# Synthesis of Hydrogel-Based Microgels and Nanogels Toward Therapeutic and Biomedical Applications
## 2 Hydrogel Materials
### Natural Hydrogels  
#### 2.2.1 Chitosan  
Chitosan-based hydrogels are among the most extensively studied natural hydrogel material, benefiting from their excellent biocompatibility, degradability, and unique physicochemical characteristics (Table 2) [17]. Chitosan is naturally derived from chitin, a polysaccharide abundantly found in the exoskeletons of shellfish like shrimps or crabs, or the cell walls of fungi. Its chemical structure includes repeating units of glucosamine and N-acetylglucosamine, which provide chemical moieties for additional modifications and crosslinking. Three major functional groups are a primary amino group at C2, and a primary and secondary hydroxyl group at C6 and C3, respectively [17; 68; 69]. The pH-dependent behavior of chitosan is governed by the amino groups which become protonated in acidic pH, enhancing solubility that subsequently allows controlled gelation. Additionally, chitosan exhibits strong antimicrobial and antifungal activities. These unique phenomena make chitosan hydrogel highly suitable for controlled drug delivery, wound healing and infection control, and tissue regeneration [53; 59; 69; 70]. Currently, chitosan hydrogels are found in emergency kits for uncontrolled bleeding, owing to their abilities to agglutinate red blood cells, activate platelets, and maintain a moist environment in the vicinity of the hydrogel. These hemostatic properties make chitosan hydrogel ideal for wound care, where timely gelation, stable adhesion, and strong antimicrobial activity are all critically important to the patient's survival [40; 66]. The chemical structure of chitosan molecules contains carboxyl and amino groups, which enable facile chemical modifications to implant functionalities via the click chemistry or EDC/NHS method [71; 72]. The water solubility of chitosan molecules varies by the degree of deacetylation (DD), where an acetyl group contributes to inducing the crystallization of chitosan molecules and dramatically decreases their solubility to water and other common solvents. The DD of chitosan is also hypothesized to be a major factor determining the antimicrobial activity of chitosan hydrogels, as the lower density of the acetyl group allows a higher level of positive net charge from the protonation of amine groups, subsequently strengthening its antibacterial activity [73; 74; 75]. Chitosan is partially water-soluble when its DD level at above 70% and becomes highly soluble at a DD above 95%. The DD level also influences the antimicrobial activity of chitosan hydrogels, as a higher degree increases the net positive charge, thereby enhancing the antibacterial effects. However, conventional techniques to chemically tune chitosan molecules are often complex and time-consuming. Moreover, intravenous applications of chitosan have been reported to induce and accelerate immune responses, emphasizing the importance of key considerations in developing new drug delivery systems [76]. The chitosan hydrogels could be formed via both physical and chemical methods. Physical crosslinking relies on ionic interactions and hydrogen bonding between functional groups, or hydrophobic interactions at a larger scale, and their non-covalent association enables a reversible and self-healing constructed gel network. On the other hand, chemical crosslinking results in the formation of stable, covalently crosslinked gel networks, by utilizing additional components like glutaraldehyde, carbodiimide, or genipin [17; 59; 68; 77]. Applications of chitosan-based hydrogels, as highlighted above, span a wide range of both academic and industrial fields. Besides the hemostatic performance, chitosan hydrogels are employed in tissue engineering as scaffolds, benefiting from the similarity of their physical and chemical properties to those of the extracellular matrix, which enables cell proliferation and differentiation.