## 7 Guidelines on Developing Biopolymeric Microparticles
### Recommended Guidelines to Prepare Microparticles Loaded with Chemical and Biological Agents
The first stage involves the dropwise addition of sodium alginate either with or without biological agent into the bath containing cations (Cu\({}^{2+}\), Ca\({}^{2+}\), or a mixture of various chemical agents, e.g., Ca\({}^{2+}\)/Mg\({}^{2+}\), since reaction with Mg\({}^{2+}\), does not occur instantly as with the above-mentioned cations. It is recommended to combine Mg\({}^{2+}\) with other divalent cations that react fast and form a strong gel, like Cu\({}^{2+}\), Ca\({}^{2+}\), or Zn\({}^{2+}\). Since donor solutions always contain anions, e.g., CuSO\({}_{4}\)x5H\({}_{2}\)O or CaCl\({}_{2}\), the inclusion of the anion into the microparticles also occurs. During the encapsulation process, anions diffuse through the microparticle into the solution, and thus, the encapsulation efficiency for anions is significantly lesser when comparing to the cations involved in the gelling process, which binds irreversibly. After the dropwise addition of sodium alginate into the bath containing divalent cations, gelling occurs, and spheres are formed. Throughout the preparation process, the dropwise addition of carrier solution (sodium alginate) can be achieved using a microdroplet generator (Figure 2). Concerning the size of the nozzle, used for the dropwise addition, the pressure, frequency of vibration (amplitude), and temperature should be considered. Higher temperatures decrease the viscosity of sodium alginate solutions, and smaller spheres may be formed via the usage of nozzles with a small diameter (e.g., 80 \(\upmu\)m). When using the bigger nozzle (1000 \(\upmu\)m), lower frequency is necessary to separate the droplets in dropwise flow, while the opposite is valid for smaller nozzles. Of course, a smaller nozzle means the need for higher pressure to eject already highly viscosity solution like sodium alginate. Figure 5 shows a schematic representation of the preparation procedure. As can be seen, in the first stage, a simple method can be used for the preparation of microspheres (gel matrix), while for the second stage of preparation, coating of microspheres, i.e., microcapsules, with chitosan, can be observed. Via the polyelectrolyte complexation, microspheres are dispersed in the acidic chitosan solution, where chitosan rapidly binds onto the surface of alginate-based microspheres by electrostatic interactions between protonated amino groups on chitosan and ionized carboxylic groups on alginate-based microspheres [144]. The main goal of the second step is to reduce the porosity of the gel matrix, improve its stability and efficiency by delaying the release of encapsulated agents. It is well documented in the literature that electrostatic interaction between chitosan and alginate tightens and stabilizes the surface of the microparticle [1; 145]. When chitosan binds to the microsphere, competing ions like H\({}^{+}\) or Na\({}^{+}\) have an insignificant influence on the stability of the polyelectrolyte complex [146], and chitosan diffusion to the inner part of microparticles is very limited [147]. This can be observed in a research paper of [PERSON] et al. [133] and [PERSON] et al. [132] where a distinct chitosan layer was formed on the surface of the microspheres, i.e., microcapsule.