## 3 Results
### Passive Cavitation Detection  
The evaluation of the acoustic cavitation on the generation of ROS after US exposure was performed through passive cavitation detection (PCD) technique. The experiments allowed us to measure the acoustic pressures reached inside the well for all the samples and the conditions tested. More details about the experiment execution and data interpretation are reported in the Materials and Method section. Figure 8 shows the area under the curve for all cycles at different US powers with and without a-TiO\({}_{2}\) based NPs (a-TiO\({}_{2}\) NPs and a-TiO\({}_{2}\)-NH\({}_{2}\) NPs) in suspension. This area is proportional to the energy release caused by the collapse of the cavitation bubbles. Figure 8 shows the obtained frequency spectra of the acoustic signals at different US intensities with and without a-TiO\({}_{2}\) based NPs (a-TiO\({}_{2}\) NPs and a-TiO\({}_{2}\)-NH\({}_{2}\) NPs) in suspension. It can be observed that only harmonics and subharmonics are present in all sample types, i.e., both water and water with pristine and amine-functionalized a-TiO\({}_{2}\) NPs when the samples were irradiated by the lowest US power (0.3 W/cm\({}^{2}\)). These signals are probably due to the oscillation of large gas bubbles trapped in the plastic wells of the sample holder. For 0.6 and 0.9 W/cm\({}^{2}\) powers, as shown in Figure 8b,c, an acoustic broadband noise was recorded in case of a-TiO\({}_{2}\) based NPs samples. This acoustic broadband corresponds to the inertial cavitation occurrence. On the other hand, in presence of water solution without NPs, the acoustic broadband noise was only detected when the sample was irradiated with the highest US power tested, 1.2 W/cm\({}^{2}\) (Figure 8d). These results allow us to conclude that the a-TiO\({}_{2}\) based NPs act as nucleation sites inducing inertial cavitation. The decrease of the threshold of US power needed to cause the collapse of the induced cavitation bubbles, enables the generation of sufficiently high temperatures and pressures to ROS production, even at low US power [12]. A comparison between these results and those obtained through EPR spectroscopy can be carried out. The US power threshold needed to induce passive cavitation, and thus the ROS generation,  
Figure 8: 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\({}_{2}\) based NPs suspensions. The analysis were made 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. is different. Indeed, the passive cavitation detection (PCD) can be considered a more sensitive technique if compared to EPR spectroscopy. Interestingly, in the case of the a-TiO\({}_{2}\)-NH\({}_{2}\) NPs coupled with lipids, the signal difference between the control sample (water without NPs) and the a-TiO\({}_{2}\)-NH\({}_{2}\)//DOPC-DOTAP NPs sample is even more pronounced (Figure 9). In this case, the acoustic broadband noise was recorded for all the tested powers (0.3, 0.6, 0.9, and 1.2 W/cm\({}^{2}\)), being more pronounced from 0.6 W/cm\({}^{2}\). These results show that the a-TiO\({}_{2}\)-NH\({}_{2}\)//DOPC-DOTAP NPs could act as a promising cavitation enhancer nano-agent, if compared to other already analyzed NPs (pristine a-TiO\({}_{2}\) NPs and a-TiO\({}_{2}\)-NH\({}_{2}\) NPs). This is a more stable suspension (see Section 3.3) and is able to decrease even more the threshold of US power needed to occur the collapse of the induced cavitation bubbles, which directly influence the generation of ROS. However, it is important to mention that the presence of the lipidic bilayer is able to scavenge the generated ROS, which are in fact not detectable by the EPR characterization.