Application of drug-loaded composite nanoparticles MSN-Ber in preparation of drugs for treating hepatic fibrosis

Abstract

The invention discloses application of a drug-loaded composite nanoparticle MSN-Ber in preparation of a drug for treating hepatic fibrosis, and belongs to the field of new application of drugs. A carbon tetrachloride induced mouse hepatic fibrosis model is adopted to prove that MSN-Ber can inhibit generation of collagenous fibers and has a good treatment effect on hepatic fibrosis, and the treatment effect is related to synthesis of lipid in lipid metabolism and a PPAR signal channel. The invention provides a basis for taking the MSN-Ber as a novel medicine for treating hepatic fibrosis.

Description

Technical Field

[0001] The present invention relates to the application of MSN-Ber in the preparation of a drug for treating liver fibrosis, and belongs to the field of new drug uses. Background Art

[0002] The liver is one of the most functionally complex organs in the human body, playing a core role in maintaining life activities. It regulates the synthesis and decomposition of carbohydrates, fats, and proteins through an efficient metabolic network. For example, it converts glucose into glycogen for storage or releases it again to stabilize blood sugar, synthesizes cholesterol and lipoproteins to participate in lipid transport, and at the same time converts amino acids into urea for excretion. It plays an inestimable role in maintaining the stability and balance of the internal environment of the body.

[0003] Liver fibrosis is an important global health problem, characterized by excessive deposition of extracellular matrix and disorder of liver structure. There are many causes of liver fibrosis, such as viral hepatitis, alcoholic liver disease, fatty liver disease, and autoimmune diseases, etc. If not treated in time, it will eventually develop to the end stage of liver diseases such as liver cirrhosis and hepatocellular carcinoma, accompanied by a relatively high incidence and mortality rate. Since there are currently no drugs for liver fibrosis or liver cirrhosis approved by the US Food and Drug Administration in clinical practice. Therefore, it is extremely important to develop drugs with good therapeutic effects against liver fibrosis.

[0004] Berberine is an isoquinoline alkaloid derived from medicinal plants such as Coptis chinensis, with a wide range of pharmacological activities. It has good therapeutic effects in aspects such as antibacterial, anti-inflammatory, and anti-tumor, and can indirectly exert multiple health effects by regulating the intestinal flora structure. Although its clinical application is limited by the problem of low oral bioavailability, its multi-target action characteristics have continuously attracted attention in the fields of infectious diseases, metabolic syndrome, and tumor prevention and treatment.

[0005] Mesoporous silica nanoparticles (MSN) are biodegradable particles, with characteristics such as simple synthesis, controllable structure, chemical tailoring, and biocompatibility. They can target and release drugs on demand, and have good clinical application prospects. Mesoporous silica nanoparticles loaded with berberine effectively improve the oral bioavailability of berberine, so as to better exert its pharmacological effects. The previous research results of our laboratory showed that compared with pure berberine hydrochloride, berberine loaded on nanoparticles had better therapeutic effects in bleomycin-induced pulmonary fibrosis in mice, but there was no relevant research on MSN-Ber in the treatment of liver fibrosis. Summary of the Invention

[0006] As a traditional Chinese medicine, berberine is widely used due to its multiple pharmacological effects, low toxicity and side effects, numerous targets, and low price. Berberine has good curative effects in many chronic inflammatory diseases and also has a therapeutic effect on liver fibrosis. Mesoporous silica nanoparticles (MSN) are good nanoplatforms with degradability and biocompatibility in vivo. Loading berberine on them can effectively improve the oral absorption rate and bioavailability of berberine, enabling berberine to better exert its anti-inflammatory and anti-fibrotic effects. MSN-Ber has been proven to have good therapeutic effects and biological safety in a bleomycin-induced pulmonary fibrosis model. Therefore, this study will provide a scientific experimental basis for the application of MSN-Ber in the treatment of liver fibrosis.

[0007] The technical solution of the present invention:

[0008] The application of the drug-loaded composite nanoparticles MSN-Ber in the preparation of a drug for treating liver fibrosis, wherein the drug-loaded composite nanoparticles MSN-Ber are prepared by combining berberine and mesoporous silica nanoparticles into drug-loaded composite nanoparticles.

[0009] Further, in the drug-loaded composite nanoparticles MSN-Ber, the mass ratio of mesoporous silica nanoparticles to berberine is 1-1.2:1.8-2.2.

[0010] Further, the dosage of the drug-loaded composite nanoparticles MSN-Ber is: orally administered 8 mg / kg body weight per day.

[0011] Further, the application of the drug-loaded composite nanoparticles MSN-Ber in the preparation of a drug for relieving liver tissue damage caused by carbon tetrachloride-induced liver fibrosis.

[0012] Further, the application of the drug-loaded composite nanoparticles MSN-Ber in the preparation of a drug for reducing collagen deposition in liver tissue and treating liver fibrosis.

[0013] Further, the application of the drug-loaded composite nanoparticles MSN-Ber in the preparation of a drug for activating the PPAR signaling pathway and affecting lipid metabolism.

[0014] Further, the preparation method of the drug-loaded composite nanoparticles MSN-Ber is specifically as follows:

[0015] Step (1): Dissolve the surfactant in water, add an inorganic base solution, ultrasonicate, heat, and stir to react; dropwise add an organic solvent containing an organosilane source monomer, react, let stand, centrifuge to obtain a precipitate, disperse the precipitate in an organic solvent, ultrasonicate, centrifuge, and dry to obtain MSN;

[0016] Step (2): Dissolve the cycloalkane and surfactant in an organic solvent to obtain a mixture. First, add the berberine solution to the mixture to form a reverse microemulsion, and stir. After adding MSN, add acetone to demulsify, centrifuge, and dry.

[0017] Further, in step (1), the surfactant includes at least one of cetyltrimethylammonium bromide or cetyltrimethylammonium chloride; the inorganic base solution includes at least one of sodium hydroxide solution or ammonia water solution; the organosilicon source monomer includes at least one of tetraethyl orthosilicate or tetramethyl orthosilicate; the organic solvent in the organic solvent containing organosilicon monomer is ethanol; the organic solvent for dispersion precipitation includes at least one of methanol, ethanol, or propanol; the mass ratio of the surfactant to the inorganic base is 0.5 - 0.7:0.1 - 0.15; the concentration of the organosilicon source monomer in the organic solvent is 2.4 - 2.6 mol / L; the mass ratio of the inorganic base to the organosilicon source monomer is 0.095 - 0.12:3.5 - 4; the heating temperature is 75 - 85 °C, the stirring reaction time is 0.5 - 1 h; the reaction time is 1.5 - 3 h; the drying temperature is 30 - 40 °C, and the drying time is 6 - 12 h.

[0018] Further, in step (2), the cycloalkane is cyclohexane; the surfactant is polyethylene glycol octyl phenyl ether; the organic solvent is ethanol; the drying temperature is 30 - 40 °C, and the drying time is 12 - 24 h; the molar ratio of the cycloalkane, surfactant, and berberine is: 70 - 71:3 - 4:2 - 3; the concentration of the berberine solution is 0.1 - 0.12 mol / L; the mass ratio of MSN to berberine is 1 - 1.2:1.8 - 2.2.

[0019] Advantages of the present invention: The present invention uses a carbon tetrachloride-induced mouse liver fibrosis model to simulate liver fibrosis in clinical practice, and proves that MSN-Ber has a good therapeutic effect on liver fibrosis. The therapeutic effect of MSN-Ber on liver fibrosis acts by activating the PPAR signaling pathway and affecting lipid metabolism. The present invention provides a basis for using MSN-Ber as a new drug for the treatment of liver fibrosis. Description of the Drawings

[0020] Figure 1 Shows the therapeutic effect of MSN-Ber on liver fibrosis and the influence on liver function. Among them, A is the histological morphology diagram of the liver tissues of the control group, model group, and MSN-Ber group. B is the body weight-time curve and liver wet weight expression level of the mice in the control group, model group, and MSN-Ber group. Compared with the control group, *** P < 0.001; compared with the model group, ###P < 0.001. C represents the expression levels of ALT and AST in the sera of mice in the control group, model group, and MSN-Ber group. Compared with the control group, *** P < 0.001; compared with the model group, ### P < 0.001.

[0021] Figure 2 Pathological staining of the liver tissues of the liver fibrosis model was performed with MSN-Ber. Among them, A is the hematoxylin-eosin staining diagram of the liver tissues of the control group, model group, and MSN-Ber group. B is the Masson staining diagram of the control group, model group, and MSN-Ber group. C is the Sirius red staining diagram of the control group, model group, and MSN-Ber group. D is the immunofluorescence staining result of α-SMA in the liver tissues of the control group, model group, and MSN-Ber group.

[0022] Figure 3 Shows the effect of MSN-Ber on lipid metabolism in liver fibrosis. Among them, A is the Oil Red O staining of the liver tissues of the control group, model group, and MSN-Ber group. B is the mRNA expression level of PPARα in the liver tissues of the control group, model group, and MSN-Ber group. C is the mRNA expression level of PPARβ / δ in the liver tissues of the control group, model group, and MSN-Ber group. D is the mRNA expression level of PPARγ in the liver tissues of the control group, model group, and MSN-Ber group. Compared with the control group, ** P < 0.01, *** P < 0.001; compared with the model group, # P < 0.05, ## P < 0.01. Specific Embodiments

[0023] The following non-limiting examples can enable those of ordinary skill in the art to more comprehensively understand the present invention, but do not limit the present invention in any way.

[0024] Example 1

[0025] 1. Experimental Scheme:

[0026] 1.1 Synthesis of MSN-Ber

[0027] 1.1.1 Synthesis of Mesoporous Silica Nanoparticle (MSN) Carrier

[0028] Dissolve 0.535 g of cetyltrimethylammonium bromide (CTAB) in 240 mL of sterile ultrapure water and ultrasonically disperse for 15 min. Then add 1.25 mL of 2 mol / L (m = 0.1 g) sodium hydroxide solution and ultrasonically disperse for 5 min. Continuously stir the liquid at 80 °C for 30 min. Add 7 mL of an ethanol solution of tetraethyl orthosilicate (m = 3.645 g) with a concentration of 2.5 mol / L to the reaction solution at a rate of 4 drops / s. After reacting for 2 h, leave the liquid at room temperature for 30 min. The liquid is layered, and the lower-layer solution is taken and centrifuged at 10000 rpm for 3 min to obtain a precipitate. Then soak the precipitate in 200 mL of ethanol solution, followed by ultrasonic dispersion and centrifugation at 10000 rpm for 3 min. The above steps are repeated 3 times. Then, transfer the sample to a vacuum drying oven and dry at 37 °C for 10 h to obtain a purified MSN sample.

[0029] 1.1.2 Synthesis of berberine-loaded mesoporous silica nanoparticles (MSN-Ber)

[0030] MSN-Ber nanoparticles were prepared by the reverse microemulsion method. Dissolve 70.85 mmol of cyclohexane and 3.27 mmol of Triton X-100 (surfactant) in 1.6 mL of ethanol (m 环己烷 = 70.85 mmol * 84.16 g / mol = 5.96 g; m triton-100 = 3.27 mmol * 646.86 g / mol = 2.12 g). Add 21.8 mL of 0.1 mol / L (n 小檗碱 = 2.18 mmol, m 小檗碱 = 2.18 mmol * 235.32 g / mol = 0.513 g) berberine solution to the mixture and magnetically stir at room temperature for 5 min to form a reverse microemulsion. Add 256.5 mg of MSN and continuously stir at room temperature for 10 min. After the reaction is completed, add 2 mL of acetone to demulsify, collect the product, and centrifuge for 20 min to remove unreacted berberine and solvents. Dry at 37 °C for 12 h to obtain the final product MSN-Ber.

[0031] 1.2 Carbon tetrachloride-induced mouse liver fibrosis model

[0032] Thirty male C57BL / 6 mice (18 - 22 g) were raised according to the SPF-level animal breeding standards. After one week of adaptive feeding, the mice were subjected to the experiment. They were randomly divided into the following three groups: the control group, the model group, and the MSN-Ber group. The mice in the control group were not exposed to toxins and were intraperitoneally injected with an equal amount of normal saline as a control. The model group was modeled using a carbon tetrachloride solution. The high-purity carbon tetrachloride solution was diluted with corn oil to a purity of 10% and a concentration of 5 ml / kg, and was intraperitoneally injected into the mice three times a week. The MSN-Ber group was given an MSN-Ber solution at a concentration of 8 mg / kg by gavage three times a week while intraperitoneally injecting the above carbon tetrachloride solution. The treatment continued for 8 weeks. After 8 weeks, the models were collected, the mice were weighed, blood was taken, and the mice were sacrificed. The livers of the mice were collected, washed with normal saline, and weighed and recorded. The therapeutic effect of MSN-Ber on fibrosis was preliminarily judged by observing the liver morphology of the mice, the body weight-time curve, the wet weight of the liver, and the liver function indicators ALT and AST. Hematoxylin-eosin staining, Masson staining, and Sirius red staining were used to judge the damage of liver tissue, the formation of collagen fibers, and the effectiveness of MSN-Ber treatment; α-SMA immunofluorescence staining was used to judge the degree of liver fibrosis and the effect of MSN-Ber on fibrosis formation. The therapeutic mechanism of MSN-Ber on liver fibrosis was obtained using RNA transcriptome sequencing results. Oil red O staining was used to observe the expression of lipids in liver tissue, and Q-PCR was used to detect the mRNA expression levels of PPARα, PPARβ / δ, and PPARγ in liver tissue.

[0033] 2. Experimental Results

[0034] 2.1 Effects of MSN-Ber on Liver Tissue and Function in Mouse Liver Fibrosis

[0035] Observing the surface morphology of the mouse liver, it was found that Figure 1 in A, the surface of the liver tissue in the control group was smooth and the color was bright. The surface of the liver in the model group was swollen, with a rough, granular, and nodular appearance, the color was deepened and unevenly distributed. Compared with the model group, the liver color in the MSN-Ber administration group was bright, and the rough and granular morphology was significantly reduced. Figure 1 in B, compared with the control group, there was no significant change in the body weight of the mice in the MSN-Ber administration group after administration, indicating that MSN-Ber did not have an adverse effect on the physiological state of the mice. The wet weight of the liver is a basic index for evaluating the change of liver status. Compared with the significant increase in the wet weight of the liver in the model group, the wet weight of the liver in the MSN-Ber administration group gradually tended to be normal after administration. Figure 1 in C, ALT and AST are key indicators for evaluating liver function. The two liver function indicators in the model group increased significantly; the indicators in the MSN-Ber administration group gradually decreased after administration, proving that MSN-Ber reduced the abnormal expression of liver function.

[0036] 2.2 Effects of MSN-Ber on liver tissue injury and fibrosis in mice with liver fibrosis

[0037] The results of hematoxylin-eosin staining showed that, as shown in Figure 2 A in it, the hepatocytes in the liver tissue of the control group were arranged neatly, showing a cord-like structure, and the hepatic lobule structure was clear. However, the hepatocytes in the model group showed swelling and were accompanied by inflammatory cell infiltration, and the hepatic lobule structure was damaged. Compared with the model group, the number of inflammatory cells in the MSN-Ber administration group decreased, and the hepatic lobule structure gradually returned to normal, indicating that MSN-Ber reduced the damage of liver tissue. The increase in collagen synthesis is one of the early signs of fibrosis. Figure 2 As shown in B-C in it, the results of Masson staining and Sirius red staining showed that the liver tissue morphology of the control group was normal, and blue and red collagen were hardly visible; while the liver tissue structure in the model group changed significantly, and a large amount of blue and red collagen fibers were generated and deposited, forming fibrous septa; after administration of the MSN-Ber administration group, the colors of blue and red collagen were significantly lightened and the area decreased, and the liver tissue morphology gradually returned to normal. In fibrotic diseases, α-SMA is an important marker for evaluating the degree of fibrosis. As Figure 2 shown in D in it: The results of α-SMA immunofluorescence staining showed that the fluorescence expression of α-SMA in the model group was more than that in the control group, the number of positive cells increased, and the intensity of green fluorescence staining increased, indicating that the expression of α-SMA began to increase in the liver tissue with liver fibrosis. After administration of the MSN-Ber administration group, the number of positive cells decreased, the intensity of green fluorescence was significantly weakened and the distribution range shrank. The immunofluorescence results indicated that MSN-Ber could slow down the degree of liver fibrosis.

[0038] Therefore, MSN-Ber can improve liver function indicators, relieve the pathological degree of liver fibrosis, reduce the generation of collagen fibers, and has a good therapeutic effect on liver fibrosis.

[0039] 2.3 MSN-Ber affects lipid metabolism in the treatment of liver fibrosis

[0040] Lipid metabolism plays an important role in liver fibrosis. Oil red O staining can detect the presence and quantity of fat droplets in tissues and is applicable to the study of lipid metabolism diseases. The results are as Figure 3As shown in A of [Figure 0], no obvious red oil droplets were seen in the liver tissues of the control group; while a large number of red lipid droplets appeared in the model group, with a darker color and wide distribution, indicating a significant increase in lipid content in the livers of liver fibrosis model mice, suggesting abnormal lipid metabolism; after administration of the MSN-Ber administration group, the color of the red lipid droplets became significantly lighter and the distribution range decreased significantly, indicating that MSN-Ber can effectively reduce the abnormally accumulated lipids in the liver. The PPARs family has become a key therapeutic target for diseases such as metabolic diseases and cancers by regulating metabolism. The expression levels of PPARα, PPARβ / δ, and PPARγ in the PPARs family are as Figure 3 shown in B-D of [Figure 0]. Compared with the control group, the expressions of PPARα, PPARβ / δ, and PPARγ were significantly decreased in the model group, weakening the anti-fibrotic function; after administration of the MSN-Ber administration group, the expression levels of PPARα, PPARβ / δ, and PPARγ increased.

[0041] The above research results indicate that MSN-Ber can reduce collagen deposition and affect lipid metabolism by activating the PPAR signaling pathway.

[0042] In summary, MSN-Ber can significantly relieve liver tissue damage and reduce collagen deposition in liver fibrosis, and can effectively treat liver fibrosis. The treatment of liver fibrosis by MSN-Ber is achieved by activating the PPAR signaling pathway and then affecting lipid metabolism. MSN-Ber can be used as a candidate drug for the treatment of liver fibrosis.

[0043] The above embodiments are only used for exemplifying and illustrating the present invention, and are not intended to limit the present invention to the scope of the described embodiments. In addition, those skilled in the art can understand that the present invention is not limited to the above embodiments, and more variations and modifications can be made according to the teachings of the present invention, and these variations and modifications all fall within the scope claimed by the present invention.

Claims

1. Application of drug-loaded composite nanoparticles MSN-Ber in the preparation of drugs for treating liver fibrosis, characterized in that: The drug-loaded composite nanoparticles MSN-Ber are prepared by combining berberine and mesoporous silica nanoparticles into drug-loaded composite nanoparticles.

2. The use according to claim 1, characterized in that: In the drug-loaded composite nanoparticles MSN-Ber, the mass ratio of mesoporous silica nanoparticles to berberine is 1-1.2:1.8-2.

2.

3. The use according to claim 1, characterized in that: The dosage of the drug-loaded composite nanoparticles MSN-Ber is: 8 mg / kg body weight per day orally.

4. The use according to claim 1, characterized in that: The drug-loaded composite nanoparticle MSN-Ber is used in preparing a drug for alleviating liver tissue damage caused by carbon tetrachloride-induced liver fibrosis.

5. The use according to claim 1, characterized in that: The drug-loaded composite nanoparticle MSN-Ber is used in a drug for reducing liver tissue collagen deposition and treating liver fibrosis.

6. The use according to claim 1, characterized in that: The drug-loaded composite nanoparticle MSN-Ber is used in drugs that activate PPAR signaling pathways and affect lipid metabolism.

7. The use according to claim 1, characterized in that: The preparation method of drug-loaded composite nanoparticles MSN-Ber is specifically as follows: Step (1): dissolving a surfactant in water, adding an inorganic alkali solution, ultrasonicating, heating, and stirring to react; dropping an organic solvent containing an organosilicon source monomer, reacting, standing, centrifuging to obtain a precipitate, dispersing the precipitate in an organic solvent, ultrasonicating, centrifuging, and drying to obtain MSN; Step (2): dissolving cycloalkane and surfactant in an organic solvent to obtain a mixed solution, first adding berberine solution to the mixed solution to form a reverse microemulsion, and stirring; After adding MSN, acetone was added to demulsify, centrifuged and dried.

8. The use according to claim 7, characterized in that: In step (1), the surfactant comprises at least one of hexadecyltrimethylammonium bromide and hexadecyltrimethylammonium chloride; the inorganic alkali solution comprises at least one of sodium hydroxide solution and ammonia solution; the organosilicon source monomer comprises at least one of ethyl orthosilicate and methyl orthosilicate; the organic solvent in the organic solvent containing the organosilicon monomer is ethanol; the organic solvent for the dispersed precipitation comprises at least one of methanol, ethanol or propanol; the mass ratio of the surfactant to the inorganic alkali is 0.5-0.7:0.1-0.15; the concentration of the organosilicon source monomer in the organic solvent is 2.4-2.6 mol / L; the mass ratio of the inorganic alkali to the organosilicon source monomer is 0.095-0.12:3.5-4; the heating temperature is 75-85°C, the stirring reaction time is 0.5-1h; the reaction time is 1.5-3h; the drying temperature is 30-40°C, and the drying time is 6-12h.

9. The use according to claim 7, characterized in that: In step (2), the cycloalkane is cyclohexane; the surfactant is polyethylene glycol octylphenyl ether; the organic solvent is ethanol; the drying temperature is 30-40°C, and the drying time is 12-24h; the molar ratio of the cycloalkane, the surfactant, and the berberine is: 70-71:3-4:2-3; the concentration of the berberine solution is 0.1-0.12 mol / L; the mass ratio of the MSN to the berberine is 1-1.2:1.8-2.2.

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