*2.7. In Vitro Drug Release Studies of Coated Pellets*

The evaluation of the drug release pattern from coated pellets was performed according to the continuous mode of the dissolution test at 37 ± 0.5 ◦C (USP dissolution apparatus I, Pharmatest, PTWS 3E, Hainburg, Germany). The amounts of weighed pellets (*n* = 6) equal to 9 mg of BU were tested in 250 mL of a medium comprising 0.25% *w*/*v* SLS at a rotation speed of 75 rpm. Dissolution tests were performed at various time intervals of 2 h (pH 1.2), 1 h (pH 6.5), 2 h (6.8), 1 h (pH 7.2) and 10 h (pH 6.8). The dissolution was also performed in the medium containing 4% *w*/*v* rat cecal content with pH 6.8 to simulate GIT media and the transit time of the pellets from various sections (i.e., stomach, duodenum, jejunum, ileum, and colon). The media supplemented by cecum content was continuously bubbled with CO2. After each incubation time for each medium, the baskets with their pellets were moved rapidly from one medium to another. At defined time intervals, samples (5 mL) were withdrawn manually and analyzed by HPLC according to the method explained in Section 2.5.1. In the case of media supplemented by cecum content, 1 mL of the medium was removed and centrifuged at 15,000 rpm at 4 ◦C for 30 min. Then, the supernatant was taken and diluted with acetonitrile at a ratio of 1:3 and the mixture was filtered using a disposable syringe filter (pore size 0.22 μm) before the sample is injected to the HPLC column [45].

### *2.8. Morphology of Coated Pellets*

The surface morphology as well as cross section of the coated BCNP were characterized using scanning electron microscope (Leo, VP1450, Neu-Isenburg, Germany). The pellets were fixed on an aluminum stub and sputter-coated (Polaron, SC7620 sputter coater, Laughton, England) with a thin layer of gold for 180 s using a sputter coating machine under argon atmosphere, and then analyzed using SEM. Voltages of 5 kV were selected for accelerating the electrons from electron gun onto the specimen. The cross-sectional samples were prepared using a razor. These samples were then covered with a thin layer of gold.

### *2.9. Animal Treatment*

To investigate the efficacy of different formulations of BU in the treatment of colonic damage, the rat model of ulcerative colitis was employed. Wistar male rats aged 10–12 weeks and weighing 250–300 g were used in this part of the study, under institutional guidelines granted by the institutional ethics committee of Mashhad University of Medical Sciences (IR.MUMS.REC.1399.011).

All animals employed in the study were kept in a clean room, with air-conditioned (22 ± 3 ◦C), controlled humidity (40 ± 5%) and light–dark cycles of 12 h; 24 h before the colitis induction, all the rats were weighed, examined to be healthy and kept fasted except for water. For induction of colitis, first of all, an intraperitoneal injection of ketamine (50 mg/kg) and xylazine (5 mg/kg) solution was used to anaesthetize rats [46,47]. Thereafter, acetic acid (2 mL 3% *v*/*v* in normal saline) was administered rectally 8 cm deep using a 2 mm diameter cannula [48]. The healthy control group received 2 mL of normal saline solution intrarectally. Rats were kept in an upside-down position for 1 min and then they

were taken back to their cages. All animals were left for 3 days without treatment to let the colitis to be developed while they were free for consumption of water and food [49]. The colitis-induced rats were then divided randomly into 7 groups, with 6 animals per group. One group of rats were used as the control group (untreated group), whilst others were treated with conventional coated pellets (coated CP), pellets containing BU loaded nanoparticles (BCNP), coated BCNP, BU nanoparticle (BCN), free drug chitosan nanoparticles (CN) and free BU powder (BU) three days after induction of colitis. Each treated group received orally an equal dose of BU (0.15 mg/day) for 7 consecutive days [50]. In the case of BCN, CN and BU, the dose was suspended in 1 mL of purified water before administration. The healthy and the untreated control groups received 1 mL of normal saline. The weight and stool consistency of the rats were evaluated during this period. The rats were killed by CO2 asphyxiation either 24 h or 6 days after the last dose administration. Then, the colon, from the cecum to the anus was cut for further studies.
