## 2 Chitosan Modifications
### Formation of Nanoparticles  
It has been proven that CS derivatives are especially effective while in the form of NPs [68]. NPs are used as a method of integration and storage of active substances and consist of macromolecular and/or molecular structures in which active substances (drugs, enzymes, metal ions) are dissolved, stored, encapsulated or even adsorbed, or maintained at the outer interface [50]. The method for producing CS NPs was described for the first time by [PERSON] et al. in 1994 [69]. The processes used to produce CS NPs can be divided into several categories:  
1. Physicochemical, in which NPs are formed by emulsification following solvent evaporation, solvent displacement, diffusion or saltingout [70];
2. The in situ chemical synthesis of macromolecules, resulting in polymerization or interphase polycondensation;
3. Mechanical processes using high-energy devices, such as high-pressure homogenizers, ultrasonic devices or high-energy wet grinding. Methods of CS NP synthesis include ionotropic gelation, emulsification and cross-linking, complexation with PE, self-assembly and drying processes. Below are some peculiarities of these methods:  
1. Ionotropic gelation:  
Ionotropic gelation is one of the most widespread methods of CS-based NP synthesis. Since CS is a cationic polysaccharide, its interaction with polyanions such as tripolyphosphate (TPP) results in gelation (Figure 8) [71]. In an acidic medium, CS can spontaneously turn into submicron-sized particles. The TPP solution is added dropwise to the CS solution underconstant stirring until an opaque suspension is formed, which indicates the formation of NPs, the mean diameter of which is controlled by the CS/TPP molar ratio [72]. The mechanism of CS NP formation, the internal structure of resulting particles and the topology of their surface are determined by the following parameters: DD, molecular weight of CS, intrinsic viscosity, concentration and molar ratio NH\({}_{3}\)+/TPP [73]. At pH > 7, deprotonation of CS occurs, which reduces the crosslinking degree [74]. Moreover, pH of TPP solution controls the swelling behavior of the resulting gels: CS modified by ionotropic crosslinking (at a pH of about 3) with TPP revealed higher swelling ability [2; 74] in comparison with gels formed at alkaline pH values of the TPP solution. Other factors such as temperature and stirring rate may also affect CS NP size and polydispersity. After synthesis, smaller particles can be separated from larger ones by usingfiltration and ultracentrifugation [75]. The resulting CS NPs allow for capturing small hydrophobic and hydrophilic molecules, and also peptides, proteins and nucleic acids. Electrostatic interactions are considered to be the main mechanism for retaining biologically active compounds inside the CS NPs. In addition toTPP, other polyanions can be used for ion gelation, e.g., adenosine triphosphate, fucodian and sodium metabisulfite (Na\({}_{2}\)S\({}_{2}\)O\({}_{5}\)) [76]. Molecules containing catechol, such as hydrocaffe acid, have demonstrated the ability to increase the mucoadhesion of various polymers, including CS. Thus, hybrid microflowers were obtained from CS and calcium pyrophosphate, which were effectively used for the removal of dyes and in enzymatic catalysis [77]. (ii) Complexation with PEs:  
The mechanism of CS NP formation in PE is based on the ionic interactions between CS and negatively charged polymers (the same principle is used for hydrogel formation [78]). Stability of PE complexes is based on several factors including the degree of ionization of each PE; charge density; concentration and proportion of PEs in the mixture; sequence of PE mixing; nature; and location of charged groups. The stability of PE complexes also correlates with the molecular weight of PEs, flexibility of polymer chains, process temperature, ionic resistance and pH of the reaction medium [79]. When the excess charges of PEs (positive or negative) are consolidated, a nonstoichiometric complex is formed.