## 3 Results and Discussion
### Influence of the CaCl\({}_{2}\) in the 5-ASA Loading  
In order to increase the drug encapsulation into the XBM and improve its hardness, the addition of CaCl\({}_{2}\) during the microparticles production was used as a strategy to promote a faster crosslinking within the xylan chains. This approach could prevent the diffusion of the 5-ASA within the continuous phase, while hardening the xylan droplet. Simultaneously, the covalent chemical crosslinking between STMP and xylan would happen during the incubation period. In addition, the CaCl\({}_{2}\) might perform a role as a crosslinker, on the sites that would not be occupied by the STMP, which may increase the number of interaction points along the polymeric chains (Figure 4). Consequently, the crosslinking and compactness of the produced microparticles would also increase, which can be observed by a reduction on the average size on the formulations containing CaCl\({}_{2}\) (XBM-Ca-F1 to XBM-Ca-F9) compared to the ones without CaCl\({}_{2}\) (XBM) (Table 1) [23]. Similarly, based on XBM, the optical microscopy of the formulation XBM-Ca-F9 (Figure 3B) shows not only a spherical shape and absence of aggregates, but also a uniform distribution particle size (Figure 3D). In addition, the CaCl\({}_{2}\) promoted a reduction of approximately 50% on the average diameter of the formulation, from 10.70 (for XBM) to 4.77 \(\upmu\)m (for XBM-Ca-F9). As can be seen in the formulations XBM-Ca-F4 and XBM-Ca-F6 (Table 1), the increase of CaCl\({}_{2}\) to 2%(w/v) promoted a decrease in the average particle size. The factorial design analysis showed that only the main effect of CaCl\({}_{2}\) and the xylan-STMP interaction were statistically significant to increase the 5-ASA encapsulation into XBM (\(p\)-value \(<\) 0.05). As shown in Figure 5, the increment of the CaCl\({}_{2}\) improved the encapsulation of 5-ASA into the microparticles. This phenomenon was related to the faster crosslinking process of the Ca\({}^{2+}\), mainly on the surface of the droplet, when compared to the STMP alone. The Ca\({}^{2+}\) could be able to keep the drug entrapped while the network between xylan an STMP was formed [24]. On the other hand, the interaction between xylan and STMP has a negative impact on the 5-ASA encapsulation. It is likely that the amount of STMP used for the crosslinking reactions was insufficient to tightly link the xylan chains. Figure 3: Optical microscopy (**A**,**B**) and particle size distribution (**C**,**D**) of XBM (**A**,**C**, respectively) and XBM-Ca-F9 (**B**,**D**, respectively), at a magnification of 10\(\upmu\)m with their respective Gaussian distribution (bold line, **C**,**D**). Previous experiments showed that the ideal condition to produce XBM was the use of 1%(w/v) of STMP. At high polymer concentrations, the formation of microparticles was hindered (data not shown). Figure 4: Scheme of the crosslinking reaction between xylan (Xyl), CaCl\({}_{2}\), and STMP. Reaction (1) represents only the chemical bond between STMP-Xyl. Reaction (2) represents the process of chemical crosslinking between Ca\({}^{2+}\)-Xyl-STMP, respectively, and Reaction (3) represents the ionic interaction between Ca\({}^{2+}\)-Xyl. Figure 5: Response surface plot. Correlation of the encapsulation of 5-ASA with xylan-STMP and CaCl\({}_{2}\) concentrations into the formulation. The STMP and xylan concentration were maintained (x-axis), while the concentration of CaCl\({}_{2}\) was increased. In (**A**) it was used 0.5%(w/v) of CaCl\({}_{2}\), in (**B**) 1.25%(w/v) and in (C) 2%(w/v). Additionally, the encapsulation of 5-ASA was enhanced at the lower level of xylan and at the higher level of STMP (Figure 5A-C). The particles formed within these concentrations likely have a smaller pore size due to the high crosslinking degree, which, consequently, improves the encapsulation of 5-ASA [25]. From the mathematical modeling of the factorial design experimental data, it was possible to retrieve a predictive model with an adjusted-R\({}^{2}\) = 0.4661 and a predictive-R\({}^{2}\) = 0.1253.