3.1.1. Measurement of PS and PDI

PS and PDI measurements were carried out for both G4.5 complexes and the empty PAMAM dendrimers G4.5 and results of PS and PDI for the G4.5 complexes and the empty PAMAM dendrimers G4.5 are demonstrated in Table 2, Figure S1A and Figure S2A, respectively. According to the manufacturer, PS of free PAMAM G4.5 is 143.0 ± 19.6 nm. Results obtained from Zetasizer demonstrated that PS of the empty PAMAM dendrimers G4.5 was 186.0 ± 2.3 nm. When loaded with RBX, the measurements revealed that PS of G4.5 complexes 1:1, 2.5:1, 5:1, and 25:1 was increased to 367.0, 416.0, 289.0, and 301.0 nm, respectively. However, G4.5 complex 25:1 possessed less PS among all studied complexes. PAMAM dendrimers possess lower PDI when compared to other nanoparticles. PDI was measured for G4.5 complexes and the empty PAMAM dendrimers G4.5 and revealed that there is no significant change in PDI measurement of the dendrimers after loading it with RBX (*p* > 0.05). However, G4.5 complex 25:1 possessed the least PDI among all studied nanoformulations (PDI ≤ 0.35).

**Table 2.** PS, PDI and ζ-potential of blank and loaded PAMAM dendrimers G4.5 and G5 (mean ± SD, *n* = 3).


\*\* *p* ≤ 0.01.

3.1.2. Measurement of ζ-Potential of G4.5 Complexes

ζ-potential measurements were carried out for G4.5 complexes and the empty PAMAM dendrimers G4.5, and results are demonstrated in Table 2. In this study, ζ-potential measurements demonstrated that ζ-potential of empty PAMAM dendrimers G4.5 is −44.0 ± 2.0 mV. After the loading process, ζ-potential measurements of G4.5 complexes 1:1, 2.5:1, 5:1 and 25:1 was −16.2, −5.1, −6.4, and −13.0 mV, respectively. In fact, the surface charge of PA-MAM dendrimers plays an important role in permeability of these nanoparticles through the negatively charged ocular barriers. In this work, it was found that the loaded G4.5 complex 25:1 have preserved its negative charge after the loading process. In summary, the previous features demonstrated with G4.5 complex 25:1 are needed for the non-invasive ocular delivery of RBX.

#### 3.1.3. Determination of DE%

The DE% of G4.5 complexes were determined and results are presented in Figure S3A. In this study, DE% of the studied nanoparticles was similar. However, G4.5 complex 25:1 showed the best DE% among all the other proposed complexes though statistically not significant.

#### 3.1.4. In Vitro Drug Release of G4.5 Complexes

Release profiles of G4.5 complexes were studied in order to evaluate the amount of RBX that will be released following the topical instillation of the nanoparticles. As indicated in Figure 1, most of the G4.5 complexes presented a sustained release of RBX. It was shown that at pH 7.4 in a period of 8 h, RBX release of G4.5 complexes 1:1, 2.5:1, and 5:1 was 47.3%, 72.5%, and 79.0%, respectively. In particular, it was observed that the RBX release rate of G4.5 complexes 25:1 was distinctly higher than all the other studied complexes. For a period of 8 h, this complex possessed an initial release of 11.9%. After that, RBX release was gradually increased in a sustained manner up to 86.1%. Moreover, the previous in vitro characterizations of the proposed complexes demonstrated that G4.5 complex 25:1 has revealed the best in vitro release as well as the highest DE% among all other proposed nanoparticles. Because of these remarkable results of G4.5 complex 25:1, this complex was chosen for the next experiments. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 9 of 23

**Figure 1.** In vitro release profiles of different complexes (mean ± SD, *n* = 6). **Figure 1.** In vitro release profiles of different complexes (mean ± SD, *n* = 6).

3.1.5. Stability Studies of G4.5 Complex 3.1.5. Stability Studies of G4.5 Complex

#### Physical Stability Physical Stability

complex 25:1.

Chemical Stability

The stability of the G4.5 25:1 was studied at daylight and in the dark in different temperature conditions ranging from (4–50 °C). For a period of 6 months, the nanoformulations were stored in colorless Eppendorfs and observed for any sign of color change, precipitation, or turbidity. Results are presented in Table S2. After the study period ended, the nanoformulations stored in the dark at 4 °C were examined by the eye inspection and did not show any signs of physical change. In daylight, the sample kept at 25 °C was visualized and showed a color change from pink to colorless while the sample kept in the dark did not show any physical changes. Moreover, a precipitate and a color change were also observed at higher temperatures, 37 °C and 50 °C, respectively. The previous investigation indicated that it is suitable to store the nanoparticles in amber containers at 4 °C. In addition, these nanoparticles were visualized using SEM to assess their shape and size. As seen in Figure 2A, the complex possessed an almost spherical shape and particle size The stability of the G4.5 25:1 was studied at daylight and in the dark in different temperature conditions ranging from (4–50 ◦C). For a period of 6 months, the nanoformulations were stored in colorless Eppendorfs and observed for any sign of color change, precipitation, or turbidity. Results are presented in Table S2. After the study period ended, the nanoformulations stored in the dark at 4 ◦C were examined by the eye inspection and did not show any signs of physical change. In daylight, the sample kept at 25 ◦C was visualized and showed a color change from pink to colorless while the sample kept in the dark did not show any physical changes. Moreover, a precipitate and a color change were also observed at higher temperatures, 37 ◦C and 50 ◦C, respectively. The previous investigation indicated that it is suitable to store the nanoparticles in amber containers at 4 ◦C. In addition, these nanoparticles were visualized using SEM to assess their shape and size. As seen in Figure 2A, the complex possessed an almost spherical shape and particle size equivalent to those measured by Zetasizer Nano ZS.

**Figure 2.** Scanning electron microscope (SEM) overview image of (**A**) G4.5 complex 25:1 and (**B**) G5

The present work is a quantitative study of the stability of RBX to assess the effect of light source and temperature overtime. This test will help in choosing the best storing conditions for the nanoparticles. Samples of G4.5 complex 25:1 containing 1 μg of RBX was stored at the dark and daylight at different temperatures (4–50 °C). Each one of these samples was kept for 1, 3, and 6 months and analyzed by UPLC MS/MS after the end of each tested period. To determine percent content of RBX for each studied sample, the remaining amount of RBX was quantified from the equation of calibration curve using peak

equivalent to those measured by Zetasizer Nano ZS.

The stability of the G4.5 25:1 was studied at daylight and in the dark in different temperature conditions ranging from (4–50 °C). For a period of 6 months, the nanoformulations were stored in colorless Eppendorfs and observed for any sign of color change, precipitation, or turbidity. Results are presented in Table S2. After the study period ended, the nanoformulations stored in the dark at 4 °C were examined by the eye inspection and did not show any signs of physical change. In daylight, the sample kept at 25 °C was visualized and showed a color change from pink to colorless while the sample kept in the dark did not show any physical changes. Moreover, a precipitate and a color change were also observed at higher temperatures, 37 °C and 50 °C, respectively. The previous investigation indicated that it is suitable to store the nanoparticles in amber containers at 4 °C. In addition, these nanoparticles were visualized using SEM to assess their shape and size. As seen in Figure 2A, the complex possessed an almost spherical shape and particle size

**Figure 1.** In vitro release profiles of different complexes (mean ± SD, *n* = 6).

3.1.5. Stability Studies of G4.5 Complex

equivalent to those measured by Zetasizer Nano ZS.

Physical Stability

**Figure 2.** Scanning electron microscope (SEM) overview image of (**A**) G4.5 complex 25:1 and (**B**) G5 complex 25:1. **Figure 2.** Scanning electron microscope (SEM) overview image of (**A**) G4.5 complex 25:1 and (**B**) G5complex 25:1.

#### Chemical Stability Chemical Stability

The present work is a quantitative study of the stability of RBX to assess the effect of light source and temperature overtime. This test will help in choosing the best storing conditions for the nanoparticles. Samples of G4.5 complex 25:1 containing 1 μg of RBX was stored at the dark and daylight at different temperatures (4–50 °C). Each one of these samples was kept for 1, 3, and 6 months and analyzed by UPLC MS/MS after the end of each tested period. To determine percent content of RBX for each studied sample, the remaining amount of RBX was quantified from the equation of calibration curve using peak The present work is a quantitative study of the stability of RBX to assess the effect of light source and temperature overtime. This test will help in choosing the best storing conditions for the nanoparticles. Samples of G4.5 complex 25:1 containing 1 µg of RBX was stored at the dark and daylight at different temperatures (4–50 ◦C). Each one of these samples was kept for 1, 3, and 6 months and analyzed by UPLC MS/MS after the end of each tested period. To determine percent content of RBX for each studied sample, the remaining amount of RBX was quantified from the equation of calibration curve using peak area values and then percent content of RBX was calculated. Results obtained from the stability studies are shown in Figure S4A. Data indicated that RBX is more stable when stored in amber containers. Upon protecting the nanoformulation from light, higher percent content of RBX was demonstrated. In addition, it was observed that the stability of RBX in PAMAM dendrimers G4.5 was slightly decreased with time. In a duration of 6 months, measurements of percent content of RBX in G4.5 complex 25:1 remain above 80.0% in this time period. Moreover, data show that RBX is more stable within PAMAM dendrimers at 4 > 25 > 37 > 50 ◦C. In fact, the highest percent content of RBX was achieved when storing the nanoparticles in the dark at 4 ◦C. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 10 of 23 area values and then percent content of RBX was calculated. Results obtained from the stability studies are shown in Figure S4A. Data indicated that RBX is more stable when stored in amber containers. Upon protecting the nanoformulation from light, higher percent content of RBX was demonstrated. In addition, it was observed that the stability of RBX in PAMAM dendrimers G4.5 was slightly decreased with time. In a duration of 6 months, measurements of percent content of RBX in G4.5 complex 25:1 remain above 80.0% in this time period. Moreover, data show that RBX is more stable within PAMAM dendrimers at 4 > 25 > 37 > 50 °C. In fact, the highest percent content of RBX was achieved when storing the nanoparticles in the dark at 4 °C.

#### TEM TEM

G4.5 complex 25:1 and the empty PAMAM dendrimers G4.5 were visualized by TEM to determine their morphology and to confirm the loading of RBX. Figure 3 displays the morphology of empty PAMAM dendrimers G4.5 as well as G4.5 complex 25:1. G4.5 complex 25:1 and the empty PAMAM dendrimers G4.5 were visualized by TEM to determine their morphology and to confirm the loading of RBX. Figure 3 displays the morphology of empty PAMAM dendrimers G4.5 as well as G4.5 complex 25:1.

**Figure 3.** Transmission electron microscope (TEM) overview images of (**A**) empty PAMAM dendrimers G4.5 and (**B**) G4.5 complex 25:1. **Figure 3.** Transmission electron microscope (TEM) overview images of (**A**) empty PAMAM dendrimers G4.5 and (**B**) G4.5 complex 25:1.

PS and PDI measurements were carried out for both G5 complexes and the empty PAMAM dendrimers, and results are demonstrated in Table 2, Figures S1B, and S2B. According to the manufacturer, the PS of free PAMAM G5 dendrimers is 224.0 ± 13.0 nm. Results obtained from Zetasizer demonstrated that PS of the empty PAMAM dendrimers G5 was 214.9 ± 8.5 nm. When loaded with RBX, the measurements revealed that PS of G5 complexes 1:1, 2.5:1, 5:1, and 25:1 was increased to 461.2, 482.4, 669.8 and 307.1 nm, respectively. Indeed, PS increase of loaded PAMAM dendrimers may be attributed to RBX entrapment within the internal cavities of PAMAM dendrimers. However, G4.5 complex 25:1 possessed the lowest PS among all studied nanoformulations. PDI was measured for the free and loaded PAMAM dendrimers and revealed that there are no significant changes in PDI of the dendrimers after loading it with RBX except for G5 complex 5:1. In fact, G5 complex 25:1 possessed the least PDI among all studied nanoformulations (PDI ≤ 0.39). However, a significant increase in PDI was demonstrated with G5 complex 5:1 (Ta-

ζ-potential provided by the manufacturer indicated that the neutral PAMAM G5 dendrimers possesses a neutral charge. In this study, ζ-potential measurements demonstrated that the surface charge of the empty PAMAM dendrimers G5 was −0.1 ± 0.0 mV. Table 2 demonstrates ζ-potential of G5 complexes. After the loading process, ζ-potential of G5 complexes 1:1, 2.5:1, 5:1, and 25:1 was 4.3, 5.5, 9.7, and −0.0 mV, respectively. Indeed,

3.2.2. Measurements of ζ-Potential of G5 Complexes

*3.2. Characterization of G5 Complexes*

ble 2 and Figure S2B).

#### *3.2. Characterization of G5 Complexes*

#### 3.2.1. Measurement of PS and PDI

PS and PDI measurements were carried out for both G5 complexes and the empty PAMAM dendrimers, and results are demonstrated in Table 2, Figure S1B, and Figure S2B. According to the manufacturer, the PS of free PAMAM G5 dendrimers is 224.0 ± 13.0 nm. Results obtained from Zetasizer demonstrated that PS of the empty PAMAM dendrimers G5 was 214.9 ± 8.5 nm. When loaded with RBX, the measurements revealed that PS of G5 complexes 1:1, 2.5:1, 5:1, and 25:1 was increased to 461.2, 482.4, 669.8 and 307.1 nm, respectively. Indeed, PS increase of loaded PAMAM dendrimers may be attributed to RBX entrapment within the internal cavities of PAMAM dendrimers. However, G4.5 complex 25:1 possessed the lowest PS among all studied nanoformulations. PDI was measured for the free and loaded PAMAM dendrimers and revealed that there are no significant changes in PDI of the dendrimers after loading it with RBX except for G5 complex 5:1. In fact, G5 complex 25:1 possessed the least PDI among all studied nanoformulations (PDI ≤ 0.39). However, a significant increase in PDI was demonstrated with G5 complex 5:1 (Table 2 and Figure S2B).

### 3.2.2. Measurements of ζ-Potential of G5 Complexes

ζ-potential provided by the manufacturer indicated that the neutral PAMAM G5 dendrimers possesses a neutral charge. In this study, ζ-potential measurements demonstrated that the surface charge of the empty PAMAM dendrimers G5 was −0.1 ± 0.0 mV. Table 2 demonstrates ζ-potential of G5 complexes. After the loading process, ζ-potential of G5 complexes 1:1, 2.5:1, 5:1, and 25:1 was 4.3, 5.5, 9.7, and −0.0 mV, respectively. Indeed, the free and loaded PAMAM G5 dendrimers possessed a neutral charge even after the loading process. Results indicated that ζ-potential of PAMAM dendrimers G5 were not affected by the drug loading process (*p* > 0.05).

#### 3.2.3. Determination of DE%

DE% of the studied nanoparticles showed similar percent content between G5 complexes. Although not statistically significant, G5 complex 25:1 showed the highest DE% among all the other proposed complexes. Results of DE% are presented in Figure S3B.

#### 3.2.4. In Vitro Drug Release of G5 Complexes

As indicated in Figure 1, nearly all G5 complexes possessed a sustained release of RBX. It was shown that at pH 7.4 in a period of 8 h, in vitro release of G5 complexes 1:1, 2.5:1, and 5:1 was 31.3%, 35.8%, and 25.3%, respectively. In particular, RBX release rate of G5 complexes 25:1 was distinctly higher than all the other studied complexes. For a period of 8 h, this complex possessed an initial release of 6.3%. After that, RBX release was gradually increased in a sustained manner up to 81.0%. In fact, among the proposed complexes, G5 25:1 has demonstrated the best in vitro characterizations. Because of these remarkable results of G4.5 complex 25:1, the next studies were manipulated for this complex.
