**1. Introduction**

Sodium alginate (SA), an irregular linear natural polysaccharide polymer derived from algae and bacteria, comprises 1,4-linked β-D-mannuronic acid (M Block) and 1,4-linked α-L-guluronic acid (G Block) with a homogeneous (poly-G, poly-M) or heterogeneous (MG) block composition [1,2], as illustrated in Figure 1. Owing to the advantages of good biocompatibility, low toxicity, non-immunogenicity, reproducibility, and plasticity, alginate is widely used in wound dressing, tissue engineering, pharmaceutical industries, food, and cosmetics [3–6]. However, as a result of the numerous carboxyl and hydroxyl groups on its molecular backbone, the hydrophilic alginate suffers from uncontrollable degradation and massive swelling properties, leading to its weak stability in biological buffers, which

**Citation:** Wang, H.; Chen, X.; Wen, Y.; Li, D.; Sun, X.; Liu, Z.; Yan, H.; Lin, Q. A Study on the Correlation between the Oxidation Degree of Oxidized Sodium Alginate on Its Degradability and Gelation. *Polymers* **2022**, *14*, 1679. https://doi.org/10.3390/ polym14091679

Academic Editors: Antonia Ressler and Inga Urlic

Received: 24 March 2022 Accepted: 13 April 2022 Published: 21 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

significantly restricts its practical application in the biomedical field [7,8]. In addition, SA with high-molecular-weight alginate hardly degrades in the body because of the lack of alginate-degrading enzymes [9,10]. The low-molecular-weight alginate with molecular weight lower than 50 kDa can be removed from the body through the kidney [11–13]. It is worth noting that the oxidation of alginate can strengthen its biodegradability and significantly reduce its molecular weight [14,15]. Simultaneously, the low molecular weight of oxidized sodium alginate (OSA) hydrogels is conducive to be used as the degradable hydrogel scaffolds for drug delivery system and tissue engineering for its functional groups (such as aldehyde groups) that can be quickly degraded in the body compared with natural alginate [14,16,17]. alginate hardly degrades in the body because of the lack of alginate-degrading enzymes [9,10]. The low-molecular-weight alginate with molecular weight lower than 50 kDa can be removed from the body through the kidney [11–13]. It is worth noting that the oxidation of alginate can strengthen its biodegradability and significantly reduce its molecular weight [14,15]. Simultaneously, the low molecular weight of oxidized sodium alginate (OSA) hydrogels is conducive to be used as the degradable hydrogel scaffolds for drug delivery system and tissue engineering for its functional groups (such as aldehyde groups) that can be quickly degraded in the body compared with natural alginate [14,16,17].

alginate suffers from uncontrollable degradation and massive swelling properties, leading to its weak stability in biological buffers, which significantly restricts its practical application in the biomedical field [7,8]. In addition, SA with high-molecular-weight

*Polymers* **2022**, *14*, x FOR PEER REVIEW 2 of 15

**Figure 1.** Molecular structure of sodium alginate. **Figure 1.** Molecular structure of sodium alginate.

In general, the oxidation of SA can be achieved via oxidizing agents including potassium permanganate, ozone, hydrogen peroxide, and periodate. Lu et al. [18] reported that SA could be oxidized by potassium permanganate under acidic conditions, and the degradation of SA increased with the increase in the amount of potassium permanganate and the decrease in pH value of the solution. Wu et al. [19] illustrated that the free radicals produced by ozone self-decomposition could cause the degradation of SA, thereby obtaining the low molecular weight of OSA. Mao et al. [20] studied the oxidation of SA using hydrogen peroxide, which could achieve the desired molecular weight of alginate by adjusting the reaction temperature, hydrogen peroxide concentration, initial concentration of SA, and pH value in the system. To note, it can be observed that there are two adjacent -OH groups at the C-2 and C-3 positions on the repetitive unit of the alginate chain, which could be specifically oxidized by the periodate to generate the aldehyde groups [21,22]. Among the above oxidation methods, using sodium periodate (NaIO4) to oxidize SA is regarded as the most commonly used method to endow alginate with active functional groups and easy degradation in drug controlled delivery [23]. The oxidation of alginate with NaIO4 to form OSA involved C2–C3 bond breakage, thus transforming the uronic acid into an open chain adduct containing aldehyde groups. Subsequently, the generated aldehyde groups will react spontaneously with hydroxyl groups present on the adjacent uronic acid in the alginate chain to form a In general, the oxidation of SA can be achieved via oxidizing agents including potassium permanganate, ozone, hydrogen peroxide, and periodate. Lu et al. [18] reported that SA could be oxidized by potassium permanganate under acidic conditions, and the degradation of SA increased with the increase in the amount of potassium permanganate and the decrease in pH value of the solution. Wu et al. [19] illustrated that the free radicals produced by ozone self-decomposition could cause the degradation of SA, thereby obtaining the low molecular weight of OSA. Mao et al. [20] studied the oxidation of SA using hydrogen peroxide, which could achieve the desired molecular weight of alginate by adjusting the reaction temperature, hydrogen peroxide concentration, initial concentration of SA, and pH value in the system. To note, it can be observed that there are two adjacent -OH groups at the C-2 and C-3 positions on the repetitive unit of the alginate chain, which could be specifically oxidized by the periodate to generate the aldehyde groups [21,22]. Among the above oxidation methods, using sodium periodate (NaIO4) to oxidize SA is regarded as the most commonly used method to endow alginate with active functional groups and easy degradation in drug controlled delivery [23]. The oxidation of alginate with NaIO<sup>4</sup> to form OSA involved C2–C3 bond breakage, thus transforming the uronic acid into an open chain adduct containing aldehyde groups. Subsequently, the generated aldehyde groups will react spontaneously with hydroxyl groups present on the adjacent uronic acid in the alginate chain to form a cyclic hemiacetal [24], as shown in Figure 2. Moreover, the carboxyl groups of alginate can be completely retained, and the oxidation degree (OD) of alginate can be also controlled by adjusting the concentration of NaIO<sup>4</sup> during the oxidation process [25].

cyclic hemiacetal [24], as shown in Figure 2. Moreover, the carboxyl groups of alginate can be completely retained, and the oxidation degree (OD) of alginate can be also controlled

by adjusting the concentration of NaIO4 during the oxidation process [25].

**Figure 2.** Schematic presentation of (**a**) the oxidation reaction of sodium alginate by NaIO4, (**b**) the formation of OSA, and (**c**) the formation of hemiacetal. **Figure 2.** Schematic presentation of (**a**) the oxidation reaction of sodium alginate by NaIO<sup>4</sup> , (**b**) the formation of OSA, and (**c**) the formation of hemiacetal.

Oxidation degree (OD) is the basic parameter of OSA, which significantly affects the physical and chemical properties of OSA-based materials such as hydrogels [26], microspheres [27], and electrospinning materials [28]. The increase in OD may make OSAbased materials possess a low crosslinking degree and network density. Additionally, OD is also related to the gelling properties of alginate. Generally, alginate and its derivatives exhibit gelling ability with divalent cations [29–32] (such as Ca2+, Ba2+, Mg2+, Sr2+, etc.). However, the strength of the ionic crosslink between the OSA polymer chain and the divalent ion are weakened with the destruction of the cooperative interaction during oxidation process [23]. Fortunately, alginate with a lower OD can still form the hydrogel [25]. Therefore, it is generally necessary to select an OSA with the suitable OD before the actual application. Although some works about oxidation reactions with NaIO4 on the hydroxyl groups of alginate uronic units have been reported [23–25], the systematic characterization of the resultant OSA by multiple testing methods and the correlation between the OD on the degradability and gelation of OSA have rarely been studied at present. A large number of studies have shown a great reduction in the molecular weight of SA after being oxidized by NaIO4, resulting in the formation of aldehyde groups with higher reactivity [24,27,28]. The resultant OSA not only retained the water solubility, low, and biocompatibility of alginate, but also exhibited good biodegradability and better molecular flexibility. Therefore, research on the correlation between the OD of OSA on its Oxidation degree (OD) is the basic parameter of OSA, which significantly affects the physical and chemical properties of OSA-based materials such as hydrogels [26], microspheres [27], and electrospinning materials [28]. The increase in OD may make OSA-based materials possess a low crosslinking degree and network density. Additionally, OD is also related to the gelling properties of alginate. Generally, alginate and its derivatives exhibit gelling ability with divalent cations [29–32] (such as Ca2+, Ba2+, Mg2+, Sr2+, etc.). However, the strength of the ionic crosslink between the OSA polymer chain and the divalent ion are weakened with the destruction of the cooperative interaction during oxidation process [23]. Fortunately, alginate with a lower OD can still form the hydrogel [25]. Therefore, it is generally necessary to select an OSA with the suitable OD before the actual application. Although some works about oxidation reactions with NaIO<sup>4</sup> on the hydroxyl groups of alginate uronic units have been reported [23–25], the systematic characterization of the resultant OSA by multiple testing methods and the correlation between the OD on the degradability and gelation of OSA have rarely been studied at present. A large number of studies have shown a great reduction in the molecular weight of SA after being oxidized by NaIO4, resulting in the formation of aldehyde groups with higher reactivity [24,27,28]. The resultant OSA not only retained the water solubility, low, and biocompatibility of alginate, but also exhibited good biodegradability and better molecular flexibility. Therefore, research on the correlation between the OD of OSA on its biodegradability and gelation is beneficial to exploring the application potential of OSA-based hydrogels in the biomedical field.

biodegradability and gelation is beneficial to exploring the application potential of OSAbased hydrogels in the biomedical field. Herein, to broaden the applicability of alginate, we prepared OSA with various theoretical ODs using NaIO4 as the oxidant. The structure and physicochemical properties of OSA was evaluated by Fourier transform infrared spectroscopy (FT-IR), 1H nuclear magnetic resonance (1H NMR), X-ray Photoelectron Spectroscopy (XPS), X-ray Diffraction (XRD), and thermogravimetric analysis (TGA). Meanwhile, gel permeation chromatography (GPC) and rheometer (DHR) were used to determine the correlation Herein, to broaden the applicability of alginate, we prepared OSA with various theoretical ODs using NaIO<sup>4</sup> as the oxidant. The structure and physicochemical properties of OSA was evaluated by Fourier transform infrared spectroscopy (FT-IR), <sup>1</sup>H nuclear magnetic resonance (1H NMR), X-ray Photoelectron Spectroscopy (XPS), X-ray Diffraction (XRD), and thermogravimetric analysis (TGA). Meanwhile, gel permeation chromatography (GPC) and rheometer (DHR) were used to determine the correlation between the ODs of oxidized sodium alginate on its biodegradability and gelation. Additionally, the cytotoxicity of the formed OSA hydrogel against the MC3T3-E1 cells was also evaluated.

between the ODs of oxidized sodium alginate on its biodegradability and gelation. Additionally, the cytotoxicity of the formed OSA hydrogel against the MC3T3-E1 cells

was also evaluated.

### **2. Materials and Methods**
