## 4 Discussion
affirmation that acidification of the treatment solution alone is insufficient to induce cancer cell death [3]. Analyzing the viability of HOB cells treated with PAM-RPMI it can be observed that the treatments had minor to no effects on it both at 2- and 24-h post-treatment. Interestingly, the viability of HOS-treated cells continued to drop after the first evaluation at 2 h after the treatment even though PAM-RPMI was not in contact with the cells, suggesting that the treatment induce in HOS cells triggers continuous long-term cytotoxic effects. Therefore, the indirect PAM-RPMI treatment produced by our custom-built CAP system is able to selectively kill HOS cells. According to [PERSON] et al., the relevant reactive species in the composition of PAM, as well as their cytotoxic effects, are present only in acidic conditions [95]. Therefore, we evaluated the dynamics of the pH levels of the PAM-RPMI solutions over a period of 3 months. Additionally, we examined the impact of storage temperature on the stability of the pH of the PAM-RPMI solutions. Figures 11 and 12 demonstrate that the acidification of RPMI 1640 due to CAP exposure remains stable for 3 months, indicating that \(-80\)\({}^{\circ}\)C is the optimal storage method of PAM-RPMI treatment due to its under 0.1 pH variation. However, given that the pH of PAM stored at \(-20\)\({}^{\circ}\)C does not undergo significant variations, this storage temperature remains a viable option, especially when considering factors such as cost or the availability of equipment in some laboratories. Analyzing the pH stability of PAM stored at different temperatures (25 \({}^{\circ}\)C, 4 \({}^{\circ}\)C, \(-20\)\({}^{\circ}\)C, and \(-80\)\({}^{\circ}\)C) for 7 days, [PERSON] et al. did not identify significant changes [96]. By comparison, in the present study, no significant variations were recorded during the first 2 months of storage, with changes being identified in the third month. CAP is known to generate a significant amount of reactive nitrogen species [11; 78], and the levels of these species in PAM solutions used for cancer cell treatment can impact the treatment's antitumor effectiveness [11; 46]. Moreover, the main contributor to the acidification of the PAM solution is attributed to the generation of nitrites (NO\({}_{2}^{-}\)) and nitrates (NO\({}_{3}^{-}\)) resulting from the interaction between the short-lived reactive nitrogen species generated by CAP and the exposed solution [3; 9]. Thus, after establishing the optimal parameters for the production of PAM with antitumor effect by using our custom-made plasma generation device, the analysis of NO\({}_{2}^{-}\) and NO\({}_{3}^{-}\) from PAM-RPMI was carried out in order to identify both NO\({}_{2}^{-}\) and NO\({}_{3}^{-}\) levels and the influence that the exposure time to CAP can have on them. The results indicated that exposure of RPMI 1640 medium to CAP resulted in a dose-dependent increase in PAM-RPMI NO\({}_{2}^{-}\) and NO\({}_{3}^{-}\) concentrations, ranging from 1.6 mM to approximately 3.6 mM for NO\({}_{2}^{-}\) and from 1.5 mM to approximately 2.3 mM for NO\({}_{3}^{-}\). The levels of NO\({}_{2}^{-}\) and NO\({}_{3}^{-}\) identified are considerably higher compared to those reported in previous studies where analyzing different solutions exposed to CAP for different periods of time and using different plasma production methods generally detected concentrations of both NO\({}_{2}^{-}\) and NO\({}_{3}^{-}\) between several \(\upmu\)M and 1 mM [3; 61; 77; 73; 75; 77; 80; 97; 98]. Moreover, apart from the observed high concentrations of NO\({}_{2}^{-}\) in PAM-RPMI, it was observed that these concentrations remained stable for up to 3 months when PAM-RPMI was stored at \(-80\) degrees Celsius.