A tripterine-loaded nanoparticle microsphere, a preparation method and application thereof

Abstract

The application discloses a tripterine self-assembled nanoparticle microsphere, a preparation method and application thereof, and belongs to the technical field of biological medicines. The tripterine self-assembled nanoparticle microsphere takes tripterine self-assembled nanoparticles as a drug core, is formed by cross-linking of a sodium alginate-pectin composite carrier through calcium ions, can significantly improve drug solubility, realizes stable protection and intestinal targeting rapid release in the gastrointestinal tract, and effectively reduces drug systemic exposure and side effect risks. The preparation process is mild and simple, the obtained preparation has high targeting and safety. Pharmacodynamic results show that the microsphere can obviously improve liver steatosis, inflammation and fibrosis related to metabolic-related fatty liver hepatitis, and has better curative effect than conventional preparations. The application provides a novel oral delivery system for preventing and treating metabolic-related fatty liver hepatitis, and has good industrial and clinical transformation prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a triptolide-loaded nanoparticle microsphere, its preparation method, and its application. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MAFLD) is the most prevalent chronic liver disease worldwide, with a global prevalence of approximately 25%–40%. The disease can progress from simple hepatic steatosis to metabolic-associated steatohepatitis (MASH), liver fibrosis, cirrhosis, and even hepatocellular carcinoma. MASH is a crucial stage for achieving reversible intervention. The pathogenesis of MAFLD is complex, primarily related to insulin resistance, oxidative stress, and inflammatory responses. Currently, the FDA has approved retinoic acid and smiglitide for the treatment of MAFLD, but these drugs have safety concerns including gastrointestinal reactions, liver damage, and cholecystitis. There are currently no domestically developed drugs for the treatment of MASH in my country; therefore, developing novel delivery systems and therapeutic agents with stronger targeting and improved safety is of significant clinical importance.

[0003] Tripterygium wilfordii, a natural active ingredient derived from the traditional Chinese medicine Tripterygium wilfordii, can regulate energy, lipid, and bile acid metabolism through multiple signaling pathways, demonstrating promising therapeutic potential in a model of metabolism-related fatty liver disease. However, its poor water solubility, uncertain in vivo target sites, and the fact that current delivery technologies primarily aim to improve oral bioavailability and increase systemic exposure, easily leading to systemic side effects, limit its clinical translation. The presence of acidic groups in its chemical structure makes it readily absorbed in the acidic environment of the stomach and the upper small intestine, causing systemic side effects and reducing its effective accumulation in the lower small intestine, thus decreasing the intensity of its local effects on the ileum / colon.

[0004] Sodium alginate and pectin are commonly used oral microsphere carrier materials, but sodium alginate alone has drawbacks such as premature drug release in the upper gastrointestinal tract and insufficient targeting, making it difficult to achieve stable intestinal targeted delivery. Therefore, constructing an intestinal targeted delivery system that can improve the solubility of triptolide, achieve dual pH / intestinal enzyme responsive drug release, reduce systemic absorption, and lower the risk of toxic side effects has become an urgent technical problem to be solved in this field. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides a triptolide nanoparticle microsphere, wherein the triptolide nanoparticle microsphere comprises triptolide self-assembled nanoparticles and a sodium alginate-pectin composite carrier, and is formed by calcium ion crosslinking; in the sodium alginate-pectin composite carrier, the mass ratio of sodium alginate to pectin is (1-6):1.

[0006] Compared with existing technologies, the triptolide nanoparticle microspheres provided by this invention, with self-assembled triptolide nanoparticles as the drug core and sodium alginate-pectin composite carrier as the framework, and formed by calcium ion cross-linking, can significantly improve the problems of poor water solubility and poor dispersibility of triptolide. Sodium alginate and pectin form a stable carrier material within a specific mass ratio range, which can effectively encapsulate and protect the nanoparticles, preventing premature drug release in the stomach and small intestine. Simultaneously, it endows the microspheres with intestinal-responsive drug release characteristics, achieving targeted intestinal delivery, effectively reducing systemic drug absorption, and lowering toxic side effects. This provides a stable and reliable formulation for the safer and more efficient use of triptolide in the prevention and treatment of metabolic-related steatohepatitis.

[0007] In one possible implementation, the mass ratio of sodium alginate to pectin is 3:2.

[0008] The present invention further limits the mass ratio of sodium alginate to pectin to 3:2, which optimizes the shapeability, structural stability and intestinal responsiveness of the composite carrier. The microspheres have regular morphology, uniform drug loading, and strong gastrointestinal stability, which is more conducive to achieving precise intestinal targeted drug release.

[0009] In one possible implementation, the mass ratio of the triptolide self-assembled nanoparticles to sodium alginate is (0.1-8):20.

[0010] This invention limits the mass ratio of triptolide self-assembled nanoparticles to sodium alginate to (0.1-8):20, which can ensure that the drug is fully loaded and evenly dispersed, avoid drug leakage or aggregation, balance drug loading and formulation stability, and improve drug utilization efficiency.

[0011] In one possible implementation, the mass ratio of the triptolide self-assembled nanoparticles to sodium alginate is 3:20.

[0012] The present invention further limits the mass ratio of nanoparticles to sodium alginate to 3:20, which can achieve the best balance of drug loading, shapeability and drug release behavior, resulting in high encapsulation efficiency and structural stability, and can maximize the intestinal targeting and therapeutic effects.

[0013] In one possible implementation, the triptolide self-assembled nanoparticles are formed by the self-assembly of triptolide under carrier-free conditions, the triptolide self-assembled nanoparticles are an aqueous dispersion, and the concentration of triptolide in the aqueous dispersion is 0.1-8 mg / mL.

[0014] This invention specifies that nanoparticles are formed by the self-assembly of triptolide under carrier-free conditions and exist in an aqueous dispersion with a concentration of 0.1-8 mg / mL. This can improve the water solubility and dispersion stability of the drug, avoid interference from carrier excipients, and provide a high-quality drug intermediate for microsphere encapsulation.

[0015] In one possible implementation, the concentration of triptolide in the aqueous dispersion is 3 mg / mL. Limiting the concentration of triptolide in the aqueous dispersion to 3 mg / mL allows for sufficient self-assembly of nanoparticles, resulting in uniform particle size and optimal stability, thus providing a drug core with the best performance for microsphere formulations.

[0016] The second objective of this invention is to provide a method for preparing triptolide-loaded nanoparticles, comprising the following steps: Step S1: Dissolve sodium alginate and pectin in water to prepare solution C; Step S2: Mix the self-assembled triptolide nanoparticles with solution C to prepare solution D; Step S3: Add solution D to calcium chloride solution for cross-linking and molding to obtain the tripterygium oleoresin-loaded nanoparticle microspheres.

[0017] Compared with existing technologies, the preparation method protected by this invention prepares triptolide-loaded nanoparticle microspheres through three steps: dissolution, mixing, and calcium ion cross-linking. The process is mild, simple to operate, and the conditions are controllable, making it suitable for industrial production. This method can uniformly disperse nanoparticles in a composite carrier and form a complete microsphere structure through calcium ion cross-linking, effectively preserving drug activity and ensuring the uniformity of microsphere morphology, particle size, and drug loading. The prepared microspheres exhibit good gastrointestinal stability and intestinal targeted drug release capability, stably achieving the goal of reducing systemic exposure and improving local intestinal efficacy.

[0018] In one possible implementation, step S1 is performed at 80°C; In step S3, solution D is added to calcium chloride solution by dropwise addition, and after static cross-linking and washing with water, tripterygium oleoresin-loaded nanoparticles are obtained.

[0019] The 80℃ temperature allows sodium alginate and pectin to fully dissolve. The dropwise addition method, static cross-linking, and water washing steps ensure that the microspheres are well-formed and have uniform particle size, remove residual cross-linking agents, and improve the safety and structural stability of the microspheres.

[0020] In one possible implementation, the triptolide self-assembled nanoparticles are prepared by the following method: Step a: Dissolve triptolide in anhydrous ethanol to prepare solution A; Step b: Under light-protected stirring conditions, inject solution A obtained in step S1 into water to obtain solution B; Step c: Place solution B in a dark environment at 45°C and remove ethanol by rotary evaporation to obtain tripterygium wilfordii self-assembled nanoparticles.

[0021] The third objective of this invention is to provide the application of triptolide-loaded nanoparticles in the preparation of drugs for the prevention and treatment of metabolic-associated fatty liver disease.

[0022] Compared with the prior art, the present invention has the following advantages: 1. The triptolide nanoparticle microspheres described herein use triptolide as the active ingredient and are formed by carrier-free self-assembly, which can significantly improve the solubility of triptolide, reduce the use of excipients, and achieve a drug-excipient integrated formulation.

[0023] 2. Using microspheres as delivery carriers, the carrier materials sodium alginate and pectin are stably encapsulated with nanoparticles through calcium ion coordination crosslinking and hydrogen bonding to construct an intestinal targeted delivery system. This system can both protect the gastrointestinal tract of triptolide nanoparticles and prevent premature drug release, and achieve dual-responsive release of intestinal pH and intestinal enzymes.

[0024] 3. The intestinal-targeted triptolide nanoparticle microspheres provided by this invention can significantly improve hepatic steatosis, reduce serum alanine aminotransferase and aspartate aminotransferase levels, inhibit the expression of hepatic inflammatory factors, chemokines and fibrosis-related factors, regulate the intestinal FXR signaling pathway and enhance the expression and secretion of GLP-1 in animal models of metabolism-related steatohepatitis, and have better efficacy than conventional triptolide suspensions.

[0025] 4. The intestinal-targeted triptolide nanoparticle microspheres provided by this invention can significantly reduce the peak blood drug concentration and the area under the curve after oral administration, achieving precise intestinal-targeted delivery and effectively reducing the systemic drug exposure level, thus improving drug safety. Attached Figure Description

[0026] Figure 1 Figure showing the experimental results of particle size (Size) of self-assembled nanoparticles of triptolide at different concentrations provided by this invention; Figure 2 The polydispersity index (PDI) experimental results of triptolide self-assembled nanoparticles at different concentrations provided by this invention are shown in the figure. Figure 3 The potential (Zeta) experimental results of self-assembled nanoparticles of triptolide at different concentrations provided by this invention; Figure 4 The images show the self-assembled triptolide nanoparticles prepared in Example 3 of this invention and the differential scanning calorimetry (DSC) spectrum of triptolide. Figure 5 This is a flowchart illustrating the preparation process of triptolide-loaded nanoparticles and microspheres according to the present invention. Figure 6 The state diagrams of triptolide nanoparticles with different ratios of sodium alginate and pectin are shown below for the present invention. Figure 7 This is a diameter distribution diagram of the triptolide nanospheres loaded with sodium alginate and pectin at a mass ratio of 1:1 according to the present invention. Figure 8 This is a diameter distribution diagram of the triptolide nanospheres loaded with sodium alginate and pectin at a mass ratio of 2:1 according to the present invention. Figure 9 This is a diameter distribution diagram of the triptolide nanospheres loaded with sodium alginate and pectin at a mass ratio of 4:1 according to the present invention. Figure 10 This is a diameter distribution diagram of the triptolide nanospheres loaded with sodium alginate and pectin at a mass ratio of 6:1 according to the present invention. Figure 11 This is a diameter distribution diagram of the triptolide nanospheres loaded with sodium alginate and pectin at a mass ratio of 3:2 according to the present invention. Figure 12 This is an infrared characterization diagram of the triptolide self-assembled nanoparticles, triptolide-loaded nanoparticle microspheres, and drug-free microspheres superimposed with triptolide self-assembled nanoparticles prepared in Example 3 of the present invention. Figure 13 The triptolide self-assembled nanoparticles, triptolide-loaded nanoparticle microspheres, and drug-free microspheres stacked with triptolide self-assembled nanoparticles prepared in Example 3 of this invention are examples of triptolide self-assembled nanoparticles and triptolide microspheres prepared in Example 3 of this invention. rattan Schematic diagram of differential scanning calorimetry characterization of erythrin and drug-free microspheres; Figure 14 This image shows the intestinal targeted release effect of triptolide self-assembled nanoparticles and triptolide nanoparticle-loaded microspheres prepared in Example 3 of the present invention. Figure 15 The graph shows the liver fat content of mice in each group of the HFD model. Figure 16 HE staining images of liver steatosis in mice of different groups of HFD model (×100). Figure 17 HE staining images of livers of mice in each group of the CDAHFD model (×100). Figure 18 For liver inflammatory factors in mice of different groups in the CDAHFD model Il-6 and fibrotic factors tgf-β Renderings; Figure 19 The image shows the effect of α-SMA labeling on liver fibrosis in mice of different groups of HFD and CDAHFD models; Figure 20 The negative regulation effect of the intestinal FXR-GLP-1 axis in mice of each group of HFD and CDAHFD models is shown. Figure 21 This is a comparison graph showing the drug concentration-time curves and relative AUC of the triptolide nanoparticle microsphere formulation of this invention and the conventional suspension formulation. Detailed Implementation

[0027] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the claims of the present invention.

[0028] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0029] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0030] In this specific embodiment, for ease of description, the components and their abbreviations are used in the following examples as follows: Lei Gong rattan Celastrol (Cel for short); Sodium alginate (ALG) Pectin (Pec for short); Particle size (Size); Potential (Zeta); Polydispersity index (PDI); Nanoparticles (NPs); Drug-free microspheres (NM); Tripterygium oleoresin nanoparticles (CNM); Tripterygium wilfordii self-assembled nanoparticles (Cel NPs).

[0031] The first aspect of this invention provides triptolide-loaded nanoparticle microspheres, which comprise triptolide self-assembled nanoparticles and a sodium alginate-pectin composite carrier, and are formed by calcium ion crosslinking. In the sodium alginate-pectin composite carrier, the mass ratio of sodium alginate to pectin is (1-6):1, and the mass ratio of triptolide self-assembled nanoparticles to sodium alginate is (0.1-8):20. The triptolide self-assembled nanoparticles are formed by triptolide self-assembly under carrier-free conditions. The nanoparticles are an aqueous dispersion, and the concentration of triptolide in the aqueous dispersion is 0.1-8 mg / mL.

[0032] The present invention also provides a method for preparing the triptolide-loaded nanoparticle microspheres, comprising the following steps: Step S1: Dissolve sodium alginate and pectin in water at 80°C to obtain solution C; Step S2: Mix the self-assembled triptolide nanoparticles with solution C to prepare solution D; Step S3: Solution D is added dropwise to calcium chloride solution, and after static cross-linking and washing with water, tripterygium oleoresin-loaded nanoparticles are obtained.

[0033] The triptolide self-assembled nanoparticles were prepared by the following method: Step a: Dissolve triptolide in anhydrous ethanol to prepare solution A; Step b: Under light-protected stirring conditions, inject solution A obtained in step S1 into water to obtain solution B; Step c: Place solution B in a dark environment at 45°C and remove ethanol by rotary evaporation to obtain tripterygium wilfordii self-assembled nanoparticles.

[0034] In a specific embodiment of the present invention, the effect of different Cel concentrations on Cel NPs was first investigated, specifically by setting the Cel concentration to 0.1-8 mg / ml. The optimal method is selected by comparing and measuring Size, PDI, and Zeta.

[0035] That is, by analyzing the three indices of Cel NPs, we can analyze their physical properties.

[0036] Example 1 This embodiment provides a triptolide self-assembled nanoparticle, which is prepared by the following method: Step S1: Dissolve triptolide in anhydrous ethanol to prepare solution A; Step S2: Under light-protected stirring conditions, the solution A obtained in step S1 is injected into water to obtain solution B; Step S3: Solution B is subjected to rotary evaporation at 45°C under light-shielding conditions to remove ethanol, thereby obtaining triptolide self-assembled nanoparticles, wherein the concentration of triptolide in the aqueous dispersion is 0.1 mg / mL.

[0037] Example 2 This embodiment provides a triptolide self-assembled nanoparticle. The only difference from Example 1 is that the concentration of triptolide in the aqueous dispersion in this embodiment is 2 mg / mL. The rest is the same as in Example 1 and will not be repeated here.

[0038] Example 3 This embodiment provides a triptolide self-assembled nanoparticle. The only difference from Example 1 is that the concentration of triptolide in the aqueous dispersion in this embodiment is 3 mg / mL. The rest is the same as in Example 1 and will not be repeated here.

[0039] Example 4 This embodiment provides a triptolide self-assembled nanoparticle. The only difference from Example 1 is that the concentration of triptolide in the aqueous dispersion in this embodiment is 4 mg / mL. The rest is the same as in Example 1 and will not be repeated here.

[0040] Example 5 This embodiment provides a triptolide self-assembled nanoparticle. The only difference from Example 1 is that the concentration of triptolide in the aqueous dispersion in this embodiment is 5 mg / mL. The rest is the same as in Example 1 and will not be repeated here.

[0041] Example 6 This embodiment provides a triptolide self-assembled nanoparticle. The only difference from Example 1 is that the concentration of triptolide in the aqueous dispersion in this embodiment is 6 mg / mL. The rest is the same as in Example 1 and will not be repeated here.

[0042] Example 7 This embodiment provides a triptolide self-assembled nanoparticle. The only difference from Example 1 is that the concentration of triptolide in the aqueous dispersion in this embodiment is 8 mg / mL. The rest is the same as in Example 1 and will not be repeated here.

[0043] Table 1 shows the results for Size, PDI, and Zeta for different concentrations of Cel NPs: surface Results of Cel NPs index at different concentrations Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Concentration (mg / ml) 0.1 2 3 4 5 6 8 Size (nm) 645.1±23.7 563.6±33.5 234.7±24.8 211.1±6.6 335.6±2.0 360.4±26.2 206.8±13.3 PDI 0.146±0.08 0.463±0.07 0.03±0.03 0.09±0.03 0.48±0.26 0.578±0.07 0.087±0.08 Zeta (mV) -29.1±0.4 -27.7±0.1 -29.5±0.1 -30.0±0.7 -28.5±0.6 -29.5±0.7 -28.8±0.3 Table 1 shows that, with a fixed total volume, as the Cel concentration increases, the size of Cel NPs first decreases and then increases, the PDI shows a trend of first increasing, then decreasing, and then increasing again, while the Zeta remains relatively consistent overall. Considering all three indicators, a Cel concentration of 3 mg / ml is the optimal concentration. The reason for this is that when the Cel concentration is too low, the triptolide self-assembled nanoparticles aggregate, resulting in insufficient self-assembly; when the Cel concentration is too high, the triptolide self-assembled nanoparticles become locally oversaturated and unevenly dispersed. Figures 1-3 The figure shows the experimental results for Cel concentrations ranging from 0.1 to 8 mg / ml. Figure 1 The figure shows the experimental results of particle size of self-assembled nanoparticles of triptolide at different concentrations according to the present invention. Figure 2 The figure shows the polydispersity index experimental results of self-assembled nanoparticles of triptolide at different concentrations in this invention. Figure 3 The diagram shows the potential experimental results of self-assembled nanoparticles of triptolide at different concentrations according to the present invention.

[0044] In summary, a Cel concentration of 3 mg / ml is the optimal solution in this invention. Figure 4 The images show the self-assembled triptolide nanoparticles prepared in Example 3 of this invention and the differential scanning calorimetry (DSC) spectrum of triptolide.

[0045] The Cel NPs can be incorporated into the microsphere material to reduce the release rate of Cel in low pH environments, thereby reducing drug absorption in the stomach, duodenum, and proximal small intestine. ALG-Pec microspheres utilize hydrogen bonding interactions and Ca... 2+ Coordination crosslinking enhances its mechanical strength, and good This reduces the release of Cel NPs and prolongs their residence time. Furthermore, the ALG-Pec microsphere material is naturally derived, resulting in low risk of hepatotoxicity and nephrotoxicity.

[0046] like Figure 5 As shown, using the triptolide self-assembled nanoparticles obtained in Example 3 as raw materials, the triptolide-loaded nanoparticle microspheres of the present invention are prepared through the following steps: Step S1: Dissolve sodium alginate and pectin in water at 80°C to obtain solution C; Step S2: Mix the self-assembled triptolide nanoparticles with solution C to prepare solution D; Step S3: Add solution D to calcium chloride solution at a rate of 1 mL / min, allow to stand for 1 hour for cross-linking, and wash with water 3-5 times to obtain tripterygium oleoresin-loaded nanoparticles. See the following example for details: Example 8 This embodiment provides a triptolide-loaded nanoparticle microsphere, which is prepared through the following steps: Step S1: Dissolve sodium alginate and pectin in water at 80°C to obtain solution C; Step S2: Mix the triptolide self-assembled nanoparticles obtained in Example 3 with solution C to obtain solution D; Step S3: Add solution D to calcium chloride solution at a rate of 1 mL / min, allow it to stand for 1 hour for cross-linking, and wash with water 3-5 times to obtain tripterygium oleoresin-loaded nanoparticle microspheres; In this embodiment: ALG:Pec=1:1; Cel NPs:ALG=3:20.

[0047] Example 9 This embodiment provides a triptolide nanoparticle microsphere, which differs from Example 8 only in that: ALG:Pec=2:1; Cel NPs:ALG=3:20. The rest is the same as in Example 8, and will not be repeated here.

[0048] Example 10 This embodiment provides a triptolide nanoparticle microsphere, which differs from Example 8 only in that: ALG:Pec=4:1; Cel NPs:ALG=3:20. The rest is the same as in Example 8, and will not be repeated here.

[0049] Example 11 This embodiment provides a triptolide nanoparticle microsphere, which differs from Example 8 only in that: ALG:Pec=6:1; Cel NPs:ALG=3:20. The rest is the same as in Example 8, and will not be repeated here.

[0050] Example 12 This embodiment provides a triptolide nanoparticle microsphere, which differs from Example 8 only in that: ALG:Pec=3:2; Cel NPs:ALG=3:20. The rest is the same as in Example 8, and will not be repeated here.

[0051] like Figure 6 The image shown is a state diagram of the triptolide-loaded nanoparticle microspheres prepared in Examples 8-12 of this invention. Figure 7 This is a diameter distribution diagram of the triptolide nanospheres loaded with sodium alginate and pectin at a mass ratio of 1:1 according to the present invention. Figure 8 This is a diameter distribution diagram of the triptolide nanospheres loaded with sodium alginate and pectin at a mass ratio of 2:1 according to the present invention. Figure 9 This is a diameter distribution diagram of the triptolide nanospheres loaded with sodium alginate and pectin at a mass ratio of 4:1 according to the present invention. Figure 10 This is a diameter distribution diagram of the triptolide nanospheres loaded with sodium alginate and pectin at a mass ratio of 6:1 according to the present invention. Figure 11This is a diameter distribution diagram of the triptolide nanospheres loaded with sodium alginate and pectin at a mass ratio of 3:2 according to the present invention.

[0052] Combination Figure 7 With an ALG:Pec ratio of 1:1 and a Cel NPs:ALG ratio of 3:20, the microsphere diameter distribution ranged from 1.5 to 2.5 mm, exhibiting a relatively wide distribution and moderate uniformity. (Combined with...) Figure 8 With an ALG:Pec ratio of 2:1 and a Cel NPs:ALG ratio of 3:20, the microsphere diameter distribution ranged from 1.8 to 3.5 mm, exhibiting a bimodal distribution and uneven distribution. (Combined with...) Figure 9 At an ALG:Pec ratio of 4:1 and a Cel NPs:ALG ratio of 3:20, the microsphere diameters ranged from 1.9 to 2.8 mm, exhibiting a wide distribution and poor uniformity, suggesting possible aggregation of multiple microspheres. (Combined with...) Figure 10 At an ALG:Pec ratio of 6:1 and a Cel NPs:ALG ratio of 3:20, the microsphere diameter distribution ranged from 2.0 to 3.8 mm, exhibiting a narrow distribution and a small number of larger microspheres. (Combined with...) Figure 11 With an ALG:Pec ratio of 3:2 and a Cel NPs:ALG ratio of 3:20, the microsphere diameters ranged from 1.9 to 2.6 mm, exhibiting a concentrated distribution and good uniformity.

[0053] The above analysis shows that changing the ALG:Pec ratio has a significant impact on the diameter of the microspheres, with ALG:Pec = 3:2 and Cel NPs:ALG = 3:20 being the preferred ratios.

[0054] The CNM under the optimized scheme was characterized by DCS, infrared spectroscopy, and in vitro release experiments.

[0055] from Figure 12 Infrared spectrum and Figure 13 The DSC spectrum shows that the infrared absorption spectrum and exothermic peak of the relevant functional groups have changed, indicating that Cel NPs have been successfully encapsulated in the microspheres.

[0056] The in vitro release behavior of Cel NPs@ALG / Pec was investigated using the dialysis bag method. The dialysis bag was placed in 30 mL of release medium and the release was carried out at the following time points: Medium A: simulated gastric fluid (SGF, pH 1.2), time 0–2 h; Medium B: simulated small intestinal fluid (SIF, pH 6.8), time 2–6 h; Medium C: simulated colonic fluid (SCF, pH 7.4), time 6–48 h. At each specified time point, 1.0 mL of dialysate was added to fresh culture medium, and the Cel content in the dialysate was determined by high-performance liquid chromatography (HPLC). The cumulative percentage of drug release was calculated.

[0057] Figure 14This image shows the intestinal targeted release effect of the triptolide self-assembled nanoparticles and triptolide-loaded nanoparticle microspheres prepared in Example 3 of this invention. As can be seen from the image, the microspheres are generally stable at pH 1.2 and pH 6.8, with a small amount of drug released slowly. At pH 7.4, the microspheres are sensitive and release the drug rapidly. Furthermore, the triptolide-loaded nanoparticle microspheres exhibit a more significant intestinal responsiveness compared to free drug and Cel NPs.

[0058] Pharmacodynamics research section: 1. Animal models and grouping: Two MASH models were established using C57BL / 6J mice: 1) HFD model: High-fat diet for 12 weeks to simulate early MASH in obese patients.

[0059] 2) CDAHFD model: choline-deficient high-fat diet was fed for 1 week to simulate early lean MASH.

[0060] The model mice were randomly divided into the following groups (n=5-6 per group): Normal control group (CON): fed with normal feed and given an equal volume of blank solvent by gavage.

[0061] HFD model control group (HFD): fed HFD diet and given an equal volume of blank solvent by gavage.

[0062] CDAHFD model control group (CDAHFD): fed CDAHFD diet and administered an equal volume of blank solvent by gavage.

[0063] Celastrol free drug group (CS, 1 mg / kg): Administered with HFD diet or CDAHFD diet and gavage with Celastrol suspension (CS, 1 mg / kg).

[0064] The present invention provides a microsphere assembly loaded with triptolide nanoparticles (CNM, 1 mg / kg, based on Cel): administered via gavage to HFD feed or CDAHFD feed, followed by administration of a microsphere suspension loaded with triptolide nanoparticles.

[0065] 2. Dosing regimen: Mice in each group were administered the drug once daily by gavage for 7 consecutive days (HFD model, therapeutic administration after modeling; CDAHFD model, prophylactic administration concurrently during modeling), and changes in mouse body weight were recorded daily.

[0066] 3. Sample collection and testing: After the last administration, the mice were euthanized by cervical dislocation, and liver and blood samples were collected.

[0067] Liver function tests: Serum was separated, and biochemical reagent kits were used to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0068] Liver lipid assay: A portion of liver tissue was homogenized, and the levels of triglycerides (TG) and total cholesterol (TC) were detected using a biochemical reagent kit.

[0069] Pathological examination: A portion of the left lobe of the liver was taken, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and subjected to hematoxylin and eosin (HE) analysis.

[0070] Molecular biological detection: RNA was extracted from liver tissue and inflammatory factors were detected by qPCR. Il-6 , Ccl2 The mRNA levels of fibrosis factors (fibrosis factor tgf-β, α-SMA) and fibrosis factors.

[0071] Western Blot analysis: A portion of liver tissue homogenate was collected, and the expression of fibrosis-related proteins such as α-SMA, Col1a1, and Col3a1 was detected by Western Blot.

[0072] 4. Experimental results of the HFD model (obese MASH): like Figure 15 , Figure 16 As shown, the livers of mice in the HFD model group showed fat accumulation and increased fat vacuoles. After treatment with the triptolide nanoparticle microsphere formulation of this invention, the liver TG content decreased and liver steatosis was alleviated. The improvement effect of the triptolide nanoparticle microsphere formulation group (HFD-CNM) was better than that of the conventional suspension drug group (HFD-CS).

[0073] 5. Experimental results of the CDAHFD model (lean MASH): 5.1 Improvement in liver pathology: like Figure 17 As shown, significant fatty degeneration was observed in the livers of mice in the CDAHFD model group. Treatment with the triptolide-loaded nanoparticle microsphere formulation of this invention significantly reduced liver fatty degeneration, with the triptolide-loaded nanoparticle microsphere formulation group (CDA-CNM) showing better improvement than the conventional suspension drug group (CDA-CS).

[0074] 5.3 Effects on the expression of inflammatory factors: like Figure 18 As shown, qPCR results indicate that liver inflammatory factors in the CDAHFD model group... Il-6 and fibrotic factors tgf-βExpression was significantly upregulated. After treatment with the triptolide nanoparticle microsphere formulation of this invention, the expression levels of both decreased significantly, with the triptolide nanoparticle microsphere formulation group (CDA-CNM) showing better inhibitory effect than the conventional suspension drug group (CDA-CS).

[0075] 6. Effects on the expression of fibrosis-related proteins in the HFD and CDAHFD models: like Figure 19 As shown, Western blotting results indicated that fibrosis in the HFD model-positive group did not change significantly and was not used as an indicator of fibrosis improvement to assess efficacy. In the CDAHFD model, α-SMA and Col1A1 were significantly upregulated in the liver of mice in the model group. After treatment with the triptolide-loaded nanoparticle microsphere formulation of this invention, the expression levels of these two markers decreased significantly, with the triptolide-loaded nanoparticle microsphere formulation (CNM) showing better inhibitory effects than the conventional suspension drug group (CS). Fibrosis in the Col3A1-positive model group did not change significantly and was not used as an indicator of fibrosis improvement to assess efficacy.

[0076] 7. Effects on the intestinal FXR signaling pathway in HFD and CDAHFD models: like Figure 20 As shown, in the HFD model and the CDAHFD model, the FXR target genes in the model group Osta , Fgf15 Significantly elevated levels of GLP-1 were observed, while the release of GLP-1 in the blood decreased. After treatment with the microsphere formulation of this invention, the expression levels of these two target genes significantly decreased, and the upward trend in blood GLP-1 levels was consistent with pharmacodynamic and signal axis regulation levels. The inhibitory effect of the triptolide-loaded nanoparticle microsphere formulation (CNM) was superior to that of the conventional suspension drug group.

[0077] The above results indicate that the intestinal-targeted triptolide nanoparticles of the present invention can effectively improve liver steatosis, inflammation, fibrosis and other indicators in HFD-induced obese CASH and CDAHFD-induced lean MASH, and inhibit the intestinal FXR-GLP-1 signaling pathway, with better effects than conventional suspension preparations.

[0078] Pharmacokinetic studies: 1. Experimental Design: Normal male C57BL / 6J mice, weighing 20-25g, were randomly divided into two groups (n=6 per group): Cel free drug group (CS): single gavage administration of Cel suspension (1 mg / kg); The present invention provides a triptolide-loaded nanoparticle microsphere formulation (CNM): a single oral administration of the triptolide-loaded nanoparticle microsphere formulation suspension prepared in Example 12 (1 mg / kg based on Cel).

[0079] 2. Blood sample collection and processing: In accordance with animal ethics requirements, a crossover sampling design was used: each group of 6 mice was further divided into 2 subgroups (3 mice each), and blood was collected from each subgroup at different time points. Subgroup 1: Blood samples of approximately 30 μL were collected from the tail at 1, 3, 5, and 7 hours after drug administration; Subgroup 2: Blood samples of approximately 30 μL were collected by tail clipping at 2, 4, 6, and 8 hours after drug administration.

[0080] Blood samples were collected in anticoagulant tubes and centrifuged at 5000 rpm for 10 min at 4°C to separate plasma. 5-10 μL of plasma sample was precisely pipetted, and 3 volumes of methanol-water solution containing internal standard (methanol:water = 4:1, v / v) were added to precipitate proteins. The mixture was vortexed for 7 min, centrifuged at 12000 rpm for 20 min at 4°C, and 80 μL of the supernatant was collected. Celsius concentration was determined by LC-MS / MS and expressed as relative abundance using the internal standard method.

[0081] 3. Experimental Results: like Figure 21 As shown, compared with the free drug group, the peak plasma concentration (Cmax) of the triptolide nanoparticle microsphere formulation of the present invention was significantly reduced by 70%, and the area under the curve (AUC0-24h) was significantly reduced by 80%, indicating that the triptolide nanoparticle microsphere formulation of the present invention significantly reduced the systemic absorption of Cel and has the characteristic of reducing the risk of adverse reactions.

[0082] In summary, this invention utilizes carrier-free self-assembly technology to prepare triptolide nanoparticles, which are then encapsulated in sodium alginate-pectin composite microspheres. Calcium ion cross-linking forms a stable intestinal-targeted delivery system of triptolide-loaded nanoparticle microspheres. These triptolide-loaded nanoparticle microspheres enable intestinal-responsive drug release, effectively reducing systemic drug absorption and improving medication safety. Animal experiments show that these triptolide-loaded nanoparticle microspheres significantly improve hepatic steatosis, inflammation, and fibrosis in metabolic-associated steatohepatitis (MAH), regulating related signaling pathways to exert therapeutic effects. The efficacy is significantly superior to conventional drug formulations, demonstrating good drugability and clinical application potential, and providing a safe and effective novel oral drug option for the prevention and treatment of MAH.

[0083] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A type of microsphere loaded with triptolide nanoparticles, characterized in that, The triptolide nanoparticle microspheres comprise triptolide self-assembled nanoparticles and a sodium alginate-pectin composite carrier, and are formed by calcium ion crosslinking; in the sodium alginate-pectin composite carrier, the mass ratio of sodium alginate to pectin is (1-6):

1.

2. The triptolide-loaded nanoparticle microspheres as described in claim 1, characterized in that, The mass ratio of sodium alginate to pectin is 3:

2.

3. The triptolide-loaded nanoparticle microspheres as described in claim 1, characterized in that, The mass ratio of the triptolide self-assembled nanoparticles to sodium alginate is (0.1-8):

20.

4. The triptolide-loaded nanoparticle microspheres as described in claim 1, characterized in that, The mass ratio of the triptolide self-assembled nanoparticles to sodium alginate is 3:

20.

5. The triptolide-loaded nanoparticle microspheres according to any one of claims 1-4, characterized in that, The triptolide self-assembled nanoparticles are formed by the self-assembly of triptolide under carrier-free conditions. The triptolide self-assembled nanoparticles are an aqueous dispersion, and the concentration of triptolide in the aqueous dispersion is 0.1-8 mg / mL.

6. The triptolide-loaded nanoparticle microspheres according to claim 5, characterized in that, The concentration of triptolide in the aqueous dispersion is 3 mg / mL.

7. A method for preparing triptolide-loaded nanoparticles as described in any one of claims 1-6, characterized in that, Includes the following steps: Step S1: Dissolve sodium alginate and pectin in water to prepare solution C; Step S2: Mix the self-assembled triptolide nanoparticles with solution C to prepare solution D; Step S3: Add solution D to calcium chloride solution for cross-linking and molding to obtain the tripterygium oleoresin-loaded nanoparticle microspheres.

8. The preparation method according to claim 7, characterized in that, Step S1 is performed at 80°C. In step S3, solution D is added to calcium chloride solution by dropwise addition, and after static cross-linking and washing with water, tripterygium oleoresin-loaded nanoparticles are obtained.

9. The preparation method according to claim 7, characterized in that, The triptolide self-assembled nanoparticles were prepared by the following method: Step a: Dissolve triptolide in anhydrous ethanol to prepare solution A; Step b: Under light-protected stirring conditions, inject solution A obtained in step S1 into water to obtain solution B; Step c: Place solution B in a dark environment at 45°C and remove ethanol by rotary evaporation to obtain tripterygium wilfordii self-assembled nanoparticles.

10. The use of triptolide-loaded nanoparticles as described in any one of claims 1-6 in the preparation of a drug for the prevention and treatment of metabolism-related steatohepatitis.

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