**1. Introduction**

Recently, much attention has been paid to hydrogels in drug delivery. In this regard, hydrogels must comply with principles such as biocompatibility, biodegradation, and non-toxicity. One common protein-based preparation used for hydrogel formation in the food industry is whey protein isolate (WPI), which we have recently begun to investigate as a hydrogel biomaterial for biomedical applications [1–4]. The major component of WPI is ß-lactoglobulin (approximate composition 74.1%) and the second major component is α-lactalbumin (23.0%) [5]. Whey proteins have been identified to have desirable properties because they consist of branched-chain amino acids which promote highly hydrated three-dimensional polymer networks in hydrogels [6]. Gelation occurs by increasing the temperature due to denaturation of native ß-lactoglobulin protein [7]. The process of whey protein aggregation consists of three stages, including conformational changes of the native

**Citation:** Mayorova, O.A.; Jolly, B.C.N.; Verkhovskii, R.A.; Plastun, V.O.; Sindeeva, O.A.; Douglas, T.E.L. pH-Sensitive Dairy-Derived Hydrogels with a Prolonged Drug Release Profile for Cancer Treatment. *Materials* **2021**, *14*, 749. https:// doi.org/10.3390/ma14040749

Academic Editor: Franz E. Weber Received: 16 December 2020 Accepted: 29 January 2021 Published: 5 February 2021

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protein structure, chemical reactions typically through disulfide bridges between intraand interchain bonds and physical interactions like hydrophobic interactions, which leads to aggregation clustering and the formation of a spatial gel network [8]. The increased comparison of ß-lactoglobulin allows to fabricate more elastic WPI hydrogels with far superior mechanical properties compered to hydrogels based on whey protein concentrate. The important functional property of a WPI hydrogels is its high ability to retain water or body fluids within its structure. The WPI denaturing permits exposure of hydrophobic regions of the protein molecule, to which the hydrophobic regions of hydrophobic drugs can bind, resulting in increased drug solubility. Cytocompatible hydrogels have been successfully used to develop drug delivery systems due to their stimulus-sensitive response to external triggers, such as pH [9]. Hence, it would be desirable to combine the ability of WPI hydrogels to solubilize and carry hydrophobic drugs with pH responsiveness.

One class of hydrophobic molecules with biological activity are tannic acids (TAs). TAs are polyphenols closely related to our daily life: They are found in many fruits and vegetables consumed by humans and are used in the food industry and herbal medicine. Hydrolyzable tannins are one of three types of TAs that are formed by a carbohydrate (glucose, quinic acid, or other), in which OH-groups are partially or completely esterified with gallic acid or related compounds [10–12]. In this context hydrolyzable means that ester hydrolysis can occur, as opposed to acid-base hydrolysis (deprotonation). Hydrolyzable tannins can be extracted from various vegetable plants and trees. As a rule, TAs are considered non-toxic in small doses [13,14] and exhibit antitumor effects [15]. The presence of TA in natural components can reduce tumor necrosis factor levels [16] and weaken the inflammatory cytokine expression [17]. Previously, it was shown that TA crosslinked into a compacting collagen gel predominantly inhibited proliferation of high-melanoma A375 cells with metastatic potential [18]. In addition, ternary composite nanofibers containing tannic acid can be used as wound dressings in the case of recessive dystrophic epidermolysis bullosa, which often leads to the development of an aggressive form of squamous cell carcinoma [19]. TA has been shown to help crosslinking of gelatin and pectin derivatives due to the presence of a large number of hydroxyl groups in the polyphenol structure due to intermolecular H-bond formation, in which the polyphenols act as electron pair donors [20]. From the physicochemical point of view, polyphenols stabilize the secondary structure of proteins, increase their thermal stability and significantly reduce their biodegradability [21]. Recently, a comparative analysis was carried out of the ability of gellan gum hydrogels enhanced with polyphenols (including the ones investigated in our research, ALSOK 02 and ALSOK 04), to enzymatic mineralization and the hydroxyapatite formation [22]. TA inclusion inhibited the growth of human osteoblast-like Saos-2 cells on substrates of mineralized gellan gum hydrogel biomaterials with calcium phosphate and did not confer antibacterial activity against *Escherichia coli*.

In this study, we combined the beneficial properties of TAs and WPI to create new pH-sensitive cytocompatible hydrogels which display an anticancer affect. Two TAs of differing molecular weight and chemical structure (polygalloyl glucoses—ALSOK 02 and polygalloyl quinic acids—ALSOK 04) were compared using swelling tests at different pH values. We hypothesized that the addition of TAs would reduce the swelling of WPI hydrogels due to the aforementioned interactions between polyphenols and proteins. To our best knowledge, this combination of components has not yet been tested for biomaterialrelated applications. We focused on the dependence of the swelling ability of hydrogels on pH of the medium, chemical structure, and concentration of TAs, which allowed a more prolonged release of TAs over several days. The behavior of hydrogels that are sensitive to external pH are especially in demand in the development of anticancer scaffolds. The cytotoxic activity of TA and WPI-based hydrogels were evaluated in vitro against the Hep-2 human laryngeal squamous carcinoma cell line (Hep-2 cells).
