# Multiresponsive Hybrid Microparticles for Stimuli-Responsive Delivery of Bioactive Compounds
## 3 Results and Discussions
### Passive and US-Triggered DOX Release Kinetics  
The non-invasiveness and the ability to regulate the depth of the penetration by tuning frequency, duty cycles, and time of exposure represent some of the advantages of using ultrasound (US) as an external trigger to cause a burst in the release of DOX [34]. For in vivo application, ultrasonic waves can reach deeper sites compared to light radiation, and the associated sonoporation phenomenon facilitates the diffusion of therapeutic molecules through the cell membrane [35]. In vitro ultrasound-induced drug delivery mechanisms have been described in detail [36; 37]. Herein, the enhancement in the DOX release after application of US cycles could be ascribed to the oscillatory motion of the surrounding fluid occurring in the absence of cavitation. Herein, two main mechanisms could be responsible for the intensification of DOX release after US application. The first is due to the oscillation of the surrounding fluid, which increases the diffusivity of the molecules; the second is related to the rupture of the carrier structure. However, the former plays a marginal role, while a complete disruption of the particles leads to an intense burst. In Figure 5 the effect of US power, application time and pH on the DOX release is resumed. Figure 4: Effect of surface modification and polymer coating on the EE of DOX under different pH levels (**A**) Three-dimensional structure of DOX. (**B**) Evaluation of the effect of pH on the encapsulation efficiency (EE) at a 1 mg/mL drug concentration. (**C**) The relationship between DOX concentration and EE at pH 5.5. For encapsulation, DOX concentration is 1 mg/mL (volume of 5 mL), and the weight ratio between DOX and carrier is 1:10. Values are reported as averages, and error bars represent the SD (n = 3). The trend illustrated in Figure 5 shows how the exposure to ultrasound increases the release between 10 and 50% with a maximum release occurring at 20 W/cm\({}^{2}\) applied for 30 s.  
In Table 1, the amounts (in \(\upmu\)g) of DOX released from 10 mg of carrier after 110 min in the medium and exposure to US cycles in different media are reported. In the brackets, the percentage released refers to the total amount (in mg) loaded. \begin{table}
\begin{tabular}{c c c c} \hline \multicolumn{4}{c}{**Total DOX Released (ug)**} \\ \hline  & \multicolumn{3}{c}{2 W/cm\({}^{2}\)} \\ \hline pH & No US & US 10 s & US 30 s \\ \hline
3 & 1276 (29\%) & 1892 (43\%) & **3828 (87\%)** \\ \hline
5.5 & 1452 (33\%) & 2112 (48\%) & **4180 (95\%)** \\ \hline
7.4 & 1628 (37\%) & 2728 (62\%) & **4268 (97\%)** \\ \hline \multicolumn{4}{c}{20 W/cm\({}^{2}\)} \\ \hline pH & No US & US 10 s & US 30 s \\ \hline
3 & 1276 (29\%) & 2684 (61\%) & **4400 (100\%)** \\ \hline
5.5 & 1452 (33\%) & 2552 (58\%) & **4268 (97\%)** \\ \hline
7.4 & 1628 (37\%) & 2816 (64\%) & **4312 (98\%)** \\ \hline \end{tabular}
\end{table} 
Table 1: Total amount of DOX (\(\upmu\)g) released from 10 mg of the carrier in different media and US conditions. Data refer to the release trend illustrated in Figure 5. Bold represents the amount of DOX released which exceed 80% of the initial load. Figure 5: Evaluation of the effect of the US in terms of power and application time on the DOX release in different media. Values are reported as averages, and error bars represent the SD (n = 3) (**A**) release at pH 3 and US power 2 W/cm\({}^{2}\); (**B**) release at pH 5 and US power 2 W/cm\({}^{2}\); (**C**) release at pH 7.4 and US power 2 W/cm\({}^{2}\); (**D**) release at pH 3 and US power 20 W/cm\({}^{2}\); (**E**) release at pH 5 and US power 20 W/cm\({}^{2}\); (**F**) release at pH 7.4 and US power 20 W/cm\({}^{2}\). The data shown in Table 1 demonstrate how the pH affects the overall amount of drug released, with or without the application of ultrasound cycles. In the absence of US, the amount of DOX released increases when the pH increases from 3 to 7.4, reaching the maximum, 37%, after 2 h of exposure. The application of US drastically increases the percentage of drug released, reaching 100%.