## 3 Chemical Modification of Hyaluronan
### Amidation of Hyaluronic Acid  
The first moiety for chemical modification is the carboxyl group, or the recognition marker for CD44 and RHAMM receptors [7]. Therefore, the chemical modification of the carboxyl may compromise the biological properties of the polysaccharide in vivo. Thus, the modification at the hydroxyl moieties is preferred. In particular, amidation is one of the most common reactions performed at the HA carboxylic moiety. The most common activator is 1-ethyl-3[-(dimethylaminoopropyl)] carbodiimide (EDC) used in combination with 1-hydroxybenzotriazole (HOBt) or N-hydroxysulfosuccinimide (NHS). The EDC coupling reaction is usually carried out in water, acidic buffers, or water mixed with an organic solvent. The reaction proceeds under mild conditions (low temperature, short reaction times) and is strongly pH dependent. Moreover, HOBt is explosive in its anhydrous form, which makes a possible scale-up difficult. The carbodiimide-mediated activation reaction includes the formation of a urea derivative. However, several side chemical reactions might occur. One of the advantages of EDC is the possibility to modify a broad range of HA molecular weights in water. In other words, without transforming HA to its salt (HA-TBA), a reaction is performed using cationic exchange reagents with consecutive degradation of the skeletal. However, if the ligand to be attached is highly hydrophobic, dimethyl sulfoxide (DMSO) is used in the reaction mixture. Furthermore, activation agents include the use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) in reactions performed in water [57]. The activation reaction mediated by diisopropylcarbodiimide (DIC)/ethyl cyanohydroximinoacetate (Oxyma) is performed in a mixture of DMSO/water due to the high hydrophobicity of functionalized cholesterol [8]. The activation of HA mediated via 2-bromo-1-ethyl pyridinium tetrafluoroborate (BEP) is carried out in DMSO [58]. Ethyl chloroform (ECF) is able to activate the carbonyl moiety of HA toward amidation reactions in DMSO as ECF reacts with water [59]. The reagent 1,1'-carbonyldimidazole (CDI) is used to modify HA-TBA salt in DMSO [60]. Figure 4 resumes several amidation reactions performed on HA. The amidation HA mediated by EDC/NHS has been performed by several groups, i.e., for attaching adipic acid dihydrazide (ADH) in deionized water with adjusted pH to 4.75 [61]. The preparation of HA-ADH was also performed in deionized water with adjusted pH (4.0-6.0) with 0.1 M diluted hydrochloric acid and used as the intermediate for the synthesis of amphiphilic derivatives [62], self-HA crosslinking [63], crosslinking with a second polymer [64; 65], or to attach a second substituent, i.e., streptomycin, for imparting antimicrobial properties to a formulation [66]. [PERSON] et al. reported the modulation of macrophages by a paeoniflorin (PF)-loaded self-crosslinked HA-ADH-HA hydrogel. The carboxylic moiety of HA was activated by EDC/NHS and reacted with ADH at pH 4.75. Three different HMW-HA sizes were tested (Mw\({}_{1}\) = 8.0 \(\times\) 10\({}^{5}\)-1.0 \(\times\) 10\({}^{6}\), Mw\({}_{2}\) = 1.3 \(\times\) 10\({}^{6}\)-1.5 \(\times\) 10\({}^{6}\), and Mw\({}_{3}\) = 1.8 \(\times\) 10\({}^{6}\)-2.2 \(\times\) 10\({}^{6}\) g/mol). The hydrogel preparedwith HA of the highest Mw presented the smallest pore size, the highest swelling, and an enhanced enzymatic resistance. HA-ADH-HA/PF of 8 wt.% gel was tested in vivo using a diabetic mice model. After 14 days, 90% of wound reduction was observed on the diabetic mice model. Interestingly, the authors evaluated as one of the controls the commercially available Intrasite gel (carboxymethylcellulose, propylene glycol). The in vitro studies showed decreased inflammation, angiogenesis, re-epithelialization, and deposition of newly formed collagen [63]. [PERSON] et al. performed the synthesis of a derivative prepared from HA (1 \(\times\) 10\({}^{6}\) g/mol) and ADH via activation mediated by EDC/HOBt in water mixed with DMSO.