*4.6. pH and Osmolarity*

The pH of the optimized rivastigmine-loaded NLC formulations produced by ultrasound technique and HPH method were adjusted to the nasal mucosa values (5.5–6.6) with a dilute HCl solution. Similarly, the osmolarity was adjusted with glycerin to the physiological range of 230–320 mOsm/kg, obtaining isotonic formulations compatible with the nasal mucosa [100]. The final values for the pH and osmolarity of the optimized rivastigmine-loaded NLC formulations were 6.22 ± 0.01 and 280 ± 1 mOsm/Kg for the ones produced by HPH method; and 6.21 ± 0.01 and 279 ± 1 for the ones produced by ultrasound technique. Furthermore, it was confirmed that the CQAs values were not altered after addition of HCl and glycerin (Supplementary Data, Section 4, Table S12).

#### *4.7. In Vitro Drug Release Studies 4.7. In Vitro Drug Release Studies*

*4.6. pH and Osmolarity* 

The release profile of rivastigmine from the NLC produced by HPH method and ultrasound technique was assessed in phosphate-buffered solution at pH 6.4 (Figure 4) and in simulated nasal electrolyte solution at pH 6.4 (Figure 5) over a period of 48 h. The release profile of rivastigmine from the NLC produced by HPH method and ultrasound technique was assessed in phosphate-buffered solution at pH 6.4 (Figure 4) and in simulated nasal electrolyte solution at pH 6.4 (Figure 5) over a period of 48 h.

altered after addition of HCl and glycerin (Supplementary Data, Section 4, Table S12).

*Pharmaceutics* **2020**, *12*, 599 17 of 27

**Table 5.** Observed and predicted response values of the two optimized rivastigmine-loaded

**Observed Responses Ultrasound Technique High-Pressure Homogenization (HPH) Method** 

**Predicted Responses Ultrasound Technique High-Pressure Homogenization (HPH) Method** 

Results presented as mean ± SD (*n* = 3); 1 Z-Ave: mean particle size; 2 PDI: polydispersity index; 3 ZP:

The pH of the optimized rivastigmine-loaded NLC formulations produced by ultrasound technique and HPH method were adjusted to the nasal mucosa values (5.5–6.6) with a dilute HCl solution. Similarly, the osmolarity was adjusted with glycerin to the physiological range of 230–320 mOsm/kg, obtaining isotonic formulations compatible with the nasal mucosa [100]. The final values for the pH and osmolarity of the optimized rivastigmine-loaded NLC formulations were 6.22 ± 0.01 and 280 ± 1 mOsm/Kg for the ones produced by HPH method; and 6.21 ± 0.01 and 279 ± 1 for the ones

Z-Ave **<sup>1</sup>** (nm) 114.000 ± 1.910 109.000 ± 0.850 PDI **<sup>2</sup>** 0.221 ± 0.003 0.196 ± 0.007 ZP **<sup>3</sup>** (mV) −30.633 ± 0.288 −30.466 ± 0.252 EE **<sup>4</sup>** (%) 96.987 ± 0.446 97.174 ± 0.297

Z-Ave **<sup>1</sup>** (nm) 155.000 124.000 PDI **<sup>2</sup>** 0.190 0.242 ZP **<sup>3</sup>** (mV) −28.400 −29.100 EE **<sup>4</sup>** (%) 95.140 97.600

nanostructured lipid carriers (NLC) formulations.

zeta potential; 4 EE: encapsulation efficiency.

**Figure 4.** Cumulative percentage of drug release in phosphate-buffered solution (pH 6.4) from rivastigmine-loaded nanostructured lipid carriers (NLC) produced by ultrasound technique and **Figure 4.** Cumulative percentage of drug release in phosphate-buffered solution (pH 6.4) from rivastigmine-loaded nanostructured lipid carriers (NLC) produced by ultrasound technique and rivastigmine-loaded NLC produced by high-pressure homogenization (HPH) method.

rivastigmine-loaded NLC produced by high-pressure homogenization (HPH) method.

From Figure 4, an initial fast drug release can be observed from both rivastigmine-loaded NLC formulations, which is related to the drug diffusion from the surface of the NLC to the dissolution From Figure 4, an initial fast drug release can be observed from both rivastigmine-loaded NLC formulations, which is related to the drug diffusion from the surface of the NLC to the dissolution medium, followed by a prolonged release [15,98]. This phenomenon can be explained by the rapid solidification of the solid lipids during the formation of the NLC, which results in nanoparticles with an internal core containing a low amount of liquid lipids, which in turn accumulate in the outermost layers. As rivastigmine is an oil, it also tends to accumulate on the surface of the NLC, meaning it is released more quickly [33,99,101].

For both optimized NLC formulations, from 4 up to 48 h, the release of rivastigmine was controlled by the diffusion rate of the drug through the lipid matrix or by the lipid matrix degradation in the dissolution medium [62,102]. For the rivastigmine-loaded NLC produced by the ultrasound technique, about 80.75 ± 7.43% of rivastigmine was released after 12 h and the maximum drug release (88.67 ± 3.45%) was observed at 48 h. In contrast, for the rivastigmine-loaded NLC produced by the HPH method, at 12 h the release of rivastigmine was lower (60.13 ± 3.12%) and the maximum drug release (89.25 ± 3.22%) was observed at 48h. Statistically significant differences between the rivastigmine-loaded NLC produced by ultrasound technique and rivastigmine-loaded NLC produced by HPH method were observed (*p* < 0.05).

Figure 5 shows that in the simulated nasal electrolyte solution, similarly to the phosphate-buffered medium (Figure 4), an initial fast drug release was observed for both rivastigmine-loaded NLC formulations, followed by a prolonged release. The release rate of rivastigmine was higher for the rivastigmine-loaded NLC produced by the HPH method in the first 15 h when compared to the one produced by ultrasound technique, and the process reverted from the 15 h up to 48 h. Nonetheless, for both formulations a sustained drug release effect was observed, showing that the drug molecules were entrapped within the lipid matrix and that there was a homogeneous distribution of the liquid lipid droplets in the solid lipids of the NLC, as described in other research studies [29,50,103]. For the rivastigmine-loaded NLC produced by ultrasound technique, a maximum drug release of 88.90 ± 8.42% was obtained at 48 h, whereas for the rivastigmine-loaded NLC produced by the HPH method the maximum drug release was 98.10 ± 7.98% at 48 h. Statistically significant differences (*p* < 0.05) between the rivastigmine-loaded NLC produced by ultrasound technique and the rivastigmine-loaded NLC produced by HPH method were observed.

released more quickly [33,99,101].

produced by HPH method were observed (*p* < 0.05).

medium, followed by a prolonged release [15,98]. This phenomenon can be explained by the rapid solidification of the solid lipids during the formation of the NLC, which results in nanoparticles with an internal core containing a low amount of liquid lipids, which in turn accumulate in the outermost layers. As rivastigmine is an oil, it also tends to accumulate on the surface of the NLC, meaning it is

For both optimized NLC formulations, from 4 up to 48 h, the release of rivastigmine was controlled by the diffusion rate of the drug through the lipid matrix or by the lipid matrix degradation in the dissolution medium [62,102]. For the rivastigmine-loaded NLC produced by the ultrasound technique, about 80.75 ± 7.43% of rivastigmine was released after 12 h and the maximum drug release (88.67 ± 3.45%) was observed at 48 h. In contrast, for the rivastigmine-loaded NLC produced by the HPH method, at 12 h the release of rivastigmine was lower (60.13 ± 3.12%) and the maximum drug release (89.25 ± 3.22%) was observed at 48h. Statistically significant differences between the

**Figure 5.** Cumulative percentage of drug release in simulated nasal electrolyte solution (pH 6.4) from rivastigmine-loaded nanostructured lipid carriers (NLC) produced by ultrasound technique and rivastigmine-loaded NLC produced by high-pressure homogenization (HPH) method. **Figure 5.** Cumulative percentage of drug release in simulated nasal electrolyte solution (pH 6.4) from rivastigmine-loaded nanostructured lipid carriers (NLC) produced by ultrasound technique and rivastigmine-loaded NLC produced by high-pressure homogenization (HPH) method.

Figure 5 shows that in the simulated nasal electrolyte solution, similarly to the phosphatebuffered medium (Figure 4), an initial fast drug release was observed for both rivastigmine-loaded Therefore, a slower release of the drug was observed for the rivastigmine-loaded NLC formulation prepared by the ultrasound technique in the two dissolution media tested.

NLC formulations, followed by a prolonged release. The release rate of rivastigmine was higher for the rivastigmine-loaded NLC produced by the HPH method in the first 15 h when compared to the one produced by ultrasound technique, and the process reverted from the 15 h up to 48 h. Nonetheless, for both formulations a sustained drug release effect was observed, showing that the drug molecules were entrapped within the lipid matrix and that there was a homogeneous distribution of the liquid lipid droplets in the solid lipids of the NLC, as described in other research studies [29,50,103]. For the rivastigmine-loaded NLC produced by ultrasound technique, a maximum drug release of 88.90 ± 8.42% was obtained at 48 h, whereas for the rivastigmine-loaded NLC produced by the HPH method the maximum drug release was 98.10 ± 7.98% at 48 h. Statistically significant differences (*p* < 0.05) between the rivastigmine-loaded NLC produced by ultrasound technique and the rivastigmine-loaded NLC produced by HPH method were observed. Therefore, a slower release of the drug was observed for the rivastigmine-loaded NLC formulation prepared by the ultrasound technique in the two dissolution media tested. After fitting the in vitro release results of the two tested dissolution media to the kinetic models, it was observed that the Korsmeyer–Peppas model presented the highest *R* <sup>2</sup> values (0.9780 and 0.9848 for phosphate-buffered solution at pH 6.4 and simulated nasal electrolyte solution, respectively) (Table 6). An *n* value between 0.599 and 0.670 indicated that the drug release follows an anomalous transport route, i.e., a combination of non-Fickian release and Fickian release, which can be explained by the initial fast release of rivastigmine followed by prolonged release, indicating a biphasic behavior. Other authors have reported similar results for in vitro drug release from NLCs across dialysis membranes. For instance, the release of teriflunomide from a NLC intranasal formulation in simulated nasal electrolyte solution followed a biphasic behavior, with 75.11% of the drug being released after 8 h. Similar values were observed for the optimized rivastigmine-loaded NLC produced by ultrasound technique (75.89%) after the same period of time and using the same dissolution media (Figure 5) [31]. Jazuli et al. conducted drug release studies with lurasidone-loaded NLCs for nose-to-brain delivery and observed fast drug release after 12 h followed by sustained drug release, with a maximum release of 92.12% after 24 h [101]. This biphasic behavior was also observed with both optimized rivastigmine-loaded NLC formulations (Figures 4 and 5), although the maximum drug release was observed after 48 h. Alam et al. observed sustained in vitro release of isradipine from a NLC, with a maximum value of 92.89% after 24 h [46], which was also observed for the rivastigmine release from the optimized NLC produced by HPH (93.55%) after 24 h (Figure 5). Garg et al. studied the in vitro release profile of thirteen aceclofenac-loaded NLC formulations [104] and observed similar patterns of biphasic drug release, with a maximum release of around 80% after 48 h. Similar patterns were observed for the optimized rivastigmine-loaded NLC prepared by ultrasound technique (88.67%) and by HPH (89.25%) (Figure 4).

In vitro drug release studies are routinely employed during the optimization of NLC formulations. However, it is important to keep in mind that these studies are limited as a means of evaluating the in vivo performance of NLC formulations. Therefore, experiments evaluating the in vitro biocompatibility in nasal cell culture models and ex vivo studies in nasal mucosa must be carried out to obtain information about the toxicity, permeability, and transport of the optimized rivastigmine-loaded NLC formulation in the nasal mucosa. In addition, in vivo tests on animals should be performed to

confirm the effectiveness of this formulation for the direct delivery of rivastigmine from the nose to the brain.

**Table 6.** Results of the curve fitting into different kinetic models for rivastigmine-loaded nanostructured lipid carriers (NLC) formulations prepared by ultrasound technique and high-pressure homogenization (HPH) method.


NLCS: rivastigmine-loaded NLC produced by ultrasound technique; NLCHPH: rivastigmine-loaded NLC produced by HPH; PBS: phosphate-buffered solution; SNE: simulated nasal electrolyte solution; *R* 2 : correlation coefficient.
