## 7 Guidelines on Developing Biopolymeric Microparticles
### Characterization of Microparticles Loaded with Chemical and Biological Agents
#### 7.3.3 Encapsulation Efficiency, Loading Capacity, and Swelling Degree  
The increase in cation concentration decreases the encapsulation efficiency, but loading with a biological agent (_T. viride_ spores) increases it. This increase is a result of the interaction of negatively charged _T. viride_ with positive ions like Cu\({}^{2+}\) or Ca\({}^{2+}\). Results for encapsulation efficiency are in correlation with the values of loading capacity [130; 131]. [PERSON] et al. [131], and this data can be related to the investigation of [PERSON] et al. [52; 133]. The decrease in encapsulation efficiency with higher cation concentration is because maximum loading capacity is reached, so lower concentrations are recommended when preparing microparticles for various applications. If higher values of cations are necessary, this can be achieved with the addition of biological agents. When observing the loading capacity, the presence of _T. viride_ spores decreases free cations, due to the binding, and thus, results may appear somewhat lower. [PERSON] et al. [150] also found that _T. viride_ may decrease the amount of Cu\({}^{2+}\) bound to organic matter by 1.1--1.2 fold. Furthermore, regarding the size of microparticles or chitosan presence, respectively, there was no significant effect on changes either on encapsulation efficiency or loading capacity. Some significant decrease in the loading capacity of chitosan-coated microparticles may be observed at lower cation concentrations. This can be explained by the fact that during the addition of the chitosan coat (second step of microparticles preparation), some of the cations from microspheres diffuse into the surrounding solution. Also, it must be noted that the mass of microparticles changes with an additional layer. Using higher cation concentrations during the preparation of microparticles solves this problem. Regarding the encapsulation efficiency of _T. viride_ spores, they are suspended in a viscous solution (sodium alginate) and are dripped in a cation-containing solution. During the formation of spheres, immediate diffusion happens for smaller molecules, but larger particles are beingimmobilized into the gel matrix. Thus, there were no observed _T. viride_ spores in the filtrate after the encapsulation procedure, revealing the \(\sim\)100% encapsulation efficiency [133]. The same was noticed in the investigation of [PERSON] et al. [123], where they reveal 100% encapsulation efficiency for _Trichoderma harzianum_ using the same ionic gelation method. Loading capacity may be used as a method to investigate the storage capabilities of microparticles. Publications of [PERSON] et al. [131,132] stimulated germination inside of microparticles during the storage period at room temperature. No significant changes in the number of spores were observed after 20 days of storage (based on our experiments even after six months), and this further confirmed that alginate-based microparticles provide an environment that is supportive to the _T. viride_ sporulation. This is in correlation with the literature data (Table 1) where, for example, an investigation of [PERSON] et al. [123] reveals preserved viability of encapsulated _Trichoderma_ spp. of above 70% even after two years of storage. The shelf life of a product is one of the most important factors which is considered when aiming for agricultural application. When discussing hydrophilic microparticles, one of the most important properties is a swelling degree [151]. When hydrophilic microparticles come in contact with water, they swell, influencing the rate-controlling release mechanism of encapsulated agents. Chitosan-coated microparticles have a higher swelling degree because of the hydrophilic nature of the formed polyelectrolyte complex [152,153,152]. The swelling degree of the complex is associated with electrostatic interactions between functional groups, and since both alginate and chitosan are weak polyelectrolytes, the degree of dissociation depends on the pH of the solution.