# Investigation of Sonosensitizers Based on Phenothiazzinium Photosensitizers
## 3 Results
### Cellular Toxicity  
The combination of light illumination and photosensitizer resulted in cellular phototoxicity. Figure 6a presents the cell morphologies from A549 lung cancer cells incubated with or without phenothiazazine derivatives before and after irradiation, and then cell death was estimated overnight. Figure 6b summarizes the concentration-dependent dark toxicity and phototoxicity cell viability plots. The A549 lung cancer cell lines display no determinable dark toxicity or phototoxicity with MB and MBOH up to a concentration of 20 uM. MB6C shows more efficient dark toxicity (with EC\({}_{50}\) at ~7 uM) and phototoxicity (with EC\({}_{50}\) at ~2.5 uM) than the others, although this compound offers a lower singlet oxygen quantum yield than MB. Figure 6a also shows that cells become highly condensed after ROS are triggered from MB6C, and the cell morphology clearly shows that in the 5 uM condition, the cells are slightly dark-toxic before irradiation. Increasing the chain length from methyl to n-hexyl results in an increase in intracellular toxicity. This means that there is no correlation between intracellular phototoxicity and ROS generation ability. We infer that the different cellular toxicities of these phenothiazazine derivatives are most likely due to cellular uptake, as shown in Figure 6c. We tried to quantitively assess the compound accumulation in the cells and found that only MB6C presents clearer intracellular fluorescent image signals, even though the emissions of these molecules are all weak and similar, as shown in Figure 3. There is no doubt that MB shows no toxicity in our experimental concentration range because it is known to present low cellular toxicity EC\({}_{50}\) values of 147 uM in dark toxicity and 54 uM in phototoxicity [42]. MBOH presents very low cellular dark toxicity and phototoxicity, which probably due to its low logP (higher hydrophilicity than MB), causing the low cellular uptake of the compound. The above results suggest that MB6C is a potential reagent for studying intracellular photosensitization, the oxidative stress of PDT. Meanwhile, we expect that MB6C should exhibit more efficient SDT than MB and MBOH. Following to the incubation of the cells with MB6C for 24 h and the exposure to ultrasonication for 20 min and then culture overnight, Figure 7a shows that the cells exhibit different morphologies and become somewhat condensed. A further cell death occurs after repeating one cycle of ultrasonication and incubation (total: ultrasonicet then incubation overnight \(\times\)2). The EC\({}_{50}\) values for SDT of MB6C are ~5 uM and ~1.5 uM for one and two cycles of ultrasonicated treatments, respectively. Herein, the ultrasound power must be adjusted to avoid damaging the cells without molecular incubation. There is no doubt that MB6C is a potential reagent for SDT. Table 1 shows the dark toxicity, phototoxicity, and sonotoxicity of the phenothiazazines in A549 cells after 12 h of incubation. As can be seen from the results in Figures 3 and 4, increasing the alkyl chain length and altering the hydrophilicity of the NR\({}_{2}\) did not change the singlet oxygen generate ability. On the other hand, the bio-assay result revealed that longer chain methylene blue MB6C showed improved intracellular phototoxicity with a higher uptake. These results are consistent with the literature [42]. Despite this, as can be seen from Figure 3c, we still need to be concerned about the intracellular aggregation problem of MB6C. Thus, as demonstrated in Figure S7, the concentration effect of these phenothiazazine derivatives was checked and it was found that the lipid concentration is higher than that of MB6C. Figure 6: (**a**) Several extracted bright-field photodamage photographs from A549 lung cancer cells incubated with or without phenothiazazine derivatives, 5 uM MB, MBOH, and MB6C and 3 uM of MB6C, for 12 h in order to check the dark toxicity.