*4.1. Materials*

Quercetin hydrate (C15H10O7·xH2O, Mw 302.24, >96% purity) and hydroxypropylβ-cyclodextrin (HP-β-CD, Mw ~1380–1500, >99.0% purity) were purchased from Tokyo Chemical Industry (Portland, OR, USA). Polyvinyl alcohol (PVA; Mw 89,000–98,000, 99+% hydrolyzed) was purchased from Sigma Aldrich (Saint Louis, MO, USA). Methanol (MeOH; HPLC grade) and dimethyl sulfoxide (DMSO; AR grade) were purchased from RCI Labscan (Bangkok, Thailand).

### *4.2. Formation of Quercetin/HP-β-CD Inclusion Complex*

Lyophilized formulations of quercetin/HP-β-CD inclusion complex were prepared by freeze-drying using the neutralization method [45]. Freeze-drying is an excellent method for preserving a wide variety of heat-sensitive materials such as proteins, microbes, pharmaceutical agents, tissues, and plasma [46]. The molar ratio of quercetin to HP-β-CD, following Lan et al., was 1:4.35 [24]. First, 10 g of HP-β-CD was dissolved in 50 mL distilled water, and 0.5 g of quercetin was dissolved in 70 mL MeOH at 60 ◦C to obtain a homogeneous solution. Then, both solutions were mixed and refluxed at 65 ◦C overnight. The solution was stirred continuously at room temperature for 5 h for complete encapsulation. Next, MeOH was removed by heating the solution at 75 ◦C for 2 h. The solution was freeze-dried after being filtered (0.45 µm) and frozen at −20 ◦C. Finally, quercetin/HP-β-CD inclusion complex was obtained after freeze-drying at −50 ◦C for 48 h. The resulting lyophilized quercetin/HP-β-CD inclusion complex powder was stored in airtight containers and protected from light until use. Freeze-dried drug–cyclodextrin complexes were used for hydrogel preparation.

### *4.3. Characterization of Quercetin/HP-β-CD Inclusion Complex*

Fourier-transform infrared spectrometry (FT-IR; Nicolet iS5 with iD7, Thermo Scientific, Waltham, MA, USA) was performed to reveal the chemical structure and group interaction of inclusion complex and analyzed in ATR mode with a framework region of 650 to 4000 cm−<sup>1</sup> with 64 scans at resolution of 4 cm−<sup>1</sup> . Samples were prepared as KBr pellets.

The crystalline structure of quercetin/HP-β-CD inclusion complex was examined by X-ray diffractometry (XRD; SmartLab, Rigaku, Japan) with diffraction angle of 2θ ranging from 5 to 50◦ at a scanning rate of 0.02◦/s.

A scanning electron microscope (SEM; JEOL, JSM-6610LV, Japan) was used to observe the morphology of quercetin/HP-β-CD inclusion complex with 15 kV accelerating voltage and magnification of 5000, 1000, and 500 times after the samples were vacuum-coated with a fine layer of gold.
