Lysosome-targeting chimera, and preparation method and application thereof

Abstract

The present application belongs to the technical field of pharmaceutical chemistry, and discloses a lysosome-targeting chimera, which is composed of an azidated mesoporous polydopamine nanoparticle carrier, an alkyne-lysosome-targeting receptor ligand and an alkyne-ALK5 inhibitor. The present application uses the mesoporous polydopamine with high biocompatibility as a carrier, constructs a safe and effective anti-oxidation nano platform, removes reactive oxygen species, reduces the oxidative stress level, and then reduces the secretion of TGF-beta by liver macrophages, simultaneously loads the ALK5 small molecule inhibitor, specifically recognizes and captures ALK5, realizes precise targeting by combining the modified M6P with the high expression CI-M6PR receptor on the surface of HSC, and provides a new technology and idea for clinically treating liver fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, and relates to a lysosome-targeting chimera, its preparation method and application, specifically to a lysosome-targeting chimera based on mesoporous polydopamine nanoparticles, its preparation method and application. Background Technology

[0002] Liver fibrosis affects the health of nearly one billion people worldwide. It is a crucial stage and central link in the progression of various chronic liver diseases to cirrhosis and liver cancer. It is characterized by the abnormal deposition of extracellular matrix components such as collagen, laminin, and hyaluronic acid, leading to structural disorders and functional impairment of the liver under the continuous stimulation of pathogenic factors such as chronic hepatitis viruses, alcohol, autoimmune diseases, and genetic metabolic disorders. Currently, there are no approved specific anti-liver fibrosis drugs in clinical practice.

[0003] Hepatic stellate cells (HSCs) are resident mesenchymal cells in the liver, located in the perisinusoidal space between sinusoidal endothelial cells and hepatocytes. After liver injury, HSCs transform from quiescent cells storing vitamin A into myofibroblast-like cells with proliferative, contractile, inflammatory, and chemotactic functions. They are characterized by excessive secretion of extracellular matrix, primarily composed of type I and type III collagen, and the formation of scar tissue, leading to fibrosis. This is widely recognized as a major driver of fibrosis in damaged human livers. Transforming growth factor β (TGF-β) plays a crucial role in HSC activation through the classical SMAD pathway. Furthermore, oxidative stress can also activate HSCs by stimulating macrophages to secrete TGF-β, and is closely linked to the TGF-β signaling pathway. Therefore, the TGF-β signaling pathway associated with HSC activation and oxidative stress can both serve as key targets for altering the development of fibrosis.

[0004] Inhibiting TGF-β signal transduction using TGF-β-targeting drugs (TGF-β antibodies, TGF-β receptor inhibitors, and recombinant proteins) has become a hot topic in basic research on anti-liver fibrosis. Among them, ALK5 (activin receptor-like kinase 5) inhibitors, namely TGF-β type I receptor inhibitors, highly specifically block the catalytic activity of ALK5, inhibiting TGF-β / SMAD signaling, preventing matrix deposition, and exerting their anti-fibrotic activity. However, ALK5i (ALK5 small molecule inhibitors) have not yet been used in clinical specific treatment of liver fibrosis, mainly due to the following limiting factors:

[0005] ① ALK5i inhibition of TGF-β receptors may have toxic side effects on multiple organs throughout the body involved in the TGF-β cascade and affect normal tissue and organ metabolism;

[0006] ② ALK5i alone can increase lipid droplet synthesis in hepatocytes, so a precise drug delivery system targeting HSCs needs to be developed.

[0007] ③ ALK5 on the cell surface is compensatorily upregulated under TGF-β induction, weakening the therapeutic effect of ALK5i.

[0008] Some studies have shown that antioxidants can exert anti-fibrotic effects. Silymarin, a plant-based drug with important antioxidant properties, can reverse liver fibrosis and stimulate liver tissue regeneration by increasing glutathione levels and reducing type III collagen production in patients with cirrhosis. It is currently used in clinical liver-protective treatment, but its treatment cycle is long and its efficacy is limited. In addition, antioxidant nanomaterials based on bioenzymes and nanozymes have been developed, such as metal-organic framework nanoparticles loaded with superoxide dismutase and catalase, and CeO2 nanoparticles. These metal nanomaterials often have low biocompatibility and long-term potential toxic side effects, which limits their clinical application. Summary of the Invention

[0009] This invention addresses the technical problems of toxic side effects and low drug delivery precision of TGF-β targeted drugs by providing a lysosomal targeted chimera. Using highly biocompatible mesoporous polydopamine as a carrier, a safe and effective antioxidant nanoplatform is constructed to scavenge reactive oxygen species, reduce oxidative stress levels, and thereby reduce the secretion of TGF-β by hepatic macrophages. At the same time, it loads an ALK5 small molecule inhibitor, which specifically recognizes and captures ALK5. By modifying M6P, it binds to the CI-M6PR receptor highly expressed on the surface of HSCs to achieve precise targeting, providing a new technology and approach for the clinical treatment of liver fibrosis.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides a lysosome-targeting chimera, which is composed of an azide-modified mesoporous polydopamine nanoparticle carrier, an alkynyl-lysosome-targeting receptor ligand, and an alkynyl-ALK5 inhibitor.

[0012] In the above technical solution, the alkynyl-lysosome targeting receptor ligand is alkynyl-mannose-6-phosphate.

[0013] In the above technical solution, the alkynyl-ALK5 inhibitor is alkynyl-R268712.

[0014] In a second aspect, the present invention provides a composition comprising the above-described lysosomal targeting chimera and a medically acceptable carrier.

[0015] Thirdly, the present invention provides a method for preparing the above-mentioned lysosome-targeting chimera, comprising the following steps: using azidated mesoporous polydopamine nanoparticles as a carrier, loading an alkynyl-lysosome-targeting receptor ligand and an alkynyl-ALK5 inhibitor onto the azidated mesoporous polydopamine nanoparticles via a Click reaction to obtain the lysosome-targeting chimera.

[0016] In the above technical solution, the grafting molar ratio of the alkynyl-lysosome-targeting receptor ligand and the alkynyl-ALK5 inhibitor is 4:1.

[0017] Fourthly, the present invention also provides the application of the above-mentioned lysosomal targeting chimera in the preparation of drugs for the treatment or adjuvant treatment of liver fibrosis.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention utilizes highly biocompatible mesoporous polydopamine as a carrier to construct a safe and effective antioxidant nanoplatform that can scavenge reactive oxygen species, reduce oxidative stress levels, and thereby decrease the secretion of TGF-β by hepatic macrophages. Simultaneously, it loads an ALK5 small molecule inhibitor, which specifically recognizes and captures ALK5. By modifying M6P, it binds to the CI-M6PR receptor highly expressed on the surface of HSCs to achieve precise targeting, and enables ALK5 to be degraded via the autologous lysosomal pathway. This solves the toxic side effects of metal nanozymes and can precisely inhibit the TGF-β / SMAD signaling pathway in HSCs, thereby alleviating liver fibrosis.

[0020] The nano-drug delivery system of this invention avoids the chain effect of inhibiting TGF-β receptors in systemic tissues and organs, and overcomes the toxic side effects of non-natural antioxidants; it downregulates TGF-β levels while inhibiting its signal transduction, thereby reducing the production of extracellular matrix and reversing liver fibrosis, providing new technologies and ideas for the clinical treatment of liver fibrosis. Attached Figure Description

[0021] Figure 1 This is the 1H NMR spectrum of p-nitrophenyl-6-phosphate-β-D-mannopyranoside (S2).

[0022] Figure 2 This is the carbon NMR spectrum of p-nitrophenyl-6-phosphate-β-D-mannopyranoside (S2).

[0023] Figure 3 This is the 1H NMR spectrum of p-aminophenyl-6-phosphate-β-D-mannopyranoside (S3).

[0024] Figure 4 This is the carbon NMR spectrum of p-aminophenyl-6-phosphate-β-D-mannopyranoside (S3).

[0025] Figure 5 The NMR spectrum of alkynyl-M6P(S4) is shown in the form of 1H NMR.

[0026] Figure 6 The image shows the carbon NMR spectrum of alkynyl-M6P(S4).

[0027] Figure 7 The 1H NMR spectrum of alkynyl-R268712 is shown.

[0028] Figure 8 The image shows the carbon NMR spectrum of alkynyl-R268712.

[0029] Figure 9 This is a schematic diagram of the MAP NPs loading process.

[0030] Figure 10 To verify the degradation effect of MAP NPs on ALK5, among which Figure 10 -A shows the Western blotting (WB) results of MAP degradation of ALK5 at different ratios. Figure 10 -B represents the statistical results of the ability of MAP to degrade ALK5 at different ratios (n=3).

[0031] Figure 11 To verify the hemolytic effect of MAP NPs, among which Figure 11 -A shows the hemolytic effects of MAP and PDA at different drug concentrations. Figure 11 -B represents the hemolysis rate concentration change curve (n=3).

[0032] Figure 12 To verify the anti-liver fibrosis effect of MAP NPs, the following was included: Figure 12 -A is a representative image of a mouse liver tissue section stained with Masson staining; Figure 12 -B represents a semi-quantitative analysis of extracellular matrix deposition in the liver tissue of mice in each group. Scale bar: 100 μm, n = 5 per group; Figure 12 -C represents fibronectin in the liver tissue of mice in each group. Figure 12 -D represents type I collagen and Figure 12 -E represents the relative mRNA level of α-smooth muscle actin, with n=5 for each group; Figure 12 -F~ Figure 12 -I represents the representative Western blot images of fibronectin, type I collagen and α-smooth muscle actin in the liver tissue of mice in each group and their corresponding quantitative analysis, with n=3 for each group; Figure 12 -J~ Figure 12 -M represents representative images of immunohistochemical staining of fibronectin, type I collagen, and α-smooth muscle actin in the liver tissue of mice in each group, along with their corresponding quantitative analysis. n=5 for each group, scale bar: 100μm. Detailed Implementation

[0033] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the test methods in the following embodiments are conventional methods; the test reagents in the following embodiments are all commercially available.

[0034] Example 1 Synthesis of Polydopamine Nanoparticles (PDANPs)

[0035]

[0036] 0.5 g of F127 (polyethylene glycol-polypropylene glycol-polyethylene glycol) and 0.25 g of dopamine hydrochloride were dissolved in a solution containing 25 mL of water and 25 mL of ethanol, and the solution was stirred vigorously to obtain a clear solution at room temperature. Then, 0.25 mL of TMB (1,3,5-trimethylbenzene) was slowly added to the solution with a stirring speed of 500 rpm for 30 min. After stirring, 2.5 mL of concentrated ammonia (NH4OH) was added dropwise to the mixture to induce the self-polymerization reaction of the dopamine oligomers. After 30 min of continuous reaction, polydopamine nanoparticles with a mesoporous structure were formed. These nanoparticles were then centrifuged, washed three times with water, and dried overnight. The surface amino group number of the PDANPs was measured to be 912 μmol / g, and the particle size was approximately 100 nm.

[0037] Example 2: Azide treatment of PDANPs surface

[0038] (1) Preparation of FSO2N3

[0039] Under light-protected conditions, a 0.50 M, 2 mL solution of NaN3 (containing 0.065 g, 1 mmol NaN3) and methyl tert-butyl ether (MTBE, 2 mL) were placed in a 10 mL plastic bottle. 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-onium trifluoromethanesulfonate (0.3939 g, 1.2 mmol) was dissolved in 0.1 mL of acetonitrile (MeCN), and the resulting viscous solution was rapidly added to the NaN3 / H2O / MTBE mixture being stirred in an ice-water bath. The bottle was then rinsed with 0.1 mL of MeCN solution, which was also added to the reaction mixture. The reaction mixture was stirred in an ice-water bath for 10 min (600 rpm), and then the mixture was poured into a glass separating funnel. The mixture was allowed to stand in the funnel at room temperature for 30 min for phase separation. The organic phase separated from the aqueous phase; this colorless organic phase can be used as a solution of FSO2N3 in MTBE without further purification.

[0040] (2) Azide reaction

[0041] In a 10 mL glass flask, add 0.03 g (0.02 mmol) of PDANPs, FSO₂N₃ solution (1 mL of 500 μL FSO₂N₃ / MTBE mixed with 500 μL DMF, containing 0.2 mmol FSO₂N₃), and KHCO₃ aqueous solution (3 M, 260 μL KHCO₃, containing 0.8 mmol KHCO₃). Stir the reaction mixture at room temperature for 5 min. After the reaction is complete, centrifuge at 10,000 rpm for 10 min and freeze-dry.

[0042] Example 3: Synthesis of alkynyl-mannose-6-phosphate (alkynyl-M6P)

[0043]

[0044] 120.0 mg of diphenyl chlorophosphate was added to a dry pyridine suspension containing 107.0 mg of p-nitrophenyl-β-D-mannopyranoside (S1) and 4.9 mg of DMAP. After stirring at room temperature for 12 h, thin-layer chromatography showed complete consumption of the starting material. The reaction was quenched with methanol, concentrated under vacuum, and purified by silica gel chromatography to obtain a colorless oil, which was the compound p-nitrophenyl-6-phosphate-β-D-mannopyranoside (S2).

[0045] In a hydrogen-filled reaction vessel under high vacuum, 138.1 mg S2 and 7.2 mg PtO2 were dissolved in 1 g / 20 mL ethanol. The reaction was maintained at room temperature and vigorously stirred under high hydrogen pressure for 12 h. Thin-layer chromatography showed complete consumption of the starting material, forming a single product. The reaction mixture was filtered through diatomaceous earth, and the solvent was removed under vacuum. The reaction mixture was purified by preparative liquid chromatography to give a brown solid (50% yield) as the compound p-aminophenyl-6-phosphate-β-D-mannopyranoside (S3).

[0046] 45.5 mg S3 was dissolved in 10 ml DMF, and 37.9 mg of 4-pentynyl succinimide and 78.8 mg of N,N-diisopropylethylamine (DIPEA) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, DMF was removed, and the product alkynyl-M6P(S4) was purified by semi-preparative HPLC.

[0047] The 1H and 1C NMR spectra of p-nitrophenyl-6-phosphate-β-D-mannopyranoside (S2) are shown below. Figure 1 and Figure 2 As shown. The 1H and 1C NMR spectra of p-aminophenyl-6-phosphate-β-D-mannopyranoside (S3) are shown below. Figure 3 and Figure 4 As shown in the figure. The 1H and 1C NMR spectra of alkynyl-M6P(S4) are shown in the figure. Figure 5 and Figure 6 As shown.

[0048] Example 4 Synthesis of alkynyl-R268712

[0049] 100.0 mg of R268712 (an ALK5 inhibitor, purchased from Shanghai Aibisin Biotechnology Co., Ltd.) and 27.0 mg of 4-pentanoic acid were dissolved in anhydrous dichloromethane (DCM). 128.0 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 6.7 mg of 4-dimethylaminopyridine (DMAP) were added to the mixture, and the mixture was stirred overnight at room temperature. The reaction was monitored by thin-layer chromatography, and the product was purified by column chromatography (yield: 85.3 mg, 70%).

[0050] The 1H and 1C NMR spectra of alkynyl-R268712 are shown below. Figure 7 and Figure 8 As shown.

[0051] Example 5

[0052] Reference Figure 9 The schematic diagram shows how MAP NPs are obtained by loading alkynyl-M6P and alkynyl-R268712 into PDANPs via the Click reaction.

[0053] Take a 2 mL finger tube, add 1 mL of water, and add 4.56 μmol (2.02 mg) of alkynyl-R268712 prepared in Example 4 and 4.56 μmol (1.97 mg) of alkynyl-M6P prepared in Example 3 at a 1:1 molar ratio. After placing the tube in a shaker for 1 min, add 10 mg of azide-treated PDANPs prepared in Example 2, 10 μL of 1 mol / L sodium ascorbate, and 1 μmol of 1 mol / L CuSO4 in sequence. React at 25 °C in a shaker for 10 h. The lower layer sample is MAPNPs, which is washed three times with anhydrous ethanol and dispersed in anhydrous ethanol for later use. Similarly, MAPNPs with alkynyl-R268712:alkynyl-M6P molar ratios of 1:2, 1:3, 1:4, and 1:5 are obtained by following the above steps.

[0054] LX-2 cells were seeded at a density of 4 × 10⁴ cells / well in 6-well culture plates and cultured overnight for adherence. After induction with TGF-β1 (5 ng / mL) for 24 h, the drug was added. MAPNPs obtained by alkynyl-R268712:alkynyl-M6P molar ratios of 1:1, 1:2, 1:3, 1:4, and 1:5 were used to treat LX-2 cells, with PBS as a negative control, to determine the effect of MAPNPs on degrading alkynyl-R268712. Cellular proteins were extracted after 24 h and analyzed by Western blotting, with GAPDH as an internal control to quantify ALK5 expression levels. Results are as follows: Figure 10As shown in the figure. The results show that the optimal molar ratio of alkynyl-R268712 to alkynyl-M6P is 1:4.

[0055] Example 6 Biosafety Experiment

[0056] To assess the biosafety of the nanomedicine, its hemolytic effect was evaluated in vitro. MAPNPs obtained in Example 5 with a molar ratio of 1:4 for alkynyl-R268712 and alkynyl-M6P were used as nanomedicines. Injection concentrations of the nanomedicine were prepared with physiological saline and diluted to concentrations of 4 μg / ml, 8 μg / ml, 16 μg / ml, 32 μg / ml, 64 μg / ml, 128 μg / ml, 256 μg / ml, and 512 μg / ml. Physiological saline and 0.5% SDS-PAGE ultrapure water were used as negative and positive controls, respectively. Fresh blood was collected from the venous plexus of mouse fundus, centrifuged at low temperature, and repeatedly washed with physiological saline to obtain blood cells. Equal volumes of drug solution, physiological saline, and ultrapure water were slowly added to the red blood cells and mixed to achieve a red blood cell concentration of approximately 2%. Incubation was then performed at 37°C with gentle shaking. After incubation, centrifugation was performed to assess the drug-induced hemolytic effect. The OD value of the supernatant at 577 nm was measured, and the hemolysis rate was calculated. Calculate the hemolysis rate (Z) using the formula:

[0057]

[0058] In the formula: At and An are the concentrations of absorbance values ​​of the experimental group and the negative group, respectively, and Ap is the absorbance value of the positive group.

[0059] The results are as follows Figure 11 As shown in the figure. The results show that neither PDA nor MAP caused significant hemolysis at high drug concentrations, indicating the superior biocompatibility of MAP nanomedicines.

[0060] Example 7: Verification of the anti-liver fibrosis effect of MAP NPs

[0061] A liver fibrosis model was established by gavage administration of CCl4 (3.2 g / kg, diluted in 20% corn oil solution) twice weekly for 4 consecutive weeks to 6-8 weeks of age. Mice were randomly divided into four groups: a blank control group (n=8), receiving corn oil (150 μL) twice weekly for 4 consecutive weeks; a CCl4 group (n=8), receiving 150 μL of physiological saline via tail vein injection every other day starting from week 3 of modeling; a PDA group (n=8), receiving PDA NPs via tail vein injection every other day starting from week 3 of modeling; and a MAP group (n=8), receiving MAP NPs via tail vein injection every other day starting from week 3 of modeling. The treatment dose for each group was maintained at 15 mg / kg. After 4 weeks, all mice were euthanized, and liver and whole blood samples were collected for further analysis.

[0062] To evaluate its anti-fibrotic effect, the mRNA levels of α-SMA, Collagen 1A1, and Fibronectin in liver tissue were first detected using standard qPCR. Subsequently, tissue proteins were extracted, and the expression levels of α-SMA, Collagen 1A1, and Fibronectin were detected using Western blot, with GAPDH as an internal control. ImageJ software was used to quantify the protein grayscale values. Masson staining and immunohistochemical staining were performed on liver tissue sections to quantify the expression of α-SMA, Collagen 1A1, and Fibronectin. All data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8.0 software to evaluate the difference between the means of the two groups. Significance levels are expressed as follows: *P<0.05; **P<0.01; ***P<0.001. Results are as follows: Figure 12 As shown.

[0063] like Figure 12 As shown, Masson staining revealed a significant amount of matrix deposition in the livers of CCl4-treated mice. MAP treatment resulted in a significant reduction in matrix deposition. Figure 12 -A, Figure 12 -B). Post-treatment analysis of fibrosis marker proteins showed that the mRNA levels of fibronectin, type I collagen, and α-smooth muscle actin were significantly reduced in MAP-treated liver tissue. Figure 12 -C~ Figure 12 -E). Western blot analysis further quantified these reductions, and the expression of these proteins was also significantly reduced in CCl4-induced liver fibrosis mice treated with MAP. Figure 12 -F~ Figure 12 -I). Furthermore, immunohistochemical staining also supports these findings; after MAP treatment, the areas expressing fibronectin, type I collagen, and α-smooth muscle actin were significantly reduced ( Figure 12 -J~ Figure 12 In summary, these results confirm the potent efficacy of MAP in slowing the progression of CCl4-induced liver fibrosis in mice.

[0064] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. A lysosome-targeting chimera, characterized in that, The lysosome-targeting chimera consists of an azide-modified mesoporous polydopamine nanoparticle carrier, an alkynyl-lysosome-targeting receptor ligand, and an alkynyl-ALK5 inhibitor.

2. The lysosome-targeting chimera according to claim 1, characterized in that, The alkynyl-lysosome-targeting receptor ligand is alkynyl-mannose-6-phosphate.

3. The lysosome-targeting chimera according to claim 1, characterized in that, The alkynyl-ALK5 inhibitor is alkynyl-R268712.

4. A composition, characterized in that, Includes the lysosomal targeting chimera as described in any one of claims 1 to 2 and a medically acceptable carrier.

5. The method for preparing the lysosomal targeting chimera according to any one of claims 1 to 3, characterized in that, The process includes the following steps: using azidized mesoporous polydopamine nanoparticles as a carrier, an alkynyl-lysosome-targeting receptor ligand and an alkynyl-ALK5 inhibitor are loaded onto the azidized mesoporous polydopamine nanoparticles via a Click reaction to obtain a lysosome-targeting chimera.

6. The preparation method according to claim 5, characterized in that, The grafting molar ratio of the alkynyl-lysosome-targeting receptor ligand to the alkynyl-ALK5 inhibitor is 4:

1.

7. The use of the lysosomal targeting chimera according to any one of claims 1 to 3 in the preparation of drugs for the treatment or adjuvant treatment of liver fibrosis.

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