*3.5. FT-IR Spectroscopy Studies*

The potential of chemical interactions between rutin and HP-β-CD in RT-NC2 was studied by recording the FT-IR spectra of rutin alone, HP-β-CD alone, their physical mixture (1:1, *w*/*w*), and the selected nanocrystal formulation (HP-β-CD-stabilized nanocrystals) and the results are shown in Figure 2. The spectrum of rutin alone shows a broad band centered at around 3430 cm−<sup>1</sup> for OH bending, a sharp band at 1654 cm−<sup>1</sup> due to C = O stretching, and a sharp band at 1594 cm−<sup>1</sup> for C = C stretching of aromatic structures which is in agreement with published reports [54]. The spectrum of HP-β-CD alone shows a broad band centered at around 3400 cm−<sup>1</sup> ascribed for vibration of free –OH groups and a band at 2927 cm−<sup>1</sup> for vibration of bound -OH groups. The spectrum of the rutin/HP-β-CD physical mixture shows as sharp band at 1652 cm−<sup>1</sup> ascribed to the stretching of rutin carbonyl groups while the stretching of rutin C = C of aromatic structures is slightly shifted to 1614 cm<sup>−</sup>1. These bands appeared at the same wavenumbers in the spectrum of RT-NC2 nanocrystals (1652 and 1614 cm−1, respectively) confirming the absence of chemical or physical interactions between rutin and HP-β-CD.

**Figure 2.** FT-IR spectra of rutin alone, HP-β-CD alone, their physical mixture (1:1, *w*/*w*), and HP-β-CD-stabilized nanocrystals (formulation RT-NC2).

### *3.6. Saturation Solubility Measurements*

RT is a hydrophobic compound with poor aqueous solubility which limits its bioavailability and clinical benefits [55,56]. The results obtained (Figure 3) show that RT solubility in phosphate buffer pH 6.5 was 1.8 ± 0.7 μg/mL. Rutin is a weak acid with *p*Ka in the range of 7.1 to 11.65 leading to a pH-dependent solubility profile [57]. Conversion of RT into NCs resulted in a significant increase in its aqueous solubility for all the tested stabilizers (Figure 3) (*p* < 0.05). For instance, NCs showed around a 102- to 202-fold increase in RT aqueous solubility that was dependent on the type of the stabilizer. The degree of solubility enhancement followed this descending order: HP-β-CD (RT-NC2) > Pluronic F127 (RT-NC1) > Tween 80 (RT-NC3) > PEG 6000 (RT-NC4). This might be related to the nanocrystal particle size where HP-β-CD-stabilized nanocrystals had the smallest particle size of 270.5 ± 16.7 nm among the tested stabilizers (Table 2). According to the Ostwald–Freundlich equation, the decrease in particle size results in increasing the particles' surface area which in turn leads to increasing rutin's aqueous solubility [58,59]. However, particle size is not the only factor influencing aqueous solubility. For example, Tween 80-stabilized nanocrystals had a bigger size than those stabilized by PEG 6000 but they had better solubility (Table 2). This is presumably attributed to the ability of Tween 80 to form micelles that encapsulate hydrophobic drugs such as rutin and increase their aqueous solubility [41,49].

The drug physical mixtures with the used stabilizers also achieved significantly higher drug aqueous solubility compared with the free drug hydrogel (*p* < 0.05) [60]. This might be attributed to the hydrophilicity of the used stabilizers which facilitates drug dissolution and solubility in water. In addition, the nanocrystals had significantly higher drug solubility compared with the corresponding physical mixture. This is probably due to the size reduction and increase in surface area achieved by the nanocrystals.

**Figure 3.** Saturation solubility of various RT-NCs in phosphate buffer pH 6.5 in comparison to free RT and various corresponding physical mixtures (PM). RT-NC1: rutin nanocrystals formulation 1, RT-NC2: rutin nanocrystals formulation 2, RT-NC3: rutin nanocrystals formulation 3, RT-NC4: rutin nanocrystals formulation 4.

### *3.7. Drug Dissolution Studies*

Figure 4 shows the percent RT dissolved as a function of time for various nanocrystal formulations in comparison to the free drug. Free RT had the slowest dissolution rate among the tested preparations where around only 25% was dissolved after 120 min. RT is known as a hydrophobic compound with a slow dissolution rate which explains this slow dissolution [54]. Interestingly, the nanocrystal formulation RT-NC2 containing HP-β-CD as a stabilizer achieved 100% drug dissolution in 30 min compared with around only 15% for the free drug. Other nanocrystal formulations had significantly faster drug dissolution rates compared with the free drug (*p* < 0.05). However, except for RT-NC1, RT-NC2 had significantly faster drug dissolution compared with the other tested RT-NC formulations after 30 min (*p* < 0.05). After 30 min, the percent of drug dissolved followed this descending order: RT-NC2 > RT-NC1 > RT-NC3 > RT-NC4. Thus, they had 2.3-, 4.9-, 6-, and 6.7-fold higher dissolution rates compared with the free drug, respectively. This is the same order observed above for the saturation aqueous solubility and is probably attributed to the effect of particle size, surface area, and micelle formation on the drug dissolution rate. Previous studies have shown that the mechanism by which a given stabilizer enhanced the drug dissolution rate might have a more important influence compared with the particle size. Thus, etodolac nanocrystals' dissolution rate was affected by the particle size, as well as the type of stabilizer [42]. The % etodolac dissolved for β-cyclodextrin-stabilized nanocrystals with a particle size of 866 nm was higher than that observed for Tween 80 stabilized nanocrystals with a particle size of 393 nm. This observation was attributed to the ability of β-cyclodextrin to form a water-soluble inclusion complex with etodolac which increased its dissolution rate [61]. The viscosity of the dissolution medium and its ability to influence the drug ionization status were also found to affect the drug dissolution rate [42].

**Figure 4.** Dissolution profiles of various RT-NCs in phosphate buffer pH 6.5 at 37 ◦C in comparison to free RT.
