2.10.6. Histological Assessment of Colitis Severity

A segment of the washed colon tissue specimens was fixed in a 10% formalin solution and inserted in paraffin. Cutting sections obtained by microtome were stained with hematoxylin and eosin (H&E) and examined by optical microscope to assess the severity of colitis. The colitis severity was scored from 0 to 4 based on the microscopic observation of cross-sections of the colon. The severity of colitis was scored 0 when no or minor inflammation was observed. When there was a trace region of focal inflammatory cell infiltration, the severity of colitis was scored 1. When a major part of the colon tissue was exposed to inflammation and sign of smooth muscle thickening was observed, the severity of colitis was scored 2. If ulcerated regions and inflammatory cell infiltration were formed in the tissue sections following the inflammation, the severity of colitis was assigned a score of 3. Finally, the highest colitis severity score (i.e., 4) was assigned when the entire tissue damage appeared as necrosis and gangrene [45].

### 2.10.7. Blood Glucose Level

The blood glucose level in rats was measured from the 12th day until the 17th day of the study by sampling the tail vein blood with an 18-gauge needle in the strip of a blood glucose meter (Accu-chek® Active, Roche, Mannheim, Germany). This experiment could provide some comparison amongst specific colon delivery potential of different formulations investigated [57,58], and also information regarding the potency of nanoparticles for accumulation in inflamed regions. Data on blood glucose levels are stated in mg/dL and compared with untreated animals [48].

### *2.11. Statistical Analysis*

To analyze the obtained data statistically, Graph Pad Prism software (Graph Pad Prism, version 7, San Diego, CA, USA) was employed. One and two-way analyses of variance (ANOVA) followed by Tukey–Kramer test was employed to compare the differences between means (*p* < 0.05 is an indication of a significant difference).

### **3. Results and Discussion**

### *3.1. Preparation and Characterization of Nanoparticles*

The anti-inflammatory effect of chitosan against IBD and UC has been reported in previous studies [59] where high-molecular-weight chitosan had better therapeutic effects in UC compared with-low molecular-weight chitosan [60]. On the other hand, an increase in the molecular weight of chitosan could result in a decline in the drug release which could be due to the greater viscosity of the gel layer created around the drug particles upon contact with the dissolution medium [61,62]. Since BU is a highly hydrophobic drug with low water solubility [63], its slow release could provide an insufficient concentration of the drug at the site of action. Furthermore, it has been reported that in the ionic gelation procedure for producing chitosan nanoparticles, medium-molecular-weight chitosan has shown more encapsulation efficacy compared with high-molecular-weight chitosan [64]. Therefore, medium-molecular-weight chitosan had been used in this study in an attempt to prepare BU nanoparticles by the ionic gelation technique [41,42]. This method has great potential for industrial application due to the lack of need for use of organic solvents, sonication or high temperatures and also the relative easiness of scaling up [65]. The production of

nanoparticles with a lower polymer content could lead to a more cost-effective formulation; therefore, in the current study, the effect of the drug-to-chitosan ratio in the preparation of nanoparticles was investigated to find out if it is possible to achieve desirable properties for nanoparticles at low concentrations of chitosan in formulations.

The size characteristics of nanoparticles have been found to affect drug accumulation in the inflamed colon [66]. Despite the fact that larger size nanoparticles (500 nm) are mainly localized in the epithelial lining [11,67], it has been demonstrated that excellent mucoadhesive properties belong to the nanoparticles with a particle size range between 200 and 300 nm [68]. In this study, the particle size of the different formulations ranged between 288 ± 7.5 and 566 ± 7.7 nm with small PDI values indicating the production of monodispersed particles. Table 1 shows that with an enhancement in the ratio of drug: chitosan (from 1:10 to 4:10), larger nanoparticles with lower encapsulation efficacy (37% to 2%, respectively) were obtained. The nanoparticles are formed through inter and intramolecular interactions between the negative groups of TPP and the positive charged amino groups of chitosan [69]. It has been found that chitosan molecules have a spread conformation in solution due to the electrostatic repulsion forces between free amino groups along the molecular chain [70]. These free amino groups are also responsible for the electrostatic interactions between the nanoparticles, helping to reduce particle sizes [70]. Upon the addition of the BU to the chitosan solution under constant stirring, oxygen atoms of BU interact non-covalently with the free amino groups of chitosan chains [41], leading to a decrease in the number of free amino groups. Since the concentration of free amino groups can have an impact on the creation of nanoparticles [62] and the encapsulation efficiency depends on the number of available sites present in the formulating material [70], the higher concentration of BU resulted in the formation of larger nanoparticles as well as lower encapsulation efficacy. Similar results have been reported in other studies [41,70,71].


**Table 1.** Size, PDI, zeta potential and EE for different nanoparticles.

All prepared formulations showed positive zeta-potential ranges from +26.6 ± 1.1 to +31.0 ± 1.2 mV. Since BU is a slightly anionic molecule [72], positive zeta potentials are due to the positively charged amino groups of chitosan molecules present at the surface of particles. It can be postulated that nanoparticles with zeta potential below −25 mV and above +25 mV can be considered stable at physiological pH [73]; therefore, all nanoparticles produced in the current study can be expected to be stable under physiological conditions.

According to data shown in Table 1, the formulation with a drug:chitosan ratio of 1:10 (BCN-1) provided the smallest particle size and highest encapsulation efficiency (37.2%). Thus, this formulation was chosen, as a representative candidate, for additional studies and was assigned as BCN in the next paragraphs. The yield of the production process of this formulation was 51.0 ± 1.2% which was considered satisfying, taking into account the initial low amounts of the processed material.

The morphology of the selected nanoparticle (BCN), as well as freeze-dried BCN, was observed by TEM and SEM, respectively. As it can be seen in Figure 1A,B, the nanoparticles were spherical with almost smooth surfaces. The microscopic images of BCN nanoparticles demonstrated a smaller average diameter in comparison with the DLS results. Such this difference which was also observed in other studies [16,74,75], could be attributed to the fact that in DLS the hydrodynamic diameter of the nanoparticles which is a combination of the diameter of nanoparticles plus the solvent layer attached to them [75] are measured, while in the microscopic image the hydration layer is not present [76]. The absence of major changes in nanoparticle diameter before and after freeze drying (TEM images in

comparisons with SEM images) could be related to the proper concentration and good functioning of the lyoprotectant used [77].

**Figure 1.** (**A**) TEM image of BCN; and (**B**) SEM images of the freeze-dried BCN.

Figure 2A shows thermograms of the drug powder as well as the chitosan, sucrose, BCN and physical mixture of the excipients used in the preparation of BCN matrix. Budesonide and sucrose are crystalline compounds. Budesonide, as stated by the literature, demonstrated a sharp endothermic melting peak at around 261 ◦C [41], and sucrose showed an endothermic melting peak at around 190 ◦C [78]. In contrast, chitosan showed a broad endothermic peak at 100 ◦C (corresponding to the loss of water) and an exothermic peak at around 310 ◦C (related to the decomposition of amine units) [79]. The peak related to the melting of sucrose as well as the decomposition peak of the chitosan could be observed in the thermogram of the physical mixture, while the chitosan decomposition peak shifted towards lower temperatures in the BCN thermogram. This shift could be ascribed to the rearrangement of an intermolecular and intramolecular network of the chitosan, due to the crosslinking with the TPP ions as reported elsewhere [41] and also probable interaction between chitosan and the drug. The characteristic peaks of BU disappeared in both BCN and physical mixture thermograms probably either due to the very low content of the drug or presenting in the molecularly dispersed state [44,57].

Figure 2B shows the XRPD spectra of the drug as well as the chitosan, sucrose and BCN. Chitosan is a semi-crystalline polymeric material with two characteristic peaks at 2θ of 11 and 21 [80]. The characteristic peaks of BU appeared at 2θ of 5.99, 11.95, 14.42, 15.36 and 15.96 which confirmed the crystalline nature of the drug have also been reported elsewhere [28]. The XRPD spectra of BCN exhibited the characteristic peak of BU with reduced intensity confirming the reduced crystallinity of the drug in nanoparticles.

FTIR analysis was performed to characterize the interactions between chitosan and TPP as well as possible interaction between chitosan and BU in nanoparticles (Figure 3). The budesonide spectrum presented peaks at 3495 and 2957 cm−<sup>1</sup> that correspond to the OH groups and CH2 groups, respectively. Additionally, at 1723 and 1667 cm−<sup>1</sup> the characteristic stretching peaks of C=O groups as well as stretching peaks of the double bond C=C at 1625 cm−<sup>1</sup> could be seen [81]. Chitosan showed absorption bands for O-H and N-H2 stretching at 3378 cm−1, C-H stretching at 2871 cm−1, amides and primary amines at 1663 and 1601 cm−1, respectively, C-N bond at 1319 cm−<sup>1</sup> and ultimately C-O bonds at 1076 and 1029 [41]. The characteristic absorbance peaks of the TPP, related to the phosphate groups presented at 898 cm−<sup>1</sup> [82], were shifted to lower values (892 cm−1) in BCN spectra. This shift could result from interactions between amino groups of chitosan and the phosphate groups of TPP [82]. Almost all characteristic peaks of the BU shifted to the lower values in the FTIR spectrum of the BCN which could account for the interaction between BU and the amino or hydroxyl groups of chitosan. Furthermore, in the BCN

spectrum, a new peak at 1565.4 cm−<sup>1</sup> could be seen that was neither observed in BU nor in chitosan spectrum. This new peak could be related to the N-H2 stretching bond and confirms the presence of hydrogen bonds between BU and chitosan in BCN and could be considered as evidence of the loading of BU into the chitosan nanoparticles.

**Figure 2.** (**A**) DSC thermograms and (**B**) XRPD spectra of budesonide, chitosan, sucrose and BCN.

**Figure 3.** FTIR spectra of chitosan, budesonide, TPP and BCN.

### *3.2. Preparation and Characterization of Pellets*

The freeze-dried BCN was loaded into the pellets in order to improve the local stability within the gastrointestinal tract and prevent premature uptake of nanoparticles. The extrusion–spheronization technique is a promising technology for converting nanoparticles into solid dosage forms [83]. Conventional pellets of BU were also made for comparison purposes. The formulation of conventional pellets was developed in our previous study [44].

Pellets containing nanoparticles (BCNP) were nearly spherical and had acceptable roundness with the mean sphericity and aspect ratios of 0.98 ± 0.06 and 1.14 ± 0.07, respectively [84]. The geometric mean diameter and standard deviation of BCNP were 1082.1 ± 1.3 μm which indicates the suitability of these formulations for consideration as multiparticulate dosage forms [85]. Additionally, the results of crushing strength (6.3 ± 0.9 N) and elastic modulus (1447.0 ± 170.2 MPa) of BCNP showed that these pellets had suitable mechanical properties for coating steps [86].

The pellets were subsequently coated with combined pH and time-dependent eudragits to prepare a colonic drug delivery system with a sustained release of the drug. Since the pH variations in the intestine could affect the efficacy of single pH-dependent drug delivery systems [29], such a combined system was selected for delivering the drug or nanoparticles to the desired site. The adopted coating process operative conditions, coating level and coating material compositions that were used in this study were previously set up by our team.

### *3.3. Release Studies*

### 3.3.1. Release of Budesonide from BCN and Uncoated BCNP

Figure 4 shows the release profiles of BU powder, budesonide-loaded nanoparticles (BCN) and uncoated BCNP in simulated gastric fluid (SGF, pH 1.2) and simulated colonic fluid (SCF, pH 6.8). It could be observed that the loading of BU in chitosan nanoparticles resulted in a decline in the drug release rate. Similar findings were observed in other studies when drugs were encapsulated in polymeric matrices [62,68]. According to the results, the release profiles of BCN were pH-dependent which is not expected as BU solubility is not pH-dependent [58] and also it dissolves completely within the first 2 h at SGF and SCF. The unexpected pH-dependency of BCN formulations could be described by the properties of chitosan which is soluble in an acidic medium and conversely, but it has poor solubility in neutral pH [87], resulting in a faster release at pH 1.2 compared with pH 6.8. Release profiles of BCN in both pH levels presented a burst release of about 45% and 30% in pH 1.2 and 6.8, respectively during the first hour, followed by a sustained release behavior for up to 12 h. Initial burst release might be due to the dissolution of the drug which is located at or close to the nanoparticle surface which is more accessible to the release medium, and the hydrophilic nature of chitosan [88]. It can be seen that after 12 h, only 74% and 67% of the encapsulated drug were released from BCN in pH 1.2 and pH 6.8, respectively. The incomplete drug release from nanoparticles could be explained by the drug release mechanism from the chitosan matrix. Upon the exposure of chitosan to the aqueous media, a gel layer is formed around the drug particles [62]. Slow drug diffusion through this high viscose gel layer and chitosan matrix makes the dissolution profile sustained and incomplete. A slower release profile from pelletized nanoparticles compared with the BCN demonstrated that nanoparticles were not adversely affected by mechanical stresses during the extrusion and spheronization process.

**Figure 4.** The drug release profile of budesonide, BCN and uncoated BCNP in (**A**) pH 1.2 and (**B**) pH 6.8.

### 3.3.2. Release of Budesonide from Coated BCNP

As it can be seen in Figure 4, uncoated BCNP released more than 50% of the loaded drug during the first 2 h in acidic pH. Thus, to avoid early drug release and deliver most of the drug into the colon, pellets were coated with a combined pH and time-dependent coating system. In this case, continuous dissolution testing was performed in a pH progression medium for 16 h for coated CP and BCNP. The coated pellets exhibited a pH-dependent release profile (Figure 5A) as expected. No drug release was observed when coated conventional pellets passed through media with pH 1.2, 6.5, and 6.8 for 2, 1, and 2 h residence times, respectively. It was observed that while coated CP released almost 90% of the drug in the medium simulating the pH of the colonic environment, in the case of pellets containing nanoparticles such a coating system was less effective for delivering most of the drug to the colon. Coated BCNP released almost 30% and 35% of the drug in simulating pH of gastric and small intestine fluid, respectively; thus, only 35% of the drug was released in the final part of the continuous dissolution test. The lower efficiency of this coating system in the colon delivery of pellets containing nanoparticles can be attributed to the swelling properties of chitosan [89] and the composition of the coating. Although the coating composition is insoluble in the acidic medium it is permeable to it due to the presence of eudragit RS. As the acidic medium penetrates the pellets, chitosan would be swelled and could damage the functional coating. The swelling ratio of chitosan at low pH values is higher than neutral pH due to repulsion between similarly charged groups [90]. It seems that by preventing the entry of the acidic medium into the pellet, it is possible to prevent the destruction of the coating so a larger amount of drug could be delivered to the colon. To prove this hypothesis, the same continuous dissolution test except for the initial 2 h in pH 1.2 was performed with and without 4% *w*/*v* rat cecal content in the last medium with pH 6.8. As illustrated in Figure 5B, when the formulation was directly placed in the medium with pH 6.5 (bypassing the acidic environment) only 30% of the drug was released before reaching the colon-simulated media. Although it seems that the drug release from coated BCNP in such conditions might be incomplete (60% during 14 h), the presence of rat cecal content in the SCF medium increased the drug dissolution (80% during 14 h). This confirms the effect of the bacteria in the rat cecal as well as the pH sensitivity of the system to the drug release. This suggests that the degradation of chitosan in the pellets' matrix might happen upon the action of the bacterial enzymes in the SCF [91], resulting in an increase in the release rate. The overall results indicated that in the case of coated BCNP, where the target of drug delivery is the colon, drug release in the stomach would be considered as a drawback [92], so more strategies such as using enteric-coated capsule [93] filled with the coated BCNP should be considered for oral delivery of this type of formulation where the target site is the colon area. However, when drug delivery is aimed at the entire GIT (Crohn's disease) coated BCNP could provide a continuous release throughout the GIT.

### *3.4. Morphological Characteristics of Coated BCNP*

Figure 6 illustrated the SEM images of the coated BCNP. These pellets were spherical and had smooth surfaces (Figure 6A). The cross-sectional image showed a uniform coat with approximately 30 μm thickness (Figure 6B).

**Figure 5.** The drug release profile of coated pellets (**A**) in the continuous mode of dissolution test and (**B**) in the continuous mode of dissolution test except for the initial 2 h in pH 1.2.

**Figure 6.** (**A**) Scanning electron microscopy of the coated BCNP and (**B**) the cross-sectional image of the coated BCNP.

### *3.5. In Vivo Therapeutic Efficacy in Rats*

The therapeutic effects of the selected formulations were characterized by determining some indexes of colonic tissue edema (colitis activity index (CAI), colon/body weight, weight/length ratio of colon, the level of MDA and amount of GSH in colon tissue) [94], as well as histological studies.

The CAI is a clinical tool for the evaluation of the severity of inflammation in living animals [14]. After inducing experimental colitis, CAI increased rapidly (Figure 7); all

animals showed loss of weight and diarrhea over the next 3 days indicating that all animals presented with colitis. All treatment groups showed a consistent decrease in CAI throughout the entire treatment period (days 5 to 11). On the day of sacrifice, all treated rats showed statistically lower CAI compared with the untreated control group (*p* < 0.05) in response to the decrease in intestinal inflammation [95]. While the group treated with free BU showed little improvement in the CAI, the coated BCNP-treated group demonstrated the most prominent decline in this index (two-fold reductions compared with the untreated group). For those rats which were not sacrificed on day 12, during the next 5 days of monitoring with no treatment (days 13 to 17), the increase in the inflammation severity was observed for both the free BU and coated CP treated groups, so that the CAI value significantly increased from day 12 to day 17, while the difference between CAI in this period was not significant for BCN, BCNP and CN treated groups. In the case of coated BCNP-treated rats, even though the administration of the drug was discontinued, surprisingly the healing process continued (Figure 7). A similar finding has been reported in another study in which the nanoparticle-treated rats continued to heal after stopping the treatment [96].

**Figure 7.** Colitis activity index for different rat groups.

As can be seen in Figure 8A, healthy rats had a colon length of 19 cm, while the colon length significantly decreased in untreated rats with values of ~11 cm. Additionally, untreated colitis rats showed signs of localized inflammation and ulceration and thickening of the colon wall. Although the conditions of the rats did not return to normal during the experiment period, it seems that the inflammatory signs were decreased in all of the treated groups when compared with the untreated group (Figure 8A). Based on the visual comparison, the most increase in colon length was observed in coated BCNP-treated group (almost 9 cm), and the colon length of this group was much close to the healthy group.

**Figure 8.** (**A**) Photographs for macroscopically examination and (**B**) colon damage score of the colon specimens resected from rats 24 h after the last drug administration.

As shown in Figure 9A,B, the colon/body weight ratio and weight/length ratio of the colon declined significantly in all of the treated groups when compared with the untreated control group (*p* < 0.05). These two indexes usually increase in response to the severity of inflammation [96]; therefore, the obtained results could indicate that all of the prescribed formulations had some anti-inflammatory effects. Regarding these two inflammatory indexes data, again, the most effective formulation for the treatment of inflammation was coated BCNP whilst free BU was less effective than others. Similar to the results obtained for CAI, BU and coated CP-treated groups showed a statistical difference when these two items (the colon/body weight ratio and weight/length ratio) were compared 6 days after the last drug administration (*p* < 0.05) while no significant differences for these items were observed in groups treated with nanoparticle-based formulations.

It is well-known that the amount of GSH is an indication of the anti-oxidative potential of the cells upon treatment and MDA is a marker indicating tissue injury [56]. A significant increase in the amount of GSH (Figure 9C), as well as a significant decrease in the level of MDA (Figure 9D), was observed in all treated groups compared with the untreated group. These results demonstrated that inflammation significantly decreased upon treatment of rats.

Figure 10 shows the histopathological characteristics of the colon's tissues and Figure 8B presents a colitis damage score. Light microscopic observation of the colon tissue of healthy rats demonstrated normal colon histology with no signs of unusual tissue architecture or abnormal tissue morphology (Figure 10A) which was scored as zero for colitis. By contrast, in untreated colitis rats the utmost tissue damage, and goblet cell depletion associated with disruption and necrotic mucosal structure was seen (Figure 10B) which corresponds to the highest score (four) of colitis. Although the histological sections of the colonic tissues in all treatment groups showed some signs of improvement compared with the untreated group (Figure 10C–H), in the colon tissue of free budesonide-treated groups signs of smooth muscle thickening were seen (Figure 10H) which was scored as two for colitis. A much closer condition to the normal colon histology was seen in rats treated with coated BCNP and coated CP (Figure 10C,E). Observation of colon tissue of other treated groups (BCNP, BCN and CN) demonstrated normal colon histology; however, trace regions of focal inflammatory cell infiltration were seen (Figure 10D,F,G) which was scored one for colitis.

**Figure 9.** (**A**) Colon/body weight ratio; (**B**) Weight/length ratio of colon; (**C**) GSH and (**D**) level of MDA; determined 1 day or 6 days after final drug administration. \* *p* < 0.05 comparison of treatment groups with untreated group after "1 day". # *p* < 0.05 comparison of each group "1 day" and "6 day" after treatment.

**Figure 10.** Histopathological characteristics of the colon tissues of the rats subjected to different treatments 24 h after the last drug administration. (**A**) Rat with normal colonic tissues; (**B**) untreated rat; (**C**) rat treated with coated CP; (**D**) rat treated with BCNP; (**E**) rat treated with coated BCNP; (**F**) rat treated with BCN; (**G**) rat treated with CN and (**H**) rat treated with Budesonide. (arrows point to area of inflammation).

Overall, results from the in vivo studies demonstrated that all prescribed formulations significantly are effective in colitis treatment. A decrease in inflammation in CN-treated rats could be related to the anti-inflammatory effects of chitosan in IBD [59].

Formulation containing nanoparticles was found to be more effective than free BU formulation. This could be due to the absorption of a higher amount of free drug in the upper sites of GIT which resulted in inadequate amounts of drug delivered to the inflamed colon [97]. The high efficacy of nanoparticles in the treatment of colitis indicates the presence of higher amounts of the drug at the site of inflammation, confirming the greater accumulation of small-sized particles at the site of inflammation [13,98] and the ease of their adherence to the thick mucus layer [99]. In addition, polymeric nanoparticles would protect drug release in the upper site of GIT and allow for a prolonged residence at the surface of the release site [68].

In this study, nanoparticles were turned into pellet dosage form to increase the clinical effects by reducing the probability of premature uptake in the small intestine. Although histological characteristics of BCN and BCNP-treated rats demonstrated similar therapeutic effects, according to the other in vivo results the superiority of BCNP is obvious. Degradation of free nanoparticles in oral delivery and during their passage in upper parts of the GIT could result in a lower drug amount delivered to the colon [6] while incorporating the nanoparticles in the matrix of pellets could improve local stability within the gastrointestinal tract [100].

During the treatment period, coated BCNP formulation showed the most anti-inflammatory effects; however, regarding the amount of GSH, level of MDA and histological assessment of colon tissues, significant differences with the coated CP were not clearly visible. The superiority of coated BCNP formulation in in vivo studies in rats could be related to the shorter and faster movement of delivery systems in the intestine in rats compared with humans [101], which could take more drugs to the site of action.

The higher efficacy of coated systems (coated BCNP and coated CP) compared with others is due to their coating system which allows the maximum delivery of the drug to the target site without early release in the stomach or upper sections of the gastrointestinal tract after taking the formulation orally.

Although it seems that with a proper coating system colon targeting of BU pellets could effectively be achieved, the advantages of using pelletized BU nanoparticles would be bolded after stopping drug administration. After treatment discontinuation, the group receiving coated CP showed a fast relapse, whereas this was not the case in rats treated with coated BCNP. Similar findings previously had been observed for PLGA nanoparticles [96,98]. These differences may suggest a higher drug accumulation in the inflamed tissue after treatment with nanoparticles and remaining drug carriers in the inflamed regions for up to several days. This claim could be confirmed by an examination of blood glucose levels.

### *3.6. Blood Glucose Level*

Glucocorticoids are known to increase blood glucose levels due to their potential for enhancing intestinal sugar uptake [58]. Thus, for determination of the potency of formulations which their aim is local drug delivery to the intestinal mucosa and accumulation in it, the level of blood glucose of the animals could be considered as an indicator. As it can be seen in Figure 11, the blood glucose level of all rats treated with a formulation containing the drug increased progressively compared with untreated rats. These results indicate that a certain amount of drug was available in the inflamed region on the test day. However, glucose levels were significantly higher in the group of rats treated with nanoparticles containing BCN and BCNP compared with the groups treated with free drugs. The increased glucose level could suggest more extent of targeted drug delivery to the site of inflammation by using nanotechnology. Similar results had been seen previously in other studies [48,57]. The blood glucose level was significantly higher in the rats treated with coated CP and coated BCNP compared with the other treated groups (110 and 109 mg/dl, respectively), which is related to the higher amounts of drug that were delivered to the colon by avoiding release in the upper parts of GIT or premature uptake of the drug due to the coating system. However, it was observed that animals treated with coated CP revealed a remarkable decrease in blood glucose concentration within 24 h after the first examination, while in animals treated with coated BCNP, this reduction become significant after four days. These results also confirm higher drug accumulation after treatment with nanoparticles in comparison with other groups which may last for up to several days and are in agreement with other in vivo results.

**Figure 11.** Blood glucose level (mg/dL) in animals on 12th day to 17th day of study. \* *p* < 0.05 comparison of treatment groups with untreated group. # *p* < 0.05 comparison of each group on different days.

### **4. Conclusions**

To overcome the limitations of oral delivery of budesonide nanoparticles in colon delivery, a novel formulation based on functional coating of pelletized nanoparticles was proposed. Although in vitro dissolution test results did not indicate the superiority of this formulation over conventional coated pellets (due to the effect of acidic conditions on chitosan), in vivo results demonstrated the supplementary effect of nanoformulation of budesonide and the encapsulation in a properly coated pellet system for colon delivery that resulted in a synergistic and long-lasting anti-inflammatory effect in comparison with coated pellets containing free drug.

**Author Contributions:** Conceptualization, F.S. (Fatemeh Sadeghi), H.A.G., A.A. and A.N.; methodology, F.S. (Fatemeh Sadeghi), H.A.G., M.G.R., H.K. and A.A.; software, A.A. and H.K.; formal analysis, F.S. (Fatemeh Soltani), F.S. (Fatemeh Sadeghi), H.A.G. and A.A.; resources, F.S. (Fatemeh Sadeghi); data curation, F.S. (Fatemeh Soltani), F.S. (Fatemeh Sadeghi) and A.A.; writing original draft preparation, F.S. (Fatemeh Soltani); writing—review and editing, F.S. (Fatemeh Sadeghi) and A.N.; supervision, F.S. (Fatemeh Sadeghi), H.A.G., A.A. and AN. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Vice Chancellor for Research and Technology of Mashhad University of Medical Sciences, grant number 981658.

**Institutional Review Board Statement:** The animal study protocol was approved by the Ethics Committee of Mashhad University of Medical Sciences (IR.MUMS.REC.1399.011) for studies involving animals.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
