*2.7. Preparation and Characterization of Free RT and RT-NCs Hydrogels*

An aqueous warm dispersion of a weighed amount of hydroxypropyl methyl cellulose (HPMC 15000) (5%, *w*/*w*) was developed with continuous stirring until plain gel was formed. This concentration of HPMC was selected based on previous work to produce hydrogels with desirable viscosity and homogeneity. The dispersion was sonicated for 15 min to remove air bubbles. RT dispersion in distilled water was added slowly to 10 mL of aqueous HPMC dispersion while stirring took place until a homogenous RT hydrogel was formed. The calculated amount of freeze-dried selected RT-NC2 formulation was incorporated into HPMC plain gel 5%, *w*/*v* by magnetic stirring and the final weight of the gel was adjusted to 10 g with distilled water. The RT concentration in the free RT and RT-NC2 hydrogels was 0.5%, *w*/*w*. The prepared free RT and RT-NC2 hydrogels were left in the fridge for further studies. The viscosity of the hydrogel was measured by a Brookfield Digital Viscometer (Model DV-II Brookfield Engineering Laboratories, Inc., Stoughton, MA, USA). The pH of the free RT and RT-NC2 hydrogels was measured using a pH meter (3500 pH meter, Jenway, UK). The RT content of the hydrogels was measured by dissolving 0.5 g in methanol and the drug concentration was measured spectrophotometrically at λmax of 359 nm.

### *2.8. In Vitro Drug Release Studies from Hydrogels*

The release of RT from free RT and RT-NC2 hydrogels was characterized using the dialysis method through a semi-permeable cellophane membrane (molecular weight cutoff 12,000–14,000, Sigma Aldrich, St. Louis, MO, USA) as mentioned previously with slight modification [35]. Briefly, the tested formulation (1 g of hydrogel equivalent to 5 mg RT) was placed over a previously soaked cellophane membrane fitted at the bottom of a glass tube open at both sides. The glass tube was immersed in a beaker containing 100 mL of phosphate buffer pH 6.5 with 0.25%, *v*/*v* ethanol. The beakers were placed in a thermostatically controlled shaking water bath, (DAIHAN Scientific Co., Seoul, South Korea) operating at 50 RPM and 37 ± 0.5 ◦C. Aliquots of 5 mL were withdrawn at intervals of 0.5, 1, 2, 4, 6, 12, and 24 h. The withdrawn samples were immediately replaced by equal volumes of the same medium. The drug content of the release samples was estimated spectrophotometrically at λmax of 359 nm. The experiments were performed in triplicate.

### *2.9. Kinetic Evaluation of the Release Data*

The data obtained from the in vitro release studies were analyzed using curve fitting to different kinetic models (zero order, first order, Higuchi diffusion model, and Korsmeyer– Peppas equation) [36]. The model that best described the data was selected based on the highest correlation coefficient (*R*2).

### *2.10. Ex Vivo Skin Permeation Study*

Skin permeation studies of RT were carried out for the selected RT-NC hydrogel formulation (RT-NC2) and free RT hydrogel using the abdominal skin of a male mouse according to previously described procedures [37]. The study protocol was approved by The Research Ethics Committee, Faculty of Pharmacy, South Valley University, Egypt (approval number P.S.V.U 125/22). The animals were sacrificed, the dorsal hair was removed and the skin was cleaned three times with phosphate buffer pH 7.4. Fresh skin specimens were stretched over one end of the open-ended glass tubes with a total base surface area of 3.14 cm2 using an elastic rubber band. The tested gel formulations (1 g of free RT or RT-NC2 hydrogels equivalent to 5 mg of RT) were placed over the skin surface. The glass tubes were dipped in a glass beaker containing 100 mL of phosphate buffer (pH 6.5 with 0.25%, *v*/*v* ethanol). The beakers were shaken at 50 RPM and 37 ± 0.5 ◦C for 24 h in a thermostatic shaker water bath. At different time intervals (0.5, 1, 2, 4, 6, 12, and 24 h), samples of 5.0 mL were withdrawn, replaced with an equal volume of the fresh release medium, and analyzed spectrophotometrically at λmax of 359 nm for RT content. The measurements were carried out in triplicate. The cumulative amount of drug permeated per unit surface area was plotted as a function of time. The slope of the linear regression line was taken as the steady state flux (*Jss*, <sup>μ</sup>g·cm−2·h−1) [38]. The apparent permeability coefficient (*P*app, cm·h<sup>−</sup>1) was calculated using the following equation:

$$P\_{\rm app} = \frac{J\_{ss}}{\mathcal{C}\_0} \tag{4}$$

where *C*<sup>0</sup> is the initial concentration of RT (μg/mL) in the donor compartment.

### *2.11. In Vivo Anti-Inflammatory Paw Edema Studies*

The acute anti-inflammatory activity for the selected hydrogel formulation was performed using a carrageenan-induced rat paw edema model [39]. The study protocol was approved by The Research Ethics Committee, Faculty of Pharmacy, South Valley University, Egypt (approval number P.S.V.U 125/22). The approximate weight of each rat was 200 g. The rats were randomly divided into four groups, each of four rats. Carrageenan (1%, *w*/*v*) in saline solution was injected subcutaneously into the left hind paw of the rats for the induction of edema. Group 1 received a placebo HPMC hydrogel and was used as an untreated control. Groups 2 and 3 received free RT and RT-NC2 hydrogels, respectively. Group 4 received a marketed diclofenac sodium gel 1% (Olfen®, Medical Union Pharmaceuticals, Cairo, Egypt) as a reference anti-inflammatory agent. The tested formulations were applied on the edematous paw 30 min post induction which was considered as the zero time of treatment. The growth in the paw thickness was determined using a vernier caliper. The measurements were performed in triplicate. The percent edema and percent edema inhibition were calculated using the following equations:

$$\text{Edema (E, \%)} = \frac{V\_l - V\_0}{V\_0} \times 100\tag{5}$$

$$\text{Edema inhibition} \left( \% \right) = \frac{E\_{\mathcal{L}} - E\_{\mathcal{l}}}{E\_{\mathcal{L}}} \times 100 \tag{6}$$

where *V*<sup>0</sup> and *Vt* are the mean paw volume before and after carrageenan injection at time *t*, respectively. *Ec* and *Et* are the edema percentages of control and treated groups at the same time interval, respectively.

### *2.12. Statistical Analyses*

The experiments were run in triplicate and the results were represented as mean ± SD. GraphPad Prism software version 8.0.1 (GraphPad Software Inc., La Jolla, CA, USA) was used to statistically analyze the data. One-way analysis of variance analysis (ANOVA) with Tukey's post-hoc test was used. A difference of *p* < 0.05 was predefined as statistically significant.

### **3. Results and Discussion**

### *3.1. RT-NCs Preparation and Characterization*

Despite the enormous advantages of nanocrystals including high drug loading and improved dissolution and saturation solubility, they suffer from poor physical stability that results from their small particle size and the associated increase in free energy leading to aggregation [40,41]. To enhance RT-NCs' stability, various stabilizers were used in this study including nonionic surfactants such as Tween 80 and nonionic polymers such as Pluronic F127, HP-β-CD, and PEG 6000. They are believed to stabilize nanocrystals through adsorption on their surface forming protective layers against particle aggregation and crystal growth [41].
