*3.8. SEM Observations*

Figure 5 shows an SEM photomicrograph of RT-NCs prepared using HP-β-CD (RT-NC2). The nanocrystals appear as homogenously distributed spherical particles with distinctive boundaries and no aggregation. The size obtained from this measurement was 111.2 ± 29.5 nm. This size is smaller than that measured by DLS (270.5 ± 16.7 nm), probably due to the dry nature of the samples measured by SEM compared to the hydrated particles measured in DLS [62]. During sample measurement in SEM, the hydrated shell collapses during drying in the high-vacuum chamber of the SEM resulting in dried nanoparticles having a smaller particle size [63].

**Figure 5.** Scanning electron microscope photomicrograph of RT NCs prepared using HP-β-CD as a stabilizer (F2).

### *3.9. Characterization of Free RT and RT-NC2 Hydrogels*

A hydrogel formulation was selected to facilitate RT-NC2 application on the skin since previous studies have shown that hydrogels were more efficacious than other vehicles in promoting drug–skin penetration from nanocrystal formulations [64,65]. Hydrogels have high water content, bioadhesive properties, and could serve as a depot system allowing for sustained drug delivery to the skin [66]. The properties of free RT and RT-NC2 hydrogels are shown in Table 5.



### *3.10. Drug Release Studies of Free RT and RT-NC2 Hydrogels*

Figure 6A shows that drug release from the free drug hydrogel was slow whereby around only 30% of the drug was released after 24 h. This is presumably due to the hydrophobic nature of the drug which limits its dissolution rate and aqueous solubility. Release of a drug suspended in a hydrogel base is believed to include two steps: drug dissolution followed by diffusion of the solubilized drug through the hydrogel matrix. In contrast, a much faster drug release was observed for RT-NC2 hydrogel whereby almost complete drug release (~97%) was observed in 12 h. These results agree well with the enhanced dissolution rate and aqueous solubility described above for RT nanocrystals in comparison to the free drug. Similar behavior was also observed previously for nanocrystals suspended in a hydrogel base and was attributed to the small particle size of the nanocrystals leading to larger surface area and smaller diffusion distance and, hence, better drug dissolution and release [67]. The drug release medium was also reported to influence the drug release rate from nanocrystal formulations [42]. For instance, the pH of the release medium was found to affect the ionization status of ionic drugs leading to an important influence on their dissolution and release rate. Moreover, stabilizers that increase the release medium viscosity in the vicinity of a nanocrystal surface decreased the drug release rate from nanocrystal formulations [42].

The release data were analyzed using various mathematical models and the correlation coefficient (*R*2) was calculated to obtain insights into the drug release mechanism (Figure 7) [68]. The *R*<sup>2</sup> values of free RT hydrogel were 0.74, 0.92, 0.87, and 0.97 for the zero order, first order, Higuchi, and Korsmeyer–Peppas models, respectively. In addition, the *R*<sup>2</sup> values of RT-NC2 hydrogel were, respectively 0.84, 0.86, 0.93, and 0.99 for the zero order, first order, Higuchi, and Korsmeyer–Peppas models (Table 6). This indicates that the drug release from both preparations followed the Korsmeyer–Peppas model. The release exponent (n) which indicates the release mechanism was 0.316 and 0.688 for the free drug hydrogel and RT-NC2 hydrogel, respectively. This confirms that the release from free drug hydrogel was governed by Fickian diffusion (case I diffusional) while that from the RT-NC2 hydrogel was governed by anomalous (non-Fickian) transport [69].

**Table 6.** Kinetic parameters of various models of RT release data from free RT and RT-NC2 hydrogels.


**Figure 6.** (**A**) Drug release profiles from RT-NC2 hydrogel in comparison to the free drug hydrogel in phosphate buffer pH 6.5 containing 0.25%, *v*/*v* ethanol at 37 ◦C. (**B**) Cumulative amount of RT permeated per unit surface area of mouse abdominal skin (μg/cm2) for free RT hydrogel and RT-NC2 hydrogel.

**Figure 7.** Plots of RT release data according to different kinetic models. (**A**) Zero order, (**B**) first order, (**C**) Higuchi diffusion model, (**D**) and Korsmeyer–Peppas equation.

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

Figure 6B shows the cumulative amount of RT permeated through mouse abdominal skin for the selected RT-NC2 hydrogel in comparison to the free drug hydrogel. RT nanocrystal hydrogel had significantly higher drug skin permeation where the cumulative drug amounts permeated after 24 h were 456.7 ± 35.5 and 1163.9 ± 33.9 <sup>μ</sup>g·cm−<sup>2</sup> for the free drug hydrogel and nanocrystal hydrogel, respectively. This indicates that the nanocrystals achieved around a 2.5-fold enhancement in the amount of drug permeated through the skin. Furthermore, the flux (*Jss*) and apparent permeability coefficient (*P*app) of the nanocrystal hydrogel were similarly enhanced by around 2.8- and 3.2-fold in comparison to the free drug hydrogel (Table 7), respectively. Similar enhancement in drug skin permeability properties was previously observed in other studies and attributed to the small particle size, enhanced dissolution, and solubility of the nanosized drug particles in comparison to the coarse drug particles. In addition, the nanocrystals might have better adhesion to the skin due to their small particle size and increased contact area with the skin which creates a positive concentration gradient between the nanocrystals and skin and ultimately leads to enhanced drug permeability [65,70,71].

**Table 7.** Ex vivo permeation parameters of RT from free drug hydrogel and RT-NC2 hydrogel through mouse abdominal skin.


<sup>a</sup> Cumulative amount of RT permeated per unit area (μg·cm−2) after 24 h. <sup>b</sup> Flux (permeation rate constant) at steady state (μg·cm−2·h<sup>−</sup>1), obtained from the slope of the regression line after plotting the cumulative amount of RT permeated per unit area vs. time. <sup>c</sup> Apparent permeability coefficient (cm·s−1) calculated from Equation (4).

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

The carrageenan-induced rat paw edema inflammatory model was used to assess the potential of HP-β-CD-stabilized RT-NCs hydrogel to enhance RT's anti-inflammatory properties in comparison to untreated control, free RT hydrogel and diclofenac sodium commercial gel (Olfen® gel) as a standard anti-inflammatory drug. The treatment was initiated 30 min post carrageenan injection and the percent edema was calculated (Figure 8A). The percent edema was highest at zero time for all of the tested preparations. Subsequently, there was a gradual decrease in the percent edema for all of the tested preparations. At any given time point, the percent edema followed this order: Control > free RT hydrogel > Olfen® (diclofenac sodium) commercial gel > RT-NC2 hydrogel. All the differences were statistically significant (*p* < 0.05).

**Figure 8.** (**A**) Percent of paw edema as a function of time in rats after treatment with the selected rutin nanocrystal formulation (RT-NC2) hydrogel in comparison to rats treated with free RT hydrogel, Olfen® gel, and untreated rats. (**B**) Percent of paw edema inhibition as a function of time in rats after treatment with the selected rutin nanocrystal formulation (RT-NC2) hydrogel in comparison to rats treated with free RT hydrogel and commercial Olfen® gel.

The percent edema inhibition was also calculated for the tested preparations and taken as a measure of their anti-inflammatory activity (Figure 8B). Both Olfen® gel and RT-NC2 hydrogel achieved significantly higher percent edema inhibition compared with the free RT hydrogel at all the studied time points (*p* < 0.05). Peak edema inhibition was achieved at 7 h post administration for both Olfen® gel and RT-NC2 hydrogel. At this time point, Olfen® gel and RT-NC2 hydrogel had around 2.2- and 2.5-fold higher edema inhibition compared with free RT hydrogel, respectively. In addition, RT-NC2 hydrogel had significantly higher percent edema inhibition at all the studied time points except at 3 and 6 h compared with Olfen® gel. The enhanced anti-inflammatory activity observed for RT-NC2 hydrogel could be explained on the basis of enhanced drug release and skin permeability (Figure 6) compared with the free drug hydrogel which might facilitate drug delivery to the inflammation site. Interestingly, the RT-NC2 hydrogel also had a better anti-inflammatory effect compared with the commercial diclofenac sodium gel (Olfen® gel) which is presumably due to the nanometric particle size of the RT crystals which enhances drug dissolution and augments its penetration through deep skin layers and eventually results in a better anti-inflammatory effect. This finding is promising since a better anti-inflammatory effect is achieved by the RT-NC2 hydrogel than by the standard non-steroidal, anti-inflammatory drug diclofenac sodium without its notorious side effects which might increase patient compliance. In addition, site-specific drug delivery through topical application is expected to further improve drug safety and efficacy. Previous studies have shown that nanocrystal preparations were able to increase the anti-inflammatory properties of several other drugs [72–74].
