## 4 Discussion  
The aim of this work was to analyze the ability of a-TiO\({}_{2}\) based NPs (a-TiO\({}_{2}\), a-TiO\({}_{2}\)-NH\({}_{2}\) NPs) to act as ultrasound responding agents to improve the generation of ROS and/or act as a cavitation enhancer for biomedical applications, namely, to fight cancer cells. The developed a-TiO\({}_{2}\) NPs were synthesized for the first time through a novel, room-temperature, base-catalyzed sol-gel protocol. Furthermore, to improve the colloidal stability of the developed NPs, a new biomimetic coating based on double lipidic bilayer, self-assembled on the NPs, was performed. Figure 9: Area under the FT curve computed from the signal recorded through a focused piezo-detector used as cavitometer to analyze the cavitation of water and a-TiO\({}_{2}\)-NH\({}_{2}\) based NPs suspensions. The analyses were performed using the following conditions: 1 MHz, 100% of DC, 1 min of irradiation with (**a**) 0.3 W/cm\({}^{2}\) of US output power; (**b**) 0.6 W/cm\({}^{2}\) of US output power; (**c**) 0.9 W/cm\({}^{2}\) of US output power; (**d**) 1.2 W/cm\({}^{2}\) output power. To analyze the generation of ROS and cavitation in the tested suspensions after US stimuli, two different techniques were used, respectively: electron paramagnetic resonance (EPR) spectroscopy and passive cavitation detection (PCD). The concentration of the NPs suspensions was kept constant only varying the output powers of US irradiation. The EPR spectroscopy and PCD results agree and both suggest that the a-TiO\({}_{2}\) and a-TiO\({}_{2}\)-NH\({}_{2}\) NPs are promising candidates to US responding agents, since the generation of ROS increases when in presence of the a-TiO\({}_{2}\) based NPs. Another studied parameter was the influence of visible light. The obtained results allow us to conclude that a synergistic effect exists between the ultrasounds and visible light when the samples are exposed to both stimuli. The colloidal stability and dispersibility improvement of the a-TiO\({}_{2}\) based NPs were also investigated, since the high aggregation tendency in water and physiological media can make the application of TiO\({}_{2}\) NPs unfeasible to biological systems [49; 50; 56]. Thus, the a-TiO\({}_{2}\)-NH\({}_{2}\) NPs were coupled with DOPC-DOTAP phospholipids and the obtained results were very encouraging since stable colloidal suspensions with very high conjugation yield (\(\approx\)88%) were achieved. Biomimetic a-TiO\({}_{2}\)-NH\({}_{2}\)/DOPC-DOTAP NPs were also analyzed through EPR and PCD. The PCD results suggest that the threshold of US power needed to cause the collapse of the induced cavitation bubbles (which influence directly the generation of ROS) is lower than what previously observed for the a-TiO\({}_{2}\) NPs and a-TiO\({}_{2}\)-NH\({}_{2}\) NPs. The absence of detected ROS by EPR measurements in the a-TiO\({}_{2}\)-NH\({}_{2}\)/DOPC-DOTAP NPs was thus attributed to the scavenging capability of the phospholipid bilayer. As mentioned above, the absence of detected ROS from lipid-coated a-TiO\({}_{2}\) NPs is not related to the cavitation effect. In contrast, these nanoparticles showed enhanced cavitation in water solutions once irradiated by US. For this reason, such biomimetic nanoparticles still represent a promising nanotherapeutic tool against cancer, as recent studies have shown anyway the cytotoxic activity induced by cavitation only once nanoparticles are present [68] and have demonstrated this therapeutic effect in absence of generated ROS. We conclude that our study proposes a modulating nanoplatform for cancer therapy where enhanced inertial cavitation or enhanced ROS production can be selectively preferred depending on the sample formulation and chemical surface functionalization. Conceptualization, J.C.M., L.C.J.P., J.C.W. and V.C. ; methodology, M.L., M.C.G. and V.C. ; software, V.V. ; validation, M.L. and V.V. ; investigation, J.C.M. and V.V. ; resources, M.C.G. and V.C. ; data curation, J.C.M., M.L. and V.V. ; writing--original draft preparation, J.C.M.