Conjugate targeting hepatic stellate cells as well as preparation method and application thereof

Abstract

The invention discloses a targeted hepatic stellate cell conjugate as well as a preparation method and application thereof, and belongs to the technical field of biological medicine and medicinal chemistry. The conjugate disclosed by the invention is RRG-TK-Ato, and the molecular formula of the conjugate is C98H144FN27O14S2. According to the present invention, the atorvastatin is covalently linked with the targeting polypeptide RRGRLKRWY through the reactive oxygen species (ROS)-sensitive thioketal (TK) connexon to form the conjugate, such that the specific targeting of the aHSCs can be achieved, the atorvastatin can be intelligently released in the hepatic fibrosis oxidative stress microenvironment, the limitation of the drug is overcome, and the efficacy-enhancing and toxicity-reducing targeting treatment of the hepatic fibrosis is achieved; the apoptosis of the activated hepatic stellate cells can be more effectively induced, the specific inhibition effect on the proliferation of the activated hepatic stellate cells is obvious, and the migration ability of the activated hepatic stellate cells can be more effectively inhibited, so that the diffusion of fibrosis is limited, and the liver function of hepatic fibrosis is improved.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and medicinal chemistry, specifically to an atorvastatin conjugate targeting activated hepatic stellate cells, its preparation method, and its application. More specifically, it relates to a conjugate RRGRLKRWY-TK-Atorvastatin (RRG-TK-Ato) formed by covalently linking atorvastatin to a targeting peptide RRGRLKRWY via a reactive oxygen species (ROS)-sensitive thioketone (TK) linker, as well as the preparation method of this conjugate and its application in the preparation of drugs for the prevention or treatment of liver fibrosis. Background Technology

[0002] Liver fibrosis is a common pathological step in the progression of various chronic liver diseases to cirrhosis and hepatocellular carcinoma. Its main characteristic lies in the imbalance between excessive deposition and degradation of the extracellular matrix (ECM). Activation of hepatic stellate cells (HSCs) is a core event in the development of liver fibrosis. In a normal liver, HSCs are in a quiescent state; when the liver is damaged, HSCs are activated, transforming into myofibroblasts, highly expressing α-smooth muscle actin (α-SMA), and secreting large amounts of ECM components such as collagen. Simultaneously, they inhibit the activity of matrix metalloproteinases (MMPs), leading to ECM synthesis far exceeding degradation, ultimately forming fibrous scars. Therefore, inducing apoptosis of activated hepatic stellate cells (aHSCs), inhibiting their activation, and suppressing ECM secretion have become key strategies in the development of anti-liver fibrosis drugs.

[0003] Statins are widely used lipid-lowering drugs in clinical practice, lowering cholesterol by inhibiting hydroxymethylglutaryl-CoA (HMG-CoA) reductase. Recent studies have shown that statins have pleiotropic effects, demonstrating potential anti-hepatic fibrosis activity. Research has found that atorvastatin (Ato) can exert its anti-fibrotic effect by acting on aHSCs: its mechanism includes downregulating the expression of the anti-apoptotic protein Bcl-2 and upregulating the expression of the pro-apoptotic protein Bax in aHSCs, thereby inducing mitochondrial membrane potential collapse and initiating the apoptosis program in aHSCs; simultaneously, atorvastatin can directly reduce the production of ECM components such as type I and type III collagen in aHSCs, and by reducing the expression of matrix metalloproteinase tissue inhibitors (TIMP-1 and TIMP-2), it relieves the inhibition of MMPs and promotes the degradation of deposited ECM. These findings provide a theoretical basis for the anti-fibrotic application of statins.

[0004] However, atorvastatin itself is not selective for aHSCs. It is widely distributed throughout the liver, acting not only on aHSCs but also affecting other cells such as hepatocytes and sinusoidal endothelial cells. This non-selective distribution leads to two main problems: first, the drug cannot reach an effective therapeutic concentration within aHSCs, limiting its anti-fibrotic efficacy; second, high doses or long-term use increase the risk of drug-induced liver injury, muscle toxicity, and other side effects. Therefore, precisely delivering atorvastatin to aHSCs for targeted therapy is crucial to improving its efficacy and reducing toxic side effects.

[0005] With in-depth research on surface biomarkers of aHSCs, several membrane proteins with specific high expression during activation have been discovered. Among them, platelet-derived growth factor receptor β (PDGFR-β) is recognized as one of the most significantly upregulated receptors. PDGFR-β is highly expressed on the surface of aHSCs, mediating cell proliferation and migration, making it an ideal drug delivery target. Previous studies have reported the development of peptides with high affinity and specificity for PDGFR-β through screening and design, such as the peptide with the sequence RRGRLKRWY, which can mimic the binding of natural ligands to PDGFR-β, thereby achieving active targeting of aHSCs.

[0006] Furthermore, the liver fibrosis microenvironment is characterized by elevated oxidative stress levels. Activated inflammatory cells and damaged hepatocytes release large amounts of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2). Utilizing this pathological characteristic, designing linkers that can selectively cleave under ROS conditions is an effective means to achieve intelligent drug release at the target site. Thioketal (TK) linkers are a class of chemical groups highly sensitive to ROS, capable of oxidative cleavage under high ROS conditions, while remaining relatively stable in normal physiological environments.

[0007] Therefore, it is of great significance to develop a delivery system that can specifically target aHSCs and intelligently release atorvastatin in the oxidative stress microenvironment of liver fibrosis, overcome the limitations of existing drugs, and achieve synergistic and toxicity-reducing targeted therapy for liver fibrosis. Summary of the Invention

[0008] This invention proposes a conjugate targeting hepatic stellate cells, its preparation method, and its application.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution: The first objective of this invention is to provide a conjugate targeting hepatic stellate cells, said conjugate being RRG-TK-Ato, with the molecular formula C. 98 H 144 FN 27 O 14 S2; its structural formula is: .

[0010] The second objective of this invention is to provide a method for preparing a conjugate targeting hepatic stellate cells, comprising the following steps: (1) Synthesis of intermediate TK-Ato: Atorvastatin was dissolved in anhydrous dichloromethane, an organic solvent, and condensing agent A and organic base were added and stirred for 30-60 min. Then 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine was added, and the reaction was carried out at room temperature for 10-24 h. The reaction solution was extracted by organic phase, concentrated under reduced pressure, and purified to obtain intermediate TK-Ato. (2) Synthesis of conjugate RRG-TK-Ato: The intermediate TK-Ato and the polypeptide were dissolved in anhydrous N,N-dimethylformamide in an organic solvent. The mixture was stirred under an ice bath and nitrogen atmosphere to obtain a mixture. Condensing agent B and organic base were added to the mixture and stirred. After activating the reaction for 30-60 min, the ice bath was removed. After reacting at room temperature for 4-8 h, the mixture was concentrated and purified under reduced pressure to obtain conjugate RRG-TK-Ato.

[0011] Its reaction formula is:

[0012] Furthermore, the condensing agent A mentioned in step (1) is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0013] Furthermore, the organic base mentioned in steps (1) and (2) is N,N-diisopropylethylamine.

[0014] Furthermore, the mass ratio of atorvastatin, condensing agent A, organic base and 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine in step (1) is 1-3:1-2:1:2-4.

[0015] Furthermore, the polypeptide mentioned in step (2) is the polypeptide fragment RRGR LKRWY.

[0016] Furthermore, the condensing agent B mentioned in step (2) is HATU, and its molecular formula is C. 10 H 15 F6N6OP.

[0017] Furthermore, in step (2), the mass ratio of intermediate TK-Ato, polypeptide, condensing agent B and organic base is 4-6:5-6:2-4:1.

[0018] The third objective of this invention is to provide the use of a conjugate targeting hepatic stellate cells in the preparation of drugs for the prevention or treatment of liver fibrosis.

[0019] Furthermore, the pulmonary fibrosis includes one or more of the following: primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, and interstitial pneumonia.

[0020] The fourth objective of this invention is to provide an application of a conjugate targeting hepatic stellate cells in the preparation of an activated hepatic stellate cell inhibitor.

[0021] The present invention relates to a conjugate targeting hepatic stellate cells, its preparation method, and its application, the beneficial effects of which are as follows: The conjugate of this invention covalently links atorvastatin to the targeting peptide RGRLKRWY via a reactive oxygen species (ROS)-sensitive thioketone (TK) linker. This conjugate can specifically target aHSCs and intelligently release atorvastatin in the oxidative stress microenvironment of liver fibrosis, overcoming the limitations of drugs and achieving targeted therapy for liver fibrosis with enhanced efficacy and reduced toxicity.

[0022] (2) The conjugate RRG-TK-Ato described in this invention can be efficiently and specifically taken up by activated hepatic stellate cells, has good active targeting ability, can more effectively induce apoptosis of activated hepatic stellate cells, has a significant specific inhibitory effect on the proliferation of activated hepatic stellate cells, can more effectively inhibit the migration ability of activated hepatic stellate cells, thereby limiting the spread of fibrosis and improving liver function in liver fibrosis. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The 1H NMR spectrum (500 MHz, CDCl3) of the intermediate TK-Ato in Example 1 of this invention is shown. Figure 2 The image shows the carbon NMR spectrum (125 MHz, CDCl3) of the intermediate TK-Ato in Example 1 of this invention. Figure 3The image shows the 1H NMR spectrum (500 MHz, DMSO) of the conjugate RRG-TK-Ato from Example 1 of this invention. Figure 4 This is the high-performance liquid chromatogram of the coupling compound RRG-TK-Ato in Example 1 of this invention after 2 hours of adding hydrogen peroxide; Figure 5 This study compares the uptake of free atorvastatin (Ato) and RRG-TK-Ato in activated hepatic stellate cells (LX-2 cells). Figure 6 The effects of free atorvastatin (Ato) and RRG-TK-Ato on the viability of activated hepatic stellate cells (LX-2 cells) were detected using the CCK-8 assay. Here, A represents the cell viability of activated hepatic stellate cells (LX-2 cells) under the free atorvastatin (Ato) condition; B represents the cell viability of activated hepatic stellate cells (LX-2 cells) under the RRG-TK-Ato conjugate obtained in Example 1. Figure 7 The effect of Annexin V-FITC / PI double staining method on the induction of apoptosis in activated LX-2 cells by free atorvastatin (Ato) and RRG-TK-Ato; Figure 8 Figure 1 shows the results of Western Blot analysis of the effects of free atorvastatin (Ato) and RRG-TK-Ato on the expression of fibrosis markers in activated LX-2 cells. Figure 9 A schematic diagram illustrating the effects of free atorvastatin (Ato) and RRG-TK-Ato on the migration ability of activated LX-2 cells using a cell scratch assay. Figure 10 A schematic diagram showing the effect of RRG-TK-Ato on serum ALT (A) and AST (B) levels in CCl4-induced liver fibrosis mice; Figure 11 A schematic diagram illustrating the effect of Masson staining on collagen deposition in liver tissue of mice with CCl4-induced liver fibrosis; Figure 12 This is a schematic diagram showing the effect of RRG-TK-Ato on the expression of α-SMA, Collagen I, and TIMP-1 in liver tissue of mice with CCl4-induced liver fibrosis as detected by ELISA. Figure 13 This is a schematic diagram illustrating the effect of RRG-TK-Ato on the hydroxyproline content in the liver tissue of mice with CCl4-induced liver fibrosis. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0026] One object of the present invention is to provide a conjugate targeting hepatic stellate cells, said conjugate being RRG-TK-Ato, with the molecular formula C 98 H 144 FN 27 O 14 S2; its structural formula is: .

[0027] A second objective of this invention is to provide a method for preparing a conjugate targeting hepatic stellate cells, comprising the following steps: (1) Synthesis of intermediate TK-Ato: Atorvastatin was dissolved in anhydrous dichloromethane, an organic solvent, and condensing agent A and organic base were added and stirred for 30-60 min. Then 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine was added, and the reaction was carried out at room temperature for 10-24 h. The reaction solution was extracted by organic phase, concentrated under reduced pressure, and purified to obtain intermediate TK-Ato. (2) Synthesis of conjugate RRG-TK-Ato: The intermediate TK-Ato and the polypeptide were dissolved in anhydrous N,N-dimethylformamide in an organic solvent. The mixture was stirred under an ice bath and nitrogen atmosphere to obtain a mixture. Condensing agent B and organic base were added to the mixture and stirred. After activating the reaction for 30-60 min, the ice bath was removed. After reacting at room temperature for 4-8 h, the mixture was concentrated and purified under reduced pressure to obtain conjugate RRG-TK-Ato.

[0028] In one embodiment, the condensing agent A in step (1) is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0029] In one embodiment, the organic base mentioned in steps (1) and (2) is N,N-diisopropylethylamine.

[0030] In one embodiment, the mass ratio of atorvastatin, condensing agent A, organic base and 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine in step (1) is 1-3:1-2:1:2-4.

[0031] In one embodiment, the polypeptide in step (2) is the polypeptide fragment RRGR LKRWY.

[0032] In one embodiment, the condensing agent B in step (2) is HATU.

[0033] In one embodiment, the mass ratio of intermediate TK-Ato, polypeptide, condensing agent B and organic base in step (2) is 4-6:5-6:2-4:1.

[0034] A third objective of this invention is to provide the use of a conjugate targeting hepatic stellate cells in the preparation of drugs for the prevention or treatment of liver fibrosis.

[0035] In this invention, pulmonary fibrosis includes one or more of the following: primary pulmonary fibrosis, secondary pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis, and interstitial pneumonia. In this embodiment, the detection and diagnostic markers for idiopathic pulmonary fibrosis are particularly specific and accurate.

[0036] A fourth objective of this invention is to provide the application of a conjugate targeting hepatic stellate cells in the preparation of an activated hepatic stellate cell inhibitor.

[0037] Example 1

[0038] A method for preparing a conjugate targeting hepatic stellate cells includes the following steps: (1) Synthesis of intermediate TK-Ato: Atorvastatin was dissolved in anhydrous dichloromethane, an organic solvent, and condensing agent A and organic base were added and stirred for 30-60 min. Then 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine was added, and the reaction was carried out at room temperature for 10-24 h. The reaction solution was extracted by organic phase, concentrated under reduced pressure, and purified to obtain intermediate TK-Ato. Specifically, atorvastatin (Ato, 1 mmol, 560 mg) was added to a single-necked round-bottom flask and dissolved in 20 mL of anhydrous dichloromethane. Condensing agent A, namely 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 1.2 mmol, 230 mg) and organic base N,N-diisopropylethylamine (DIPEA, 1.2 mmol, 155 mg), were added dropwise to the system. After stirring for 30 minutes, a dichloromethane solution of 2,2'-(propane-2,2-diylbis(sulfonamide dimethyl))diethylamine (2.5 mmol, 487 mg) was added dropwise. After reacting at room temperature for 12 hours, the reaction was quenched with 0.1 M dilute hydrochloric acid. The reaction solution after quenching was extracted with dichloromethane, concentrated under reduced pressure, and then purified by column chromatography to obtain 572 mg of white solid, which was the intermediate TK-Ato, with a yield of 78%. Its chemical structure was confirmed by 1H NMR and MS. (2) Synthesis of conjugate RRG-TK-Ato: The intermediate TK-Ato and the polypeptide were dissolved in anhydrous N,N-dimethylformamide, an organic solvent, and stirred in an ice bath under a nitrogen atmosphere to obtain a mixture. Condensing agent B and organic base were added to the mixture and stirred. After activating the reaction for 30-60 min, the ice bath was removed. After reacting at room temperature for 4-8 h, the mixture was concentrated and purified under reduced pressure to obtain the conjugate RRG-TK-Ato. Specifically, the intermediate TK-Ato (0.5 mmol, 365 mg) obtained in step (1) and the polypeptide fragment RRGR LKRWY (0.3 mmol, 387 mg) were dissolved in anhydrous N,N-dimethylformamide (DMF), cooled in an ice bath, and stirred under a nitrogen atmosphere to obtain a mixture. Then, the condensing agent HATU (0.5 mmol, 192 mg) and the organic base DIPEA (0.5 mmol, 65 mg) were added to the mixture, and the mixture was activated by stirring at 0°C for 30 minutes. The ice bath was then removed, the mixture was brought to room temperature, and the reaction was continued to be stirred for 6 hours. The reaction progress was monitored by high performance liquid chromatography (HPLC). After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography. After lyophilization, 253 mg of white powder solid was obtained, which was the conjugate RRG-TK-Ato, with a yield of 42%. Its final structure was confirmed by proton nuclear magnetic resonance spectroscopy and high resolution mass spectrometry.

[0039] In step (2), gradient elution is used in the high performance liquid chromatography purification. The mobile phase is a water / acetonitrile gradient system containing 0.1% trifluoroacetic acid. The fraction corresponding to the target product is collected. The gradient of the eluent is: 10% acetonitrile / 90% water → 80% acetonitrile / 20% water in the first 0-30 min. The peak time range of the target product is from 11.05 to 11.32 min. The fraction is collected during this period.

[0040] The specific compound formula is as follows: .

[0041] The spectra and data of intermediate TK-Ato and conjugate RRG-TK-Ato were obtained by proton nuclear magnetic resonance (¹H NMR) and high-resolution mass spectrometry (MS). The spectrum of intermediate TK-Ato is shown below. Figures 1-2 As shown in Figure 3; the spectrum of the conjugate RRG-TK-Ato is shown in Figure 3; where The data for intermediate TK-Ato is as follows: 1 H NMR (500 MHz, Chloroform- d ) δ 7.64 – 7.58 (m,2H), 7.56 – 7.50 (m, 2H), 7.38 – 7.21 (m, 7H), 7.12 – 7.01 (m, 3H), 4.76 (d, J = 4.9 Hz, 1H), 4.68 (d, J = 4.9 Hz, 1H), 4.42 (t, J= 5.5 Hz, 1H), 4.21 –4.10 (m, 3H), 4.07 (d, J = 12.3 Hz, 1H), 3.90 (d, J = 4.9 Hz, 1H), 3.78 (d, J = 4.9 Hz, 1H), 3.66 – 3.59 (m, 1H), 3.59 – 3.53 (m, 2H), 3.05 (s, 2H), 2.98(s, 2H), 2.66 (d, J = 12.3 Hz, 1H), 2.25 (d, J = 0.7 Hz, 2H), 2.19 – 2.04 (m,3H), 1.38 (s, 3H), 1.32 (s, 3H), 1.23 (s, 3H), 1.18 (s, 3H). 13 C NMR (125 MHz, Chloroform- d ) δ 172.58, 163.81, 163.32, 141.22, 138.95, 138.70, 137.96,130.29, 130.09, 130.00, 129.01, 128.48, 127.41, 125.60, 125.33, 121.95,121.25, 115.79, 68.50, 67.77, 65.34, 62.98, 56.40, 46.81, 45.00, 43.19,37.03, 35.88, 34.50, 29.27, 26.96, 22.83. HR-MS (ESI): m / z C 40 H 51 FN4O4S2, [M+H + Calcd 735.3409, found 735.3416.

[0042] The data for the conjugate RRG-TK-Ato are as follows: 1 H NMR (500 MHz, DMSO- d 6) δ 10.80 (s, 1H), 10.39 (s, 1H), 9.10 (s, 1H), 8.34 (s, 4H), 8.18 (d, J = 15.8 Hz, 2H), 8.04(s, 1H), 7.99 (d, J= 15.8 Hz, 2H), 7.92 (s, 1H), 7.87 – 7.80 (m, 3H), 7.70 –7.62 (m, 3H), 7.62 – 7.50 (m, 7H), 7.38 – 7.25 (m, 8H), 7.25 – 7.19 (m, 2H),7.19 – 7.11 (m, 3H), 7.11 – 7.02 (m, 3H), 6.81 (s, 4H), 6.67 (d, J = 7.8 Hz,6H), 6.47 (s, 1H), 5.85 (s, 1H), 4.85 (d, J = 4.9 Hz, 1H), 4.77 (d, J = 4.9Hz, 1H), 4.56 (d, J = 10.9 Hz, 2H), 4.28 (s, 1H), 4.16 (d, J = 12.2 Hz, 1H),4.11 – 4.04 (m, 5H), 3.85 – 3.74 (m, 4H), 3.70 (d, J = 5.2 Hz, 1H), 3.55 (s,1H), 3.47 – 3.32 (m, 4H), 3.27 – 3.04 (m, 12H), 2.96 (d, J = 12.4 Hz, 1H),2.90 – 2.83 (m, 2H), 2.79 (d, J = 12.4 Hz, 1H), 2.72 – 2.60 (m, 3H), 2.42 –2.31 (m, 2H), 2.29 (d, J = 12.4 Hz, 1H), 2.18 (d, J = 12.4 Hz, 1H), 2.05 (s,2H), 1.86 (d, J = 12.5 Hz, 1H), 1.82 – 1.66 (m, 11H), 1.65 – 1.53 (m, 7H),1.52 – 1.40 (m, 4H), 1.39 – 1.33 (m, 4H), 1.34 – 1.27 (m, 4H), 1.25 – 1.16(m, 7H), 0.92 (s, 3H), 0.87 (s, 3H). HR-MS (ESI): C 98 H144 FN 27 O 14 S2, [M+6H] 6+ m / zcalcd 335.5214, found 335.5219.

[0043] Example 2

[0044] A method for preparing a conjugate targeting hepatic stellate cells includes the following steps: (1) Synthesis of intermediate TK-Ato: Avastatin was added to a single-necked round-bottom flask and dissolved in 20 mL of anhydrous dichloromethane. Condensing agent A, namely 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and organic base N,N-diisopropylethylamine (DIPEA) were added under ice bath. After stirring for 30 minutes, a dichloromethane solution of 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine was added dropwise to the system. After reacting at room temperature for 12 hours, the reaction was quenched with 0.1 M dilute hydrochloric acid. The reaction solution after quenching was extracted with dichloromethane, concentrated under reduced pressure, and then purified by column chromatography to obtain a white solid, which is the intermediate TK-Ato; the mass ratio of atorvastatin, condensing agent A, organic base and 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine is 3:1.5:1:3. (2) Synthesis of conjugate RRG-TK-Ato: The intermediate TK-Ato obtained in step (1) and the polypeptide fragment RRGRLKRWY were dissolved in anhydrous N,N-dimethylformamide (DMF), cooled in an ice bath, and stirred under a nitrogen atmosphere to obtain a mixture. Then, the condensing agent HATU and the organic base DIPEA were added to the mixture, and the mixture was stirred and activated at 0°C for 30 minutes. The ice bath was removed, the mixture was brought to room temperature, and the reaction was continued to be stirred for 6 hours. The reaction process was monitored by high performance liquid chromatography (HPLC). After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography. The mobile phase was a water / acetonitrile gradient system containing 0.1% trifluoroacetic acid. The fraction corresponding to the target product was collected, and after freeze-drying, a white powder solid was obtained, which is the conjugate RRG-TK-Ato. The mass ratio of intermediate TK-Ato, polypeptide, condensing agent B and organic base was 5:6:3:1. In step (2), gradient elution is used in the high performance liquid chromatography purification. The mobile phase is a water / acetonitrile gradient system containing 0.1% trifluoroacetic acid. The fraction corresponding to the target product is collected. The gradient of the eluent is: 10% acetonitrile / 90% water → 80% acetonitrile / 20% water in the first 0-30 min. The peak time range of the target product is from 11.05 to 11.32 min. The fraction is collected during this period.

[0045] Example 3

[0046] A method for preparing a conjugate targeting hepatic stellate cells includes the following steps: (1) Synthesis of intermediate TK-Ato: Avastatin was added to a single-necked round-bottom flask and dissolved in 20 mL of anhydrous dichloromethane. Condensing agent A, namely 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and organic base N,N-diisopropylethylamine (DIPEA) were added under ice bath. After stirring for 30 minutes, a dichloromethane solution of 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine was added dropwise to the system. After reacting at room temperature for 12 hours, the reaction was quenched with 0.1 M dilute hydrochloric acid. The reaction solution after quenching was extracted with dichloromethane, concentrated under reduced pressure, and then purified by column chromatography to obtain a white solid, which is the intermediate TK-Ato; wherein the mass ratio of atorvastatin, condensing agent A, organic base and 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine is 1:1:1:2. (2) Synthesis of conjugate RRG-TK-Ato: The intermediate TK-Ato obtained in step (1) and the polypeptide fragment RRGRLKRWY were dissolved in anhydrous N,N-dimethylformamide (DMF), cooled in an ice bath, and stirred under a nitrogen atmosphere to obtain a mixture. Then, the condensing agent HATU and the organic base DIPEA were added to the mixture, and the mixture was stirred and activated at 0°C for 30 minutes. The ice bath was removed, the mixture was brought to room temperature, and the reaction was continued to be stirred for 6 hours. The reaction process was monitored by high performance liquid chromatography (HPLC). After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography. The mobile phase was a water / acetonitrile gradient system containing 0.1% trifluoroacetic acid. The fraction corresponding to the target product was collected, and after freeze-drying, a white powder solid was obtained, which is the conjugate RRG-TK-Ato. The mass ratio of intermediate TK-Ato, polypeptide, condensing agent B and organic base was 4:5:2:1. In step (2), gradient elution is used in the high performance liquid chromatography purification. The mobile phase is a water / acetonitrile gradient system containing 0.1% trifluoroacetic acid. The fraction corresponding to the target product is collected. The gradient of the eluent is: 10% acetonitrile / 90% water → 80% acetonitrile / 20% water in the first 0-30 min. The peak time range of the target product is from 11.05 to 11.32 min. The fraction is collected during this period.

[0047] Example 4

[0048] A method for preparing a conjugate targeting hepatic stellate cells includes the following steps: (1) Synthesis of intermediate TK-Ato: Avastatin was added to a single-necked round-bottom flask and dissolved in 20 mL of anhydrous dichloromethane. Condensing agent A, namely 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and organic base N,N-diisopropylethylamine (DIPEA) were added under ice bath. After stirring for 30 minutes, a dichloromethane solution of 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine was added dropwise to the system. After reacting at room temperature for 12 hours, the reaction was quenched with 0.1 M dilute hydrochloric acid. The reaction solution after quenching was extracted with dichloromethane, concentrated under reduced pressure, and then purified by column chromatography to obtain a white solid, which is the intermediate TK-Ato; wherein the mass ratio of atorvastatin, condensing agent A, organic base and 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine is 3:2:1:4. (2) Synthesis of conjugate RRG-TK-Ato: The intermediate TK-Ato obtained in step (1) and the polypeptide fragment RRGRLKRWY were dissolved in anhydrous N,N-dimethylformamide (DMF), cooled in an ice bath, and stirred under a nitrogen atmosphere to obtain a mixture. Then, the condensing agent HATU and the organic base DIPEA were added to the mixture, and the mixture was stirred and activated at 0°C for 30 minutes. The ice bath was removed, the mixture was brought to room temperature, and the reaction was continued to be stirred for 6 hours. The reaction process was monitored by high performance liquid chromatography (HPLC). After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography. The mobile phase was a water / acetonitrile gradient system containing 0.1% trifluoroacetic acid. The fraction corresponding to the target product was collected, and after freeze-drying, a white powder solid was obtained, which is the conjugate RRG-TK-Ato. The mass ratio of intermediate TK-Ato, polypeptide, condensing agent B and organic base was 6:6:4:1. In step (2), gradient elution is used in the high performance liquid chromatography purification. The mobile phase is a water / acetonitrile gradient system containing 0.1% trifluoroacetic acid. The fraction corresponding to the target product is collected. The gradient of the eluent is: 10% acetonitrile / 90% water → 80% acetonitrile / 20% water in the first 0-30 min. The peak time range of the target product is from 11.05 to 11.32 min. The fraction is collected during this period.

[0049] Example 5

[0050] Validation of the drug release behavior of the conjugate RRG-TK-Ato of this invention Verification method: The conjugate RRG-TK-Ato obtained in Example 1 was added to acetonitrile to obtain an acetonitrile solution. Hydrogen peroxide was added to the acetonitrile solution of conjugate RRG-TK-Ato, wherein the molar ratio of hydrogen peroxide to RRG-TK-Ato was 10 / 1. The mixture was then shaken for 5 minutes and allowed to stand.

[0051] The drug release behavior was detected by high-performance liquid chromatography (HPLC), and the results were as follows: Figure 4 As shown; from Figure 4 As shown in the high-performance liquid chromatography (HPLC) chromatogram, after adding hydrogen peroxide (H2O2) for 2 hours, the thioclase linkage (TK) in the molecular structure of the conjugate RRG-TK-Ato undergoes an oxidation reaction, leading to the breakage of the linkage and the release of the therapeutic drug atorvastatin. Only a small amount of the compound RRG-TK-Ato is present at a retention time of 11.03 minutes, with most of it converted to the drug atorvastatin (Rt = 7.72 min). Figure 4 This indicates that atorvastatin can be released from RRG-TK-Ato.

[0052] Example 6

[0053] Validation of the activated hepatic stellate cell targeting of the conjugate RRG-TK-Ato of this invention An activated hepatic stellate cell model was constructed by stimulating the human hepatic stellate cell line LX-2 with transforming growth factor-β (TGF-β, 10 ng / mL) for 24 hours. The activated LX-2 cells were co-incubated with an equimolar concentration (10 μM) of the conjugate RRG-TK-Ato obtained in Example 1 or free atorvastatin (Ato) for 12 hours. After incubation, the cells were collected, washed three times with pre-cooled PBS buffer to remove surface-adsorbed drug, and intracellular drug was extracted by ultrasonic disruption after adding an appropriate amount of acetonitrile (containing 0.1% formic acid). The supernatant was collected by centrifugation, and the drug content was determined by high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: Agilent Zorbax SB-C18 column (4.6 × 250 mm, 5 μm); mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution (60:40, v / v); detection wavelength: 245 nm; flow rate: 1.0 mL / min; column temperature: 30℃; injection volume: 20 μL. The results are as follows: Figure 5 As shown, compared with the free atorvastatin group, the RRG-TK-Ato treatment group had a significantly higher drug accumulation in activated LX-2 cells, with a relative uptake rate of approximately 7.3 times that of free atorvastatin (7.13 ± 0.15 vs 0.98 ± 0.21, p < 0.0001). This indicates that the conjugate RRG-TK-Ato of the present invention can be efficiently and specifically taken up by activated hepatic stellate cells and has good active targeting ability.

[0054] Example 7

[0055] Verification of the specific inhibitory effect of the conjugate RRG-TK-Ato on activated hepatic stellate cells 7.1 The effect of the conjugate RRG-TK-Ato on the viability of activated hepatic stellate cells (LX-2 cells) was detected by the CCK-8 assay.

[0056] Human hepatic stellate cell line LX-2 was seeded at 5 × 10³ cells per well in 96-well plates and cultured overnight. Then, culture medium containing transforming growth factor-β (TGF-β, 10 ng / mL) was added for 24 hours to establish an activated hepatic stellate cell model. The supernatant was then discarded, and culture medium containing gradient concentrations (0, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, 160 μM) of free atorvastatin (Ato) or the conjugate RRG-TK-Ato obtained in Example 1 was added, with three replicates per group. Incubation continued for 48 hours. After incubation, 10 μL of CCK-8 solution was added to each well, and the plates were incubated at 37°C for 2 hours. The absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated, and dose-response curves were fitted using GraphPad Prism software to calculate the half-maximal inhibitory concentration (IC50).

[0057] ; in, Control group: No cells added; Control group: Contains LX-2 cells but without the addition of atorvastatin (Ato) or RRG-TK-Ato; Drug group: containing LX-2 cells and supplemented with atorvastatin (Ato) or RRG-TK-Ato.

[0058] The results are as follows Figure 6 As shown, the IC50 value of free atorvastatin against activated LX-2 cells was 6.3 ± 1.3 μM, while the IC50 value of RRG-TK-Ato was 1.0 ± 0.2 μM, which was about 1 / 6 of that of free atorvastatin. This indicates that RRG-TK-Ato has a significantly stronger inhibitory effect on the proliferation of activated hepatic stellate cells than free atorvastatin.

[0059] 7.2 The effect of the compound on induced apoptosis in activated LX-2 cells was detected by Annexin V-FITC / PI double staining combined with flow cytometry.

[0060] LX-2 cells were distributed at a rate of 2 × 10⁶ cells per well. 5Cells were seeded in 6-well plates and cultured overnight. Afterward, the cells were stimulated for 24 hours with medium containing TGF-β (10 ng / mL) to establish an activation model. Then, equimolar concentrations (10 μM) of free Ato or RRG-TK-Ato were added, with a blank control group (no drug added), and incubation continued for another 24 hours. Cells (including floating cells) were collected, washed twice with pre-cooled PBS, resuspended in Binding Buffer, and incubated with Annexin V-FITC and PI staining solution at room temperature in the dark for 15 minutes. Immediately after incubation, flow cytometry was used to analyze the apoptosis rate (the sum of early and late apoptosis) using FlowJo software.

[0061] The results are as follows Figure 7 As shown, the apoptosis rate of the blank control group was 3.2%, the apoptosis rate of the free Ato treatment group was 14.1%, while the apoptosis rate of the RRG-TK-Ato treatment group was as high as 50.1%, which is about 3.6 times that of free Ato, indicating that RRG-TK-Ato can more effectively induce apoptosis of activated hepatic stellate cells.

[0062] 7.3 The effect of the compound on the expression of fibrosis-related proteins in activated LX-2 cells was detected by Western blotting.

[0063] LX-2 cells were distributed at a rate of 3 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates and cultured overnight. Then, medium containing TGF-β (10 ng / mL) was added for 24 hours to establish an activation model. Equimolar concentrations (10 μM) of free Ato or RRG-TK-Ato were then added, while a model control group (TGF-β stimulation only) was set up. Figure 8 2) and the blank control group (without TGF-β stimulation, i.e. Figure 8 (1) Continue incubation for 48 hours. Collect cells, extract total protein with RIPA lysis buffer, and determine protein concentration using the BCA method. Take an equal amount of protein for SDS-PAGE electrophoresis, transfer to a PVDF membrane, block with 5% skim milk powder, and add primary antibodies against α-smooth muscle actin (α-SMA), type I collagen (Collagen I), type III collagen (Collagen III), tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), and internal control GAPDH, respectively. Incubate overnight at 4°C. The next day, add HRP-labeled secondary antibody and incubate at room temperature for 1 hour. Develop with ECL chemiluminescence, analyze the band gray values ​​using ImageJ software, and calculate the relative expression level of the target protein. The methods for extracting total protein with RIPA lysis buffer and determining protein concentration using the BCA method in this embodiment are conventional methods used by those skilled in the art, and their steps and principles will not be elaborated again.

[0064] The results are as follows Figure 8As shown, compared with the model control group, the expression of α-SMA, Collagen I, Collagen III, and TIMP-1 in the free Ato treatment group was decreased, while the expression level of the above proteins in the RRG-TK-Ato treatment group was more significantly decreased, indicating that RRG-TK-Ato can more effectively inhibit the activation phenotype of activated hepatic stellate cells, reduce extracellular matrix synthesis, and relieve the inhibition of matrix metalloproteinases.

[0065] 7.4 The effect of the compound on the migration ability of activated LX-2 cells was detected by cell scratch assay.

[0066] LX-2 cells were spaced at 5 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates and cultured to confluence. After 24 hours of stimulation with medium containing TGF-β (10 ng / mL), an activated hepatic stellate cell model was established. Vertical scratches were made on the monolayer of cells using a 200 μL sterile pipette tip, and detached cells were removed by washing with PBS. Serum-free medium containing an equimolar concentration (10 μM) of free atorvastatin (Ato) or RRG-TK-Ato was then added to form drug groups: the TGF-β + Ato group and the TGF-β + RRG-TK-Ato group. A blank control group and a model control group (TGF-β group) (PBS group) were also included. The cells were incubated at 37°C in a 5% CO2 incubator. Scratch healing was observed and photographed under an inverted microscope on days 0 and 3.

[0067] The results are as follows Figure 9 As shown, the control group exhibited strong cell migration, with the scratches largely closed by day 3. Cell migration in the free Ato treatment group was somewhat inhibited, with some unclosed areas still visible on day 3. In contrast, cell migration in the RRG-TK-Ato treatment group was significantly inhibited, with the scratch width on day 3 remaining similar to that on day 0, and no significant healing observed. These results indicate that RRG-TK-Ato can more effectively inhibit the migration ability of activated hepatic stellate cells, thereby limiting the spread of fibrosis.

[0068] Example 8

[0069] In vivo anti-liver fibrosis verification of the conjugate RRG-TK-Ato of this invention 8.1 A mouse model of liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl4) to evaluate the in vivo anti-liver fibrosis efficacy of the conjugate RRG-TK-Ato.

[0070] Animal model establishment: Male C57BL / 6 mice were randomly divided into four groups: sham-operated group (Sham), model control group (CCl4+PBS), free atorvastatin group (CCl4+Ato), and RRG-TK-Ato group (CCl4+RRG-TK-Ato), with six mice in each group. Except for the sham-operated group, the mice in the other groups were intraperitoneally injected with 10% CCl4 olive oil solution (2 mL / kg) twice a week for 6 consecutive weeks to establish a liver fibrosis model.

[0071] II. Experimental Treatment Simultaneously with model establishment, the treatment groups received a tail vein injection of free atorvastatin (5 mg / kg) or the conjugate RRG-TK-Ato obtained in Example 1 (5 mg / kg based on atorvastatin), while the model control group received an equal volume of PBS. This was administered three times a week for six consecutive weeks. Twenty-four hours after the last administration, blood was collected from the orbital cavity, serum was separated, and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using an automated biochemical analyzer.

[0072] The results are as follows Figure 10 As shown, compared with the sham-operated group, the serum ALT and AST levels in the model control group were significantly increased (ALT: 51.2 ± 2.5 U / L vs 435.3 ± 45.8 U / L; AST: 74.1 ± 10.6 U / L vs 496.1 ± 49.5 U / L), indicating that the liver fibrosis model was successfully established. Compared with the model control group, the ALT and AST levels in the free atorvastatin group were decreased (ALT: 279.5 ± 23.8 U / L; AST: 259.6 ± 15.9 U / L), while the ALT and AST levels in the RRG-TK-Ato group were further significantly decreased (ALT: 167.2 ± 36.1 U / L; AST: 177.3 ± 20.3 U / L), and significantly lower than those in the free atorvastatin group, indicating that RRG-TK-Ato can more effectively improve liver function in mice with liver fibrosis.

[0073] 8.2 Masson staining was used to assess collagen deposition in liver tissue.

[0074] After euthanizing the mice, left lobe liver tissue was harvested, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 μm. After dewaxing and hydration, the sections were stained with Masson's compound staining solution for 5 minutes, rinsed with 0.2% glacial acetic acid solution, treated with 5% phosphotungstic acid for 5 minutes, counterstained with aniline blue for 5 minutes, dehydrated with anhydrous ethanol, cleared with xylene, and mounted with neutral resin. The sections were observed and photographed under an optical microscope; collagen fibers appeared blue, while muscle fibers and cytoplasm appeared red.

[0075] The results are as follows Figure 11As shown, in the sham-operated group, only a small amount of blue collagen fibers were visible around the blood vessel walls in the liver tissue; in the model control group, a large amount of blue collagen fibers were deposited in the liver tissue, forming obvious fibrous septa, and the collagen volume fraction was significantly increased; in the free atorvastatin group, collagen deposition was reduced and the fibrous septa were thinned; while in the RRG-TK-Ato group, collagen deposition was significantly reduced, with only a small amount of thin collagen fibers visible, and the collagen volume fraction was significantly lower than that in the free atorvastatin group, indicating that RRG-TK-Ato can more effectively inhibit CCl4-induced collagen deposition in liver fibrosis.

[0076] 8.3 The expression levels of α-smooth muscle actin (α-SMA), type I collagen (Collagen I), and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in liver tissue were detected by enzyme-linked immunosorbent assay (ELISA).

[0077] Approximately 50 mg of liver tissue from each group of mice was collected, homogenized with RIPA lysis buffer, centrifuged at 4°C, and the supernatant was collected. The total protein concentration was determined using the BCA method. Following the ELISA kit instructions, the contents of α-SMA, Collagen I, and TIMP-1 were measured, and expressed as per milligram of total protein.

[0078] The results are as follows Figure 12 As shown, compared with the sham-operated group, the expression of α-SMA, Collagen I, and TIMP-1 in the liver tissue of the model control group was significantly increased (α-SMA: 2.06 ± 0.20 ng / mg vs 74.6 ± 24.1 ng / mg; Collagen I: 3.29 ± 0.70 ng / mg vs 21.6 ± 3.8 ng / mg; TIMP-1: 18.3 ± 3.5 pg / mg vs 374.1 ± 87.6 pg / mg), indicating that hepatic stellate cells were activated, ECM synthesis was increased, and degradation was inhibited. Compared with the model control group, the expression of α-SMA, Collagen I, and TIMP-1 in the free atorvastatin group was decreased (α-SMA: 43.0 ± 12.2 ng / mg; Collagen I: 11.6 ± 2.2 ng / mg; TIMP-1: 155.5 ± 59.2 pg / mg), while the above indicators in the RRG-TK-Ato group were further significantly reduced (α-SMA: 8.65 ± 1.60 ng / mg; Collagen I: 5.05 ± 1.55 ng / mg; TIMP-1: 49.6 ± 4.1 pg / mg), and were significantly lower than those in the free atorvastatin group. This indicates that RRG-TK-Ato can more effectively inhibit hepatic stellate cell activation, reduce extracellular matrix synthesis, and relieve the inhibition of matrix metalloproteinases.

[0079] 8.4 The content of hydroxyproline in liver tissue was determined by alkaline hydrolysis to assess the total amount of collagen deposition.

[0080] Approximately 100 mg of liver tissue from each group of mice was accurately weighed and placed in a hydrolysis tube. 1 mL of 6 mol / L hydrochloric acid was added, and the mixture was incubated at 95°C for 5 hours. After cooling, the pH of the hydrolysate was adjusted to 6.0-7.0 with 10 mol / L NaOH, and the volume was brought to 10 mL with distilled water. The mixture was then filtered. 1 mL of the filtrate was taken, and 0.5 mL of chloramine T solution was added. Oxidation was carried out at room temperature for 10 minutes, followed by the addition of 0.5 mL of p-dimethylaminobenzaldehyde solution. The mixture was then incubated at 60°C for 20 minutes for color development. After cooling, the absorbance was measured at 560 nm. The hydroxyproline content was calculated based on the standard curve, and the results were expressed as micrograms per gram of liver tissue.

[0081] The results are as follows Figure 13 As shown, the hydroxyproline content in the liver tissue of the sham-operated group was low (41.4 ± 9.2 μg / g). The hydroxyproline content in the model control group was significantly increased (626.7 ± 92.5 μg / g), indicating a large amount of collagen deposition in the liver tissue. The hydroxyproline content in the free atorvastatin group decreased (516.7 ± 99.4 μg / g), but there was no statistically significant difference compared with the model control group. However, the hydroxyproline content in the RRG-TK-Ato group was significantly reduced to 226.1 ± 88.4 μg / g, significantly lower than that in the model control group and the free atorvastatin group, indicating that RRG-TK-Ato can more effectively reduce the total amount of collagen deposition in the liver tissue and exert an anti-liver fibrosis effect.

[0082] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A conjugate targeting hepatic stellate cells, characterized in that: The coupling agent is RRG-TK-Ato, and its structural formula is: 。 2. A method for preparing a conjugate targeting hepatic stellate cells according to claim 1, characterized in that: Includes the following steps: (1) Synthesis of intermediate TK-Ato: Atorvastatin was dissolved in anhydrous dichloromethane, an organic solvent, and condensing agent A and organic base were added and stirred for 30-60 min. Then 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine was added, and the reaction was carried out at room temperature for 10-24 h. The reaction solution was extracted by organic phase, concentrated under reduced pressure, and purified to obtain intermediate TK-Ato. (2) Synthesis of conjugate RRG-TK-Ato: The intermediate TK-Ato and the polypeptide were dissolved in anhydrous N,N-dimethylformamide in an organic solvent. The mixture was stirred under an ice bath and nitrogen atmosphere to obtain a mixture. Condensing agent B and organic base were added to the mixture and stirred. After activating the reaction for 30-60 min, the ice bath was removed. After reacting at room temperature for 4-8 h, the mixture was concentrated and purified under reduced pressure to obtain conjugate RRG-TK-Ato.

3. The method for preparing the conjugate targeting hepatic stellate cells according to claim 2, characterized in that: The condensing agent A mentioned in step (1) is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

4. The method for preparing the conjugate targeting hepatic stellate cells according to claim 2, characterized in that: The organic base mentioned in steps (1) and (2) is N,N-diisopropylethylamine.

5. The method for preparing the conjugate targeting hepatic stellate cells according to claim 2, characterized in that: The mass ratio of atorvastatin, condensing agent A, organic base and 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine in step (1) is 1-3:1-2:1:2-4.

6. The method for preparing the conjugate targeting hepatic stellate cells according to claim 2, characterized in that: The polypeptide mentioned in step (2) is the polypeptide fragment RRGR LKRWY.

7. The method for preparing the conjugate targeting hepatic stellate cells according to claim 2, characterized in that: The condensing agent B mentioned in step (2) is HATU.

8. The method for preparing the conjugate targeting hepatic stellate cells according to claim 2, characterized in that: The mass ratio of intermediate TK-Ato, polypeptide, condensing agent B and organic base in step (2) is 4-6:5-6:2-4:

1.

9. The use of a conjugate targeting hepatic stellate cells according to claim 1 in the preparation of a drug for the prevention or treatment of liver fibrosis.

10. The use of a conjugate targeting hepatic stellate cells according to claim 1 in the preparation of an activated hepatic stellate cell inhibitor.

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