# Multiresponsive Hybrid Microparticles for Stimuli-Responsive Delivery of Bioactive Compounds
## 3 Results and Discussions
### Microparticles Characterization  
Figure 1 shows the effects of surface modification, the conjugation with the amphiphilic PLACA-g-CS and the media pH on the average diameter and the \(\zeta\)-potential of the microparticles in solution. In the preparation medium (phosphate buffer pH 5.5), the introduction of the organic layer shifts the microparticle size from 800 nm to 1 \(\upmu\)m, while it increases 2-fold, reaching the dimension of 2 \(\upmu\)m, after coating. Surface modification and coating strongly affect the \(\zeta\)-potential value. IO particles have \(\zeta\)-potential close to zero, as no ionizable groups are displaced on the surface. Following the modification with 4-carboxybenzenediamineonium tosylate, the presence of carboxylic groups in the ionic state (pH 5.5 and 7.4) cause a drop in the \(\zeta\)-potential, which assumes a negative value. Further conjugation with the amphiphilic chitosan increases the \(\zeta\)-potential to positive values (pH 2 and 5.5), ensuring stability in the solution. At pH 7.4, the \(\zeta\)-potential drops to a negative value due to the deprotonation of the amino groups. The influence of the surface modification and the further conjugation with the PLACA-g-CS polymer on the magnetic properties of the microparticles was investigated by vibrating-sample magnetometer, using IO as control [12]. The saturation magnetization value (M\({}_{8}\)) was 78 emu/g in IO,  
Figure 1: The effects of surface modification (**A**) Influence of the surface modification and polymeric shell on the average diameter and \(\zeta\)-potential of the microparticles in different media. (**B**) Magnetization curve. Iron oxide (IO) microparticles have been used as a control to evaluate the effect of surface modification (ZV) and the presence of the polymeric shell (core–shell). (**C**,**D**) Microparticle stability in terms of variation of average diameter versus time (**C**) in preparation medium pH 5.5 and (**D**) at physiological condition (pH 7.4). Values are reported as averages, and the error bars represent the SD (n = 3). and no significant variation in M\({}_{\rm s}\) was detected after the introduction of the organic layer. Conversely, addition of the polymer shell caused a 20% reduction in the magnetic response (Figure 1B). As previously described [27; 29], an Ms value of 16.3 emu/g is necessary to separate IO microparticles in aqueous solution by employing an external magnetic field [30]. With this supposition, the adopted set-up can be considered optimal for preserving the magnetic response. Figure 2 shows TEM micrographs of IO, ZV and core-shell microparticles. As can be seen in Figure 2A,B, IO and ZV microparticles tend to aggregate in a string-like structure, which means low stability in solution [31; 32; 33]. The TEM micrograph reported in Figure 2B shows that not enough carboxylic groups are present on the surface of the microparticles in order to stabilize them and avoid the aggregation phenomena. Conversely, after conjugation with PLACA-g-CS (Figure 2C), no string-like structure is observed; this is due to an increase in the microparticles' stability in solution, which can be ascribed to electrostatic repulsion between the positively charged microparticles. The presence of the PLACA-g-CS has been proved by ATR-FTIR spectroscopy, using ZV microparticles as control to demonstrate the formation of the new amide bond. Figure 3 shows the FTIR-ATR spectra relative to ZV and core-shell microparticles. In the spectra of ZV microparticles (Figure 3, Track A), the peaks at 2974, 2900 and 1689 cm\({}^{-1}\) are ascribed to the presence of carboxylic groups, while those at 1603 and 1585 cm\({}^{-1}\) to the benzene ring [26; 27]. In the spectra referring to the core-shell particles (Figure 3, Track B), the peaks at 3000 and 2950 cm\({}^{-1}\) are related to carboxylic groups and methyl groups (-CH\({}_{3}\)) belonging to the PLACA structure.