Theoretical screening of QRC solubility in different liquid lipids
In order to select the best possible liquid lipid to solubilize QRC, theoretical screening was employed using HSP. The euclidean distance between QRC and the different liquid lipids alongside the parameters of all materials are mentioned in table 2 Each material using its parameters is plotted in a 3D diagram (fig. 1) using Matlab version 2021a (Mathworks, Inc., Natick, MA, USA) to visualize the distance between QRC and each liquid lipid.
Table 2 HSP of QRC and different liquid lipids as well as the euclidean distance between QRC and each liquid lipid
Material
|
δD
|
δP
|
δH
|
Euclidean Distance
|
QRC
|
21
|
10.6
|
13.7
|
-
|
Capryol 90
|
16.39
|
4.98
|
8.41
|
8.99
|
Capryol PGMC
|
16.33
|
4.42
|
7.1
|
10.18
|
Labrafil M 1944 CS
|
16.27
|
3.63
|
6.64
|
10.99
|
Labrafil M 2125 CS
|
16.17
|
3.69
|
6.19
|
11.3
|
Oleic Acid
|
16.4
|
3
|
5.5
|
12.09
|
Captex 200P
|
16.23
|
2.99
|
4.04
|
13.19
|
Labrafac PG
|
16.25
|
2.83
|
3.92
|
13.36
|
Labrafac Lipophile WL 1349
|
16.42
|
3.06
|
3.58
|
13.43
|
Miglyol 810
|
16.43
|
2.99
|
3.51
|
13.51
|
Captex 355
|
16.45
|
2.83
|
3.38
|
13.7
|
Ethyl Oleate
|
16.1
|
2.3
|
2.90
|
14.48
|
Isopropyl Myristate
|
15.9
|
2.1
|
2.80
|
14.73
|
Among the different liquid lipids assessed, Capryol® 90 exhibited the lowest euclidean distance while isopropyl myristate showed the highest euclidean distance.
QRC loaded polymeric nanocapsules characterization
Characterization is considered to be one of the critical steps in order to identify the physicochemical properties of the prepared nanoparticles[27]. The results of the prepared formulations are summarized in table 3.
Table 3 Summary of the results obtained for all the prepared formulae
Formula
|
Particle Size (nm)
|
PDI
|
Zeta Potential (mV)
|
EE%
|
F1
|
184.6 ± 4.4
|
0.19 ± 0.015
|
-14.1 ± 0.14
|
79.1 ± 2.3
|
F2
|
262.3 ± 1.6
|
0.268 ± 0.017
|
-15 ± 0.57
|
83.9 ± 1.6
|
F3
|
227.8 ± 11.9
|
0.466 ± 0.023
|
-17.5 ± 0.01
|
92.5 ± 1.9
|
F4
|
278.1 ± 3.7
|
0.371 ± 0.018
|
-18.7 ± 1.70
|
92.7 ± 2.1
|
All of the prepared nanocapsules demonstrated a size in the nano scale ranging from 184.6 nm for F1 to 278.1 nm for F4. PDI values ranged from 0.19 for F1 to 0.466 for F3. These PDI values indicated good homogeneity for all of the prepared formulations as it was previously reported that the ideal PDI value is less than 0.5[28]. Regarding zeta potential, the prepared nanocapsules exhibited values ranging from -14.1 mV for F1 to -18.7 mV for F4. All of the prepared formulations are considered to be stable as it was previously reported that zeta potential values between -15 mV and -30 mV are considered ideal for nanoparticles stabilization[29] as well as the presence of stearic stabilization[30]. As for EE%, the values ranged between 79.1% for F1 to 92.7% for F4.
Based on these results, F3 was chosen to be subjected to further investigations.
The morphology of the chosen formula was evaluated using TEM. As shown in (Fig. 2), TEM image shows well defined, spherical shaped nanoparticles that appear to be unilamellar.
Thermal analysis of QRC, PCL, and the chosen formula using DSC (Fig. 3). QRC displayed a sharp endothermic peak at 322oC similar to what was previously reported by Li et al.[31]. PCL displayed a sharp endothermic peak at 65.26oC close to what was reported by González-Reza et al.[32]. However, the chosen formula displayed a broad peak 290.3oC with the disappearance of both peaks of QRC and PCL.
XRD was conducted for pure QRC, PCL, and the chosen formula (Fig. 4). QRC displayed its highly crystalline structure[33] through exhibiting sharp peaks at 2θ of 10.73o, 12.45o, 13.06o, 14.1o, 15.76o, 17.16o, 22.09o, 26.49o, and 26.99o. PCL displayed its semi crystalline structure[34] by exhibiting two peaks at 2θ of 21.49o, and 23.86o. The chosen formula exhibited amorphous structure with the complete disappearance of QRC’s peaks indicating the drug change in structure from crystalline to amorphous.
In vitro Release Study
Release pattern of both the chosen formulation of QRC loaded polymeric nanocapsules and QRC dispersion are illustrated in (Fig. 5). As shown in the figure, the prepared formulation resulted in a much slower release pattern over the course of the experiment compared to QRC dispersion. After 6 hours, the cumulative percent release of the prepared nanocapsules was 36.06% compared to 53.91% of QRC dispersion. The release of the prepared nanocapsules continued to exhibit slower release after 24 hours with cumulative release of 63.38% for the prepared nanocapsules compared to 82.71% for the QRC dispersion. In vitro release of QRC loaded polymeric nanocapsules exhibited a biphasic release with an initial burst release followed by a sustained release pattern with the release of both, the prepared nanocapsules and QRC dispersion, reaching almost complete release after 48 hours.
Through analyzing the kinetic models applied to the release data of QRC loaded polymeric nanocapsules (table 4), it was possible to determine that the release profile followed Higuchi model (r2 adjusted= 0.9947). The n value of Korsmeyer–Peppas model was 0.48 with a r2 adjusted of 0.9957.
Table 4. Summary of different release models and the corresponding r2 adjusted
Model
|
Equation
|
R2 adjusted
|
Zero order
|
Q = Q0 + K0t
|
0.3838
|
First order
|
dC/dT = −Kt
|
0.7834
|
Higuchi
|
Q = KH t1/2
|
0.9947
|
Hixson–Crowell
|
Q01/3 – Qt1/3 = KHC t
|
0.6777
|
⁂ Q is the amount of drug released or dissolved, Q0 is the initial amount of drug in solution, t is time, K0 is the zero order release constant, dC is the concentration derivative, dT is the time derivative, K is the first order rate constant, KH is the Higuchi dissolution rate constant, Qt is the remaining weight of solid at time t, KHC is the Hixson–Crowell dissolution rate constant.
Animal Studies
The anxiolytic effect was assessed by two behavioral assessments which are open field and elevated plus-maze. The results of both assessments are present in (Fig. 6) When analyzing the open field results, intranasal QRC loaded polymeric nanocapsules significantly increased the time spent in the center of the field compared to the control group (P < 0.05), oral QRC dispersion group (P < 0.05), and intranasal QRC dispersion (P < 0.05).
Regarding elevated plus-maze, intranasal QRC loaded polymeric nanocapsules significantly increased the time spent in the open arms compared to the control group (P < 0.05), oral QRC dispersion group (P < 0.01), and intranasal QRC dispersion (P < 0.01).
Safety Studies
In order to ensure the safety of our prepared formulation, safety studies were conducted using both observational nasal irritation test and histopathological examination. Visual observation demonstrated that the animals administered intranasally with both, QRC loaded polymeric nanocapsules as well as QRC dispersion, did not exhibit any signs of nasal mucosal irritation compared to animals administered with intranasal saline. This resulted in classifying both QRC loaded polymeric nanocapsules as well as QRC dispersion in the no irritation level on the nasal mucosa irritation index.
Histopathological examination of the negative control group showed intact epithelial lining, average submucosa with average blood vessels and cellularity, and average nasal cartilage. While the positive control group demonstrated ulcerated epithelial lining, submucosa with marked hypercellularity, marked inflammatory infiltrate, and marked edema. Regarding our chosen formula, histopathological examination revealed intact epithelial lining, submucosa with average cellularity, average nasal cartilage, and minute edema compared to the positive control group (Fig. 7).