## 4 Discussion  
While NIPP-induced damage to bacterial cell membranes has been extensively studied [22; 23], the impact of NIPP on the cytoplasmic membrane of eukaryotic cells, particularly the effects on permeability to low molecular weight compounds, is poorly investigated. Recent studies have demonstrated increased cytoplasmic membrane permeability after NIPP treatment of skeletal sarcoma cells [6; 24; 25] and NIPP-induced changes in the  
\begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \multirow{2}{*}{**Cells**} & \multirow{2}{*}{**Assay**} & \multirow{2}{*}{**Neg Ctrl**} & \multirow{2}{*}{**Pos Ctrl**} & \multicolumn{3}{c}{**Ctrl**} & \multicolumn{3}{c}{**NIPP**} \\ \cline{4-10}  & & & & **24 h** & **48 h** & **72 h** & **24 h** & **48 h** & **72 h** \\ \hline \multirow{4}{*}{**MCF-7**} & \multirow{2}{*}{**Casp-37**} & \(1.5\times 10^{-1}\) & \(2.1\times 10^{-1}\) & \(1.9\times 10^{-1}\) & \(2.5\times 10^{-1}\) & \(2.6\times 10^{-1}\) & \(4.4\times 10^{-1}\) & \(5.9\times 10^{-1}\) & \(5.9\times 10^{-1}\) \\  & & \(\pm 9.8\times 10^{-2}\) & \(\pm 3.2\times 10^{-1}\) & \(\pm 1.2\times 10^{-2}\) & \(\pm 4.2\times 10^{-2}\) & \(\pm 1.7\times 10^{-1}\) & \(\pm 4.6\times 10^{-2}\) & \(\pm 1.4\times 10^{-1}\) & \(\pm 3.5\times 10^{-1}\) \\ \cline{2-10}  & & & & & & & & & \\ \cline{2-10}  & & & & & & & & & & \\ \hline \multirow{4}{*}{**MDA-MB-231**} & \multirow{2}{*}{**Casp-37**} & \(8.3\times 10^{-2}\) & \(1.4\times 10^{0}\) & \(2.8\times 10^{-1}\) & \(3.2\times 10^{-1}\) & \(2.3\times 10^{-1}\) & \(5.7\times 10^{-1}\) & \(9.7\times 10^{-1}\) & \(6.1\times 10^{-1}\)  
membrane potential of keratinocytes [26]. Moreover, characterization by Raman imaging identified significant alteration of the molecular fingerprint of phospholipids following NIPP treatment with different plasma devices [27]. The present study revealed that low molecular weight compounds (\(\leq\)10 kDa) could pass unimpaired through the cytoplasmic membrane of breast cancer cells after NIPP treatment. This effect on the molecule passing seems dose-independent as NIPP treatment times of 20 and 60 s demonstrated no dose-dependent variation on permeability. Changes in membrane integrity after NIPP treatment has been previously reported in osteosarcoma cells [25]. FDA efflux could be shown in osteosarcoma cells after NIPP treatment in a concentration-dependent manner. The discrepancy between the sensitivity to different NIPP concentrations indicates a possible tumor entity-specific NIPP efficiency. The release of small molecules such as those of ATP has been associated with NIPP-induced damage-associated molecular patterns, enhancing cellular visibility to the immune system [28]. In this study, NIPP treatment resulted in an efflux of endogenously synthesized ATP, ruling out artificial and toxic effects of the fluorescent dye incubation. This ATP release was also demonstrated in malignant cells of the skin and the pancreas [29]. Although ATP can also be liberated from apoptotic cells and induce macrophage activation [30; 31], apoptosis-induced ATP release can be excluded in the present study. NIPP-induced ATP efflux could be detected immediately after cancer cell treatment. However, NIPP-induced apoptosis would not occur until many hours after treatment, so even early apoptotic processes cannot be responsible for ATP release [32]. In contrast to the incubation experiments with fluorescent dyes, NIPP-induced ATP release exhibited a clear positive correlation with treatment duration. Whether the chemical structure of the released molecules plays a role in the accelerated transfer through the cytoplasmic membrane or whether the incubation with dyes can lead to artificial effects cannot be ruled out at this point. One explanation for the enhanced extracellular ATP concentration could be the increased cell permeability mediated by NIPP-induced ROS including H\({}_{2}\)O\({}_{2}\)[33]. ROS are the primary mediators of the effects of NIPP on cancer cells [34; 35; 36; 37], and their formation depends on carrier gas flow rate and treatment duration.