# Cold Atmospheric Plasma Cancer Treatment, a Critical Review
## 2 Treatment Based on CAP-Activated Solutions  
Conventional CAP-based cancer treatment is performed in a chemically based manner, including indirect CAP treatment and direct CAP treatment strategies. The basic principle of chemical-based indirect CAP treatment is using CAP-treated or -activated solution (PAS) or medium (PAM) to affect the growth of cancer cells via reactive species and other possible plasma-generated products in an aqueous solution [25]. Such an approach is not novel. The use of CAP-treated water or underwater discharge is a long-standing approach, and leads to the death of bacteria or other microorganisms due to the cytotoxicity of reactive species [10; 26; 27; 28; 29; 30; 31; 32]. If these CAP-originated chemical factors are toxic to microorganisms, it is not fully unexpected that these can also be toxic to cancer cells. A more attractive feature of PAS/PAM is the selective anti-cancer performance in many cell lines [33; 34; 35]. However, this selectivity is solely determined by the sensitivity of cancer cells or normal cells to long-lived reactive species or other formed components. PAS/PAM can be derived using a CAP treatment above the surface of the solution (Figure 2). Contact between the bulk plasma and the medium is necessary. Several long-lived reactive species, such as H\({}_{2}\)O\({}_{2}\), NO\({}_{2}\)\({}^{-}\), NO\({}_{3}\)\({}^{-}\), and ONOO\({}^{-}\), have been observed in the aqueous solutions after treatment [36; 37]. The reaction products of CAP and original components also form in other solutions, such as lactate solution [38]. In terms of cell culture medium, CAP will result in complex reactions between ROS/RNS and medium components, particularly amino acids, such as cysteine, methionine, and tryptophan [39; 40]. PAS can also be derived using the discharge in solution [9; 34]. The largest advantage of PAS is thought to be its ability to serve independently from the CAP source once PAS has been derived [39; 41; 42; 43]; that is, indirect treatment can be used as a pharmacological modality. However, due to its pharmacological nature, a clear understanding of its chemical processes and mechanisms needs to be obtained before it can be accepted by the market. To date, only some long-lived reactive species, such as H\({}_{2}\)O\({}_{2}\), NO\({}_{2}\)\({}^{-}\), NO\({}_{3}\)\({}^{-}\), and ONOO\({}^{-}\), in addition to a small number of other components in PAS, have been regarded as being effective anti-cancer components [39; 42; 44]. In general, a single chemical factor cannot explain the observed cytotoxicity. This is demonstrated by a synergistic use of H\({}_{2}\)O\({}_{2}\)/NO\({}_{2}\)\({}^{-}\), or H\({}_{2}\)O\({}_{2}\)/NO\({}_{2}\)\({}^{-}\)/NO\({}_{3}\)\({}^{-}\). Varying ratios of these chemical combinations can achieve similar results to those seen after an indirect CAP treatment [45; 46; 47]. To date, the uniqueness of indirect treatment compared with a simple long-lived reactive species treatment has not been clearly explained or distinguished. Figure 1: CAP sources used in plasma medicine. (**a**) A schematic illustration of typical component of CAP and reactive species formed in the CAP-treated liquid (reproduced with permission from Tumor Biology 37.6, 7021–7031 (2016). Copyright 2016 Springer Nature). (**b**) A volume DBD source. (**c**) A surface discharge source. (**d**) A CAP jet source. Volume and flow rate of jet can be modulated by the nozzle. An additional concern regarding practical implementation is the stability of PAS/PAM during storage. As a pharmacological modality, it must be stably stored under conditions which have relatively low-cost requirements. The extremely cold temperature (\(-\)196 \({}^{\circ}\)C) of liquid nitrogen is an ideal storage condition, but this storage strategy has a high cost and is not practical when a large amount of PAM or PAS is needed. The ideal storage temperature should be either 2-8 \({}^{\circ}\)C, \(-\)20 \({}^{\circ}\)C, or room temperature.