An oral colon-targeted delivery system of budesonide, preparation method and application

Abstract

The present application relates to the technical field of medicine, and in particular to a budesonide-loaded oral colon-targeted delivery system, a preparation method and application. The delivery system takes Pickering emulsion-encapsulated budesonide as the core, and is sequentially coated with a low-ester pectin-calcium ion crosslinked inner layer and a low-ester pectin-chitosan polyelectrolyte complex outer layer. The preparation process is realized by combining an emulsion template method with layer-by-layer self-assembly technology. The obtained microcapsules have a uniform spherical structure, a particle size of about 400 μm, and a double-layer three-dimensional network structure. The delivery system remains stable in the gastrointestinal environment, and only releases drugs rapidly under colon pH conditions. In vitro simulation experiments show that the drug release rate is less than 5% in the stomach / small intestine stage, and is rapidly released in the colon stage. Animal experiments confirm that the effect of treating ulcerative colitis is equivalent to that of mesalamine, while the systemic toxicity is significantly reduced, the drug retention time at the colon site is more than 24 hours, and there is no accumulation in non-target organs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to an oral colon-targeted delivery system loaded with budesonide, its preparation method, and its application. Background Technology

[0002] Ulcerative colitis (UC), a chronic relapsing inflammatory bowel disease, has long relied on aminosalicylic acid derivatives, glucocorticoids, and immunosuppressants for clinical treatment, but all have significant limitations. Aminosalicylic acid derivatives are only suitable for mild to moderate cases, and long-term use easily leads to drug resistance; they are also insufficiently effective for severe cases. Although glucocorticoids can quickly control acute inflammation, long-term use leads to systemic side effects such as osteoporosis and metabolic disorders, making them unsuitable for maintenance therapy. Biologics, represented by anti-TNF-α monoclonal antibodies, can improve moderate to severe symptoms, but they suffer from high failure-to-respond rates, inconvenient administration methods, and high treatment costs.

[0003] Budesonide (BUD), a second-generation glucocorticoid, has the advantages of good anti-inflammatory effects and few side effects. However, its oral administration route is limited by the first-pass effect of the liver, with approximately 90% of the drug being metabolized and cleared before entering systemic circulation, resulting in extremely low oral bioavailability. To maintain effective blood drug concentrations, patients need to take the medication multiple times a day, significantly increasing the medication burden.

[0004] The development of oral colon-targeted delivery systems (OCDDS) still faces multiple challenges. Single-response carriers, such as pH-dependent materials (Eudragit S100), are significantly affected by individual differences in gastrointestinal transit time; time-delay formulations (such as HPMC capsules) exhibit significant release lag fluctuations. While pickering emulsions possess good biocompatibility and stimuli responsiveness, they are prone to significant droplet coalescence in pepsin-rich environments. Although natural polysaccharide carriers can be degraded by colonic enzymes, their drug release lag fluctuates considerably due to individual differences in enzyme activity; in vitro simulation studies show significant drug release in the early stages.

[0005] While composite OCDDS attempts to combine multiple response mechanisms, they generally suffer from compatibility issues with carrier materials. Multi-step encapsulation processes significantly reduce drug loading, and large-scale production requires the use of organic solvents, posing a potential risk of solvent residue. Research data shows that long-term use of traditional drugs easily leads to drug resistance, biologics have high primary / secondary loss-of-response rates, composite carriers may cause burst-release effects, drug release from non-inflammatory sites can cause toxic side effects, and organic solvent residues may exceed safety limits. Therefore, there is an urgent need to develop novel colon-targeted delivery systems that combine stability, precise targeting, and low toxicity. Summary of the Invention

[0006] In view of this, the present invention proposes an oral colon-targeted delivery system loaded with budesonide, its preparation method and application.

[0007] The technical solution of this invention is implemented as follows:

[0008] In a first aspect, the present invention provides a method for preparing an oral colon-targeted delivery system loaded with budesonide, comprising the following steps:

[0009] S1. Budesonide is dispersed in the oil phase, and then a 5wt%-6.5wt% calcium ion aqueous dispersion is mixed with the oil phase. After shearing and homogenization, a water-in-oil Pickering emulsion is formed. The calcium ions are one of calcium chloride, calcium carbonate, and calcium alginate. The oil phase is one or more of soybean oil, rapeseed oil, corn oil, peanut oil, palm oil, rapeseed oil, and olive oil.

[0010] S2. The Pickering emulsion is added dropwise to a 0.5wt%-3wt% low-ester pectin solution, the pH is adjusted to 3.5-4.5, and crosslinked for 10-30 minutes to obtain gel beads;

[0011] S3. Add the gel beads to a 1wt%-3wt% chitosan solution, crosslink for 20-60 min to obtain microcapsules, and dry them to obtain the oral colon-targeted delivery system loaded with budesonide.

[0012] In some specific embodiments, in step S1, the mass-to-volume ratio of budesonide to the oil phase is 5-30 mg: 1 L.

[0013] In some specific embodiments, in step S1, the volume ratio of the calcium ion aqueous dispersion to the oil phase is (4.5-5.5):1.

[0014] In some specific embodiments, in step S2, the volume ratio of the Pickering emulsion to the low-ester pectin solution is (75-125):1.

[0015] In some specific embodiments, in step S3, the mass-to-volume ratio of the gel beads to the chitosan solution is 1:(5-10).

[0016] In some specific embodiments, the degree of esterification of the low-ester pectin is 25%-35%.

[0017] In some specific embodiments, the number-average molecular weight (Mn) of the chitosan is 300,000 to 1,100,000 Daltons.

[0018] Secondly, the present invention provides an oral colon-targeted delivery system for budesonide prepared by any of the above-described preparation methods, which has a core-shell structure, including a core structure, an inner shell, and an outer shell; the core is budesonide loaded with Pickering emulsion, the inner shell is formed by cross-linking low-ester pectin with calcium ions, and the outer shell is formed by electrostatic assembly (composite) of low-ester pectin and chitosan through polyelectrolytes.

[0019] Thirdly, the present invention provides the application of the preparation method described in any of the above-mentioned claims in the preparation of anti-colitis drugs.

[0020] In some specific embodiments, the colitis is ulcerative colitis.

[0021] The beneficial effects of the present invention include at least the following:

[0022] This invention utilizes budesonide, an anti-inflammatory drug encapsulated in Pickering emulsion, as its core component, along with low-ester pectin and Ca... 2+ Cross-linking forms the inner shell, while low-ester pectin and chitosan are combined via polyelectrolytes to form the outer shell, thus providing an oral colon-targeted delivery system for budesonide (BUD-CG). BUD-CG possesses a distinct, uniformly sized spherical structure (approximately 400 μm), and SEM characterization confirms its dual-layer three-dimensional network structure. This structure exhibits controllable swelling properties in the gastrointestinal environment, providing a stable physical basis for sustained drug release. In vitro drug release experiments show that BUD-CG has a release rate of <5% in simulated gastric and small intestinal fluids, but rapid release in colonic fluid, demonstrating its pH sensitivity and enabling precise colon-targeting.

[0023] The BUD-CG provided by this invention, in a colitis model, showed comparable results to the positive control drug (Mesalamine) in terms of DAI score, colon length, stool morphology, and organ weight ratio. It also effectively repaired mucosal integrity and reduced crypt damage and goblet cell loss. BUD-CG significantly reduced neutrophil and macrophage infiltration in colonic tissue and inhibited the secretion of pro-inflammatory factors.

[0024] Furthermore, compared to BUD alone, BUD-CG significantly reduces cytotoxicity, exhibits fluorescence intensity in the colon for ≥24 hours, and shows no significant accumulation in non-target organs (heart, liver, spleen, lung, kidney), indicating that it prolongs local drug action time and reduces systemic exposure risk through a colon-responsive release mechanism, demonstrating good biosafety and targeting properties. Experimental data fully validate that BUD-CG has structural stability and also possesses colon-targeted delivery, sustained-release synergistic effects, and inflammatory microenvironment regulation functions, providing a novel, highly efficient, and low-toxicity delivery system for ulcerative colitis with significant clinical translational potential.

[0025]

Terminology Explanation

[0026] In some specific embodiments of the present invention, the meanings of the relevant terms include the following:

[0027] Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 A is a schematic diagram of BUD-CG preparation; Figure 1 B is a diagram illustrating the therapeutic principle of BUD-CG for colitis;

[0030] Figure 2 Pickering emulsion characterization: Figure 2 A represents the apparent (O:W) of Pickering emulsions with different oil-to-water ratios. Figure 2 B represents the microstructure of Pickering emulsions with different oil-to-water ratios; Figure 2 C is the particle size distribution of Pickering with the optimal oil-water ratio (1:5); Figure 2 D is the Zeta potential distribution of the Pickering emulsion with the optimal oil-to-water ratio (1:5);

[0031] Figure 3 A is a Pickering emulsion formed from 5%-6.5% calcium carbonate; Figure 3 B consists of microcapsules formed from 5%-6.5% calcium carbonate; Figure 3 C is a Pickering emulsion formed from 5-30 mg of budesonide; Figure 3 D is prepared by using an emulsion to LMP solution volume ratio of 1:(50-125) for CG;

[0032] Figure 4 A1-4A3 are the microscopic morphology and finished product appearance of microcapsules formed by LMP solution and Pickering emulsion with a volume ratio of 1:100. Figure 4 B1-4B3 are SEM (scanning electron microscope) images of microcapsules of LMP solution and Pickering emulsion at different magnifications with a volume ratio of 1:100. Figure 4 C represents the FT-IR (Fourier transform infrared) spectra of CS, LMP, and CG microcapsules. Figure 4 D is the standard curve for BUD; Figure 4 E represents the BUD release behavior of the BUD-CG microcapsules;

[0033] Figure 5 Cell viability analysis: Figure 5 A is the empty CG preparation. Figure 5 B is pure BUD medicine. Figure 5 C is a BUD-CG formulation;

[0034] Figure 6 A shows colon photos of each group; Figure 6 B represents the mouse's weight change in the morning; Figure 6 C represents the change in mouse body weight in the afternoon; Figure 6 D represents the weight distribution in the colon; Figure 6 E is the colon weight / length ratio; Figure 6 F is the spleen weight / body weight ratio; Figure 6 G is the kidney weight / body weight ratio (**P<0.01, *P<0.05 vs DSS model group).

[0035] Figure 7 A is a representative H&E staining image of colon sections from each group of mice (scale bar: 100μm). Figure 7 B represents the histological score of H&E staining images of the colon of mice in each group; Figure 7 C is the immunohistochemical assay for MPO (scale bar: 100 μm).

[0036] Figure 8 A is an immunofluorescence staining image of macrophages in colon tissue (scale bar: 100 μm). Figure 8 B represents the IL-2 level in colon tissue; Figure 8 C represents the IL-6 level in colon tissue; Figure 8 D represents the TNF-α level in colon tissue;

[0037] Figure 9 Images show the biodistribution of microcapsules in visceral organs and the gastrointestinal tract (GIT) at different time points after oral administration. Figure 9A represents the main organ distribution of the microcapsules; Figure 9 B is the image of the separated GIT. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0039] In some specific embodiments of the present invention, the main materials and reagents include the following:

[0040] Low-ester pectin (LMP) (DE value 26.1%) was purchased from Andre Pectin Ltd. Chitosan (CS), with a number-average molecular weight of 1,087,000 Da, was provided by Jinko Biochemical Co., Ltd. Budesonide (BUD), sodium dextran sulfate (DSS), and sodium carboxymethyl cellulose were purchased from Aladdin Biochemical Technology Co., Ltd. Soybean oil was purchased from Yihai Kerry Arawana Food Group Co., Ltd. Mesalamine granules were purchased from Shanghai Aifa Pharmaceutical Co., Ltd., heavy calcium carbonate was purchased from Sinopharm Chemical Reagent Co., Ltd., concentrated hydrochloric acid was purchased from Xinyang Chemical Reagent Factory, sodium hydroxide was purchased from Sinopharm Chemical Reagent Co., Ltd., and sodium dextran sulfate was purchased from Aladdin Reagent Co., Ltd. ELISA kits were purchased from Xinbosheng Biotechnology Co., Ltd. All water used was deionized water, and all other reagents were standard reagents.

[0041] Example 1

[0042] 1. Preparation of BUD-CG microcapsules

[0043] (1) Obtaining Pickering emulsion

[0044] Calcium carbonate powder was dispersed in deionized water to prepare a 5.5% (w / v) calcium carbonate aqueous dispersion. 0.1 g of BUD was weighed and dispersed in 5 mL of soybean oil, stirred until homogeneous, to prepare an oil phase containing 2% (w / v) BUD. 5 mL of the calcium carbonate aqueous dispersion (aqueous phase) and 1 mL of the BUD-containing oil phase (the oil-water ratio in Example 2 is the volume ratio of the oil phase to the water phase) were added sequentially to a 10 mL centrifuge tube, mixed by shaking, and homogenized for 5 min to obtain an O / W type Pickering emulsion.

[0045] (2) Obtaining pectin calcium gel beads

[0046] Add the O / W Pickering emulsion to 200 mL of 2.5% (w / v) LMP solution under stirring at a volume ratio of 100:1. After dispersing evenly, slowly add 1 mol / L acetic acid to adjust the pH to 4, and continue stirring for 30 min. Wash with deionized water and filter through an 80-mesh sieve to obtain pectin calcium gel beads.

[0047] (3) Obtaining BUD-CG microcapsule particles

[0048] Disperse 20 mL of pectin calcium gel beads into 200 mL of 1% (w / v) CS colloidal solution while stirring, and continue stirring for 30 min to crosslink. Wash successively with deionized water and anhydrous ethanol, and filter through an 80-mesh sieve to obtain BUD-CG wet particles. Air dry at room temperature to obtain BUD-CG microcapsule particles.

[0049] 2. Preparation of CG microcapsules

[0050] (1) Obtaining Pickering emulsion

[0051] Calcium carbonate powder was dispersed in deionized water to prepare a 5.5% (w / v) calcium carbonate aqueous dispersion. 5 mL of the calcium carbonate aqueous dispersion and 1 mL of soybean oil were added sequentially to a 10 mL centrifuge tube, mixed by shaking, and homogenized for 5 min to obtain an O / W type Pickering emulsion.

[0052] (2) Obtaining pectin calcium gel beads

[0053] Add the O / W Pickering emulsion to 200 mL of 2.5% (w / v) LMP solution under stirring at a volume ratio of 100:1. After dispersing evenly, slowly add 1 mol / L acetic acid to adjust the pH to 4, and continue stirring for 30 min. Wash with deionized water and filter through an 80-mesh sieve to obtain pectin calcium gel beads.

[0054] (3) Obtaining CG microcapsule particles

[0055] Disperse 20 mL of pectin calcium gel beads into 200 mL of 1% (w / v) CS colloidal solution while stirring, and continue stirring for 30 min for crosslinking. Wash successively with deionized water and anhydrous ethanol, and filter through an 80-mesh sieve to obtain CG wet particles. Air dry at room temperature to obtain CG microcapsule particles.

[0056] Example 2

[0057] 1. Method

[0058] Pickering emulsions with different oil-water ratios were prepared according to the preparation steps of BUD-CG microcapsules in Example 1.

[0059] Pickering emulsions of calcium carbonate aqueous dispersions with different concentrations (w / v=5, 5.5, 6 or 6.5) were prepared according to the preparation steps of BUD-CG microcapsules in Example 1.

[0060] The morphology of different Pickering emulsions was observed and photographed using an electron microscope. The particle size distribution and zeta potential of the samples were determined using a nanoparticle size potentiostat. The specific methods are as follows:

[0061] The dispersant was deionized water with a refractive index of 1.33, and the temperature was 25℃. 1 μL of the emulsion was diluted with 10 mL of distilled water, and the particle size distribution of the diluted sample in pure water was measured using a nanoparticle size analyzer. Each sample was measured three times, and the average value was taken. 1 mL of the emulsion was diluted with 10 mL of distilled water, and the zeta potential of the diluted sample in pure water was measured using a zeta potential analyzer. Each sample was measured three times, and the average value was taken. The morphology of Pickering emulsions formed with different calcium carbonate concentrations was observed using an electron microscope.

[0062] 2. Results

[0063] The effect of different oil-water ratios (V:V = 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, or 1:7) on the properties of Pickering emulsions is as follows: Figure 2 As shown in Figures A and 2B, with the increasing proportion of the aqueous phase, the volume fraction of the emulsion phase in the emulsion first increases and then decreases. When the oil-to-water ratio is less than 1:5, the proportion of water in the system is in excess, and after emulsification, there is an unemulsified aqueous phase in the system. When the oil-to-water ratio is equal to 1:5, the proportions of the oil phase, aqueous phase, and stabilizer in the system reach equilibrium. After shearing treatment, the system exhibits a homogeneous and stable emulsion state. When the oil-to-water ratio is greater than 1:5, the proportion of oil in the system is in excess, and after emulsification, there is an unemulsified oil phase. Therefore, based on the stability and the state of the emulsion under a microscope (…),… Figure 2 B) It can be seen that when the oil-water ratio is 1:5, the Pickering emulsion has a better morphology.

[0064] Depend on Figure 2 As shown in C, the optimal Pickering emulsion has an average particle size of 231.1 nm, a particle size distribution range of 150-500 nm, and a PDI of 0.300, indicating uniform particle size. This experiment used rod-shaped calcium carbonate, with a potential of +1.2 mV. Compared to rod-shaped calcium carbonate in pure water, the average Zeta potential in the calcium carbonate-stabilized Pickering emulsion is -1.04 mV (e.g., ...). Figure 2D, Pickering2.1 is a Pickering emulsion with an oil-to-water ratio of 1:5, and Pickering2.2 is rod-shaped calcium carbonate); this indicates the presence of ion exchange, or the addition of soybean oil molecules, which reduces the electric field strength in the emulsion, thus slightly decreasing the Zeta potential value on the emulsion surface.

[0065] The detection results for Pickering emulsions prepared from calcium carbonate aqueous dispersions of different concentrations (w / v=5, 5.5, 6, or 6.5) are as follows: Figure 3 A and 3B indicate that excessively low or high calcium carbonate concentrations can cause poor Pickering emulsion morphology, resulting in locally depressed ellipsoidal emulsions, which in turn leads to poor encapsulation and leakage of contents. From Figure 3 C indicates that the optimal concentration of the calcium carbonate aqueous dispersion is 5.5% (w / v). The Pickering emulsion becomes denser with increasing drug concentration, demonstrating that the drug penetrates the emulsion.

[0066] Example 3

[0067] 1. Method

[0068] Following the preparation steps of BUD-CG microcapsules in Example 1, BUD-CG microcapsules were prepared using LMP solution and Pickering emulsion at different volume ratios (V / V = 1:50, 1:75, 1:100, or 1:125). The morphology of the microcapsules was observed and photographed using an electron microscope. The morphology of the freeze-dried microcapsule samples was studied using a scanning electron microscope. After gold sputtering, the samples were observed at different magnifications under an accelerating voltage of 10 kV.

[0069] 2. Results

[0070] like Figure 3 As shown in Figure D, both LMP solution and Pickering emulsion with a volume ratio of 1:100 can form well-defined microcapsules. Figure 4 A1 shows that the microcapsules are uniform in size and densely packed. Figure 4 As shown in A2-A3, the BUD-CG microcapsules exhibit distinct granular structures with well-defined morphology and a uniform particle size distribution, approximately 400 μm in diameter. The microstructure of the BUD-CG microcapsules was characterized using SEM at different magnifications. Figure 4 B1 clearly showed that the BUD-CG microcapsules were uniformly sized spheres with dense encapsulation. Figure 4 B2, by observing the boundary between the inner and outer layers, clearly showed structural differences between the outer and inner microcapsule layers, confirming that the BUD-CG microcapsules formed a bilayer structure. Both the inner and outer layers are interconnected three-dimensional network structures. Figure 4B3) facilitates swelling in the gastrointestinal environment, providing a structural basis for slow drug release.

[0071] Example 4

[0072] 1. Method

[0073] Fourier transform infrared (FT-IR) spectroscopy analysis was performed on freeze-dried BUD-CG microcapsule samples (with single-component LMP and CS as control samples, respectively) using potassium bromide pelleting. The freeze-dried microcapsule samples were dried under an infrared lamp. A small amount of potassium bromide was placed in a mortar, and the sample was added at a ratio of 1:100 (sample:potassium bromide). The mixture was carefully ground, and the ground sample was placed in a pelleting device to obtain a transparent sample sheet. This sheet was mounted on a solid sample holder and placed in the sample cell of the infrared spectrometer, from 4000-800 cm⁻¹. -1 Wavenumber scanning was performed to obtain the infrared absorption spectrum.

[0074] 2. Results

[0075] like Figure 4 As shown in C, in the infrared spectrum of LMP, at 3397 cm⁻¹ -1 The nearby peak is the stretching vibration peak of OH- and -NH2, 2931 cm⁻¹. -1 The nearby peak is the stretching vibration peak of -CH, at 1700 cm⁻¹. -1 -1600 cm -1 The peak is the C=O vibration peak; 1421 cm⁻¹ -1 The peak is the CH bending vibration peak, 1200 cm⁻¹. -1 -1000cm -1 The peaks are ROR and ring CC stretching vibration peaks; in the CS infrared spectrum, the peak at 3415 cm⁻¹ is... -1 The peak value for the stretching vibration of NH is 2921 cm⁻¹. -1 The peak of the stretching vibration of CH is 1631 cm⁻¹. -1 The peak of the bending vibration of NH is located at 1390 cm⁻¹. -1 The point is a tensile vibration of CN, 1031 cm. -1 The position corresponds to CO tensile vibration. The microcapsule infrared spectrum shows a position of 3390 cm⁻¹. -1 3009cm -1 2854cm -1 The peaks at 1745 and 1605 correspond to the stretching vibrations of OH- and CH, respectively. The peak at 1420 cm⁻¹ is the stretching vibration peak of C=O, and the peak at 1605 is the vibration peak of C=O. -1 The peak is the CH bending vibration peak, 1200 cm⁻¹. -1 -1000cm -1The vibration peaks are the ROR and the ring CC stretching vibration peaks. Figure 4 C shows that the infrared spectrum of the microcapsule formulation only shows changes and shifts in peak intensity, and no new absorption characteristic peaks were found, indicating that BUD-CG microcapsules are simply physical cross-linked and no new substances are generated based on the raw materials.

[0076] Example 5

[0077] 1. Method

[0078] Weigh 10 mg of BUD standard into a 100 mL amber volumetric flask, dissolve and dilute to volume with anhydrous ethanol. Transfer 5 mL of this solution to a 10 mL amber volumetric flask and dilute to volume to obtain the BUD stock solution. Accurately pipette 0, 1, 2, 3, 4, and 5 μL of the BUD stock solution from the 10 mL amber volumetric flasks, add anhydrous ethanol to dilute to 100 mL, shake thoroughly, and measure at a wavelength of λ = 245 nm. A standard curve can be obtained based on the absorbance value A and the BUD concentration.

[0079] A certain mass of air-dried BUD-CG at room temperature was weighed into an Erlenmeyer flask. 50 mL of simulated gastric fluid (SGF) at pH 1.2 was added to the flask. Samples were removed at regular intervals, and the same volume of SGF was added to the flask. After 2 hours, the trough medium was changed to simulated small intestinal solution (SSIS) at pH 6.8. After 3 hours, the trough medium was changed to simulated colonic solution (SCS) at pH 7.4, and samples were taken as described above. The samples were detected using UV at 245 nm. The cumulative drug release rate of the microcapsules at different time points was calculated.

[0080] Among them, the simulated gastric juice, simulated small intestinal solution, and simulated colon solution are prepared using concentrated hydrochloric acid and sodium hydroxide.

[0081] according to: The formula is used for calculation; where C n V represents the drug concentration at the nth sampling, V is the single sampling volume, V0 is 50 mL, and W is the drug loading of the microcapsule.

[0082] 2. Results

[0083] By comparing single-layer Ca 2+ -LMP microcapsules and bilayer Ca 2+ -LMP-CS microcapsule drug release behavior (e.g. Figure 4 (D) indicates that the double-layered microcapsules are better able to achieve targeted release in the colon.

[0084] To further investigate the effect of LMP concentration on the in vitro release behavior of BUD-CG, the cumulative release of BUD-CG at three LMP concentrations in a simulated gastrointestinal environment was tested (e.g., Figure 4 (E) In the first 2 hours, the drug release rate in each group in simulated gastric fluid (pH 1.2) was less than 5% of the total drug amount. When the release environment was changed to simulated small intestinal solution (SSIS) at pH 6.8, the drug release rate increased. After 5 hours, when the dissolution environment was changed to simulated colonic solution (SCS) at pH 7.4, the drug release rate in each group significantly accelerated. The results indicate that BUD-CG's release behavior is significantly pH-sensitive. Furthermore, by comparing the release rates of each formulation, it was demonstrated that 2.5% LMP BUD-CG has a better sustained-release effect, delivering more drug to the colon to exert its therapeutic effect.

[0085] Example 6

[0086] 1. Method

[0087] The MTT assay was used to evaluate the cytotoxicity of the blank formulation (blank CG microcapsule formulation), pure BUD, and BUD-CG against RAW264.7 cells. The specific methods are as follows:

[0088] RAW264.7 cells were grown at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of 10 cells / well in 96-well plates. The cell culture plates were incubated in DMEM medium at 37°C with 5% CO2 for 24 h. After incubation, the medium was removed, and the cells were washed three times with PBS buffer. Three groups were then divided into three groups, each receiving 100 μL of different concentrations (0, 5, 10, 20, 40, 80 µg / mL) of blank carrier, BUD, and BUD-CG solution, respectively. After incubation for another 24 h, the medium was removed, and 90 μL of DMEM medium and 10 μL of MTT solution (5 mg / mL) were added. Incubation was continued for another 4 h. The medium was then completely removed, and 150 μL of DMSO was added. The plates were placed at room temperature until the purple crystals completely dissolved, and the plates were shaken to ensure uniform staining. Cell viability was calculated by measuring the OD value at 490 nm using an ELISA reader.

[0089] 2. Results

[0090] like Figure 5As shown in Figure A, the survival rate of RAW264.7 cells was higher than 95% at concentrations ranging from 0 to 80 μg / mL for the blank CG microcapsule formulation, indicating that the blank CG microcapsule formulation did not cause significant cytotoxicity to the cell lines at normal concentrations and possessed excellent biocompatibility. (Comparison) Figure 5 B and 5C showed significantly reduced cell viability when treated with pure BUD compared to the BUD-CG formulation, indicating that the BUD-CG microcapsule formulation can reduce the cytotoxicity of BUD to some extent.

[0091] Example 7

[0092] 1. Method

[0093] C57BL / 6 mice (8 weeks old) were selected as experimental mice and randomly divided into six groups: Blank normal group, DSS modeling group, CG empty preparation group, BUD pure drug group, Mesalamine positive drug group, and BUD-CG preparation group, with eight mice in each group.

[0094] After five days of acclimatization in a clean animal facility with alternating 12-hour light and 12-hour dark cycles, the experiment began. The blank group had free access to food and water daily without any treatment. The other groups were administered 0.6 g / mL DSS by gavage in the morning at a dose of 8 g / kg / day, and in the afternoon by gavage of 0.5% (w / v) CMC-Na, 0.168 mg / kg / day CG, 100 mg / kg / day Mesalamine, 0.168 mg / kg / day BUD solution, and 0.168 mg / kg / day BUD-CG, respectively. The blank preparation and BUD-CG microcapsules were dispersed in 0.5% CMC-Na solution.

[0095] Mice were monitored and their fecal blood loss was recorded daily (twice a day, morning and afternoon). Five days after drug administration, mice were euthanized, blood was collected, colon length was measured, and the spleen, kidneys, and colon were weighed. Colon sections from fixed sites were excised and immersed in fixative for subsequent histopathological examination. The following histopathological methods were used to evaluate the pathological changes in the colitis model and the treatment effect:

[0096] Hematoxylin and eosin (H&E) staining (servicebio+G1076).

[0097] MPO immunochemical sections: The degree of neutrophil infiltration was detected by MPO (servicebio+GB120016) labeling. MPO is an important iron-containing lysosome, present in aniline blue granules of myeloid cells, and is a specific marker of myeloid cells, which can be used as an indicator to evaluate the severity of inflammation in mice.

[0098] Immunofluorescence staining: Macrophage infiltration was located by F4 / 80 (servicebio+GB113373) protein labeling combined with DAPI nuclear staining, and the location was observed using a fluorescence microscope.

[0099] The serum levels of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor-α, i.e., IL-6 and TNF-α) were measured according to the corresponding method in the ELISA kit.

[0100] Disease Activity Index (DAI) scoring: On day 5, the rats were observed for fecal characteristics, bloody stools, and occult blood, and their weight changes were recorded. Scoring was performed according to the mouse DAI scoring standard table (Table 1), and a comprehensive score was calculated for each mouse (indicators such as individual weight loss rate, fecal characteristics, and bloody stools were scored separately, and the scores of each group were added together and divided by 3; the comprehensive DAI score ranged from 0 to 4 points), which was used to evaluate the degree of lesions in the colonic mucosa.

[0101] Colonic histopathological scoring: Colonic histopathological scoring was performed on H&E stained sections according to Table 2.

[0102] Table 1: DAI Scoring Results (See Table 3 for scoring results)

[0103]

[0104] Table 2: Colonic histopathological scoring (scoring results are shown in Table 2) Figure 7 B)

[0105]

[0106] 2. Results

[0107] (1) DAI score (Table 3)

[0108] As shown in Table 3, the DSS group and the empty preparation group had the highest scores, indicating the most severe inflammation. The BUD-CG group and the positive drug group (Mesalamine) had lower scores that were close to the normal group, indicating that the treatment of UC was effective.

[0109] Table 3: DAI scores (x±s, n=7).

[0110]

[0111] (2) Colon morphology and stool state

[0112] When an inflammatory response occurs in an organism, the length of the colon in mice decreases sharply, and phenomena such as unformed stool and bloody stool appear. Therefore, changes in colon length and stool morphology are used to evaluate the treatment effect. Colon examination results are as follows: Figure 6As shown in Figure A, the average colon length of healthy mice was 6.1 cm and their feces were formed, while the colon length of the DSS group was significantly reduced and the feces were unformed. Each drug administration group showed some effect in maintaining the length of the mouse colon and the integrity of the feces. The colon and fecal condition of the BUD-CG group and the positive control group were closest to that of normal mice, indicating that the BUD-CG group had a better therapeutic effect. Figure 6 D (colon length) and Figure 6 The results for E (colon weight / length ratio) also indirectly support this conclusion.

[0113] (3) Weight changes

[0114] Changes in mouse body weight are also an important indicator for evaluating colitis. For example... Figure 6 As shown in B and 6C, the body weight of mice in the normal group fluctuated very little within 5 days, while the body weight of the DSS group and the blank preparation group showed a significant downward trend, indicating successful modeling and severe inflammation. This also indicates that the blank preparation had no therapeutic effect on inflammation. For the other three treatment groups, the BUD-CG group and the positive control group showed a slower rate of body weight loss.

[0115] (4) Organ weight ratio

[0116] When an inflammatory response occurs in mice, a large number of immune cells accumulate in the spleen and kidneys, leading to an increase in organ weight; Figure 6 F and 6G showed that the spleen and kidney weight / body weight ratios in the DSS group were higher than those in the normal group, indicating significant inflammation. Compared with the normal group, there was no significant difference in the spleen and kidney weight / body weight ratios between the BUD-CG group and the positive drug group, indicating that the BUD-CG group had a good anti-inflammatory effect.

[0117] Therefore, BUD released by BUD-CG microcapsules can significantly improve DSS-induced colitis and show unique advantages in protecting the colon.

[0118] (5) H&E staining

[0119] H&E staining results are as follows: Figure 7 As shown in Figure A, the intestinal mucosa of mice in the normal group was intact, with tightly packed cells, orderly goblet cells, and obvious intestinal crypts. Mice in the DSS and CG groups showed severe intestinal mucosal damage, extremely poor structural integrity, damaged and necrotic intestinal crypt cells, a significant reduction in goblet cells, and extensive inflammatory cell infiltration in the mucosa. This indicates that the DSS group successfully established the model, and CG had no therapeutic effect on colitis. Comparison of the degree of damage to the colonic crypts, the extent of lesions, the number of goblet cells, and the integrity of the mucosa among the groups showed that the BUD-CG group had a significantly better therapeutic effect than the BUD group, and its mucosal morphology was closer to that of the Blank group, indicating that the BUD-CG group had a good therapeutic effect.

[0120] In addition, the histopathological scoring results of H&E stained sections of colon tissue ( Figure 7 B) It can be seen that each treatment group differed significantly from the DSS group. Among them, the BUD-CG group and the Mesalamine group had scores closer to the Blank group and lower inflammation levels. These results indicate that BUD-CG can effectively reduce the degree of DSS damage and significantly alleviate inflammation in colonic tissue.

[0121] (6) MPO immunochemical sections

[0122] Immunochemical sectioning results of colon tissue from each group of mice are as follows: Figure 7 As shown in Figure C, the brownish substance indicated by the black arrow is MPO. In the DSS group and the blank preparation group, cells infiltrated with MPO showed a strong brownish-red positivity. The MPO content in the BUD group was reduced to some extent, and the entire section showed a weak yellowish positivity. The MPO staining patterns of the BUD-CG group and the Mesalamine group were closest to the normal group. This indicates that BUD-CG can effectively reduce the MPO content in the colonic tissue of mice, thereby alleviating inflammation.

[0123] (7) Immunofluorescence staining of macrophages

[0124] Figure 8 Image A shows an immunofluorescence staining image of macrophages in colonic tissue. F4 / 80 protein is a cell surface glycoprotein. Its expression is significantly altered during macrophage maturation and activation, resulting in blue fluorescence when stained with DAPI. Therefore, F4 / 80 staining can be used to study the degree of macrophage infiltration and the severity of colitis. As shown in the figure, compared with the normal group, macrophage infiltration was significantly more severe in the DSS group and the blank preparation group, indicating a more severe degree of inflammation in the colonic tissue. Compared with the DSS group, the BUD-CG group significantly improved macrophage infiltration and colonic inflammation. These results indicate that the BUD-CG preparation has a good therapeutic effect on colitis.

[0125] (8) Levels of inflammatory factors

[0126] The severity of inflammation is related to the amount of pro-inflammatory cytokines. For example... Figure 8B-8D represents the levels of IL-2, IL-6, and TNF-α in colonic tissue. In colonic tissue, the DSS group had the highest concentrations of IL-2, IL-6, and TNF-α, reaching 188.04, 335.42, and 491.18 pg / mL, respectively. The average concentrations of IL-2, IL-6, and TNF-α in the normal group were 75.82, 90.53, and 127.01 pg / mL, respectively. Compared with the normal group, the DSS group and the empty preparation group showed significantly increased secretion of inflammatory factors, while the BUD-CG group, the positive control group, and the pure drug group showed significantly decreased levels of inflammatory factors compared with the DSS group. In the positive control group, the BUD-CG group had inflammatory factor levels similar to the normal group and much lower than the pure drug group. In conclusion, the BUD-CG preparation can promote drug absorption at the site of inflammation and has a good effect on improving inflammatory factors in colitis.

[0127] The above results indicate that the DAI score, weight loss trend, and organ weight ratio of the BUD-CG group were comparable to those of Mesalamine and significantly better than those of the pure drug group (BUD). The BUD-CG preparation significantly improved DSS-induced colitis in mice and had a protective effect on the colon, helping to maintain colon length and improve fecal morphology, and reducing inflammatory edema by decreasing the colon weight / length ratio.

[0128] BUD-CG formulations also significantly inhibit inflammatory cell infiltration and tissue damage by reducing neutrophil and macrophage infiltration and promoting the restoration of colonic mucosal integrity. Furthermore, BUD-CG formulations exert anti-inflammatory effects by inhibiting inflammatory signaling pathways and significantly reducing the levels of pro-inflammatory factors. By enhancing drug retention at the site of inflammation and improving BUD bioavailability, BUD-CG formulations simultaneously protect the drug from degradation by the intestinal environment, reducing the risk of systemic exposure. Organ weight ratio data confirm that it has no systemic toxicity and exhibits good stability and safety.

[0129] Example 8

[0130] 1. Method

[0131] In this embodiment, to study the in vivo distribution of orally administered BUD-CG microcapsules, the hydrophobic fluorescent dye DiR was first encapsulated in the microcapsules. Mice were orally administered free DiR and DiR-CG formulations via gavage, and the entire gastrointestinal tract and major organs were dissected at predetermined time intervals for fluorescence imaging. The specific procedures are as follows:

[0132] The hydrophobic fluorescent dye DiR was dispersed in 1 mL of DMSO, and then dispersed together with budesonide in a Pickering emulsion. Following the experimental method in Example 1, the mixture was encapsulated in CG microcapsules to study the distribution of DiR-CG microcapsules in tissues. Following the method in Example 7, a model was established at a dosage of 8 g / kg and 0.6 g / mL DSS. After successful modeling, the animal was administered 20 μg of DiR fluorescent dye by gavage. The gastrointestinal tract and major organs were dissected at predetermined time intervals and photographed using a live imaging device. The excitation wavelength was 800 nm.

[0133] 2. Results

[0134] like Figure 9 As shown in Figure A, in isolated organs, the free DiR group showed weak fluorescence in the liver at 1 hour, while no significant fluorescence was observed in the heart, liver, spleen, lungs, and kidneys at other time points. This indicates that there was no DiR or DiR-CG accumulation in these organs. Figure 9 Figure B shows the changes in fluorescence intensity in the gastrointestinal tract at 1, 2, 3, 5, 7, 10, 12, and 24 hours after administration to each group. When free DiR was administered orally, it distributed rapidly in the gastrointestinal tract, but the fluorescence intensity weakened rapidly over time, indicating that free DiR was quickly eliminated from the body. Only weak fluorescence was visible after 5 hours, and the fluorescence disappeared after 12 hours. In contrast, the DiR-CG group still showed significant fluorescence in the colon 24 hours after administration. These findings suggest that oral DiR-CG can accumulate in large quantities in the colon for at least 24 hours, which is beneficial for exerting its therapeutic effect.

[0135] The above results indicate that BUD-CG microcapsules significantly reduce drug distribution in non-target organs through a colonic environment-responsive release mechanism. Simultaneously, they exhibit a long-lasting retention characteristic of ≥24 h in the colon, effectively extending the local drug action window. This further validates the feasibility of BUD-CG formulations improving the therapeutic effect of ulcerative colitis by enhancing colonic drug bioavailability and reducing systemic toxicity.

[0136] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an oral colon-targeted delivery system loaded with budesonide, characterized in that, Includes the following steps: S1. Budesonide is dispersed in the oil phase, and then a 5.5 wt% calcium ion aqueous dispersion is mixed with the oil phase. After shearing and homogenization, a water-in-oil Pickering emulsion is formed. The calcium ion is one of calcium chloride, calcium carbonate, or calcium alginate. The oil phase is one or more of soybean oil, rapeseed oil, corn oil, peanut oil, palm oil, canola oil, or olive oil. The volume ratio of the calcium ion aqueous dispersion to the oil phase is 5:

1. S2. The Pickering emulsion is added dropwise to a 0.5wt%-3wt% low-ester pectin solution, the pH is adjusted to 3.5-4.5, and crosslinking is performed for 10-30 minutes to obtain gel beads; the volume ratio of the Pickering emulsion to the low-ester pectin solution is 100:

1. S3. Add the gel beads to a 1wt%-3wt% chitosan solution, crosslink for 20-60 min to obtain microcapsules, and dry them to obtain the oral colon-targeted delivery system loaded with budesonide.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass-to-volume ratio of budesonide to the oil phase is 5-30 mg: 1 L.

3. The preparation method according to claim 1, characterized in that, In step S3, the volume ratio of the gel beads to the chitosan solution is 1:(5-10).

4. The preparation method according to claim 1, characterized in that, The degree of esterification of the low-ester pectin is 25%-35%.

5. The preparation method according to claim 1, characterized in that, The number-average molecular weight of the chitosan is 300,000 to 1,100,000 Daltons.

6. The oral colon-targeted delivery system for budesonide loaded with budesonide prepared by the method of any one of claims 1-5, characterized in that, It has a core-shell structure, including a core structure, an inner shell, and an outer shell; the core is budesonide loaded with Pickering emulsion, the inner shell is formed by cross-linking low-ester pectin with calcium ions, and the outer shell is formed by combining low-ester pectin and chitosan through polyelectrolytes.

7. The use of the oral colon-targeted delivery system loaded with budesonide prepared by the preparation method of any one of claims 1-5 in the preparation of anticolitis drugs.

8. The application as described in claim 7, characterized in that, The colitis mentioned is ulcerative colitis.

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