# Investigation of Sonosensitizers Based on Phenothiazzinium Photosensitizers
## 3 Results
### Cellular Toxicity
Then cells were irradiated with an average 18 J-cm\({}^{-2}\) of light source (510 nm lp) for 20 min and cell deaths were estimated after a further 12 h. (**b**) Dark toxicity and phototoxicity cell viability plots of A549 cells treated with variable concentrations of phenothiazazine derivatives, as in the experiment in (**a**). Each datapoint is the average from three separate experiments. (**c**) Fluorescent photodamage images extracted from experiment (**a**) to check the cellular uptake for every compound in (**a**). Figure 7: (**a**) Bright-field sonodamage images from A549 lung cancer cells incubated with or without phenothiazazine derivatives, 5 uM MB, MBOH, and MB6C and 3 uM MB6C, for 12 h and then ultrasonicated for 20 min (conditions are as described in the experimental section, scale bar: 50 μm). (**b**) Cell sonotoxicity plots of A549 cells treated with variable concentrations of phenothiazazine derivatives, as in experiment (**a**) but ultrasonicated twice; each datapoint is the average of three individual experiments. that more hydrophilic MBOH seems to have no apparent aggregation, while MB showed more obvious aggregation (wavelength 610 nm) at high concentration. In contrast, based on specific absorption spectral shifts and patterns, accompanied with the almost complete quenching of emission, we inferred that MB6C was able to aggregate through a different mechanism, which could cause the inactivation of optical behaviors, such as emission and singlet oxygen generation. Furthermore, the SOSG assay in Figure S8 pointed out that the compound MB6C actually presented a weaker ability for singlet oxygen generation in aqueous solution. The preceding discussion seems to indicate that MB6C is not suitable for applying PDT in water condition, let alone SDT. However, our bioassay results presented the positive responses. MB6C implemented intracellular fluorescence images, better cellular uptake, phototoxicity, and ultrasonic toxicity over MB and MBOH. These phenomena suggested that MB6C is unlikely to stay in a hydrophilic environment in the cells so that it could exhibit above optical behavior. Proposing the cell death mechanism for MB6C-induced SDT, Figure S9 shows the double stain result from Hoechst/PI and we observed, during the current period of SDT execution, apparent blue emission enhancement in the nucleus but no red fluorescence. It was inferenced that DNA condensed while the nuclear member is intact. The nucleus then dramatically presented red fluorescence enhancement from PI after the second ultrasonication proceeding. This is a symptom of the rupture of the nuclear membrane damage during a later period of sonication. That is, this cell death pathway is more or less related to pro/anti-proteins in mitochondrial [47], and this phenomenon seems to be consistent with the inferences above, in the early stage of cell death (staining with the two probes, immediately after ultrasonication). We prepared and studied phenothiazze derivatives, the analogs of MB, and evaluated their ROS to improve phototoxicity and sonotoxicity against cancer cells in vitro. Compound MB6C is more efficient than other compounds with particular cellular toxicity. Based on our studies, spectral photo- and sonodynamic activity levels correlate with the cellular phenothiazze levels and subcellular behaviors of these photosensizers or sonosensitizers. All these MB analogs are localized in the cytoplasm, with MB6C presenting the highest uptake. We infer that this is why MB6C exhibits better PDT and SDT. However, upon exposure to light, MB6C does not relocalize into the nucleus, as MB does [39]. This phenomenon also does not occur in ultrasonicated systems. Therefore, MB6C, a more hydrophobic MB analog, shows significantly improved PDT and SDT. In addition, this compound makes it possible to avoid the problems of PDT/SDT-induced photosensitizer/sonosensitizer relocation to the nuclear, which causes mutagenicity.