**3. Sonodynamic therapy**

Sonodynamic Therapy (SDT) has recently gained much attention as a new anticancer treatment strategy that is relatively cheap, minimally invasive, and possesses deep penetration power. In this therapy, ultrasound waves activate the sonosensitizers (sound-sensitive agents), killing tumor cells by producing ROS [22]. The use of ultrasound offers some advantages in comparison to the use of light in cancer treatment which includes sonoporation (cell permeabilization mediated by ultrasound waves) and deeper penetration (depending on the frequency of ultrasound) which could be up to 15 cm in soft tissues [23–25]. Sonosensitizers refer to the use of chemical compounds that could increase the cytotoxicity of ultrasound. Nano-sonosensitizers are considered potent sonosensitizers, as compared to conventional organic sonosensitizing agents, owing to their high bioavailability achieved by improved pharmacokinetics, pharmacodynamics, and

biodistribution properties. Generally, nano-sonosensitizers can be categorized into two main types: (1) nanoparticles which include TiO2, and (2) nanoparticles assisted sonosensitizers, consisting of nanoparticles loaded with organic molecules with controlled release at the target site [26]. Among many nanoparticles, the use of TiO2 NPs is preferred because of their inert behavior in the biological system, easy fabrication, and cost-effectiveness. TiO2 is a semiconductor with a large energy band gap, allowing for electron transitions from the valence to the conduction band when exposed to UV light. This facilitates the generation of free radicals, including the enormously reactive singlet oxygen. However, UV radiations are not clinically ideal due to low penetration power. Using ultrasound can overcome this due to its greater in vivo penetration ability with low frequency [27]. Various studies have reported the use of TiO2 NPs as anticancer agents in vitro and in vivo systems, especially when combined with ultrasound irradiation.

TiO2 NPs, in association with high-intensity ultrasound waves, were used for sonodynamic therapy of squamous cell carcinoma cells (HSC-2). The authors reported that the toxicity of TiO2 with ultrasound was much higher than that of TiO2 or ultrasound alone, which increased with the increase in intensity and exposure time [28]. SDT with TiO2 NPs was evaluated for the treatment of melanoma. C32 (melanoma cell line) was treated with ultrasound waves of 1 MHz frequency. The apoptotic effect was more significantly observed in the TiO2-based SDT than in either treatment alone. In addition, the apoptotic percentage of cells was increased by 2.73 times than untreated cells [29]. Aksel et al. reported that TiO2 NPs mediated sonodynamic, photodynamic, and Sono-Photodynamic (SPDT) Therapy for prostate cancer. SDPT combines sonodynamic therapy and photodynamic therapy along with TiO2 NPs as sensitizers. The results showed a reduction in cancer cell viability after TiO2-mediated sono-photodynamic therapy. The production of singlet oxygen affects the intrinsic pathway, which might be responsible for producing antiapoptotic effects [30].
