**1. Introduction**

Targeting drug delivery is advantageous for the treatment of inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis (UC) [1–3]. An appropriate drug delivery system could allow delivery of the sufficient amounts of drugs with appropriate release rates to the site of its action [4]. Budesonide (BU) is a topical anti-inflammatory steroid, which has proven to be effective in the treatment of IBD [5]. Therefore, targeting this drug at the site of inflammation is an interesting perspective for clinical applications.

Nanoparticles have shown an encouraging and promising outlook for drug or gene delivery in IBD treatment [6–8]. The design of drug-loaded nanoparticles for delivery to the colon via oral administration has been used as a strategy to further amplify drug uptake

**Citation:** Soltani, F.; Kamali, H.; Akhgari, A.; Ghasemzadeh Rahbardar, M.; Afrasiabi Garekani, H.; Nokhodchi, A.; Sadeghi, F. Preparation and Characterization of a Novel Multiparticulate Dosage Form Carrying Budesonide-Loaded Chitosan Nanoparticles to Enhance the Efficiency of Pellets in the Colon. *Pharmaceutics* **2023**, *15*, 69. https:// doi.org/10.3390/pharmaceutics 15010069

Academic Editor: Ana Isabel Fernandes

Received: 10 November 2022 Revised: 14 December 2022 Accepted: 20 December 2022 Published: 26 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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into the inflamed tissue of the colon [1,9]. Nanoparticles have a great chance to reach and accumulate in inflamed tissues due to the loosening of tight junctional complexes [10]. A study on the rat intestinal loop model demonstrated that nanoparticles in the size range of 100 nm allow better penetration in the submucosal layers compared with those in the size range of 500 nm which mainly showed localized targeting to the epithelial lining [11]. Additionally, it has been reported that nano-sized particles showed favored uptake by immune cells whose numbers are high in inflamed tissue [12].

In some studies, triggering drug release into the colon was reported by encapsulation of prednisolone in nanocarriers [13]. Additionally, 5-aminosalicylic acid nanoparticles demonstrated a significant improvement in targeting inflamed tissue in a mouse with UC [14]. Many studies demonstrated that budesonide-loaded nanoparticles have shown promising results for targeted delivery to intestinal mucosa with inflammation [15–17].

Limitations such as burst drug release, premature nanoparticle uptake or lack of pH sensitivity have been noticed as obstacles to the efficient transport of drugs to the colon upon oral administration of nanoparticles [4]. For example, the orally administered tacrolimus-nanoparticles showed minor therapeutic effects due to the slow rate of drug release, degradation in an enzymatic environment of the upper parts of the GI tract, or uptake into the systemic circulation followed by hepatic metabolism [1,12].

Based on this consideration, additional strategies such as surface charge-dependent nanoparticles [18], PEGylation-dependent nanoparticles [19], pH-dependent nanoparticles [1,20], hydrogel-based targeting [21], ligand-receptor-mediated targeting [22] and reactive oxygen species [23] are being explored to enhance drug delivery to the areas of inflammation and achieve maximal retention time in diseased tissues for orally administered nanoparticles. Amongst the methods mentioned above, the design of a pH-sensitive nano-delivery system is one of the simplest [24] and most commonly used approaches for the selective delivery of nanoparticles to the site of inflammation [6,25,26]. Many studies demonstrated that pH-dependent BU nanoparticles could alleviate the colitis better than plain nanoparticles [27–29].

Despite the promising results observed for pH-dependent nanoparticles in targeting the colon, some concerns including the inter- and intra-individual discrepancies in pH in GIT and the disease-related variations in luminal pH could be obstacles to the successful performance of these systems. Encapsulation of nanoparticles in dual pH and time-dependent polymers has been applied as a strategy to decrease initial drug release and activity in the upper parts of GIT in comparison with plain nanoparticles [29]. While such systems demonstrated better therapeutic effects than single pH or time-dependent systems, premature drug absorption in the upper region of GI due to their small size should also be considered [30]. It should also be mentioned that the systems described to date such as entrapped nanoparticles in enteric microparticles require two steps of emulsification and solvent evaporation techniques for preparation [31,32]. On the other hand, the use of organic solvents for solubilizing pH-sensitive polymers could probably increase the leaching of the drug and result in a decrease in the drug content of the nanoparticles [33], and a lack of reproducibility [34]. To overcome these limitations researchers embedded BU lipid-based nanoparticles in enteric-coated pellets to reach the exact site of action [30].

Since lipid-based nanoparticles might be unstable in the GI tract [35], and also due to the superiority of polymeric to lipid-based formulations of BU nanoparticles in the treatment of IBD [36], polymer-based nanocarriers for specific delivery of BU to the colon would be desirable.

Chitosan (CS) as a cationic natural polysaccharide with desirable biodegradability, biocompatibility and mucoadhesive properties [16] has been extensively used in colon targeting delivery systems [37–39]. Chitosan-based nanoparticles could adhere to the surface of intestinal mucosa in inflamed tissues via the interaction between the positively charged nanoparticles and the negatively charged intestinal mucosa [40] and for drugs such as BU with local action this perspective might be beneficial.

In this study, to overcome the limitations of orally administered nanoparticles for delivery of BU to the colon, an attempt was made to design a coated multiparticulate dosage form (pellets) carrying budesonide-loaded chitosan nanoparticles. The coating was composed of combined pH and time-dependent eudragits to minimize the early drug release and absorption in the upper sections of the GI tract and maximize delivery of the drug-loaded nanoparticles to the colon.

### **2. Materials and Methods**

### *2.1. Materials*

Budesonide (BU) was acquired from Jaber Ebne Hayyan Pharmaceutical Company (Tehran, Iran). Lactose monohydrate and Avicel pH 102 were obtained from Merck, Frankfurt, Germany. Polyvinylpyrrolidone (PVP K30) (Rahavard Tamin, Saveh, Iran), eudragit S PO, (Evonik Industries AG, Hanau, Germany), eudragit L PO, (Evonik Industries AG, Hanau, Germany), eudragit RS PO, (Evonik Industries AG, Hanau, Germany), talc, (Merck, Frankfurt, Germany), triethyl citrate (TEC) (Merck, Frankfurt, Germany), chitosan (mediummolecular-weight, 75–85% deacetylated) (Sigma-Aldrich, St. Louis, USA), three polyphosphate (Merk, Frankfurt, Germany), acetic acid (Dr. Mojallaly, Tehran, Iran), ethanol (Dr. Mojallaly, Tehran, Iran), isopropyl alcohol (2-propanol) (Dr. Mojallaly, Tehran, Iran) and sodium lauryl sulfate (SLS) (Scharlau, Barcelona, Spain) were utilized. All other reagents and solvents were of analytical grades.

### *2.2. Preparation of Nanoparticles*

Budesonide-loaded chitosan nanoparticles (BCN) were made based on the ionotropic gelation technique [41]. First, 100 mg of chitosan was dispersed and dissolved in 50 mL of deionized water containing 1% *v*/*v* acetic acid (pH 4.7) to obtain the homogeneous solution (2 mg/mL). Second, ethanol was employed to dissolve BU (the minimum volume of ethanol was used). The BU ethanolic solution was added to chitosan solution at a different drug: chitosan ratios (1:10, 2:10, 3:10 and 4:10 *w*/*w*), followed by magnetic stirring at 700 rpm for 30 min (Heidolph Instruments, MR Hei-Tec, Schwabach, Germany). This solution was heated to 60 ◦C for 10 min. An aqueous solution of three polyphosphate (TPP) (1 mg/mL at 8 ◦C), was added dropwise to the warm chitosan solution under continuous stirring (700 rpm) at room temperature. The weight ratio of chitosan to TPP in all formulations was kept at 3:1 based on previous studies [42,43]. After the addition of TPP, stirring was continued for 1 more hour at room temperature. The resulting suspension was centrifuged at 21,000 rpm at 4 ◦C for 30 min (Sigma, 3–30 K, Schnelldorf, Germany) to separate nanoparticles. The nanoparticles were washed with deionized water and then freeze-dried for 48 h (Heto, Dw 3, Allerd, Denmark) using sucrose 1.5% *w*/*v* as lyoprotectant. The freeze-dried nanoparticles were maintained in a closed container for further use. The same procedure was followed for the preparation of drug-free chitosan nanoparticles (CN) for comparison purposes.
