# Sonoporation, a Novel Frontier for Cancer Treatment: A Review of the Literature
## 4 Discussion and Conclusions  
The present paper provides a thorough analysis of the available literature regarding the possible application of sonoporation for a novel therapeutic strategy in cancer treatment, analyzing the available data relative to the preclinical application of sonoporation in different cancer types and different cancer models. However, it is necessary to point out the main challenges and limitations inherent in this issue:  
* Ultrasound, especially at high acoustic intensities, can induce collateral damages that are not easy to predict; this is why low-intensity applications must be promoted. * The penetration capability of ultrasounds depends on the frequency adopted for the treatment. This leads to technical difficulties related to the need to adapt the experimental setups in relation to frequency, resulting in different configurations of the relative position between the probe and the sample. Sonoporation has a limitation in specificity, as it can cause undesired effects in neighboring cells. Consequently, it is imperative to further investigate the intensities and frequencies used in sonoporation in order to achieve an optimal compromise between the effectiveness of stimulation and the mitigation of potential damage to healthy cells. Moreover, there is a low correlation between elastic modules and acoustic intensity, pressure, and frequency, as shown in the graphs in Figure 8-10, respectively. A considerable variability in the data collected is evident; however, it is important to emphasize that this lack of correlation is due to substantial differences in the study protocols adopted. As reported in the results, some of the articles are based on in vivo models while others are based on in vitro models, and some use three-dimensional tissues and others use single-layered tissues. These methodological differences may lead to apparently conflicting results but instead represent the different applications of a complex phenomenon. Figure 8: Acoustic intensity related to elastic modulus. The authors of this paper argue for the need to characterize in depth the fundamental mechanical variables that describe both 2D single-layer cells and 3D cultures. It is important to note that the latter may not exhibit the same mechanical behavior as the former. This in-depth study is crucial to establish an effective correlation between the stiffness of the stimulated sample and the applied intensity. In particular, the characterization of Young's modulus, stress-strain curves, and resonance frequency emerge as relevant aspects. The determination of Young's modulus for three-dimensional cultures, which, by their nature, are immersed in multivell containers rather than on slides or plates, presents considerable technical difficulties. The AFM investigation method works relatively well with 2D samples; instead, for the best characterization of mechanical properties of 3D samples [40], the data could undergo a deep analysis with the support of specific software to simulate the complex structure and the interaction between cells and the extracellular matrix. Furthermore, as far as the resonance frequency is concerned, finding it for cancerous tissue could clarify whether it is close to the stimulation frequencies used to induce the sonoporation phenomenon in the samples. Indeed, it is also possible that these frequencies are correlated with the tissue type, which this analysis would lead us to understand. Additionally, it is pivotal to highlight the limited number of publications available on this topic of research due to the complexity of sonoporation. The scarcity of scientific literature can be a significant challenge, as it limits access to established knowledge. However, this scarcity also offers a huge opportunity for new discoveries, so it would be interesting to increase the efforts devoted to its analysis.