Polyporus polysaccharide microneedle composition with CD47 protein degradation function, microneedle as well as preparation method and application of polyporus polysaccharide microneedle composition

Abstract

The invention provides a polyporus polysaccharide microneedle composition with a CD47 protein degradation function. The polyporus polysaccharide microneedle composition comprises a lysosome targeting chimera for degrading CD47 protein, polyporus polysaccharide and hyaluronic acid. The invention also provides a preparation method of the lysosome targeting chimera for degrading the CD47 protein. The invention also provides a microneedle prepared from the polyporus polysaccharide microneedle composition. The invention also provides a preparation method of the polyporus polysaccharide microneedle with the CD47 protein degradation function and application of the polyporus polysaccharide microneedle in preparation of medicines for treating tumors. According to the microneedle, on one hand, CD47 protein in melanoma tumor tissue can be efficiently degraded, then the phagocytosis effect of macrophages on tumor cells is enhanced, on the other hand, polyporus polysaccharide in the microneedle can promote the macrophages to be polarized towards anti-tumor types, and the anti-tumor activity of the macrophages is further enhanced. The immunosuppressive microenvironment is remodeled while CD47 is degraded, and the efficient and synergistic anti-tumor effect of the two drugs is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a pharmaceutical composition, specifically a polyporus polysaccharide microneedle composition with CD47 protein degradation function, a microneedle and a preparation method and application thereof. Background Art

[0002] Melanoma is a malignant tumor caused by melanocytes. It is highly invasive, easy to metastasize and has poor prognosis. The recurrence rate is as high as 70%, and the 5-year survival rate of patients in the late stage is only 23%. Melanoma is mainly treated by surgical resection in the early stage, but it can only remove the primary tumor and is difficult to deal with tumor metastasis. Immune checkpoint inhibitors represented by PD-1 / L1 have made breakthrough progress in the treatment of melanoma in recent years. These drugs activate the patient's cytotoxic T cells to enable them to recognize and kill tumor cells. However, the existing immune checkpoint inhibitors still have the limitation of low response rate. As an important part of innate immunity, macrophages can recognize and clear foreign, aging, damaged and dead cells in tissues, and play a key role in maintaining the body's homeostasis. Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor stroma. Among them, M1 TAMs can recognize and phagocytose tumor cells in the early stages of tumor development, thereby exerting anti-tumor effects and promoting adaptive immune responses. As the tumor microenvironment forms, M1 TAMs will gradually polarize into M2 TAMs that promote tumor development. In addition to the fact that the phenotype of macrophages affects their ability to clear tumor cells, signaling proteins on the surface of tumor cells also affect the phagocytic function of macrophages. CD47 is a transmembrane protein expressed on the surface of many tumor cells. It can interact with SIRPα on macrophages to transmit the "don't eat me" immunosuppressive signal, thereby achieving immune escape. At present, CD47 has become a new potential target for immunotherapy, and multiple CD47 antibodies are in clinical trials or preclinical studies. However, CD47 antibody treatment often affects the homeostasis of red blood cells, platelets, and hematopoietic stem cells, leading to severe "on-target toxicity", including red blood cell coagulation, anemia, and platelet aggregation. Therefore, how to block CD47 on the surface of tumor cells without affecting the normal function of blood cells has become a new research hotspot.

[0003] Targeted protein degradation (TPD) is an emerging disease treatment model that mainly exerts degradation effects through the proteasome pathway or the lysosome pathway. Among them, lysosome-targeting chimaera (LYTAC) provides the possibility of targeted degradation of extracellular proteins and membrane proteins, and has gradually been applied to new drug research and development. Since there are no organelles in mature red blood cells, LYTAC can avoid degradation of CD47 on the red blood cell membrane, and thus it will not be recognized and cleared by macrophages, which is expected to overcome the side effects such as anemia caused by CD47 antibody treatment.

[0004] Microneedles are a new type of minimally invasive transdermal drug delivery system. They can achieve sustained drug release in the skin without damaging blood vessels or touching nerve endings, significantly improving patient compliance. They have been widely used in the diagnosis and treatment of superficial tumors (such as melanoma, etc.). Chinese herbal polysaccharides have the advantages of high structural stability, good biocompatibility, and biodegradability, and are natural materials for preparing microneedles. Among them, Polyporus polysaccharide (PPS), as one of the main active ingredients of the Chinese herbal medicine Polyporus, has been approved by the China Food and Drug Administration as an immunopotentiator for chemotherapy in cancer patients. It has been reported that PPS can promote macrophage polarization to M1 type, thereby enhancing the anti-tumor effect of macrophages, so it has the potential to synergize with CD47 therapy.

[0005] Therefore, based on the lysosome-targeted chimera that degrades CD47 protein and Polyporus polysaccharide, the present invention designs and constructs Polyporus polysaccharide microneedles with CD47 protein degradation function, which can achieve tumor site administration, release the lysosome-targeted chimera that degrades CD47 protein, achieve CD47 degradation, and release Polyporus polysaccharide, promote the polarization of M2-type TAMs to M1-type, further enhance the ability of M1-type TAMs to eliminate tumor cells, and synergistically enhance the immunotherapy of melanoma. There are no similar reports so far. Summary of the invention

[0006] In view of the above-mentioned problems in the prior art, the present invention provides a polyporus polysaccharide microneedle composition, a microneedle, and a preparation method and application thereof with CD47 protein degradation function. The polyporus polysaccharide microneedle composition, a microneedle, and a preparation method and application thereof with CD47 protein degradation function are intended to solve the technical problem of poor effect of melanoma immunotherapy in the prior art.

[0007] The present invention provides a polyporus polysaccharide microneedle composition with CD47 protein degradation function, comprising raw materials in parts by weight:

[0008] Polyporus polysaccharide 100-300;

[0009] Lysosomal targeting chimera 5-20 for degradation of CD47 protein;

[0010] Hyaluronic acid 100-300.

[0011] Furthermore, the structure of the lysosome-targeted chimera for degrading CD47 protein is as follows:

[0012]

[0013] Furthermore, the synthesis process of the lysosomal targeting chimera for degrading CD47 protein is described as follows:

[0014] 1) dripping acetic anhydride into a pyridine solution of mannose at 0°C, stirring at room temperature overnight, adding anhydrous ethanol to quench excess acetic anhydride, concentrating the sample under reduced pressure, pouring the compound into ice water, and filtering to obtain fully acetylated mannose M6P-1; 2) under nitrogen protection, dripping boron trifluoride ether solution into a dichloromethane solution of azidoethanol and M6P-1 at 0°C, moving to room temperature and stirring overnight after 1 to 2 hours, adding dichloromethane to dilute, washing with saturated sodium bicarbonate solution, drying the organic phase, filtering and concentrating under reduced pressure, and purifying by column chromatography to obtain M6P-2; preferably, the molar ratio of azidoethanol, M6P-1, and boron trifluoride is 1.5:1:4.0.

[0015] 3) M6P-2 was dissolved in a methanol solution of sodium methoxide with a mass percentage concentration of 25% to 35%, and after reaction at room temperature, the pH was adjusted to neutral using a cation exchange resin, and the crude product was concentrated by suction filtration. The crude product was dissolved in pyridine, triphenylmethane was added, and stirred at 45 to 55° C. for 3 to 5 hours, and acetic anhydride was added at room temperature to react overnight, and the excess acetic anhydride was quenched with anhydrous ethanol. After concentrating under reduced pressure, ice water was added, and the precipitated white solid was dissolved in an acetic acid aqueous solution, stirred at 55 to 65° C. for 1 to 2 hours, and ice water was added to precipitate the solid, which was filtered and washed with water, and the solid was purified by column chromatography to obtain M6P-3;

[0016] 4) Under nitrogen protection, N,N-diisopropylphosphoramidite di-tert-butyl ester is added to an anhydrous dichloromethane solution of M6P-3 and 1H-tetrazole at 0°C, stirred for 2 to 3 hours, tert-butyl hydroperoxide is added at 0°C, stirred for 5 minutes, and then moved to room temperature for reaction for 2 to 3 hours, saturated sodium thiosulfate solution is added to quench the reaction, extracted with dichloromethane, the organic phases are combined, filtered and concentrated under reduced pressure, and purified by column chromatography to obtain M6P-4; preferably, the molar ratio of N,N-diisopropylphosphoramidite di-tert-butyl ester, M6P-3, and 1H-tetrazole is 1.5:1.0:2.0.

[0017] 5) Take M6P-4 and add a methanol solution of sodium methoxide with a mass percentage concentration of 25% to 35%, react at room temperature for 2 to 3 hours, use a 50-100 mesh cation exchange resin to adjust the pH to neutral, filter the resin and concentrate the filtrate under reduced pressure, dissolve the residue in a dichloromethane solution containing trifluoroacetic acid with a mass percentage concentration of 8% to 12%, stir at room temperature for 1 to 2 hours, and then concentrate under reduced pressure to obtain M6P;

[0018] 6) A peptide conjugate RS17-(PβA)3 was obtained by solid phase synthesis method, using Rink Amide MBHA amino resin as a carrier and ethyl 2-oxime cyanoacetate / N,N'-diisopropylcarbodiimide as a condensation system; the amino acid sequence of RS17 is: H-RRYKQDGGWSHWSPWSS-NH2 (SEQ ID NO.1);

[0019] 7) A copper-catalyzed click chemistry reaction was used with cuprous sulfate / sodium ascorbate as the catalyst system, and the polypeptide conjugate RS17-(PβA)3, M6P, cuprous sulfate and sodium ascorbate were added to a mixed solvent of methanol and pure water. After stirring and reacting at room temperature in the dark, the reaction was purified and freeze-dried to obtain a lysosomal-targeted chimera that degraded CD47 protein.

[0020] Preferably, the molar ratio of RS17-(PβA)3, M6P, cuprous sulfate, and sodium ascorbate is 1:4.5:4.5:9.0.

[0022]

[0023] Furthermore, the lysosome-targeted chimera for degrading CD47 protein can degrade CD47 protein on melanoma cells, block their immune escape function, and promote macrophages to recognize and kill melanoma cells.

[0024] The present invention also provides a polyporus polysaccharide microneedle with CD47 protein degradation function, which is prepared from the raw materials of the polyporus polysaccharide microneedle composition with CD47 protein degradation function.

[0025] Furthermore, the Polyporus polysaccharide microneedle with CD47 protein degradation function comprises a substrate and a needle body located on the substrate; wherein the needle body is prepared from Polyporus polysaccharide and a lysosome-targeted chimera that degrades CD47 protein as raw materials, and the microneedle substrate is composed of hyaluronic acid.

[0026] The present invention also provides a method for preparing the above-mentioned Polyporus polysaccharide microneedle with CD47 protein degradation function, comprising the following steps:

[0027] 1) Weighing a lysosome-targeted chimera for degrading CD47 protein and Polyporus polysaccharide, adding them to pure water to prepare a mixed aqueous solution containing 5-20 mg / mL of the lysosome-targeted chimera for degrading CD47 protein and 100-300 mg / mL of Polyporus polysaccharide, and swelling them at 4° C. overnight;

[0028] 2) Pour the mixed aqueous solution of the lysosome-targeted chimera that degrades CD47 protein and Polyporus polysaccharide into the groove of the microneedle mold as the needle body matrix material of the microneedle, centrifuge at 4000 rpm for 5 minutes at 4°C, and rotate 180°

[0029] Repeat the centrifugation once to recover the excess material solution in the groove.

[0030] 3) preparing an aqueous solution with a hyaluronic acid concentration of 100-300 mg / mL as the base material of the microneedle, pouring it into a mold and placing it in a 55° C. oven to dry overnight, and then demolding to obtain the microneedle.

[0031] The present invention also provides the use of the above-mentioned Polyporus polysaccharide microneedle with CD47 protein degradation function in the preparation of drugs for treating tumor diseases.

[0032] Specifically, the tumor is melanoma.

[0033] The present invention also provides a lysosome-targeted chimera for degrading CD47 protein, and its structural formula is shown below:

[0034] The present invention also provides a method for preparing the above-mentioned lysosome-targeted chimera for degrading CD47 protein, comprising the following steps:

[0035] 1) Add acetic anhydride dropwise to a pyridine solution of mannose at 0°C, stir at room temperature overnight, add anhydrous ethanol to quench the excess acetic anhydride, concentrate the sample under reduced pressure, pour the compound into ice water, and filter to obtain fully acetylated mannose M6P-1;

[0036] 2) Under nitrogen protection, a boron trifluoride ether solution is added dropwise to a dichloromethane solution of azidoethanol and M6P-1 at 0°C. After 1 to 2 hours, the mixture is moved to room temperature and stirred overnight. Dichloromethane is added to dilute the mixture, and the mixture is washed with a saturated sodium bicarbonate solution. The organic phase is dried, filtered, and concentrated under reduced pressure. M6P-2 is purified by column chromatography. Preferably, the molar ratio of azidoethanol, M6P-1, and boron trifluoride is 1.5:1:4.0.

[0037] 3) M6P-2 was dissolved in a methanol solution of sodium methoxide with a mass percentage concentration of 25% to 35%, and after reaction at room temperature, the pH was adjusted to neutral using a cation exchange resin, and the crude product was concentrated by suction filtration. The crude product was dissolved in pyridine, triphenylmethane was added, and stirred at 45 to 55° C. for 3 to 5 hours, and acetic anhydride was added at room temperature to react overnight, and the excess acetic anhydride was quenched with anhydrous ethanol. After concentrating under reduced pressure, ice water was added, and the precipitated white solid was dissolved in an acetic acid aqueous solution, stirred at 55 to 65° C. for 1 to 2 hours, and ice water was added to precipitate the solid, which was filtered and washed with water, and the solid was purified by column chromatography to obtain M6P-3;

[0038] 4) Under nitrogen protection, N,N-diisopropylphosphoramidite di-tert-butyl ester is added to an anhydrous dichloromethane solution of M6P-3 and 1H-tetrazole at 0°C, stirred for 2 to 3 hours, tert-butyl hydroperoxide is added at 0°C, stirred for 5 minutes, and then moved to room temperature for reaction for 2 to 3 hours, saturated sodium thiosulfate solution is added to quench the reaction, extracted with dichloromethane, the organic phases are combined, filtered and concentrated under reduced pressure, and purified by column chromatography to obtain M6P-4; preferably, the molar ratio of N,N-diisopropylphosphoramidite di-tert-butyl ester, M6P-3, and 1H-tetrazole is 1.5:1.0:2.0.

[0039] 5) Take M6P-4 and add a methanol solution of sodium methoxide with a mass percentage concentration of 25% to 35%, react at room temperature for 2 to 3 hours, use a 50-100 mesh cation exchange resin to adjust the pH to neutral, filter the resin and concentrate the filtrate under reduced pressure, dissolve the residue in a dichloromethane solution containing trifluoroacetic acid with a mass percentage concentration of 8% to 12%, stir at room temperature for 1 to 2 hours, and then concentrate under reduced pressure to obtain M6P;

[0040] 6) 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 polypeptide conjugate RS17-(PβA)3; the amino acid sequence of RS17 is: H-RRYKQDGGWSHWSPWSS-NH2 (SEQ ID NO.1);

[0041] 7) Through a copper-catalyzed click chemistry reaction, using cuprous sulfate / sodium ascorbate as a catalyst system, the polypeptide conjugate RS17-(PβA)3, M6P, cuprous sulfate and sodium ascorbate were added to a mixed solvent of methanol and pure water, and after reacting at room temperature in the dark, the reaction was purified and lyophilized to obtain a lysosomal targeting chimera that degrades CD47 protein.

[0042] Preferably, the molar ratio of RS17-(PβA)3, M6P, cuprous sulfate, and sodium ascorbate is 1:4.5:4.5:9.0.

[0043] .

[0045] The polyporus polysaccharide microneedle with CD47 protein degradation function of the present invention is to dissolve rapidly at the tumor site after the needle body is partially pierced into the skin, and releases a lysosome-targeted chimera and polyporus polysaccharide that degrades CD47 protein. The lysosome-targeted chimera that degrades CD47 protein consists of three parts: (1) a polypeptide sequence targeting CD47; (2) a connecting chain; (3) a glycopeptide sequence targeting the non-cationic mannose-6-phosphate receptor (CI-M6PR). The targeted CD47 polypeptide sequence (H-RRYKQDGGWSHWSPWSS-NH2) in the lysosome-targeted chimera can specifically bind to CD47, which is derived from literature reports [Wang, Xinmin et al. An antitumor peptide RS17-targeted CD47, design, synthesis, and antitumor activity. Cancer medicine, 2021, 10, 2125-2136.]. The intermediate connecting chain is polyethylene glycol (PEG3); the glycopeptide sequence targeting CI-M6PR (H-Pra(M6P)-βAla-Pra(M6P)-βAla-Pra(M6P)-βAla-NH2) can specifically bind to CI-M6PR, 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.]. The lysosome-targeted chimera for degrading CD47 protein can mediate the degradation of membrane protein CD47 through the endocytosis-lysosome pathway of melanoma cells, thereby enhancing the phagocytosis of tumor cells by macrophages. At the same time, the released Polyporus polysaccharide can improve the tumor immunosuppressive microenvironment, promote the polarization of M2 macrophages to M1 macrophages, and further enhance the ability of M1 macrophages to clear tumor cells. The invention can effectively inhibit the tumor growth of melanoma-bearing mice, exert an anti-tumor effect, and can be used to prepare anti-melanoma drugs.

[0046] The present invention constructs a polyporus polysaccharide microneedle with CD47 protein degradation function, which is loaded with a lysosome-targeted chimera for degrading CD47 protein and polyporus polysaccharide, and activates the body's anti-tumor immunity through transdermal administration.

[0047] The present invention has the advantages that:

[0048] 1. The present invention constructs a soluble Poria polysaccharide microneedle with good biosafety. After entering the tumor site, it can quickly dissolve and release the lysosome-targeted chimera and Poria polysaccharide that degrade CD47, thereby achieving the synergy of immune checkpoint CD47 degradation and remodeling of the macrophage-related immunosuppressive microenvironment.

[0049] 2. The lysosome-targeted chimera for degrading CD47 in the microneedle composition of the present invention can degrade CD47 of B16F10 melanoma cells.

[0050] 3. The lysosome-targeted chimera that degrades CD47 in the microneedle composition of the present invention can significantly improve the recognition and phagocytosis of melanoma cells by macrophages, and the combination with Polyporus polysaccharide can further synergistically enhance the phagocytic effect of macrophages.

[0051] 4. The polyporus polysaccharide in the microneedle composition of the present invention can significantly promote the polarization of M2 macrophages to M1 macrophages, and further enhance the ability of M1 macrophages to eliminate melanoma cells.

[0052] 5. The Poria polysaccharide microneedles with CD47 protein degradation function of the present invention can significantly inhibit the growth of B16F10 melanoma transplanted tumors in C57BL / 6 mice, and exert a more significant anti-tumor effect than blank microneedles and microneedles loaded with drugs alone, and have good application prospects.

[0053] 6. The Poria polysaccharide microneedles with CD47 protein degradation function of the present invention can significantly degrade the level of CD47 protein in melanoma tumors in vivo.

[0054] 7. The Poria polysaccharide microneedles with CD47 protein degradation function of the present invention can significantly increase the ratio of M1 / M2 macrophages in in vivo experiments and improve the macrophage-related immunosuppressive microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The invention provides a synthetic route for the lysosome-targeted chimera for degrading CD47 protein in the polyporus polysaccharide microneedle composition of the present invention.

[0056] Figure 2 It is a mass spectrum of the lysosome-targeted chimera for degrading CD47 protein in the Polyporus polysaccharide microneedle composition of the present invention.

[0057] Figure 3 The present invention characterizes the morphology and mechanical strength of the Polyporus polysaccharide microneedles.

[0058] Figure 4 It is the CD47 degradation effect of the lysosome-targeted chimera that degrades CD47 protein in the Polyporus polysaccharide microneedle composition of the present invention.

[0059] Figure 5The present invention relates to the effect of the lysosome-targeted chimera for degrading CD47 protein in the polyporus polysaccharide microneedle composition of the present invention on red blood cells.

[0060] Figure 6 This is the in vitro polarization effect of the Polyporus polysaccharide in the Polyporus polysaccharide microneedle composition of the present invention on macrophages.

[0061] Figure 7 The polyporus polysaccharide microneedle composition of the present invention promotes the uptake of melanoma cells by macrophages.

[0062] Figure 8 This is the in vivo anti-tumor effect of the Poria polysaccharide microneedle of the present invention.

[0063] Fig. 9 This is the regulatory effect of the Polyporus polysaccharide microneedle administration on macrophages in the body. DETAILED DESCRIPTION

[0064] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0065] Example 1 Synthesis of the lysosome-targeted chimera for degrading CD47 protein provided by the present invention.

[0066] Synthesis steps:

[0067] Acetic anhydride (30 mL) was added dropwise to a pyridine (50 mL) solution of D-(+)-mannose (5.4 g, 30 mmol, 1 eq.) at 0°C and stirred overnight at room temperature. Thin-layer chromatography (TLC) (petroleum ether PE: ethyl acetate EA = 3:1) was used for detection and ethanolic sulfuric acid was used for color development. After the reaction was complete, anhydrous ethanol was added to react the excess acetic anhydride and the mixture was concentrated under reduced pressure. Ice water was added to the residue, filtered, and the upper solid precipitate was collected to obtain fully acetylated mannose M6P-1.

[0068] Dissolve ethanol azido (1.0 g, 11.5 mmol, 1.5 eq.) and M6P-1 (3.0 g, 7.69 mmol, 1.0 eq.) in 40 mL of dichloromethane (DCM), and slowly add boron trifluoride ether solution (mass percent concentration of 48%) (9.1 mL, 30.76 mmol, 4.0 eq.) at 0 ° C under nitrogen protection. Warm to room temperature after 1 hour and stir overnight. Thin layer chromatography TLC (PE: EA = 1: 1) detection, sulfuric acid ethanol color. After the reaction is complete, dichloromethane is added to dilute, and ice water and saturated sodium bicarbonate solution are used for washing. Collect the organic phase, dry it over anhydrous sodium sulfate, filter it and concentrate it under reduced pressure. The residue is purified by column chromatography (PE: EA = 5: 1 → 3: 1) to obtain M6P-2.

[0069] M6P-2 (2.512 g, 6.02 mmol, 1.0 eq.) and sodium methoxide (1.625 g, 30.1 mmol, 5.0 eq.) were dissolved in 30 mL of methanol and reacted at room temperature for 2 hours. TLC (DCM: methanol MeOH = 10: 1) was used for detection, and sulfuric acid ethanol was used for color development. The pH of the reaction solution was adjusted to neutral by cation exchange resin, and the solution was filtered and concentrated to obtain a crude product. The crude product was dissolved in pyridine (15 mL), triphenylmethane (1.762 g, 6.32 mmol, 1.05 eq.) was added, and stirred at 50 ° C for 4 hours. TLC (PE: EA = 2: 1) was used for detection. After the reaction was complete, it was moved to room temperature, acetic anhydride (10 mL) was added, and the reaction was allowed to proceed overnight at room temperature. TLC (PE: EA = 2: 1) was used for detection. After the reaction was complete, the residue was dissolved in anhydrous ethanol, concentrated under reduced pressure to remove pyridine, added to ice water, a white solid was precipitated, and the crude product was obtained by filtration. The crude product was dissolved in acetic acid and water (12 mL of acetic acid and 8 mL of water) and stirred at 60°C for 1 hour. After the reaction was complete, ice water was added to the reaction solution to precipitate a white solid, which was filtered and washed with water. The solid was purified by column chromatography (PE:EA=5:1→2:1) to obtain M6P-3.

[0070] M6P-3 (200 mg, 0.4 mmol, 1.0 eq.) and 1H-tetrazolyl (56 mg, 0.8 mmol, 2.0 eq.) were dissolved in anhydrous DCM and protected by nitrogen. N,N-diisopropylphosphoramidite di-tert-butyl ester (166 mg, 0.6 mmol, 1.5 eq.) was slowly added dropwise at 0°C and continued to stir for 2 hours. TLC (PE:EA=2:1) ​​was used for detection. After the reaction was complete, tert-butyl hydroperoxide (5.5 mol / L) (145 μL, 0.8 mmol, 2.0 eq.) was slowly added at 0°C. After stirring for 5 minutes, the mixture was moved to room temperature and stirred for 2 hours. TLC (PE:EA=1:3) was used for detection. After the reaction was complete, a saturated sodium thiosulfate solution was added to quench the reaction, and the mixture was extracted with DCM for 3 times. The organic phases were combined and washed with a saturated sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA=2:1→1:1) to obtain M6P-4.

[0071] Take M6P-4 (204 mg, 0.36 mmol, 1.0 eq.) and add a methanol solution of sodium methoxide (mass percentage concentration of 30%) (65 μL, 0.36 mmol, 3.0 eq.) and react at room temperature for 2 hours. After TLC (DCM: MeOH = 10: 1) detection, after the reaction is complete, adjust the pH to neutral with a 50-100 mesh cation exchange resin, filter the resin, and concentrate the filtrate under reduced pressure. The residue is dissolved in a dichloromethane solution (2 mL) containing a mass percentage concentration of 10% trifluoroacetic acid (Trifluoroacetic acid, TFA), and stirred at room temperature for 1 hour. After TLC detection, after the reaction is complete, concentrate under reduced pressure to obtain M6P.

[0072] According to the solid phase synthesis method, the polypeptide conjugate RS17-(PβA)3 was obtained; the amino acid sequence of RS17 is: H-RRYKQDGGWSHWSPWSS-NH2; the above-mentioned Fmoc solid phase synthesis method is a prior art and will not be repeated here.

[0073] The peptide conjugate RS17-(PβA)3 (10.0 mg, 10.6 μmol, 1 eq.) was dissolved in 300 μL of water, CuSO4 (7.5 mg, 47.7 μmol, 4.5 eq.) was dissolved in 100 μL of water, L-sodium ascorbate (18.9 mg, 95.4 μmol, 9.0 eq.) was dissolved in 100 μL of water, and M6P (15.7 mg, 47.7 μmol, 4.5 eq.) was dissolved in 100 μL of methanol. RS17-(PβA)3, M6P, CuSO4 and L-sodium ascorbate solution were added to a 5 mL reaction bottle, stirred at room temperature overnight, and then purified and lyophilized to obtain the lysosomal targeting chimera RS17-M6P3 that degrades CD47 protein. The synthesis process is shown in Figure 1

[0074] Its structure is as follows:

[0075]

[0076] The primary structure of the lysosomal targeting chimera RS17-M6P3 that degrades CD47 protein was confirmed by mass spectrometry. Figure 2 , the data is as follows:

[0077]

[0078]

[0079] 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.

[0080] Example 2 Preparation of Polyporus polysaccharide microneedles with CD47 protein degradation function of the present invention

[0081] Preparation steps:

[0082] Weigh the lysosome-targeted chimera that degrades CD47 protein and Polyporus polysaccharide, add them into pure water, prepare a mixed aqueous solution containing 10 mg / mL of the lysosome-targeted chimera that degrades CD47 protein and 200 mg / mL of Polyporus polysaccharide, and swell overnight at 4°C;

[0083] A mixed aqueous solution of the lysosome-targeted chimera that degrades CD47 protein and Polyporus polysaccharide was poured into the groove of the microneedle mold as the needle body matrix material of the microneedle, centrifuged at 4000 rpm at 4°C for 5 minutes, rotated 180° horizontally and centrifuged again to recover the excess material solution in the groove.

[0084] An aqueous solution with a hyaluronic acid concentration of 200 mg / mL was prepared as the base material of the microneedle, poured into a mold and placed in a 55° C. oven to dry overnight, and then demolded to obtain the microneedle.

[0085] Example 3 Characterization of the morphology and mechanical strength of the polyporus polysaccharide microneedles with CD47 protein degradation function of the present invention

[0086] Experimental methods:

[0087] After the microneedles were placed in an ion sputtering coating machine for surface gold coating, the morphology of the microneedles was observed by scanning electron microscopy. FITC-labeled lysosomal targeting chimera RS17-M6P3 that degrades CD47 protein and Cy5-labeled Polyporus polysaccharide were added to the microneedles, and the drug distribution in the microneedles was observed by laser confocal microscopy. The results showed that RS17-M6P3 and Polyporus polysaccharide could be evenly dispersed in the microneedle body. The mechanical strength was analyzed by a texture analyzer. When the probe (a stainless steel cylinder with a diameter of 10 mm) contacted the sample (when the trigger force reached 0.05N), it was pressed down at a speed of 30 mm / min to deform the sample by 40%, and then the probe returned to the starting position. The force-displacement diagram was obtained. The microneedles were inserted into the skin of SD rats and removed at different time points, and the morphology of the microneedles was observed by optical microscopy.

[0088] Figure 3 To characterize the morphology and mechanical strength of Poria polysaccharide microneedles with CD47 protein degradation function. Figure 3 A) The morphology of microneedles in different groups under scanning electron microscopy. Figure 3 B) The force-displacement curve shows that the microneedle has the mechanical strength to pierce the skin. Figure 3 C) The drug-loaded polysaccharide microneedles can completely dissolve and release the drug within 5 minutes. Figure 3 D) Laser confocal microscopy was used to observe the drug distribution in the microneedles.

[0089] Example 4 CD47 degradation effect of the lysosome-targeted chimera for degrading CD47 protein provided by the present invention in melanoma cells B16F10

[0090] Experimental methods:

[0091] B16F10 cells were cultured at 3×10 5 Cells were inoculated in 6-well plates, cultured in a 37°C incubator with 5% CO2 for 24 hours, and then different concentrations of RS17-M6P3 (0.1μM, 0.3μM, 1μM, 3μM, 10μM) were added. After 24 hours of action, the supernatant was discarded and 1mL of pre-cooled PBS was added to each well to wash the cells. 60μL of NP-40 lysis buffer containing full enzyme inhibitors was added to each well and lysed in an ice bath for 30 minutes. After scraping the cells with a scraper, they were transferred to a 1.5mL centrifuge tube and centrifuged at 4°C and 15000rpm for 15 minutes. The cell supernatant was collected and 5× loading buffer was added in proportion, boiled at 100°C for 10 minutes, the boiled protein was centrifuged, and stored in a -20°C refrigerator. The level of CD47 protein was detected by protein immunoblotting.

[0092] In vitro CD47 degradation assay Figure 4 :Depend on Figure 4 It can be seen that the lysosome-targeted chimera for degrading CD47 protein of the present invention can be dose-dependent ( Figure 4 A) and time dependence ( Figure 4 B) Degradation of CD47 protein in B16F10 cells, ***p<0.001.

[0093] Example 5 The lysosome-targeted chimera for degrading CD47 protein provided by the present invention promotes macrophage uptake of melanoma cells

[0094] Experimental methods:

[0095] Extract and culture mouse bone marrow-derived macrophages (BMDM). Female mice (Seven-week-old C57BL / 6 mice) were killed by cervical dislocation and soaked in 75% alcohol for 15 minutes. In the sterile environment of the clean bench, all femurs and tibias were surgically removed, and the muscle tissue around the bones was removed. The bone marrow cavity was repeatedly rinsed, and the debris and tissue were filtered out with a 200-mesh nylon mesh. Filter, 400g, centrifuge for 5 minutes, and discard the supernatant. Add red blood cell lysis buffer (3mL), let stand at room temperature for 5 minutes, then add 1640 complete medium to terminate the reaction, centrifuge at 1200rpm for 5 minutes, and discard the supernatant. Resuspend the cells to 5×10 in BMDM medium containing 20ng / mL M-CSF. 6 / mL, transfer to a 10 cm dish. Prepare fresh culture medium containing 40 ng / mL M-CSF, change the medium half every two days, gently scrape the macrophages after culture adherence, centrifuge at 400g for 5 minutes, add serum-free culture medium to resuspend, and count. 6 100 ng / mL LPS and 20 ng / mL IFN-γ were added to induce macrophage polarization to M1 type and incubated for 24 hours. After incubation, the macrophages were gently scraped, centrifuged at 400g for 5 minutes, resuspended in serum-free medium with CFDASE (10μM), and incubated at room temperature in the dark for 10 minutes. After incubation, centrifuged at 400g for 5 minutes and counted.

[0096] B16F10 cells were treated with different concentrations of RS17-M6P3 (0.1μM, 0.3μM, 1μM), and PPS (500μg / mL) and RS17-M6P3 (1μM) for 8 hours. The cells were trypsinized, centrifuged at 800rpm for 3 minutes, resuspended in serum-free medium containing deepred cell tracker (1μM), and incubated at 37℃ in the dark for 30 minutes. After incubation, the cells were centrifuged at 800rpm for 3 minutes and counted.

[0097] The stained BMDM were divided into 5 × 10 4 / hole and B16F10 with 2×10 5 100 μL / well of cells were plated in a 12-well plate and incubated for 2 hours. After incubation, cells were washed 3 times with PBS, resuspended in 300 μL PBS, and analyzed on a flow cytometer.

[0098] Figure 5 Flow cytometry was used to detect the phagocytic effect of macrophages on B16F10 cells treated with RS17-M6P3 and RS17. Figure 5 A), and phagocytosis of B16F10 cells by macrophages after combined treatment with PPS (500 μg / mL) and RS17-M6P3 (1 μM) ( Figure 5 B). *p<0.05, **p<0.01, ***p<0.001.

[0099] It can be seen that the lysosome-targeted chimera that degrades CD47 in the microneedle composition of the present invention can significantly improve the recognition and phagocytosis of B16F10 cells by macrophages, and the combination with Polyporus polysaccharide can further synergistically enhance the phagocytic effect of macrophages.

[0100] Example 5 Effect of the lysosome-targeted chimera for degrading CD47 protein provided by the present invention on erythrocytes.

[0101] Experimental methods:

[0102] Collect fresh whole blood from mice in anticoagulation tubes, centrifuge at 4°C and 1000g for 30 minutes, remove the supernatant, obtain red blood cell pellets, resuspend and dilute red blood cells with 0.9% sodium chloride solution (10:1), inoculate red blood cells in 6-well plates, culture in a 37°C incubator with 5% CO2 for 12 hours, add different concentrations of RS17-M6P3 (0.1μM, 0.3μM, 1μM, 3μM, 10μM). After 12 hours of action, discard the supernatant, add 1mL pre-cooled PBS to each well to wash the cells. Add 60μL PBS containing full enzyme inhibitors to each well, and swell in an ice bath for 30 minutes. Scrape the cells with a scraper and transfer to a 1.5mL centrifuge tube, centrifuge at 4°C and 15000rpm for 15 minutes, collect the cell supernatant and add 5× loading buffer in proportion, boil at 100°C for 10 minutes, centrifuge the boiled protein, and store in a -20°C refrigerator. Western blotting was used to detect the level of CD47 protein.

[0103] Add 100 μL of red blood cell suspension to each well of a 96-well plate. Add different concentrations of RS17-M6P3 (0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM) for 12 hours. The control group was a 0.9% sodium chloride solution, and the positive group was a 0.9% sodium chloride solution containing 0.1% TritonX-100. Each group had 3 replicate wells, and 100 μL was added to each well. Incubate in a 37°C constant temperature oscillator for 1 hour. After incubation, the 96-well plate was centrifuged at 4°C and 1000g for 15 minutes, and 100 μL of the cell supernatant was aspirated into another 96-well plate, and the absorbance at 570 nm was detected with an ELISA reader.

[0104] After treating erythrocytes with 1 μM RS17-M6P3 for 8 hours, the cells were centrifuged at 2000 rpm for 3 minutes, resuspended in serum-free medium containing deepredcell tracker (1 μM), and incubated at 37°C in the dark for 30 minutes. After incubation, the cells were centrifuged at 2000 rpm for 3 minutes and counted.

[0105] The stained BMDM were divided into 5 × 10 4 / well and red blood cells at 2×10 5 100 μL / well of cells were plated in a 12-well plate and incubated for 2 hours. After incubation, cells were washed 3 times with PBS, resuspended in 300 μL PBS, and analyzed on a flow cytometer.

[0106] Figure 6 The effect of RS17-M6P3, a lysosomal targeting chimera that degrades CD47 protein, on erythrocytes. RS17-M6P3 has no degradation effect on CD47 on erythrocytes ( Figure 6 A), and no obvious hemolytic side effects ( Figure 6 B), meanwhile, macrophages did not phagocytose red blood cells treated with the lysosome-targeting chimera RS17-M6P3 ( Figure 6 C) Example 7 Effect of Polyporus polysaccharide in the Polyporus polysaccharide microneedle composition of the present invention on polarization of macrophages in vitro

[0107] Experimental methods:

[0108] After one week of culture, M0 BMDM cells were cultured at 5×10 5 The cells were seeded at a density of 1 / well in a 12-well plate and cultured for 24 hours to allow the cells to adhere to the wall. A blank control group was set up as a group without any stimulating factors.

[0109] M2 macrophage repolarization: IL-4 (20 ng / mL) was added to induce the polarization of M0 macrophages to M2 macrophages. PPS (500 g / mL) was added to the polysaccharide group on this basis.

[0110] Polarization of M1 macrophages: LPS (8 ng / mL) and IFN-γ (8 ng / mL) were added to induce the polarization of M0 macrophages to M1 macrophages; on this basis, PPS (500 g / mL) was added to the polysaccharide group.

[0111] Protective effect of M1 macrophages: First, M1 macrophages were induced by incubating with LPS (8ng / mL) and IFN-γ (8ng / mL) for 12 hours, and then the culture conditions were replaced with a culture medium containing IL-4 (20ng / ml) to allow M1 macrophages to differentiate into M2 types under environmental stimulation. PPS (500μg / mL) was added during this process. After the administration, the cells in the well plate were collected in a centrifuge tube and centrifuged at 400g for 5 minutes at 4°C. The supernatant was discarded, 1mL of ice PBS was added to wash the cells, and the supernatant was discarded after centrifugation. 100μL of 3% BSA was added to block on ice for 15 minutes, and the cells were washed with PBS. 100 μL of staining buffer containing Anti-Mouse-CDl1b-FITC, Anti-Mouse-F4 / 80-Alexa'Fluor647, and Anti-Mouse-CD86-PE antibodies was added and stained on ice for 30 minutes. Subsequently, the cells were permeabilized and fixed using a cell permeabilization and fixation kit for 30 minutes, and then washed with 1× wash buffer, and 100 μL of staining buffer containing Anti-Mouse CD206-PE-Cy7 antibody was added for staining on ice for 30 minutes. After washing with PBS, 300 μL of staining buffer was added to resuspend the cells, and the cells were filtered into a flow tube using a 70 mm cell strainer, and the macrophages were analyzed by flow cytometry.

[0112] Figure 7 The results show that PPS can repolarize M2 macrophages to M1 macrophages. Figure 7 A, B), promote macrophage polarization to M1 type ( Figure 7 C), and protects M1 macrophages from the effects of the immunosuppressive microenvironment ( Figure 7 D). **p<0.01, ***p<0.001.

[0113] Example 7 In vivo anti-melanoma effect of the polyporus polysaccharide microneedle of the present invention

[0114] 7.1 Model building

[0115] Female C57BL / 6 mice were inoculated with B16F10 cells (5×10 5 ), and tumors formed in about 6 days.

[0116] 7.2 Evaluation of the anti-melanoma effect of Poria polysaccharide microneedles in vivo

[0117] Experimental groups: ① blank control group; ② blank microneedle (Blank@MN) group; ③ poria polysaccharide microneedle (PPS@MN) group; ④ poria polysaccharide microneedle (RS17-M6P3@MN) group; ⑤ poria polysaccharide microneedle combined with lysosome-targeted chimera for CD47 degradation (PPS / RS17-M6P3@MN) group, 5 mice in each group. Evaluation indicators: ① tumor volume 2000mm 3 ① The mouse tumors were dissected out as the endpoint and the tumor sizes of each group were compared; ② The general health status of the mice, changes in diet, drinking water and body weight during the experiment were tracked and recorded, and side effects were compared; and at the end point of the experiment, Western blotting experiments and immunofluorescence section staining were performed on the tumor tissues to examine the changes in CD47 protein levels.

[0118] Figure 8 In vivo anti-melanoma growth of Polyporus polysaccharide microneedles. Figure 8 A) B16F10 tumor volume growth curve; ( Figure 8 B) and ( Figure 8 C) B16F10 tumor pictures and tumor weight statistics. Compared with other groups, PPS / RS17-M6P3@MN has the strongest inhibitory effect on tumor growth (n=5). Figure 8 D) and ( Figure 8E) CD47 protein levels in B16F10 tumor tissues and their statistical graphs. Compared with other groups, groups ④ and ⑤ containing RS17-M6P3 can significantly reduce CD47 levels in tumor sites (n=3). *p<0.05, **p<0.01, ***p<0.001.

[0119] Example 8 The regulatory effect of the lysosomal targeting chimera for degrading CD47 on macrophages in vivo after administration of the polyporus polysaccharide microneedle

[0120] 8.1 Model building

[0121] Female C57BL / 6 mice were inoculated with B16F10 cells (5×10 5 ), and tumors formed in about 6 days.

[0122] 8.2 Evaluation of the anti-melanoma effect of Poria polysaccharide microneedles in vivo

[0123] Experimental groups: ① blank control group; ② blank microneedle (Blank@MN) group; ③ poria polysaccharide microneedle (PPS@MN) group; ④ poria polysaccharide microneedle (RS17-M6P3@MN) group; ⑤ poria polysaccharide microneedle combined with lysosome-targeted chimera for CD47 degradation (PPS / RS17-M6P3@MN) group, 5 mice in each group. Evaluation index: tumor volume 2000mm 3 As the endpoint, the mouse tumors were dissected on the 13th day of tumor bearing and the 16th day of endpoint, and single cell suspensions were prepared. Flow cytometry was used to detect the changes in macrophage polarization in the tumor site.

[0124] Fig. 9 The in vivo regulatory effect of PPS / RS17-M6P3@MN on macrophage polarization, compared with other groups, on the 13th day of tumor loading and the 16th day of endpoint, the groups loaded with PPS, such as the ③PPS@MN group and the ⑤PPS / RS17-M6P3@MN group, can significantly promote the polarization of macrophages in the tumor site to M1 type and inhibit M2 type polarization (n=3). ***p<0.001.

Claims

1. A polyporus polysaccharide microneedle composition with CD47 protein degradation function, characterized in that: Contains the following raw materials in parts by weight: Polyporus polysaccharide 100-300; Lysosomal targeting chimera 5-20 for degradation of CD47 protein; Hyaluronic acid 100-300.

2. The polyporus polysaccharide microneedle composition with CD47 protein degradation function according to claim 1, characterized in that: The structure of the lysosome-targeted chimera for degrading CD47 protein is as follows:

3. The polyporus polysaccharide microneedle composition with CD47 protein degradation function according to claim 1, characterized in that: The method for preparing the lysosome-targeted chimera for degrading CD47 protein comprises the following steps: 1) Add acetic anhydride dropwise into a pyridine solution of mannose at 0°C, stir at room temperature overnight, add anhydrous ethanol to quench the excess acetic anhydride, concentrate the sample under reduced pressure, pour the residue into ice water, and filter to obtain fully acetylated mannose M6P-1; 2) Under nitrogen protection, add a solution of boron trifluoride in ether to a solution of ethyl azide and M6P-1 in dichloromethane at 0°C, move to room temperature after 1 to 2 hours and stir overnight, add dichloromethane to dilute, wash with saturated sodium bicarbonate solution, dry the organic phase, filter and concentrate under reduced pressure, and purify by column chromatography to obtain M6P-2; 3) M6P-2 was dissolved in a methanol solution of sodium methoxide with a mass percentage concentration of 25% to 35%, and after reaction at room temperature, the pH was adjusted to neutral using a cation exchange resin, and the crude product was concentrated by suction filtration. The crude product was dissolved in pyridine, triphenylmethane was added, and stirred at 45 to 55° C. for 3 to 5 hours, and acetic anhydride was added at room temperature to react overnight, and the excess acetic anhydride was quenched with anhydrous ethanol. After concentrating under reduced pressure, ice water was added, and the precipitated white solid was dissolved in an acetic acid aqueous solution, stirred at 55 to 65° C. for 1 to 2 hours, and ice water was added to precipitate the solid, which was filtered and washed with water, and the solid was purified by column chromatography to obtain M6P-3; 4) Under nitrogen protection, N,N-diisopropylphosphoramidite di-tert-butyl ester was added to an anhydrous dichloromethane solution of M6P-3 and 1H-tetrazole at 0°C, stirred for 2 to 3 hours, tert-butyl hydroperoxide was added at 0°C, stirred, and then moved to room temperature for 2 to 3 hours, saturated sodium thiosulfate solution was added to quench the reaction, extracted with dichloromethane, the organic phases were combined, filtered and concentrated under reduced pressure, and purified by column chromatography to obtain M6P-4; 5) Take M6P-4 and add a methanol solution of sodium methoxide with a mass percentage concentration of 25% to 35%, react at room temperature for 2 to 3 hours, use a 50-100 mesh cation exchange resin to adjust the pH to neutral, filter the resin and concentrate the filtrate under reduced pressure, dissolve the residue in a dichloromethane solution containing trifluoroacetic acid with a mass percentage concentration of 8 to 12%, stir at room temperature for 1 to 2 hours, and then concentrate under reduced pressure to obtain M6P; 6) A peptide conjugate RS17-(PβA)3 was obtained by solid phase synthesis method, using Rink Amide MBHA amino resin as a carrier and ethyl 2-oxime cyanoacetate / N,N'-diisopropylcarbodiimide as a condensation system; the amino acid sequence of RS17 is: H-RRYKQDGGWSHWSPWSS-NH2; 7) Using copper-catalyzed click chemistry reaction, with cuprous sulfate / sodium ascorbate as the catalyst system, the peptide conjugate RS17-(PβA)3, M6P, cuprous sulfate, and sodium ascorbate were added to a mixed solvent of methanol and pure water, reacted at room temperature in the dark, and then purified and freeze-dried to obtain a lysosomal targeting chimera that degrades CD47 protein; 4. A polyporus polysaccharide microneedle with CD47 protein degradation function, characterized in that: The composition is prepared from the raw materials of the polyporus polysaccharide microneedle composition with CD47 protein degradation function according to any one of claims 1 to 3.

5. The polyporus polysaccharide microneedle with CD47 protein degradation function according to claim 4, characterized in that: The microneedle comprises a substrate and a needle body located on the substrate; wherein the needle body is prepared from the polyporus polysaccharide of any one of claims 1 to 3 and a lysosome targeting chimera for degrading CD47 protein as raw materials, and the microneedle substrate is composed of hyaluronic acid.

6. The method for preparing the polyporus polysaccharide microneedle with CD47 protein degradation function according to claims 4-5, characterized in that: The steps include: 1) Weighing a lysosome-targeted chimera for degrading CD47 protein and Polyporus polysaccharide, adding them to pure water to prepare a mixed aqueous solution containing 5-20 mg / mL of the lysosome-targeted chimera for degrading CD47 protein and 100-300 mg / mL of Polyporus polysaccharide, and swelling them at 4° C. overnight; 2) Pour a mixed aqueous solution of the lysosome-targeted chimera that degrades CD47 protein and Polyporus polysaccharide into the groove of the microneedle mold as the needle body matrix material of the microneedle, centrifuge at 4°C, rotate 180° horizontally, and repeat the centrifugation once to recover the excess material solution in the groove; 3) preparing an aqueous solution with a hyaluronic acid concentration of 100-300 mg / mL as the base material of the microneedle, pouring it into a mold and placing it in an oven at 50-60° C. to dry overnight, and then demolding to obtain the microneedle.

7. Use of the polyporus polysaccharide microneedles with CD47 protein degradation function as described in claims 4-6 in the preparation of drugs for treating tumors.

8. The use according to claim 7, characterized in that: The tumor is melanoma.

9. A lysosomal targeting chimera for degrading CD47 protein, characterized in that: Its structure is as follows:

10. The method for preparing the lysosome-targeted chimera for degrading CD47 protein according to claim 9, characterized in that: The steps include: 1) Add acetic anhydride dropwise to a pyridine solution of mannose at 0°C, stir at room temperature overnight, add anhydrous ethanol to quench the excess acetic anhydride, concentrate the sample under reduced pressure, pour the compound into ice water, and filter to obtain fully acetylated mannose M6P-1; 2) Under nitrogen protection, add a solution of boron trifluoride in ether to a solution of ethyl azide and M6P-1 in dichloromethane at 0°C, move to room temperature after 1 to 2 hours and stir overnight, add dichloromethane to dilute, wash with saturated sodium bicarbonate solution, dry the organic phase, filter and concentrate under reduced pressure, and purify by column chromatography to obtain M6P-2; 3) M6P-2 was dissolved in a methanol solution of sodium methoxide with a mass percentage concentration of 25% to 35%, and after reaction at room temperature, the pH was adjusted to neutral using a cation exchange resin, and the crude product was concentrated by suction filtration. The crude product was dissolved in pyridine, triphenylmethane was added, and stirred at 45 to 55° C. for 3 to 5 hours, and acetic anhydride was added at room temperature to react overnight, and the excess acetic anhydride was quenched with anhydrous ethanol. After concentrating under reduced pressure, ice water was added, and the precipitated white solid was dissolved in an acetic acid aqueous solution, stirred at 55 to 65° C. for 1 to 2 hours, and ice water was added to precipitate the solid, which was filtered and washed with water, and the solid was purified by column chromatography to obtain M6P-3; 4) Under nitrogen protection, N,N-diisopropylphosphoramidite di-tert-butyl ester was added to an anhydrous dichloromethane solution of M6P-3 and 1H-tetrazole at 0°C, stirred for 2 to 3 hours, tert-butyl hydroperoxide was added at 0°C, stirred, and then moved to room temperature for 2 to 3 hours, saturated sodium thiosulfate solution was added to quench the reaction, extracted with dichloromethane, the organic phases were combined, filtered and concentrated under reduced pressure, and purified by column chromatography to obtain M6P-4; 5) Take M6P-4 and add a methanol solution of sodium methoxide with a mass percentage concentration of 25% to 35%, react at room temperature for 2 to 3 hours, use a 50-100 mesh cation exchange resin to adjust the pH to neutral, filter the resin and concentrate the filtrate under reduced pressure, dissolve the residue in a dichloromethane solution containing trifluoroacetic acid with a mass percentage concentration of 8 to 12%, stir at room temperature for 1 to 2 hours, and then concentrate under reduced pressure to obtain M6P; 6) 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 polypeptide conjugate RS17-(PβA)3; the amino acid sequence of RS17 is: H-RRYKQDGGWSHWSPWSS-NH2; 7) Through a copper-catalyzed click chemistry reaction, using cuprous sulfate / sodium ascorbate as a catalyst system, the peptide conjugate RS17-(PβA)3, M6P, cuprous sulfate, and sodium ascorbate were added to a mixed solvent of methanol and pure water, reacted at room temperature in the dark, and then purified and lyophilized to obtain a lysosomal-targeted chimera that degrades CD47 protein;

Images