A composite drug-loaded microsphere system and its application in enteritis

Abstract

The application relates to a composite drug-loaded microsphere system and application thereof in intestinal inflammation. A nanoscale gallium-based metal organic framework (GaMOF) nanoparticle is prepared, an anti-inflammatory drug curcumin is loaded on the GaMOF nanoparticle as a carrier, and a drug-loaded composite GaMOF@Cur is constructed. Subsequently, the GaMOF@Cur and alginate polysaccharides with a gastrointestinal microenvironment response are jointly coated on the surface of Escherichia coli (EcN). Through an electrostatic spraying technology, the composite is dropped into a calcium chloride solution at a certain flow rate, a chelation effect of alginate and calcium ions is utilized, and a gelatinous protective framework is formed on the surface of the EcN. The morphology is characterized, the performance is determined, and in a DSS-induced intestinal inflammation mouse model, the body weight change of the mouse before and after treatment of the probiotic microsphere drug-loaded system, the inflammatory factor and the intestinal flora change are compared.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and nanomaterials technology, specifically relating to a composite drug-loaded microsphere system and its application in enteritis. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of diseases that cause chronic inflammation in the colon and small intestine. It primarily includes ulcerative colitis (UC) and Crohn's disease (CD). IBD is characterized by its chronic nature, lack of a cure, young age of onset, and low mortality rate, thus its prevalence gradually increases over time. IBD patients often experience frequent abdominal pain, diarrhea, nausea, and rectal bleeding. Studies have also found that 25-40% of IBD patients develop extraintestinal manifestations, affecting almost all organ systems, such as muscles, bones, skin, pancreas, eyes, kidneys, and lungs. These extraintestinal inflammatory manifestations further exacerbate the severity of the disease. Therefore, IBD not only damages the patient's intestinal health but also poses a significant risk to overall health. Oral administration is one of the traditional methods of drug delivery. Due to its convenience and excellent patient compliance, oral administration has become the primary route of administration for the treatment of many diseases. Furthermore, given the crucial role of the gut microbiota in the etiology of IBD, probiotic supplements, which regulate the microbiota, reduce inflammation, and promote gut homeostasis, have garnered significant attention in IBD treatment. Consequently, various oral medications and traditional probiotic therapies have become integral parts of the prevention and treatment of numerous gastrointestinal diseases.

[0003] However, many drugs suffer from poor stability, low solubility, and weak targeting, resulting in low bioavailability and poor therapeutic effects when taken orally. For example, curcumin has shown excellent efficacy in the prevention and treatment of ulcerative colitis. However, the nutritional and pharmaceutical applications of curcumin are often limited by its low oral bioavailability and susceptibility to oxidative degradation (especially photoinduced degradation). Furthermore, probiotics, when taken orally, are easily affected by various adverse factors in the gastrointestinal tract, such as the low pH environment of gastric acid, the action of digestive enzymes, and the destruction by bile acids, all of which reduce their bioavailability. All these factors limit the effective realization of their intended therapeutic effects.

[0004] Therefore, designing and developing a novel probiotic microsphere drug delivery system is crucial for the effective treatment of inflammatory bowel disease. Summary of the Invention

[0005] To address the challenges of oral drug delivery, this invention utilizes a carrier to improve drug stability, enhance bioavailability and therapeutic efficacy, while maintaining an appropriate drug concentration range and reducing side effects. A nanoscale gallium-based metal-organic framework (GaMOF) nanoparticle is synthesized, which can serve as a carrier to load curcumin (GaMOF@Cur) with anti-inflammatory effects. Addressing the targeting issue, probiotics, as a potential carrier, can colonize the intestinal mucosa surface, are less likely to trigger a strong immune response, and possess certain immunomodulatory functions. The curcumin-loaded GaMOF (GaMOF@Cur) is modified onto the surface of probiotics to synthesize EcN / GaMOF@Cur. Simultaneously, alginate polysaccharides with gastrointestinal microenvironment responsiveness are modified onto the surface of EcN / GaMOF@Cur. Alginate polysaccharides, by chelating calcium ions, can construct a gel-like protective framework on the surface of probiotics, effectively resisting gastric acid erosion after oral administration. Upon reaching the intestine, this coating gradually dissolves in a neutral pH environment, releasing the loaded probiotics and therapeutic drugs, thereby achieving a synergistic therapeutic effect for enteritis.

[0006] To achieve the above effects, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a composite drug-loaded microsphere system, comprising: probiotics; gallium-based metal-organic framework nanoparticles loaded with anti-inflammatory drugs (GaMOF@Cur); and a calcium alginate protective coating on the outermost layer; wherein the GaMOF@Cur is modified on the surface of the probiotics to form an EcN / GaMOF@Cur composite, and the calcium alginate coating is wrapped around the outside of the composite.

[0008] Furthermore, the probiotic is Escherichia coli Nissle 1917 (EcN).

[0009] Furthermore, the anti-inflammatory drug is curcumin.

[0010] Furthermore, the gallium-based metal-organic framework nanoparticles are synthesized from gallium nitrate and 1,3,5-benzenetricarboxylic acid via a solvothermal reaction, and have a particle size of 50-70 nm.

[0011] Furthermore, the calcium alginate protective coating is formed by cross-linking sodium alginate and calcium chloride through electrostatic spraying, and the microspheres have a particle size of 200-280 μm.

[0012] Furthermore, the system can protect the survival rate of probiotics in simulated gastric fluid significantly higher than that of free probiotics.

[0013] Secondly, the present invention provides the application of the composite drug-loaded microsphere system in the preparation of drugs for treating enteritis.

[0014] Furthermore, the drug is used to reduce enteritis-related weight loss, decrease the level of the pro-inflammatory factor IL-1β, and regulate gut microbiota homeostasis.

[0015] Furthermore, the regulation of gut microbiota homeostasis includes increasing the abundance of Lactobacillus, decreasing the abundance of Helicobacter pylori, increasing the abundance of Firmicutes, and decreasing the abundance of Campylobacterota.

[0016] Curcumin was loaded into gallium-based metal-organic framework (GaMOF) nanoparticles to improve its stability and oral bioavailability. It was then delivered to the intestines via probiotics and encapsulated with calcium alginate microspheres to avoid the probiotics being affected by adverse factors in the gastrointestinal tract, thereby achieving the goal of synergistic treatment of enteritis.

[0017] The technical effects achieved by this invention are:

[0018] (1) Improve the oral bioavailability of curcumin: By loading curcumin into GaMOF nanoparticles and modifying the surface of probiotics, the oxidative degradation of curcumin in gastric juice is avoided, allowing it to reach the intestine smoothly for release, thus significantly improving bioavailability.

[0019] (2) Protecting probiotic activity: The calcium alginate coating remains stable in gastric juice (pH 2.5), protecting EcN from strong acid and pepsin damage; after incubation in simulated gastric juice for 2 hours, the survival rate of EcN in EcN / GaMOF@Cur microspheres is significantly higher than that of free EcN.

[0020] (3) Targeted delivery and controlled release: The calcium alginate coating gradually dissolves in neutral intestinal fluid, achieving targeted delivery and drug release to the site of inflammation.

[0021] (4) Significantly relieves colitis symptoms: In a DSS-induced colitis mouse model, oral administration of EcN / GaMOF@Cur microspheres significantly reduced weight loss and decreased plasma levels of the pro-inflammatory factor IL-1β.

[0022] (5) Regulation of gut microbiota homeostasis: The Shannon and Simpson indices of gut microbiota in the treatment group mice increased, the abundance of Lactobacillus and Firmicutes increased, and the abundance of Helicobacter pylori and Campylobacterota decreased, indicating that the system promotes gut microecological balance and repairs the intestinal barrier. Attached Figure Description

[0023] Figure 1Transmission electron microscope image and EDS energy dispersive spectroscopy image of GaMOF;

[0024] Figure 2 This is a particle size distribution diagram of GaMOF;

[0025] Figure 3 Transmission electron microscope image and EDS energy dispersive spectroscopy image of GaMOF@Cur;

[0026] Figure 4 The particle size distribution of GaMOF@Cur is shown.

[0027] Figure 5 Transmission electron microscope image and EDS energy dispersive spectroscopy image of EcN / GaMOF@Cur;

[0028] Figure 6 Potential diagrams for GaMOF, GaMOF@Cur, EcN, and EcN / GaMOF@Cur;

[0029] Figure 7 Electron microscope image of EcN / GaMOF@Cur microspheres;

[0030] Figure 8 The particle size distribution of EcN / GaMOF@Cur microspheres;

[0031] Figure 9 The survival of EcN and EcN / GaMOF@Cur microspheres in gastric juice is shown, where E / Ga@Cms represents EcN / GaMOF@Cur microspheres.

[0032] Figure 10-11 To validate the in vivo therapeutic effect of EcN / GaMOF@Cur microspheres. Figure 10 The changes in body weight of mice in different treatment groups over 12 days; Figure 11 Comparison of IL-1β levels in mice under different treatment groups;

[0033] Figure 12-13 The Shannon and Sipmson indices represent the gut microbiota of mice.

[0034] Figure 14-15 The relative abundance of genera and phyla in the mouse gut microbiota;

[0035] Figure 16-19 The relative abundance of Helicobacter, Lactobacillus, Firmicutes, and Campylobacterota in the mouse gut microbiota. Detailed Implementation

[0036] The technical solution of the present invention will be further described below through embodiments and in conjunction with the accompanying drawings.

[0037] Example 1: Synthesis of GaMOF

[0038] According to one embodiment of this application, 20 mL of N,N-dimethylformamide (259 mmol) was measured into a 50 mL beaker using a graduated cylinder, and 96 μL of concentrated hydrochloric acid was accurately transferred in using a pipette to obtain the reaction solvent. Then, 0.24 g of gallium(III) nitrate (0.87 mmol) hydrate and 0.11 g of 1,3,5-benzenetricarboxylic acid (0.53 mmol) were weighed using an electronic balance and transferred to the solvent, and stirred to dissolve. The resulting solution was transferred to a 50 mL polytetrafluoroethylene reaction vessel liner (pre-soaked overnight in aqua regia, soaked in saturated sodium carbonate solution, and then washed with ultrapure water), and placed in the reaction vessel. The reaction was carried out in a solvothermal oven at 120°C for 300 min. After the solvothermal reaction is complete, allow it to cool to room temperature before removing it. Pour it into centrifuge tubes and centrifuge at 4000 rpm for 4 minutes. Then, discard the supernatant and wash the solid twice with water, using the same centrifuge speed and time settings as before. Store it in a refrigerator. TEM images are shown below. Figure 1 The results show that GaMOFs are small, uniformly sized spheres. Figure 2 The particle size shown is approximately 53.48 ± 7.30 nm.

[0039] Example 2, Synthesis of GaMOF@Cur

[0040] According to one embodiment of this application, GaMOF and curcumin were dissolved in an equal proportion in a certain amount of anhydrous ethanol, and the mixture was vortexed at room temperature in the dark for 2 hours. After the vortexing, the mixture was poured into centrifuge tubes and centrifuged at 6000 rpm for 4 minutes. The supernatant was then discarded, and the solid was washed twice, first with anhydrous ethanol and then with water, using the same centrifuge speed and time settings as before. The solid was then stored in a refrigerator. TEM images are shown below. Figure 3 The results show that GaMOF@Cur forms small, uniformly sized spheres. Figure 4 The particle size shown is approximately 60.90 ± 10.62 nm. This variation in particle size clearly indicates that curcumin was successfully loaded onto GaMOF. Furthermore, the presence of Ga in the EDS spectrum further supports this finding.

[0041] Example 3, Synthesis of EcN / GaMOF@Cur

[0042] According to one embodiment of this application, 1×10 9CFU / mL bacterial cells and 0.5 mg / mL GaMOF@Cur were dissolved in sterile PBS and vortexed at room temperature in the dark for 1 h. After vortexing, the mixture was centrifuged at 8000 rpm for 4 min, the supernatant was discarded, and the precipitate was washed once with sterile PBS by centrifugation. The centrifugation speed and time were set the same as before. TEM images are shown below. Figure 5 The results show that the EcN / GaMOF@Cur is rod-shaped and uniform in size. EDS spectroscopy reveals the presence of Ga elements, indicating that GaMOF@Cur has been successfully modified onto the EcN surface.

[0043] Figure 6 The zeta potential diagrams for various materials show that the changes in potential also prove that GaMOF@Cur has been successfully modified onto the EcN surface.

[0044] Example 4: Synthesis of EcN / GaMOF@Cur microspheres

[0045] According to one embodiment of this application, EcN / GaMOF@cur was dissolved in a 1% sodium alginate solution, and the mixture was then added dropwise at a constant rate to a 1% calcium chloride solution using an electrostatic spraying method. Electron microscope images are shown below. Figure 7 The EcN / GaMOF@cur microspheres are shown to be small and uniform in size. Figure 8 As shown, its particle size is approximately 252.38 ± 25.23 μm.

[0046] Example 5: Survival of EcN and EcN / GaMOF@Cur microspheres in gastric juice.

[0047] According to one embodiment of this application, simulated gastric fluid (SGF) is prepared as follows: 3.84 mL of hydrochloric acid is diluted with 800 mL of ultrapure water, 10 g of pepsin is added, and ultrapure water is added to bring the volume to 1 L, adjusting the pH to 2.5. 1×10 g of pepsin is added to 1 mL of SGF. 8 CFU EcN and EcN / GaMOF@Cur microspheres were sampled at 0 h, 1.0 h, and 2.0 h, centrifuged at 8000 rpm for 4 min to remove the supernatant, washed once with PBS, and then plated for bacterial counting, with three replicates per group. Figure 9 As shown, after 1 h and 2 h of incubation, the survival rate of EcN in SGF EcN / GaMOF@Cur microspheres under the protection of calcium alginate microspheres was significantly higher than that of bare EcN. This proves that the calcium alginate coating significantly enhances the tolerance of EcN / GaMOF@Cur to strong acid gastric juice and has a protective effect on probiotics in harsh environments.

[0048] Example 6: Validation of the in vivo therapeutic effect of EcN / GaMOF@Cur microspheres

[0049] Mice were randomly divided into 3 groups (n = 5): normal group, DSS group, and EcN / GaMOF@Cur microsphere group. The treatment methods for each group of mice are as follows: (1) Control: allowed to drink tap water; (2) DSS: allowed to drink tap water containing DSS (days 1-2: 3%, w / v; days 3-5: 5%, w / v; day 6: 3%, w / v) from day 0 to day 6; (3) EcN / GaMOF@Cur microspheres: drank tap water containing DSS (days 1-2: 3%, w / v; days 3-5: 5%, w / v; day 6: 3%, w / v) from day 0 to day 6, and were given EcN / GaMOF@Cur microspheres (10) by gavage from day 7 to day 12. 6 CFU / mouse / day;

[0050] Mouse body weight changes were recorded daily during the experiment. The concentrations of IL-1β and TNF-α in mouse plasma were detected using an ELISA kit.

[0051] Depend on Figure 10 It can be seen that, compared with the DSS group, EcN / GaMOF@Cur microsphere treatment significantly alleviated the weight loss in colitis mice.

[0052] The levels of inflammatory factors in mice are a key indicator of their immune status. Mouse plasma was used to detect the levels of inflammation-related cytokines using an ELISA (enzyme-linked immunosorbent assay) kit. The test results were analyzed, such as... Figure 11 As shown, EcN / GaMOF@Cur microsphere treatment significantly reduced the concentration of the pro-inflammatory factor interleukin-1β (IL-1β) in the blood of mice.

[0053] Example 7: EcN / GaMOF@Cur microspheres regulate gut microbiota homeostasis

[0054] The gut microbiota plays a crucial role in maintaining the integrity of the intestinal barrier and defending against pathogen invasion. However, intestinal infections often lead to gut microbiota imbalance. Given EcN's ability to regulate the gut microbiota, it is hypothesized that EcN / GaMOF@Cur microspheres may repair the intestinal barrier by restoring gut microbiota balance. Therefore, cecal contents from mice treated with EcN / GaMOF@Cur microspheres were rapidly frozen in liquid nitrogen and sent to the company for gut microbiota analysis based on 16S rRNA gene sequencing. Figure 12-13The figures represent the Shannon and Simpson indices of the mouse gut microbiota. As can be seen from the figure, compared with the DSS group, the group treated with EcN / GaMOF@Cur microspheres showed increased Shannon and Simpson indices, indicating enhanced species diversity and evenness in the treatment group. Further analysis of the gut microbiota at the genus and phylum levels was also conducted across different groups. Figure 14-19 As we can see, in the treatment group, EcN / GaMOF@Cur microspheres effectively increased the abundance of Lactobacillus and decreased the abundance of Helicobacter. Lactobacillus' core functions include lactic acid production to acidify the intestinal environment, inhibiting harmful bacteria, regulating immunity, promoting nutrient absorption, and maintaining the mucosal barrier; it is a well-known probiotic in the gut microbiota. Helicobacter, on the other hand, is associated with gastritis, peptic ulcers, and gastric cancer, and is classified as a Group 1 carcinogen by the WHO. The decrease in abundance helps repair the intestinal barrier and inhibit intestinal inflammation. The increase in beneficial gut bacteria indicates that EcN / GaMOF@Cur microspheres improve intestinal mucosal and metabolic functions by regulating the gut microbiota. Furthermore, EcN / GaMOF@Cur microspheres increased the abundance of Firmicutes, most of which are associated with anti-inflammation, suggesting that EcN / GaMOF@Cur microspheres enhance the intestinal anti-inflammatory capacity. Simultaneously, the abundance of Campylobacterota decreased. Campylobacter is the primary culprit in acute bacterial enteritis, while Helicobacter pylori indirectly exacerbates intestinal inflammation. A decrease in their abundance is beneficial in alleviating intestinal inflammation. Analysis of the gut microbiota indicates that EcN / GaMOF@Cur microspheres not only promote the recovery of intestinal mucosa and metabolic function but also promote the balance of microorganisms crucial for intestinal homeostasis, reducing DSS-induced intestinal inflammatory damage.

Claims

1. A composite drug-loaded microsphere system, characterized in that, include: Probiotics; Gallium-based metal-organic framework nanoparticles GaMOF@Cur loaded with anti-inflammatory drugs; and a calcium alginate protective coating on the outermost layer; the GaMOF@Cur is modified on the surface of probiotics to form an EcN / GaMOF@Cur complex, and the calcium alginate coating is wrapped around the outside of the complex.

2. The composite drug-loaded microsphere system according to claim 1, characterized in that, The probiotic is Escherichia coli Nissle 1917.

3. The composite drug-loaded microsphere system according to claim 1, characterized in that, The anti-inflammatory drug is curcumin.

4. The composite drug-loaded microsphere system according to claim 1, characterized in that, The gallium-based metal-organic framework nanoparticles are synthesized from gallium nitrate and 1,3,5-benzenetricarboxylic acid via a solvothermal reaction, and have a particle size of 50-70 nm.

5. The composite drug-loaded microsphere system according to claim 1, characterized in that, The calcium alginate protective coating is formed by cross-linking sodium alginate and calcium chloride through electrostatic spraying, and the microspheres have a particle size of 200-280 μm.

6. The composite drug-loaded microsphere system according to claim 1, characterized in that, The system can protect the survival rate of probiotics in simulated gastric fluid significantly higher than that of free probiotics.

7. The use of the composite drug-loaded microsphere system according to any one of claims 1 to 6 in the preparation of drugs for treating enteritis.

8. The application according to claim 7, characterized in that, The drug is used to reduce enteritis-related weight loss, lower the level of the pro-inflammatory factor IL-1β, and regulate gut microbiota homeostasis.

9. The application according to claim 8, characterized in that, The regulation of intestinal flora homeostasis includes increasing the abundance of lactic acid bacteria, decreasing the abundance of Helicobacter pylori, increasing the abundance of Firmicutes, and decreasing the abundance of Campylobacteria.

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