# Multiresponsive Hybrid Microparticles for Stimuli-Responsive Delivery of Bioactive Compounds
## 3 Results and Discussions
### Cytotoxicity and Hemolysis Assessment of Micorpatrices  
The MTT has been performed to determine (i) the cytotoxicity of the bare microparticles and the impact of the surface properties on the in vitro biocompatibility; (ii) the advantages of using a microcarrier in the delivery of DOX compared to the free drug, considering the in vitro cytotoxicity enhancement in particular; and (iii) DOX cytotoxicity, extended over time. IO microparticles have been used as the control to evaluate the effect of the surface modification and the polymer coat on the cytocompatibility of the system. Figure 6 shows cell viability evaluation for all formulations. NIH/3T3 cells were treated with a solution containing 50 ug of microparticles, and the viability was determined after 24, 48 and 72 h (Figure 6). The toxic effect was taken into consideration when the  
Figure 6: Cell viability evaluation for all formulations with and without application of US (concentration of particles: 50 ug/mL): (**A–C**) 2 W/cm\({}^{2}\) and (**D–F**) 20 W/cm\({}^{2}\). The cells were incubated with the core–shell samples at the concentration of 50 ug/mL and treated with five US cycles within 110 min (the timing used for the release was followed). Values are reported as averages, and error bars represent the SD (n = 3). viability was below 80%. The control, containing only cells, was treated with ultrasound to determine any possible contribution of US on the cell viability. Results reported in Figure 6 demonstrate the advantages of the core-shell design in improving DOX cytotoxicity. According to the results, the presence of the polymer shell could also play a certain role by interacting with the cell membrane and favoring the uptake. In fact, DOX cytotoxicity is higher when loaded in the core-shell structure than in ZV; in the case of ZV, the release begins immediately after contact with the medium, leading to the release of a certain amount of the drug in the solution before the internalization of the particles. The cell viability tends to decrease over time when exposed to core-shell microparticles, conversely to the observed increase in viability 48 and 72 h after exposure to free DOX, even if the core-shell viability value is maintained as much lower in comparison. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolzolium bromide (MTT) test was performed in the presence of US using the same power, number of cycles and application time used in the release studies. The application of US at 2 and 20 W/cm\({}^{2}\) power did not affect the cell viability but might reversibly alter the structure of the membrane of the cell by sonoporation, favoring the entry of the microparticles and free DOX. In the case of core-shell microparticles, the positive \(\zeta\)-potential represents an additional advantage, as it promotes the interactions with the negatively charged cell membrane. The use of 5 \(\times\) 10 s and 5 \(\times\) 30 s US cycles did not cause any significant increase in temperature, with an observed variation of less than 1 \({}^{\circ}\)C. This is important to determine, as the temperature affects the shell structure and, consequently, the release rate and mechanism. Moreover, an increase in temperature affects the cell viability. The graph in Figure 6 shows how the cytotoxicity of the free DOX and DOX-loaded ZV particles is lower than that of the DOX loaded in the core-shell particles due to the slower release of the drug. As reported in the release studies subsection, the US cycles cause a burst in the DOX release, meaning that a large amount of drug is released in a short period of time; such an event, in combination with sonoporation, leads to a higher permeation of the drug into the cell, with a decrease in the viability. The increased in vitro cytotoxicity of DOX resulting from the use the US is provided by the synergic effects of US and DOX. US acts either directly and indirectly, by sonoporation and by causing a burst in the DOX release. Moreover, the possibility to turn the burst on and off is only provided by the core-shell systems.