# Alginate Modification and Lectin-Conjugation Approach to Synthesize the Mucoadhesive Matrix
## 4 Alginate Modification and Coupling Strategies
### Hydroxyl Groups
#### 4.2.4 Oxidation and Reduction  
The oxidation of alginate has been significantly studied in the field of soft tissue engineering. Several oxidation agents have been used to carry out the reaction, but sodium meta periodic (NaIO\({}_{4}\)) is the reagent most frequently used to serve this purpose. The oxidation can be performed in pure water or ethanol/water dispersion in a subduing environment. The ring-opening leads to the formation of di-aldehyde, the so-called alginate dialdehyde (ADA). A decrease of molecular weight and gelation ability is expected, depending on the degree of oxidation [67; 90]. ATR-FTIR spectroscopy is used to characterize the newly formed aldehyde, although the band is weak, in some cases, due to the formation of hemicatals of the free aldehyde group [48]. The proton at 90 \({}^{\circ}\)C or carbon NMR spectroscopy is another option for confirming the new group formation [11].  
The newly formed aldehyde groups are characterized mainly by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy. A new characteristic band of aldehyde is observed in the range of 1725-1751 cm\({}^{-1}\)[48; 90]. This band is weak and, in some cases, not even detected. The phenomenon is related to the hemiacetal formation of a high concentration of aldehyde groups in oxidized urinates [47; 48]. The presence of aldehyde can also be investigated with \({}^{13}\)C NMR, and the aldehyde signal clearly appears at 90-95 ppm when the degree of oxidation is 19 mol\(\%\) or above [47; 91].  
The degree of oxidation is defined as a percentage of oxidized uronic acid residues in the ADA. Such a degree can be determined by UV-Vis spectrophotometry or potentiometric titration [92; 93]. The degree of oxidation controls the physicochemical properties of the ADA and resultant hydrogels. Because the oxidation reaction significantly affects the galvanonic acid residues, only ADA with a maximum 10\(\%\) degree of oxidation can form hydrogels with calcium ions [47].  
Without further modification, alginate dialdehyde has been used to synthesize hydrogels with gelatin in the presence of borax [55], while [PERSON] et al. studied the physical properties of these hydrogels by controlling the concentration of alginate solution and oxidation condition [90]. With the addition of 2-hydroxyethyl methacrylate (HEMA) and the presence of hydroxyapatite (HAp), a porous alginate hydrogel is synthesized [94]. The hydrogel is synthesized with the cryogelation method, where the additional compounds enhance the mechanical properties and biocompatibility.  
Figure 6: Structural resemble between sulfated alginate with heparin.  
ADA-GEL (gelatin) hydrogel is also reported as bioink, where the gelatinization is obtained by crosslinking with divalent ion [95]. The ADA with a 30% degree of oxidation is utilized to produce stable ADA-GEL scaffolds, where barium is present in the cross-linker solution. Nevertheless, ADA-GEL stiffness can be tuned by controlling the concentration of ADA, type of crosslinker, and the ratio of divalent ions in the crosslinker. The general procedure of periodate oxidation followed by reductive amination on the MGM fragment of alginate is depicted in Figure 7.  
The advantage of oxidation alginate derivatives is their highly reactive alginate dilidehyde moieties compared to the original sodium alginate. This now available active site of alginate derivative brings an opportunity for further modification to achieve the required characteristics for specific applications. The reductive amination is most explored as the following procedure to modify the alginate dialdehyde. Sodium cyanoborohydride (NaBH\({}_{3}\)CN), among other hydrides, is the reagent most employed for reductive amination to avoid reducing the unreacted aldehyde. Other reagents, such as picoline borane complex (pic-BH\({}_{3}\)) or \(\alpha\)-picoline borane, are also reported as mild and efficient reductive agents [96; 97].