Polypeptide coupled imidazole quinoline compound as well as synthesis method and application thereof

Abstract

The invention belongs to the field of biological medicines and materials, and particularly relates to a polypeptide coupled imidazole quinoline compound as well as a synthesis method and application thereof. According to the compound, polypeptide and a TLR agonist R848 are accurately coupled at active sites of the compound. The single molecule not only has tumor killing activity, but also has low non-specific toxicity; and after further coordination with metal iridium, the anti-tumor activity can be enhanced. Besides, the compound can respond to the level of glutathione in a tumor microenvironment, controllable release of the immunologic adjuvant R848 is achieved, and therefore the anti-tumor effect is achieved through the synergistic mechanism of chemical treatment and immunoregulation. The compound can effectively integrate chemical killing and immunotherapy, kill tumor cells through multiple ways and multiple mechanisms and remodel a tumor immune microenvironment. The compound is further delivered by adopting a nano preparation, so that the accumulation efficiency of the compound in tumor tissues can be improved, microenvironment responsive release is realized, and high-efficiency and low-toxicity tumor treatment is expected to be realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and materials, and relates to a polypeptide-conjugated imidazoquinoline compound, its synthesis method, and its application. Background Technology

[0002] Some chemotherapeutic drugs improve the tumor microenvironment by eliminating stromal cells and inducing immunogenic death of tumor cells, thereby enhancing the tumor penetration of immunotherapeutic drugs such as immune checkpoint inhibitors and synergistically activating anti-tumor immune responses, showing promising application prospects in tumor treatment. However, the mechanisms of action of chemotherapeutic drugs focus on targets such as DNA, RNA, and proteins related to cell proliferation, leading to severe non-selective toxic side effects and tumor drug resistance. Limited by the low permeability and drug resistance of tumor tissues, most chemotherapeutic drugs still induce apoptosis with low immunogenicity, and the resistance of tumor cells to apoptosis driven by multiple factors has become a serious challenge in cancer treatment. In addition, commonly used immunotherapeutic drugs, such as immune checkpoint inhibitors of biological macromolecules, have limited penetration and distribution in tumor tissues, and the individual response rate of patients is generally low. These limitations have prevented current combined chemoimmunotherapy strategies from achieving breakthrough progress in tumor treatment. Therefore, there is an urgent need to develop tumor chemoimmunotherapy strategies with novel mechanisms of action to overcome the limitations of existing treatments. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a peptide-conjugated imidazoquinoline compound, its synthesis method, and its applications. Through a tumor microenvironment-responsive release conjugation technology and an albumin nanoparticle (or cell membrane-encapsulated PLGA nanoparticles forming biomimetic nanoparticles) delivery system, a nanomedicine of a membrane-active pro-infarction peptide-Toll-like receptor (TLR) agonist conjugate (PISC) is constructed. This nanomedicine integrates multiple functions such as chemical killing, tumor microenvironment remodeling, and immune activation to achieve highly efficient chemoimmunotherapy for tumors. Based on the above conjugation method and nanodelivery system, the conjugate achieves efficient accumulation at the tumor target site, followed by the controllable release of the membrane-active pro-infarction peptide and TLR7 / 8 agonist, thereby reducing systemic toxicity. The pro-infarction peptide achieves deep distribution in tumor tissue and induces immunogenic death of tumor cells and stromal cells through its membrane-active characteristics. It synergistically regulates the tumor stromal barrier and immunosuppressive microenvironment with a TLR7 / 8 agonist capable of reprogramming immunosuppressive cells, and activates an anti-tumor immune response. By delving into the chemoimmunosynergistic regulatory mechanism of the two molecules and comparing their therapeutic effects under different combination strategies, this study aims to provide optimal combination strategies for tumor treatment and offer theoretical and experimental basis for the design of highly effective and low-toxicity tumor therapeutic drugs.

[0004] The technical solution provided by this invention is as follows:

[0005] A polypeptide-conjugated imidazoquinoline compound, the structure of which is as follows:

[0006] ;

[0007] Where R is CH2 or S, and R1 is C0 to C2. 20 Straight-chain or branched alkyl groups or R2-NHCO-R3, R2-(OCH2CH2) n - Any one of R3, where n is 1~10, and R2 and R3 are each independently selected from C0~C 20 Straight-chain or branched alkyl groups.

[0008] Furthermore, the peptide-conjugated imidazoquinoline compound is any one of SM7, SM8, SM10, SM11, SM14, and SM15, and the structures of SM7, SM8, SM10, SM11, SM14, and SM15 are as follows:

[0009] ;

[0010] ;

[0011]

[0012] .

[0013] The present invention also provides a metal-peptide-imidazoquinoline complex, which is obtained by coupling the above-mentioned peptide with an imidazoquinoline compound and coordinating with a metal, wherein the metal is one or more of Ir, Co, Pt, and Ni.

[0014] The present invention also provides a nanoparticle, wherein the albumin nanoparticle is obtained by conjugating the above-mentioned polypeptide with imidazoquinoline compounds through albumin or by covering a hybrid cell membrane with PLGA as the core.

[0015] The present invention also provides amino acid residue-containing compounds derived from the above-mentioned polypeptide-conjugated imidazoquinoline compounds.

[0016] The present invention also provides a method for synthesizing the above-mentioned polypeptide-conjugated imidazoquinoline compounds, characterized by employing any one of the following steps:

[0017] R848 and N,N-carbonyldiimidazole react with diols to give compound 1; compound 1, azidoacetic acid, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine are mixed and reacted to give compound 2; compound 2 and a 5-alkynylhexanoic acid-modified polypeptide are mixed and reacted to give compound 3.

[0018] R848 and N,N-carbonyldiimidazole react with diols to give compound 1; compound 1, succinic anhydride, N,N-diisopropylethylamine, and 4-dimethylaminopyridine are mixed and reacted to give SM12; SM12, azide-propanediamine hydrochloride, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine are mixed and reacted to give SM13; SM13 and a 5-alkynylhexanoic acid-modified peptide are mixed and reacted to give a peptide-coupled imidazoquinoline compound.

[0019] The present invention also provides the use of the above-mentioned peptide-conjugated imidazoquinoline compounds, the above-mentioned metal-peptide-imidazoquinoline complexes, or the above-mentioned nanoparticles in the preparation of tumor-inhibiting drugs.

[0020] Furthermore, the aforementioned peptides conjugated with imidazoquinoline compounds are assembled into nanoparticles via albumin or into biomimetic nanoparticles with PLGA as the core and covered by a hybrid cell membrane, and then delivered to the tumor microenvironment.

[0021] Furthermore, the nanoparticles are delivered to the tumor microenvironment either through direct local application or through systemic application.

[0022] Furthermore, the medication is administered alone or in combination with other therapeutic agents.

[0023] Furthermore, the tumor is one or more of melanoma, pancreatic cancer, breast cancer, gastric cancer, and colorectal cancer.

[0024] Furthermore, the peptide conjugated with imidazoquinoline compounds is used for cancer cell apoptosis and tumor immune microenvironment remodeling.

[0025] Beneficial effects

[0026] This invention constructs a chemical reaction to couple a membrane-active pro-infarction peptide with a Toll-like receptor (TLR) agonist to generate an organic molecule with multiple functions including chemical killing, tumor microenvironment remodeling, and immune activation. Nanoparticles (hybridized cell membrane-encapsulated PLGA-encapsulated PISC core biomimetic nanoparticles) are assembled from albumin and delivered to the tumor microenvironment. Through a tumor microenvironment-responsive release coupling technology and an albumin nanodelivery system, the conjugates achieve efficient accumulation in the tumor, followed by the controllable release of the membrane-active pro-infarction peptide and TLR7 / 8 agonist, thereby reducing systemic toxicity.

[0027] Through organic synthesis, a slit-membrane peptide is precisely coupled with a Toll-like receptor (TLR) agonist (R848) to form an organic molecule with a precise molecular weight. This molecule, when used alone, exhibits tumor-killing toxicity, which is further enhanced upon coordination with iridium. Simultaneously, it functions as a GSH-responsive immune adjuvant to the tumor microenvironment, releasing R848 and forming a synergistic mechanism for chemoimmunotherapy in tumor treatment. Its precise molecular weight offers the advantages of small-molecule drug formulation, providing significant convenience and advantages for drug therapy control, biological research, and subsequent clinical studies. It effectively integrates synergistic effects of chemotoxicity and immune adjuvant, achieving multi-pathway and multi-mechanism killing and remodeling of the tumor immune microenvironment. Furthermore, nano-formulations are used for efficient accumulation and responsive release within the tumor microenvironment, achieving highly effective and low-toxicity tumor treatment. Attached Figure Description

[0028] Figure 1 Synthetic route diagrams for products SM7, SM8, SM10, SM11, SM14, and SM15;

[0029] Figure 2 The structural diagrams are for PLGA, SM7, SM8, SM10, SM11, SM14, and SM15.

[0030] Figure 3 The image shows the proton NMR spectrum of product SM3.

[0031] Figure 4 The image shows the proton NMR spectrum of product SM4.

[0032] Figure 5 The image shows the proton NMR spectrum of product SM5.

[0033] Figure 6 High-resolution image of charge 4 for product SM7 prepared by method 5;

[0034] Figure 7 High-resolution image of charge 5 of product SM7 prepared by method 5;

[0035] Figure 8 The proton NMR spectrum of product SM6 prepared by method 4;

[0036] Figure 9 High-resolution image of charge 4 for product SM8 prepared by method 6;

[0037] Figure 10 High-resolution image of charge 5 of product SM8 prepared by method 6;

[0038] Figure 11 High-resolution image of product SM12 prepared by method 9;

[0039] Figure 12The hydrogen NMR spectrum of product SM13 is shown below.

[0040] Figure 13 High-resolution image of product SM14 prepared by method 11;

[0041] Figure 14 The graph shows the glutathione release response of compounds SM7, SM8, SM10, and SM11.

[0042] Figure 15 The image shows the water content and particle size distribution of biomimetic nanoparticles SM8 and SM11, where A represents SM8 and B represents SM11.

[0043] Figure 16 The image shows the water content and particle size distribution of albumin nanoparticles of SM8 and SM11, where A represents SM8 and B represents SM11.

[0044] Figure 17 The results of the cytotoxicity experiments of SM7, SM8, SM10, and SM11 on mouse breast cancer cells (4T1) are shown, where A is SM7, B is SM10, C is SM8, and D is SM11.

[0045] Figure 18 A graph showing the apoptosis of mouse breast cancer cells (4T1) induced by SM8 at different time points;

[0046] Figure 19 A graph showing the apoptosis of mouse breast cancer cells (4T1) induced by SM11 at different time points;

[0047] Figure 20 Image showing the maturation of BMDCs induced by the supernatant of mouse breast cancer cells (4T1) induced by SM7, SM8, SM10, and SM11 apoptosis.

[0048] Figure 21 Hemolysis diagrams of free compounds of SM7, SM8, SM10, SM11, SM14, and SM15 with biomimetic nanoparticles;

[0049] Figure 22 The average growth curves of tumors in each group of the 4T1 subcutaneous tumor model are shown.

[0050] Figure 23 A statistical chart showing the body weight of mice in each group of the 4T1 subcutaneous tumor model;

[0051] Figure 24 The average growth curves of tumors with SM11 biomimetic nanoparticles and albumin nanoparticles in the 4T1 subcutaneous tumor model are shown.

[0052] Figure 25 The average growth curves of tumors with SM11 biomimetic nanoparticles and albumin nanoparticles in the B16F10 subcutaneous tumor model are shown.

[0053] Figure 26 A map of mature dendritic cells in the draining lymph nodes after tumor treatment with SM7, SM8, SM10, and SM11 biomimetic nanoparticles in a 4T1 subcutaneous tumor model;

[0054] Figure 27 A map showing the ratio of M1 to M2 macrophages in the tumor microenvironment after tumor treatment with SM7, SM8, SM10, and SM11 biomimetic nanoparticles in a 4T1 subcutaneous tumor model.

[0055] Figure 28 A map showing the percentage of myeloid-derived inhibitory cells in the tumor microenvironment after tumor treatment with SM7, SM8, SM10, and SM11 biomimetic nanoparticles in a 4T1 subcutaneous tumor model.

[0056] Figure 29 A map showing the percentage of T cells infiltrating the tumor microenvironment after tumor treatment with SM7, SM8, SM10, and SM11 biomimetic nanoparticles in a 4T1 subcutaneous tumor model.

[0057] Figure 30 SM11 hybrid cell membrane biomimetic nanoparticles combined with PD-1 were used in a 4T1 subcutaneous tumor model for the treatment of large tumors. Detailed Implementation

[0058] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0059] Example 1

[0060] 1. Synthesis of compound SM3: R848 (8 mmol) and N,N-carbonyldiimidazole (9.2 mmol) were dissolved in 50 mL dichloromethane in a 100 mL flask and stirred at room temperature for 12 h. 1,6-hexanediol (SM1, 16 mmol) was added, and the reaction continued for 24 h. The solvent was evaporated to dryness under pressure, and purified by column chromatography (ethyl acetate) to give a yellow solid SM3 (yield, 80%). 1 H spectrum Figure 3As shown. 1 H NMR (400 MHz, CDCl3) δ 8.14 (dd, J = 7.8, 2.9 Hz, 2H), 7.59 (t, J= 7.6 Hz, 1H), 7.46 (dd, J = 11.2, 4.2 Hz, 1H), 4.85 (d, J = 53.4 Hz, 4H), 4.28 (t, J = 6.5 Hz, 2H), 3.71–3.58 (m, 4H), 1.80–1.70 (m, 2H), 1.65–1.55 (m,2H), 1.46 (dd, J = 7.6, 4.4 Hz, 4H), 1.33 (s, 6H), 1.25 (t, J = 7.0 Hz, 3H).

[0061] 2. Synthesis of compound SM4: R848 (8 mmol) and N,N-carbonyldiimidazole (9.2 mmol) were dissolved in 50 mL dichloromethane in a 100 mL flask and stirred at room temperature for 12 h. 2,2'-dithiodiethanol (SM2, 16 mmol) was added, and the reaction continued for 24 h. The solvent was evaporated to dryness under pressure, and purified by column chromatography (ethyl acetate) to give a yellow solid SM4 (yield, 85%). 1 H spectrum Figure 4 shown. 1 H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 8.2 Hz, 1H), 8.05 (s,1H), 7.56 (t, J = 7.6 Hz, 1H), 7.45 (t, J = 7.2 Hz, 1H), 4.89 (s, 2H), 4.76(s, 2H), 4.49 (t, J = 6.4 Hz, 2H), 3.91 (t, J = 6.2 Hz, 2H), 3.61 (q, J = 7.0Hz, 2H), 3.05 (t, J = 6.4 Hz, 2H), 2.96 (t, J = 6.2 Hz, 2H), 1.47–1.11 (m,9H).

[0062] 3. Synthesis of compound SM5: In a 100 mL flask, SM3 (4 mmol), azidoacetic acid (4.8 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (4.8 mmol), and N,N-diisopropylethylamine (8 mmol) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 24 h. 100 mL of water was added, and the mixture was extracted with dichloromethane (50 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, evaporated to dryness under pressure, and purified by column chromatography (ethyl acetate:petroleum ether 2:1) to give a yellow solid SM5 (yield, 78%). 1 H spectrum Figure 5 As shown. 1 H NMR (400 MHz, cdcl3) δ8.14 (d, J = 8.5 Hz, 2H), 7.60 (t, J = 7.8 Hz, 1H), 7.47 (t, J = 7.8 Hz, 1H), 4.92 (s, 2H), 4.79 (s, 2H), 4.28 (t, J = 6.5 Hz, 2H), 4.21 (t, J = 6.6 Hz, 2H), 3.87 (s, 2H), 3.67 (q, J = 7.0 Hz, 2H), 1.81 – 1.66 (m, 4H), 1.46 (d, J= 10.6 Hz, 4H), 1.28 (dd, J = 21.3, 14.4 Hz, 9H).

[0063] 4. Synthesis of compound SM6: In a 100 mL flask, SM4 (4 mmol), azidoacetic acid (4.8 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (4.8 mmol), and N,N-diisopropylethylamine (8 mmol) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 24 h. 100 mL of water was added, and the mixture was extracted with dichloromethane (50 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, evaporated to dryness under pressure, and purified by column chromatography (ethyl acetate: petroleum ether 2:1) to give a yellow solid SM6 (yield, 82%). 1 H spectrum Figure 8 As shown. 1H NMR (400 MHz, CDCl3) δ8.15 (t, J = 8.2 Hz, 2H), 7.61 (t, J = 7.8 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 4.85 (d, J = 50.1 Hz, 4H), 4.54 (t, J = 6.4 Hz, 2H), 4.48 (t, J = 6.4 Hz, 2H), 3.92 (s, 2H), 3.66 (q, J = 6.9 Hz, 2H), 3.06 (t, J = 6.5 Hz, 2H), 3.00(t, J = 6.4 Hz, 2H), 1.28 (m, 9H).

[0064] 5. Synthesis of compound SM7: In a 50 mL single-necked flask, SM-5 (1 mmol), the 5-hypoxanoic acid-modified polypeptide (5-Hexynoicacid-HRRRRRRRRH-amide, 1.2 mmol), copper sulfate pentahydrate (1 mmol), and sodium ascorbate (1.1 mmol) were dissolved in 5 mL of dimethyl sulfoxide. The mixture was stirred at room temperature for 48 h under nitrogen protection. 2 mL of water was added to completely dissolve the SM7. The solution was then lyophilized by preparative liquid chromatography to obtain a white solid, SM7 (yield, 60%). The high-resolution mass spectra of SM7 are shown below. Figure 6 , 7 As shown. High-resolution mass spectrometry (electrospray ionization) C 92 H 154 N 46 O 17 [M+4H] 4+ Theoretical molecular weight 544.8223, measured molecular weight 544.8227; C 92 H 154 N 46 O 17 [M+5H] 5+ The theoretical molecular weight is 436.0593, and the measured molecular weight is 436.0598.

[0065] 6. Synthesis of compound SM8: In a 50 mL single-necked flask, SM-6 (1 mmol), the 5-hydantoin-modified polypeptide (5-Hexynoicacid-HRRRRRRRRH-amide, 1.2 mmol), copper sulfate pentahydrate (1 mmol), and sodium ascorbate (1 mmol) were dissolved in 5 mL of dimethyl sulfoxide. The mixture was stirred at room temperature for 48 h under nitrogen protection. 2 mL of water was added to completely dissolve the SM8. The solution was then lyophilized by preparative liquid chromatography to obtain a white solid, SM8 (yield, 40%). The high-resolution mass spectra of SM8 are shown below. Figure 9 ,10 As shown. High-resolution mass spectrometry (electrospray ionization) C 90 H 150 N 46 O 17 S2 [M+4H] 4+ Theoretical molecular weight 554.0513, measured molecular weight 554.0502; C 92 H 154 N 46 O 17 [M+5H] 5+ The theoretical molecular weight is 443.2418, and the measured molecular weight is 443.2419.

[0066] 7. Synthesis of compound SM10: The mother liquors of SM9 ([Ir(ppy)2(H2O)2]Otf, purchased from Suzhou Nakai Technology Co., Ltd.) and SM7 were diluted to 2000 μM with 50 mM tris-HCl at pH 7.4. Equimolar concentrations of SM9 and SM7 were mixed at a volume ratio of 1:1, vortexed for 2 min, and mixed overnight in a shaking incubator at 37℃ to obtain SM10.

[0067] 8. Synthesis of compound SM11: The mother liquors of SM9 ([Ir(ppy)2(H2O)2]OTf) and SM8 were diluted to 2000 μM with 50 mM tris-HCl at pH 7.4. Equimolar concentrations of SM9 and SM7 were mixed at a volume ratio of 1:1, vortexed for 2 min, and mixed overnight in a constant temperature shaking incubator at 37℃ to obtain SM11.

[0068] 9. Synthesis of compound SM12: In a 100 mL flask, SM4 (5 mmol), succinic anhydride (20 mmol), N,N-diisopropylethylamine (25 mmol), and 4-dimethylaminopyridine (1 mmol) were dissolved in 50 mL of dichloromethane and stirred at room temperature for 12 h. The mixture was extracted three times with ethyl acetate (50 mL x 3), and the organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to give a yellow solid SM12 (yield, 68%). The mass spectrum of SM12 is shown below. Figure 11 As shown. High-resolution mass spectrometry (electrospray ionization) C 26 H 34 N4O8S2 [M+H] + The theoretical molecular weight is 595.1896, and the measured molecular weight is 595.1880.

[0069] 10. Synthesis of compound SM13: In a 100 mL flask, SM12 (4 mmol), azide azide hydrochloride (4.8 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (4.8 mmol), and N,N-diisopropylethylamine (12 mmol) were dissolved in 20 mL of dichloromethane and stirred at room temperature for 24 h. 100 mL of water was added, and the mixture was extracted with ethyl acetate (50 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, evaporated to dryness under pressure, and purified by column chromatography (ethyl acetate) to give a yellow solid SM13 (yield, 79%). 1 H spectrum Figure 12 As shown. 1 H NMR (400 MHz, CDCl3) δ8.14 (dd, J = 25.2, 8.0 Hz, 2H), 7.61 (t, J = 7.7 Hz, 1H), 7.49 (t, J = 7.1Hz, 1H), 6.26 (d, J = 50.5 Hz, 1H), 4.93 (s, 2H), 4.79 (s, 2H), 4.53 (t, J =6.5 Hz, 2H), 4.36 (t, J = 6.4 Hz, 2H), 3.66 (q, J = 7.0 Hz, 2H), 3.37 – 3.21(m, 6H), 3.07 (dd, J = 17.0, 10.5 Hz, 2H), 2.97 (t, J = 6.4 Hz, 2H), 2.66 (t,J = 6.8 Hz, 2H), 2.46 (dd, J = 13.2, 6.3 Hz, 3H), 1.81 – 1.66 (m, 4H), 1.34(s, 5H), 1.26 (t, J = 7.0 Hz, 4H).

[0070] 11. Synthesis of compound SM14: In a 50 mL single-necked flask, SM13 (1 mmol), the 5-hypoacetic acid-modified polypeptide (5-Hexynoicacid-HRRRRRRRRH-amide, 1.2 mmol), copper sulfate pentahydrate (1 mmol), and sodium ascorbate (1 mmol) were dissolved in 5 mL of dimethyl sulfoxide. The mixture was stirred at room temperature for 48 h under nitrogen protection. 2 mL of water was added to completely dissolve the SM13. The solution was then lyophilized by preparative liquid chromatography to obtain a white solid, SM14 (yield, 30%). The high-resolution mass spectra of SM14 are shown below. Figure 13 As shown. High-resolution mass spectrometry (electrospray ionization) C 95 H 159 N47 O 18 S2 [M+4H] 4+ The theoretical molecular weight is 578.8184, and the measured molecular weight is 578.8171. (C) 95 H 159 N 47 O 18 S2 [M+3H] 3+ The theoretical molecular weight is 771.4221, and the measured molecular weight is 771.4195.

[0071] 12. Synthesis of compound SM15: The mother liquors of SM9 ([1r(ppy)2(H2O)2]OTf) and SM14 were diluted to 2000 μM with 50 mM tris-HCl at pH 7.4. Equimolar concentrations of SM9 and SM14 were mixed at a volume ratio of 1:1, vortexed for 2 min, and mixed overnight in a constant temperature shaking incubator at 37℃ to obtain SM15.

[0072] Example 2

[0073] Glutathione-responsive apoptosis peptide immunoadjuvant-responsive release assay

[0074] Prepare a 10 mM pH 7.4 phosphate buffer solution, add GSH to prepare a 10.0 mM receiving buffer, and pipette 0.1 mL of dimethyl sulfoxide dissolved in SM7, SM8, SM10, and SM11 (containing 5.0 mg of R848) into 2 mL medium EP tubes. Set up the following groups: free R848, SM7 + 10 mM glutathione pH 7.4 buffer, SM7 + 10 mM glutathione pH 7.4 buffer, SM10 + 10 mM glutathione pH 7.4 buffer, SM11 + 10 mM glutathione pH 7.4 buffer, and SM8 + pH 7.4 buffer. Incubate at 37°C and 180 rpm on a shaker. Repeat each group for 3 samples. At 0, 1, 2, 4, 6, 8, 12, 16, 24, and 48 h, pipette 20 μL of the solution, remove the excess liquid, and add 980 μL of dimethyl sulfoxide solution (20000 mL). Centrifuge at rpm for 5 minutes and perform liquid chromatography analysis. Chromatographic conditions were as follows: column, InertSustain® C18 (4.6 mm × 150 mm, 5 μm); column temperature, 30℃; flow rate, 1.0 mL / min; injection volume, 10 μL; diode array detector detection wavelength, 254 nm; run time, 40 min; dilution solution, acetonitrile:water (V:V) = 60:40; injection wash, methanol; mobile phase A, 0.1% trifluoroacetic acid aqueous solution; mobile phase B, acetonitrile; elution gradient (%A, 0 min, 90%; 5 min, 90%; 25 min, 10%; 33 min, 10%; 35 min, 90%; 40 min, 90%; %B, 0 min, 10%; 5 min, 10%; 25 min, 90%; 33 min, 90%; 35 min, 10%; 40 min, 10%). The in vitro GSH-responsive release of free R848 in response to SM7, SM8, SM10, and SM11 is shown in the figure below. Figure 14 As shown, SM8 and SM11 exhibited a cumulative R848 release of over 60% within 10 hours at a GSH concentration of 10 mM, demonstrating extremely rapid response rates. SM7 and SM10 showed a cumulative R848 release of less than 6% within 48 hours at a GSH concentration of 10 mM. SM8 showed a cumulative R848 release of less than 6% within 48 hours in a pH 7.4 buffer solution. These results indicate that the compounds are highly stable in normal tissues, possess high safety, and exhibit no off-target toxicity.

[0075] Example 3

[0076] Preparation of biomimetic nanoparticles from PISC

[0077] Dissolve PLGA (polylactic acid-glycolic acid copolymer, x=50, y=50, left, molecular weight 24000~38000 Da) in dichloromethane to prepare a solution containing 100 mg of PLGA per mL. Add 100 mL of the above solution to 1 mL of the prepared solution, using a buffer-coordinated concentration of SM10, SM11, and SM15 (20-5 mg / mL; SM7, SM8, and SM14 are prepared by dissolving in dimethyl sulfoxide to achieve this concentration). Sonicate the solution for 1 min using an ultrasonic homogenizer (40% power, 5 s sonication followed by 5 s intermittent sonication). Then, add polyvinyl alcohol (PVA) aqueous solution (20 mg / mL): PLGA dichloromethane solution at a volume ratio of 1:2.5, and sonicate for 1.5 min. Gradually add the sonicated sample dropwise to a 2.5% PVA aqueous solution (w / v) under stirring, and stir overnight. After the dichloromethane has completely evaporated, centrifuge and discard the supernatant to obtain nanoparticles with a theoretical diameter of 100-200 nm. Resuspend the nanovaccine in 3 mL of pure water. Collect blood from mouse eyeballs, add 3 times the volume of erythrocyte lysis buffer to the blood, lyse on ice for 20 min, centrifuge at 3500 g for 5 min, collect the supernatant, centrifuge at 2000 g for 25 min, collect the precipitate, add PBS for reselection, wash three times until the precipitate turns white, and quantify using a BCA kit. Culture 4T1 cells in a numbering dish, collect the culture medium with a cell scraper into a 50 mL centrifuge tube, and centrifuge (500 g, 5 min, 4℃). Discard the supernatant, disperse the precipitate with washing buffer, transfer to another tube, centrifuge (500 g, 5 min, 4℃), and wash three times. Discard the supernatant, add Tris buffer to disperse the precipitate, and aliquot into 10 mL EP tubes (approximately 3 mL / tube, ensuring no air bubbles). Rinse the ultrasonic cell disruptor. Place the EP tube in an ice box and connect it to the machine. Perform sonication for 3-5 seconds with a 3-6 second interval, repeating 15-50 times. Reset the cycle twice, gradually adjusting the power to 60-100 W. After completion, reduce the sonication power to minimum, remove the EP tube, and replace it with the next one. Rinse the ultrasonic machine after the operation. Transfer the sonicated solution to a 50 mL EP tube and centrifuge (3200 g, 5 min, 4℃). Collect the supernatant; a very small amount of precipitate will be visible on the wall. Centrifuge again (20000 g, 25 min, 4℃), again collecting the supernatant. Transfer the solution to an adapter tube, balance, and centrifuge (100000 g, 45 min, 4℃). Discard the supernatant. Dissolve the precipitate in water and the remaining protease inhibitor (50 mL: 1 tablet), approximately 1 mL (300 μL). Measure the concentration using a BCA kit.The above-mentioned nanoparticles and cell membranes (nanoparticle to cell membrane mass ratio 1:5, erythrocyte membrane to tumor cell membrane mass ratio 1:1) were placed in 15 mL centrifuge tubes and sonicated in an ice bath (ultrasonic power 60-100 W, time 3-5 s, interval time 3-6 s, number of cycles 10-30) to prepare PLGA nanoparticles coated with hybrid membranes. A portion of the nanoparticles were resuspended in ultrapure water, and the size, morphology, and particle size distribution index of the nanoparticles were detected by dynamic light scattering and transmission electron microscopy. The results of dynamic light scattering showed that ( ). Figure 15 The prepared nanoparticles have a particle size of 100-200 nm, a particle size dispersion index of <0.3, and a relatively uniform particle size distribution.

[0078] Example 4

[0079] Preparation of albumin nanoparticles by PISC (using SM8 and SM11 as representative compounds).

[0080] SM11 was added to ultrapure water to obtain a 500 μM SM11 solution. Human serum albumin was dissolved in water to prepare a human serum albumin solution of the same concentration. The SM11 solution and human serum albumin solution were mixed at a molar ratio of 1:1, and an equal volume of phosphate buffer (1×PBS) was added. The mixture was incubated at room temperature for 2 h, and then 1 volume of 1×PBS was added to obtain SM11-HSA nanoparticles. The results of dynamic light scattering analysis showed that ( Figure 16 The prepared nano-vaccines have a particle size of 100-200 nm, a vesicle structure with a core of water and a shell of lipid, a particle size dispersion index of <0.3, and a relatively uniform particle size distribution.

[0081] Example 5

[0082] Cytotoxicity assays of SM7, SM8, SM10, and SM11

[0083] Dissolve SM7 and SM8 in dimethyl sulfoxide (DMSO), and prepare a 1000 μM stock solution using UV quantification. Prepare SM10 and SM11 using the same stock solution, and calculate the final concentrations according to the coordination method. Add 10, 5, 2.5, 1.25, 0.625, 0.312, 0.15, 0.075, 0.036, 0.018, 0.009, and 0.0045 μM of the above drugs to 8000 wells of 4T1 cells / well, with each concentration replicated in triplicate. Incubate at 37°C with 5% CO2 for 24 hours, then discard the solution. Add CCK8 and incubate for 2 hours. Measure the absorbance at 450 nm using a microplate reader. Results are as follows: Figure 17 As shown, the nano-vaccines do not exhibit significant cytotoxicity. Experimental results indicate that the IC50 values ​​for SM7, SM8, SM10, and SM11 are... 50The concentrations were 0.94 μM, 8.45 μM, 0.62 μM, and 4.68 μM, respectively. The toxicity of all compounds was in the micromolar range, indicating sufficiently high cytotoxicity against tumor cells; the IC50 values ​​for SM7 and SM8 were... 50 The results showed that the greater the hydrophobicity, the greater the toxicity, with a difference of about one order of magnitude. Furthermore, the toxicity of the compound before and after coordination differed by approximately two-fold, with coordination further increasing the toxicity.

[0084] Example 6

[0085] Assay of SM8 and SM11

[0086] 4T1 cells were dispersed in DMEM high-glucose medium containing 10% FBS at a cell concentration of 2 × 10⁻⁶ cells / mL. 5 Add 1 mL of cell suspension per 1 mL to a 20 mm diameter glass culture dish and incubate overnight at 37°C with 5% CO2. Wash twice gently with pH 7.4 1× PBS. Add 1 mL of standard DMEM medium containing PI (10 μg / mL) to each well, then add IC50 solution to each well. 50 Cells were incubated with SM8 and SM11 at concentrations, and immediately observed using a laser confocal microscope. Images were acquired every 40 seconds. The results are as follows: Figure 18 and 19 As shown. Figure 18 Experimental results showed that cell death occurred approximately 17 minutes after administration of SM8 (SM8 is non-fluorescent, so cell dynamics can only be tracked using PI dye). PI entered the cytoplasm and nucleus, indicating that cell membrane permeability was altered due to the action of schistosomes. Massive cell death occurred at 25 minutes, and around 30 minutes, substantial changes in nuclear membrane permeability occurred, with a large amount of PI entering the nucleus. The SM11 apoptosis phenomenon was similar to that of SM8. Because SM11 is fluorescent, the dynamics of drug entry into cells could be observed simultaneously.

[0087] Example 7

[0088] Dendritic cells promote maturation

[0089] C57 mice were euthanized by dislocation and immersed in 75% ethanol for 10 min. The thigh was dissected and immersed in 75% ethanol for 2 min, then placed in PBS. Sterile scissors were used to cut open both ends of the leg bone in a laminar flow hood. The bone was rinsed into a culture dish with PBS until it turned white (approximately 10 mL PBS). Bone marrow from three femurs and tibias was collected, dispersed by pipetting, filtered through a 70 μm cell filter into 50 mL centrifuge tubes, and centrifuged at 1500 rpm for 6 min. After centrifugation, the supernatant was discarded, and 4 mL of erythrocyte lysis buffer was added. Lysis was performed for 3 min, and the cells were transferred to 15 mL centrifuge tubes. 6 mL of PBS was added to terminate lysis, and the cells were centrifuged at 1500 rpm for 3 min. After centrifugation, the supernatant was discarded, and the cells were washed twice with 4 mL of PBS. The cells were then gently resuspended in 3 mL of 1640 complete culture medium to form a cell suspension. Four cell types were evenly divided into three 75 mm cell culture dishes. 9 mL of 1640 complete culture medium was added, along with 4 μL of GM-CSF (50 μg / mL) to achieve a concentration of 20 ng / mL. After 3 days of culture, the culture medium was transferred to 50 mL centrifuge tubes and centrifuged at 800 rpm for 6 min. Differential centrifugation was used to remove contaminating cells. Half of the supernatant was slowly aspirated and discarded. The mixture was then dispersed into each culture dish, and 5 mL of 1640 complete culture medium was added, along with 2 μL of GM-CSF (50 μg / mL). The medium was partially changed, and the cells were cultured for another 7-10 days. Adherent cells were detached by pipetting, and the cell culture medium was collected. The cells were centrifuged at 1500 rpm for 6 min, and the supernatant was discarded. The cells were resuspended in 12 mL of 1640 complete culture medium. Dendritic cells were seeded into 24-well plates at 2 x 10⁶ cells per well. 5 Each cell.

[0090] Take another 48-well plate, each well containing 5×10 4Mouse breast cancer cells (4T1) were incubated overnight at 37°C with 5% CO2. DMEM medium containing PBS, SM7 (2 μM), SM8 (2 μM), SM10 (2 μM), and SM11 (2 μM) was added and incubated for 24 h. 200 μL of the supernatant from each group was added to dendritic cells and incubated for 24 h. LPS (1 μ / mL) was added as a positive control and co-incubated with the dendritic cells. Dendritic cells from each group were collected, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 1 mL of cell staining buffer, washed once by centrifugation at 1500 rpm for 5 min, and then resuspended in 200 μL of cell staining buffer. Anti-mouse CD11c-FITC, anti-mouse CD80-PE, and anti-mouse CD86-APC staining solutions were added at a ratio of 1 μL per million cells. The cells were incubated on ice in the dark for 15 min, washed three times with 1 mL of cell staining buffer, and finally resuspended in 500 μL of cell staining buffer. Flow cytometry analysis was performed, and the results are shown below. Figure 20 As shown, the maturation rate in the PBS group was approximately 12.5%. The maturation rate increased by about 25% after adding the tumor cell apoptosis supernatant from SM7 and SM10, indicating that tumor apoptosis also has an effective maturation-promoting effect on BMDCs. When SM8 and SM11 induced tumor apoptosis, the disulfide bonds in the compounds simultaneously broke, releasing the immune adjuvant R848. Its maturation-promoting rate was approximately 1.2 times higher than that of SM7 and SM10, indicating a good synergistic effect between tumor antigens and immune adjuvants in chemoimmunization.

[0091] Example 8

[0092] Hemolytic effect of SM7, SM8, SM10, SM11, SM14, and SM15 free drugs with PLGA hybrid cell membrane biomimetic nanoparticles

[0093] Blood was collected from the eyes of mice and transported to an anticoagulant blood collection tube containing EDTA. 1 mL of blood was transferred to 3 mL of 1×pH 7.4 PBS, mixed thoroughly, and centrifuged (1000 rpm, 5 min). This process was repeated twice, followed by centrifugation to remove the supernatant PBS. Fresh PBS was then added, and the blood cell count was performed to achieve a concentration of 4×10⁶ cells / mL. 8 / mL. Eight groups were set up: blank control group, negative control group, positive control group, and sample group, with 3 replicates per group. Blank control group: 140 μL of 1×PBS was added to each well; negative control group: 70 μL of red blood cell suspension and 70 μL of 1×PBS were added to each well; positive control group: 1 mL of red blood cell suspension was centrifuged to remove PBS, 1 mL of pure water was added to obtain red blood cell lysis buffer, and 70 μL of red blood cell lysis buffer and 70 μL of pure water were added to each well; sample group: 70 μL of red blood cell suspension and 70 μL of sample were added to each well, with final sample concentrations of 10, 20, 50, 100, and 200 μM. Each group was placed in a U-shaped 96-well plate and incubated at 37°C and 87 rpm for 2 h. The 96-well plates were then centrifuged (3000 rpm, 5 min), and 90 μL of the supernatant was transferred to a flat-bottomed 96-well plate. The absorbance of each group at 405 nm was measured using a microplate reader. Figure 21 As shown in the figure. Experimental results indicate that when the concentration of free cleavage peptide compounds is greater than 50 μM, hemolysis exceeds 20%, and the hemolytic activity further increases with increasing concentration. However, when the compounds are made into nanoparticles, no hemolysis occurs at concentrations below 200 μM.

[0094] Example 9

[0095] Tumor suppression experiment of a 4T1 subcutaneous model using SM7, SM8, SM10, SM11, SM14, and SM15 PLGA hybrid cell membrane biomimetic nanoparticles.

[0096] Babl / c female mice aged 6-8 weeks were selected and subcutaneously injected with 50 μL of 4T1 cells in the logarithmic growth phase. The tumors grew to 100 mm. 3 For four consecutive days, mice were injected intravenously with 100 μL of SM7, SM8, SM10, SM11, SM14, and SM15 nanoparticles (nanoparticle concentration 500 μM). Tumor volume and body weight were measured every three days. The results are as follows: Figure 22 As shown, when compound SM11 was administered alone, due to its poor targeting and easy degradation, the tumor inhibition rate was approximately 30% compared to the PBS group. The nanoparticle formulation of SM7 increased drug stability and long-term circulating effects, effectively targeting tumors and increasing its tumor inhibition rate to approximately 75%. Although the toxicity of SM10 increased after coordination, its therapeutic effect was not significantly different from that of SM7. SM8, SM11, SM14, and SM15, while performing chemical killing, released immune adjuvants to activate the immune system, effectively synergistically curing tumors. In vivo experiments showed that apoptosis peptides alone have a good inhibitory effect on tumor treatment. When apoptosis peptides perform chemical killing, the tumor microenvironment GSH responds by releasing R848, activating the immune system and synergistically curing tumors.

[0097] Example 10

[0098] Tumor suppression experiment of a 4T1 subcutaneous model using SM11PLGA hybrid cell membrane biomimetic nanoparticles and albumin nanoparticles.

[0099] Babl / c female mice aged 6-8 weeks were selected and subcutaneously injected with 50 μL of 4T1 cells in the logarithmic growth phase. The tumors grew to 100 mm. 3 For four consecutive days, mice were injected intravenously with 100 μL of SM11 biomimetic nanoparticles and albumin nanoparticles (nanoparticle concentration 500 μM). Tumor volume and body weight were measured every three days. The results are as follows: Figure 24 As shown, both SM11 biomimetic nanoparticles and albumin nanoparticles can cure mice.

[0100] Example 11

[0101] Tumor suppression experiment using a B16F10 subcutaneous model of SM11PLGA hybrid cell membrane biomimetic nanoparticles and albumin nanoparticles.

[0102] Female C57bl / 6J mice aged 6-8 weeks were selected and subcutaneously injected with 50 μL of B16F10 cells in the logarithmic growth phase. The tumors grew to 100 mm. 3 For four consecutive days, 100 μL of SM11 biomimetic nanoparticles and albumin nanoparticles (nanoparticle concentration 500 μM) were injected via the tail vein. Tumor volume was monitored every three days. The results are as follows: Figure 25 As shown, both SM11 biomimetic nanoparticles and albumin nanoparticles can cure mice.

[0103] Example 12

[0104] Biocompatibility of SM7, SM8, SM10, SM11, SM14, and SM15 PLGA hybrid cell membrane biomimetic nanoparticles

[0105] BalB / c mice, aged 6-8 weeks and weighing 16-18 g, were injected via tail vein with 100 μL each of 500 μM nanoparticles (SM7, SM8, SM10, SM11, SM14, and SM15) for 4 consecutive days, and their body weight was measured. The trend of body weight change after tail vein injection of each sample in mice is shown below. Figure 23 As shown, the nanoparticles have no significant toxicity.

[0106] Example 13

[0107] SM7, SM8, SM10, and SM11 hybrid cell membrane biomimetic nanoparticles regulate the tumor immune microenvironment.

[0108] Babl / c female mice aged 6-8 weeks were selected and subcutaneously injected with 50 μL of 4T1 cells in the logarithmic growth phase. The tumors grew to 100 mm. 3 Mice were injected intravenously with 100 μL of SM11 biomimetic nanoparticles and albumin nanoparticles (nanoparticle concentration 500 μM) for four consecutive days. Three days later, mice were sacrificed, and their draining lymph nodes and tumors were separated into single cells. The Zombie Aqua™ Fixable Viability Kit was then used to stain for live and dead cells, and to block CD16 / CD32; subsequently, CD80, CD86, CD11c, CD45, CD3, F4 / 80, CD206, and Ly-6G / Ly-6C (Gr-1) were stained. The results showed that the slit-cell peptide effectively promoted the maturation of dendritic cells in the draining lymph nodes. When the slit-cell peptide was used in combination with R848 (the GSH-corresponding drug), the maturation rate of dendritic cells increased by 2.5 times compared to the slit-cell peptide alone, indicating a significant synergistic effect between the slit-cell peptide and R848 (e.g., [missing information]). Figure 26 As shown). Tests in its tumor microenvironment revealed that the slit-membrane peptide effectively polarized M2 macrophages to M1 type. When the slit-membrane peptide was combined with the GSH-corresponding R848, the polarization effect was further enhanced to 2.5 times (e.g., ...). Figure 27 As shown). Myeloid-derived suppressor cells were also significantly downregulated (e.g. Figure 28 (As shown). It can also effectively increase T cell entry; the slit-membrane peptide group and the slit-membrane peptide combined with immune adjuvant group showed increases of 1.8 times and 4 times, respectively, compared to the blank control group (as shown). Figure 29 (As shown in the image). This demonstrates that the combination of slit-membrane peptides and immunoadjuvant nanoparticles can effectively remodel the tumor immune microenvironment.

[0109] Example 14

[0110] SM11 hybrid cell membrane biomimetic nanoparticles combined with PD-1 for the treatment of large tumors

[0111] Babl / c female mice aged 6-8 weeks were selected and subcutaneously injected with 50 μL of 4T1 cells in the logarithmic growth phase. The tumors grew to 300 mm. 3 Tumors were administered 100 μL of SM11 biomimetic nanoparticles and PD-1 monoclonal antibody via tail vein injection for four consecutive days. Tumor volume was monitored every three days. The results were as follows: Figure 30 As shown, the combination of SM11 biomimetic nanoparticles and PD-1 monoclonal antibody has a good synergistic effect and a good therapeutic effect on large tumors.

Claims

1. A polypeptide-conjugated imidazoquinoline compound, characterized in that, The structure of the polypeptide-conjugated imidazoquinoline compound is as follows: ； Where R is CH2 or S, and R1 is C0 to C2. 20 Straight-chain or branched alkyl groups, R2-NHCO-R3, R2-(OCH2CH2) n - Any one of R3, where n is 1~10, and R2 and R3 are each independently selected from C0~C 20 Straight-chain or branched alkyl groups.

2. The polypeptide-conjugated imidazoquinoline compound according to claim 1, characterized in that, The polypeptide conjugated with an imidazoquinoline compound is any one of SM7, SM8, SM10, SM11, SM14, and SM15, and the structures of SM7, SM8, SM10, SM11, SM14, and SM15 are as follows: ； ； 。 3. A metal-peptide-imidazoquinoline complex, characterized in that, The product is obtained by coupling the polypeptide of claim 1 with an imidazoquinoline compound and coordinating it with a metal, wherein the metal is one or more of Ir, Co, Pt, Ni, Ru, Ti, and Au.

4. A nanoparticle, characterized in that, The nanoparticles are obtained by loading the polypeptide-coupled imidazoquinoline compound of claim 1 onto proteins, polymers, biological membranes, or inorganic nanoparticles.

5. The amino acid residue-containing compound derived from the polypeptide-conjugated imidazoquinoline compound of claim 1.

6. The method for synthesizing polypeptide-conjugated imidazoquinoline compounds according to claim 1, characterized in that, Take any of the following steps: R848 and N,N-carbonyldiimidazole react with diols to give compound 1; compound 1, azidoacetic acid, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine are mixed and reacted to give compound 2; compound 2 and a 5-alkynylhexanoic acid-modified polypeptide are mixed and reacted to give compound 3. R848 and N,N-carbonyldiimidazole react with diols to give compound 1; compound 1, succinic anhydride, N,N-diisopropylethylamine, and 4-dimethylaminopyridine are mixed and reacted to give SM12; SM12, azide-propanediamine hydrochloride, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine are mixed and reacted to give SM13; SM13 and a 5-alkynylhexanoic acid-modified peptide are mixed and reacted to give a peptide-coupled imidazoquinoline compound.

7. The use of the polypeptide-conjugated imidazoquinoline compound of claim 1 or 2, the metal-peptide-imidazoquinoline complex of claim 3, or the nanoparticle of claim 4 in the preparation of antitumor drugs.

8. The application according to claim 7, characterized in that, The polypeptide conjugated with imidazoquinoline compounds or their nanoparticles as described in claim 1 is delivered to the tumor microenvironment.

9. The application according to claim 7, characterized in that, The peptide-conjugated imidazoquinoline compound is used for cancer cell death and tumor immune microenvironment remodeling.

10. A pharmaceutical composition, characterized in that, The product comprises one or more of the following: a polypeptide-conjugated imidazoquinoline compound of claim 1 or 2, a metal-peptide-imidazoquinoline complex of claim 3, or a nanoparticle of claim 4, and one or more pharmaceutically acceptable carriers or excipients.

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