# Investigation of Sonosensitizers Based on Phenothiazzinium Photosensitizers
###### Abstract  
The main advantage of sonodynamic therapy (SDT), the combining of ultrasound with a sonosensitizer, over photodynamic therapy (PDT) is that ultrasound penetrates deeper into tissues to activate the sonosensitizer, which offers noninvasive therapy for tumors in a site-oriented approach. In this study, we synthesized two symmetrical phenothiazze derivatives in which the methyl groups of MB (methylene blue) have been replaced by a hexyl and hydroxyethyl chains, named 3,7-bis(dihexylamino)-phenothiazzin-5-ium iodide (MB6C) and 3,7-bis(di(2-hydroxyethyl)amino)-phenothiazz-5-ium iodide (MBOH), respectively. We explore the efficiency differences between PDT and SDT induced by these phenothiazze derivatives based on the standard of methylene blue (MB). Spectral studies indicate that these MB analogs exhibit sonosensitization ability with a similar tendency to the photosensitization ability. This means that MB, MBOH, and MB6C can be potential photosensizers and sonosensitizers. After biological evaluation, we conclude that compound MB6C is a potential PDT and SDT candidate because it exhibits higher uptake, efficient intracellular phototoxicity and sonotoxicity over MB and MBOH, with IC\({}_{50}\) values of \(\sim\)2.5 uM and \(\sim\)5 uM, respectively.  
photodynamic therapy; photosensitizer; sonodynamic therapy; sonosensitizer; reactive oxygen species +
Footnote †: journal: Medical  
between SDT and PDT for activating the sensitizer; one utilizes ultrasound and the other uses light. Ultrasound penetrates deeper into tissues and can be focused tightly even through tens of centimeters into soft tissues when compared to PDT, which is limited by the short penetration depth of light [12; 13]. The ultrasonically activated sonosensitizers can produce reactive oxygen species (ROS), such as superoxide anion (O2*\({}^{-}\)), hydroxy radical (\(\bullet\)OH), hydrogen peroxide (H2O2) or singlet oxygen (1O2) [9; 14]. When discussing the underlying mechanisms of SDT-induced apoptosis or necrosis, sonosensitizers are considered the most critical factor to maximize effectiveness. We focused on organic small molecule sonosensitizers and found that the most commonly used are porphyrin derivatives, such as hematoporphyrin (Hp), protoporphyrin IX (PpIX), chlorin e6 (Ce6), and hematoporphyrin monomethyl ether (HMME) [15; 16; 17]. The xanthene derivatives erythrosine B (EB) [18; 19] and rose bengal (RB) [20; 21] have also been found to exhibit a high relationship with ultrasonication. Other small molecule agents, such as hypocrellin B (HB) [22; 23], IR780 [24; 25], indocyanine green (ICG) [26; 27], curcumin [28; 29], and 5-aminolevulinic acid (a natural porphyrin precursor) [30; 31], have also been reported as effective sonosensitizers. Most notably, the anticancer drug doxorubicin was also found to act as a sonosensitizer and showed relatively high SDT efficiency [32; 33]. Thus, we know that most sonosensitizers are derived from photosensizers, which may cause apparent phototoxicity and offer undesirable biological profiles.  
The photosensitizer methylene blue (MB) [34; 35], a low-toxicity phenothiazze molecule, has been approved for antifungal, antibacterial [36], and antimalarial activity [37] and in the staining of living organisms for clinical use. The literature has reported that MB could be a new sonosensitizer and cause ultrasound-induced cell death [38; 39; 40]. However, the specific cellular mechanism of MB-induced SDT on tumors is unclear. The purpose of this study is to evaluate the SDT efficiencies of MB, its hydrophilic derivative MBOH, and its lipophilic derivative MB6C (as shown in Scheme 1). Molecular synthesis, spectra, and ROS measurements and the cellular assays of these molecules were performed and discussed for SDT development.