*3.2. Swelling Kinetics of WPI Hydrogels*

The swelling characteristics play an important role in the absorption of body fluids and the transfer of nutrients and cellular metabolites. One of the main strategies for releasing captured drugs is controlled hydrogel swelling. It is known that an osmotic pressure is also defined as the measure of the tendency of a solution to take in pure solvent by osmosis. Under an action of a solvent diffusion and hydrogel network osmotic pressure, an increase of the pore size is observed that results in mixing between the solvent and the WPI segments and, as a consequence, swelling of hydrogels [35]. The swelling degree of hydrogels depends on the stretching of the polymer chains, which exert a pressure inside the hydrogel through their elasticity.

A swelling test was performed for WPI hydrogels containing different amounts of TAs and a control hydrogel without TAs in PBS solution (pH 7) for six repetitions within 48 h. The swelling degree of hydrogels depends on the hydrogel composition and the surrounding aqueous medium, as well as the degree of protein–protein, protein–water or protein–polyphenol interactions. [7] The increase of the mass increasing (MI) was observed for all hydrogels at the first 1 h of the swelling experiment (Figure 2). It indicates that all hydrogels absorbed and retained a certain amount of water in their structure. According to two-way analysis of variance (ANOVA), the swelling data of WPI-TA hydrogels are statistically significantly different (*p* < 0.05) between hydrogels with different TA/WPI ratio compared to the control hydrogels without TA. (Table S1).

**Figure 2.** Mass increase (MI) of WPI hydrogels with TAs (ALSOK 02 (**left**), ALSOK 04 (**right**)) in PBS (pH 7). WPI hydrogel (control); TA/WPI ratios are 0.0375/0.075/0.15/0.30. *p* < 0.05 compared with the control groups (within each pH-dependence swelling group).

As shown in Figure 2, the presence of TAs which are bound to WPI proteins by noncovalent electrostatic interaction in the hydrogel structure significantly reduces its swelling ability. The inability of TAs to absorb water reasons for the decrease in the MI of hydrogels thereby preventing swelling. Thus, a high polyphenol content in hydrogels can inhibit the penetration of various proteins and, therefore, it is believed that bioactive drugs will be protected from premature degradation due to the hindrance of enzyme diffusion into pores in the hydrogels. Also, the correlation between the swelling ratio of the hydrogel and the TA concentration will allow hindrance of drug diffusion into the body and, as a consequence, slow the kinetics of drug release [36]. The highest MI was observed for hydrogels with the lowest TAs/WPI ratio—0.0375.

In general, for hydrogels containing TAs with a high content of hydroxyl groups (ALSOK 02), the MI is higher than for hydrogels with the same concentration of polygalloylquinic acids (ALSOK 04). This is primarily due to the chemical structure of the added compounds. Addition of greater numbers of the hydroxyl groups to the hydrogel network allows an increase in the number of formed intermolecular H-bonds. As a rule, such bonds are labile and are easily stretched and broken by exposure to external stimuli. The osmotic pressure generated during the swelling process can be responsible for such spatial changes in the hydrogel networks.

An increase of the TA concentration in the WPI hydrogels reduces and limits the mobility of the hydrogel network, which leads to resistance to diffusion and water uptake. [37] So the smallest MI is observed for the hydrogels containing the maximum amount of ALSOK 02 and ALSOK 04. For hydrogels with a maximum TA content (TA/WPI ratio 0.30), complete swelling by water is observed 24 h after the incubation start. After 48 h, the MI decrease is observed (Figure 2) due to the subsequent reduction in the hydrogel mass, provoked, probably, by TA release from the hydrogel networks.

#### *3.3. pH-Dependent Swelling Behaviors and TA Release from WPI Biohydrogels*

We also focused on studying the pH dependence of hydrogel swelling. The prepared hydrogel compositions were immersed in acidic (pH 5, Figure 3 above) and basic (pH 9, Figure 3 below) phosphate buffered saline (PBS), incubated for 48 h at room temperature.

**Figure 3.** Mass increase (MI) of WPI hydrogels with TAs (ALSOK 02 (**left, up**) ALSOK 04 (**right, up**)) in PBS (pH 5). Mass increase (MI) of WPI hydrogels with TAs (ALSOK 02 (**left, down**) ALSOK 04 (**right, down**)) in PBS (pH 9). *p* < 0.05 compared with the control groups (within each pH-dependence swelling group).

For 48 h after a storage, the solutions became more opaque in the basic state (pH 9), but transparent at acidic medium (pH 5). This is due to TA hydrolysis and the subsequent oxidation by decarboxylation of the hydrolysis products in the presence of base. Usually, hydrogels formed from amphoteric polyelectrolytes (for example, WPI) have a small MI at a pH equal to their isoelectric point (pI of native ß-lactoglobulin is 5.1) [38]. The presence of a high TAs content affects the diffusion of ions, reducing the elasticity of the hydrogel network. Such a low ability of hydrogels to take up water is associated with less interaction or absence of WPI hydrophilic sites with water due to the formation of numerous bonds between the protein and TAs. Due to this, the formation of denser and more rigid structures occurs, which leads to a decrease in the flexibility of protein chains. In PBS solutions, the swelling capacity of hydrogels is lower compared to the values in distilled water. This can be explained by the uneven distribution of ions in the hydrogel network and solution. This causes a decrease in the equilibrium water absorption of the hydrogel and a swelling decrease over time.

It is interesting to note the behavior of hydrogels in the basic medium (Figure 4 left, down; right, down). The MI value for hydrogels at pH 9 is higher than at pH 2 during the first hour of the experiment. So the higher the pH, the more surface charges, the higher the electrostatic repulsive force, and higher MI value [30,39]. For the control WPI sample that does not contain TAs, the MI value continues to grow throughout the duration of the experiment. However, the presence of TA in the hydrogel results in lower MI values. According to two-way analysis of variance (ANOVA), statistically significant differences (*p* < 0.05) in the swelling data of hydrogels are observed between hydrogels with different TA/WPI ratio compared to the control hydrogels without TA (Tables S2 and S3). A decrease of MI values is observed with increasing TA concentration in the hydrogels. Due to the hydrolysis of TAs under basic conditions and partial deprotonization, the destruction of intermolecular H-bonds is possible and, as a consequence, the release of TA hydrolysis products from hydrogels with subsequent weight loss. We do not exclude the possibility

that WPI material may be diffusing out of the hydrogels too. Future work will investigate the possible simultaneous release of hydrogel material.

**Figure 4.** Histograms of the released TA amount (ALSOK 02 (**left**) ALSOK 04 (**right**)) from the WPI hydrogel at 48 h after incubation. Error bars show standard errors.

Targeted drug release from hydrogels in combination with a controlled release rate is a desirable property of pH-sensitive hydrogels. To confirm its hypothesis, the TA release from hydrogels was studied at different pH. Figure 4 shows the TA release profiles from hydrogels 48 h after their incubation in PBS solution at pH 5, 7, and 9, respectively.

According to Figure 4, TA release was the smallest when the samples were immersed in a neutral medium (pH 7). It is believed that the strongest ionic interaction between polyphenols and protein occurs in the solution at pH was close to the isoelectric point of native whey proteins (pI 5.1) [40], which leads to the formation of a denser hydrogels.

The highest TAs release 48 h after incubation is observed for hydrogels in the basic medium (pH 9), which is consistent with the swelling test data. An increase in pH will lead to deprotonation of WPI and TAs. As a result, a large TA release percentage is observed, which is associated with a violation of intermolecular H-bonds [41]. For hydrogels containing a small TA weight (TAs/WPI ratio—0.0375) the TA release percentage reaches high values, up to 80%. However, for WPI hydrogels with the highest TA content (TAs/WPI ratio—0.30), only 40% of the TA initially present is released from the hydrogel network. It leads to the formation of a denser hydrogel. We do not exclude the possibility that WPI material may be diffusing out of the hydrogels. Our future work will investigate the possibility of simultaneous release of hydrogel material. This aspect is important for the development of hydrogel scaffold with controlled release of drugs and nutrients, as well as the case of wound healing, absorption of wound exudates.

In an acidic medium (pH 5), a high TAs release value is observed, which is also associated with protein dissociation and protonation. This may be a positive sign for effective cancer therapy, since the local and endosomal pH is significantly lower than that of normal tissue [42].

Thus, the pH-dependent drug release from hydrogels allows hydrogels to be used locally, as anticancer scaffolds for the treatment or palliative treatment of serious gastrointestinal malignancies where pH values range from acidic (in the stomach) to basic (in the intestine).

#### *3.4. Anticancer Activity of WPI Hydrogels Containing TA*

Cytotoxicity of WPI hydrogels was estimated on the laryngeal cancer cell line (Hep 2) using the Alamar Blue assay, which measures the metabolic activity of cells.

The cultivation of Hep 2 cells during 48 h in the presence of WPI hydrogel discs without and with the addition of TAs (ALSOK 02, ALSOK 04) showed that samples without TAs exerted an inconsiderable cytotoxic effect on the cell line whereas hydrogels contained TAs caused a significant inhibition of metabolic processes (Figure 5).

**Figure 5.** Results of cytotoxicity tests of WPI hydrogels without and with addition of TAs (ALSOK 02 and ALSOK 04) on the Hep 2 cell line. The Hep 2 cells were cultured in the presence of WPI hydrogels containing TAs (blue—ALSOK 02, green—ALSOK 04), hydrogels without TAs (brown). Cell culture without adding WPI hydrogels (black) was the control throughout the experiment. *p* < 0.05 compared with the control groups without adding TA.

Hydrogels with TA/WPI ratio 0.0375 produced a similar effect in comparison to pure hydrogel samples. The increase of TAs concentration led to more significant cytotoxic effects, correspondingly. Samples with maximum TA/WPI ratio 0.3 after 24 h incubation exhibited to 50% inhibition of metabolic processes whereas after 48 h this value increased to 80%. Previously, the ability of polyphenol derivatives to induce apoptosis and cell cycle termination was shown for cancer cell lines in vitro [43,44]. However, the cytotoxic effect of hydrogels with ALSOK 02 was higher than for the sample containing ALSOK 04 (Figure 5). Significant differences in cell viability between WPI hydrogels with different TA/WPI ratio were observed (*p* < 0.05) compared with the control groups without adding TA for each one of TA types (Table S4). In previous work on mineralized gellan gum hydrogels containing ALSOK 02 and ALSOK 04, greater cytotoxicity towards osteosarcoma-derived Saos-2 cells was observed after2h[22]. Thus, the use of WPI hydrogels containing TAs at 3 mg per mL (TA/WPI ratio 0.075) concentration is the most promising for provision of a prolonged anti-cancer effect.
