Inulin gels loaded with polypyrrole nanoparticles and pirfenidone and uses thereof

Abstract

This invention discloses an inulin gel loaded with polypyrrole nanoparticles and pirfenidone, and its applications. The inulin gel serves as a drug delivery system, loading polypyrrole nanoparticles and pirfenidone. The inulin gel of this invention exhibits excellent reactive oxygen species scavenging capabilities, can regulate intestinal flora, and inhibit colonic fibroblast proliferation, making it suitable for alleviating intestinal inflammatory diseases and their complications, including intestinal fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation and biomedicine, specifically relating to a method for preparing an inulin gel loaded with polypyrrole nanoparticles and pirfenidone for the treatment of colitis and its complication intestinal fibrosis. Background Technology

[0002] Inflammatory bowel disease (IBD) is described as an idiopathic, chronic, relapsing, and remission-prevailing inflammatory bowel disease. Crohn's disease (CD) and ulcerative colitis (UC) are two major types of IBD. Its pathogenesis is complex, actually a related complex disease caused by a combination of genetic, environmental, microbial, and immune factors. The incidence of IBD has been gradually increasing over the past few decades. Despite continuous progress in its treatment, the global prevalence of IBD increased from 79.5 per 105 people in 1990 to 84.3 per 105 people in 2017. Many prescription and over-the-counter medications are currently available for treating IBD, including anti-inflammatory drugs, immunomodulators, reactive oxygen species scavengers, wound healing agents, and microbiome modulators. However, IBD is difficult to cure completely and is prone to relapse. Long-term, repeated intestinal wall inflammation can lead to abnormal deposition of extracellular matrix, causing intestinal wall fibrosis, which can then lead to intestinal stenosis, obstruction, or fistulas.

[0003] Although the etiologies of IBD are complex, oxidative stress in the intestinal epithelium is considered to play a key role in the pathogenesis of intestinal inflammation. In an inflamed gut, the immune response to normal bacterial antigens becomes dysregulated. This uncontrolled activation of the immune system leads to the sustained overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS), resulting in excessive amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS include superoxide radicals, hydroxyl radicals, and hydrogen peroxide. Excessive ROS / RNS interact with various molecular complexes, inducing oxidative damage to cells, affecting the synthesis of lipids, proteins, and nucleic acids, leading to lipid peroxide formation, various enzyme dysfunctions, and DNA strand breaks. Simultaneously, excessive ROS can activate inflammatory factors, triggering an immune response, affecting the expression of multiple inflammatory signaling pathways and related proteins, and exacerbating tissue damage. Excessive ROS causes significant damage to the intestinal epithelium, impairing the intestinal mucosal barrier. The gut microbiota plays an indispensable role, and some of their metabolites, such as short-chain fatty acids like butyrate, can stimulate immune cells (such as regulatory T cells) to reduce inflammation. Butyrate has been repeatedly described as a fundamental energy source for colon cell proliferation and maintaining the intestinal barrier, thus short-chain fatty acids can increase the integrity of the intestinal barrier and mucus production.

[0004] Strategies that can effectively eliminate ROS and improve gut microbiota have received widespread attention. Summary of the Invention

[0005] Inspired by the mechanisms of scavenging reactive oxygen species and regulating gut microbiota, this invention develops an inulin gel loaded with polypyrrole nanoparticles and pirfenidone, which can scavenge reactive oxygen species in inflamed gut, regulate imbalanced gut microbiota, and treat IBD while avoiding intestinal fibrosis caused by recurrent inflammation.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention first discloses an inulin gel loaded with polypyrrole nanoparticles and pirfenidone. The inulin gel serves as a drug delivery system, loading polypyrrole nanoparticles with excellent reactive oxygen species (ROS) scavenging capabilities and pirfenidone (PFD), a small compound with broad-spectrum anti-fibrotic activity. Polypyrrole is one of the most widely used inherently conductive polymers in the biomedical field and other fields such as sensors; however, its antioxidant properties are often overlooked. The alternating single and double carbon-carbon bonds in polypyrrole delocalize π electrons, making it more easily polarized. By providing electrons or active hydrogen atoms, it neutralizes free radicals, thus scavenging ROS. In addition to its drug delivery function, the inulin gel itself acts as a prebiotic, which can be utilized by beneficial microorganisms in the colon, thereby improving the intestinal environment.

[0008] The inulin gel containing polypyrrole nanoparticles and pirfenidone described in this invention has good injectability.

[0009] The preparation method of the inulin gel loaded with polypyrrole nanoparticles and pirfenidone according to the present invention is as follows:

[0010] Pirfenidone was dissolved in a small amount of methanol, and then a deionized aqueous dispersion of polypyrrole nanoparticles and inulin were added. The mixture was stirred until homogeneous to obtain a reaction solution. The reaction solution was then placed in a water bath at 70–90°C and stirred for 5–15 minutes. Finally, it was allowed to stand at room temperature for 10–12 hours to obtain an inulin gel loaded with polypyrrole nanoparticles and pirfenidone.

[0011] Preferably, the mass ratio of pirfenidone, polypyrrole nanoparticles, and inulin is 10–20 mg: 1–4 mg: 0.6 g, and the ratio of inulin to deionized water in the reaction solution is 0.6 g / mL. The ratio of inulin to deionized water is a key factor influencing the formation of inulin gel.

[0012] Preferably, the stirring speed is 800-1000 rpm.

[0013] The inulin gel containing polypyrrole nanoparticles and pirfenidone of this invention has the following characteristics: the gel is simple to prepare; it has good biocompatibility at the cellular and animal levels; it has the ability to scavenge reactive oxygen species and reactive nitrogen species; the contained pirfenidone has a good inhibitory effect on fibrosis; the inulin gel system can regulate the intestinal flora, be utilized by beneficial microorganisms, promote the growth of beneficial bacteria, and thus improve the intestinal environment; it can inhibit the proliferation of colonic fibroblasts and inhibit collagen production. In addition, the gel can increase the retention time of nanoparticles and the drug pirfenidone in the intestine, allowing them to exert their effects better at sites of intestinal inflammation.

[0014] The inulin gel containing polypyrrole nanoparticles and pirfenidone described in this invention can be used to prepare a pharmaceutical agent for treating colitis and its complication, intestinal fibrosis, by regulating intestinal cells and the intestinal environment. The agent can be in the form of an orally administered gel. The mechanism of action is as follows: polypyrrole nanoparticles can neutralize free radicals by donating electrons or active hydrogen atoms, thereby scavenging various reactive oxygen species and reactive nitrogen species. Pirfenidone prevents intestinal fibrosis, a complication of colitis, by inhibiting the proliferation of colonic fibroblasts and the TGF-β signaling pathway. As a drug carrier, the inulin gel, in addition to extending the drug's retention time in the intestine, also acts as a prebiotic, promoting the growth of beneficial bacteria and regulating the intestinal flora.

[0015] The beneficial effects of this invention are reflected in:

[0016] 1. In the gel of this invention: the presence of polypyrrole nanoparticles provides the ability to scavenge various reactive oxygen species and reactive nitrogen species, which can target various inflammatory diseases; the presence of pirfenidone gives the gel the potential to inhibit lipid peroxidation of intestinal epithelial cells and inhibit the proliferation of colonic fibroblasts; the presence of inulin allows the drug to remain in the intestine for a longer time, while regulating the intestinal flora. The synergistic effect between the components makes the gel of this invention useful for alleviating intestinal inflammatory diseases and their complications, such as intestinal fibrosis: in DSS (dextrose sulfate sodium salt)-induced prevention and delayed colitis models, the gel of this invention significantly alleviated the occurrence of colitis; in a long-term DSS-induced fibrosis model, the gel of this invention significantly alleviated the occurrence of intestinal fibrosis.

[0017] 2. This invention synthesizes inulin gel loaded with polypyrrole nanoparticles and pirfenidone by heating and cooling. The method is simple, and the resulting gel has good injectability, good biocompatibility and oral applicability, which is beneficial for clinical use. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the synthesis of the present invention.

[0019] Figure 2The image shows a transmission electron microscope (TEM) image of the polypyrrole nanoparticles prepared in Example 1.

[0020] Figure 3 The image shows a scanning electron microscope image of the inulin gel loaded with polypyrrole nanoparticles and pirfenidone prepared in Example 1.

[0021] Figure 4 Image of the inulin gel loaded with polypyrrole nanoparticles and pirfenidone prepared in Example 1.

[0022] Figure 5 The in vitro scavenging diagram of DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals in the inulin gel loaded with polypyrrole nanoparticles and pirfenidone prepared in Example 1.

[0023] Figure 6 This is a comparison diagram of the release of pirfenidone and the active pharmaceutical ingredient pirfenidone in the inulin gel loaded with polypyrrole nanoparticles and pirfenidone prepared in Example 1.

[0024] Figure 7 This is a comparison image of the retention of inulin gel loaded with polypyrrole nanoparticles and pirfenidone prepared in Example 1 and polypyrrole nanoparticles alone in the intestine, where: Figure 7 (a) Distribution of materials in the mouse intestines at different time points after oral administration of Cy5.5-labeled polypyrrole nanoparticles alone and inulin gel loaded with Cy5.5-labeled polypyrrole nanoparticles and pirfenidone, as captured by small animal imaging software. Figure 7 (b) is done using small animal imaging software. Figure 7 (a) Comparison of the results of quantitative analysis of fluorescence intensity.

[0025] Figure 8 The image shows the MTT assay of the inulin gel containing polypyrrole nanoparticles and pirfenidone prepared in Example 1.

[0026] Figure 9 This image shows the cellular reactive oxygen species staining of polypyrrole nanoparticles at different concentrations.

[0027] Figure 10 The image shows a statistical diagram of colon length in mice after preventive treatment with inulin gel containing polypyrrole nanoparticles and pirfenidone, prepared in Example 1, following treatment for colitis.

[0028] Figure 11 The image shows a statistical diagram of colon length in mice after delayed treatment with inulin gel containing polypyrrole nanoparticles and pirfenidone, prepared in Example 1, for colitis.

[0029] Figure 12 The image shows a statistical diagram of the thickness of the colonic muscularis propria after treating fibrotic mice with inulin gel loaded with polypyrrole nanoparticles and pirfenidone, prepared in Example 1. Detailed Implementation

[0030] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to examples. The following content is merely an example and illustration of the concept of the present invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not depart from the inventive concept or exceed the scope defined by the claims, all of which should fall within the protection scope of the present invention.

[0031] Example 1

[0032] In this embodiment, inulin gel loaded with polypyrrole nanoparticles and pirfenidone was prepared by the following method:

[0033] (1) Preparation of polypyrrole nanoparticles: 1.5 g of polyvinyl alcohol was weighed and dissolved in 20 mL of deionized water. The solution was stirred at 60 °C until completely dissolved. After cooling to room temperature, 1.2434 g of ferric chloride hexahydrate was added and stirred at room temperature for 1 h. At 5 °C, 140 μL of pyrrole was added and stirred for 4 h. After the reaction was completed, the mixture was centrifuged at 15000 rpm for 20 min, washed three times with water, and lyophilized to obtain polypyrrole nanoparticle powder. A standard curve of concentration-absorbance of polypyrrole nanoparticle powder was prepared using a UV spectrophotometer to determine the concentration of polypyrrole nanoparticles in each system in subsequent experiments. The polypyrrole nanoparticles were dispersed in deionized water to obtain a dispersion with a concentration of 4 mg / mL and stored at 4 °C.

[0034] (2) Synthesis of inulin gel loaded with polypyrrole nanoparticles and pirfenidone:

[0035] Weigh 20 mg of pirfenidone, add 50 μL of methanol to dissolve it completely, then add 1 mL of deionized water dispersion of 4 mg / mL polypyrrole nanoparticles and 0.6 g of inulin, and stir to mix evenly; then place it in an 80℃ water bath and stir at 800 rpm for 10 min, and finally let it stand at room temperature for 12 h to obtain inulin gel loaded with polypyrrole nanoparticles and pirfenidone.

[0036] The morphology and properties of the inulin gel containing polypyrrole nanoparticles and pirfenidone obtained in this embodiment were characterized as follows:

[0037] I. Morphological Characterization

[0038] Figure 2 The image shows a transmission electron microscope (TEM) image of the polypyrrole nanoparticles obtained in this embodiment. The characterization method was as follows: an aqueous dispersion of polypyrrole nanoparticles was dropped onto a copper grid of a TEM, dried, and then observed within the TEM. The image shows that the nanosheets have a diameter of 60–80 nm.

[0039] Figure 3 This is a scanning electron microscope image of the inulin gel containing polypyrrole nanoparticles and pirfenidone obtained in this embodiment. The characterization method involved cutting a cross-section of the lyophilized powder with a knife. An ordered gel network structure can be observed in the image.

[0040] The inulin gel containing polypyrrole nanoparticles and pirfenidone obtained in this embodiment was loaded into syringes of different sizes and then injected into water to verify its injectability. The results are as follows: Figure 4 As shown, the gel can be injected with needles of different sizes, with the smallest needle having an outer diameter of 0.4 mm and an inner diameter of 0.21 mm, demonstrating excellent injectability.

[0041] II. Performance test for in vitro DPPH clearance

[0042] Figure 5 This is a diagram showing the in vitro DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging effect of the inulin gel loaded with polypyrrole nanoparticles and pirfenidone obtained in this embodiment. DPPH is a very stable nitrogen-centered free radical that dissolves in ethanol, appears purple, and has a characteristic UV absorption peak at 519 nm. The DPPH scavenging effect was detected by detecting the UV absorption peak at 519 nm after the material was co-incubated with DPPH. The specific experimental steps were as follows: 1 mg of DPPH was dissolved in 20 mL of anhydrous ethanol and sonicated until completely dissolved. Inulin gel loaded with different concentrations of polypyrrole nanoparticles and an equal amount of pirfenidone (the concentration was adjusted by adjusting the mass of the polypyrrole nanoparticles during preparation) was added and reacted with DPPH for 30 min (the final concentration of polypyrrole nanoparticles in the system were 3.75, 7.5, 15, 30, or 60 μg / mL, respectively). DPPH + water was set as a positive control. Finally, the sample was centrifuged at 15000 rpm for 10 min, and the supernatant was then measured for UV absorption at 519 nm. The figure shows that the gel system containing 60 μg / mL polypyrrole nanoparticles has an DPPH scavenging capacity of approximately 60%.

[0043] III. Drug Release Performance Test

[0044] Figure 6 This is a comparison of the release of pirfenidone and the active pharmaceutical ingredient (PFD) in the inulin gel (PPy / PFDgel) containing polypyrrole nanoparticles and pirfenidone obtained in this embodiment. The characterization method is as follows: pirfenidone has a characteristic UV absorption peak at 320 nm. By preparing standard solutions of pirfenidone at concentrations of 5, 10, 20, 30, and 40 μg / mL, and measuring their UV absorbance values, a standard curve on the concentration-absorbance value was obtained, with the equation: y = 0.0268x + 0.0599, R 2 =0.9994.

[0045] Free drug release: Weigh 20 mg of pirfenidone and add it to 4 mL of PBS buffer containing 0.5% Tween 80 (pH = 1.5, 6.0, 7.4, 8.0). Sonicate until completely dissolved. Take 1 mL of the solution and place it in a dialysis bag with a molecular weight of 1000. Add 30 mL of PBS containing 0.5% Tween 80. Place the bag on a shaker at 37°C and 100 rpm. Take 1 mL of the dissolution medium at 5, 10, 20, 40, 60, 120, and 180 min, and simultaneously add 1 mL of PBS buffer containing 0.5% Tween 80 at the corresponding pH. Then, measure the amount of pirfenidone in the dissolution medium at each time point using a UV spectrophotometer.

[0046] Drug release from inulin gel loaded with polypyrrole nanoparticles and pirfenidone: The prepared gel was placed in a dialysis bag with a molecular weight of 1000, and 30 mL of PBS containing 0.5% Tween 80 (pH = 1.5, 6.0, 7.4, 8.0) was added. The experiment was conducted on a shaker at 37℃ and 100 rpm. At 0.5, 1, 2, 4, 8, 12, 24, and 48 h, 1 mL of dissolution medium was collected, and 1 mL of PBS buffer containing 0.5% Tween 80 at the corresponding pH was added simultaneously. The amount of pirfenidone in the dissolution medium at each time point was then measured using a UV spectrophotometer.

[0047] from Figure 6 It can be seen that the release of the active pharmaceutical ingredient (API) and the release of the drug loaded in the gel are basically unaffected by pH. The API can be completely released in about 2 hours, while the complete release time of the drug loaded in inulin gel is about 24 hours, which achieves a sustained-release effect.

[0048] IV. Drug Retention Performance Test

[0049] First, polypyrrole nanoparticles were labeled with Cy5.5N-hydroxysuccinimide: a deionized aqueous dispersion of 4 mg / mL polypyrrole nanoparticles was prepared, and 40 μL of 0.2 mg / mL Cy5.5N-hydroxysuccinimide was added per mL. The mixture was incubated on a shaker at 37°C for 12 h, and then dialyzed for 24 h using a dialysis bag with a molecular weight of 14000 to remove excess Cy5.5N-hydroxysuccinimide. A gel was then prepared using the method described in Example 1. Mice were orally administered polypyrrole nanoparticles alone and inulin gel containing polypyrrole nanoparticles and pirfenidone via gavage. Mice were dissected at 0, 2, 4, 8, 12, and 24 h, and their small intestine to colon was harvested. Fluorescence distribution was captured using small animal imaging software, and the fluorescence intensity was quantitatively analyzed.

[0050] Figure 7This is a comparison diagram of the retention of inulin gel loaded with polypyrrole nanoparticles and pirfenidone in the intestine compared with that of polypyrrole nanoparticles alone, obtained in this embodiment. Figure 7 (a) Distribution of materials in the mouse intestines at different time points after oral administration of Cy5.5-labeled polypyrrole nanoparticles alone and inulin gel loaded with Cy5.5-labeled polypyrrole nanoparticles and pirfenidone, as captured by small animal imaging software. Figure 7 (b) is done using small animal imaging software. Figure 7 (a) Comparison of the results of quantitative analysis of fluorescence intensity. As can be seen from the figure, the pure Cy5.5-labeled nanoparticles reached their peak in the intestine at 4 h, while the inulin gel loaded with Cy5.5-labeled polypyrrole nanoparticles and pirfenidone reached its peak in the intestine at 8 h. Furthermore, at 24 h, the fluorescence intensity of the inulin gel loaded with Cy5.5-labeled polypyrrole nanoparticles and pirfenidone was stronger than that of the pure Cy5.5-labeled nanoparticle group, indicating a longer retention time in the colon.

[0051] V. MTT Experiment

[0052] The following different groups of materials were prepared using serum-free 1640 medium:

[0053] Control: Fresh culture medium;

[0054] Pirfenidone group (PFD): Pirfenidone was added to the culture medium at a concentration of 1 mg / mL.

[0055] Inulingel preparation: 0.6 g of inulin was added to 1 mL of deionized water, then placed in an 80°C water bath and stirred at 800 rpm for 10 min. The mixture was then allowed to stand at room temperature for 12 h to obtain the inulin gel. 0.2 mL of the prepared inulin gel was transferred to a 15 mL centrifuge tube, and 4 mL of serum-free 1640 culture medium was added. The mixture was repeatedly pipetted to prepare a suspension for later use.

[0056] Pirfenidone-containing inulin gel (PFDgel): Weigh 20 mg of pirfenidone, add 50 μL of methanol to completely dissolve it, then add 1 mL of deionized water and 0.6 g of inulin, and stir to mix evenly; then place in an 80℃ water bath and stir at 800 rpm for 10 min, and finally let stand at room temperature for 12 h to obtain the pirfenidone-containing inulin gel. Take 0.2 mL of the prepared pirfenidone-containing inulin gel (the concentration of pirfenidone in the gel is 20 mg / mL) into a 15 mL centrifuge tube, add 4 mL of serum-free 1640 culture medium, and repeatedly pipette to prepare a suspension for later use.

[0057] Polypyrrole nanoparticles (PPy): A polypyrrole nanoparticle solution with a concentration of 200 μg / mL was prepared using serum-free 1640 medium.

[0058] Inulin gel containing polypyrrole nanoparticles (PPygel): 1 mL of 4 mg / mL polypyrrole nanoparticles was mixed with 0.6 g of inulin until homogeneous. The mixture was then placed in an 80℃ water bath and stirred at 800 rpm for 10 min. Finally, it was allowed to stand at room temperature for 12 h to obtain the inulin gel containing polypyrrole nanoparticles. 0.2 mL of the prepared inulin gel containing polypyrrole nanoparticles (concentration of polypyrrole nanoparticles 4 mg / mL) was transferred to a 15 mL centrifuge tube, and 4 mL of serum-free 1640 culture medium was added. The mixture was repeatedly pipetted to prepare a suspension for later use.

[0059] Inulin gel containing polypyrrole nanoparticles and pirfenidone (PPy / PFDgel): Take 0.2 mL of the inulin gel containing polypyrrole nanoparticles prepared in Example 1 (the concentration of polypyrrole nanoparticles in the gel is 4 mg / mL and the concentration of pirfenidone is 20 mg / mL) into a 15 mL centrifuge tube, add 4 mL of serum-free 1640 culture medium, and repeatedly pipette to prepare a suspension for later use.

[0060] Figure 8 The MTT assay diagram is shown below. The characterization method is as follows: 100 μL (1 × 10⁻⁶) is seeded into each well. 5 NCM460 cells were incubated for 24 hours. The supernatant was aspirated, and 100 μL of the material from each group was added to each well, with incubation for 24 hours and 48 hours respectively. After incubation, 20 μL of LMT (5 mg / mL) was added to each well, and the cells were incubated for 4 hours. Finally, the supernatant was aspirated, and 150 μL of LDMSO was added to each well to terminate the culture. The cells were incubated at 37°C in the dark for 10 minutes, and the absorbance was measured at 490 nm. The graph shows that the cell viability of all groups was higher than 85%, indicating good biocompatibility of the material.

[0061] VI. Experiments on the scavenging of intracellular reactive oxygen species

[0062] The following groups of materials were prepared using serum-free 1640 medium: Control (fresh medium); 1 mM H2O2; 100 μg / mL PPy; 200 μg / mL PPy; 400 μg / mL PPy; 1 mM H2O2 + 100 μg / mL PPy; 1 mM H2O2 + 200 μg / mL PPy; 1 mM H2O2 + 400 μg / mL PPy.

[0063] To verify the scavenging effect of polypyrrole nanoparticles on intracellular reactive oxygen species, 100 μL (1 × 10⁻⁶) was seeded into each well of a 96-well plate. 5NCM460 cells were incubated for 24 hours. The supernatant was aspirated, and 100 μL of the prepared solution was added to each well. Incubation was continued for 4 hours. After incubation, the supernatant was aspirated, and the cells were washed three times with PBS. 10 μM DCFH-DA staining solution prepared in PBS was added to each well, and the cells were incubated at 37°C in the dark for 30 minutes. The cells were then washed three times with PBS and photographed using a fluorescence microscope. Figure 9 The images show the cellular reactive oxygen species staining of polypyrrole nanoparticles at different concentrations in this embodiment. As can be seen from the images, the H2O2+400μg / mLPPy group showed no obvious green fluorescence, indicating that it has a good ability to scavenge intracellular reactive oxygen species.

[0064] VII. Performance Test for Relieving Colitis

[0065] 1. To verify the ability of the inulin gel loaded with polypyrrole nanoparticles and pirfenidone obtained in this embodiment to alleviate colitis, the following test was conducted: In a colitis prevention and treatment model, normal mice were randomly divided into 8 groups (n=8 per group), and the drug administration methods for each group were as follows:

[0066] (1) Control group: PBS + normal water; (2) DSS group: PBS + 3% DSS; (3) Inulingel group: inulin gel + 3% DSS; (4) PFD group: pirfenidone (200 mg / kg) + 3% DSS; (5) PFDgel group: inulin gel containing pirfenidone (200 mg / kg) + 3% DSS; (6) PPy group: polypyrrole nanoparticles (40 mg / kg) + 3% DSS; (7) PPygel group: inulin gel containing polypyrrole nanoparticles (40 mg / kg) + 3% DSS; (8) PPy / PFDgel group: inulin gel loaded with polypyrrole nanoparticles (40 mg / kg) and pirfenidone (200 mg / kg) + 3% DSS.

[0067] The experimental group mice were fed 3% DSS for 7 consecutive days, followed by drinking water. The drug-treated groups were administered the drug via gavage on days 1, 3, 5, and 7. Mice were sacrificed on day 9, and the colon length of each group was recorded. Figure 10 It can be seen that the colon length of mice containing polypyrrole nanoparticles in groups (6), (7), and (8) is close to 9 cm, which is not much different from the colon length of mice in the normal group.

[0068] 2. To further verify the ability of the inulin gel loaded with polypyrrole nanoparticles and pirfenidone obtained in this embodiment to alleviate colitis, the following test was conducted: In a delayed-treatment colitis model, normal mice were randomly divided into 9 groups (n=8 per group), and the drug administration methods for each group were as follows:

[0069] (1) Control group: PBS + normal water; (2) DSS group: PBS + 3% DSS; (3) 5-ASA group: 5-aminosalicylic acid + 3% DSS; (4) Inulingel group: inulin gel + 3% DSS; (5) PFD group: pirfenidone (200mg / kg) + 3% DSS; (6) PFDgel group: inulin gel containing pirfenidone (200mg / kg) + 3% DSS; (7) PPy group: polypyrrole nanoparticles (40mg / kg) + 3% DSS; (8) PPygel group: inulin gel containing polypyrrole nanoparticles (40mg / kg) + 3% DSS; (9) PPy / PFDgel group: inulin gel loaded with polypyrrole nanoparticles (40mg / kg) and pirfenidone (200mg / kg) + 3% DSS.

[0070] Mice in the experimental group were fed 3% DSS for 6 consecutive days, followed by drinking water. Mice in the drug-treated group were administered the drug orally on days 7, 9, 11, and 13. Mice were sacrificed on day 15, and the colon length of each group was recorded. Figure 11 It can be seen that the colon length of mice containing polypyrrole nanoparticles in groups (7), (8), and (9) is close to 8.5 cm, which is not much different from the colon length of mice in the normal group.

[0071] 3. To further verify the ability of the inulin gel loaded with polypyrrole nanoparticles and pirfenidone obtained in this embodiment to alleviate intestinal fibrosis, the following test was conducted: In the fibrosis model, normal mice were randomly divided into 8 groups (n=8 per group):

[0072] (1) Control group: PBS + normal water; (2) DSS group: PBS + DSS; (3) Inulingel group: inulin gel + DSS; (4) PFD group: pirfenidone (200 mg / kg) + DSS; (5) PFDgel group: inulin gel containing pirfenidone (200 mg / kg) + DSS; (6) PPy group: polypyrrole nanoparticles (40 mg / kg) + DSS; (7) PPygel group: inulin gel containing polypyrrole nanoparticles (40 mg / kg) + DSS; (8) PPy / PFDgel group: inulin gel loaded with polypyrrole nanoparticles (40 mg / kg) and pirfenidone (200 mg / kg) + DSS.

[0073] Mice in the experimental group were fed DSS in stages over a long period. From days 0-5, they were fed 1.5% DSS; from days 10-15, 2% DSS; and from days 20-25, 2.5% DSS. The remaining days were given normal water. The drug-treated groups were administered the drug daily by gavage from day 15 to day 30. Mice were sacrificed on day 35, and the colon length of each group was recorded. Figure 12It can be seen that the thickness of the colonic muscularis propria in mice in groups (3), (5), and (8) containing pirfenidone was similar to that in the normal group.

[0074] 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, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A inulin gel loaded with polypyrrole nanoparticles and pirfenidone, characterized in that: The inulin gel is used as a drug delivery system to load polypyrrole nanoparticles and pirfenidone; the inulin gel loaded with polypyrrole nanoparticles and pirfenidone is injectable.

2. A method for preparing the inulin gel loaded with polypyrrole nanoparticles and pirfenidone as described in claim 1, characterized in that: Pirfenidone was dissolved in methanol, and then a deionized water dispersion of polypyrrole nanoparticles and inulin were added. The mixture was stirred until homogeneous to obtain a reaction solution. The reaction solution was then placed in a water bath at 70-90°C and stirred for 5-15 minutes. Finally, it was allowed to stand at room temperature for 10-12 hours to obtain an inulin gel loaded with polypyrrole nanoparticles and pirfenidone. The mass ratio of pirfenidone, polypyrrole nanoparticles and inulin was 10-20 mg: 1-4 mg: 0.6 g, and the ratio of inulin to deionized water in the reaction solution was 0.6 g / mL.

3. The preparation method according to claim 2, characterized in that: The stirring speed is 800-1000 rpm.

4. Use of the inulin gel loaded with polypyrrole nanoparticles and pirfenidone according to claim 1, characterized by the fact that it is used as a drug for the treatment of liver diseases. This is used to prepare agents that regulate intestinal cells and the intestinal environment to treat colitis and its complications, including intestinal fibrosis.

5. Use according to claim 4, characterized in that: The inulin gel loaded with polypyrrole nanoparticles and pirfenidone has the following functions: scavenging reactive oxygen species; regulating intestinal flora; inhibiting colonic fibroblast proliferation; and inhibiting collagen production.

Images