## 7 Guidelines on Developing Biopolymeric Microparticles
### Characterization of Microparticles Loaded with Chemical and Biological Agents
#### 7.3.4. In Vitro Release of Chemical and Biological Agents  
One of the most important features of microparticles is the release of encapsulated agents. Their physicochemical characteristics determine the release behavior, and with regard to that, the preparation method is a crucial step. The main factors which determine the properties of microparticles are characteristics of biopolymer (sodium alginate), its concentration and type, and the concentration of crosslinking cation [161]. With this in mind, it is possible to tailor microparticles with desirable release capabilities, which are important when applying them in ecological agriculture. Some mechanisms need to be considered when observing the release of an active agent from biopolymer-based microparticles. These include (i) surface wetting, (ii) glassy-to-rubbery-phase transition of polymers, (iii) penetration of water molecules into the core of microparticles, (iv) diffusion of agents through the matrix and surface layers, (v) desorption from the surface of the microparticle, (vi) disintegration and (vii) dissolution and/or erosion of microparticle structure [52]. Kinetics and mechanisms of chemical and biological agents released from biopolymer-based microparticles (microspheres and microcapsules) mainly depend on the characteristics of microparticles composition and the type of active agent. The most important rate-controlling release mechanisms from hydrophilic microparticles are diffusion, erosion, and swelling [151]. For optimal development of microparticles, knowledge of mechanisms involved in the release of encapsulated material is crucial. The precise cation concentration used to prepare microparticles loaded with a biological agent is important for synergistic effect (i.e., positive effect on the growth and behavior). Respectively, cation release from microparticles shows rapid initial release followed by slower release obeying the power-law equation. From the results of [PERSON] et al. [130], the number of released cations depends on microparticle size and loaded agent. When microparticles are prepared under the same conditions, but with a variation in size, the thickness of the matrix will be different. With this in mind, it is possible to correlate microparticle release properties with microparticle surface-to-volume ratio [158]. As discussed above in the previous section, with the increase in microparticles diameter, the chitosan layer gets thinner, causing changes in the mechanical and permeability properties of particles. In general, the release of cations from larger microparticles is somewhat slower when comparing to the smaller microparticles. This may be attributed to the fact that cations in the core need more time to diffuse through the matrix and to the surface of the particle. Results of [PERSON] et al. [130] indicate that the release process of cations is controlled by the diffusion through the microparticle. When discussing microspheres loaded with _T. viride_ spores, the fraction of released Cu\({}^{2+}\) is diminished [130]. The fraction of released biological agents (_T. viride_ spores) was found to be higher than the chemical agent. Keeping in mind the interactions between the alginate gel matrix and _T. viride_ spores, as well as the physical entanglement of spores in the matrix, the release of spores should be slower. But, because of the supportive environment, _T. viride_ sporulates, and growth can be observed (released fraction is detected to be well above 1). An increase in the fraction of spores may be ascribed to the two facts, (i) the release from microparticles and germination of _T. viride_ and (ii) the formation of germ tube biomass in the surrounding medium. With regards to the Cu-alginate microspheres, the amount of released Cu\({}^{2+}\) from microparticles prepared with low Cu\({}^{2+}\) concentration was somewhat insufficient to stimulate germination. Generally speaking, chitosan-coated microparticles (microapsules) have a slower release rate of both cations and _T. viride_ spores [130]. Microcapsules without _T.