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

The cumulative amount of released quercetin/HP-β-CD from hydrogels was measured using an immersion method according to Chuysinuan et al. [51] with minor modifications. The disc-shaped specimens (2.8 cm in diameter) were immersed in phosphate-buffered saline (PBS, pH = 7.4) and incubated at 37 ◦C under continuous stirring. The sample solution (1 mL) was withdrawn at specific time intervals, and an equal amount of fresh release medium was added. The absorbance of released quercetin was measured with a UV-Vis spectrophotometer (LAMBDA 850+, PerkinElmer, Waltham, MA, USA) at a wavelength of 370 nm. The obtained data were back calculated from the predetermined quercetin calibration curve (R<sup>2</sup> = 0.9944) to determine the amounts of quercetin released from the

hydrogel samples. The cumulative release of quercetin from each hydrogel at different time points (with measurement carried out in triplicate at each time point) was reported as parts per million of the amounts of quercetin released (WQuercetin released) divided by the weight of hydrogels (Whydrogel):

$$\text{Cumulative release} \left( \% \right) = \frac{W\_{\text{Quercetin released}}}{W\_{\text{hydroel}}} \times 10^6 \tag{5}$$

A release kinetics study was used to ascertain the release mechanism, and the quercetin release data were fitted to Korsmeyer–Peppas and zero-order models. The first 60% of the drug release data was used to fit the models.

The Korsmeyer–Peppas model is a simple model that describes drug release from polymer nanoparticle systems [52,53]:

$$\mathbf{M}\_{\mathrm{lt}}/\mathbf{M}\_{\infty} = \mathbf{k}\mathbf{t}^{\mathrm{n}}$$

where Mt/M<sup>∞</sup> is the quercetin release fraction at time t, k is a constant incorporating geometric structural features, and n is the release exponent indicating the release rate mechanism. The value of n indicates the mechanism of the release; a value around 0.45 indicates case I (Fickian) diffusion, between 0.45 and 0.89 indicates anomalous (non-Fickian) diffusion, and between 0.89 and 1 indicates case II transport (zero-order kinetics).

A zero-order kinetic model is used to describe drugs that are released slowly with a constant concentration and can be characterized as ideal kinetic model in that it maintains constant drug levels during the delivery process [54,55]:

$$\mathbf{M}\_{\mathbf{l}}/\mathbf{M}\_{\infty} = \mathbf{k}\mathbf{t}$$

where Mt/M<sup>∞</sup> is the quercetin release fraction at time t, with zero-order rate constant, and t is investigation time.

### *4.10. In Vitro Antioxidant Characterization*

The antioxidant activity of free quercetin and quercetin/HP-β-CD inclusion complex was evaluated using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay following [3]. First, quercetin and quercetin/HP-β-CD in various concentrations (15, 30, 60, and 120 µg/mL) were dissolved in methanol. Then, the samples (1 mL) were mixed with 3 mL DPPH solution (0.1 mM in methanol). The mixture was incubated for 30 min in the dark. Finally, the absorbance of the reaction mixture was measured using a microplate reader at 517 nm. Radical scavenging activity (%) was calculated using the following equation. Ascorbic acid was used as a standard and all analyses were performed in triplicate:

$$\text{DPPH radicalscavenging activity} \left( \% \right) = \frac{\left( \text{Absorbance of control} - \text{Absorbance of tested sample} \right)}{\text{Absorbance of control}} \times 100 \tag{6}$$

### *4.11. Cytotoxicity Evaluation*

While CDs are chosen for pharmaceutical formulations to increase the solubility, bioavailability, and stability of many medications, their structure and cytotoxic capability are crucial for more effective drug delivery [56]. To evaluate the potential biomedical applications of PVA hydrogels loaded with various amounts of quercetin/HP-β-CD inclusion complex, their biocompatibility in terms of indirect cytotoxicity toward NCTC 929 clone cells (ATCC-CCL-1, Rockville, MD, USA (17th passage) was evaluated in accordance with the ISO10993-5 standard test method [57]. First, cells were cultured in Dulbecco's Modified Eagle Medium (DMEM (1×); GIBCO, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; GIBCO, USA) and 1% antibiotic–antimycotic agent (GIBCO, Waltham, MA, USA). Then, the cells were seeded into 96-well tissue-culture polystyrene (TCPS) plates (SPL Lifescience, Pochon, Korea) at 8000 cells/well and then incubated at 37 ◦C in a humidified

atmosphere containing 95% air and 5% CO2. Next, the hydrogel samples were sterilized by exposure to UV radiation for 30 min/side and were immersed in serum-free medium (SFM, containing DMEM and 1% antibiotic–antimycotic agent) in a 96-well TCPS plate. The samples were incubated for 24 h to produce sample extraction at five ratios of extraction medium (0.5, 5, 10, 25, and 50 mg/mL). NCTC 929 clone cells were cultured separately in culture medium in wells of TCPS plates at 8000 cells/well for 24 h to allow cells to attach to the well surface. After that, the medium was replaced with an extraction medium, and mouse fibroblast L929 cells were incubated for a further 24 h.

The viability of cells cultured in each extraction medium was determined with 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After treatment, the medium was removed, and the samples were washed with PBS; then, MTT solution (0.5 mg/mL) was added, and samples were incubated for 3 h. After that, the MTT solution was removed from the well and replaced by DMSO (Labscan, Bangkok, Thailand) to dissolve the formazan crystals. Finally, the absorbance of the solutions was measured at 570 nm using a microplate reader (BioTek Instruments, Winooski, VT, USA) to investigate cell viability. The viability of cells cultured with fresh SFM was used as a control.

**Author Contributions:** Conceptualization, supervision, and funding acquisition, P.S.; Data curation, formal analysis, investigation, and methodology, N.W., C.C., S.C., O.S. and P.C.; Project administration, P.E.; Data curation, validation, and manuscript review, O.S., P.C. and S.T.; Visualization, N.W., C.C. and S.C.; Writing—original draft, N.W., C.C. and S.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the 90th Anniversary of Chulalongkorn University Scholarship Batch 50: GCUGR1125643048M No. 2–14 and Fundamental Fund 2565: FF65, Chulalongkorn University, Thailand.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This work was conducted with the support of the Herbal Extract-Infused Advanced Wound Dressing Research Unit, Rachadaphiseksomphot Endowment Fund, Chulalongkorn University, Thailand.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **Abbreviations**


