# Investigation of Sonosensitizers Based on Phenothiazzinium Photosensitizers
## 4 Conclusions  
We synthesized two symmetrical phenothiazze derivatives, in which the methyl groups of methylene blue were substituted by hexyl and hydroxyethyl chains. Spectral studies indicate that the intrinsic photosensitizing ability was not altered. Here, the first conclusion is that these MB analogs exhibit a sonosensitizing ability with a similar tendency to photosensitizing ability. This means that MB, MBOH, and MB-6C can be potential photosensizers and sonosensitizers. However, in terms of the in vitro phototoxicity and sonotoxicity after cell-assays, a more hydrophobic MB6C, implemented intracellular fluo \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multirow{2}{*}{**LogP**} & \multicolumn{4}{c}{**Toxicity (IC50, \(\upmu\)M)**} \\ \cline{2-5}  & & **Dark-** & **Photo-** & **Sono- * \\ \hline MB & \(-\)0.1 & \textgreater{}20 & \textgreater{}20 & \textgreater{}20 \\ MBOH & \(-\)0.54 & \textgreater{}20 & \textgreater{}20 & \textgreater{}20 \\ MB6C & \textless{}1.3 & \textgreater{}7 & \textasci{}2.5 & \textasci{}(\times 1)\), \(1.5\) (\(\times 2\)) \\ \hline \hline \end{tabular}  
* The EC\({}_{50}\) values for SDT of MB6C is \(-\)5 \(\upmu\)M and \(-\)1.5 \(\upmu\)M for one and two cycles of US treatments, respectively.  
\end{table} 
Table 1: LogP (octanol: buffer partition coefficients) values, dark-, photo- and sonotoxicities of the phenothiazze derivatives after 12 h incubation in A549 cancer cells. rescence images, better cellular uptake, phototoxicity, and ultrasonic toxicity over MB and MBOH can result in the improved PDT and SDT of methylene blue derivatives. Eventually, potential PDT and SDT candidate MB6C exhibits efficient intracellular phototoxicity and sonotoxicity with IC\({}_{50}\) values of \(\sim\)2.5 \(\upmu\)M and \(\sim\)5 \(\upmu\)M, respectively. In addition, MB6C is excluded from the nucleus, thus reducing the mutagenic potential of PDT and SDT. Investigations utilizing animal testing are currently underway. The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3390/app12157819/s1](https://www.mdpi.com/article/10.3390/app12157819/s1)]([https://www.mdpi.com/article/10.3390/app12157819/s1](https://www.mdpi.com/article/10.3390/app12157819/s1)), Figure S1: H\({}^{1}\) NMR spectrum of MBOH in DMSO-\(d_{6}\) solution (400 MHz). Figure S2: C\({}^{13}\) NMR spectrum of MBOH in DMSO-\(d_{6}\) solution (400 MHz). Figure S3: H\({}^{1}\) NMR spectrum of MB6C in DMSO-\(d_{6}\) solution (400 MHz). Figure S4: C\({}^{13}\) NMR spectrum of MBOH in DMSO-\(d_{6}\) solution (400 MHz). Figure S5: Electrospray ionization mass spectra (ESI-MS) of MBCO (M/Z). Figure S6: Electrospray ionization mass spectra (ESI-MS) of MB6C (M/Z). Figure S7: Concentration effect absorption (a) and emission (b,c) spectra of compound MB (top), MBOH (middle) and MB6C (bottom). The emission spectra was collected by exciting dimer form (b) and monomer form (c), respectively. Figure S8: The emission spectra recorded for the Singlet Oxygen Sensor Green (SOSG) in the presence of three MB derivatives as a function of illumination time. Following the experimental condition in Section 2.6.1 of manuscript, and the excitation wavelength for the SOSG was 490 nm. The result also shows the singlet oxygen generation plots, and the rate of singlet oxygen generation by compounds is as follows: MB- MBOH \(>\) MB6C. Figure S9: Following the experimental condition of Figure 7: (a) Before ultrasonication (no Hoechst or PI treatment). (b) Cells were treated with Hoechst and PI once after ultrasonication. (c) second cycle of ultrasonication and then overnight. The images of Hoechst-stained cells were collected by exciting with a blue light which was guided through a 370 \(\pm\) 10 nm bp filter, and the emission wavelength rang was collected through a 450 nm lp filter (A cube). The images of PI-stained cells were excited by a green light which was guided through a 530 \(\pm\) 20 nm bp filter, and the emission wavelength rang was collected through a 590 nm lp filter (N cube).