7.5.2. Drug Entrapment Efficiency

The drug entrapment efficiency of the mirtazapine nanoemulsion formulation was measured using the UV visible spectroscopic method. Each 1 mL sample was cooling centrifuged at 3500 rpm for 30 min. After centrifuge, the supernatant transparent layer was taken and diluted with 10 mL distilled water. The samples were measured at 228 nm using the UV-VIS spectroscopic method. Results were taken in triplicate, and the average was taken into consideration.

### 7.5.3. In Vitro Drug Diffusion Studies

A Franz diffusion cell and a dialysis membrane were used in an in vitro drug diffusion study (Hi Media, molecular weight 5000 Daltons). Simulated nasal fluid (SNF) was used to saturate the dialysis membrane overnight. The dialysis membrane was then attached to the Franz diffusion cell's upper (donor) and lower (receptor) compartments. On the donor compartment side of the dialysis membrane, the formulation corresponding to 7.5 mg of the dose was administered. An amount of simulated nasal fluid (SNF) equal to 18 mL was placed in the receptor compartment of the Franz diffusion cell. The diffusion cells (Remi, India) were maintained at 37 ± 0.5 ◦C throughout the experiment, with stirring happening at 600 rpm. At fixed time intervals, 1 mL of aliquots was withdrawn every 15 min, 30 min, 45 min, 60 min, 60 min, 120 min, 180 min, 240 min, 300 min, 360 min, and 420 min from the receptor compartment through a side tube, and an equal volume of SNF was replaced to maintain the sink condition [17–19] The samples withdrawn were analyzed through UV-visible spectrophotometry at 280 nm.

By dividing the slope of the steady state section of the line in the plot of drug amount penetrated per unit area of dialysis membrane verses time, the drug flux (g/hr/cm<sup>2</sup> ) at the steady state was estimated.

### 7.5.4. Ex Vivo Drug Diffusion Studies

Sheep nasal mucosa that had just been excised was used in a Franz diffusion cell for the ex vivo drug diffusion investigations of the mirtazapine nanoemulsion.

Sheep's nasal mucosa was the subject of experiments on drug diffusion that were done ex vivo. The sheep nasal mucosa was taken from a freshly removed sheep's nose at the butcher shop. Simulated nasal fluid (SNF) was used to soak this sheep's nasal mucosa before it was glued to a Franz diffusion cell with a 3.14 cm<sup>2</sup> diffusion area and then clamped. In the donor compartment, a formulation corresponding to a 7.5 mg dose was applied to the nasal mucosa, and simulated nasal fluid was placed in the receptor compartment. The diffusion cell was kept at body temperature throughout the experiment, and the stirring speed was kept at 600 rpm (Remi, India). At predetermined intervals, such as 15 min, 30 min, 45 min, 60 min, 120 min, 180 min, 240 min, 300 min, 360 min, and 420 min, the 1 mL aliquots were removed from the receptor compartment.

To keep the sink condition, the amounts of samples that were extracted were promptly replaced with equivalent amounts of SNF. At 228 nm, these samples were examined using a UV-visible spectrophotometer [19–21].

### **8. Fourier Transform Infrared Spectroscopy (FTIR) Studies**

To evaluate any potential interactions that might have arisen during formulation between mirtazapine and other excipients, FTIR analysis was carried out. The infrared spectra of mirtazapine and thermotriggered nanoemulsion were acquired using a Bruker Alpha E, FTIR spectrometer (Bruker Alpha E, Opus-7.0.122), equipped with an ATR. They are typically evaluated using the single-reflection ATR accessories (attenuated total reflectance). The spectra were scanned in transmission mode spanning the 4000–650 cm−<sup>1</sup> wavenumber range at room temperature.

### *8.1. Differential Scanning Calorimeter*

To determine the physical condition of the medication in the nanoemulsion formulation, DSC experiments were carried out. A differential scanning calorimeter was used for the DSC measurements (Shimadzu, Kyoto, Japan, Thermal Analyzer DSC 60). Heat of fusion (enthalpy) (Hf) and peak transition temperature (Tm) were calculated and used in the analysis. For routine calibration, indium (Tm = 159.2 ◦C; Hf = 28.8 J/g) was utilized as the standard. Nitrogen (purity > 99.99%) was employed as the purge gas, and an empty aluminium pan served as the reference. Samples of 2.4–2.8 mg were weighed, put in open aluminium pans, and scanned over the temperature range of 30–350 ◦C at a rate of 10 ◦C/min.

### *8.2. Transmission Electron Microscopy*

Transmission electron microscopy (TEM) (H-7500, Hitachi, Kyoto, Japan) was used to examine the morphology of the improved formulation in order to research the morphology of the resulting nanoemulsion. The nanoemulsion was placed on copper grids for observation after being dyed with 1% (*w*/*v*) phosphotungstic acid, and by examining the TEM images, the globule's shape was discovered [22].
