*2.1. Preparation of Rutin Nanocrystals (RT-NCs)*

RT-NCs were prepared by modifying the anti-solvent nanoprecipitation–ultrasonication method reported in the literature [28]. ln brief, RT was dissolved in ethanol to prepare the organic phase. The anti-solvent phase was prepared by dissolving the stabilizers (Pluronic F-17, Tween 80, HP-β-CD, or PEG 6000) at a concentration of 0.2%, *w/v* in distilled water with 1 mL of PEG 200 as a co-stabilizer (Table 1). The organic phase was added drop-wise by a syringe into the specified volume of anti-solvent phase and the dispersion was stirred on a magnetic stirrer at a speed of 3000 RPM for 2 h at room temperature to remove the organic solvent. The obtained suspension was subjected to ultrasonication using a probe sonicator (Cole-Parmer, Vernon Hills, IL, USA) at 5 s pauses, 5 s ON at an amplitude of 45% to form nanosized particles [29]. A drug suspension without a stabilizer was prepared for comparison studies.



## *2.2. Particle Size, Polydispersity Index, and Zeta Potential Measurements*

The mean particle size, polydispersity index (PDI), and zeta potential were determined by dynamic light scattering using a Malvern Zetasizer Nanoseries ZS® instrument (Malvern Instruments, Malvern, UK) equipped with a backscattered light detector operating at 173◦. The measurements were performed in triplicate at room temperature.

### *2.3. Determination of Percent Drug Entrapment Efficiency (%EE)*

The entrapment efficiency of RT nanocrystals was evaluated indirectly by estimating the unentrapped RT. Briefly, the unentrapped drug was separated from nanocrystals by centrifugation at 15,000 RPM for 30 min at 4 ± 0.5 ◦C using cooling ultracentrifuge. The concentration of the drug in the supernatant was measured spectrophotometrically at λmax of 359 nm (LISCO GmbH, Bargteheide, Germany) [30] and using a calibration curve (y = 0.0151x + 0.1129, where y is the absorbance and x is rutin concentration, *R*<sup>2</sup> = 0.9992). The drug entrapment efficiency (%EE) was determined by applying the following equation:

$$\text{EE}(\%) = \frac{(\text{Total drug } - \text{Drug in supernatant})}{\text{Total drug}} \times 100\tag{1}$$

### *2.4. Stability Studies for RT-NCs*

### 2.4.1. Assessment of RT-NCs Physical Stability

RT-loaded NC dispersions prepared with different stabilizers were stored in a dark place at an ambient temperature for up to three weeks. The physical stability of RT-NC formulations was evaluated by visual appearance and settlement volume ratio (*F*). The settlement volume ratio is the ratio of volume or height before and after sedimentation for a given period [31,32]. It was calculated using the following equation:

$$F = \frac{V}{V\_0} = \frac{H}{H\_0} \tag{2}$$

where *H*<sup>0</sup> is the height of suspension before sedimentation and *H* is the height of sediment surface after sedimentation. *V* and *V*<sup>0</sup> are the suspension volumes after and before sedimentation, respectively.

### 2.4.2. Storage Stability

An aqueous dispersion of the selected formulation (RT-NC2) was transferred into sealed brown glass bottles and stored at two different storage conditions: the room temperature and refrigerated conditions of (4 ◦C) for 60 days. At various time intervals, aliquots were withdrawn and analyzed for their particle size, polydispersity index, and percent drug entrapment efficiency.

### 2.4.3. Storage Chemical Photostability

Free RT and RT-NC2 aqueous dispersions were transferred into transparent glass vials and sealed by rubber stoppers. The vials were exposed to sunlight for 1 month at room temperature. At various time intervals, aliquots were withdrawn and methanol was added to dissolve RT followed by filtration to remove undissolved materials [33]. The RT concentration in the filtrate was measured spectrophotometrically at 359 nm.

The percent of RT remaining after different light exposure times was calculated using the following equation:

$$\text{RT remaining } (\%) = \frac{\text{C}\_{\text{f}}}{\text{C}\_{0}} \times 100 \tag{3}$$

where *C*<sup>0</sup> and *Ct* are the concentrations of RT at zero time and various time intervals, respectively.

### *2.5. Lyophilization of RT-NCs*

RT-NCs were lyophilized to convert them to a dry form. The formulations were transferred to glass flasks then frozen over night at −80 ± 1 ◦C and lyophilized over a period of 48 h using a FreeZone freeze drier (Labconco Inc., Kansas City, MO, USA). The dried nanocrystals were stored in a desiccator until further investigations.

### *2.6. Characterization of Freeze-Dried Powder of RT-NCs*

### 2.6.1. Fourier Transform Infrared Spectroscopy (FT-IR) Studies

The FT-IR spectra of RT alone, HP-β-CD alone, their physical mixture (1:1, *w*/*w*), and selected RT–nanocrystal formulation (RT-NC2) were recorded using a Shimadzu IR-470 spectrophotometer (Shimadzu, Seisakusho Ltd., Kyoto, Japan) at a wavenumber range of 4000–400 cm−1. The potassium bromide (KBr) disc method was used. The

samples were ground, mixed thoroughly with KBr, and compressed into discs using an IR compression machine.

### 2.6.2. Saturation Solubility

An excess amount of sample (equivalent to 5 mg) was placed in a screw-capped glass vial containing 500 mL of phosphate buffer pH 6.5 and shaken in a thermostatically controlled shaking water bath (DAIHAN Scientific Co., Seoul, Republic of Korea) at 50 RPM and 37 ◦C for 48 h until equilibrium was attained [34]. The suspensions were filtered using a membrane disc filter (0.45 μm) and the drug concentration in the filtrate was determined spectrophotometrically at λmax of 359 nm.

### 2.6.3. In Vitro Drug Dissolution Studies

Dissolution studies were performed in pH 6.5 phosphate buffer containing 0.25% *v*/*v* ethanol using an USP XXIV type II dissolution apparatus. Free RT and freeze-dried RT-NCs were dispersed into 500 mL of the dissolution medium and stirred at 50 RPM at 37 ± 0.5 ◦C. At various time intervals (0, 5, 15, 30, 60, 90, and 120 min), an aliquot (5 mL) was withdrawn and replaced immediately with the same volume of fresh dissolution medium. The drug concentration in the withdrawn samples was measured spectrophotometrically at 359 nm. The dissolution experiments were conducted in triplicate.

### 2.6.4. Scanning Electron Microscopy (SEM) Studies

The surface morphology of RT-NCs was examined using scanning electron microscopy (SEM) (Jeol, JSM-5200, Tokyo, Japan). A sample of selected formulation (RT-NC2) was prepared by applying a droplet of RT-NCs onto an aluminum specimen stub, dried overnight, and sputter-coated with gold prior to imaging. An acceleration voltage of 15 kV was utilized.
