## 3 Chemical Modification of Hyaluronan
### Exterification of HA at the Hydroxyl
Similarly, for MA-HA, DS of 54.1 \(\pm\) 2.6% and 85.5 \(\pm\) 4.9% were achieved by using 15 and 20 molar excess of maleic anhydride. The reinforcing effect of inorganic fillers was demonstrated after mineralization. The compressive stress was two-fold for ME-HA compared to MA-HA, and the reinforcement was related to DS [101]. [PERSON] et al. prepared a derivative of maleic anhydride with HA (1.5 \(\times\) 10\({}^{6}\) g/mol) by one-step synthesis. The reaction takes place in an anhydrous DMSO for 24 h at 50 \({}^{\circ}\)C. At the end of the reaction, the pH was adjusted to 8-9 with a NaHCO solution (conversion of the carboxylic acid to its sodium salt). The product was precipitated with acetone, dissolved in water, and dialyzed. After dialysis, the precipitate is removed from the solution by centrifugation and lyophilized. The derivative was used for crosslinking with thiol-terminated poly(ethylene glycol) [102]. The reaction takes place in a suspension due to the insolubility of HA in DMSO. Disadvantages of this reaction for scaling up could be prolonged heating, wherein the molecular weight of HA decreased, and the use of an expensive solvent. The authors mentioned the potential use of the derivative for tissue engineering. [PERSON] and collaborators prepared physically and chemically crosslinked hydrogels, which the authors considered potential as wound dressings. The hydrogels were obtained by copolymerizing methacrylated HA (HA-MA) and carboxy betaine methacrylamide (CBMAA), which was prepared by reacting acrylic acid and N-(3-(dimethylamino)propyl) methyl acrylamide (DMAPMA). The physicochemical interaction of the positively charged CBMAA and negative HA reinforced the network. The chemical modification of HA (1.0 to 1.5 \(\times\) 10\({}^{5}\) g/mol) with glycidyl methacrylate was performed under basic catalysis. The interpenetration network was obtained under radical polymerization mediated by Irgacure 2959. The prepared hydrogels were not cytotoxic to L929 cells after 24 h of contact. The hemolysis ratio of tested compositions was less than 5% [103]. However, the use of acrylates is not adequate for human use. The reagent 1,1\({}^{\prime}\)-carbonyldiimidazole (CDI) can be used for the activation of carboxylic acids of several ligands; however, it reacts with water and is sensitive to degradation due to atmospheric moisture; thus, the reaction with HA occurs in solvents such as DMSO. This type of reaction can be used for crosslinking. For example, [PERSON] et al. studied the activation of ferulic acid (FA) with CDI. Two reactive intermediates, mono-imidazolide and bis-imidazolide, can be obtained [104]. This activation reaction can be used to attach fluorescent dyes to track the fate of HA during skin permeation. For example, [PERSON] et al. reported the HA esterification with activated cypate using CDI in the presence of imidazole as a base at 60 \({}^{\circ}\)C for 24 h in DMSO. The authors described that neither the use of a different base nor prolonged reaction time increased the degree of HA modification. The advantage of CDI to the activation of highly hydrophobic molecules is effective in reactive moieties that can be quenched by more reactive activators such as ethyl chloroformate or benzoyl chloride [105]. Though, these types of reactions are performed in DMSO, DMF, or formamide. Due to the insolubility of HA in organic solvents, HA has to be converted to tetrabutylammonium salt (TBA) or acid form (HA-H), so that it becomes soluble. Unfortunately, this conversion increases the risk of chain fragmentation. The use of organic solvents is also necessary for the covalent bonding of highly hydrophobic molecules. By using organic solvents, the process becomes highly expensive and less convenient for industrial applications. On the other hand, the activation of carboxylic moieties of biologically active molecules such as fatty acids [29], steroids [112], and vitamins [113] can be easily performed by mixed anhydrides methodology.