pH-Sensitive Dairy-Derived Hydrogels with a Prolonged Drug Release Profile for Cancer Treatment

A novel versatile biocompatible hydrogel of whey protein isolate (WPI) and two types of tannic acid (TAs) was prepared by crosslinking of WPI with TAs in a one-step method at high temperature for 30 min. WPI is one common protein-based preparation which is used for hydrogel formation. The obtained WPI-TA hydrogels were in disc form and retained their integrity after sterilization by autoclaving. Two TA preparations of differing molecular weight and chemical structure were compared, namely a polygalloyl glucose-rich extract-ALSOK 02-and a polygalloyl quinic acid-rich extract-ALSOK 04. Hydrogel formation was observed for WPI solutions containing both preparations. The swelling characteristics of hydrogels were investigated at room temperature at different pH values, namely 5, 7, and 9. The swelling ability of hydrogels was independent of the chemical structure of the added TAs. A trend of decrease of mass increase (MI) in hydrogels was observed with an increase in the TA/WPI ratio compared to the control WPI hydrogel without TA. This dependence (a MI decrease-TA/WPI ratio) was observed for hydrogels with different types of TA both in neutral and acidic conditions (pH 5.7). Under alkaline conditions (pH 9), negative values of swelling were observed for all hydrogels with a high content of TAs and were accompanied by a significant release of TAs from the hydrogel network. Our studies have shown that the release of TA from hydrogels containing ALSOK04 is higher than from hydrogels containing ALSOK 02. Moreover, the addition of TAs, which display a strong anti-cancer effect, increases the cytotoxicity of WPI-TAs hydrogels against the Hep-2 human laryngeal squamous carcinoma (Hep-2 cells) cell line. Thus, WPI-TA hydrogels with prolonged drug release properties and cytotoxicity effect can be used as anti-cancer scaffolds.

ing both preparations. The swelling characteristics of hydrogels were investigated at room temper-23 ature at different pH values, namely 5, 7 and 9. The swelling ability of hydrogels was independent 24 of the chemical structure of the added TAs. A trend of decrease of mass increase (MI) in hydrogels 25 was observed with an increase in the TA / WPI ratio compared to the control WPI hydrogel without 26 TA. This dependence (a MI decrease -TA / WPI ratio) was observed for hydrogels with different 27 types of TA both in neutral and acidic conditions (pH 5.7). Under alkaline conditions (pH 9), nega-28 tive values of swelling were observed for all hydrogels with a high content of TAs and were accom-29 panied by a significant release of TAs from the hydrogel network. Our studies have shown that the 30 release of ТА from hydrogels containing ALSOK04 is higher than from hydrogels containing AL-31 SOK 02. Moreover, the addition of TAs, which display a strong anti-cancer effect, increases the cy-32 totoxicity of WPI-TAs hydrogels against the Hep-2 human laryngeal squamous carcinoma (Hep-2

Introduction 38
Recently, much attention has been paid to hydrogels in drug delivery. In this regard, 39 hydrogels must comply with principles such as biocompatibility, biodegradation and 40 non-toxicity. One common protein-based preparation used for hydrogel formation in the 41 food industry is whey protein isolate (WPI), which we have recently begun to investigate 42 as a hydrogel biomaterial for biomedical applications. [1][2][3][4] The major component of WPI 43 is ß-lactoglobulin (approximate composition 74.1%) and the second major component is 44 α-lactalbumin (23.0%). [5] Whey proteins have been identified to have desirable proper-45 ties because they consist of branched-chain amino acids which promote highly hydrated 46 three-dimensional polymer networks in hydrogels. [6] Gelation occurs by increasing the 47 temperature due to denaturation of native ß-lactoglobulin protein. [7] The process of 48 whey protein aggregation consists of three stages, including conformational changes of 49 the native protein structure, chemical reactions typically through disulphide bridges be-50 tween intra-and interchain bonds and physical interactions like hydrophobic interactions, 51 which leads to aggregation clustering and the formation of a spatial gel network. [8] The 52 increased comparison of ß-lactoglobulin allows to fabricate more elastic WPI hydrogels 53 with far superior mechanical properties compered to hydrogels based on whey protein 54 concentrate. The important functional property of a WPI hydrogels is its high ability to 55 retain water or body fluids within its structure. Also the WPI denaturing permits exposed 56 hydrophobic regions of the protein molecule, to which the hydrophobic regions of hydro-57 phobic drugs can bind, resulting in increased drug solubility. Cytocompatible hydrogels 58 have been successfully used to develop drug delivery systems due to their stimulus-sen-59 sitive response to external triggers, such as pH. [9] Hence, it would be desirable to com-60 bine the ability of WPI hydrogels to solubilize and carry hydrophobic drugs with pH re-61 sponsiveness.
62 One class of hydrophobic molecules with biological activity are tannic acids (TAs). 63 TAs are polyphenols closely related to our daily life: they are found in many fruits and 64 vegetables consumed by humans and are used in the food industry and herbal medicine. 65 Hydrolyzable tannins are one of three types of TAs that are formed by a carbohydrate 66 (glucose, quinic acid or other), in which OH-groups are partially or completely esterified 67 with gallic acid or related compounds. [10][11][12] In this context hydrolyzable means that 68 ester hydrolysis can occur, as opposed to acid-base hydrolysis (deprotonation). Hydro-69 lyzable tannins can be extracted from various vegetable plants and trees. As a rule, TAs 70 are considered non-toxic in small doses [13,14] and exhibit antitumor effects. [15] The 71 presence of TA in natural components can reduce tumor necrosis factor levels [16] and 72 weaken the inflammatory cytokine expression. [17] Previously, it was shown that TA 73 crosslinked into a compacting collagen gel predominantly inhibited proliferation of high-74 melanoma A375 cells with metastatic potential. [18] In addition, ternary composite nano-75 fibers containing tannic acid can be used as wound dressings in the case of recessive dys-76 trophic epidermolysis bullosa, which often leads to the development of an aggressive 77 form of squamous cell carcinoma. [19] TA has been shown to help crosslinking of gelatin 78 and pectin derivatives due to the presence of a large number of hydroxyl groups in the 79 polyphenol structure due to intermolecular H-bond formation, in which the polyphenols 80 act as electron pair donors. [20] From the physicochemical point of view, polyphenols sta-81 bilize the secondary structure of proteins, increase their thermal stability and significantly 82 reduce their biodegradability. [21] Recently, a comparative analysis was carried out of the 83 ability of gellan gum hydrogels enhanced with polyphenols (including the ones investi-84 gated in our research, ALSOK 02 and ALSOK 04), to enzymatic mineralization and the 85 hydroxyapatite formation. [22] TA inclusion inhibited the growth of human osteoblast-86 like Saos-2 cells on substrates of mineralized gellan gum hydrogel biomaterials with cal-87 cium phosphate and did not confer antibacterial activity against E.Coli. 88 In this study, we combined the beneficial properties of TAs and WPI to create new 89 pH-sensitive cytocompatible hydrogels which display an anticancer affect. Two TAs of 90 differing molecular weight and chemical structure (polygalloyl glucoses -ALSOK 02 and 91 polygalloyl quinic acids -ALSOK 04) were compared using swelling tests at different pH 92 values. We hypothesized that the addition of TAs would reduce the swelling of WPI hy-93 drogels due to the aforementioned interactions between polyphenols and proteins. To our 94 best knowledge, this combination of components has not yet been tested for biomaterial-95 related applications. We focused on the dependence of the swelling ability of hydrogels 96 on pH of the medium, chemical structure and concentration of TAs, which allowed a more 97 prolonged release of TAs over several days.  5,7,9). The desired basic and acidic pH values were obtained by pH 129 adjustment using NaOH and HCl solutions, respectively. To measure the swelling, after 130 autoclaving, samples of the excised hydrogel discs (diameter 3 mm) were dried at 80 °C 131 for 1 hour, then a dried sample with known weight was placed in 24-well plates and in-132 cubated in a solution (1: 10). The swelling process took place at room temperature for up 133 to 48 hours. Swollen gels were periodically (1, 24 and 48 hours) removed, blotted on dry 134 filter paper to remove excess water and immediately weighed. Then, the mass increase 135 (MI) was calculated as: 136 137 where Mt is the weight of the hydrogel at a certain time, Mo is the initial hydrogel 138 weight. All experiments were carried out with n = 6.. 139

Fourier transform infrared (FTIR) spectroscopy
140 The chemical structure of the synthesized WPI hydrogels was investigated by using 141 Fourier Transform Infrared spectroscopy using a Fourier-Transform Infrared (FTIR) spec-142 trophotometer (Agilent Technology, UK) in Attenuated Total Reflectance (ATR) mode.
143 Spectra were collected in the 500 -4000 cm -1 spectral range with a resolution of 4 cm -1 and 144 an average of 8 scans. 145

In vitro release studies
146 The TA release from WPI hydrogels was measured using a spectrophotometer 147 (Multi-Mode Reader Synergy H1) at 48 hours after incubation. A dried hydrogel sample 148 was weighed accurately and then incubated in PBS at room temperature for up to 48 149 hours. At the indicated time, a few drops of 0.5 N iron(III) chloride were added to the 150 selected aliquot, and the optical density of the solutions was measured at 586 nm ( Fig. S1, 151 S2). [24] The tests were conducted on six independent replicates. 152

Cell viability test
153 Cells were seeded in 96-well plates at the density described in the individual experi-154 ments. The following day, the excised hydrogel discs (diameter 3 mm) were added to trip-155 licate wells. Fresh medium was added to each of 96 wells. Subsequently, the cells were 156 incubated (Innova CO-170, New Brunswick Scientific) at 37 °C for 48 hours, together with 157 the added materials. In the last step, 10 μL of AlamarBlue dye was added to each well and 158 the intensity was measured using a spectrophotometer (Multi-Mode Reader Synergy H1).
159 The experiment showed the capability of metabolically active cells to convert the Alamar-160 Blue reagent into a fluorescent and colorimetric indicator. 166 The data on the kinetics of swelling of hydrogels loaded with TA incubated in PBS at 167 different pH values were plotted as "mean ± standard error" (n = 6). The viability of Hep2 168 cells incubated for 24 and 48 hours with hydrogels containing different TA/WPI ratio was 169 presented as "mean ± standard error" (n = 4). Differences between treatments were ana-170 lyzed using two-way analysis of variance (ANOVA). [26] Calculations were carried out 171 using Microsoft Excel software. Values of P ≤ 0.05 were considered significant (Tables S1-172  S4). 173

Preparation and characterization of WPI hydrogels containing TAs.
175 WPI is a promising cross-linking component for the preparation of hydrogels con-176 taining various biologically active compounds. Previously, hydrogels based on various 177 WPI concentrations were synthesized and their properties were studied. [6] Two types of 178 TAs (polygalloyl glucoses -ALSOK 02, polygalloyl quinic acids -ALSOK 04) were used 179 for the fabrication of the WPI hydrogels. The main differences in these preparations are-180 varying amounts of hydroxyl groups and chemical structure. Based on the literature data, 181 TA concentrations in WPI hydrogels were selected and hydrogels with differing TAs con-182 tents were synthesized 1.5; 3.0; 6.0 and 12.0 mg per mL, which corresponds to the TA / 183 WPI ratios were 0.0375 / 0.075 / 0.15 / 0.30 in the hydrogels. [27,28] Hydrogels were ob-184 tained by heating the solution to 90 °C for 30 minutes. Such a short exposure to high tem-185 peratures does not lead to pathological changes in the TA structure. [29] 186 The gelification process of WPI-TAs solutions was carried out at pH 7 in deionized 187 water. It is assumed that the incorporation of an additional small TAs amount into the 188 WPI hydrogel structure (maximum TA / WPI ratio of 0.30) does not affect the hydrogel pI, 189 since WPI is the prevailing constituent of hydrogels. According to previously published 190 studies [30] the pI of hydrogels obtained at a pH above the native protein pI (pI 5.2) shifts 191 to a more acidic range (pI 4.1) due to the electrostatic repulsion of negatively charged 192 groups of glutamic and aspartic acids and corresponding deprotonation of lysine amino 193 acid residues.
194 To understand the functional properties of WPI-TA hydrogels, it is necessary to de-195 termine their structure and identify the binding nature of the protein and polyphenols.
196 FTIR measurements are a sensitive tool for detecting conformational changes in the sec-197 ondary structure of a protein. [31] In the present study FTIR-spectra of WPI-TAs hydro-198 gels were measured from a solid dried condition to exclude pronounced stretching vibra-199 tions of water molecules in the 3673-2942 cm -1 range and a deformation band of water in 200 the 1644 cm -1 region. Figure 1 shows the FTIR spectra of unmodified WPI hydrogel and 201 hydrogels with various TA concentrations. In the spectrum of the unmodified WPI hy-202 drogel (burgundy line), we observed three strong bands at 3208, 1673 and 1545 cm -1 , which 203 correspond to vibrations for amide A, amide I and amide II, respectively. [32] In the vi-204 brational spectrum region of Amide I, stretching vibrations of the COOof the Asn and 205 Gln side residues and NH3 + deformation vibrations of amino acids containing additional 206 NH2-groups in the side chain (Asp, Glu, Lys and Arg) are manifested. This overlap of the 207 amino acid residues absorption bands with the Amide I absorption band makes it very 208 sensitive to the intermolecular H-bonds manifestation. A signal change of the Amide I 209 absorption band makes it possible to determine the conformational protein change.
210 FTIR spectra of hydrogels with different TA contents showed similar bands to that 211 of the WPI hydrogel control spectrum. It indicates that new covalent bonds were not cre-212 ated. A similar result was reported by Ferraro, et al. (2015), who studied the nature of the 213 interaction between rosmarinic acid (natural polyphenol) and milk whey proteins 214 through non-covalent bonds in detail. [33] 215 226 The spectral lines of hydrogels with TAs revealed broadening of the vibrational sig-227 nal at 3208 cm -1 , which indicates the formation of intermolecular H-bonds (Fig. 1c, d). For 228 hydrogels containing polygalloyl glucose (ALSOK 02) the broadening of the symmetric 229 vibration signal of -NH and -OH groups into Amide A is more pronounced than for hy-230 drogels with the same content of the polygalloyl quinine acid (ALSOK 04). H-bonds are 231 the main binding force of WPI and hydrophilic substances. [34] Vibrational signals of Am-232 ide I and Amide II are considered the basis of the WPI signal and confirm the presence of 233 whey proteins. A change in the secondary structure of the protein is usually explained by 234 broadening of Amide I and a shift of Amide II. When more ALSOK 02 is added into hy-235 drogels, the peaks of Amide I bending vibrations are shifted by 7 cm -1 (from 1545 cm -1 to 236 1538 cm -1 ) towards a lower wavenumber (Fig. 1c). This indicates a change in the nature of 237 the side amino group vibrations of Asp, Glu, Lys and Arg due to the formation of inter-238 molecular H-bonds with the polyphenols. The same phenomenon occurred for Amide II; 239 the maximum shift was observed from 1673 cm -1 to 1657 cm -1 for a hydrogel with ALSOK 240 02 / WPI ratio 0.30 (Fig. 1b, d). For hydrogels containing ALSOK 04, the shifts of stretching 241 vibrations of Amide I and Amide II groups were more significant than for hydrogels with 242 ALSOK 02, perhaps due to the contribution of closely spaced signals of stretching vibra-243 tions of carboxyl groups and stretching of the C=C aromatic bonds of uncrosslinked AL-244 SOK 04. The maximum shift was up to 25 cm -1 and was observed also for hydrogels with 245 ALSOK 04 / WPI ratio 0.30 (Fig. 1d). The shift of Amide I and Amide II indicates the pres-246 ence of an electrostatic interaction between WPI and TA, and not chemical reactions. [31] 247 For the WPI-ALSOK 02 complex, the formation of intermolecular H-bonds is more char-248 acteristic than for the WPI-ALSOK 04, which directly depends on the chemical structure 249 of TAs and their ability to ionize in water. Thus, a hydrolysable polygalloyl glucose (AL-250 SOK 02) with a large number of hydroxyl groups interacts better with protein than 251 polygalloyl quinic acid (ALSOK 04). Thus, in all cases, non-specific binding between pol-252 yphenols and WPI is confirmed, without additional covalent bond formation during the 253 hydrogels' preparation. 254 255 3.2. Swelling kinetics of WPI hydrogels 256 The swelling characteristics play an important role in the absorption of body fluids 257 and the transfer of nutrients and cellular metabolites. One of the main strategies for re-258 leasing captured drugs is controlled hydrogel swelling. It is known that an osmotic pres-259 sure is also defined as the measure of the tendency of a solution to take in pure solvent by 260 osmosis. Under an action of a solvent diffusion and hydrogel network osmotic pressure, 261 an increase of the pore size is observed that results in mixing between the solvent and the 262 WPI segments and, as a consequence, swelling of hydrogels. [35] The swelling degree of 263 hydrogels depends on the stretching of the polymer chains, which exert a pressure inside 264 the hydrogel through their elasticity. 265 A swelling test was performed for WPI hydrogels containing different amounts of 266 TAs and a control hydrogel without TAs in PBS solution (pH 7) for six repetitions within 267 48 hours. The swelling degree of hydrogels depends on the hydrogel composition and the 268 surrounding aqueous medium, as well as the degree of protein-protein, protein-water or 269 protein-polyphenol interactions. [7] The increase of the mass increasing (MI) was ob-270 served for all hydrogels at the first 1 hour of the swelling experiment ( Figure 2). It indi-271 cates that all hydrogels absorbed and retained a certain amount of water in their structure. 272 According to two-way analysis of variance (ANOVA), the swelling data of WPI-TA hy-273 drogels are statistically significantly different (P <0.05) between hydrogels with different 274 TA / WPI ratio compared to the control hydrogels without TA. (Table S1) 280 281 As shown in fig. 2, the presence of TAs which are bound to WPI proteins by non-282 covalent electrostatic interaction in the hydrogel structure significantly reduces its swell-283 ing ability. The inability of TAs to absorb water reasons for the decrease in the MI of hy-284 drogels thereby preventing swelling. Thus, a high polyphenol content in hydrogels can 285 inhibit the penetration of various proteins and, therefore, it is believed that bioactive 286 drugs will be protected from premature degradation due to the hindrance of enzyme dif-287 fusion into pores in the hydrogels. Also, the correlation between the swelling ratio of the 288 hydrogel and the TA concentration will allow hindrance of drug diffusion into the body 289 and, as a consequence, slow the kinetics of drug release. [36] The highest MI was observed 290 for hydrogels with the lowest TAs / WPI ratio -0.0375. 291 In general, for hydrogels containing TAs with a high content of hydroxyl groups (AL-292 SOK 02), the MI is higher than for hydrogels with the same concentration of polygal-293 loylquinic acids (ALSOK 04). This is primarily due to the chemical structure of the added 294 compounds. Addition of greater numbers of the hydroxyl groups to the hydrogel network 295 allows an increase in the number of formed intermolecular H-bonds. As a rule, such bonds 296 are labile and are easily stretched and broken by exposure to external stimuli. The osmotic 297 pressure generated during the swelling process can be responsible for such spatial 298 changes in the hydrogel networks. 299 An increase of the TA concentration in the WPI hydrogels reduces and limits the mo-300 bility of the hydrogel network, which leads to resistance to diffusion and water uptake. 301 [37] So the smallest MI is observed for the hydrogels containing the maximum amount of 302 ALSOK 02 and ALSOK 04. For hydrogels with a maximum TA content (TA / WPI ratio 303 0.30), complete swelling by water is observed 24 hours after the incubation start. After 48 304 hours, the MI decrease is observed (Fig. 2) due to the subsequent reduction in the hydrogel 305 mass, provoked, probably, by TA release from the hydrogel networks. 306 307 3.3. pH-dependent swelling behaviors and TA release from WPI biohydrogels. 308 We also focused on studying the pH dependence of hydrogel swelling. The prepared 309 hydrogel compositions were immersed in acidic (pH 5, Fig. 3  316 317 318 For 48 hours after a storage, the solutions became more opaque in the basic state (pH 319 9), but transparent at acidic medium (pH 5). This is due to TA hydrolysis and the subse-320 quent oxidation by decarboxylation of the hydrolysis products in the presence of base. 321 Usually, hydrogels formed from amphoteric polyelectrolytes (for example, WPI) have a 322 small MI at a pH equal to their isoelectric point (pI of native ß-lactoglobulin is 5.1). [38] 323 The presence of a high TAs content affects the diffusion of ions, reducing the elasticity of 324 the hydrogel network. Such a low ability of hydrogels to take up water is associated with 325 less interaction or absence of WPI hydrophilic sites with water due to the formation of 326 numerous bonds between the protein and TAs. Due to this, the formation of denser and 327 more rigid structures occurs, which leads to a decrease in the flexibility of protein chains. 328 In PBS solutions, the swelling capacity of hydrogels is lower compared to the values in 329 distilled water. This can be explained by the uneven distribution of ions in the hydrogel 330 network and solution. This causes a decrease in the equilibrium water absorption of the 331 hydrogel and a swelling decrease over time.
332 It is interesting to note the behavior of hydrogels in the basic medium (Fig. 4 left, 333 down; right, down). The MI value for hydrogels at pH 9 is higher than at pH 2 during the 334 first hour of the experiment. So the higher the pH, the more surface charges, the higher 335 the electrostatic repulsive force, and higher MI value. [30,39] For the control WPI sample 336 that does not contain TAs, the MI value continues to grow throughout the duration of the 337 experiment. However, the presence of TA in the hydrogel results in lower MI values. Ac-338 cording to two-way analysis of variance (ANOVA), statistically significant differences (P 339 <0.05) in the swelling data of hydrogels are observed between hydrogels with different 340 TA / WPI ratio compered to the control hydrogels without TA. (Tables S2, S3). A decrease 341 of MI values is observed with increasing TA concentration in the hydrogels. Due to the 342 hydrolysis of TAs under basic conditions and partial deprotonization, the destruction of 343 intermolecular H-bonds is possible and, as a consequence, the release of TA hydrolysis 344 products from hydrogels with subsequent weight loss. We do not exclude the possibility 345 that WPI material may be diffusing out of the hydrogels too. Future work will investigate 346 the possible simultaneous release of hydrogel material. 347 Targeted drug release from hydrogels in combination with a controlled release rate 348 is a desirable property of pH-sensitive hydrogels. To confirm its hypothesis, the TA re-349 lease from hydrogels was studied at different pH. Figure 4  356 357 According to Figure 4, TA release was the smallest when the samples were immersed 358 in a neutral medium (pH 7). It is believed that the strongest ionic interaction between 359 polyphenols and protein occurs in the solution at pH was close to the isoelectric point of 360 native whey proteins (pI 5.1) [40], which leads to the formation of a denser hydrogels. 361 The highest TAs release 48 hours after incubation is observed for hydrogels in the 362 basic medium (pH 9), which is consistent with the swelling test data. An increase in pH 363 will lead to deprotonation of WPI and TAs. As a result, a large TA release percentage is 364 observed, which is associated with a violation of intermolecular H-bonds. [41] For hydro-365 gels containing a small TA weight (TAs / WPI ratio -0.0375) the TA release percentage 366 reaches high values, up to 80%. However, for WPI hydrogels with the highest TA content 367 (TAs / WPI ratio -0.30), only 40% of the TA initially present is released from the hydrogel 368 network. It leads to the formation of a denser hydrogel. We do not exclude the possibility 369 that WPI material may be diffusing out of the hydrogels. Our future work will investigate 370 the possibility of simultaneous release of hydrogel material. This aspect is important for 371 the development of hydrogel scaffold with controlled release of drugs and nutrients, as 372 well as the case of wound healing, absorption of wound exudates. 373 In an acidic medium (pH 5), a high TAs release value is observed, which is also asso-374 ciated with protein dissociation and protonation. This may be a positive sign for effective 375 cancer therapy, since the local and endosomal pH is significantly lower than that of nor-376 mal tissue. [42] 377 Thus, the pH-dependent drug release from hydrogels allows hydrogels to be used 378 locally, as anticancer scaffolds for the treatment or palliative treatment of serious gastro-379 intestinal malignancies where pH values range from acidic (in the stomach) to basic (in 380 the intestine). 394 395 396 Hydrogels with TA/WPI ratio 0.0375 produced a similar effect in comparison to pure 397 hydrogel samples. The increase of TAs concentration led to more significant cytotoxic ef-398 fects, correspondingly. Samples with maximum TA/WPI ratio 0.3 after 24 hours' incuba-399 tion exhibited to 50% inhibition of metabolic processes whereas after 48 hours this value 400 increased to 80%. Previously, the ability of polyphenol derivatives to induce apoptosis 401 and cell cycle termination was shown for cancer cell lines in vitro. [43,44] However, the 402 cytotoxic effect of hydrogels with ALSOK 02 was higher than for the sample containing 403 ALSOK 04 ( Figure 5). Significant differences in cell viability between WPI hydrogels with 404 different TA/WPI ratio were observed (P< 0.05) compared with the control groups without 405 adding TA for each one of TA types (Table S4). In previous work on mineralized gellan 406 gum hydrogels containing ALSOK 02 and ALSOK 04, greater cytotoxicity towards osteo-407 sarcoma-derived Saos-2 cells was observed after 2 h. [22] Thus, the use of WPI hydrogels 408 containing TAs at 3 mg per mL (TA/WPI ratio 0.075) concentration is the most promising 409 for provision of a prolonged anti-cancer effect. 410

Conclusions and Outlook 411
WPI hydrogels containing two types of TA have been produced, which withstand 412 autoclaving. The greatest influence on the swelling change is exerted by the amount of 413 TAs contained in the WPI hydrogels. An increase of the TA / WPI ratio in the hydrogels 414 to 0.30 (for ALSOK 02 and ALSOK 04 both) leads to a significant decrease in MI compared 415 with the control hydrogel without TA in neutral conditions (pH 7). The pH lowering leads 416 to a MI decrease and an increase in the amount of released TAs by 1.5-2 times compared 417 with incubation at neutral pH (pH 7) for all WPI hydrogels with and without TAs. The 418 maximum TAs release was observed for hydrogels with the TA / WPI ratio 0.0375 (for 419 ALSOK 02 and ALSOK 04 both) in alkaline pH (pH 9) and amounted to almost 80% 48 420 hours after the incubation start. According to the swelling data, at this time point, the 421 hydrogels begin to destruct, since their MI have negative values at 48 hours. Future work 422 will investigate the possible simultaneous release of hydrogel material. Also, measure-423 ments of the pH and zeta potential of the hydrogel dependence on pH gelification will be 424 investigated in our future work. All obtained hydrogels containing TAs have cytotoxic