**1. Introduction**

At some point, everyone will experience an injury that involves skin or body tissue damage, and it is important to care for it properly. There are many wound care products on the market, and several factors need to be considered when choosing an appropriate dressing for each type of wound, including the color and depth of the wound, degree of infection, amount of exudate, and condition of the peri-wound skin [1]. Normally, an ideal dressing should have the following properties: it should maintain a moist environment, protect from bacterial invasion, accelerate the formation of epithelialization, provide thermal insulation, and be easy to remove without debris after healing [2].

Today, wound dressings have been developed to facilitate biological functions, such as antibacterial, antifungal, and antiviral functions, compared with traditional wound dressings, which just cover wounds. Herbal drugs from medicinal plants are widely

**Citation:** Wangsawangrung, N.; Choipang, C.; Chaiarwut, S.; Ekabutr, P.; Suwantong, O.; Chuysinuan, P.; Techasakul, S.; Supaphol, P. Quercetin/Hydroxypropyl-β-Cyclodextrin Inclusion Complex-Loaded Hydrogels for Accelerated Wound Healing. *Gels* **2022**, *8*, 573. https://doi.org/ 10.3390/gels8090573

Academic Editors: Kiat Hwa Chan and Yang Liu

Received: 8 August 2022 Accepted: 6 September 2022 Published: 8 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**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/).

incorporated in wound dressing materials to improve the healing properties [3,4]. Quercetin is an interesting component of the flavonoid family that is found in onions, apples, berries, tea, and tomatoes [5,6]. In addition, it has physicochemical features and absorption ability, is a source of dietary nutrition, and principally impacts inflammation and immunological function. Research has shown that ingesting 50 to 800 mg of quercetin per day (quercetin accounts for 75%) may have potential health advantages owing to its easy absorption in the organs responsible for metabolism and elimination [7]. The antioxidant, anti-inflammatory, antibacterial, antiviral, anticancer, antitoxic, and immunomodulatory effects of quercetin prove that it has potential therapeutic value for wound healing.

However, the major problems that limit its biological activity for wound healing are poor solubility, low bioavailability, and hydrophobicity, which means it generally dissolves more readily in organic solution [8,9]. To overcome the disadvantages of quercetin, a drug-delivery system can be used, for example, that forms an inclusion complex with host molecules such as cyclodextrins (CDs), which is one of the most popular techniques [10].

Cyclodextrins, generated by the action of an enzyme on starch, are cyclic oligosaccharides linked by α-1,4 glycosidic bonds that are normally composed of six (α-CD), seven (β-CD), or eight (γ-CD) α-D-glucopyranose units. Because cyclodextrins enhance hydrophobic–hydrophilic interactions between proteins and other molecules, they are employed in the food processing industry to create reduced-cholesterol products and increase the bioavailability of desired molecules [11]. In general, CDs have a cone-shaped structure, with a hydrophilic outer surface and a hydrophobic inner cavity. Due to the hydrophobic cavity, CDs can trap hydrophobic molecules to form host–guest complexes for use in the pharmaceutical industry [12]. CDs are used to encapsulate odors and flavors to avoid food discoloration and minimize off-flavors and can be applied in food packaging [13]. In addition, CDs are used in many medical applications, such as wound dressings. Inclusion complexes containing curcumin and hydroxypropyl–cyclodextrin have been added to bacterial cellulose hydrogels to enhance wound healing [14]. However, mainly hydroxypropyl-β-cyclodextrin (HP-β-CD) is employed as a complexing agent to improve the water solubility and bioavailability of poorly soluble medicines and enhance their stability [15].

Hydrogels are three-dimensional natural or synthetic polymers that contain more than 90% water. The polymer chains are crosslinked to form a network to maintain the hydrogel structure, and the crosslinking process can be either physical or chemical. Hydrogels have a hydrophilic porous structure that can absorb a certain amount of water depending on many factors such as the pore size or the polymer type [16]. Hydrogels are popular for wound dressing because of several benefits including the ability to maintain a moist environment and absorb exudates, good biocompatibility, low adhesion to wound tissue, reduced pressure and shear forces, protection from bacterial invasion, and thermal insulation [16,17]. Hydrogels also have the ability to cool and soothe the skin, which is helpful for pain relief of burns or painful wounds [18]. Polyvinyl alcohol (PVA) is a biodegradable and watersoluble hydrophilic polymer that is produced by hydrolyzing polyvinyl acetate (PVAc). This polymer has good mechanical properties, excellent thermal stability, low toxicity, and high gas barrier properties, so it is widely used in many applications, including biomedical, food packaging, paper coating, and textile sizing and as a thickener and emulsion stabilizer, or it can be blended with other polymers to increase the overall properties. PVA hydrogel wound dressings can be produced by physical crosslinking through the cyclic freeze–thaw process [19]. Due to the PVA structure, they can form H-bonding between −OH groups during freezing, leading to crosslinking of their polymer chains (crystalline zone). However, the freezing conditions, including temperature, time, and number of cycles, could affect the degree of physical crosslinking or crystallinity of the obtained hydrogels. At lower freezing temperature, there are greater interactions between PVA chains and more crystal formation. Small water crystals are formed and sublimated at low freezing temperatures, resulting in a porous structure [20], whereas more freeze–thaw cycles lead to further crystal formation and a higher degree of physical crosslinking [21].

The purpose of this work was to form an inclusion complex by encapsulating quercetin into HP-β-CD to overcome its limitations and to develop new hydrogels incorporating the inclusion complex to accelerate wound healing. Drug-loaded hydrogels were evaluated for their physical properties in terms of gelation, swelling, and compression. The quercetin released from the prepared hydrogels and the cytotoxic effects on an NCTC 929 clone cells were also investigated regarding the biological abilities of the drug-loaded hydrogels. ing the inclusion complex to accelerate wound healing. Drug-loaded hydrogels were evaluated for their physical properties in terms of gelation, swelling, and compression. The quercetin released from the prepared hydrogels and the cytotoxic effects on an NCTC 929 clone cells were also investigated regarding the biological abilities of the drug-loaded hydrogels. **2. Results and Discussion** 

The purpose of this work was to form an inclusion complex by encapsulating quercetin into HP-β-CD to overcome its limitations and to develop new hydrogels incorporat-

ture, time, and number of cycles, could affect the degree of physical crosslinking or crystallinity of the obtained hydrogels. At lower freezing temperature, there are greater interactions between PVA chains and more crystal formation. Small water crystals are formed and sublimated at low freezing temperatures, resulting in a porous structure [20], whereas more freeze–thaw cycles lead to further crystal formation and a higher degree of physical

### **2. Results and Discussion** *2.1. Characterization of Quercetin/HP-β-CD Inclusion Complex*

crosslinking [21].

### *2.1. Characterization of Quercetin/HP-β-CD Inclusion Complex* FTIR spectroscopy was used to investigate the successful encapsulation of quercetin

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FTIR spectroscopy was used to investigate the successful encapsulation of quercetin into HP-β-CD (Figure 1). The FTIR spectrum of quercetin (Figure 1a) showed –OH groups stretching at 3395 and 3268 cm−<sup>1</sup> ; C=O aryl ketone stretching at 1663 cm−<sup>1</sup> ; C=C aromatic ring stretching at 1605, 1560, and 1518 cm−<sup>1</sup> ; and C–O aryl ether ring stretching, C–O phenol stretching, and C–CO–C ketone stretching and bending at 1256, 1194, and 1164 cm−<sup>1</sup> , respectively [22]. For HP-β-CD (Figure 1b), the characteristic broad peak of OH group stretching was shown at 3344 cm−<sup>1</sup> ; C–H stretching was detected at 2926 cm−<sup>1</sup> ; and C-O-C glucose unit stretching occurred at 1020 cm−<sup>1</sup> [23]. The FTIR spectrum of the physical mixture of the two (Figure 1c) showed characteristic peaks of quercetin, including C=O aryl ketone stretching (1663 cm−<sup>1</sup> ), C=C aromatic ring stretching (1609, 1560, and 1520 cm−<sup>1</sup> ), and C–CO–C ketone stretching and bending (1165 cm−<sup>1</sup> ), as well as C–O–C glucose unit stretching of HP-β-CD. When quercetin was encapsulated, its characteristic peaks seemed to be masked (Figure 1d). The C=O aryl ketone stretching (1663 cm−<sup>1</sup> ), C=C aromatic ring stretching (1605, 1560, and 1518 cm−<sup>1</sup> ), and C–CO–C ketone stretching and bending (1164 cm−<sup>1</sup> ) peaks of quercetin were absent in the inclusion complex, which may be because the quercetin was entrapped in the HP-β-CD cavities [24]. Signals of the characteristic bands of quercetin shifted from 3282 cm−<sup>1</sup> to 3142 cm−<sup>1</sup> O–H bending signals showing the formation of quercetin/HP-β-CD inclusion complex [25]. into HP-β-CD (Figure 1). The FTIR spectrum of quercetin (Figure 1a) showed –OH groups stretching at 3395 and 3268 cm−1; C=O aryl ketone stretching at 1663 cm−1; C=C aromatic ring stretching at 1605, 1560, and 1518 cm−1; and C–O aryl ether ring stretching, C–O phenol stretching, and C–CO–C ketone stretching and bending at 1256, 1194, and 1164 cm−1, respectively [22]. For HP-β-CD (Figure 1b), the characteristic broad peak of OH group stretching was shown at 3344 cm−1; C–H stretching was detected at 2926 cm−1; and C-O-C glucose unit stretching occurred at 1020 cm−1 [23]. The FTIR spectrum of the physical mixture of the two (Figure 1c) showed characteristic peaks of quercetin, including C=O aryl ketone stretching (1663 cm−1), C=C aromatic ring stretching (1609, 1560, and 1520 cm−1), and C–CO–C ketone stretching and bending (1165 cm−1), as well as C–O–C glucose unit stretching of HP-β-CD. When quercetin was encapsulated, its characteristic peaks seemed to be masked (Figure 1d). The C=O aryl ketone stretching (1663 cm−1), C=C aromatic ring stretching (1605, 1560, and 1518 cm−1), and C–CO–C ketone stretching and bending (1164 cm−1) peaks of quercetin were absent in the inclusion complex, which may be because the quercetin was entrapped in the HP-β-CD cavities [24]. Signals of the characteristic bands of quercetin shifted from 3282 cm−1 to 3142 cm−1 O–H bending signals showing the formation of quercetin/HP-β-CD inclusion complex [25].

**Figure 1.** FTIR spectra of (**a**) quercetin, (**b**) HP-β-CD, (**c**) quercetin/HP-β-CD physical mixture, and (**d**) quercetin/HP-β-CD inclusion complex. **Figure 1.** FTIR spectra of (**a**) quercetin, (**b**) HP-β-CD, (**c**) quercetin/HP-β-CD physical mixture, and (**d**) quercetin/HP-β-CD inclusion complex.

The XRD patterns provide further evidence of the successful encapsulation of quercetin in HP-β-CD (Figure 2). Characteristic diffraction peaks (Figure 2a) indicated that quercetin existed in a crystalline form, with peaks at three 2θ positions (10.66◦ , 12.34◦ , and 27.26◦ ) [26], while HP-β-CD (Figure 2b) showed broad peaks of amorphous materials caused by random X-ray scattering [27]. After the complexation of quercetin and HP-β-CD, there were no noticeable crystalline peaks of quercetin in the inclusion complex (Figure 2d) because the CD cavities hindered its crystalline structure, unlike the physical mixture, which showed

crystalline and amorphous behavior (Figure 2c) of quercetin and HP-β-CD, respectively. These results suggest that quercetin was successfully encapsulated into HP-β-CD cavities, leading to the loss of characteristic peaks. Similarly, Zhu et al. encapsulated l-menthol, which is crystalline (2θ = 8.0◦ , 14.0◦ , 20.5◦ , and 21.6◦ ), into amorphous HP-β-CD and observed no sharp peaks of l-menthol [28]. This result agrees with Kim et al., who reported similar XRD patterns of some selective flavonoids in hydroxypropyl-β-cyclodextrin inclusion complexes, which was indicative of the amorphous nature of the complex [29]. β-CD, respectively. These results suggest that quercetin was successfully encapsulated into HP-β-CD cavities, leading to the loss of characteristic peaks. Similarly, Zhu et al. encapsulated l-menthol, which is crystalline (2θ = 8.0°, 14.0°, 20.5°, and 21.6°), into amorphous HP-β-CD and observed no sharp peaks of l-menthol [28]. This result agrees with Kim et al., who reported similar XRD patterns of some selective flavonoids in hydroxypropyl-β-cyclodextrin inclusion complexes, which was indicative of the amorphous nature of the complex [29].

The XRD patterns provide further evidence of the successful encapsulation of quercetin in HP-β-CD (Figure 2). Characteristic diffraction peaks (Figure 2a) indicated that quercetin existed in a crystalline form, with peaks at three 2θ positions (10.66°, 12.34°, and 27.26°) [26], while HP-β-CD (Figure 2b) showed broad peaks of amorphous materials caused by random X-ray scattering [27]. After the complexation of quercetin and HP-β-CD, there were no noticeable crystalline peaks of quercetin in the inclusion complex (Figure 2d) because the CD cavities hindered its crystalline structure, unlike the physical mixture, which showed crystalline and amorphous behavior (Figure 2c) of quercetin and HP-

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**Figure 2.** XRD patterns of (**a**) quercetin, (**b**) HP-β-CD, (**c**) quercetin/HP-β-CD physical mixture, and (**d**) quercetin/HP-β-CD inclusion complex. **Figure 2.** XRD patterns of (**a**) quercetin, (**b**) HP-β-CD, (**c**) quercetin/HP-β-CD physical mixture, and (**d**) quercetin/HP-β-CD inclusion complex.

The morphologies of quercetin, HP-β-CD, and quercetin/HP-β-CD physical mixture and inclusion complex were observed at different magnifications on SEM (Figure 3). Quercetin (Figure 3a) presented a strip-like structure with a smooth surface [30], while HP-β-CD (Figure 3b) appeared as rough spheres [31]. In the physical mixture (Figure 3c), both quercetin and HP-β-CD kept their own structure, with particles of HP-β-CD embedded with quercetin particles [32]. On the other hand, the morphology and shape of quercetin/HP-β-CD inclusion complex obtained from the solvent evaporation method were completely different from those of the original product (Figure 3d). Our results were consistent with Pradhan et al. [33] in that the morphology of Berberis anthocyanin-loaded CD inclusion complex revealed the rod shape. The encapsulation of quercetin with CD demonstrated the electrostatic and hydrogel bond interactions between quercetin and CD. The morphologies of quercetin, HP-β-CD, and quercetin/HP-β-CD physical mixture and inclusion complex were observed at different magnifications on SEM (Figure 3). Quercetin (Figure 3a) presented a strip-like structure with a smooth surface [30], while HP-β-CD (Figure 3b) appeared as rough spheres [31]. In the physical mixture (Figure 3c), both quercetin and HP-β-CD kept their own structure, with particles of HP-β-CD embedded with quercetin particles [32]. On the other hand, the morphology and shape of quercetin/HP-β-CD inclusion complex obtained from the solvent evaporation method were completely different from those of the original product (Figure 3d). Our results were consistent with Pradhan et al. [33] in that the morphology of Berberis anthocyanin-loaded CD inclusion complex revealed the rod shape. The encapsulation of quercetin with CD demonstrated the electrostatic and hydrogel bond interactions between quercetin and CD.

## *2.2. Encapsulation Efficiency and Loading Capacity of Quercetin in the Inclusion Complex*

Cyclodextrins are nontoxic cyclic oligosaccharides consisting of α1,4-glycosidic bonds. CDs are distinguished by hydrophilic surfaces and hydrophobic cavities. The cavities are suitable for inclusion complexes with hydrophobic drugs (quercetins) to improve the aqueous solubility and yield Van der Waals and hydrophobic forces [34,35]. Quercetin/HPβ-CD inclusion complex was formed with optimum loading and encapsulation efficacy, achieved at a mass ratio of HP-β-CD to quercetin of 4.35:1. Loading capacity (LC) is the ratio of mass of encapsulated compound to mass of the polymer (Equation (1)), and encapsulation efficacy (EE%) refers to the ratio of the mass of the encapsulated compounds to the total mass of the compounds (Equation (2)). Encapsulation is considered to be an excellent carrier of compounds when LC id ≥ 5%, and %EE in the range of 70–100% indicates excellent encapsulation capacity.

**Figure 3.** SEM images of (**a**) quercetin, (**b**) HP-β-CD, (**c**) quercetin/HP-β-CD physical mixture, and (**d**) quercetin/HP-β-CD inclusion complex. **Figure 3.** SEM images of (**a**) quercetin, (**b**) HP-β-CD, (**c**) quercetin/HP-β-CD physical mixture, and (**d**) quercetin/HP-β-CD inclusion complex.

*2.2. Encapsulation Efficiency and Loading Capacity of Quercetin in the Inclusion Complex*  Cyclodextrins are nontoxic cyclic oligosaccharides consisting of α1,4-glycosidic bonds. CDs are distinguished by hydrophilic surfaces and hydrophobic cavities. The cavities are suitable for inclusion complexes with hydrophobic drugs (quercetins) to improve The absorbance of ~1 mg/mL inclusion complex in DMSO was 0.68917 ± 0.01715, which can be calculated back to the concentration of quercetin using the predetermined calibration curve (R<sup>2</sup> = 0.99758). The estimated total quercetin content in the inclusion complex was 0.4554 ± 0.0089 g.

the aqueous solubility and yield Van der Waals and hydrophobic forces [34,35]. Quercetin/HP-β-CD inclusion complex was formed with optimum loading and encapsulation efficacy, achieved at a mass ratio of HP-β-CD to quercetin of 4.35:1. Loading capacity (LC) is the ratio of mass of encapsulated compound to mass of the polymer (Equation (1)), and encapsulation efficacy (EE%) refers to the ratio of the mass of the encapsulated compounds to the total mass of the compounds (Equation (2)). Encapsulation is considered to be an excellent carrier of compounds when LC id ≥ 5%, and %EE in the range of 70–100% indicates excellent encapsulation capacity. The absorbance of ~1 mg/mL inclusion complex in DMSO was 0.68917 ± 0.01715, which can be calculated back to the concentration of quercetin using the predetermined calibration curve (R2 = 0.99758). The estimated total quercetin content in the inclusion complex was 0.4554 ± 0.0089 g. The %EE and %LC of quercetin were quantified with UV-Vis spectrophotometry at a wavelength of 379 nm and calculated using Equations (1) and (2), respectively. The %EE of the obtained quercetin/HP-β-CD inclusion complex was 90.50 ± 1.84%, and the %LC was 4.67 ± 0.13%. The %LC of the inclusion complex seemed to be quite low but was not. The molecular weight of HP-β-CD was greater than that of quercetin by around 4.6 times. We also used the excess molar ratio of HP-β-CD to prepare the inclusion complex. That explains why when we calculated %LC, which compares the drug in the complexation (or inclusion complex) in terms of weight, the molar ratio seemed to be low. This was similar to the results of Hadian et al., who prepared four formulations of geraniol/β-CD inclusion complexes by varying the mole ratio of GR:β-CD (0.44:0.13, 0.44:0.2, 0.44:0.4, and 0.44:1), and showed that %LC gradually decreased with increasing mole ratio (by 7.8 ± 0.70, 6.9 ± 0.1 6.6 ± 0.28 and 6.5 ± 0.6, respectively) [36].

### The %EE and %LC of quercetin were quantified with UV-Vis spectrophotometry at *2.3. Gelation of PVA Hydrogels Loaded with Quercetin/HP-β-CD Inclusion Complex*

a wavelength of 379 nm and calculated using Equations (1) and (2), respectively. The %EE of the obtained quercetin/HP-β-CD inclusion complex was 90.50 ± 1.84%, and the %LC was 4.67 ± 0.13%. The %LC of the inclusion complex seemed to be quite low but was not. The molecular weight of HP-β-CD was greater than that of quercetin by around 4.6 times. We also used the excess molar ratio of HP-β-CD to prepare the inclusion complex. That explains why when we calculated %LC, which compares the drug in the complexation (or The gelation of neat and all prepared drug-loaded hydrogels in PBS (pH = 7.4) at 37 ◦C for 24 h was calculated using Equation (3). The experiments were repeat five times, and values were averaged. The gelation of neat PVA hydrogel was 97.40 ± 0.62%, while the gelation of drug-loaded hydrogels ranged from 74.52 ± 0.86% to 94.36 ± 0.80%, as shown in Table 1.

inclusion complex) in terms of weight, the molar ratio seemed to be low. This was similar to the results of Hadian et al., who prepared four formulations of geraniol/β-CD inclusion complexes by varying the mole ratio of GR:β-CD (0.44:0.13, 0.44:0.2, 0.44:0.4, and 0.44:1), **Table 1.** Gelation value and mass content of PVA hydrogels containing various amounts of quercetin/HP-β-CD inclusion complex. Data shown as mean ± standard deviation (*n* = 5). IC as quercetin/HP-β-CD inclusion complex.


in Table 1.


The percent gelation of drug-loaded hydrogel clearly gradually decreased with in-

**IC PVA** 

**Table 1.** Gelation value and mass content of PVA hydrogels containing various amounts of quercetin/HP-β-CD inclusion complex. Data shown as mean ± standard deviation (n = 5). IC as querce-

**Sample Gelation (%) Mass Content (%)** 

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**Table 1.** *Cont.* PVAIC0 97.40 ± 0.62 0.00 100.00

tin/HP-β-CD inclusion complex.

The percent gelation of drug-loaded hydrogel clearly gradually decreased with increasing inclusion complex content in the hydrogels. The reduction in hydrogel weight may have occurred for the following reasons. The first is that hydrogel swells when immersed in PBS, leading to a loose structure, so the quercetin can dissolve and be released out of the hydrogels [20]. The second reason is that when loading the quercetin/HP-β-CD inclusion complex into the hydrogels, the inclusion complex may disrupt the crosslink formation of the hydrogel, so some PVA that did not participate in crystallite formation could have dissolved into the solution [21]. creasing inclusion complex content in the hydrogels. The reduction in hydrogel weight may have occurred for the following reasons. The first is that hydrogel swells when immersed in PBS, leading to a loose structure, so the quercetin can dissolve and be released out of the hydrogels [20]. The second reason is that when loading the quercetin/HP-β-CD inclusion complex into the hydrogels, the inclusion complex may disrupt the crosslink formation of the hydrogel, so some PVA that did not participate in crystallite formation could have dissolved into the solution [21].

### *2.4. Swelling Ratio of PVA Hydrogels Loaded with Quercetin/HP-β-CD Inclusion Complex 2.4. Swelling Ratio of PVA Hydrogels Loaded with Quercetin/HP-β-CD Inclusion Complex*  The swelling ratio of neat and drug-loaded hydrogels in PBS (pH = 7.4) at 37 °C at 10,

The swelling ratio of neat and drug-loaded hydrogels in PBS (pH = 7.4) at 37 ◦C at 10, 30, 60, 180, and 360 min was calculated using Equation (4). The experiment was repeated three times at each release time point, and values were averaged. The results show that the swelling ratio of hydrogels with various drug amounts was slightly higher than that of neat PVA hydrogel, but there was not much difference (Figure 4). After the first 60 min of the experiment, the swelling ratio of all hydrogels dramatically increased to around 4.3%, then gradually increased to around 5.8% at 180 min and became relatively stable at around 6.4% after immersion in PBS for 360 min. The incorporation of quercetin/HP-β-CD inclusion complex into the PVA hydrogels had no effect on the swelling ratio, which may be caused by the similar conditions of the freeze–thaw process that we used to prepare the hydrogels. Therefore, the number and temperature of freezing–thawing cycles could be the most important factors controlling the swelling ratio of hydrogels. We can also add other polymers or materials, such as gelatin or chitosan, to increase the swellability of PVA hydrogels [20]. 30, 60, 180, and 360 min was calculated using Equation (4). The experiment was repeated three times at each release time point, and values were averaged. The results show that the swelling ratio of hydrogels with various drug amounts was slightly higher than that of neat PVA hydrogel, but there was not much difference (Figure 4). After the first 60 min of the experiment, the swelling ratio of all hydrogels dramatically increased to around 4.3%, then gradually increased to around 5.8% at 180 min and became relatively stable at around 6.4% after immersion in PBS for 360 min. The incorporation of quercetin/HP-β-CD inclusion complex into the PVA hydrogels had no effect on the swelling ratio, which may be caused by the similar conditions of the freeze–thaw process that we used to prepare the hydrogels. Therefore, the number and temperature of freezing–thawing cycles could be the most important factors controlling the swelling ratio of hydrogels. We can also add other polymers or materials, such as gelatin or chitosan, to increase the swellability of PVA hydrogels [20].

**Figure 4.** Swelling ratios of PVA hydrogels containing 0% (PVAIC0), 0.5% (PVAIC0.5), 1.0% (PVAIC1.0), 1.5% (PVAIC1.5), 2.5% (PVAIC2.5), and 5.0% (PVAIC5.0) quercetin/HP-β-CD inclusion complex. Data shown as mean ± standard deviation (n = 3). **Figure 4.** Swelling ratios of PVA hydrogels containing 0% (PVAIC0), 0.5% (PVAIC0.5), 1.0% (PVAIC1.0), 1.5% (PVAIC1.5), 2.5% (PVAIC2.5), and 5.0% (PVAIC5.0) quercetin/HP-β-CD inclusion complex. Data shown as mean ± standard deviation (*n* = 3).

### *2.5. Mechanical Test of PVA Hydrogels Loaded with Quercetin/HP-β-CD Inclusion Complex*

The compressive strength of neat and all prepared drug-loaded hydrogels was tested, and the experiment was repeated seven times to obtain average values. The stiffness and compressive modulus of neat PVA hydrogel were 27,318 ± 831 N/m and 1.19 ± 0.07 MPa,

respectively, while the values for drug-loaded hydrogels were 19,520 ± 1066 to 26,199 ± 1275 N/m and 0.88 ± 0.04 to 1.12 ± 0.08 MPa, respectively (Table 2). As the amount of inclusion complex in the hydrogels increased, stiffness and compressive modulus seemed to gradually decrease. This may have resulted from the molecules of the inclusion complex being distributed in the PVA structure, leading to a lower crystalline zone [20] and decreased crosslink density of the PVA hydrogel [37], so it was not able to withstand the compressive force compared with lower drug content hydrogels. Water content is another factor that can affect the mechanical properties of hydrogels because when the water in hydrogel evaporates, the hydrogel becomes stronger, so it will have stronger resistance against compressive force than softer or lower-water-content hydrogels [38]. Qing et al. prepared PVA hydrogel dressings that incorporated N-succinyl chitosan (NSCS) and lincomycin, and the results revealed that the introduction of 10% NSCS reduced the compression strength of pure PVA hydrogel from 0.33 to 0.25 MPa because NSCS increases the distance between PVA chains and weakens the molecular interaction force. However, when they further increased NSCS content, hydrogel bonding occurred, and the hydrogel became stronger, leading to enhanced compression strength (0.75 MPa). As NSCS content was further increased to 40 and 50%, the compression strength in turn decreased due to the low crosslinking density resulting from the decreased gel fraction [39].

**Table 2.** Mechanical test of PVA hydrogels containing various amounts of quercetin/HP-β-CD inclusion complex. Data shown as mean ± standard deviation (*n* = 7).


*2.6. In Vitro PVA Hydrogels Loaded with Quercetin/HP-β-CD Inclusion Complex Dissolution and Release Kinetics Study*

The characteristics of quercetin release from the prepared hydrogels were investigated using the total immersion method over a period of 48 h in PBS (pH = 7.4) and incubated at 37 ◦C under continuous stirring. The cumulative release of quercetin from all hydrogels was calculated using Equation (5) and reported in parts per million. As shown in Figure 5, the cumulative amount of quercetin released from the hydrogels increased rapidly with increasing immersion time for the first 5 or 8 h of the experiment, then gradually increased and reached a plateau. After a period of 48 h, the cumulative quercetin quantities released from the quercetin/HP-β-CD inclusion complex PVA hydrogels were 81.66 ± 17.42, 142.07 ± 30.43, 167.64 ± 23.30, 211.74 ± 27.78, and 286.10 ± 27.71 ppm for PVAIC0.5, PVAIC1.0, PVAIC1.5, PVAIC2.5, and PVAIC5.0, respectively. The greatest cumulative amounts of CIP released were observed when the quercetin concentration of PVAIC5.0 was used, and therefore, it can be concluded that when a greater concentration of quercetin is introduced to the nanoparticles, the drug release will increase. In the USA, the mean quercetin intake was approximately 14.90 to 16.39 mg per day [40]. Thus, the release characteristics of quercetin for the PVAIC5.0 hydrogels (close to 28.6 mg/100 g of sample) was found to be about 1.9-times higher than the oral solution of the quercetin intake per day.

**Figure 5.** Release profiles of quercetin from PVA hydrogels containing 0.5% (PVAIC0.5), 1.0% (PVAIC1.0), 1.5% (PVAIC1.5), 2.5% (PVAIC2.5), and 5.0% (PVAIC5.0) of quercetin/HP-β-CD inclusion complex. Data shown as mean ± standard deviation (*n* = 3). **Figure 5.** Release profiles of quercetin from PVA hydrogels containing 0.5% (PVAIC0.5), 1.0% (PVAIC1.0), 1.5% (PVAIC1.5), 2.5% (PVAIC2.5), and 5.0% (PVAIC5.0) of quercetin/HP-β-CD inclusion complex. Data shown as mean ± standard deviation (*n* = 3).

The drug-release kinetics of quercetin from PVA hydrogels loaded with quercetin/HP-β-CD inclusion complex were fitted with Zero-order and Korsmeyer–Peppas models to reveal the kinetics of drug release. Release constant (k) and release exponent (*n*) were calculated to identify the release mechanism, and the fitted model parameters and correlation coefficients (r2) for the release profiles are given in Table 3. An r2 coefficient of determination over 0.95 was considered to reflect the goodness of fit of the release model [41]. The release data from PVA hydrogels loaded with quercetin/HP-β-CD inclusion complex fit with the Korsmeyer–Peppas model. The release exponent (n) values can be used to indicate the release rate mechanism. The PVAIC0.5, PVAIC1.0, PVAIC1.5, PVAIC2.5, and PVAIC5.0 hydrogels had n values of 1.31, 0.77, 0.68, 0.58, and 0.77, respectively. The n values between 0.89 and 1 indicate that the release of quercetin from PVAIC1.0 to PVAIC5.0 were controlled by the mechanism of anomalous (non-Fickian) diffusion. The diffusion exponent for PVAIC0.5 is in the range of 0.89 and 1, which denotes the case II transport mechanism (zero-order kinetics). The drug-release kinetics of quercetin from PVA hydrogels loaded with quercetin/HPβ-CD inclusion complex were fitted with Zero-order and Korsmeyer–Peppas models to reveal the kinetics of drug release. Release constant (k) and release exponent (*n*) were calculated to identify the release mechanism, and the fitted model parameters and correlation coefficients (r<sup>2</sup> ) for the release profiles are given in Table 3. An r<sup>2</sup> coefficient of determination over 0.95 was considered to reflect the goodness of fit of the release model [41]. The release data from PVA hydrogels loaded with quercetin/HP-β-CD inclusion complex fit with the Korsmeyer–Peppas model. The release exponent (n) values can be used to indicate the release rate mechanism. The PVAIC0.5, PVAIC1.0, PVAIC1.5, PVAIC2.5, and PVAIC5.0 hydrogels had n values of 1.31, 0.77, 0.68, 0.58, and 0.77, respectively. The n values between 0.89 and 1 indicate that the release of quercetin from PVAIC1.0 to PVAIC5.0 were controlled by the mechanism of anomalous (non-Fickian) diffusion. The diffusion exponent for PVAIC0.5 is in the range of 0.89 and 1, which denotes the case II transport mechanism (zero-order kinetics).


**Table 3.** Modeling results of quercetin released from PVA hydrogels loaded with quercetin/HP-β-CD inclusion complex fitting with Zero-order and Korsmeyer–Peppas models. **Table 3.** Modeling results of quercetin released from PVA hydrogels loaded with quercetin/HP-β-CD inclusion complex fitting with Zero-order and Korsmeyer–Peppas models.

### The encapsulation of quercetin within cyclodextrin increases the antioxidant activity *2.7. In Vitro Antioxidant Activity: DPPH-Radical Scavenging Ability Assay*

compared with free molecules [42]. The antioxidant activity of quercetin/HP-β-CD inclusion complex is widely used in 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay in comparison with free counterparts and the results were shown in Figure 6. A concentration of 120 µg/mL quercetin/HP-β-CD inclusion complex was determined to The encapsulation of quercetin within cyclodextrin increases the antioxidant activity compared with free molecules [42]. The antioxidant activity of quercetin/HP-β-CD inclusion complex is widely used in 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging

assay in comparison with free counterparts and the results were shown in Figure 6. A concentration of 120 µg/mL quercetin/HP-β-CD inclusion complex was determined to have 74.80% DPPH inhibition compared with free quercetin, with 61.23% of inhibition, and ascorbic acid with 74.80%. HP-β-CD inclusion complex showed significant antioxidant activity compared with quercetin alone (Figure 6) in a dose-dependent manner. The results indicate that HP-β-CD inclusion complex is associated with the sustained release of quercetin and increases the stability of incorporated bioactive compounds such as quercetin, significantly improving its antioxidant activity [43]. have 74.80% DPPH inhibition compared with free quercetin, with 61.23% of inhibition, and ascorbic acid with 74.80%. HP-β-CD inclusion complex showed significant antioxidant activity compared with quercetin alone (Figure 6) in a dose-dependent manner. The results indicate that HP-β-CD inclusion complex is associated with the sustained release of quercetin and increases the stability of incorporated bioactive compounds such as quercetin, significantly improving its antioxidant activity [43].

*Gels* **2022**, *8*, 573 9 of 17

**Figure 6.** Scavenging activity of ascorbic acid, quercetin and quercetin/ HP-β-CD as determined by DPPH assay. Values expressed as mean ± standard deviation (*n* = 5). **Figure 6.** Scavenging activity of ascorbic acid, quercetin and quercetin/ HP-β-CD as determined by DPPH assay. Values expressed as mean ± standard deviation (*n* = 5).

### *2.8. Cytotoxicity Evaluation 2.8. Cytotoxicity Evaluation*

applications.

The indirect cytotoxicity evaluation of all samples toward NCTC 929 clone cells investigated the potential use of drug loaded PVA hydrogels as wound dressing material. Figure 7 shows the viability of cells obtained from MTT assay after being cultured with various concentrations of media extracted from neat PVA hydrogel as blank PVA, and PVA hydrogels loaded with quercetin/HP-β-CD inclusion complex (PVAIC0.5, PVAIC1.0, PVAIC1.5, PVAIC2.5, and PVAIC5.0) compared with that obtained after cells were cultured with fresh SFM. According to the in vitro cytotoxicity standard, a reduction of cell viability by more than 30% is considered a cytotoxic effect [44]. The viability of NCTC 929 clone cells with all extraction medium concentrations, i.e., neat PVA, PVAIC0.5, PVAIC1.0, PVAIC1.5, PVAIC2.5, and PVAIC5.0, were 92.7–96.3, 76.8–88.2, 80.0–97.5, 74.7– 92.9, 87.6–96.8, and 78.3–85.3%, respectively. Cell viability was higher than 70% in all prepared hydrogels loaded with quercetin/HP-β-CD inclusion complex. Therefore, all hydrogels loaded with quercetin/HP-β-CD inclusion complex with extraction medium concentrations of 0.5, 5, 10, 25, and 50 mg/mL had cell viability of more than 70%, indicating that these conditions were non-cytotoxic, and the hydrogels have potential in wound dressing The indirect cytotoxicity evaluation of all samples toward NCTC 929 clone cells investigated the potential use of drug loaded PVA hydrogels as wound dressing material. Figure 7 shows the viability of cells obtained from MTT assay after being cultured with various concentrations of media extracted from neat PVA hydrogel as blank PVA, and PVA hydrogels loaded with quercetin/HP-β-CD inclusion complex (PVAIC0.5, PVAIC1.0, PVAIC1.5, PVAIC2.5, and PVAIC5.0) compared with that obtained after cells were cultured with fresh SFM. According to the in vitro cytotoxicity standard, a reduction of cell viability by more than 30% is considered a cytotoxic effect [44]. The viability of NCTC 929 clone cells with all extraction medium concentrations, i.e., neat PVA, PVAIC0.5, PVAIC1.0, PVAIC1.5, PVAIC2.5, and PVAIC5.0, were 92.7–96.3, 76.8–88.2, 80.0–97.5, 74.7–92.9, 87.6–96.8, and 78.3–85.3%, respectively. Cell viability was higher than 70% in all prepared hydrogels loaded with quercetin/HP-β-CD inclusion complex. Therefore, all hydrogels loaded with quercetin/HP-β-CD inclusion complex with extraction medium concentrations of 0.5, 5, 10, 25, and 50 mg/mL had cell viability of more than 70%, indicating that these conditions were non-cytotoxic, and the hydrogels have potential in wound dressing applications.

**Figure 7.** Indirect cytotoxicity of PVA hydrogels loaded with various amounts of quercetin/HP-β-CD inclusion complex with extraction medium: neat PVA, 0.5, 5, 10, 25, and 50 mg/mL cultured with NCTC 929 clone cells. **Figure 7.** Indirect cytotoxicity of PVA hydrogels loaded with various amounts of quercetin/HP-β-CD inclusion complex with extraction medium: neat PVA, 0.5, 5, 10, 25, and 50 mg/mL cultured with NCTC 929 clone cells.

### **3. Conclusions 3. Conclusions**

We successfully prepared PVA hydrogels containing quercetin/HP-β-CD inclusion complex that achieved efficient drug loading and effective encapsulation. The quercetin/HP-β-CD inclusion complex was prepared with an excess molar ratio of HP-β-CD via the solvent evaporation method. The successful encapsulation of quercetin in HP-β-CD was confirmed by FT-IR and XRD, in which characteristic peaks of quercetin could not be observed after encapsulation due to the hindering of CD cavities. As observed on SEM, the quercetin/HP-β-CD inclusion complex demonstrated rod-shaped particles. The prepared quercetin/HP-β-CD inclusion complex had 90.50 ± 1.84% encapsulation efficiency and 4.67 ± 0.13% loading capacity. It was incorporated into PVA hydrogels in various amounts (0, 0.5, 1.0, 1.5, 2.5, and 5.0% MIC/Vsolvent ratio). The hydrogels were evaluated for their potential use as wound dressing in terms of gelation, swelling, compression, release characteristics, antioxidant properties, and in vitro non-cytotoxicity. The incorporation of quercetin/HP-β-CD inclusion complex into PVA hydrogels had no effect on the swelling ratio of the hydrogels. The gelation and compressive strength of hydrogels decreased with increasing inclusion complex content, whereas the cumulative release of quercetin from We successfully prepared PVA hydrogels containing quercetin/HP-β-CD inclusion complex that achieved efficient drug loading and effective encapsulation. The quercetin/HPβ-CD inclusion complex was prepared with an excess molar ratio of HP-β-CD via the solvent evaporation method. The successful encapsulation of quercetin in HP-β-CD was confirmed by FT-IR and XRD, in which characteristic peaks of quercetin could not be observed after encapsulation due to the hindering of CD cavities. As observed on SEM, the quercetin/HP-β-CD inclusion complex demonstrated rod-shaped particles. The prepared quercetin/HP-β-CD inclusion complex had 90.50 ± 1.84% encapsulation efficiency and 4.67 ± 0.13% loading capacity. It was incorporated into PVA hydrogels in various amounts (0, 0.5, 1.0, 1.5, 2.5, and 5.0% MIC/Vsolvent ratio). The hydrogels were evaluated for their potential use as wound dressing in terms of gelation, swelling, compression, release characteristics, antioxidant properties, and in vitro non-cytotoxicity. The incorporation of quercetin/HP-β-CD inclusion complex into PVA hydrogels had no effect on the swelling ratio of the hydrogels. The gelation and compressive strength of hydrogels decreased with increasing inclusion complex content, whereas the cumulative release of quercetin from hydrogels increased with higher amounts of inclusion complex.

hydrogels increased with higher amounts of inclusion complex. Hydrogels loaded with quercetin/HP-β-CD inclusion complex investigated for their cytotoxicity in vitro by MTT assay were shown to be nontoxic toward mouse fibroblast NCTC 929 clone cells at the test concentrations. The results showed significantly increased antioxidant properties of quercetin/HP-β-CD inclusion complex compared with free quercetin. These findings suggest that the use of quercetin/HP-β-CD inclusion complex ensures non-cytotoxicity and potential antioxidant activity. Hence, hydrogel with quercetin/HP-β-CD inclusion complex is an attractive candidate for the encapsulation of bioac-Hydrogels loaded with quercetin/HP-β-CD inclusion complex investigated for their cytotoxicity in vitro by MTT assay were shown to be nontoxic toward mouse fibroblast NCTC 929 clone cells at the test concentrations. The results showed significantly increased antioxidant properties of quercetin/HP-β-CD inclusion complex compared with free quercetin. These findings suggest that the use of quercetin/HP-β-CD inclusion complex ensures non-cytotoxicity and potential antioxidant activity. Hence, hydrogel with quercetin/HP-β-CD inclusion complex is an attractive candidate for the encapsulation of bioactive compounds for biomedical applications.

### **4. Materials and Methods**

tive compounds for biomedical applications.
