*2.3. Physicochemical Characterization of Nanoparticles*

### 2.3.1. Particle Size Distribution Study

The particle size distribution and zeta potential of nanoparticles were assessed by dynamic light scattering (DLS) (Zetasizer 5000, Malvern, UK). The obtained nanoparticles were diluted 10 times by adding 900 μL of distilled water to 100 μL of the solution containing nanoparticles before measurement. All measurements were carried out in triplicate at room temperature.

### 2.3.2. Encapsulation Efficiency and Yield

The amount of BU entrapped within the nanoparticles was measured directly by dispersing 20 mg of nanoparticles in a solvent mixture containing 5 mL of acetic acid 1% *v*/*v* (pH 4.7) and 5 mL of ethanol. This suspension was shaken at 50 rpm by a mechanical shaker (TUV-NORD, Tehran, Iran) for 72 h at room temperature, and then filtered via a disposable syringe filter (pore size 0.22 μm). The concentration of BU in the filtrate was examined by a UV spectrophotometer at 246 nm (Shimadzu, UV/1204, Tokyo, Japan). Specimens were prepared in triplicate, and encapsulation efficiency (%) (EE) was analyzed by Equation (1) below:

$$\text{Encapsulation efficiency } \left( \% \right) \text{ } \frac{\text{Amount of buckets in NPs}}{\text{Amount of buckets in initially added}} \times 100 \tag{1}$$

Additionally, the production yield (%) for the selected formulation was calculated using the following equation:

$$\text{Yield } (\%) = \frac{\text{Weight of nonparticles}}{\text{Initial weight of polymer and drug}} \times 100\tag{2}$$

### 2.3.3. Transmission Electron Microscopy (TEM)

In order to observe the morphology of the selected nanoparticles (nanoparticles with 1:10 drug:chitosan) transmission electron microscopy (TEM) (Jeol JEM-1400, JEOL Ltd.; Tokyo, Japan) was employed. A drop of the aqueous dispersion of the washed nanoparticles was deposited in a mesh copper grid and air-dried at room temperature. Then, the grid was subjected to a 60 kV acceleration voltage.

### 2.3.4. Scanning Electron Microscopy (SEM)

To evaluate the external morphology of the selected nanoparticles after freeze-drying scanning electron microscope (Leo, VP1450, Neu-Isenburg, Germany) was employed. Freeze-dried nanoparticles were spread on a black steel grid and then coated with a thin film of gold using a gold sputter (Polaron, SC7620 sputter coater, Laughton, England) for 180 s under argon atmosphere. Voltages of 5 kV were selected for accelerating the electrons from electron gun onto the specimen.

### 2.3.5. Differential Scanning Calorimetry (DSC)

To evaluate the thermal property of nanoparticles, a differential scanning calorimeter calibrated with indium standard was used (Mettler Toledo, DSC 822e, Greifensee, Switzerland). The samples (3–5 mg) were hermetically sealed in DSC pans and were scanned in the temperature range of 25–300 ◦C at a rate of 10 ◦C/min under a nitrogen flow of 80 mL/min.

### 2.3.6. X-ray Powder Diffraction (XRPD)

XRPD study of nanoparticles with 1:10 drug:chitosan was carried out by employing an X-ray powder diffractometer (GNR, Explorer, Milan, Italy). The instrument was operated at 40 kV and 30 mA in the range (2θ) of 5 to 55◦ using a step size of 0.01◦ 2θ and step-time of 3 s, with a non-stop mode.

### 2.3.7. FTIR

Selected nanoparticles (1:10 drug:chitosan) were analyzed with an FTIR spectrometer (Thermo Nicolet, AVATAR 370, Walthman, USA) from 400 to 4000 cm−<sup>1</sup> at room temperature by the KBr disc method.

### 2.3.8. In Vitro Drug Release Studies

The release pattern of BU (powder), as well as the selected BCN (1:10 drug:chitosan) corresponding to 9 mg of BU (*n* = 6) were studied in 250 mL of simulated gastric fluid (SGF) pH 1.2 and simulated colonic fluid (SCF) pH 6.8 comprising 0.25% *w*/*v* SLS. USP dissolution apparatus I (Pharmatest, PTWS 3E, Hainburg, Germany) was employed to study the release pattern of BU from the selected formulations. The dissolution was operated under the rotation speed of 75 rpm, at 37 ± 0.5 ◦C. In the case of BU powder and freeze-dried BCN, an accurately weighed sample was gently dispersed in the medium. At pre-defined time intervals, samples (5 mL) were withdrawn from the release medium and filtered through

a disposable syringe filter (pore size 0.22 μm). Subsequently, the amount of BU in 1 mL of filtrate sample was quantified using HPLC (Shimadzu, Kyoto, Japan) equipped with a Teknokroma column (BRISA LC2 C18 250 mm × 4.6 mm, 5 μm). The mobile phase consisted of an acetate buffer (pH 3.9) and acetonitrile mixture (35:65) flowing at a rate of 1.5 mL/min and BU was measured at 240 nm.

### *2.4. Pelletization of Budesonide Nanoparticles*

The extrusion–spheronization technique was used to load selected BCN (the formulation with the smallest size and highest encapsulation efficiency) into the pellet formulation. For the preparation of pellets containing budesonide-loaded nanoparticles (BCNP) with 1% *w*/*w* of the drug, a powder blend containing 25% *w*/*w* freeze-dried selected BCN, 2% *w*/*w* PVP K30, 56% *w*/*w* Lactose mono hydrate and 17% *w*/*w* Avicel® PH 102, were mixed by a kitchen mixer (FUMA, Fu-1877 Hand Mixer, Tokyo, Japan) for twenty minutes. The mixture was wetted by adding distilled water. The wet mass was passed through an axial screw extruder (Dorsa Tech, EX-01, Tehran, Iran). The extrusion was performed under the rotation speed of 100 rpm using flat sieves of 1 mm aperture size. Then, the extrudates were spheronized (Dorsa Tech, EX-01, Tehran, Iran) for 5 min using a cross-hatched friction plate rotated at 1200 rpm. The obtained pellets were dried in an oven (24 h at 40 ◦C) and followed by sifting through 1180 and 850 μm. The sifted pellets between 1180 and 850 μm were collected.

Conventional BU pellets (CP) were also manufactured according to the method established in our previous study [44]. In brief, all powdered components, including 1.5% *w*/*w* BU, 5% *w*/*w* PVP K30, 68.5% *w*/*w* lactose mono hydrate and 25% *w*/*w* Avicel® PH 102 were mixed for 20 min and then turned to cohesive mass using distilled water. The wet mass was extruded and spheronized according to the methods described for BCNP.

### *2.5. Evaluation of Pellets*

### 2.5.1. In Vitro Drug Release Studies

The release profiles of uncoated BCNP corresponding to 9 mg of BU (*n* = 6) were studied as described in Section 2.3.8.

### 2.5.2. Pellet Morphology Studies

The morphology of BCNP and CP was analyzed by measuring the sphericity and aspect ratio of pellets. Pictures were taken by a stereomicroscope (Kyowa, Tokyo, Japan) equipped with a computer system and video camera (Sony, Tokyo, Japan) and then analyzed by an image analyzing software (ImageJ 1/50 for windows).

### 2.5.3. Particle Size Analysis of Pellets

The particle size of BCNP and CP was analyzed using the sieve method. A sample of 25 g of pellets was shaken on top of the series of standard sieves (150, 180, 250, 425, 850, 1000, and 1180 μm) using a sieve shaker (Azmun test, 50410, Tehran, Iran) for 10 min. The mass of pellets that remained on each sieve was determined and used to calculate the geometric mean particle size (dg) and geometric standard deviation (σg) from the plot of the cumulative percentage of undersize on the probability scale versus the log of particle diameter.

### 2.5.4. Pellet Mechanical Properties

The mechanical characteristics of BCNP and CP were examined by testing twenty pellets in the size range of 850–1180 μm using a Material Testing Machine (Hounsfield, H50KS, London, England). Force–displacement graphs were prepared by a computer program connected to the device (Hounsfield, QMAT, London, England). The 1 kN load cell was set up at a moving speed of 1 mm/min to determine the pellet crushing strengths and elastic modulus.

### *2.6. Coating of Pellets*

CP and BCNP were coated using a Wurster column fluid bed coater (Haltingen-Binzen, UNI-Glatt, Binzen, Germany). A solution of 48% *w*/*w* eudragit S, 12% *w*/*w* eudragit L and 40% *w*/*w* eudragit RS was prepared in a mixture of isopropanol and distilled water (9:1) under agitation. Triethyl citrate (TEC) was included as a plasticizer (10% *w*/*w* based on the weight of polymers) and stirred for 1 h. Talc was then added as an antiadhesion agent (5% *w*/*w* based on polymer weight). The coating solution was utilized onto 50 g pellets where the inlet air and outlet air temperatures were adjusted at 43–40 ◦C and 39–36 ◦C, respectively. In this experiment, the atomization pressure and spray rate were set up at 2 bar and 0.5 g/min, respectively. The coating process was continued till the coating mass reached 10% (*w*/*w*).
