that H₂O₂ alone cannot account for the toxicity of He plasma. Indeed, NO₂⁻, but not NO₃⁻, acts in synergy with H₂O₂ to enhance cell death in normal and tumour cell lines to a level similar to that observed after plasma treatment. Our findings suggest that the efficiency of plasma treatment strongly depends on the combination of H₂O₂ and NO₂⁻ in determined concentrations. We also show that the interaction of the He plasma jet with the ambient air is required to generate NO₂⁻ and NO₃⁻ in solution.

Absolute densities of singlet delta oxygen (SDO) molecules were measured using infrared optical emission spectroscopy in the flowing effluents of two different atmospheric-pressure plasma jets (APPJs): a capacitively coupled radio-frequency-driven jet (rf-APPJ) and a lower frequency kilohertz-driven dielectric barrier discharge jet. The plasma jets were operated in helium, with small admixtures of molecular oxygen (O₂ < 2%). High absolute SDO densities of up to 6.2 × 10¹⁵ cm⁻³ were measured at approximately 10 cm downstream. The rf-APPJ seems to be much more efficient in producing SDO. The influence of different parameters, such as gas flows and mixtures and power coupled to the plasmas, on the production of SDO by the two APPJs has been investigated. Despite the considerable differences between the two plasma jets (excitation frequency, electric field direction, inter-electrode distance, plasma propagation), similar dependencies on the oxygen admixture and on the dissipated power were found in both APPJs. However, opposite trends were observed for the gas flow dependence. The results presented in this paper show that the control of the external operating conditions of each APPJ enables the tailoring of the SDO composition of both plasma effluents. This provides scope to tune the plasma jets for desired applications, e.g., in biomedicine.

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.

(1) Plasma-activated liquids (PAL) have been extensively studied for their anti-cancer properties. Two treatment modalities can be applied to the cells, direct and indirect plasma treatments, which differ by the environment to which the cells are exposed. For direct plasma treatment, the cells covered by a liquid are present during the plasma treatment time (phase I, plasma ON) and the incubation time (phase II, plasma OFF), while for indirect plasma treatment, phase I is cell-free and cells are only exposed to PAL during phase II. The scope of this work was to study these two treatment modalities to bring new insights into the potential use of PAL for cancer treatment. (2) We used two models of head and neck cancer cells, CAL27 and FaDu, and three models of normal cells (1Br3, NHK, and RPE-hTERT). PBS was used as the liquid of interest, and the concentration of plasma-induced H₂O₂, NO₂⁻ and NO₃⁻, as well as pH change, were measured. Cells were exposed to direct plasma treatment, indirect plasma treatment or reconstituted buffer (PBS adjusted with plasma-induced concentrations of H₂O₂, NO₂⁻, NO₃⁻ and pH). Metabolic cell activity, cell viability, lipid peroxidation, intracellular ROS production and caspase 3/7 induction were quantified. (3) If we showed that direct plasma treatment is slightly more efficient than indirect plasma treatment and reconstituted buffer at inducing lipid peroxidation, intracellular increase of ROS and cancer cell death in tumor cells, our data also revealed that reconstituted buffer is equivalent to indirect plasma treatment. In contrast, normal cells are quite insensitive to these two last treatment modalities. However, they are extremely sensitive to direct plasma treatment. Indeed, we found that phase I and phase II act in synergy to trigger cell death in normal cells and are additive concerning tumor cell death. Our data also highlight the presence in plasma-treated PBS of yet unidentified short-lived reactive species that contribute to cell death. (4) In this study, we provide strong evidence that, in vitro, the concentration of RONS (H₂O₂, NO₂⁻ and NO₃⁻) in combination with the acidic pH are the main drivers of plasma-induced PBS toxicity in tumor cells but not in normal cells, which makes ad hoc reconstituted solutions powerful anti-tumor treatments. In marked contrast, direct plasma treatment is deleterious for normal cells in vitro and should be avoided. Based on our results, we discuss the limitations to the use of PAL for cancer treatments.