Heparin-modified emodin nano-drug for treating acute pancreatitis as well as preparation method and application of heparin-modified emodin nano-drug

Abstract

The invention discloses a heparin-modified emodin nano-drug for treating acute pancreatitis and a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations, the preparation method comprises the following steps: preparing a mixed solution containing 2-methylimidazole and emodin, dropwise adding a zinc source solution into the mixed solution, stirring for reaction, filtering, washing, and drying to obtain the heparin-modified emodin nano-drug for treating acute pancreatitis. And centrifuging and washing the reaction solution to obtain emodin-loaded ZIF-8 nanoparticles, mixing and stirring the aqueous dispersion of the emodin-loaded ZIF-8 nanoparticles and a heparin aqueous solution, centrifuging, and washing to obtain the heparin-modified emodin nano-drug for treating acute pancreatitis. The heparin-modified emodin nano-drug for treating acute pancreatitis can specifically target inflammatory pancreatic tissues, release emodin in an acidic inflammatory microenvironment to recover mitochondrial membrane potential damaged by AP, relieve oxidative stress and significantly reduce the level of inflammatory cytokines.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a heparin-modified rheumatoid arthritis nanomedicine and a preparation method and application thereof. Background Art

[0002] Acute pancreatitis (AP) is a common acute abdominal inflammatory disease, characterized by pancreatic self-digestion, acinar cell necrosis, and possible systemic inflammatory response. It is characterized by rapid onset, complex and variable disease, and common causes include biliary disease, alcohol abuse, hyperlipidemia, etc. Despite its great clinical significance, there is currently no way to completely cure AP. Commonly used drugs in clinical practice include analgesics, antibiotics, and pancreatic enzyme inhibitors, etc. Therefore, there is an urgent need to develop new effective drugs for the treatment of acute pancreatitis.

[0003] In recent years, the application research of nano drugs in the treatment of acute pancreatitis has made significant progress. Researchers have been committed to solving the problems of poor water solubility, poor targeting, and fast metabolism of traditional drugs by designing multifunctional nano carriers. For example, the Chinese patent document with publication number CN110063945A discloses a bilirubin nanoparticle for the treatment of acute pancreatitis and a preparation method thereof. The invention dissolves bilirubin, genipin and ceramide in a mixed organic reagent, and then dissolves silk fibroin in an aqueous solution, and then slowly drips the mixed solution of bilirubin, genipin and ceramide into the silk fibroin solution, continuously stirs, dialyzes to remove the organic solvent, and dilutes the obtained nanoparticle suspension with pure water to obtain bilirubin nanoparticles. The bilirubin nanoparticles are effective in treating acute pancreatitis and have no toxic side effects. The Chinese patent document with the publication number CN119345380A discloses a natural product-based nano antioxidant for the treatment of acute pancreatitis and a preparation method thereof. The invention dissolves zein / and ferulic acid FA in an ethanol-deionized water solution to obtain a zein / FA mixed solution, and the zein / FA mixed solution is mixed with a chondroitin sulfate CS solution for dialysis to obtain a natural product-based nano antioxidant FA@Zein-CS, which can significantly reduce pancreatic structural damage and restore pancreatic function. The Chinese patent document with the publication number CN111700909A discloses the use of Prussian blue in the preparation of a drug for the treatment of acute pancreatitis. The invention uses an iron source and an organic substance with a reducing and stabilizing effect to prepare Prussian blue or its analog nanoparticles. Prussian blue or its analog nanoparticles can simultaneously reduce inflammation and inhibit the TLRs / NFkB signaling pathway, so that the microenvironment of acute pancreatitis gradually returns to the microenvironment of a normal pancreas, thereby achieving the purpose of treating pancreatitis.

[0004] Emodin is a natural anthraquinone compound found in traditional Chinese medicines such as rhubarb and knotweed. It has significant anti-inflammatory and antioxidant properties and has shown the potential to reduce the severity of acute pancreatitis. However, its clinical application is limited by poor water solubility, short half-life, poor targeting ability and insufficient pancreatic retention. Combining nanotechnology or structural modification will help promote the clinical transformation of emodin drugs. Summary of the invention

[0005] The present invention provides a heparin-modified rheumatoid arthritis nanomedicine for treating acute pancreatitis and a preparation method thereof, which can specifically target inflamed pancreatic tissue, release rheumatoid arthritis in an acidic inflammatory microenvironment to restore mitochondrial membrane potential damaged by AP, alleviate oxidative stress and significantly reduce the level of inflammatory cytokines.

[0006] The specific technical solutions adopted are as follows: A method for preparing a heparin-modified rheumatoid arthritis nanomedicine for treating acute pancreatitis comprises the following steps: A mixed solution of 2-methylimidazole and rheum is prepared, a zinc source solution is added dropwise into the mixed solution, the mixture is stirred for reaction, the reaction solution is centrifuged and washed to obtain rheum-loaded ZIF-8 nanoparticles, an aqueous dispersion of the rheum-loaded ZIF-8 nanoparticles is mixed and stirred with a heparin aqueous solution, and the heparin-modified rheum nanomedicine for treating acute pancreatitis is obtained after centrifugation and washing.

[0007] The present invention has developed a heparin-modified emollient nanodrug EMO@ZIF-8 / Heparin (HEZ), which aims to specifically target inflammatory pancreatic tissue by recognizing CD44 receptors overexpressed on activated macrophages. In an in vitro AP cell model, the nanodrug HEZ was enhanced by macrophages in the inflammatory microenvironment, restored the mitochondrial membrane potential damaged by AP, alleviated oxidative stress, and significantly reduced the levels of key cytokines including amylase, TNF-α, IL-6 and IL-1β. In addition, through the mouse AP model verification, the nanodrug HEZ showed the effect of targeting pancreatic tissue and was enriched in pancreatic tissue, which effectively interrupted the further development of systemic inflammation, reduced pancreatic and lung tissue pathological damage, reduced pancreatic cell apoptosis, and improved the 15-day survival rate of AP mice. Mechanism studies have shown that the nanodrug HEZ restored the mitochondrial function of macrophages by inhibiting the JNK pathway. In short, this multifunctional nanodrug HEZ provides an upstream treatment strategy by targeting macrophages, providing an effective method for improving the treatment of AP.

[0008] Preferably, in the mixed solution of the components including 2-methylimidazole and rheumatoid artemisia, the concentration of 2-methylimidazole is 0.1-1 mmol / mL, and the concentration of rheumatoid artemisia is 0.01-0.1 mmol / mL.

[0009] Preferably, the zinc source in the zinc source solution is Zn(NO3)2·6H2O, and the concentration of the zinc source in the zinc source solution is 0.1-1 mmol / mL.

[0010] Further preferably, the components include a mixed solution of 2-methylimidazole and rhamnosine and a zinc source solution, and the solvent is methanol.

[0011] Preferably, when preparing ZIF-8 nanoparticles loaded with rhamnosin, the reaction is stirred at 0-4°C for 0.5-1.5 h. Too high a temperature may result in unsuccessful loading.

[0012] Preferably, when the aqueous dispersion of the ZIF-8 nanoparticles loaded with rheum-emodin and the aqueous heparin solution are mixed and stirred, the mass ratio of the ZIF-8 nanoparticles loaded with rheum-emodin to the heparin is 1:1-10.

[0013] More preferably, the molecular weight of heparin is 4000-6000 Da, and the concentration of the heparin aqueous solution is 5-15 mg / mL.

[0014] The present invention also provides the heparin-modified emodin nano-drug for treating acute pancreatitis prepared by the preparation method of the heparin-modified emodin nano-drug for treating acute pancreatitis.

[0015] The heparin-modified rheumatoid arthritis nanoparticles have a particle size range of 100-1000 nm and include rheumatoid arthritis-loaded ZIF-8 nanoparticles and heparin modified on the surface thereof.

[0016] The present invention also provides a drug for treating acute pancreatitis, comprising the heparin-modified emodin nano drug for treating acute pancreatitis.

[0017] Preferably, the dosage of the heparin-modified emodin nanodrug for treating acute pancreatitis is 0.5-1.5 mg / kg.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) Heparin modification enables the emodin nanodrug to target macrophages. The heparin-modified emodin nanodrug for the treatment of acute pancreatitis has a high anti-inflammatory effect and low toxicity to normal tissues.

[0019] (2) Rheum officinale is highly hydrophobic. The present invention uses ZIF-8 nanoparticles to carry Rheum officinale molecules, which greatly improves the solubility of Rheum officinale in water, making its in vivo application possible.

[0020] (3) Since the emodin nanodrug is modified with heparin, it can specifically target the inflamed pancreatic tissue by recognizing the CD44 receptor overexpressed on activated macrophages. In the acidic inflammatory microenvironment, acid-induced degradation of ZIF-8 will gradually release the loaded emodin, thereby restoring the mitochondrial membrane potential damaged by AP, reducing oxidative stress, and significantly reducing the levels of key cytokines including amylase, TNF-α, IL-6 and IL-1β, effectively interrupting the further development of systemic inflammation. It has good application prospects in the treatment of acute pancreatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Transmission electron microscopy images of ZIF-8 nanoparticles, ZIF-8 nanoparticles EZ loaded with emodin in Example 1, and heparin-modified emodin nanomedicine HEZ for treating acute pancreatitis.

[0022] Figure 2 The graphs show the particle size measurements of ZIF-8 nanoparticles, ZIF-8 nanoparticles EZ loaded with emodin in Example 1, and heparin-modified emodin nanomedicine HEZ for treating acute pancreatitis.

[0023] Figure 3 These are potential measurement diagrams of ZIF-8 nanoparticles, ZIF-8 nanoparticles EZ loaded with emodin in Example 1, and heparin-modified emodin nanomedicine HEZ for treating acute pancreatitis.

[0024] Figure 4 This is a graph showing the levels of inflammatory factors in the blood of mice after administration of the heparin-modified emodin nanodrug HEZ for the treatment of acute pancreatitis in Example 1. ** indicates p <0.01 ， ***express p <0.001, discrete points represent data values ​​from parallel experiments.

[0025] Figure 5 The pancreatic tissue section staining image (scale is 100 μm) and the scoring image of mice after administration of the heparin-modified emodin nanodrug HEZ for treating acute pancreatitis in Example 1 ， **express p <0.01 ， ****express p <0.0001, discrete points represent data values ​​from parallel experiments. DETAILED DESCRIPTION

[0026] Example 1 (1) Preparation of heparin-modified rheumatoid arthritis nanoparticles 2-Methylimidazole (3960 mg, 48.30 mmol) was completely dissolved in 120 mL of methanol solution, and then rheum officinale (240 mg, 0.89 mmol) was added and stirred for 15 min to obtain a mixed solution containing 2-methylimidazole and rheum officinale. Next, Zn(NO3)2·6H2O (1800 mg, 6.04 mmol) was dissolved in 60 mL of methanol solution to obtain a zinc source solution, which was added dropwise to the mixed solution, stirred at 4°C for 60 min, and the reaction solution was centrifuged at 14000 rpm / min for 30 min to obtain a crude product, which was washed with methanol three times, and centrifuged at 14000 rpm / min for 30 min each time to finally obtain rheum officinale loaded ZIF-8 nanoparticles EZ. The aqueous dispersion of ZIF-8 nanoparticles loaded with emodin (10 mg / mL, 10 mL) was added to an aqueous heparin solution (molecular weight of heparin is 4000-6000 Da, 10 mg / mL, 10 mL) and further stirred at room temperature for 4 h. Then, the solid product was collected by centrifugation and washed three times with methanol and deionized water to finally obtain the heparin-modified emodin nanodrug HEZ for the treatment of acute pancreatitis.

[0027] Example 2

[0028] Preparation of heparin-modified rheumatoid artemisia nanodrug: 2-methylimidazole (3960 mg, 48.30 mmol) was completely dissolved in 120 mL of methanol solution, and then rheumatoid artemisia (240 mg, 0.89 mmol) was added and stirred for 15 min to obtain a mixed solution containing 2-methylimidazole and rheumatoid artemisia. Next, Zn(NO3)2·6H2O (1800 mg, 6.04 mmol) was dissolved in 60 mL of methanol solution to obtain a zinc source solution, which was added dropwise to the mixed solution, stirred at 4°C for 60 min, and the reaction solution was centrifuged at 14000 rpm / min for 30 min to obtain a crude product, which was washed with methanol three times, and centrifuged at 14000 rpm / min for 30 min each time to finally obtain rheumatoid artemisia ZIF-8 nanoparticles EZ. The aqueous dispersion of ZIF-8 nanoparticles loaded with emodin (10 mg / mL, 10 mL) was added to an aqueous heparin solution (molecular weight of heparin is 4000-6000 Da, 10 mg / mL, 50 mL) and further stirred at room temperature for 4 h. Then, the solid product was collected by centrifugation and washed three times with methanol and deionized water to finally obtain the heparin-modified emodin nanodrug HEZ for the treatment of acute pancreatitis.

[0029] Example 3

[0030] Preparation of heparin-modified rheumatoid artemisia nanodrug: 2-methylimidazole (3960 mg, 48.30 mmol) was completely dissolved in 120 mL of methanol solution, and then rheumatoid artemisia (240 mg, 0.89 mmol) was added and stirred for 15 min to obtain a mixed solution containing 2-methylimidazole and rheumatoid artemisia. Next, Zn(NO3)2·6H2O (1800 mg, 6.04 mmol) was dissolved in 60 mL of methanol solution to obtain a zinc source solution, which was added dropwise to the mixed solution, stirred at 4°C for 60 min, and the reaction solution was centrifuged at 14000 rpm / min for 30 min to obtain a crude product, which was washed with methanol three times, and centrifuged at 14000 rpm / min for 30 min each time to finally obtain rheumatoid artemisia ZIF-8 nanoparticles EZ. The aqueous dispersion of ZIF-8 nanoparticles loaded with emodin (10 mg / mL, 10 mL) was added to an aqueous heparin solution (molecular weight of heparin is 4000-6000 Da, 10 mg / mL, 20 mL) and further stirred at room temperature for 4 h. Then, the solid product was collected by centrifugation and washed three times with methanol and deionized water to finally obtain the heparin-modified emodin nanodrug HEZ for the treatment of acute pancreatitis.

[0031] Sample analysis Figure 1-Figure 3 The transmission electron microscope images, particle size images and potential images of ZIF-8 nanoparticles, ZIF-8 nanoparticles EZ loaded with rheum in Example 1 and heparin-modified rheum nanodrug HEZ for treating acute pancreatitis are shown. The results show that the particle size range of HEZ is 100-1000 nm, and its structure includes ZIF-8 nanoparticles loaded with rheum and heparin modified on its surface. The zeta potential of HEZ is negative and it carries a negative charge.

[0032] Efficacy verification: In the cholecystokinin-induced acute pancreatitis model, mice were intraperitoneally injected with cholecystokinin (50 μg / kg / h), once every hour, for seven consecutive injections, and the last injection was co-administered with lipopolysaccharide (LPS; 10 mg / kg; Solebol). The mice were killed 12 hours after the first injection, and blood and tissue samples were collected. The mice were divided into 5 experimental groups (control group, model group, blank vector group, emodin group and nanodrug group). The control group was a healthy control, and the model group, blank vector group, emodin group and nanodrug group were treated by tail vein injection of PBS, blank vector ZIF8 nanoparticles, free emodin drug, and heparin-modified emodin nanodrug HEZ for the treatment of acute pancreatitis in Example 1. The blood of the mice was then collected to detect the levels of related inflammatory factors ( Figure 4 ), and pancreatic tissue was isolated for histological evaluation ( Figure 5 , where the scale bar is 100 μm). The results showed that heparin-modified rhubarb nanodrug HEZ could significantly reduce the levels of key cytokines including amylase Amyalse, TNF-α, lipase and IL-1β, and had an anti-pancreatitis effect.

[0033] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a heparin-modified rheumatoid arthritis nanomedicine for treating acute pancreatitis, characterized in that: The following steps are involved: A mixed solution of 2-methylimidazole and rheum is prepared, a zinc source solution is added dropwise into the mixed solution, the mixture is stirred for reaction, the reaction solution is centrifuged and washed to obtain rheum-loaded ZIF-8 nanoparticles, an aqueous dispersion of the rheum-loaded ZIF-8 nanoparticles is mixed and stirred with a heparin aqueous solution, and the heparin-modified rheum nanomedicine for treating acute pancreatitis is obtained after centrifugation and washing.

2. The method for preparing the heparin-modified rheumatoid arthritis nanomedicine for treating acute pancreatitis according to claim 1, characterized in that: In the mixed solution of components including 2-methylimidazole and rheum officinale, the concentration of 2-methylimidazole is 0.1-1 mmol / mL, and the concentration of rheum officinale is 0.001-0.01 mmol / mL.

3. The method for preparing the heparin-modified rheumatoid arthritis nanomedicine for treating acute pancreatitis according to claim 1, characterized in that: The zinc source in the zinc source solution is Zn(NO3)2·6H2O, and the concentration of the zinc source in the zinc source solution is 0.1-1 mmol / mL.

4. The method for preparing the heparin-modified rheumatoid arthritis nanomedicine for treating acute pancreatitis according to claim 1, characterized in that: The components include a mixed solution of 2-methylimidazole and rhamnosine and a zinc source solution, and the solvent is methanol.

5. The method for preparing the heparin-modified rheumatoid arthritis nanomedicine for treating acute pancreatitis according to claim 1, characterized in that: When preparing ZIF-8 nanoparticles loaded with rhamnosine, the reaction was stirred at 0-4 °C for 0.5-1.5 h.

6. The method for preparing the heparin-modified rheumatoid arthritis nanomedicine for treating acute pancreatitis according to claim 1, characterized in that: When the aqueous dispersion of the ZIF-8 nanoparticles loaded with rheum-emodin is mixed and stirred with the heparin aqueous solution, the mass ratio of the ZIF-8 nanoparticles loaded with rheum-emodin to the heparin is 1:1-10.

7. The method for preparing the heparin-modified rheumatoid arthritis nanomedicine for treating acute pancreatitis according to claim 1, characterized in that: The molecular weight of heparin is 4000-6000 Da, and the concentration of heparin aqueous solution is 5-15 mg / mL.

8. The heparin-modified emodin nanodrug for treating acute pancreatitis obtained according to the preparation method of the heparin-modified emodin nanodrug for treating acute pancreatitis according to any one of claims 1 to 7.

9. The heparin-modified rhamnosine nanodrug for treating acute pancreatitis according to claim 8, characterized in that: The heparin-modified rheumatoid arthritis nanoparticles have a particle size range of 100-1000 nm and include rheumatoid arthritis-loaded ZIF-8 nanoparticles and heparin modified on their surfaces.

10. A drug for treating acute pancreatitis, comprising the heparin-modified emodin nanomedicine for treating acute pancreatitis according to claim 8 or 9.

Images