*3.2. Particle Size, Polydispersity Index, and Zeta Potential Measurements*

Table 2 shows the particle size, polydispersity index, zeta potential, and percent drug entrapment efficiency (%EE) for RT-NCs prepared using various stabilizers. The particle sizes ranged from 270.5 ± 16.7 to 505.8 ± 20.5 nm. The smallest size was detected for HP-β-CD RT-NCs while Tween 80 formed the largest particles with the differences being statistically significant at *p* < 0.05 except RT-NC1 versus RT-NC2. The particle size of nanocrystals is controlled by several factors, such as the method of preparation, eventual presence of stabilizers, and the type of stabilizer. The generation of nanocrystals is associated with an enormous increase in surface area due to the production of a large number of small particles and a vast decrease in particle size. This is associated with increasing the system Gibb's free energy leading to thermodynamic instability [42]. These nanoparticles will eventually agglomerate in an attempt to minimize their total energy [43]. Stabilizers (e.g., surfactants and polymers) are thus required to minimize the system free energy and prevent agglomeration. A successful stabilizer should be able to control the particle growth during the production of uniform nanoparticles [42]. The larger size for Tween 80-stabilized nanocrystals might be related to their weak ability to sterically stabilize the nanoparticles, allowing them to grow in size during preparation. On the other hand, the smaller size detected for HP-β-CD-stabilized nanocrystals might be due to their ability to perfectly coat the newly formed nanoparticles which sterically stabilized them and prevented their aggregation and increase in size. These results are in agreement with previous work which showed a larger particle size of Tween 80-stabilized nanocrystals compared to those stabilized by HP-β-CD [41]. The PDI values were in the range of ~0.3 to 0.5, thus indicating the acceptable size distribution of the nanocrystals. A PDI value of 0.05 or smaller indicates a monodispersed population while heterogeneous nanoparticles have a PDI more than 0.7 [44].

The zeta potential was measured for all of the prepared rutin nanocrystal formulations due to its paramount importance for nanoparticle colloidal stability; it represents the electrostatic barrier that prevents nanoparticle aggregation and agglomeration [42,45]. Table 2 shows that the zeta potential of rutin nanocrystals ranged from −12.4 ± 1.0 to −28.8 ± 1.0 mV with the differences being statistically significant at *p* < 0.05 except RT-NC2 versus RT-NC3. RT-NCs had negative zeta potential values, probably due to the adsorption

of water hydroxide ions at the nanocrystal surface [46,47]. It was previously shown that an absolute zeta potential value of around 30 mV is required for good colloidal stability [45]. However, this applies when the stabilization depends on pure electrostatic forces only without contributions from steric stabilization [42]. For instance, it was previously shown that nanosuspensions stabilized by non-ionic polymers and surfactants showed good colloidal stability while having zeta potential values much lower than the suggested value of 30 mV [28,41,48].

**Table 2.** Particle size, polydispersity index, zeta potential, and percent drug encapsulation efficiency of various RT-NCs formulations.


All data are presented as mean ± SD.

### *3.3. Percent Drug Entrapment Efficiency (%EE) Measurements*

The percent drug entrapment efficiency (%EE) ranged from 65.7 ± 0.7% for RT-NCs prepared with Tween 80 to 75.5 ± 0.9% for those prepared with HP-β-CD. The differences were statistically significant at *p* < 0.05 except for those between RT-NC4 and either RT-NC1 or RT-NC3. The %EE was measured by an indirect method where the nanocrystals were separated by centrifugation and the drug content in the supernatant was measured. Thus, the highest %EE for HP-β-CD-stabilized nanocrystals is probably due to its ability to effectively coat and stabilize the nanoparticles which prevented their escape in the supernatant. On the other hand, the relatively lower %EE detected for Tween 80 (non-ionic surfactant) and Pluronic F127 (non-ionic polymer) might be due to their ability to partially solubilize the drug in water through micelle formation which might have facilitated its escape in the supernatant, thus decreasing the %EE [41,49].

In light of the above results RT-NCs with HP-β-CD as a stabilizer were selected for further studies since they showed the smallest particle size making them the most promising candidate to enhance rutin's anti-inflammatory properties and penetration into the skin [18,25]. In addition, these RT-NCs also had the highest %EE of 75.5 ± 0.9%, thus limiting the needed excipients and maximizing the drug/excipient ratio. They also had the highest zeta potential of −28.8 ± 1.0 mV which suggests better colloidal stability compared with other RT-NC preparations.

### *3.4. Stability Studies*

### 3.4.1. Physical Stability of RT-NCs

The settlement volume ratio (*F*), the ratio between the volume or height of the nanocrystal suspension after and before sedimentation for a given period of time, is usually used as an indicator of nanocrystal suspension physical stability [31]. Table 3 shows that the *F* values were in the range of 0.15 to 0.95 with formulation RT-NC2 containing HP-β-CD showing the highest *F* values at all the studied time points. There was a general trend of a decrease in *F* values with time for all the studied preparations. At any time point, the *F* values followed this order: RT-NC2 (HP-β-CD) > RT-NC1 (Pluronic F127) > RT-NC3 (Tween 80) > RT-NC4 (PEG 6000). All the differences were statistically significant (*p* < 0.05). The lowest *F* values were detected for the nanocrystals with PEG 6000 as a stabilizer. Thus, a value of 0.21 ± 0.01 was measured for freshly prepared nanocrystals that gradually decreased to 0.15 ± 0.01 after three weeks indicating poor colloidal stability. In contrast, RT-NC2 containing HP-β-CD had the best colloidal stability as indicated by the highest *F* values among the tested preparations. A value of 0.95 ± 0.03 that was measured for freshly

prepared samples decreased to 0.89 ± 0.05 after three weeks with no significant difference (*p* > 0.05). This high stability might be related to the relatively higher zeta potential of −28.8 ± 1.0 mV for HP-β-CD-stabilized nanocrystals in addition to their ability to sterically stabilize the nanocrystals. Similar high stability was previously observed for HP-β-CDstabilized daidzein nanocrystals confirming its ability to efficiently coat the nanocrystals and prevent their agglomeration over time [41]. These results support the selection of formulation RT-NC2 for further studies.

**Table 3.** Settlement volume ratios for various RT nanocrystals after storage at room temperature for various time periods.


### 3.4.2. Storage and Photostability

To further characterize the stability of RT nanocrystals, the selected formulation (RT-NC2 stabilized by HP-β-CD) was stored at room temperature (25 ◦C) and refrigerated conditions (4 ◦C) for 60 days and their particle size, polydispersity index, and percent drug entrapment efficiency were determined. Table 4 shows that there was a gradual decrease in the percent drug entrapment efficiency with time. Thus, after 60 days of storage the %EE decreased from 75.53 ± 0.91 to 71.23 ± 1.07 and 70.00 ± 1.00 for the samples stored at 4 ◦C and 25 ◦C, respectively. The %EE after 60 days was significantly smaller than that of either zero time or 30 days of storage (*p* < 0.05). Furthermore, the storage temperature had no important influence on the %EE as evidenced by a non-significant difference between the samples stored at 4 ◦C and 25 ◦C. The small decrease in %EE over time might be attributed to the drug solubilization over time by HP-β-CD which converts the drug from a nanocrystal to a solubilized form and facilitates its escape to the surrounding bulk medium.

**Table 4.** Effect of storage at room temperature (25 ◦C) and 4 ◦C on the percent drug entrapment efficiency (%EE), particle size (nm), and polydispersity index (PDI) of RT nanocrystals formulation RT-NC2.


Regarding the particle size, there was a general size increase over time regardless of the storage temperature, albeit the increase at 25 ◦C was higher than at 4 ◦C. Thus, the size after 60 days of storage at 25 ◦C was significantly bigger compared to that of all other tested samples (*p* < 0.05). This indicates that storage in refrigerated conditions is advisable for these nanocrystals. A similar bigger particle size at a higher storage temperature was observed in other pieces of research and attributed to the increase in nanoparticle kinetic energy at higher temperatures leading to a higher probability of particle collisions and subsequently increasing the particle size [41,50,51]. Similarly, there was a general increase in the PDI values over time. However, the differences were not significant compared with the freshly prepared nanocrystals (*p* > 0.05).

Concerning RT photostability, previous studies have shown that RT is susceptible to photodegradation where exposure to UVB radiation for 120 min resulted in a decrease

of 13.6% in RT content [52]. Our results show that light exposure caused progressive degradation of free RT (Figure 1). Thus, after 4 weeks of light exposure, only 42.7 ± 0.7% was remaining for free RT. In contrast, RT-NC2 had much better stability against light exposure. For instance, after the same time, the remaining RT for the nanocrystals was 95.1 ± 3.4%. This confirms that the nanocrystals stabilized by HP-β-CD had around 2.3-fold better RT photostability. This much better stability for the nanocrystals might be attributed to the protection effect offered by HP-β-CD where it covers the drug nanocrystals. These results are in agreement with previous reports showing better photostability of RT when formulated into nanoparticles [53].

**Figure 1.** Percent remaining of RT as a function of exposure time to light for free RT and RT nanocrystals formulation RT-NC2.
