Kombucha polysaccharide microneedle composition with cd47 protein degradation function, microneedle and preparation method and application thereof

Abstract

The present application provides a kuzhuang polysaccharide microneedle composition with CD47 protein degradation function, comprising a lysosome-targeting chimera degrading CD47 protein, kuzhuang polysaccharide and hyaluronic acid. The present application also provides a preparation method of the lysosome-targeting chimera degrading CD47 protein. The present application also provides a microneedle prepared from the kuzhuang polysaccharide microneedle composition. The present application also provides a preparation method of the kuzhuang polysaccharide microneedle with CD47 protein degradation function and application thereof in preparing a medicine for treating tumors. The microneedle of the present application can efficiently degrade CD47 protein in melanoma tumor tissues, thereby enhancing the phagocytosis of macrophages on tumor cells, and on the other hand, the kuzhuang polysaccharide in the microneedle can promote the polarization of macrophages to an anti-tumor type, further enhancing the anti-tumor activity of macrophages. The present application can degrade CD47 and reshape the immunosuppressive microenvironment at the same time, realizing the efficient synergistic anti-tumor effect of the two drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a pharmaceutical composition, specifically a microneedle composition of Poria cocos polysaccharide with CD47 protein degradation function, the microneedles, the preparation method thereon, and their applications. Background Technology

[0002] Melanoma is a malignant tumor caused by melanocytes, characterized by its aggressiveness, high metastasis rate, and poor prognosis, with a recurrence rate as high as 70% and a 5-year survival rate of only 23% for advanced-stage patients. Early-stage melanoma is primarily treated with surgical resection; however, this only removes the primary tumor and is insufficient to address metastasis. Immune checkpoint inhibitors, represented by PD-1 / L1 inhibitors, have made significant progress in melanoma treatment in recent years. These drugs activate the patient's cytotoxic T cells, enabling them to recognize and kill tumor cells. However, existing immune checkpoint inhibitors still have limitations, such as low response rates. As an important component of innate immunity, macrophages recognize and clear foreign, aging, damaged, and dead cells from tissues, playing a crucial role in maintaining homeostasis. Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor stroma. M1-type 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 develops, M1-type tumor markers (TAMs) gradually polarize into M2-type TAMs, which promote tumor development and progression. Besides the macrophage phenotype affecting their ability to clear tumor cells, signaling proteins on the surface of tumor cells also influence macrophage phagocytic function. CD47, a transmembrane protein expressed on the surface of various tumor cells, interacts with SIRPα on macrophages, transmitting an immunosuppressive signal ("don't eat me"), thus enabling immune escape. Currently, CD47 has become a novel potential target for immunotherapy, with several CD47 antibodies undergoing clinical trials or preclinical studies. However, CD47 antibody therapy often affects the homeostasis of erythrocytes, platelets, and hematopoietic stem cells, leading to severe "on-target toxicity," including erythrocyte 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 therapeutic modality that primarily exerts its degradation effects through the proteasome or lysosome pathway. Lysosome-targeting chimaeras (LYTACs) offer the potential for targeted degradation of extracellular and membrane proteins and are increasingly being used in drug development. Since mature erythrocytes lack organelles, LYTACs can avoid degrading CD47 on the erythrocyte membrane, thus preventing recognition and clearance by macrophages and potentially overcoming side effects such as anemia associated with CD47 antibody therapy.

[0004] Microneedles, as a novel minimally invasive transdermal drug delivery system, can achieve continuous drug release within 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). Traditional Chinese medicine polysaccharides possess advantages such as high structural stability, good biocompatibility, and biodegradability, making them a natural material for microneedle preparation. Among them, Polyporus polysaccharide (PPS), one of the main active ingredients of the traditional Chinese medicine Polyporus umbellatus, has been approved by the China Food and Drug Administration as an immune enhancer for chemotherapy in cancer patients. Reports have shown that PPS can promote macrophage polarization towards the M1 type, thereby enhancing the anti-tumor effect of macrophages, thus possessing the potential for synergistic effects with CD47 therapy.

[0005] Therefore, this invention designs and constructs a microneedle containing *Polyporus umbellatus* polysaccharide, based on a lysosomal targeting chimera that degrades CD47 protein and *Polyporus umbellatus* polysaccharide, which enables CD47 protein degradation. This microneedle allows for drug delivery to the tumor site, releasing the lysosomal targeting chimera that degrades CD47 protein, thus achieving CD47 degradation. It also releases *Polyporus umbellatus* polysaccharide, promoting the polarization of M2-type tumor markers (TAMs) towards M1-type, further enhancing the ability of M1-type TAMs to clear tumor cells, and synergistically enhancing the immunotherapy of melanoma. No similar reports have been found to date. Summary of the Invention

[0006] To address the aforementioned problems in the prior art, this invention provides a microneedle composition of *Polyporus umbellatus* polysaccharide with CD47 protein degradation function, microneedles, their preparation method, and applications. This microneedle composition of *Polyporus umbellatus* polysaccharide with CD47 protein degradation function, microneedles, their preparation method, and applications aim to solve the technical problem of poor efficacy in melanoma immunotherapy in the prior art.

[0007] This invention provides a microneedle composition of *Polyporus umbellatus* polysaccharide with CD47 protein degradation function, comprising the following raw materials in parts by weight:

[0008] Polyporus umbellatus polysaccharide 100-300;

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

[0010] Hyaluronic acid 100-300.

[0011] Furthermore, the structure of the lysosomal targeting chimera that degrades CD47 protein is as follows:

[0012]

[0013] Furthermore, the synthesis process of the lysosomal targeting chimera that degrades CD47 protein is described below:

[0014] 1) Acetic anhydride was added dropwise to a pyridine solution of mannose at 0°C, stirred overnight at room temperature, and excess acetic anhydride was quenched with anhydrous ethanol. The sample was concentrated under reduced pressure, the compound was poured into ice water, and filtered to obtain fully acetylated mannose M6P-1; 2) Under nitrogen protection, a boron trifluoride diethyl ether solution was added dropwise to a solution of azidoethanol and M6P-1 in dichloromethane at 0°C. After 1-2 hours, the solution was moved to room temperature and stirred overnight. The solution was diluted with dichloromethane, washed with saturated sodium bicarbonate solution, dried, filtered, concentrated under reduced pressure, and purified 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) Dissolve M6P-2 in a methanol solution of sodium methoxide with a mass percentage concentration of 25%–35%. After reacting at room temperature, adjust the pH to neutral using a cation exchange resin. Filter and concentrate the crude product. Dissolve the crude product in pyridine, add triphenylchloromethane, and stir at 45–55°C for 3–5 hours. Then, add acetic anhydride at room temperature and react overnight. Quench excess acetic anhydride with anhydrous ethanol, concentrate under reduced pressure, and add ice water. Dissolve the precipitated white solid in an aqueous acetic acid solution, stir at 55–65°C for 1–2 hours, add ice water, and precipitate the solid. Filter and wash with water. Purify the solid by column chromatography to obtain M6P-3.

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

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

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

[0019] 7) Using a 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. After stirring at room temperature in the dark, the mixture was purified and lyophilized to obtain the lysosomal targeted chimera that degrades 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.

[0021]

[0022] Furthermore, the lysosomal targeting chimera that degrades CD47 protein can degrade CD47 protein on melanoma cells, block their immune escape function, and promote macrophage recognition and killing of melanoma cells.

[0023] The present invention also provides a microneedle of *Polyporus umbellatus* polysaccharide with CD47 protein degradation function, which is prepared from the raw materials of the above-mentioned microneedle composition of *Polyporus umbellatus* polysaccharide with CD47 protein degradation function.

[0024] Furthermore, the *Polyporus umbellatus* polysaccharide microneedles with CD47 protein degradation function comprise a substrate and a needle body located on the substrate; wherein, the needle body is prepared from *Polyporus umbellatus* polysaccharide and a lysosomal targeting chimera that degrades CD47 protein as raw materials, and the microneedle substrate is composed of hyaluronic acid.

[0025] This invention also provides a method for preparing the above-mentioned microneedles of *Polyporus umbellatus* polysaccharide with CD47 protein degradation function, comprising the following steps:

[0026] 1) Weigh the lysosomal targeting chimera that degrades CD47 protein and the polysaccharide of Poria cocos, add them to pure water, and prepare a mixed aqueous solution containing 5-20 mg / mL of the lysosomal targeting chimera that degrades CD47 protein and 100-300 mg / mL of the polysaccharide of Poria cocos. Let it swell overnight at 4°C.

[0027] 2) Pour a mixed aqueous solution of lysosomal targeting chimera degraded with CD47 protein and poria cocos polysaccharide into the groove of the microneedle mold as the microneedle matrix material. Centrifuge at 4000 rpm for 5 minutes at 4°C and rotate 180° horizontally.

[0028] Then repeat the centrifugation once more to recover any excess material solution in the groove.

[0029] 3) Prepare an aqueous solution of hyaluronic acid with a concentration of 100-300 mg / mL as the base material for microneedles. Pour the solution into a mold and dry it overnight in an oven at 55°C. Then, demold the microneedles to obtain the microneedles.

[0030] This invention also provides the application of the above-mentioned microneedles containing CD47 protein degradation function of Poria cocos polysaccharide in the preparation of drugs for treating tumor diseases.

[0031] Specifically, the tumor in question is melanoma.

[0032] The present invention also provides a lysosomal targeting chimera for degrading CD47 protein, the structural formula of which is shown below:

[0033] This invention also provides a method for preparing the above-mentioned lysosomal targeting chimera that degrades CD47 protein, comprising the following steps:

[0034] 1) Acetic anhydride was added dropwise to a pyridine solution of mannose at 0°C. After stirring overnight at room temperature, excess acetic anhydride was quenched with anhydrous ethanol. The sample was concentrated under reduced pressure, and the compound was poured into ice water and filtered to obtain fully acetylated mannose M6P-1.

[0035] 2) Under nitrogen protection, a boron trifluoride diethyl ether solution was added dropwise to a solution of azidoethanol and M6P-1 in dichloromethane at 0°C. After 1–2 hours, the mixture was moved to room temperature and stirred overnight. The solution was diluted with dichloromethane, washed with saturated sodium bicarbonate solution, dried, filtered, and concentrated under reduced pressure. M6P-2 was purified by column chromatography. Preferably, the molar ratio of azidoethanol, M6P-1, and boron trifluoride is 1.5:1:4.0.

[0036] 3) Dissolve M6P-2 in a methanol solution of sodium methoxide with a mass percentage concentration of 25%–35%. After reacting at room temperature, adjust the pH to neutral using a cation exchange resin. Filter and concentrate the crude product. Dissolve the crude product in pyridine, add triphenylchloromethane, and stir at 45–55°C for 3–5 hours. Then, add acetic anhydride at room temperature and react overnight. Quench excess acetic anhydride with anhydrous ethanol, concentrate under reduced pressure, and add ice water. Dissolve the precipitated white solid in an aqueous acetic acid solution, stir at 55–65°C for 1–2 hours, add ice water, and precipitate the solid. Filter and wash with water. Purify the solid by column chromatography to obtain M6P-3.

[0037] 4) Under nitrogen protection, N,N-diisopropylphosphonamide di-tert-butyl ester was added to an anhydrous dichloromethane solution of M6P-3 and 1H-tetrazole at 0°C. After stirring for 2-3 hours, tert-butyl hydroperoxide was added at 0°C and stirred for 5 minutes. The mixture was then moved to room temperature and reacted for 2-3 hours. The reaction was quenched by adding saturated sodium thiosulfate solution. The mixture was extracted with dichloromethane, and the organic phases were combined, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain M6P-4. Preferably, the molar ratio of N,N-diisopropylphosphonamide di-tert-butyl ester, M6P-3, and 1H-tetrazole is 1.5:1.0:2.0.

[0038] 5) Add M6P-4 to a methanol solution of sodium methoxide with a mass percentage concentration of 25% to 35%, react at room temperature for 2 to 3 hours, adjust the pH to neutral using a 50-100 mesh cation exchange resin, filter the resin, concentrate the filtrate under reduced pressure, dissolve the residue in a dichloromethane solution containing a mass percentage concentration of 8% to 12% trifluoroacetic acid, stir at room temperature for 1 to 2 hours, concentrate under reduced pressure to obtain M6P;

[0039] 6) Based on the 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 polypeptide conjugate RS17-(PβA)3 was obtained; the amino acid sequence of RS17 is: H-RRYKQDGGWSHWSPWSS-NH2 (SEQ ID NO.1);

[0040] 7) Through copper-catalyzed click chemistry, using 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. After reacting at room temperature in the dark, the conjugate was purified and lyophilized to obtain the lysosomal targeted chimera that degrades CD47 protein.

[0041] Preferably, the molar ratio of RS17-(PβA)3, M6P, cuprous sulfate, and sodium ascorbate is 1:4.5:4.5:9.0.

[0042]

[0043] The *Polyporus umbellatus* polysaccharide microneedle of the present invention, which has CD47 protein degradation function, rapidly dissolves at the tumor site after the needle body is inserted into the skin, releasing a lysosomal targeting chimera that degrades CD47 protein and *Polyporus umbellatus* polysaccharide. The lysosomal targeting chimera that degrades CD47 protein consists of three parts: (1) a polypeptide sequence targeting CD47; (2) a linker chain; and (3) a glycopeptide sequence targeting the non-cationic mannose-6-phosphate receptor (CI-M6PR). The CD47 targeting polypeptide sequence (H-RRYKQDGGWSHWSPWSS-NH2) in the lysosomal targeting chimera can specifically bind to CD47, as reported in the literature [Wang, Xinmin et al. An antitumor peptide RS17-targeted CD47, design, synthesis, and antitumor activity. Cancer medicine, 2021, 10, 2125-2136.]. The intermediate linker 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. The specific binding of M6P to CI-M6PR is reported in the literature [Banik, Steven M et al. Lysosome-targeting chimaeras for degradation of extracellular proteins. Nature, 2020, 584, 291-297.]. This lysosomal targeting chimera that degrades CD47 protein can mediate the degradation of the membrane protein CD47 via the endocytosis-lysosomal pathway in melanoma cells, thereby enhancing the phagocytosis of tumor cells by macrophages. Simultaneously released *Polyporus umbellatus* 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. This invention can effectively inhibit tumor growth in melanoma-bearing mice, exert anti-tumor effects, and can be used to prepare anti-melanoma drugs.

[0044] This invention constructs a microneedle of *Polyporus umbellatus* polysaccharide with CD47 protein degradation function, loaded with a lysosomal targeting chimera that degrades CD47 protein and *Polyporus umbellatus* polysaccharide, which activates the body's anti-tumor immunity through transdermal administration.

[0045] The advantages of this invention are:

[0046] 1. This invention constructs soluble poria cocos polysaccharide microneedles with good biocompatibility. After entering the tumor site, they can rapidly dissolve and release lysosomal targeting chimeras that degrade CD47 and poria cocos polysaccharide, thereby achieving synergistic effects of immune checkpoint CD47 degradation and remodeling of the macrophage-associated immunosuppressive microenvironment.

[0047] 2. The lysosomal targeting chimera that degrades CD47 in the microneedle composition of the present invention can degrade CD47 in B16F10 melanoma cells.

[0048] 3. The CD47-degraded lysosomal targeting chimera in the microneedle composition of the present invention can significantly improve the recognition and phagocytosis of melanoma cells by macrophages, and the combination with poria cocos polysaccharide can further synergistically enhance the phagocytic activity of macrophages.

[0049] 4. The polysaccharide in the microneedle composition of the present invention can significantly promote the polarization of M2 macrophages to M1 type, and further enhance the ability of M1 macrophages to clear melanoma cells.

[0050] 5. The poria cocos polysaccharide microneedles of the present invention with CD47 protein degradation function can significantly inhibit the growth of B16F10 melanoma xenografts in C57BL / 6 mice, and exert a more significant anti-tumor effect than blank microneedles and drug-loaded microneedles alone, and have good application prospects.

[0051] 6. The poria cocos polysaccharide microneedles of the present invention, which have CD47 protein degradation function, can significantly degrade the level of CD47 protein in melanoma tumors in vivo.

[0052] 7. The poria cocos polysaccharide microneedles of the present invention, which have CD47 protein degradation function, can significantly increase the proportion of M1 / M2 macrophages in in vivo experiments and improve the macrophage-related immunosuppressive microenvironment. Attached Figure Description

[0053] Figure 1 This is the synthetic route for the lysosomal targeting chimera that degrades CD47 protein in the poria cocos polysaccharide microneedle composition of the present invention.

[0054] Figure 2 This is a mass spectrum of the lysosomal targeting chimera of degraded CD47 protein in the poria cocos polysaccharide microneedle composition of the present invention.

[0055] Figure 3 Morphological and mechanical strength characterization of the poria cocos polysaccharide microneedles of the present invention.

[0056] Figure 4 This invention demonstrates the CD47 degradation effect of the lysosomal targeting chimera in the poria cocos polysaccharide microneedle composition on CD47 protein degradation.

[0057] Figure 5This invention relates to the effect of the lysosomal targeting chimera of degraded CD47 protein in the poria cocos polysaccharide microneedle composition on erythrocytes.

[0058] Figure 6 This invention relates to the in vitro polarization effect of poria cocos polysaccharide on macrophages in the poria cocos polysaccharide microneedle composition.

[0059] Figure 7 The present invention relates to a polysaccharide microneedle composition from *Polyporus umbellatus* that promotes the uptake of melanoma cells by macrophages.

[0060] Figure 8 This invention relates to the in vivo antitumor effect of poria cocos polysaccharide microneedles.

[0061] Figure 9 This invention relates to the regulatory effect of poria cocos polysaccharide microneedles on macrophages in vivo. Detailed Implementation

[0062] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0063] Example 1 Synthesis of the lysosomal targeting chimera for degrading CD47 protein provided by the present invention.

[0064] Synthesis steps:

[0065] 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 the mixture was stirred overnight at room temperature. Thin-layer chromatography (TLC) (petroleum ether PE: ethyl acetate EA = 3:1) was used for detection, with sulfuric acid and ethanol as the colorimetric indicator. After the reaction was complete, anhydrous ethanol was added to react with excess acetic anhydride, and the mixture was concentrated under reduced pressure. The residue was added to ice water, filtered, and the upper solid precipitate was collected to obtain fully acetylated mannose M6P-1.

[0066] Azide ethanol (1.0 g, 11.5 mmol, 1.5 eq.) and M6P-1 (3.0 g, 7.69 mmol, 1.0 eq.) were dissolved in 40 mL of dichloromethane (DCM). Under nitrogen protection, boron trifluoride diethyl ether solution (48% by mass) (9.1 mL, 30.76 mmol, 4.0 eq.) was slowly added dropwise at 0 °C. After 1 hour, the mixture was brought to room temperature and stirred overnight. Thin-layer chromatography (TLC) (PE:EA = 1:1) was used for detection, with sulfuric acid and ethanol as the colorimetric indicator. After the reaction was complete, the mixture was diluted with dichloromethane and washed with ice water and 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 = 5:1 → 3:1) to obtain M6P-2.

[0067] 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:methanolMeOH = 10:1) was performed, with sulfuric acid and ethanol as the colorimetric indicator. The pH of the reaction solution was adjusted to neutral using a cation exchange resin, and the solution was concentrated by filtration to obtain the crude product. This crude product was dissolved in pyridine (15 mL), and triphenylchloromethane (1.762 g, 6.32 mmol, 1.05 eq.) was added. The mixture was stirred at 50 °C for 4 hours. TLC (PE:EA = 2:1) was performed. After the reaction was complete, the mixture was moved to room temperature, acetic anhydride (10 mL) was added, and the mixture was incubated overnight at room temperature. TLC (PE:EA = 2:1) was performed. After the reaction was complete, the residue was dissolved in anhydrous ethanol, concentrated under reduced pressure to remove pyridine, and then added to ice water. A white solid precipitated, which was then filtered to obtain the crude product. The crude product was dissolved in acetic acid and water (12 mL acetic acid, 8 mL water) and stirred at 60 °C for 1 hour. TLC (PE:EA = 2:1) was used for detection. After the reaction was complete, ice water was added to the reaction solution, and a white solid precipitated. The solid was filtered and washed with water, and then purified by column chromatography (PE:EA = 5:1 → 2:1) to obtain M6P-3.

[0068] M6P-3 (200 mg, 0.4 mmol, 1.0 eq.) and 1H-tetrazole (56 mg, 0.8 mmol, 2.0 eq.) were dissolved in anhydrous DCM under nitrogen protection. N,N-diisopropylphosphonamide di-tert-butyl ester (166 mg, 0.6 mmol, 1.5 eq.) was slowly added dropwise at 0 °C, and stirring was continued for 2 hours. The reaction was monitored by TLC (PE:EA = 2:1). 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, and the mixture was stirred for 5 minutes. The mixture was then cooled to room temperature and stirred for 2 hours. The reaction was monitored by TLC (PE:EA = 1:3). After the reaction was complete, saturated sodium thiosulfate solution was added to quench the reaction. The mixture was extracted three times with DCM, and the combined organic phases were washed with 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.

[0069] M6P-4 (204 mg, 0.36 mmol, 1.0 eq.) was added to a 30% sodium methoxide methanol solution (65 μL, 0.36 mmol, 3.0 eq.) and reacted at room temperature for 2 hours. TLC (DCM:MeOH = 10:1) was used for detection. After the reaction was complete, the pH was adjusted to neutral using a 50-100 mesh cation exchange resin. The resin was filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in a 2 mL solution of dichloromethane containing 10% trifluoroacetic acid (TFA) and stirred at room temperature for 1 hour. TLC was used for detection. After the reaction was complete, the solution was concentrated under reduced pressure to obtain M6P.

[0070] The polypeptide conjugate RS17-(PβA)3 was obtained by solid-phase synthesis. The amino acid sequence of RS17 is: H-RRYKQDGGWSHWSPWSS-NH2. The Fmoc solid-phase synthesis method described above is existing technology and will not be described in detail here.

[0071] 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-ascorbic acid sodium (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. The RS17-(PβA)3, M6P, CuSO4, and L-ascorbic acid sodium solutions were added to a 5 mL reaction flask and stirred overnight at room temperature. The resulting lysosomal targeting chimera RS17-M6P3, which degrades CD47 protein, was then purified and lyophilized. The synthetic procedure is described in [link to synthetic procedure]. Figure 1

[0072] Its structure is as follows:

[0073]

[0074] The primary structure of the lysosomal targeting chimera RS17-M6P3, which degrades CD47 protein, was confirmed by mass spectrometry. (Mass spectrum shown below) Figure 2 The data is shown in the table below:

[0075]

[0076]

[0077] All reagents used in the examples were commercially available analytical grade, and the solvents used in the preparation of liquid phases were chromatographic grade.

[0078] Example 2: Preparation of Poria cocos polysaccharide microneedles with CD47 protein degradation function according to the present invention

[0079] Preparation steps:

[0080] Weigh the lysosomal targeting chimera that degrades CD47 protein and the polysaccharide from *Polyporus umbellatus*, add them to pure water, and prepare a mixed aqueous solution containing 10 mg / mL of the lysosomal targeting chimera that degrades CD47 protein and 200 mg / mL of the polysaccharide from *Polyporus umbellatus*. Let it swell overnight at 4°C.

[0081] A mixed aqueous solution of lysosomal targeting chimera degraded CD47 protein and poria cocos polysaccharide was poured into the groove of the microneedle mold as the matrix material of the microneedle. The microneedle was centrifuged at 4000 rpm for 5 minutes at 4°C, rotated 180° and centrifuged again to recover the excess material solution in the groove.

[0082] An aqueous solution of hyaluronic acid with a concentration of 200 mg / mL was prepared as the base material for microneedles. After being poured into a mold, the microneedles were dried overnight in an oven at 55°C and then demolded to obtain the microneedles.

[0083] Example 3: Morphological and mechanical strength characterization of the *Polyporus umbellatus* polysaccharide microneedles with CD47 protein degradation function according to the present invention.

[0084] Experimental methods:

[0085] After surface gold plating in an ion sputtering coating machine, the morphology of the microneedles was observed using scanning electron microscopy. FITC-labeled lysosomal targeting chimera RS17-M6P3 (degrading CD47 protein) and Cy5-labeled *Polyporus umbellatus* polysaccharide were added to the microneedles. Drug distribution within the microneedles was observed using laser confocal microscopy, showing that RS17-M6P3 and *Polyporus umbellatus* polysaccharide were uniformly dispersed within the microneedle body. Mechanical strength was analyzed using a texture analyzer. When the probe (a 10 mm diameter stainless steel cylinder) contacted the sample (with a trigger force of 0.05 N), it was pressed downwards at a speed of 30 mm / min to deform the sample by 40%, and then the probe returned to its initial position. Force-displacement maps were obtained. The microneedles were inserted into the skin of SD rats, and their morphology was observed using an optical microscope after removal at different time points.

[0086] Figure 3 Morphological and mechanical strength characterization of *Polyporus umbellatus* polysaccharide microneedles with CD47 protein degradation function. Figure 3 A) Microneedle morphology of different groups under scanning electron microscopy. Figure 3 B) The force-displacement curve shows that the microneedles possess the mechanical strength to pierce the skin. Figure 3 C) The drug-loaded *Polyporus umbellatus* polysaccharide microneedles completely dissolve within 5 minutes, releasing the drug. Figure 3 D) Observe the drug distribution in the microneedles using laser confocal microscopy.

[0087] Example 4: CD47 degradation in B16F10 melanoma cells by the lysosomal targeting chimera provided in this invention.

[0088] Experimental methods:

[0089] B16F10 cells were divided into 3×10 groups. 5 Cells were seeded per well in 6-well plates and incubated at 37°C with 5% CO2 for 24 hours. Different concentrations of RS17-M6P3 (0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM) were then added. After 24 hours of incubation, the supernatant was discarded, and 1 mL of pre-chilled PBS was added to each well for washing. 60 μL of NP-40 lysis buffer containing the holoenzyme inhibitor was added to each well, and the cells were lysed on ice for 30 minutes. Cells were scraped off with a spatula and transferred to 1.5 mL centrifuge tubes, centrifuged at 4°C and 15000 rpm for 15 minutes, and the cell supernatant was collected. 5× loading buffer was added proportionally, and the cells were boiled at 100°C for 10 minutes. The boiled protein was centrifuged and stored at -20°C. CD47 protein levels were detected using a protein immunoblotting assay.

[0090] In vitro CD47 degradation experiment (see) Figure 4 :Depend on Figure 4 It is evident that the lysosomal targeting chimera for degrading CD47 protein of the present invention is dose-dependent ( Figure 4 A) and time dependence ( Figure 4 B) Degradation of CD47 protein in B16F10 cells, ***p<0.001.

[0091] Example 5: The lysosomal targeting chimera that degrades CD47 protein provided by the present invention promotes macrophage uptake of melanoma cells.

[0092] Experimental methods:

[0093] Mouse bone marrow-derived macrophages (BMDM) were extracted and cultured. Female mice (seven-week-old C57BL / 6 mice) were euthanized by cervical dislocation and soaked in 75% ethanol for 15 minutes. Under sterile conditions in a laminar flow hood, all femurs and tibias were surgically removed, and surrounding muscle tissue was excised. The bone marrow cavity was repeatedly flushed, and debris and tissue were filtered through a 200-mesh nylon mesh. The cells were then centrifuged at 400g for 5 minutes, and the supernatant was discarded. Red blood cell lysis buffer (3 mL) was added, and the mixture was incubated at room temperature for 5 minutes. Then, 1640g of complete culture medium was added to terminate the reaction, and the cells were centrifuged at 1200rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in BMDM medium containing 20 ng / mL M-CSF to a final volume of 5 × 10⁶ cells / mL. 6Transfer macrophages at 1×10⁶ cells / mL to 10cm dishes. Prepare fresh medium containing 40 ng / mL M-CSF. Perform half-medium medium changes every two days using BMDM. Gently scrape off adherent macrophages, centrifuge at 400g for 5 minutes, resuspend in serum-free medium, and count. 6 Macrophages were seeded per well in 12-well plates and incubated for 24 hours. After incubation, 100 ng / mL LPS and 20 ng / mL IFN-γ were added to induce macrophage polarization to the M1 type, and incubation continued for another 24 hours. After incubation, macrophages were gently scraped off, centrifuged at 400g for 5 minutes, resuspended in serum-free medium (10 μM CFDASE), and incubated at room temperature in the dark for 10 minutes. After incubation, macrophages were centrifuged at 400g for 5 minutes and counted.

[0094] B16F10 cells were treated with different concentrations (0.1 μM, 0.3 μM, 1 μM) of RS17-M6P3, and after treatment with a combination of PPS (500 μg / mL) and RS17-M6P3 (1 μM) for 8 hours, the cells were digested with trypsin, centrifuged at 800 rpm for 3 minutes, resuspended in serum-free medium containing 1 μM of deepred cell tracker, and incubated at 37°C in the dark for 30 minutes. After incubation, the cells were centrifuged at 800 rpm for 3 minutes and counted.

[0095] The stained BMDM was subjected to 5×10 4 Each hole and B16F10 in 2×10 5 Cells were seeded per well in 12-well plates and incubated for 2 hours. After incubation, the cells were washed three times with PBS, resuspended in 300 μL PBS, and analyzed by flow cytometry.

[0096] Figure 5 To detect the phagocytic effect of macrophages on B16F10 cells treated with RS17-M6P3 and RS17 by flow cytometry. Figure 5 A), and the phagocytic effect of macrophages on B16F10 cells 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.

[0097] It is evident that the CD47-degraded lysosomal targeting chimera in the microneedle composition of the present invention can significantly enhance the recognition and phagocytosis of B16F10 cells by macrophages, and the combination with Polyporus umbellatus polysaccharide can further synergistically enhance the phagocytic activity of macrophages.

[0098] Example 5: Effect of the lysosomal targeting chimera that degrades CD47 protein provided by the present invention on erythrocytes.

[0099] Experimental methods:

[0100] Fresh mouse whole blood was collected in anticoagulant tubes and centrifuged at 1000g for 30 minutes at 4°C. The supernatant was discarded, and red blood cell pellets were obtained. The red blood cells were resuspended and diluted with 0.9% sodium chloride solution (10:1). The red blood cells were seeded into 6-well plates and incubated at 37°C with 5% CO2 for 12 hours. Different concentrations of RS17-M6P3 (0.1μM, 0.3μM, 1μM, 3μM, 10μM) were added. After incubation for 12 hours, the supernatant was discarded, and 1 mL of pre-chilled PBS was added to each well to wash the cells. 60 μL of PBS containing the holoenzyme inhibitor was added to each well, and the cells were swollen on ice for 30 minutes. The cells were scraped off with a spatula and transferred to 1.5 mL centrifuge tubes. The cells were centrifuged at 15000 rpm for 15 minutes at 4°C. The cell supernatant was collected, and 5× loading buffer was added proportionally. The cells were boiled at 100°C for 10 minutes. The boiled protein was centrifuged and stored at -20°C. The level of CD47 protein was detected by protein immunoblotting.

[0101] 100 μL of red blood cell suspension was added to each well of a 96-well plate. Different concentrations of RS17-M6P3 (0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM) were added and incubated for 12 hours. The control group used 0.9% sodium chloride solution, and the positive group used 0.9% sodium chloride solution containing 0.1% Triton X-100. Each group was set up in triplicate, with 100 μL added to each well. The plates were incubated at 37°C with a shaker for 1 hour. After incubation, the 96-well plates were centrifuged at 1000g for 15 minutes at 4°C. 100 μL of the cell supernatant was transferred to another 96-well plate, and the absorbance at 570 nm was measured using a microplate reader.

[0102] After treating erythrocytes with 1 μM RS17-M6P3 for 8 hours, centrifuge at 2000 rpm for 3 minutes, resuspend in serum-free medium containing 1 μM deepredcell tracker, and incubate at 37°C in the dark for 30 minutes. After incubation, centrifuge at 2000 rpm for 3 minutes and count the cells.

[0103] The stained BMDM was subjected to 5×10 4 Each well and red blood cells at 2 × 10 5 Cells were seeded per well in 12-well plates and incubated for 2 hours. After incubation, the cells were washed three times with PBS, resuspended in 300 μL PBS, and analyzed by flow cytometry.

[0104] Figure 6 The effect of the lysosomal targeting chimera RS17-M6P3 on erythrocytes to degrade CD47 protein. RS17-M6P3 has no degrading effect on CD47 on erythrocytes. Figure 6 A), and there were no obvious hemolytic side effects ( Figure 6 B), and simultaneously, macrophages did not phagocytose erythrocytes treated with the lysosome-targeting chimera RS17-M6P3. Figure 6 C). Example 7: In vitro polarization effect of poria cocos polysaccharide in the microneedle composition of the present invention on macrophages.

[0105] Experimental methods:

[0106] M0 type BMDM cells, cultured for one week, were then used at 5×10⁻⁶ 5 Cells were seeded at a density of / well in 12-well plates and cultured for 24 hours to allow cell adhesion. A blank control group (Blank) was set up, which received no stimulating factors.

[0107] M2 macrophage repolarization: IL-4 (20 ng / mL) was added to induce M0 macrophages to polarize into M2 macrophages. In the polysaccharide group, PPS (500 g / mL) was added on top of this.

[0108] M1 macrophage polarization: LPS (8 ng / mL) and IFN-γ (8 ng / mL) were added to induce M0 macrophages to polarize into M1 macrophages; PPS (500 g / mL) was added to the polysaccharide group in addition to the above.

[0109] Protective effect on M1 macrophages: M1 macrophages were induced by incubation with LPS (8 ng / mL) and IFN-γ (8 ng / mL) for 12 hours. The culture conditions were then changed to medium containing IL-4 (20 ng / mL) to stimulate M1 macrophage differentiation into M2 macrophages. PPS (500 μg / mL) was added during this process. After drug administration, cells were collected from the wells of the plate and centrifuged at 400g for 5 minutes at 4°C. The supernatant was discarded, and the cells were washed with 1 mL of ice-cold PBS, centrifuged again, and the supernatant was discarded. 100 μL of 3% BSA was added for blocking on ice for 15 minutes, followed by washing with PBS. 100 μL of staining buffer containing Anti-Mouse-CD11b-FITC, Anti-Mouse-F4 / 80-Alexa Fluor647, and Anti-Mouse-CD86-PE antibodies was added for staining on ice for 30 minutes. Cells were then permeabilized and fixed using a cell permeabilization and fixation kit for 30 minutes. After washing the cells with 1× wash buffer, 100 μL of staining buffer containing Anti-Mouse CD206-PE-Cy7 antibody was added, and the cells were stained on ice for 30 minutes. After washing with PBS, the cells were resuspended in 300 μL of staining buffer and filtered into flow cytometry tubes using a 70 mm cell filter. Macrophages were then analyzed by flow cytometry.

[0110] Figure 7 The study investigated the effect of polysaccharide PPS (Polyporus umbellatus polysaccharide) in the microneedle composition on the in vitro polarization phenotype of macrophages. Results showed that PPS could repolarize M2 macrophages to M1 type (…). Figure 7 A, B), promote macrophage polarization towards M1 type ( Figure 7 C), and has a protective effect on M1 macrophages, avoiding the influence of the immunosuppressive microenvironment on them. Figure 7 D). **p<0.01, ***p<0.001.

[0111] Example 7: In vivo anti-melanoma effect of the present invention's *Polyporus umbellatus* polysaccharide microneedles

[0112] 7.1 Model Establishment

[0113] Female C57BL / 6 mice were inoculated with B16F10 cells (5 × 10⁻⁶) on the right side of their backs. 5 It takes about 6 days for a tumor to form.

[0114] 7.2 Evaluation of the in vivo anti-melanoma effect of Poria cocos polysaccharide microneedles

[0115] Experimental groups: ① Blank control group; ② Blank microneedle (Blank@MN) group; ③ Microneedles loaded with Polyporus umbellatus polysaccharide alone (PPS@MN) group; ④ Microneedles loaded with lysosome-targeted chimeric microneedles that degrade CD47 alone (RS17-M6P3@MN) group; ⑤ Microneedles loaded with Polyporus umbellatus polysaccharide that degrades CD47 in combination (PPS / RS17-M6P3@MN) group, 5 mice in each group. Evaluation index: ① Tumor volume of 2000 mm 3 The experiment aimed to: ① dissect mouse tumors to identify the endpoint and compare tumor sizes in each group; ② track and record the general health status of mice during the experiment, including changes in diet, water intake, and weight, and compare side effects; and ③ perform immunoblotting and immunofluorescence staining on tumor tissues at the experimental endpoint to examine changes in CD47 protein levels.

[0116] Figure 8 This study investigated the in vivo anti-melanoma growth effect of microneedles containing *Polyporus umbellatus* polysaccharide. Figure 8 A) B16F10 tumor volume growth curve; Figure 8 B) and ( Figure 8 C) Images and tumor weight statistics of B16F10 tumors. Compared with other groups, PPS / RS17-M6P3@MN showed the strongest inhibitory effect on tumor growth (n=5). Figure 8 D) and ( Figure 8E) CD47 protein levels and their statistical distribution in B16F10 tumor tissue. Compared with other groups, groups ④ and ⑤ containing RS17-M6P3 significantly reduced CD47 levels in the tumor site (n=3). *p<0.05, **p<0.01, ***p<0.001.

[0117] Example 8: Regulatory effect of the microneedle administration of the lysosomal-targeting chimeric polysaccharide of *Polyporus umbellatus* (a type of polysaccharide) that degrades CD47 on macrophages in vivo.

[0118] 8.1 Model Establishment

[0119] Female C57BL / 6 mice were inoculated with B16F10 cells (5 × 10⁻⁶) on the right side of their backs. 5 It takes about 6 days for a tumor to form.

[0120] 8.2 Evaluation of the in vivo anti-melanoma effect of Poria cocos polysaccharide microneedles

[0121] Experimental groups: ① Blank control group; ② Blank microneedle (Blank@MN) group; ③ Microneedles loaded with Polyporus umbellatus polysaccharide alone (PPS@MN) group; ④ Microneedles loaded with lysosome-targeted chimeric microneedles that degrade CD47 alone (RS17-M6P3@MN) group; ⑤ Microneedles loaded with Polyporus umbellatus polysaccharide that degrades CD47 in combination (PPS / RS17-M6P3@MN) group, 5 mice in each group. Evaluation index: tumor volume of 2000 mm². 3 As the endpoint, mouse tumors were dissected on day 13 of tumor bearing and day 16 of the endpoint, and prepared into single-cell suspensions. Flow cytometry was used to detect changes in macrophage polarization at the tumor site.

[0122] Figure 9 To investigate the in vivo regulatory effect of PPS / RS17-M6P3@MN on macrophage polarization, compared with other groups, at day 13 of tumor bearing and the endpoint day 16, the PPS-loaded groups, such as group ③PPS@MN and group ⑤PPS / RS17-M6P3@MN, significantly promoted M1 polarization of macrophages in the tumor site and inhibited M2 polarization (n=3). ***p<0.001.

Claims

1. A microneedle composition of *Polyporus umbellatus* polysaccharide with CD47 protein degradation function, characterized in that, The ingredients comprise the following parts by weight: Polyporus umbellatus polysaccharide 100-300; Lysosomal targeting chimera 5-20 for degrading CD47 protein; Hyaluronic acid 100-300; The structure of the lysosomal targeting chimera that degrades CD47 protein is as follows: 。 2. The *Polyporus umbellatus* polysaccharide microneedle composition with CD47 protein degradation function according to claim 1, characterized in that, The method for preparing the lysosomal targeting chimera that degrades CD47 protein includes the following steps: 1) Acetic anhydride is added dropwise to a pyridine solution of mannose at 0°C, wherein the structural formula of mannose is: After stirring overnight at room temperature, excess acetic anhydride was quenched with anhydrous ethanol, the sample was concentrated under reduced pressure, the residue was poured into ice water, and filtered to obtain fully acetylated mannose M6P-1; the structural formula of M6P-1 is as follows: ; 2) Under nitrogen protection, a boron trifluoride diethyl ether solution was added dropwise to a solution of azide ethanol and M6P-1 in dichloromethane at 0°C. After 1-2 hours, the mixture was moved to room temperature and stirred overnight. The solution was diluted with dichloromethane, washed with saturated sodium bicarbonate solution, dried, filtered, and concentrated under reduced pressure. M6P-2 was purified by column chromatography. The structural formula of M6P-2 is [insert structural formula here]. ; 3) Dissolve M6P-2 in a methanol solution of sodium methoxide (25%–35% by mass). After reacting at room temperature, adjust the pH to neutral using a cation exchange resin. Filter and concentrate the crude product. Dissolve this crude product in pyridine, add triphenylchloromethane, and stir at 45–55°C for 3–5 hours. Then, add acetic anhydride and react overnight at room temperature. Quench excess acetic anhydride with anhydrous ethanol, concentrate under reduced pressure, and add ice water. Dissolve the precipitated white solid in an aqueous acetic acid solution, stir at 55–65°C for 1–2 hours, add ice water, and precipitate the solid. Filter and wash with water. Purify the solid by column chromatography to obtain M6P-3. The structural formula of M6P-3 is [insert structural formula here]. ; 4) Under nitrogen protection, N,N-diisopropylphosphonamide di-tert-butyl ester was added to an anhydrous dichloromethane solution of M6P-3 and 1H-tetrazole at 0°C. After stirring for 2-3 hours, tert-butyl hydroperoxide was added at 0°C and stirred. The mixture was then moved to room temperature and reacted for 2-3 hours. The reaction was quenched with saturated sodium thiosulfate solution, and the mixture was extracted with dichloromethane. The organic phases were combined, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain M6P-4. The structural formula of M6P-4 is [insert structural formula here]. ; 5) Add M6P-4 to a methanol solution containing 25%–35% sodium methoxide by mass, react at room temperature for 2–3 hours, adjust the pH to neutral using a 50–100 mesh cation exchange resin, filter through the resin, concentrate the filtrate under reduced pressure, dissolve the residue in a dichloromethane solution containing 8–12% trifluoroacetic acid by mass, stir at room temperature for 1–2 hours, concentrate under reduced pressure to obtain M6P. The structural formula of M6P is [insert structural formula here]. ; 6) Using a solid-phase synthesis method, with Rink Amide MBHA amino resin as the carrier and ethyl 2-oxime cyanoacetate / N,N'-diisopropylcarbodiimide as the condensation system, the polypeptide conjugate RS17-(PraβA)3 was obtained; RS17-(PraβA)3 is specifically... Pra is propargylglycine. 7) Using a copper-catalyzed click chemistry reaction with cuprous sulfate / sodium ascorbate as the catalyst system, the peptide conjugate RS17-(PraβA)3, M6P, cuprous sulfate, and sodium ascorbate were added to a mixed solvent of methanol and pure water. After reacting at room temperature in the dark, the resulting product was purified and lyophilized to obtain a lysosomal targeting chimera that degrades CD47 protein.

3. A microneedle of *Polyporus umbellatus* polysaccharide with CD47 protein degradation function, characterized in that... It is prepared from the raw materials of the poria cocos polysaccharide microneedle composition with CD47 protein degradation function as described in any one of claims 1-2.

4. The *Polyporus umbellatus* polysaccharide microneedles with CD47 protein degradation function according to claim 3, characterized in that, The microneedle comprises a substrate and a needle body located on the substrate; wherein the needle body is prepared from the polysaccharide of any one of claims 1-2 and the lysosomal targeting chimera that degrades CD47 protein as raw materials, and the microneedle substrate is composed of hyaluronic acid.

5. The method for preparing *Polyporus umbellatus* polysaccharide microneedles with CD47 protein degradation function as described in claim 4, characterized in that... Includes the following steps: 1) Weigh the lysosomal targeting chimera that degrades CD47 protein and the polysaccharide of Poria cocos, add them to pure water, and prepare a mixed aqueous solution containing 5-20 mg / mL of the lysosomal targeting chimera that degrades CD47 protein and 100-300 mg / mL of the polysaccharide of Poria cocos. Let it swell overnight at 4°C. 2) Pour the mixed aqueous solution of CD47 protein-degrading lysosomal targeting chimera and poria cocos polysaccharide into the groove of the microneedle mold as the microneedle matrix material. Centrifuge at 4°C, rotate horizontally 180° and repeat centrifugation once to recover the excess material solution in the groove. 3) Prepare an aqueous solution of hyaluronic acid with a concentration of 100-300 mg / mL as the base material for microneedles. After pouring it into a mold, dry it overnight in an oven at 50-60℃ and then demold to obtain microneedles.

6. The use of the poria cocos polysaccharide microneedles with CD47 protein degradation function as described in claim 3 or 4 in the preparation of a drug for treating melanoma.

7. A lysosomal-targeting chimera for degrading CD47 protein, characterized in that, Its structure is as follows: 。 8. The method for preparing the lysosomal targeting chimera for degrading CD47 protein according to claim 7, characterized in that, Includes the following steps: 1) Acetic anhydride is added dropwise to a pyridine solution of mannose at 0°C, wherein the structural formula of mannose is: After stirring overnight at room temperature, excess acetic anhydride was quenched with anhydrous ethanol, the sample was concentrated under reduced pressure, the residue was poured into ice water, and filtered to obtain fully acetylated mannose M6P-1; the structural formula of M6P-1 is as follows: ; 2) Under nitrogen protection, a boron trifluoride diethyl ether solution was added dropwise to a solution of azide ethanol and M6P-1 in dichloromethane at 0°C. After 1-2 hours, the mixture was moved to room temperature and stirred overnight. The solution was diluted with dichloromethane, washed with saturated sodium bicarbonate solution, dried, filtered, and concentrated under reduced pressure. M6P-2 was purified by column chromatography. The structural formula of M6P-2 is [insert structural formula here]. ; 3) Dissolve M6P-2 in a methanol solution of sodium methoxide (25%–35% by mass). After reacting at room temperature, adjust the pH to neutral using a cation exchange resin. Filter and concentrate the crude product. Dissolve this crude product in pyridine, add triphenylchloromethane, and stir at 45–55°C for 3–5 hours. Then, add acetic anhydride and react overnight at room temperature. Quench excess acetic anhydride with anhydrous ethanol, concentrate under reduced pressure, and add ice water. Dissolve the precipitated white solid in an aqueous acetic acid solution, stir at 55–65°C for 1–2 hours, add ice water, and precipitate the solid. Filter and wash with water. Purify the solid by column chromatography to obtain M6P-3. The structural formula of M6P-3 is [insert structural formula here]. ; 4) Under nitrogen protection, N,N-diisopropylphosphonamide di-tert-butyl ester was added to an anhydrous dichloromethane solution containing M6P-3 and 1H-tetrazole at 0°C. After stirring for 2-3 hours, tert-butyl hydroperoxide was added at 0°C and stirred. The mixture was then moved to room temperature and reacted for 2-3 hours. The reaction was quenched with saturated sodium thiosulfate solution, and the mixture was extracted with dichloromethane. The organic phases were combined, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain M6P-4. The structural formula of M6P-4 is [insert structural formula here]. ; 5) Add M6P-4 to a methanol solution containing 25%–35% sodium methoxide (w / w), react at room temperature for 2–3 hours, adjust the pH to neutral using a 50–100 mesh cation exchange resin, filter through the resin, concentrate the filtrate under reduced pressure, dissolve the residue in a dichloromethane solution containing 8–12% trifluoroacetic acid (w / w), stir at room temperature for 1–2 hours, and concentrate under reduced pressure to obtain M6P. The structural formula of M6P is [insert structural formula here]. ; 6) Using a solid-phase synthesis method, with Rink Amide MBHA amino resin as the carrier and ethyl 2-oxime cyanoacetate / N,N'-diisopropylcarbodiimide as the condensation system, the polypeptide conjugate RS17-(PraβA)3 was obtained; RS17-(PraβA)3 is specifically... Pra is propargylglycine. 7) Using a copper-catalyzed click chemistry reaction with cuprous sulfate / sodium ascorbate as the catalyst system, the peptide conjugate RS17-(PraβA)3, M6P, cuprous sulfate, and sodium ascorbate were added to a mixed solvent of methanol and pure water. After reacting at room temperature in the dark, the resulting product was purified and lyophilized to obtain a lysosomal targeting chimera that degrades CD47 protein.

Images