Lysosome targeted chimera self-assembled micelle for degrading CTGF (cytotoxic T growth factor) as well as preparation method and application of lysosome targeted chimera self-assembled micelle

Abstract

The invention provides a polypeptide lysosome targeting chimera for degrading CTGF (cytotoxic growth factor) protein, and the structural formula of the polypeptide lysosome targeting chimera is as shown in the specification, namely # imgabs0 #. The invention also provides a lysosome targeted chimera self-assembled micelle for degrading the CTGF protein, and the structure of the lysosome targeted chimera self-assembled micelle is as follows: # imgabs 1 #. The invention also provides a preparation method of the lysosome targeted chimera self-assembled micelle for degrading the CTGF protein and application of the lysosome targeted chimera self-assembled micelle in preparation of medicines for treating tumors. The CTGF in the triple negative breast cancer tumor tissue can be efficiently degraded, tumor growth, lung metastasis and bone metastasis are inhibited, and then the anti-tumor effect is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a lysosome-targeted chimera self-assembly micelle for targeted degradation of CTGF, and a preparation method and application thereof. Background Art

[0002] Triple-negative breast cancer (TNBC) is highly invasive, prone to recurrence, and has a poor prognosis. First-line chemotherapy is ineffective and prone to drug resistance, and the response rate to emerging immunotherapy is low. There is an urgent need to explore effective targeted treatment strategies. Multiple analyses have revealed the complex relationship between the highly heterogeneous tumor microenvironment of TNBC and cancer progression, metastasis, and treatment response. Studies have shown that the extracellular matrix protein connective tissue growth factor (CTGF) is overexpressed in TNBC tissues and is highly correlated with the growth and metastasis of TNBC. As a multifunctional protein, CTGF can interact with functional proteins in the tumor microenvironment to promote tumor proliferation, migration, angiogenesis, and metastasis. Therefore, antagonists targeting CTGF are extremely valuable in anti-TNBC treatment. At present, the monoclonal antibody FG-3019 / pamrevlumab combined with chemotherapy for CTGF has completed phase I clinical trials. However, because the flexible hinge region of CTGF is easily hydrolyzed, and its hydrolyzed domains can still play a cancer-promoting function respectively, antibody drugs that only target a single domain of CTGF are difficult to completely inhibit CTGF activity, limiting the efficacy of antibody drugs.

[0003] In recent years, targeted protein degradation (TPD) technology has become a promising tumor treatment method. This technology can specifically identify and degrade target pathogenic proteins to achieve complete inhibition of protein function. Among them, lysosome-targeting chimera (LYTAC) technology is a new strategy for targeted degradation of extracellular proteins and membrane proteins, which has gradually been applied to new drug research and development. The development of new drugs targeting CTGF based on the LYTAC strategy can overcome the challenges faced by inhibitors or antibody drugs and has important application prospects. In addition, compared with LYTAC with antibody as the target head, LYTAC based on peptide target head has the advantages of easy design and synthesis, small molecular weight and high biosafety. However, peptide drugs have low stability and short half-life under physiological conditions, which limits their in vivo application.

[0004] Nano drug delivery systems have the advantages of improving the in vivo stability and biodistribution of peptide drugs, prolonging the in vivo circulation time of encapsulated drugs, reducing drug toxicity and side effects, and thus improving their therapeutic effects. They are currently widely used in targeted delivery of tumor drugs. Since the concentration of glutathione in the tumor microenvironment (2-10mM) is 100 times higher than that in normal tissues, connecting the drug to the micelle material through a glutathione-responsive disulfide bond can achieve selective release of the drug at the tumor site, reduce systemic toxicity and side effects, and enhance the efficacy.

[0005] Therefore, the present invention designs and constructs a self-assembled micelle NanoCLY with reduction responsiveness based on LYTAC that degrades CTGF. The micelle can circulate in vivo for a long time and specifically accumulate in the tumor site of triple-negative breast cancer. Under the condition of relatively high concentration of glutathione in the tumor microenvironment, the polypeptide LYTAC CL8-M6P3 is released to achieve CTGF degradation and effectively inhibit the in vivo growth, lung metastasis and bone metastasis of triple-negative breast cancer. There is no similar report so far. Summary of the invention

[0006] In view of the above-mentioned technical problems in the prior art, the present invention provides a lysosome-targeted chimera self-assembled micelle for degrading CTGF and a preparation method and application thereof. The lysosome-targeted chimera self-assembled micelle for degrading CTGF and a preparation method and application thereof are intended to solve the technical problem that the prior art has poor effect in treating triple-negative breast cancer with drugs.

[0007] The present invention provides a polypeptide-based lysosomal targeting chimera for degrading CTGF protein, the structural formula of which is shown below:

[0008]

[0009] The present invention also provides a method for preparing the above-mentioned polypeptide-based lysosomal targeting chimera for degrading CTGF protein, comprising the following steps:

[0010] (1) According to the solid phase synthesis method, Rink Amide MBHA amino resin was used as a carrier and ethyl 2-oxime cyanoacetate / N,N'-diisopropylcarbodiimide was used as a condensation system to obtain a resin connected with the polypeptide CL8-(PβA)3;

[0011] The amino acid sequence of CL8-(PβA)3 is:

[0012] H-CPSAAHYL-PEG3-Pra-βAla-Pra-βAla-Pra-βAla; where

[0013] The Pra-βAla-Pra-βAla-Pra-βAla repeat sequence provides alkyne groups for subsequent click chemistry reactions to achieve mannose-6-phosphate modification;

[0014] (2) Through a copper-catalyzed click chemistry reaction, using cuprous sulfate / tri(benzyltriazolylmethyl)amine / sodium ascorbate as a catalyst system, the resin connected to the polypeptide CL8-(PβA)3, azido-mannose-6-phosphate, cuprous sulfate, tri(benzyltriazolylmethyl)amine and sodium ascorbate were added to a mixed solvent of N,N-dimethylformamide and pure water, and stirred at room temperature in the dark for reaction (20 to 30 hours), followed by cutting, purification and lyophilization to obtain a polypeptide-based lysosomal targeting chimera LYTAC CL8-M6P3 for degrading CTGF protein;

[0015] The amino acid sequence of the polypeptide LYTAC CL8-M6P3 is:

[0016] H-CPSAAHYL-PEG3-Pra(M6P)-βAla-Pra(M6P)-βAla-Pra(M6P)-βAla-NH2;

[0017] The structural formula is shown below:

[0018]

[0019] Furthermore, the molar ratio of CL8-(PβA)3, mannose-6-phosphate azide, cuprous sulfate, tris(benzyltriazolylmethyl)amine and sodium ascorbate is 1:4.5:4.5:4.5:5.

[0020] The present invention provides a lysosome-targeted chimeric self-assembled micelle for degrading CTGF protein, and its structure is as follows:

[0021]

[0022] Furthermore, the self-assembled micelle is a double-layer structure, the inner layer is dodecyl, and the outer layer is the polypeptide LYTAC described in claim 1 connected by a disulfide bond.

[0023] The present invention also provides a method for preparing the above-mentioned lysosome-targeted chimeric self-assembled micelles for degrading CTGF, comprising the following steps:

[0024] (1) According to the solid phase synthesis method, Rink Amide MBHA amino resin was used as a carrier and ethyl 2-oxime cyanoacetate / N,N'-diisopropylcarbodiimide was used as a condensation system to obtain a resin connected with the polypeptide CL8-(PβA)3;

[0025] The amino acid sequence of CL8-(PβA)3 is:

[0026] H-CPSAAHYL-PEG3-Pra-βAla-Pra-βAla-Pra-βAla; where

[0027] The Pra-βAla-Pra-βAla-Pra-βAla repeat sequence provides alkyne groups for subsequent click chemistry reactions to achieve mannose-6-phosphate modification;

[0028] (2) Through a copper-catalyzed click chemistry reaction, using cuprous sulfate / tri(benzyltriazolylmethyl)amine / sodium ascorbate as a catalyst system, the resin connected to the polypeptide CL8-(PβA)3, azido-mannose-6-phosphate, cuprous sulfate, tri(benzyltriazolylmethyl)amine and sodium ascorbate were added to a mixed solvent of N,N-dimethylformamide and pure water, and stirred at room temperature in the dark for reaction (20 to 30 hours), followed by cutting, purification and lyophilization to obtain a polypeptide-based lysosomal targeting chimera LYTAC CL8-M6P3 for degrading CTGF protein;

[0029] The amino acid sequence of the polypeptide LYTAC CL8-M6P3 is:

[0030] H-CPSAAHYL-PEG3-Pra(M6P)-βAla-Pra(M6P)-βAla-Pra(M6P)-βAla-NH2;

[0031] The structural formula is shown below:

[0032]

[0033] (3) Dissolve 2,2'-dithiodipyridine in methanol, add 1-dodecanethiol solution dropwise under stirring, stir at room temperature in the dark for 3 seconds, purify to remove excess 2,2'-dithiodipyridine, and freeze-dry to obtain 1-dodecanethiol

[0034] -Pure thiopyridine;

[0035] (4) dissolving 1-dodecane-thiopyridine in N,N-dimethylformamide, adding the polypeptide LYTAC CL8-M6P3 aqueous solution dropwise under stirring, reacting under stirring at room temperature in the dark, extracting to remove excess 1-dodecane-thiopyridine, and then freeze-drying to obtain pure 1-dodecane-CL8-M6P3;

[0036] (5) 1-Dodecane-CL8-M6P3 was dissolved in phosphate buffered saline and sonicated at room temperature to obtain LYTAC self-assembled micelles that degrade CTGF protein.

[0037] Furthermore, the molar ratio of CL8-(PβA)3, mannose-6-phosphate azide, cuprous sulfate, tris(benzyltriazolylmethyl)amine and sodium ascorbate is 1:4.5:4.5:4.5:5.

[0038] Furthermore, the molar ratio of 1-dodecanethiol to 2,2'-dithiodipyridine is 1:2.

[0039] Furthermore, the molar ratio of LYTAC CL8-M6P3 to 1-dodecane-thiopyridine is 1:2.

[0040] The present invention also provides the use of the above-mentioned lysosome-targeted chimera self-assembly micelles for degrading CTGF in the preparation of drugs for treating tumors.

[0041] The present invention also provides the use of the above-mentioned lysosome-targeted chimera self-assembly micelles for degrading CTGF combined with paclitaxel in the preparation of drugs for treating tumors.

[0042] Specifically, the tumor is triple-negative breast cancer.

[0043] The polypeptide LYTAC CL8-M6P3 of the present invention is composed of three amino acid sequences: (1) a polypeptide sequence targeting CTGF; (2) a connecting chain; and (3) a glycopeptide sequence of a non-cationic mannose-6-phosphate receptor (CI-M6PR).

[0044] The targeting CTGF polypeptide sequence (H-CPSAAHYL-NH2) in the polypeptide LYTAC CL8-M6P3 can specifically bind to CTGF, which is derived from literature reports [Kawaki, Harumi et al. "Design and utility of CCN2 anchor peptide aptamers." Biochimie, 2010, 92, 1010-1015]. The intermediate connecting chain is polyethylene glycol (PEG3); the non-cationic mannose-6-phosphate receptor (CI-M6PR) glycopeptide ligand sequence (H-Pra(M6P)-βAla-Pra(M6P)-βAla-Pra(M6P)-βAla-NH2) can specifically bind to CI-M6PR. As a transporter, CI-M6PR can mediate the entry of proteins carrying mannose-6-phosphate (M6P) into lysosomes, initiate the endocytosis-lysosomal pathway to achieve target protein degradation, and the specific binding of M6P to CI-M6PR is derived from literature reports [Banik, Steven M et al. "Lysosome-targeting chimaeras for degradation of extracellular proteins." Nature, 2020, 584, 291-297].

[0045] The present invention connects the hydrophilic polypeptide LYTAC CL8-M6P3 with the hydrophobic dodecyl group through a disulfide bond to form a self-assembled micelle, and constructs a new type of nano drug LYTAC self-assembled micelle NanoCLY for degrading CTGF, so as to achieve the purpose of specifically degrading CTGF in the tumor site of triple-negative breast cancer. The present invention can efficiently degrade CTGF in triple-negative breast cancer tumor tissue, inhibit tumor growth and metastasis, and then play an anti-tumor effect.

[0046] The present invention has the advantages that:

[0047] 1. The lysosome-targeted chimeric self-assembled micelle NanoCLY for degrading CTGF of the present invention can degrade the extracellular protein CTGF and exhibit anti-proliferation and anti-migration activities in triple-negative breast cancer cell lines BT549 and MDA-MB-231.

[0048] 2. The accumulation of the lysosome-targeted chimeric self-assembled micelle NanoCLY for degrading CTGF in tumor tissue is significantly increased compared with the free drug.

[0049] 3. The lysosome-targeted chimeric self-assembled micelle NanoCLY for degrading CTGF of the present invention can significantly inhibit the growth and metastasis of orthotopic triple-negative breast cancer transplanted tumors in BALB / c nude mice, and exerts a more significant anti-tumor effect than the positive drug CTGF antibody FG-3019.

[0050] 4. The lysosome-targeted chimeric self-assembled micelle NanoCLY for degrading CTGF of the present invention can significantly downregulate the activation of inflammatory tumor-associated fibroblasts, inhibit the secretion of inflammatory cytokines and chemokines, and regulate the inflammatory tumor microenvironment.

[0051] 5. The lysosome-targeted chimeric self-assembled micelle NanoCLY for degrading CTGF of the present invention can cooperate with the chemotherapy drug paclitaxel to significantly enhance the anti-tumor efficacy.

[0052] 6. The present invention achieves tumor tissue-specific targeted degradation of CTGF, while achieving direct anti-tumor activity and inhibiting the tumor inflammatory microenvironment, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Mass spectra of the polypeptide lysosome targeting chimera CL8-M6P3 (A) and 1-dodecane-CL8-M6P3 (B). Figure 2 Transmission electron microscopy (A), dynamic light scattering particle size (B) and zeta potential (C) of LYTAC self-assembled micelles NanoCLY for degrading CTGF.

[0054] Figure 3 Inhibitory effects of lysosome-targeted chimeric self-assembled micelles NanoCLY for CTGF degradation on triple-negative breast cancer BT549 (A) and MDA-MB-231 cells (B).

[0055] Figure 4 The lysosome-targeted chimeric self-assembled micelle NanoCLY for CTGF degradation affects the extracellular CTGF protein degradation in triple-negative breast cancer BT549 (A) and MDA-MB-231 cells (B).

[0056] Figure 5 Tumor targeting of lysosomal-targeted chimeric self-assembled micelles NanoCLY for CTGF degradation.

[0057] Figure 6 The lysosome-targeted chimeric self-assembled micelle NanoCLY for the degradation of CTGF inhibits the growth and metastasis of orthotopic transplanted triple-negative breast cancer tumors.

[0058] Figure 7The lysosome-targeted chimeric self-assembled micelle NanoCLY for the degradation of CTGF combined with the chemotherapy drug paclitaxel inhibits the growth and metastasis of orthotopic transplanted tumors of triple-negative breast cancer.

[0059] Figure 8 The lysosome-targeted chimeric self-assembled micelle NanoCLY for the degradation of CTGF combined with the chemotherapy drug paclitaxel inhibits the bone metastasis of triple-negative breast cancer. DETAILED DESCRIPTION

[0060] Product sources: polypeptide LYTAC CL8-M6P3 (Example 1), 1-dodecanethiol (Shanghai Titan Technology Co., Ltd.).

[0061] Example 1 Synthesis of the polypeptide LYTAC CL8-M6P3 of the present invention

[0062] Synthesis steps:

[0063] The amino acid sequence of the lysosome-targeted chimera (polypeptide LYTAC CL8-M6P3) for degrading CTGF protein of the polypeptide of the present invention is: H-CPSAAHYL-PEG3-Pra(M6P)-βAla-Pra(M6P)-βAla-Pra(M6P)-βAla-NH2.

[0064] First, according to the solid phase synthesis method, 667 mg of Rink Amide MBHA amino resin (substitution degree 0.3 mmol / g) was weighed and placed in a peptide synthesis tube, 5 mL of dichloromethane was added at room temperature to swell for 20 minutes, and then the solvent was pumped out with a water pump. After adding 5 mL of N,N-dimethylformamide to the above synthesis tube to wash the resin, 7 mL of 20% piperidine / N,N-dimethylformamide solution was added to the synthesis tube to remove the Fmoc protecting group on the amino resin, and the reaction was shaken at 35°C for 10 minutes, and the reaction liquid was pumped out. Then 7 mL of 20% piperidine / N,N-dimethylformamide solution was added to the synthesis tube, and the reaction was shaken at 35°C for 10 minutes, and the reaction liquid was pumped out. Repeat the above washing of the resin, that is, 3 times of N,N-dimethylformamide, 3 times of dichloromethane, and 3 times of N,N-dimethylformamide, and the dosage and method are as described above. Weigh Fmoc-βAla-OH (1mmol) and put it into a 10mL centrifuge tube. Then add ethyl 2-oxime cyanoacetate (142mg, 1mmol) and N,N'-diisopropylcarbodiimide (155μL, 1mmol) to the above centrifuge tube, add 7mL N,N-dimethylformamide, mix well and activate for one minute. Add the first activated Fmoc-βAla-OH amino acid to the solid phase synthesis tube, react at 60℃ for 20min and then wash. Then remove the Fmoc protecting group of the first amino acid, using the same method as above. We used the above method to sequentially access Fmoc-Pra-OH, Fmoc-βAla-OH, Fmoc-Pra-OH, Fmoc-βAla-OH, Fmoc-Pra-OH, Fmoc-NH-PEG3-CH2CH2COOH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, and Fmoc-Cys(Trt)-OH. A resin connected to the polypeptide CL8-(PβA)3 is obtained; the amino acid sequence of the CL8-(PβA)3 is: H-CPSAAHYL-PEG3-Pra-βAla-Pra-βAla-Pra-βAla; wherein the Pra-βAla-Pra-βAla-Pra-βAla repeating sequence provides an alkynyl group for subsequent click chemistry reaction to achieve mannose-6-phosphate (M6P) modification;

[0065] Through copper-catalyzed click chemistry reaction, cuprous sulfate / tri(benzyltriazolylmethyl)amine / sodium ascorbate was used as the catalyst system. 0.1mmol of the resin connected with the peptide CL8-(PβA)3 was placed in a round-bottom flask. 3mL of azide mannose-6-phosphate (0.45mmol), cuprous sulfate (0.45mmol), tri(benzyltriazolylmethyl)amine (0.45mmol) and sodium ascorbate (0.5mmol) N,N-dimethylformamide mixed aqueous solution were added at room temperature. The reaction was stirred for 24 hours and the reaction solution was drained. The above washing of the resin was repeated, i.e., 3 times of N,N-dimethylformamide, 3 times of dichloromethane, and 3 times of N,N-dimethylformamide, and the dosage and method were as described above. 15mL of the cleavage reagent (95% trifluoroacetic acid, 2.5% water, 2.5% triisopropylsilane) was added and shaken at room temperature for 2 hours. The cutting reagent was filtered into a 50 mL centrifuge tube, and the resin was rinsed with 5 mL trifluoroacetic acid, and the trifluoroacetic acid in the centrifuge tube was blown dry with argon. Then 35 mL of ice ether was added to the centrifuge tube, oscillated and precipitated, centrifuged at 3500 rpm for 3 minutes, the supernatant solution was poured out, and the above precipitation process was repeated three times, and the crude peptide was obtained by air drying. Finally, the peptide was purified by preparative liquid phase and freeze-dried to obtain a lysosomal targeting chimera (polypeptide LYTAC CL8-M6P3) for degrading CTGF protein, and its structure is as follows:

[0066]

[0067] The primary structure of peptide LYTAC CL8-M6P3 was confirmed by mass spectrometry. Figure 1 A, the data is as follows:

[0068]

[0069]

[0070] The reagents used in the examples were all commercially available analytical grade, and the solvents used in the preparation of the liquid phase were chromatographic grade.

[0071] Example 2 Preparation of Lysosome-Targeted Chimera Self-Assembled Micelle for Degrading CTGF of the Present Invention (LYTAC Self-Assembled Micelle NanoCLY for Degrading CTGF)

[0072] Synthesis steps:

[0073] 2,2'-dithiodipyridine (1 mmol) was dissolved in 1 mL of methanol. Under stirring, 1-dodecanethiol (0.5 mmol) in 1 mL of methanol solution was slowly added dropwise to the solution. The mixture was protected from light and stirred at room temperature for 3 h. The excess 2,2'-dithiodipyridine was purified by column chromatography (petroleum ether: ethyl acetate = 100:1→500:1), and the mixture was lyophilized to obtain pure 1-dodecane-thiopyridine.

[0074] 1-Dodecane-thiopyridine (0.84 mmol) was dissolved in 1 mL N,N-dimethylformamide. Under stirring, 1 mL aqueous solution of the polypeptide LYTAC CL8-M6P3 (0.42 mmol) of Example 1 was slowly added dropwise to the solution. The mixture was stirred at room temperature for 16 h. The excess 1-dodecane-thiopyridine was removed by extraction with petroleum ether. The pure 1-dodecane-CL8-M6P3 was obtained by freeze-drying. The structure of the pure product is as follows:

[0075]

[0076] The primary structure of 1-dodecane-CL8-M6P3 was confirmed by mass spectrometry. Figure 1 B, the data is as follows:

[0077]

[0078] 1-Dodecane-CL8-M6P3 (1 mg) was dissolved in 363.7 μL of phosphate buffered saline and sonicated at room temperature for 0.5 h to obtain LYTAC self-assembled micelles NanoCLY that degrade CTGF protein.

[0079] Figure 2 A is a transmission electron microscopy image of LYTAC self-assembled micelle NanoCLY that degrades CTGF protein. Figure 2 B is the dynamic light scattering particle size of 128.50nm, Figure 2 C is the zeta potential -26.30 mV.

[0080] Example 3 Inhibitory effect of the CTGF-degrading LYTAC self-assembled micelle NanoCLY on triple-negative breast cancer BT549 and MDA-MB-231 cells

[0081] Experimental methods:

[0082] The real-time cell analysis (RTCA) function of the xCELLigence system was used to test the anti-proliferative activity of CTGF-degrading LYTAC self-assembled micelles NanoCLY on triple-negative breast cancer cells BT549 and MDA-MB-231.

[0083] First, BT549 and MDA-MB-231 cells were cultured at 5×10 3 The cells were seeded at a density of 1000 cells / mL in an E-Plate 16 plate and placed in the xCELLigence Cell Function Analyzer for overnight culture.

[0084] We then replaced the original culture medium with a basal culture medium containing self-assembled micelles NanoCLY. We set up three replicate wells in each group and continued to place the cells in the xCELLigence cell function analyzer for continuous monitoring. We used RTCA software to collect cell indexes, normalized the data, and used GraphPad Prism 8.0 to perform statistics on the data.

[0085] In vitro antiproliferative activity assay Figure 3 :Depend on Figure 3 It can be seen that the micelle NanoCLY of the present invention can inhibit BT549 ( Figure 3 A) and MDA-MB-231 cells ( Figure 3 B) proliferation. ***p<0.001. Example 4 The degradation effect of the LYTAC self-assembled micelle NanoCLY for degrading CTGF of the present invention on the extracellular CTGF protein of triple-negative breast cancer BT549 and MDA-MB-231 cells.

[0086] Experimental method: ELISA experiment was used to test the degradation effect of LYTAC self-assembled micelles NanoCLY on extracellular CTGF protein in triple-negative breast cancer BT549 and MDA-MB-231 cells.

[0087] 2000 μL of BT549 and MDA-MB-231 cell suspensions were prepared in 6-well plates, and the plates were pre-cultured in a 37°C, 5% CO2 incubator for 24 hours. Micellar NanoCLY was diluted to the desired concentration using basal culture medium.

[0088] The 6-well plate was taken out of the incubator, and the supernatant was discarded with a pipette. 2000 μL of culture medium containing different concentrations of micelle NanoCLY was added to each well, and the culture was continued for 12 hours. The culture medium was collected into a 1.5 mL centrifuge tube, centrifuged at 4°C and 3000 rpm for 10 minutes, and 50 μL was added to the 96-well plate of the ELISA kit. The content of the target protein CTGF in the supernatant was detected according to the instructions of the ELISA kit, and the data were statistically analyzed using GraphPad Prism 8.0.

[0089] Figure 4 To degrade CTGF, LYTAC self-assembled micelles NanoCLY were used to synthesize BT549 ( Figure 4 A) and MDA-MB-231 cells ( Figure 4 B) Degradation of extracellular CTGF protein, which can degrade CTGF protein in a dose-dependent manner. *p<0.05, **p<0.01, ***p<0.001.

[0090] Example 5 Tumor targeting of the LYTAC self-assembled micelle NanoCLY for degrading CTGF of the present invention.

[0091] 5.1 Model establishment

[0092] Female BALB / c nude mice were inoculated with MDA-MB-231 cells (5×10 6 ), and tumors formed in about 6 days.

[0093] 5.2 Evaluation of the targeted accumulation of CTGF-degrading LYTAC self-assembled micelles NanoCLY in tumor tissues

[0094] Experimental groups: ① free DiR iodide group; ② DiR iodide-encapsulated self-assembled micelle NanoCLY group. Experimental methods: Free DiR iodide PBS solution or DiR iodide-encapsulated self-assembled micelle NanoCLY PBS solution was injected into the subcutaneous MDA-MB-231 triple-negative breast cancer orthotopic tumor model of BALB / c nude mice (DiR 2.5mg / kg, solution volume 100μL) through the tail vein, and the enrichment of DiR iodide in the tumor site was observed using the VISQUE InVivo Elite small animal in vivo real-time imaging system. Evaluation index: Fluorescence intensity of DiR iodide in the tumor area.

[0095] Figure 5 The accumulation results of LYTAC self-assembled micelles NanoCLY for degrading CTGF in the tumor area. Compared with the free DiR iodide group, the accumulation of LYTAC self-assembled micelles NanoCLY for degrading CTGF in the tumor tissue was significantly higher, indicating that the micelles NanoCLY of the present invention can significantly increase the accumulation of polypeptide LYTAC CL8-M6P3 in the tumor site. ***p<0.001.

[0096] Example 6 Effect of the LYTAC self-assembled micelle NanoCLY that degrades CTGF on the growth and lung metastasis of triple-negative breast cancer orthotopic transplanted tumors

[0097] 6.1 Model building

[0098] Female BALB / c nude mice were inoculated with MDA-MB-231 cells (5×10 6 ), and tumors formed in about 6 days.

[0099] 6.2 Evaluation of the in vivo anti-tumor growth and lung metastasis effects of LYTAC self-assembled micelles NanoCLY that degrade CTGF

[0100] Experimental groups: ① PBS group; ② prototype peptide CL8 group; ③ LYTAC self-assembled micelle NanoCLY group that degrades CTGF; ④ antibody FG-3019 positive control group, 6 mice in each group. Evaluation indicators: ① tumor volume 1500mm 3 As the endpoint, the mouse tumors were dissected out and the tumor size and lung metastasis of each group were compared; ② The general health status of the mice, diet, drinking water and body weight changes during the experiment were tracked and recorded, and the side effects were compared.

[0101] Figure 6 The self-assembled micelles NanoCLY significantly inhibited the growth of orthotopic MDA-MB-231 triple-negative breast cancer in BALB / c nude mice. (A) MDA-MB-231 tumor volume growth curve; (B) and (C) MDA-MB-231 tumor images and tumor weight statistics. Compared with other groups, the self-assembled micelles NanoCLY had the strongest inhibitory effect on tumor growth (n=6). (D) and (E) MDA-MB-231 lung metastasis H&E images and metastasis area ratio statistics. Compared with other groups, the self-assembled micelles NanoCLY had the strongest inhibitory effect on tumor lung metastasis (n=3). (F) Mouse weight monitoring (n=6). *p<0.05, **p<0.01, ***p<0.001.

[0102] Example 7 The LYTAC self-assembled micelle NanoCLY for degrading CTGF of the present invention combined with the chemotherapy drug paclitaxel inhibits the growth of orthotopic transplanted tumors and lung metastasis of triple-negative breast cancer.

[0103] 7.1 Model building

[0104] Female BALB / c nude mice were inoculated with MDA-MB-231 cells (5×10 6 ), and tumors formed in about 6 days.

[0105] 7.2 Evaluation of the effect of CTGF-degrading LYTAC self-assembled micelles NanoCLY combined with the chemotherapy drug paclitaxel in inhibiting the growth of triple-negative breast cancer orthotopic xenografts and lung metastasis

[0106] Experimental groups: ① PBS group; ② paclitaxel (PTX) group; ③ LYTAC self-assembled micelles NanoCLY group that degrades CTGF; ④ self-assembled micelles NanoCLY combined with paclitaxel (PTX) group, 6 mice in each group. Evaluation indicators: ① Tumor volume 1500mm 3 ① As the endpoint, the mouse tumors were dissected out and the tumor sizes of each group were compared; ② The general health status of the mice, diet, drinking water and body weight changes during the experiment were tracked and recorded, and the side effects were compared.

[0107] Figure 7The self-assembled micelles NanoCLY combined with paclitaxel significantly inhibited the growth of orthotopic MDA-MB-231 triple-negative breast cancer in BALB / c nude mice. (A) MDA-MB-231 tumor volume growth curve; (B) and (C) MDA-MB-231 tumor pictures and tumor weight statistics. Compared with other groups, the self-assembled micelles NanoCLY combined with paclitaxel had the strongest inhibitory effect on tumor growth (n=6). (D) and (E) MDA-MB-231 lung metastasis H&E pictures and metastasis area ratio statistics. Compared with other groups, the combination group had the strongest inhibitory effect on tumor lung metastasis (n=3). (F) Mouse weight monitoring (n=6). *p<0.05, **p<0.01, ***p<0.001.

[0108] Example 8 The effect of the LYTAC self-assembled micelle NanoCLY for degrading CTGF combined with the chemotherapy drug paclitaxel on inhibiting bone metastasis of triple-negative breast cancer.

[0109] 8.1 Model building

[0110] Female BALB / c nude mice were injected with triple-negative breast cancer bone metastasis MDA-Bom-1833-Luc cells (1×10 5 ), bone metastasis occurs in about 7 days.

[0111] 8.2 Evaluation of the effect of CTGF-degrading LYTAC self-assembled micelles NanoCLY combined with the chemotherapy drug paclitaxel in inhibiting bone metastasis of triple-negative breast cancer

[0112] Experimental groups: ①PBS group; ②paclitaxel (PTX) group; ③CTGF-degrading LYTAC self-assembled micelles NanoCLY group; ④self-assembled micelles NanoCLY combined with paclitaxel (PTX) group, 5 mice in each group. Evaluation indicators are: ①In vivo imaging of small animals was performed on the 7th, 14th, and 21st days of modeling to observe the fluorescence intensity of mouse bone metastasis; ②The general health status of mice, diet, drinking water and body weight changes during the experiment were tracked and recorded, and side effects were compared.

[0113] Figure 8 The self-assembled micelles NanoCLY combined with paclitaxel significantly inhibited the bone metastasis of triple-negative breast cancer MDA-Mom-1833-Luc in BALB / c nude mice. Compared with the PBS group, paclitaxel (PTX) and self-assembled micelles NanoCLY can significantly inhibit the bone metastasis of triple-negative breast cancer. Compared with single-drug treatment, the self-assembled micelles NanoCLY combined with paclitaxel had the strongest effect in inhibiting the bone metastasis of triple-negative breast cancer (n=5). **p<0.01, ***p<0.001, ****p<0.0001.

[0114] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and additions without departing from the method of the present invention. These improvements and additions should also be regarded as within the scope of protection of the present invention.

Claims

1. A polypeptide-based lysosomal targeting chimera for degrading CTGF protein, the structural formula of which is shown below:

2. The method for preparing the lysosomal targeting chimera of the polypeptide for degrading CTGF protein according to claim 1, characterized in that The steps include: (1) According to the solid phase synthesis method, Rink Amide MBHA amino resin is used as a carrier and ethyl 2-oxime cyanoacetate / N,N'-diisopropylcarbodiimide is used as a condensation system to obtain a resin connected to the polypeptide CL8-(PβA)3; the amino acid sequence of the CL8-(PβA)3 is: H-CPSAAHYL-PEG3-Pra-βAla-Pra-βAla-Pra-βAla; (2) Through a copper-catalyzed click chemistry reaction, using cuprous sulfate / tri(benzyltriazolylmethyl)amine / sodium ascorbate as a catalyst system, the resin connected to the polypeptide CL8-(PβA)3, azido-mannose-6-phosphate, cuprous sulfate, tri(benzyltriazolylmethyl)amine and sodium ascorbate were added to a mixed solvent of N,N-dimethylformamide and pure water, and after stirring at room temperature in the dark, the reaction was performed, followed by cutting, purification and lyophilization to obtain the polypeptide LYTAC CL8-M6P3; the amino acid sequence of the polypeptide LYTAC CL8-M6P3 is: H-CPSAAHYL-PEG3-Pra(M6P)-βAla-Pra(M6P)-βAla-Pra(M6P)-βAla-NH2; The structural formula is shown below:

3. The method for preparing a lysosomal targeting chimera of a polypeptide for degrading CTGF protein according to claim 2, characterized in that: The molar ratio of CL8-(PβA)3, mannose-6-phosphate azide, cuprous sulfate, tris(benzyltriazolylmethyl)amine and sodium ascorbate is 1:4.5:4.5:4.5:

5.

4. A lysosome-targeted chimeric self-assembled micelle for degrading CTGF, characterized in that: Its structure is as follows:

5. The lysosome-targeted chimeric self-assembled micelle for degrading CTGF according to claim 4, characterized in that: The self-assembled micelle is a double-layer structure, the inner layer is dodecyl, and the outer layer is connected to the polypeptide of claim 1 through a disulfide bond.

6. The method for preparing a lysosome-targeted chimeric self-assembled micelle for degrading CTGF according to claim 4, characterized in that The steps include: (1) According to the solid phase synthesis method, Rink Amide MBHA amino resin is used as a carrier and ethyl 2-oxime cyanoacetate / N,N'-diisopropylcarbodiimide is used as a condensation system to obtain a resin connected to the polypeptide CL8-(PβA)3; the amino acid sequence of the CL8-(PβA)3 is: H-CPSAAHYL-PEG3-Pra-βAla-Pra-βAla-Pra-βAla; (2) using a copper-catalyzed click chemistry reaction, with cuprous sulfate / tri(benzyltriazolylmethyl)amine / sodium ascorbate as the catalyst system, the resin connected to the polypeptide CL8-(PβA)3, azido-mannose-6-phosphate, cuprous sulfate, tri(benzyltriazolylmethyl)amine and sodium ascorbate were added to a mixed solvent of N,N-dimethylformamide and pure water, stirred at room temperature in the dark, and cut, purified and freeze-dried to obtain the polypeptide LYTAC CL8-M6P3; the amino acid sequence of the polypeptide LYTAC CL8-M6P3 is: H-CPSAAHYL-PEG3-Pra(M6P)-βAla-Pra(M6P)-βAla-Pra(M6P)-βAla-NH2; The structural formula is shown below: (3) dissolving 2,2'-dithiodipyridine in methanol, adding 1-dodecanethiol solution under stirring, reacting under stirring at room temperature in the dark, purifying to remove excess 2,2'-dithiodipyridine, and lyophilizing to obtain pure 1-dodecane-thiopyridine; (4) dissolving the pure 1-dodecane-thiopyridine in N,N-dimethylformamide, adding the polypeptide LYTAC CL8-M6P3 aqueous solution dropwise under stirring, reacting under room temperature in the dark, extracting to remove excess 1-dodecane-thiopyridine, and then freeze-drying to obtain the pure 1-dodecane-CL8-M6P3; (5) 1-Dodecane-CL8-M6P3 was dissolved in phosphate buffered saline and sonicated at room temperature to obtain lysosome-targeted chimeric self-assembled micelles that degrade CTGF.

7. The method for preparing a lysosome-targeted chimeric self-assembled micelle for degrading CTGF according to claim 6, characterized in that: The molar ratio of CL8-(PβA)3, mannose-6-phosphate azide, cuprous sulfate, tris(benzyltriazolylmethyl)amine and sodium ascorbate is 1:4.5:4.5:4.5:

5.

8. The method for preparing a lysosome-targeted chimeric self-assembled micelle for degrading CTGF according to claim 6, characterized in that: The molar ratio of 1-dodecanethiol to 2,2'-disulfide dipyridine is 1:

2.

9. The method for preparing a lysosome-targeted chimeric self-assembled micelle for degrading CTGF according to claim 6, characterized in that: The molar ratio of LYTAC CL8-M6P3 to 1-dodecane-thiopyridine is 1:

2.

10. Use of the lysosome-targeted chimeric self-assembled micelles for degrading CTGF according to claim 4 in the preparation of drugs for treating tumors.

11. Use of the lysosome-targeted chimeric self-assembled micelles for degrading CTGF according to claim 4 in combination with paclitaxel in the preparation of a drug for treating tumors.

12. The use according to claim 10 or 11, characterized in that: The tumor is triple-negative breast cancer.

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