2.4.1. Physicochemical Properties

All the developed nanoemulsion formulations were physically examined and were confirmed to be clear and transparent. The pH of all the formulations was measured using a pH meter (Remi Equipment Pvt. Ltd., Kolkata, India) and was found to be close to nasal pH, indicating an acceptable range, as evident from Table 1.


**Table 1.** Physicochemical properties of nanoemulsion formulations.

Thermotriggered nanoemulsion gel formation: The thermotriggered nanoemulsion formulation was added dropwise into distilled water by heating. A consistent thermal gel was formed at 32 ◦C and was stable.

### 2.4.2. In Vitro Drug Diffusion Studies

For the created formulations, in vitro release tests were carried out utilizing Franz diffusion cells and diffusion experiments. The study was carried out for up to 6 h for developed thermotriggered nanoemulsion (RD1), mucoadhesive nanoemulsion (RD2), and water-based nanoemulsion (RD3). The results are graphically represented in Figure 3. It can be observed that optimal formulation, i.e., thermotriggered nanoemulsion (RD1), has shown 71.42% drug release after 6 h, whereas mucoadhesive nanoemulsion (RD2) and water-based nanoemulsion (RD3) showed drug releases of 66.12% and 60%, respectively, after 6 h. This indicated that the thermotriggered nanoemulsion (RD1) was better among the three formulations. The high release may be because of the poloxamer 407, which is helping the formulation to form in situ gel as soon as it is sprayed in the nose, while the mucoadhesive properties of carbopol are keeping the nanoemulsion adhered to the mucosal lining. Formulation RD2 lacks poloxamer 407, hence it doesn't get sufficient time for contact with the mucosal area and drains. The least release with RD3 formulation is due to the absence of a mucoadhesive polymer as well as a thermosensitive polymer.

### 2.4.3. Ex Vivo Permeation Studies

Figure 4 illustrates how mirtazapine thermotriggered nanoemulsions (RD1), mucoadhesive nanoemulsions (RD2), and water-based nanoemulsions (RD3) permeate the nasal mucosa. From the figure, it is evident that pure drug dispersion could show only 25% drug difussion in 6 h, whereas the nanoemulsion formulation showed greatly improved drug diffusion compared with the pure drug. Formulation RD1 showed drug diffusion of more than 80%, whereas RD2 and RD3 only showed drug diffusion in the range of 70–72%. Due

[13].

to a lack of mucoadhesion and thermosensitive characteristics, the drug in the water-based nanoemulsion (RD3) demonstrated the lowest permeability. As the contact time with the mucosal area was extended, drug penetration in mucoadhesive and thermotriggered nanoemulsions improved. The high transcellular uptake, high solubilization capacity, possibility for improved absorption by gel formation by mucoadhesion, and presence of a poloxamer may all contribute to the higher permeability. Carbopol 934 P may have also improved drug absorption; this may be because it opened tight junctions, which made it easier for pharmaceuticals to go through paracellular pathways [13]. *Gels* **2023**, *9*, x FOR PEER REVIEW 5 of 15 **Figure 3.** In vitro diffusion profiles of the formulations.

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**Figure 3.** In vitro diffusion profiles of the formulations. **Figure 3.** In vitro diffusion profiles of the formulations.

**Figure 4.** Ex vivo diffusion profiles of the formulations. **Figure 4.** Ex vivo diffusion profiles of the formulations.

**Figure 4.** Ex vivo diffusion profiles of the formulations.

### 2.4.4. Fourier Transform Infrared Spectroscopy (FTIR) 2.4.4. Fourier Transform Infrared Spectroscopy (FTIR)

FTIR studies were performed to determine the compatibility between pure drug and excipients. FTIR spectrum of pure mirtazapine, thermotriggered formulation, and poloxamer 407 characteristic peaks are given in Figure 5. From the results, it was concluded that there is no considerable change in the drug when mixed with the excipients. It can be concluded that there is no drug excipient incompatibility. FTIR studies were performed to determine the compatibility between pure drug and excipients. FTIR spectrum of pure mirtazapine, thermotriggered formulation, and poloxamer 407 characteristic peaks are given in Figure 5. From the results, it was concluded that there is no considerable change in the drug when mixed with the excipients. It can be concluded that there is no drug excipient incompatibility.

**Figure 5.** IR Spectra of (**A**) mirtazapine, (**B**) formulation, (**C**) oleic acid, (**D**) Carbapol934P, (**E**) poloxamer 407, and (**F**) PEG 6000. **Figure 5.** IR Spectra of (**A**) mirtazapine, (**B**) formulation, (**C**) oleic acid, (**D**) Carbapol934P, (**E**) poloxamer 407, and (**F**) PEG 6000.

### 2.4.5. Differential Scanning Calorimetry (DSC) 2.4.5. Differential Scanning Calorimetry (DSC)

DSC studies were performed for pure mirtazapine, thermotriggered nanoemulsion, poloxamer 407, and other excipients used in the formulation to know the thermal behavior and the physical state of the drug in the formulation (crystalline/amorphous) through characteristic melting points. DSC studies were performed for pure mirtazapine, thermotriggered nanoemulsion, poloxamer 407, and other excipients used in the formulation to know the thermal behavior and the physical state of the drug in the formulation (crystalline/amorphous) through characteristic melting points.

The DSC thermogram of mirtazapine, thermotriggered nanoemulsion, and other excipients are shown in Figure 6. The thermogram of pure mirtazapine showed melting endotherm at 116 °C. The thermogram of thermotriggered nanoemulsion showed an The DSC thermogram of mirtazapine, thermotriggered nanoemulsion, and other excipients are shown in Figure 6. The thermogram of pure mirtazapine showed melting endotherm at 116 ◦C. The thermogram of thermotriggered nanoemulsion showed an endotherm peak at 162.85 ◦C, and poloxamer 407 showed an endotherm peak at 60.53 ◦C, which indicates that there is no significant change in the melting endotherm of the pure drug. The slightly broad peak in the formulation indicates the change in the state of the drug to an amorphous state. which indicates that there is no significant change in the melting endotherm of the pure drug. The slightly broad peak in the formulation indicates the change in the state of the drug to an amorphous state.

endotherm peak at 162.85 °C, and poloxamer 407 showed an endotherm peak at 60.53 °C,

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**Figure 6.** Reports of DSC thermograms of pure drug, formulation, and excipients. (**A**) Poloxamer 407, (**B**) Oleic acid, (**C**) Formulation, (**D**) Mirtazapine. **Figure 6.** Reports of DSC thermograms of pure drug, formulation, and excipients. (**A**) Poloxamer 407, (**B**) Oleic acid, (**C**) Formulation, (**D**) Mirtazapine.

2.4.6. Measurement of Particle Size and Zeta Potential 2.4.6. Measurement of Particle Size and Zeta Potential

The average sizes of the globules of RD1, RD2, and RD3 were found to be 42.6 nm, 135.2 nm, and 52.44 nm, respectively, as seen in Table 2 and depictated in Figure 7. The percentage of the intensity of distribution of the 42.64 nm-sized globules of thermotrig-The average sizes of the globules of RD1, RD2, and RD3 were found to be 42.6 nm, 135.2 nm, and 52.44 nm, respectively, as seen in Table 2 and depictated in Figure 7. The percentage of the intensity of distribution of the 42.64 nm-sized globules of thermotriggered nanoemulsion was high, i.e., 90.2%, whereas the average globule size and distribution percentage of RD2 and RD3 were found to be 50.8 and 60.5, respectively.


gered nanoemulsion was high, i.e., 90.2%, whereas the average globule size and distri-

bution percentage of RD2 and RD3 were found to be 50.8 and 60.5, respectively.

**Table 2.** Particle size and zeta potential for developed formulations. **Table 2.** Particle size and zeta potential for developed formulations.

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**Figure 7.** Particle size data of thermotriggered nanoemulsion (RD1), (**A**) size distribution and (**B**) zeta potential.

#### 2.4.7. Transmission Electron Microscopy (TEM) 2.4.7. Transmission Electron Microscopy (TEM)

zeta potential.

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From the images obtained by TEM analysis, it is evident that thermotriggered nanoemulsion (RD1) showed highly uniform and spherical shapes globules. White, nearly spherical globules were seen against a dark background created by negative staining with phosphotungstic acid, as evident from Figure 8. From the images obtained by TEM analysis, it is evident that thermotriggered nanoemulsion (RD1) showed highly uniform and spherical shapes globules. White, nearly spherical globules were seen against a dark background created by negative staining with phosphotungstic acid, as evident from Figure 8.

**Figure 7.** Particle size data of thermotriggered nanoemulsion (RD1), (**A**) size distribution and (**B**)

**Figure 8.** TEM Photographs of Thermotriggered Nanoemulsion (RD1). **Figure 8.** TEM Photographs of Thermotriggered Nanoemulsion (RD1).

#### **3. Stability Studies 3. Stability Studies**

Nanoemulsions are considered to be thermodynamically stable systems that are formed with a particular concentration of oil, surfactant, and water, with no phase separation, creaming, or cracking. Selected formulations from the phase diagram were subjected to different stress stability tests, such as a heating-cooling cycle, centrifugation, and a freeze-thaw cycle. All nanoemulsion formulation cycles were found to be stable. Nanoemulsions are considered to be thermodynamically stable systems that are formed with a particular concentration of oil, surfactant, and water, with no phase separation, creaming, or cracking. Selected formulations from the phase diagram were subjected to different stress stability tests, such as a heating-cooling cycle, centrifugation, and a freeze-thaw cycle. All nanoemulsion formulation cycles were found to be stable.

### **4. Statistical Analysis 4. Statistical Analysis**

The results of different formulations were analyzed by one-way ANOVA. The values were considered to be statistically significant when the *p* value was less than 0.05. It was observed that the *p* value of all the responses was found to be below 0.05, hence considered as significant. The results of different formulations were analyzed by one-way ANOVA. The values were considered to be statistically significant when the *p* value was less than 0.05. It was observed that the *p* value of all the responses was found to be below 0.05, hence considered as significant.

### **5. Conclusions 5. Conclusions**

Thermotriggered intranasal gel for the antidepressant mirtazapine was successfully developed to treat depression in patients. Three different nanoemulsions, including thermotriggered nanoemulsion (RD1), mucoadhesive nanoemulsion (RD2), and plain nanoemulsion, were developed, and comparative studies were done. After comparison of these three formulations for various physicochemical and analytical parameters, it was concluded that the thermotriggered nanoemulsion (RD1) consisting of carbopol and PEG 6000 was best suitable for intranasal delivery in terms of properties like excellent globule size, shape, zeta potential, polydispersity index, and percentage of drug release. From the results obtained in ex vivo studies conducted on freshly excised sheep nasal mucosa, it can be hypothesized that the thermotriggered nanoemulsion may have still better absorption capacity through the olfactory and trigeminal nerves, as it shows good para-Thermotriggered intranasal gel for the antidepressant mirtazapine was successfully developed to treat depression in patients. Three different nanoemulsions, including thermotriggered nanoemulsion (RD1), mucoadhesive nanoemulsion (RD2), and plain nanoemulsion, were developed, and comparative studies were done. After comparison of these three formulations for various physicochemical and analytical parameters, it was concluded that the thermotriggered nanoemulsion (RD1) consisting of carbopol and PEG 6000 was best suitable for intranasal delivery in terms of properties like excellent globule size, shape, zeta potential, polydispersity index, and percentage of drug release. From the results obtained in ex vivo studies conducted on freshly excised sheep nasal mucosa, it can be hypothesized that the thermotriggered nanoemulsion may have still better absorption capacity through the olfactory and trigeminal nerves, as it shows good paracellular and transcellular transport and can be efficiently used to treat major depressive disorder. To support the hypothesis, in vivo animal studies must be done; these studies would give a clear idea on brain-targeting efficiency of the formulation.

### **6. Materials**

Mirtazapine (CAS: 85650-52-8) was procured as a kindly gifted sample from Wockhardt Pvt. Ltd., Aurangabad, Maharashtra, India. Poloxamer 407 (CAS: 691397-13-4) was obtained as a gifted sample from BASF India, Ltd., Mumbai, India. Labrafil M1944 CS (CAS: 69071-70-1), Capryol PGMC (CAS: 31565-12-5), Peceol, and Labrosol, were gift samples from Gattefosse India, Pvt. Ltd. (Mumbai, India), while Campul MCM C8 EP (CAS: 26402-26-6) and Acconon MC82E/NF (CAS: 85536-01-8) were a generous gifts from Abitech Ltd. Finar Chemicals, Hyderabad, India supplied the Tween 80, Tween 20, Span 80, propylene glycol, polyethylene glycol 400, and oleic acid. The remaining reagents were all of analytical grade.

### **7. Methods**

### *7.1. Saturation Solubility Studies*

The solubility studies of mirtazapine were carried out in different solvents and buffers, like double-distilled water, ethanol, dichloromethane, Labrafil M1944 CS, Caproyl PGMC, Peceol, Acconon MC82E/NF, Labrosol, Campul MCMC8EP, propylene glycol, PEG 400, Tween 80, Tween 20, Span 80, and oleic acid. In order to create saturated solutions of mirtazapine, excess amounts of the drug were added to 5 mL of each chosen vehicle and stirred on a rotary shaker for 48 h at 25 ◦C. After equilibrium was reached, samples were taken and centrifuged for 15 min at 10,000 rpm. Following the collection and proper dilution of an additional 100 L of supernatant with dichloromethane, mirtazapine samples were analyzed using UV-visible spectrophotometry at 228 nm [14].

### *7.2. Construction of Pseudo-Ternary Phase Diagram*

Oleic acid was chosen as the oil phase, Tween 80 as the surfactant, and ethanol as the co-surfactant based on the solubility experiments. Tween 80 and ethanol were measured in the weight ratios (1:1, 1:2, 1:3, 2:1, and 3:1) of surfactant to co-surfactant (Smix). The pseudo-ternary phase diagrams were produced at room temperature by the water titration method. For each pseudo-ternary phase diagram, oil and Smix mixtures were created with weight ratios (*w*/*w*) of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. When the combination reached a particular point and began to break down into a macroemulsion, double-distilled water was gradually added to each ratio of Smix to oil while the mixture was being stirred magnetically. In order to complete the pseudo-ternary phase diagrams, the component concentrations were noted. The oil, surfactant, co-surfactant, and water contents were then chosen at the proper weight ratios based on the stability and transparency of the resulting microemulsions [14,15].

### *7.3. Development of Nanoemulsion Formulation*

The criteria for selection of the microemulsion zone were carried out by observing the stability of the formulation from the pseudo-ternary phase diagrams. The microemulsion formulations were composed of Smix (1:3) and oil. In accordance with the pseudoternary phase diagrams, a Smix ratio of 1:9 was used. Oleic acid was used as oil in the microemulsion [14,15].

### 7.3.1. Formulation of Thermotriggered Nanogel

Thermotriggered nanogel was prepared by a cold method, different concentrations of polymers were used, such as poloxamer 407 (10.5–20.5% *w*/*v*) Carbopol 934 P (0.1–0.5% *w*/*w*), and PEG 6000 (0.1–0.3% *w*/*v*) to determine the optimum concentration of poloxamer 407 and other polymers required for thermotriggered gelling. After a series of combinations, it was found that poloxamer 407 (18.5%), Carbopol (0.1%), and PEG 6000 (0.3%) were found to have optimum concentration, which formed thermotriggered gel at a temperature of 32 ◦C.

Thermotriggered nanogel was prepared by dissolving 0.1% Carbopol 934 P and 0.3% PEG 6000 in the aqueous phase by continuously stirring on a magnetic stirrer at 400 rpm. To the aqueous phase, 18.5% of poloxamer 407 was slowly added under continuous stirring in

cold conditions [16]. This aqueous phase was then added dropwise to the nanoemulsions. After complete solubilization of poloxamer 407, the solution was stored at 4 ◦C overnight to get the thermotriggered nanogel.

Carbopol 934P was used as the mucoadhesive agent for its mucoadhesive properties to make the formulation adhere to the mucosal system; poloxamer 407 was used as the thermosensitive agent to form in situ gel after spraying into the nostrils. The active drug was added in the formulations depending upon the amount of formulation being delivered by the spray pump. Table 3 shows the composition of the nanoemulsions.


### **Table 3.** Composition of nanoemulsion formulations.

### 7.3.2. Determination of Reproducibility of Dosage

The developed formulations were loaded into the 5 mL clean sterile glass container and were fitted with a spray actuator and crimpled.

The spray actuator was analyzed for determination of the reproducibility of the dosage. The formulation was sprayed in a test tube and weighed. The empty weight of the test tube was subtracted from the total weight. This was repeated a number of times, and the average weight of the dose was taken [17].

### *7.4. Characterization of Nanoemulsion*

### 7.4.1. Measurement of Droplet Size and Zeta Potential

Using the zetasizer, a dynamic light scattering or photon correlation spectroscopy technique was used to measure the mean droplet size and zeta potential (Malvern Instruments version 7.01). Using filtered, twice-distilled water, each was diluted to the proper concentration. Analysis of globule size was carried out at 25 ◦C and with a detection angle of 90 ◦C. Nanoemulsions' size and polydispersity index were directly measured by the equipment [16–18].

### 7.4.2. pH Measurement

A pH meter was used to determine the pH values of the nanoemulsion samples (Remi Equipment Pvt. Ltd., Kolkata, India). Before each usage, the pH meter was calibrated using buffer solutions having pH values of 4.0, 7.0, and 9.0. The formulation's pH was measured three times, and the means of the measurements were computed.

### 7.4.3. Measurement of Viscosity

A Brookfield viscometer with spindle no. DV-II+ PRO was used to measure the viscosities of nanoemulsions. The spindle code for LV1 is 61. The spindle was dipped in the preparation and revolved for 5 min at 100 rpm at room temperature.
