**1. Introduction**

Hydrogels are a three-dimensional hydrophilic polymer capable of absorbing large amounts of water [1]. Due to their unique properties, hydrogels are widely used in pharmaceutical, tissue engineering, biomedical, cosmetic, and drug delivery systems [2–6]. Natural polymers and synthetic polymers are used to manufacture hydrogels. Compared to synthetic-based polymers, natural polymers tend to be environmentally friendly, reproducible, and biocompatible [7]. Among them, natural polysaccharides derived from microorganisms are very ideal candidates for the fabrication of new hydrogels because they have biodegradability, versatility, and biocompatibility [8]. In many studies, bacterial polysaccharides such as chitosan [9], starch [10], xanthan gum [11], gellan gum [12], and alginate [7] have been reported as hydrogel components. Particularly, acidic bacterial polysaccharide-based hydrogels have a very wide application field because they can successfully perform drug delivery depending on the pH [13].

Succinoglycan is an acidic exopolysaccharide (EPS) derived from soil microorganisms *Sinorhizobium* and *Agrobacterium* [14,15]. It plays an important role in the development of

**Citation:** Shin, Y.; Kim, D.; Hu, Y.; Kim, Y.; Hong, I.K.; Kim, M.S.; Jung, S. pH-Responsive Succinoglycan-Carboxymethyl Cellulose Hydrogels with Highly Improved Mechanical Strength for Controlled Drug Delivery Systems. *Polymers* **2021**, *13*, 3197. https://doi.org/10.3390/ polym13183197

Academic Editor: Luis García-Fernández

Received: 31 August 2021 Accepted: 17 September 2021 Published: 21 September 2021

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the root nodule between bacteria and the Alfalfa legume [14]. Succinoglycan is a polysaccharide with repeating octasaccharides composed of seven glucose residues and one galactose residue. They have succinate, pyruvate, and acetate groups as non-carbohydrate substituents [16]. Due to the functional groups with a carboxyl group, it is easy to crosslink with metal cations such as Fe3+ and Cr3+ [17], and is very sensitive to pH [18]. In addition, succinoglycan can maintain the physical consistency of its physical properties even under extreme conditions such as high temperature, high shear rate, high salinity, or ionic concentration [19,20]. In particular, the high heat stability of succinoglycan showed mass stability of about 60%, even at 600 ◦C, as measured using thermogravimetric analysis (TGA) [17].

Carboxmethyl cellulose (CMC), a derivative of cellulose, is synthesized by the reaction between cellulose and chloroacetic acid [21]. Unlike cellulose, CMC is relatively soluble in water and can absorb large amounts of water. Due to these characteristics, CMC has numerous potentials as a superabsorbent hydrogel, such as controlled fertilizer and agrochemicals, drug delivery, wound dressing, and tissue engineering [22–24]. However, despite these advantages, CMC hydrogels are limited in their application due to the critical disadvantage of weak mechanical strength. Therefore, various multi-component CMC hydrogel systems have been reported for the purpose of improving mechanical strength [25]. For example, the addition of polyacrylamide, acrylic acid, and carboxymethyl β-cyclodextrin increased the mechanical strength of CMC-based hydrogels [26–28]. However, these synthetic-based polymers have disadvantages of low biodegradability, versatility, and biocompatibility compared to natural polysaccharides derived from microorganisms [8]. In addition, since they are not acidic polysaccharides, there is a limit to pH-dependent drug delivery. Therefore, CMC-based hydrogels using acidic polysaccharides have been reported, but there is a disadvantage of being in the form of beads or modified forms [29–32].

To our knowledge, there have been no reports of non-beaded CMC-based hydrogels capable of pH-responsive drug delivery using succinoglycan, an unmodified bacterial polysaccharide to significantly improve the mechanical strength of hydrogels while maintaining characteristic superabsorbency. We hypothesized that bacterial succinoglycan could increase the mechanical strength of CMC-based hydrogels because succinoglycan can maintain a stable consistency of physical properties even in extreme environments [7,17].

Here, succinoglycan (SG), an unmodified natural polysaccharide, was successfully used to increase the mechanical strength of the CMC hydrogel. Since polysaccharide-based IPN (interpenetrating polymer network) hydrogels provide excellent biocompatibility, mechanical strength, and excellent phase stability [33,34], an SG/CMC IPN hydrogel (SG/CMC gel) was fabricated by cross-linking the hydroxyl groups and carboxyl groups present in both SG and CMC through Fe3+ ions. These structures were characterized using Fourier Transform Infrared (FTIR) Spectroscopy, Thermogravimetric Analysis (TGA), Field Emission Scanning Electron Microscopy (FE-SEM), a rheology test, and a compressive test. We also investigated the pH-responsive drug release properties using 5-fluorouracil as a model drug.

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

### *2.1. Materials*

The bacterial strain (*Sinorhizobium meliloti* Rm1021) was supplied by the Microbial Carbohydrate Resource Bank (MCRB) at Konkuk University (Seoul, Korea). CMC (Mw = 250,000 g/mol with degree of substitution 0.7–based on manufacturer's data) was obtained from Sigma Aldrich (St. Louis, MO, USA). Iron (III) chloride hexahydrate (97.5%) was purchased from Daejung Chemicals & Metals Co., Ltd. (Siheung-si, Korea) and 5-fluorouracil (5-FU) purchased from Sigma Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade and used without further purification.

### *2.2. Growth Conditions and Production and Preparation of Succinoglycan*

The isolation and purification of succinoglycan from *S. meliloti* Rm1021 was performed as previously described [18]. Bacteria were cultured in medium comprised of d-mannitol (10 g/L), glutamic acid (1.5 g/L), K2HPO<sup>4</sup> (5 g/L), KH2PO<sup>4</sup> (5 g/L), MgSO4·7H2O (0.2 g/L), and CaCl2·2H2O (0.04 g/L), which was adjusted to a pH of 7.00 at 30 ◦C for 7 days with shaking (180 rpm). After, cells were centrifuged at 8000× *g* for 15 min at 4 ◦C and the supernatant was collected. To obtain succinoglycan, three volumes of ethanol were added to the supernatant. Furthermore, the precipitated succinoglycan was dissolved in distilled water and dialyzed (MWCO 12–14 kDa, distilled water for 3 days). After collection, succinoglycan purified via dialysis was lyophilized for later use. The molecular weights of succinoglycan were estimated via gel permeation chromatography (GPC) analysis. GPC was performed using a Waters Breeze System equipped with a Waters 1525 Binary pump and a Waters 2414 refractive index detector and was performed at 30 ◦C with a flow rate of 0.8 mL min−<sup>1</sup> using 0.02 N sodium nitrate as a solvent. The molecular weight (Mw) of succinoglycan, as estimated via GPC, is 1.8 × 105 Da.
