Preparation and application of hepatic stellate cell targeted nano-drug delivery system

Abstract

The invention discloses preparation and application of a hepatic stellate cell targeted nano-drug delivery system, and belongs to the technical field of medicines, and the nano-drug delivery system is composed of a nano-carrier with enzyme-like activity, an antioxidant drug and a hepatic stellate cell targeted ligand. Liver fibrosis is a dynamic development process, hepatic stellate (HSC-T6) cells in a resting state are activated under the stimulation of a large amount of active oxygen generated by oxidative stress, so that the balance between synthesis and degradation of an extracellular matrix (ECM) is destroyed, a large amount of ECM is deposited, and finally the hepatic fibrosis is caused. Therefore, mesoporous polydopamine with SOD-like enzyme and CAT-like enzyme is selected as a carrier, the anti-oxidation drug curcumin is loaded, and the surface of the anti-oxidation drug curcumin is coated with hepatic stellate cell targeting ligand hyaluronic acid, so that the nano drug delivery system Cur / CMPH is formed. The nano-drug delivery system can remove active oxygen and inhibit activation of hepatic stellate cells, thereby inhibiting development of hepatic fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the preparation of a nanodelivery system with targeting function of hepatic stellate cells, and the application of the nanodelivery system in inhibiting the development of liver fibrosis. Background Technology

[0002] The liver is the largest metabolic organ in the human body, responsible for vital functions such as bile synthesis, fat regulation, glycogen metabolism, and amino acid synthesis, playing an irreplaceable role in the body's metabolism. Liver fibrosis is a dynamic process, initially manifesting as liver inflammation. Under repeated inflammatory stimulation, quiescent hepatic stellate cells (HSC-T6) are activated, disrupting the balance between extracellular matrix (ECM) synthesis and degradation, leading to massive ECM deposition and ultimately liver fibrosis. The manifestation of liver fibrosis is highly regulated by various cells and cytokines, and the activation of HSC-T6 cells is a key step in its development. HSC-T6 cells are hepatic mesenchymal cells. Under normal physiological conditions, HSC-T6 cells are in a quiescent state. When the liver is injured, HSC-T6 cells are activated by inflammatory mediators, differentiating into myofibroblasts and inducing the secretion of extracellular matrix proteins, thereby initiating the liver tissue remodeling process.

[0003] Currently, one key approach to treating liver fibrosis is to block the activation and transformation of hepatic stellate cells to prevent ECM deposition. Given that liver fibrosis is characterized by over-differentiation of cluster 44 (CD44), its receptor overexpression in activated hepatic stellate cells allows for maximal drug delivery to target cells without harming healthy cells through binding to ligands such as hyaluronic acid and chondroitin sulfate. The second key approach is to reduce reactive oxygen species (ROS)-mediated oxidative stress. Excessive ROS, such as superoxide anion (O2), can lead to oxidative stress. ·- Hydrogen peroxide (H2O2) and hydroxyl radicals (·OH) are closely related to the development of liver fibrosis. Reactive oxygen species (ROS) play an important role in amplifying inflammatory responses and stimulating the production of pro-fibrotic mediators. Elevated ROS levels can not only directly promote the development of liver fibrosis, but also interact with oxidative stress-induced inflammatory responses, thereby synergistically exacerbating liver fibrosis. Therefore, clearing ROS and reducing oxidative stress in liver tissue can effectively reverse liver fibrosis. However, traditional antioxidants lack specificity in clearing ROS and have drawbacks such as low efficacy and high toxicity. Developing a ROS-clearing drug with high liver-targeting ability to treat liver fibrosis is very challenging.

[0004] To date, there are no specific drugs for treating liver fibrosis in clinical practice. Therefore, it is urgent to develop strategies for treating liver fibrosis. Summary of the Invention

[0005] The purpose of this invention is to construct a hyaluronic acid-coated mesoporous polydopamine nanomedicine delivery system that possesses both excellent antioxidant and enzyme-like activities. By scavenging reactive oxygen species within hepatic stellate cells and inhibiting their activation, this system aims to suppress the development of liver fibrosis.

[0006] The technical solution adopted in this invention is as follows: The hepatic stellate cell-targeted nanomedicine delivery system of this invention consists of a nanocarrier with enzyme-like activity, an antioxidant drug, and a hepatic stellate cell-targeting ligand.

[0007] Preferably, the enzyme-like nanocarrier is formed by the oxidative polymerization of metal ions and dopamine, wherein the metal ion is Cu. 2+ Zn 2+ Fe 2+ Fe 3+ and Mn 2+ One of them.

[0008] Preferably, the antioxidant is one of curcumin, resveratrol, and quercetin.

[0009] Preferably, the hepatic stellate cell targeting ligand is one of hyaluronic acid (HA) and chondroitin sulfate.

[0010] The preparation method of the hepatic stellate cell-targeted nanodrug delivery system of the present invention includes the following steps:

[0011] (1) Preparation of metal-doped mesoporous polydopamine nanocarriers: Prönnick F127 was dissolved in a mixed solution of ethanol and water, and the mixture was ultrasonically dispersed with a mesitylene (TMB) probe. Tris solution, dopamine hydrochloride and metal ions were added in sequence. After stirring for a period of time, the prepared nanocarriers were dispersed in a mixed solvent of acetone and ethanol. After removing the template, the black solid obtained by centrifugation was the metal-doped mesoporous polydopamine nanocarriers.

[0012] (2) Preparation of drug-loaded nanocarriers: Antioxidant drugs were dissolved in ethanol solution and added dropwise to metal-doped mesoporous polydopamine nanocarriers. After reacting for a period of time, the drug-loaded nanoparticles were obtained by centrifugation.

[0013] (3) Preparation of hyaluronic acid-coated nanocarriers: Drug-loaded nanoparticles are uniformly dispersed in an aqueous solution, hepatic stellate cell targeting ligands are added, and after a period of reaction, the product obtained by centrifugation is the hepatic stellate cell-targeted nanodrug delivery system.

[0014] In the above preparation method, wherein:

[0015] Preferably, in step (1), the molar ratio of the metal ion to dopamine hydrochloride is 4:1 to 1:2, the mass ratio of Pranic F127 to TMB is 2:1 to 1:4, the reaction environment is alkaline with a pH of 8 to 10, the reaction temperature is 25-30℃, the volume ratio of water to ethanol is 1:1 to 1:2, and the reaction time is 1 to 6 hours; the metal ion is preferably Cu. 2+ .

[0016] Preferably, in step (2), the mass ratio of the antioxidant drug to the metal-doped mesoporous polydopamine nanocarrier is 4:1 to 2:1, the reaction time is 3 to 8 hours, the reaction temperature is 40 to 60 degrees Celsius, and the drug is Cur.

[0017] Preferably, in step (3), the mass ratio of the hepatic stellate cell targeting ligand to the drug-loaded nanocarrier is 1:2 to 2:1, the reaction time is 8 to 24 hours, and the hepatic stellate cell targeting ligand is preferably HA.

[0018] The specific preparation steps are as follows:

[0019] Preferably, step (1) is as follows: Pronic F127 is dissolved in a mixed solution of ethanol and water, a TMB probe is added and sonicated until it is completely dispersed and uniform, then Tris solution is added to adjust the pH, dopamine hydrochloride and CuCl2 solution are added, the reaction system is stirred in a 30°C water bath, and then centrifuged at 11000 rpm for 15 min to obtain the reaction precipitate. The reaction precipitate is washed three times with distilled water, and then dispersed in a mixed solution of ethanol and acetone (v / v = 2:1) and sonicated for 10 min. This operation is repeated three times, and centrifuged at 8000 rpm for 8 min to obtain a black solid, which is the copper-doped mesoporous polydopamine nanocarrier (CMP).

[0020] Preferably, step (2) specifically involves: uniformly dispersing CMP in ethanol, adding the ethanol dispersion of CMP to the ethanol solution of Cur and dispersing it evenly, sonicating in a water bath for 30 minutes, then magnetically stirring at 50°C for 4 hours, and centrifuging at 8000 rpm for 8 minutes to obtain a black solid, which is the drug-loaded nanoparticle Cur / CMP.

[0021] Preferably, step (3) specifically involves: dispersing Cur / CMP in an aqueous solution, adding HA solution dropwise, centrifuging at 11000 rpm for 15 min after the reaction is complete, and obtaining a solid Cur / CMP nanomedicine delivery system Cur / CMPH coated with HA.

[0022] This invention uses metal ion-doped mesoporous polydopamine nanoparticles as a carrier, loads the antioxidant drug Cur, and coats the carrier surface with the hepatic stellate cell targeting ligand HA to form a multifunctional nanomedicine delivery system for hepatic stellate cell targeting.

[0023] Among them, Cu is used 2+ Cu was used as a dopant ion to construct CMP nanocarriers with a particle size of approximately 200 nm. 2+ The doping of CMP nanocarriers endows them with excellent SOD-like and CAT-like activities, free radical scavenging capabilities, and in vivo degradation capacity. Furthermore, CMP nanocarriers, through their SOD-like and CAT-like properties, convert superoxide anion free radicals and hydrogen peroxide into water and oxygen, scavenging reactive oxygen species within hepatic stellate cells and reducing oxidative damage. Cur, as an antioxidant, can inhibit hepatic stellate cell activation by suppressing the TGF-β1 pathway and can also remove excess reactive oxygen species in the liver. HA, as a targeting ligand for hepatic stellate cells, can enhance the biosafety of the nanocarriers and increase their uptake by hepatic stellate cells by targeting the CD44 receptor.

[0024] This nanomedicine delivery system targets hepatic stellate cells by encapsulating hyaluronic acid (HA) to recognize and target these cells, which are overexpressed on the surface of stellate cells. The HA is then hydrolyzed by intracellular hyaluronidase, releasing the drug Cur, thereby scavenging reactive oxygen species (ROS) and inhibiting stellate cell activation. Once inside the stellate cells, the nanocarrier exhibits SOD-like and CAT-like enzyme activities, catalyzing the formation of water and oxygen from superoxide anions and hydrogen peroxide, thus scavenging ROS and reducing oxidative damage to the liver. By targeting stellate cells and scavenging ROS within them to inhibit stellate cell activation, this nanomedicine delivery system achieves a therapeutic effect in inhibiting the development of liver fibrosis.

[0025] The beneficial effects of this invention are:

[0026] (1) The CMP nanocarrier constructed in this invention removes reactive oxygen species such as superoxide anion and hydrogen peroxide through the activity of SOD-like enzyme and CAT-like enzyme, and has excellent free radical scavenging ability, which can effectively treat problems caused by oxidative stress in the body.

[0027] (2) This invention uses Cur as a model drug, which has excellent antioxidant capacity and other effects. Cur can inhibit the activation of hepatic stellate cells by inhibiting the TGF-β1 pathway, and because it is rich in phenolic hydroxyl groups, it can carry out redox reactions to remove excess reactive oxygen species in the liver.

[0028] (3) This invention uses mesoporous polydopamine, which has SOD-like and CAT-like enzymes, as a carrier to load the antioxidant drug curcumin and coat the surface with hyaluronic acid, a targeting ligand for hepatic stellate cells, to form the Cur / CMPH nanomedicine delivery system. This nanomedicine delivery system can scavenge reactive oxygen species and inhibit the activation of hepatic stellate cells, thereby inhibiting the development of liver fibrosis. Attached Figure Description

[0029] Figure 1 Transmission electron microscopy images of CMP prepared in Example 1 and CMPH prepared in Example 2.

[0030] Figure 2 The particle size distribution and zeta potential of CMP prepared in Example 1 and CMPH prepared in Example 2 are shown in Figure A, where particle size is shown and zeta potential is shown in Figure B.

[0031] Figure 3 The image shows the activity of SOD-like and CAT-like enzymes of CMP prepared in Example 1.

[0032] Figure 4 Differential calorimetry (DC) spectra of CMPH prepared in Example 2, active pharmaceutical ingredient Cur, Cur / CMPH prepared in Example 4, and physical mixtures of active pharmaceutical ingredient and blank carrier.

[0033] Figure 5 This document verifies the scavenging abilities of CMPH prepared in Example 2, the active pharmaceutical ingredient Cur, and the Cur / CMPH prepared in Example 4 against DPPH, ABTS radicals, and superoxide anions. Figure A shows the DPPH scavenging ability, Figure B shows the ABTS scavenging ability, and Figure C shows the superoxide anion scavenging ability.

[0034] Figure 6 The in vitro release curves for the drug-loaded system Cur / CMPH prepared in Example 4 are shown. These include releases under pH 7.4 PBS and pH 6.8 PBS conditions.

[0035] Figure 7 This study validates the ability of CMPH prepared in Example 2, Cur / CMP prepared in Example 3, and Cur / CMPH prepared in Example 4 to scavenge ROS at the cellular level.

[0036] Figure 8 The cytotoxicity of the active pharmaceutical ingredient Cur and the Cur / CMPH prepared in Example 4 are shown in Figure A and Figure B, respectively.

[0037] Figure 9 The effects of the active pharmaceutical ingredient Cur, CMPH prepared in Example 2, Cur / CMP prepared in Example 3, and Cur / CMPH prepared in Example 4 on cell migration.

[0038] Figure 10 The values ​​represent the ALT and AST levels in the serum of the active pharmaceutical ingredient Cur and the serum of each group in Examples 2, 3, and 4. Where A represents the ALT level and B represents the AST level.

[0039] Figure 11The content of active pharmaceutical ingredient Cur and the content of Hyp in liver tissue of each group in Examples 2, 3 and 4. Detailed Implementation

[0040] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0041] Example 1

[0042] Preparation and characterization of CMP

[0043] (1) Preparation of CMP nanocarriers

[0044] Dissolve 360 ​​mg of Pranic F127 in 100 mL of a mixture of ethanol and water (V 水 :V 乙醇 =1:1), add 420 μL of mesitylene, sonicate evenly with a probe, add 4 mL of Tris solution (200 mg / mL) and 60 mg of dopamine hydrochloride dropwise, then add 30 mg of CuCl2, and react at 30 °C for 1 h. After the reaction is complete, centrifuge at 11000 rpm for 15 min to collect the precipitate, wash three times with distilled water, and then evenly disperse in a mixed solution of ethanol and acetone (V 乙醇 :V 丙酮 =2:1), sonicate for 10 min to remove the template agent, repeat this operation 3 times, and finally obtain a black solid as CMP nanocarrier.

[0045] (2) Characterization of CMP nanocarriers

[0046] The particle size of the CMP nanocarriers was measured using transmission electron microscopy and dynamic light scattering method. The results are shown in the attached figure. Figure 1 A and Appendix Figure 2 As shown in Figure A, the nanozyme has a particle size of approximately 200 nm, a uniform particle size distribution, and a uniform spherical morphology. Obvious mesoporous channels can be observed, proving that the CMP nanocarrier was successfully prepared.

[0047] Example 2

[0048] 10 mg of CMP nanocarrier was dispersed in 10 mL of distilled water, and 10 mL of 1 mg / mL hyaluronic acid solution was added dropwise. After stirring at room temperature for 8 hours, the solid precipitate was obtained by centrifugation, which is the CMPH nanocarrier.

[0049] The particle size of the CMPH nanocarrier was measured using transmission electron microscopy and dynamic light scattering method. The results are shown in the attached figure. Figure 1 B and appendix Figure 2As shown in Figure A, the nanozyme has a particle size of approximately 220 nm, a uniform particle size distribution, and a uniform spherical morphology.

[0050] The zeta potential of the CMPH surface was measured, and the results are attached. Figure 2 As shown in Figure B, compared with the CMP nanocarrier, the surface potential of the CMPH nanocarrier decreased from -21.2mV to -28.0mV, proving that hyaluronic acid was successfully coated on the surface of the CMP nanocarrier.

[0051] Example 3

[0052] Preparation of Cur / CMP

[0053] Take 3 mg of CMP prepared in Example 1, disperse it in 3 mL of anhydrous ethanol, add 9 mg of Cur, sonicate for 30 min, and continue stirring in a 50 °C water bath for 4 h. The product is obtained by centrifugation, which is Cur / CMP.

[0054] Example 4

[0055] 10 mg of Cur / CMP prepared in Example 3 was dispersed in 10 mL of distilled water, and 10 mL of 1 mg / mL hyaluronic acid solution was added dropwise. After stirring at room temperature for 8 h, the solid precipitate obtained by centrifugation was the Cur / CMPH nano-drug delivery system.

[0056] Example 5

[0057] In the experimental group, 0.3 mL of NBT solution, 1.5 mL of methionine solution, and 0.3 mL of riboflavin solution were added to 5 mL EP tubes. Then, 0.05, 0.15, 0.25, 0.35 mL, and 0.45 mL of the test sample were added, respectively. Sodium phosphate buffer was added to bring the final volume to 3 mL, and the mixture was thoroughly mixed. After irradiation for 30 min, the supernatant was collected, and the absorbance was measured at 560 nm. In the control group, 0.9 mL of sodium phosphate buffer, 0.3 mL of NBT solution, 1.5 mL of methionine solution, and 0.3 mL of riboflavin solution were added to 5 mL EP tubes, and the absorbance was measured after the same procedure. The scavenging rate represents the activity of superoxide anion radical scavenging, i.e., SOD-like enzyme activity.

[0058] Add 0, 10, 20, 40, 60 μL and 80 μL of the sample solution to be tested to a 96-well plate, respectively, and add 20 μL of H2O2 solution and 60 μL of disodium ethylenediaminetetraacetate solution. Make up the final volume of each well to 200 μL with pH 7.4 PBS. After reacting at 37°C in the dark for 2 h, add 34 μL of terephthalic acid (TA) solution and react at 37°C in the dark for 1 h. Then measure the fluorescence intensity of each well.

[0059] The activity graphs of the SOD-like and CAT-like enzymes of the prepared CMP are shown in the attached figure. Figure 3As shown.

[0060] Example 6

[0061] Differential scanning calorimetry (DSC) was performed on the Cur / CMPH prepared in Example 4, the active pharmaceutical ingredient Cur, the blank carrier CMPH prepared in Example 2, and a physical mixture of the active pharmaceutical ingredient and the blank carrier to examine the change in drug crystallinity after drug loading. Detailed results are shown in the appendix. Figure 4 .

[0062] Example 7

[0063] DPPH scavenging capability verification

[0064] Curl, CMPH (Example 2), and Curl / CMPH (Example 4) were prepared at different concentrations (each system contained the same amount of Curl). 1 mL of sample was added to a centrifuge tube, followed by 1 mL of DPPH reaction solution and 1 mL of anhydrous ethanol. The reaction solution was mixed thoroughly and reacted in the dark for 30 min. The absorbance was measured at 515 nm. Anhydrous ethanol solution was used as a negative control instead of the sample solution, and ethanol was used as a blank control instead of the DPPH reaction solution. The scavenging activity of DPPH free radical scavenging was expressed as the scavenging rate. The results are attached. Figure 5 As shown in Figure A, Cur, CMPH, and Cur / CMPH all exhibit DPPH scavenging activity, and Cur / CMPH shows better scavenging ability under the same concentration conditions, with scavenging ability exhibiting concentration dependence.

[0065] ABTS clearance capability verification

[0066] Curl, CMPH (Example 2), and Curl / CMPH (Example 4) were prepared at different concentrations (each system contained the same amount of Curl). 1 mL of sample was added to each centrifuge tube, followed by 1 mL of ABTS reaction solution and 1 mL of anhydrous ethanol. The mixture was thoroughly mixed and reacted in the dark for 7 min. The absorbance at 734 nm was then measured. Anhydrous ethanol was used as a negative control instead of the sample solvent, and ethanol was used as a blank control instead of the ABTS reaction solution. The scavenging rate was used to represent the ABTS free radical scavenging activity. The results are attached. Figure 5 As shown in B, Cur, CMPH, and Cur / CMPH all exhibit ABTS scavenging activity, with Cur / CMPH showing better scavenging ability under the same concentration conditions, and the scavenging ability is concentration-dependent.

[0067] The superoxide anion radical scavenging ability was verified in the same way as in Example 5, and the results are attached. Figure 5 As shown in C, Cur, CMPH, and Cur / CMPH all exhibit superoxide anion radical scavenging activity, and Cur / CMPH shows better scavenging ability under the same concentration conditions, with the scavenging ability showing a concentration-dependent relationship.

[0068] Example 8

[0069] In vitro release study of drug delivery system

[0070] 5 mL of Cur / CMPH dispersion was placed in a 10 mL centrifuge tube. Dispersion media were prepared using pH 7.4 PBS and pH 6.8 PBS (both containing 0.5% Tween 80 as a solubilizer), respectively. The tubes were placed in a constant temperature shaker (37℃) and shaken in the dark. The absorbance of the supernatant at 425 nm was measured at specific time points (0.5, 1, 2, 4, 6, 10, 12, 24 h, 36 h). After sampling, an equal volume of the original dispersion medium was added to the original sample. The in vitro release curve of the Cur / CMPH drug delivery system is attached. Figure 6 As shown.

[0071] Example 9

[0072] Intracellular ROS scavenging capacity assay

[0073] Hepatic stellate cells were seeded into 24-well plates and cultured for 12 h before the culture medium was discarded. Experimental groups were set as (1) blank control group, (2) CMPH group, (3) Cur / CMP group, and (4) Cur / CMPH group. The culture medium solutions prepared for each nanosystem were added to each group, and the cells were incubated together for 12 h. After washing the cells with PBS, they were incubated with dichlorofluorescein diacetate (DCFH-DA) solution (20 μM, 10 μL) for 30 min. The cells were washed three times with PBS. The fluorescence of 2,7-dichlorofluorescein (DCF) was observed using CLSM. The relevant results are shown in the appendix. Figure 7 .

[0074] Example 10

[0075] Cytotoxicity detection

[0076] HSC-T6 cells and L02 cells (1×10⁻⁶) were used to... 4 Cells were seeded per well into 96-well plates, with 200 μL of PBS added to each of the 36 wells at the edge as a blank control. The plates were incubated for 24 hours. The old culture medium was discarded, and then serum-free medium was used to prepare gradient concentrations of Cur and Cur / CMPH sample solutions, which were added to the corresponding wells. Wells incubated with serum-free medium served as control groups. After incubation, 50 μL of pre-prepared MTT solution (2 mg / mL) was added to each well. -1Add the culture medium to each well and incubate in a cell culture incubator (37℃, 4h). Discard the old culture medium and add 150μL of DMSO to each well, shaking in the dark for 10min. Measure the absorbance of each well at 570nm using a microplate reader, and calculate the cell viability using the following formula: Cell viability = (Number of viable cells / Total number of cells) × 100%. See the attached table for relevant results. Figure 8 .

[0077] Example 11

[0078] Cell migration ability detection

[0079] HSC-T6 cells in the logarithmic growth phase were harvested at a concentration of 1 x 10⁻⁶. 6 Cells were seeded at a density of cells / well in 6-well plates and cultured overnight. After cell attachment, three straight lines were drawn in each well using a 10 μL sterile pipette tip, and the cells were washed off with PBS. CMPH, Cur / CMP, Cur / CMPH, and free Cur solutions were added, and the plates were incubated at 37°C and 5% CO2. The scratches were observed and photographed under a microscope at 0 h and 24 h after drug addition, and the changes in scratch width were recorded. The relevant results are shown in the appendix. Figure 9 .

[0080] Example 12

[0081] In vivo anti-liver fibrosis effect

[0082] SPF-grade healthy male C57BL / 6 mice were randomly divided into 6 groups (n=5 per group):

[0083] (1) Blank control group: Olive oil was injected intraperitoneally at a dose of 2 mL / kg for a total of 6 weeks;

[0084] (2) Model group: 20% CCl4 was injected intraperitoneally at a dose of 2 mL / kg for a total of 6 weeks;

[0085] (3) Cur group: 20% CCl4 was injected intraperitoneally at a dose of 2 mL / kg for a total of 6 weeks. Starting from the fourth week, 2 mg / kg of Cur was injected via the tail vein.

[0086] (4) CMPH group: 20% CCl4 was injected intraperitoneally at a dose of 2 mL / kg for a total of 6 weeks. Starting from the fourth week, CMPH (containing 2 mg / kg Cur) was injected via the tail vein.

[0087] (5) Cur / CMP group: 20% CCl4 was injected intraperitoneally at a dose of 2 mL / kg for a total of 6 weeks. Starting from the fourth week, Cur / CMP (containing 2 mg / kg Cur) was injected via the tail vein.

[0088] (6) Cur / CMPH group: 20% CCl4 was injected intraperitoneally at a dose of 2 mL / kg for a total of 6 weeks. Starting from the fourth week, Cur / CMPH (containing 2 mg / kg Cur) was injected via the tail vein.

[0089] (7) MPH (8) Cur / MP (9) Cur / MPH is the in vivo anti-liver fibrosis effect of a carrier without copper ion doping.

[0090] After the last administration, mice were fasted for 12 hours but allowed free access to water. They were then anesthetized with an intraperitoneal injection of urethane. The eyeballs were quickly removed by forceps, and blood was collected in a 1.5 ml sterile centrifuge tube. The tube was incubated at 37°C for 3 hours until the blood coagulated and separated into layers. The cells were then centrifuged (4°C, 3000 rpm, 15 min), and the supernatant serum was collected. The levels of alanine aminotransferase (AST) and aspartate aminotransferase (ALT) in the serum were measured according to the kit instructions. The results are as follows: Figure 10 As shown. Mice were euthanized and dissected, livers were removed, and the hydroxyproline (Hyp) content in the liver tissue was measured according to the kit instructions. The results are shown below. Figure 11 As shown.

[0091] The above description is only the best specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the invention.

Claims

1. A hepatic stellate cell-targeted nanomedicine delivery system, characterized in that, The nanomedicine delivery system consists of an enzyme-like nanocarrier, an antioxidant drug, and a hepatic stellate cell targeting ligand.

2. The hepatic stellate cell-targeted nanomedicine delivery system according to claim 1, characterized in that, The enzyme-like nanocarrier is formed by the oxidative polymerization of metal ions and dopamine, wherein the metal ions are Cu. 2+ Zn 2+ Fe 2+ Fe 3+ and Mn 2+ One of them.

3. The hepatic stellate cell-targeted nanomedicine delivery system according to claim 1, characterized in that, The antioxidant is one of curcumin, resveratrol, and quercetin.

4. The hepatic stellate cell-targeted nanomedicine delivery system according to claim 1, characterized in that, The hepatic stellate cell targeting ligand is one of hyaluronic acid and chondroitin sulfate.

5. A method for preparing the hepatic stellate cell-targeted nanodrug delivery system according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Dissolve Pronic F127 in a mixed solution of ethanol and water, add mesitylene and ultrasonically disperse it evenly, add tris(hydroxymethyl)aminomethane solution, dopamine hydrochloride and metal ions, stir for a period of time, and centrifuge to obtain metal-doped mesoporous polydopamine. (2) Dissolve the antioxidant drug in an ethanol solution, add the metal-doped mesoporous polydopamine nanocarrier obtained in step (1), react for a period of time, and centrifuge to obtain drug-loaded nanoparticles. (3) Disperse the drug-loaded nanoparticles obtained in step (2) uniformly in an aqueous solution, add hepatic stellate cell targeting ligands, and after reacting for a period of time, the product obtained by centrifugation is the hepatic stellate cell targeting nanodrug delivery system.

6. The preparation method according to claim 5, characterized in that, In step (1), the molar ratio of the metal ion to dopamine hydrochloride is 4:1 to 1:2, the mass ratio of Pranic F127 to mesitylene is 2:1 to 1:4, the reaction environment is alkaline with a pH of 8 to 10, the volume ratio of water to ethanol is 1:1 to 1:2, and the reaction time is 1 to 6 hours; the metal ion is Cu. 2+ .

7. The preparation method according to claim 5, characterized in that, In step (2), the mass ratio of the antioxidant drug to the metal-doped mesoporous polydopamine nanocarrier is 4:1 to 2:1, the reaction time is 3 to 8 hours, the reaction temperature is 40 to 60°C, and the drug is curcumin.

8. The preparation method according to claim 5, characterized in that, In step (3), the mass ratio of the hepatic stellate cell targeting ligand to the drug-loaded nanocarrier is 1:2 to 2:1, and the reaction time is 8 to 24 hours.

9. The application of the hepatic stellate cell-targeted nanomedicine delivery system according to any one of claims 1-4 as a drug delivery system combining anti-oxidation and hepatic stellate cell targeting.

10. The application of the hepatic stellate cell-targeted nanomedicine delivery system according to claim 9, characterized in that, It has enzyme-like activity, can scavenge various free radicals, and inhibit the activation of hepatic stellate cells, thereby inhibiting the development of liver fibrosis.

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