Lysosome targeting chimera as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicinal chemistry, and discloses a lysosome targeting chimera, which is composed of an azidation mesoporous polydopamine nanoparticle carrier, an alkynyl-lysosome targeting receptor ligand and an alkynyl-ALK5 inhibitor. Mesoporous polydopamine with high biocompatibility is used as a carrier, a safe and effective anti-oxidation nano platform is constructed, active oxygen is removed, the oxidative stress level is reduced, then TGF-beta secreted by liver macrophages is reduced, meanwhile, an ALK5 small-molecule inhibitor is loaded, ALK5 is specifically recognized and captured, and the anti-oxidation nano platform has a good anti-oxidation effect on liver macrophages. Through combination of modified M6P and a CI-M6PR receptor highly expressed on the surface of HSC, a precise targeting effect is realized, and a new technology and thought are provided for clinical treatment of hepatic fibrosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and relates to a lysosome-targeted chimera and a preparation method and application thereof, and specifically relates to a lysosome-targeted chimera based on mesoporous polydopamine nanoparticles and a preparation method and application thereof. Background Art

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

[0003] Hepatic stellate cells (HSCs) are resident mesenchymal cells in the liver, located in the perisinusoidal space between the hepatic sinusoidal endothelial cells and hepatocytes. After liver injury, HSCs are transdifferentiated from quiescent cells that store vitamin A into myofibroblast-like cells with proliferative, contractile, inflammatory and chemotactic properties. They are characterized by excessive secretion of extracellular matrix mainly composed of type I collagen and type III collagen, and the formation of scar tissue, which leads to fibrosis. They have been recognized as the main driving factor of fibrosis in damaged human livers. Transforming growth factor β (TGF-β) plays a key role in activating HSCs through signal transduction of the classical SMAD pathway. In addition, oxidative stress can also activate HSCs by stimulating macrophages to secrete TGF-β, and is closely related to the TGF-β signaling pathway. Therefore, the TGF-β signaling pathway and oxidative stress related to HSC activation can serve as key targets for changing the outcome of fibrosis development.

[0004] The use of TGF-β targeted drugs (TGF-β antibodies, TGF-β receptor inhibitors and recombinant proteins) to inhibit its signal transduction has become a hot topic in basic research on anti-liver fibrosis. Among them, ALK5 (activin receptor-like kinase 5) inhibitors, i.e., TGF-β type I receptor inhibitors, highly specifically block the catalytic activity of ALK5, inhibit TGF-β / SMAD signal transduction, prevent matrix deposition, and exert its anti-fibrotic activity. However, ALK5i (ALK5 small molecule inhibitor) has not yet been applied to clinical specific liver fibrosis treatment. The main limiting factors are as follows:

[0005] ① ALK5i inhibits TGF-β receptors, which may cause toxic side effects on multiple organs involved in the TGF-β cascade and affect the metabolism of normal tissues and organs;

[0006] ② The use of ALK5i alone can increase the synthesis of lipid droplets in hepatocytes, and it is necessary to develop a precise drug delivery system for HSCs.

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

[0008] Some studies have shown that antioxidants can play an anti-fibrotic role. Silybin is a plant medicine with important antioxidant properties. It can reverse liver fibrosis and stimulate liver tissue regeneration by increasing glutathione levels and reducing the production of type III collagen in patients with cirrhosis. It has been used in clinical liver protection treatment, but its treatment cycle is long and the efficacy is limited. In addition, antioxidant nanomaterials based on bioenzymes and nanozymes have been developed one after another, such as metal organic framework nanoparticles loaded with superoxide dismutase and catalase and CeO 2 Nanoparticles, etc. These metal nanomaterials often have low biocompatibility and potential long-term toxic side effects, which limits their clinical application. Summary of the invention

[0009] In order to solve the technical problems of toxic side effects and low drug delivery accuracy of TGF-β targeted drugs, the present invention provides a lysosome-targeted chimera, which uses highly biocompatible mesoporous polydopamine as a carrier to construct a safe and effective antioxidant nanoplatform to remove reactive oxygen species, reduce oxidative stress levels, and thereby reduce the secretion of TGF-β by liver macrophages. At the same time, it loads ALK5 small molecule inhibitors to specifically recognize and capture ALK5, and achieves precise targeting by modifying M6P to bind to the CI-M6PR receptor highly expressed on the surface of HSCs, providing new technologies and ideas for the clinical treatment of liver fibrosis.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

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

[0012] In the above technical solution, the alkynyl-lysosomal 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-mentioned lysosome targeting chimera and a medically acceptable carrier.

[0015] In a third aspect, the present invention provides a method for preparing the above-mentioned lysosome targeting chimera, comprising the following steps: using azidized mesoporous polydopamine nanoparticles as carriers, and loading alkynyl-lysosome targeting receptor ligands and alkynyl-ALK5 inhibitors onto azidized mesoporous polydopamine nanoparticles through a Click reaction to obtain a lysosome targeting chimera.

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

[0017] In a fourth aspect, the present invention also provides the use of the above-mentioned lysosome targeting chimera in the preparation of drugs for treating or assisting in the treatment of liver fibrosis.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention uses highly biocompatible mesoporous polydopamine as a carrier to construct a safe and effective antioxidant nanoplatform, which can remove reactive oxygen, reduce the level of oxidative stress, and thereby reduce the secretion of TGF-β by liver macrophages; at the same time, it loads an ALK5 small molecule inhibitor, specifically recognizes and captures ALK5, and achieves precise targeting by modifying M6P to bind to the CI-M6PR receptor highly expressed on the surface of HSC, and degrades ALK5 through the autolysosomal pathway, thus solving the toxic side effects of metal nanozymes and accurately inhibiting the TGF-β / SMAD signaling pathway in HSC, thereby playing a role in alleviating liver fibrosis.

[0020] The nano drug delivery system of the present invention avoids the chain effect of inhibiting TGF-β receptors in tissues and organs throughout the body, and overcomes the toxic side effects produced by non-natural antioxidants; it downregulates TGF-β levels while inhibiting its signal transmission, thereby reducing the production of extracellular matrix and reversing liver fibrosis, providing new technology and ideas for the clinical treatment of liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0025] Figure 5 This is the H NMR spectrum of alkynyl-M6P(S4).

[0026] Figure 6 This is the C NMR spectrum of alkynyl-M6P(S4).

[0027] Figure 7 This is the H NMR spectrum of alkynyl-R268712.

[0028] Figure 8 This is the C NMR spectrum of alkynyl-R268712.

[0029] Fig. 9 Schematic diagram of the MAP NPs loading process.

[0030] Fig.10 To verify the degradation effect of MAP NPs on ALK5, Fig.10 -A is the WB effect diagram of ALK5 degradation by MAP at different ratios. Fig.10 -B is the statistical result of the ability of different ratios of MAP to degrade ALK5 (n=3).

[0031] Fig.11 To verify the hemolytic effect of MAP NPs, Fig.11 -A is the hemolytic effect diagram of MAP and PDA at different drug concentrations, Fig.11 -B is the hemolysis rate concentration change curve (n=3).

[0032] Fig.12 To verify the anti-liver fibrosis effect of MAP NPs, including: Fig.12 -A is a representative image of Masson staining of mouse liver tissue sections; Fig.12 -B is a semi-quantitative analysis of extracellular matrix deposition in liver tissue of mice in each group, scale bar: 100 μm, n = 5 per group; Fig.12 -C is the fibronectin in the liver tissue of mice in each group, Fig.12 -D is type I collagen and Fig.12 -E is the relative mRNA level of α-smooth muscle actin, n = 5 per group; Fig.12 -F~ Fig.12 -I are representative Western blot images of fibronectin, type I collagen, and α-smooth muscle actin in the liver tissues of mice in each group and their corresponding quantitative analysis, n = 3 per group; Fig.12 -J~ Fig.12 -M are representative images of immunohistochemical staining of fibronectin, type I collagen, and α-smooth muscle actin in the liver tissues of mice in each group and their corresponding quantitative analysis, n = 5 in each group, scale bar: 100 μm. DETAILED DESCRIPTION

[0033] The following examples 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 examples are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified; the test reagents in the following examples 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 mixture and stirred vigorously to obtain a clear solution at room temperature. Then, 0.25 mL of TMB (1,3,5-trimethylbenzene) was slowly injected into the solution at a stirring speed of 500 rpm and stirred for 30 min. After stirring, 2.5 ml of concentrated ammonia (NH 4 OH) was added dropwise to the above mixture to induce the self-polymerization reaction of dopamine oligomers. After continuous reaction for 30 minutes, polydopamine nanoparticles with a mesoporous structure were formed, which were then centrifuged and washed three times with water and then dried overnight. The number of amino groups on the surface of PDANPs was measured to be 912 μmol / g, and the particle size was about 100 nm.

[0037] Example 2 Surface azidation of PDANPs

[0038] (1)FSO 2 N 3 Preparation

[0039] Use NaN in dark conditions 3 Aqueous solution (0.50M, 2ml, containing 0.065g, 1mmol NaN 3 ) and methyl tert-butyl ether (MTBE, 2 ml) were placed in a 10 ml plastic bottle. 1-(Fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate (0.3939 g, 1.2 mmol) was dissolved in 0.1 ml of acetonitrile (MeCN), and the resulting viscous solution was quickly added to NaN stirred in an ice-water bath. 3 / H 2 O / MTBE mixture. Then 0.1 ml of MeCN solution was taken to rinse the vial, 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 separation funnel. The mixture was placed in the funnel at room temperature for 30 min to allow phase separation. The organic phase was separated from the aqueous phase, and the colorless organic phase was used as FSO without further purification.2 N 3 Solution in MTBE.

[0040] (2) Azide reaction

[0041] In a 10 ml glass flask, 0.03 g (0.02 mmol) of PDANPs, FSO 2 N 3 Solution (take FSO 2 N 3 / MTBE 500ul, add DMF 500μl, a total of 1ml, containing 0.2mmol FSO 2 N 3 ) and KHCO 3 Aqueous solution (3M, 260 μl KHCO 3 , containing 0.8mmol KHCO 3 The reaction mixture was stirred at room temperature for 5 min. After the reaction was completed, it was centrifuged at 10000 rpm for 10 min and freeze-dried.

[0042] Example 3 Synthesis of Alkynyl-Mannose-6-Phosphate (Alkynyl-M6P)

[0043]

[0044] 120.0 mg of diphenyl chlorophosphate was added to a stirred suspension of 107.0 mg of p-nitrophenyl-β-D-pyranoside (S1) and 4.9 mg of DMAP in dry pyridine. After stirring at room temperature for 12 hours, thin layer chromatography showed that the starting material was completely consumed. The reaction was quenched with methanol, concentrated in vacuo, and purified by silica gel chromatography to obtain a colorless oil, which was the compound p-nitrophenyl-6-phospho-β-D-pyranoside (S2).

[0045] In a reaction vessel filled with hydrogen under high vacuum conditions, 138.1 mg S2 and 7.2 mg PtO 2 Dissolved in 1g / 20ml ethanol. The reaction was kept at room temperature and stirred vigorously under a large pressure of hydrogen for 12h, after which thin layer chromatography showed that the raw material was completely consumed to form a single product. The reaction mixture was filtered through diatomaceous earth and the solvent was removed in vacuo. The reaction mixture was purified by preparative liquid chromatography to obtain a tan solid (yield 50%), which was the compound p-aminophenyl-6-phospho-β-D-pyranoside (S3).

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

[0047] The H-NMR and C-NMR spectra of p-nitrophenyl-6-phospho-β-D-pyranoside (S2) are shown in Figure 1 and Figure 2 The H-NMR and C-NMR spectra of p-aminophenyl-6-phospho-β-D-pyranoside (S3) are shown in Figure 3 and Figure 4 The H NMR spectrum and C NMR spectrum of alkynyl-M6P(S4) are shown as follows: Figure 5 and Figure 6 shown.

[0048] Example 4 Synthesis of Alkynyl-R268712

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

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

[0051] Example 5

[0052] Reference Fig. 9 As shown in the schematic diagram, alkynyl-M6P and alkynyl-R268712 were loaded onto PDANPs through Click reaction to obtain MAP NPs.

[0053] Take a 2 mL finger tube, add 1 mL of water and add alkynyl-R268712 (4.56 μmol, 2.02 mg) prepared in Example 4 and alkynyl-M6P (4.56 μmol, 1.97 mg) prepared in Example 3 in a 1:1 molar ratio, place in an oscillator for 1 min, and then add 10 mg of azidated PDANPs prepared in Example 2, 10 μL of 1 mol / L sodium ascorbate and 1 μmol of 1 mol / L CuSO 4, reacted in an oscillator at 25°C for 10 h, the lower layer sample was MAPNPs, washed three times with anhydrous ethanol, and dispersed in anhydrous ethanol for later use. Similarly, MAPNPs with molar ratios of alkynyl-R268712:alkynyl-M6P=1:2, 1:3, 1:4 and 1:5 were obtained according to the above steps.

[0054] LX-2 cells were seeded in 6-well culture plates at a density of 4×104 / well, cultured overnight to adhere, and then induced with TGF-β1 (5ng / mL) for 24h before adding the drug. LX-2 cells were treated with MAPNPs obtained with a molar ratio of alkynyl-R268712:alkynyl-M6P = 1:1, 1:2, 1:3, 1:4, and 1:5, and PBS was used as a negative control group to determine the effect of MAPNPs in degrading alkynyl-R268712. Cell proteins were extracted after 24h and analyzed by Western blotting, with GAPDH as an internal reference to quantify the expression level of ALK5. The results are shown in Fig.10 The results showed that the optimal molar ratio of alkynyl-R268712:alkynyl-M6P was 1:4.

[0055] Example 6 Biosafety Experiment

[0056] In order to evaluate the biosafety of nanomedicine, its hemolytic effect was evaluated in vitro. MAPNPs obtained with a molar ratio of 1:4 between alkynyl-R268712 and alkynyl-M6P in Example 5 were used as nanomedicines. The injection concentration of the nanomedicine was prepared with normal saline and diluted to 4μg / ml, 8μg / ml, 16μg / ml, 32μg / ml, 64μg / ml, 128μg / ml, 256μg / ml, and 512μg / ml concentrations. Normal saline and 0.5% SDS ultrapure water were used as negative and positive controls. Fresh blood was collected from the venous plexus of the mouse fundus, and blood cells were obtained by repeated washing with normal saline after low-temperature centrifugation. Equal volumes of drug solution, normal saline and ultrapure water were slowly added to the red blood cells and mixed to make the red blood cell concentration about 2%. Subsequently, incubation and gentle shaking were performed at 37°C. Centrifugation was performed after incubation to evaluate the hemolytic effect caused by the drug. The OD value of the supernatant at 577nm was measured to calculate the hemolysis rate. The hemolysis rate (Z) was calculated according to the formula:

[0057]

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

[0059] The results are as follows Fig.11 The results showed that PDA and MAP did not cause significant hemolysis at high drug concentrations, indicating the superior biocompatibility of MAP nanomedicine.

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

[0061] Male C57B / 6J mice aged 6-8 weeks weighing approximately 20 g were fed CCl twice a week for 4 consecutive weeks. 4 (3.2 g / Kg, diluted to 20% corn oil solution) was intragastrically administered to establish a liver fibrosis model. The mice were randomly divided into 4 groups: a blank control group (n = 8), which was intragastrically administered with corn oil (150 μL) twice a week for 4 consecutive weeks; 4 The mice in the PDA group (n=8) were injected with 150 μL of normal saline via the tail vein every other day starting from the third week of modeling; the PDA group (n=8) were injected with PDA NPs via the tail vein every other day starting from the third week of modeling; the MAP group (n=8) were injected with MAP NPs via the tail vein every other day starting from the third week of modeling. The treatment dose of each treatment group was maintained at 15 mg / kg. After 4 weeks, all mice were euthanized, and their liver and whole blood samples were collected for further analysis.

[0062] In order to evaluate its anti-fibrotic effect, the mRNA levels of α-SMA, Collagen 1A1, and Fibronectin in liver tissue were first detected using the qPCR standard procedure. Tissue proteins were then extracted, and the expression levels of α-SMA, Collagen 1A1, and Fibronectin proteins were detected using Western blot technology. GAPDH was used as an internal reference, and the grayscale value of the protein was quantified using ImageJ software. Masson staining and immunohistochemical staining were performed on liver tissue sections to quantitatively analyze the expression of α-SMA, Collagen 1A1, and Fibronectin. All data were expressed as mean ± standard deviation, and GraphPad Prism 8.0 software was used for statistical analysis to evaluate the differences in the mean values ​​between the two groups. The significance levels are expressed as follows: *P<0.05; **P<0.01; ***P<0.001. The results are shown in Fig.12 shown.

[0063] like Fig.12 As shown, Masson staining showed that CCl 4 There was considerable matrix deposition in the livers of treated mice. MAP treatment resulted in a significant reduction in matrix deposition ( Fig.12 -A, Fig.12 -B). Post-treatment analysis of fibrosis marker proteins showed that mRNA levels of fibronectin, type I collagen, and α-smooth muscle actin were significantly decreased in MAP-treated liver tissues ( Fig.12 -C~ Fig.12 -E). These reductions were further quantified by Western blot analysis. 4The expression of these proteins was also significantly reduced in mice with induced liver fibrosis after MAP treatment ( Fig.12 -F~ Fig.12 In addition, immunohistochemical staining also supported these findings, showing that the areas expressing fibronectin, type I collagen, and α-smooth muscle actin were significantly reduced after MAP treatment ( Fig.12 -J~ Fig.12 -M). Taken together, these results confirm the role of MAP in slowing down CCl 4 Strong therapeutic effect on the progression of liver fibrosis induced in mice.

[0064] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in 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 azidated mesoporous polydopamine nanoparticle carrier, an alkynyl-lysosome targeting receptor ligand and an alkynyl-ALK5 inhibitor.

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

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

4. A composition, characterized in that It comprises the lysosomal targeting chimera according to 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 method comprises the following steps: using azidized mesoporous polydopamine nanoparticles as carriers, loading alkynyl-lysosome targeting receptor ligands and alkynyl-ALK5 inhibitors onto azidized mesoporous polydopamine nanoparticles through Click reaction to obtain lysosome targeting chimeras.

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

1.

7. Use of the lysosome targeting chimera according to any one of claims 1 to 3 in the preparation of a drug for treating or assisting in the treatment of liver fibrosis.

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