A protein-coupled imidazole-quinoline compound and its synthesis method and application

Abstract

The present invention belongs to the field of biomedicine and materials, and specifically relates to a protein-coupled imidazoquinoline compound and a synthesis method and application thereof. The present invention aims at the problem that imidazoquinoline agonists have large toxic side effects and are prone to cause cytokine storms when used in vivo. By coupling the active amino group of the imidazoquinoline agonist with albumin, which has low human immunogenicity, good biosafety and accumulation advantage at tumor sites, through a reduced glutathione-responsive linker, the targeting and responsive release at the tumor site are achieved, thereby reducing systemic nonspecific toxicity, reducing the possibility of causing cytokine storms, improving the immunosuppressive microenvironment of the tumor, activating anti-tumor immunity, and achieving better therapeutic effects and biosafety.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and materials, and specifically relates to a protein-coupled imidazole-quinoline compound and a synthesis method and application thereof. Background Art

[0002] The tumor microenvironment is closely related to the growth, development and metastasis of tumors. The tumor microenvironment of hot tumors usually shows more immune cell infiltration, which makes them have a higher response rate to immunotherapy, while cold tumors lack immune cell infiltration, express lower levels of immunostimulatory molecules, and accumulate a large number of immunosuppressive cells such as regulatory T cells (Tregs) and tumor-associated macrophages (TAMs) in their microenvironment, making them resistant to immunotherapy. Reshaping cold tumors to transform them from "cold" to "hot" is helpful for tumor immunotherapy.

[0003] Toll-like receptors (TLRs) are a class of transmembrane proteins located on the cell membrane surface and intracellular organelles. They are usually expressed on antigen presenting cells (APCs), such as dendritic cells (DCs) and macrophages, to recognize structurally conserved microbial sequences. Agonists targeting intracellular receptors TLR3, TLR7, TLR8, and TLR9 are widely used in cancer treatment research. Among them, the imidazoquinoline TLR7 agonist imiquimod (R837) and the TLR7 / 8 agonist resiquimod (R848) have better immune activation and tumor microenvironment improvement capabilities compared to other types of TLR agonists, including recruiting DCs and natural killer cells (NK cells) to lymph nodes, increasing the activity of NK cells, promoting the maturation of immature DCs, and activating CD4 + and CD8 + Effector T cells, and the activated T cells have stronger proliferation ability, and the generated memory T cells against tumors are also more sensitive. In addition, they promote immune cells to secrete pro-inflammatory factors such as interferon-γ (IFN-γ) and interleukin-2 (IL-2). At the same time, studies have shown that imidazoquinoline agonists have a significant improvement effect on the tumor microenvironment. Previous researchers have determined a variety of compounds related to macrophage polarization recorded in the literature through single-cell high-throughput polarization analysis. The screening results show that imidazoquinoline agonists have the best effect in polarizing human and mouse immunosuppressive M2 macrophages into M1 macrophages that are beneficial to immunotherapy. Studies have shown that R848 reshaped the tumor immune microenvironment in a mouse pancreatic cancer model, and lymphoid structures were formed at the tumor site and CD3 + 、CD8 + and CD3 + 、CD4 + The infiltration of T cells increased and the proportion of regulatory T cells decreased.

[0004] Although imidazoquinoline TLR agonists have good potential for anti-cancer immunotherapy, their main toxic side effects include inducing severe systemic inflammatory responses and even cytokine storms, which limit their application in vivo. Some researchers have reduced its systemic toxic side effects by reacting with the active amino group of R848 to form the prodrug R848-N3, and used radiation to disintegrate the prodrug at the tumor site to release R848. Although the toxicity of R848 is reduced, the increased radiotherapy toxicity needs to be taken into consideration; some researchers have tried to load R848 into the pores of mesoporous silica to reduce its toxic side effects, but there is a possibility that R848 will fall off from the pores of mesoporous silica.

[0005] Albumin has good biosafety, low immunogenicity, multiple modification sites and hydrophobic drug binding sites, and is negatively charged under physiological conditions. As a nutrient, albumin is preferentially internalized by tumor cells. Pancreatic cancer cells have been shown to grow indefinitely in a culture medium lacking essential amino acids in the presence of physiological albumin. Albumin can pass through the vascular endothelium through GP60-SPARC. Nanoparticles made of albumin can also accumulate in tumors by enhancing the permeability retention effect. Although albumin has certain tumor targeting, it is impossible to achieve responsive release of drugs at the tumor site by simply loading drugs with albumin, and it is impossible to achieve the purpose of selectively killing tumors. Summary of the invention

[0006] The present invention aims at the problem that imidazoquinoline agonists have great toxic and side effects and are prone to cause cytokine storms when used in vivo. By coupling the active amino group of the imidazoquinoline agonists with albumin, which has low human immunogenicity, good biosafety and an accumulation advantage in tumor sites, through a reduced glutathione-responsive linker, the targeting and responsive release of the tumor sites are achieved, thereby reducing systemic nonspecific toxicity, reducing the possibility of causing cytokine storms, improving the immunosuppressive microenvironment of the tumor, activating anti-tumor immunity, and achieving better therapeutic effects and biosafety.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A protein-coupled imidazole-quinoline compound, the structure of which is shown in formula (I):

[0009]

[0010] Formula (I)

[0011] In formula (I), R is S, n is an integer between 1 and 20, and A is albumin.

[0012] The present invention also provides a method for synthesizing the protein-coupled imidazole-quinoline compound, wherein R848, ‌N,N-carbonyldiimidazole and 2,2'-dithiodiethanol are reacted to obtain R848-SS-OH; R848-SS-OH, succinic anhydride, N,N-diisopropylethylamine and 4-dimethylaminopyridine are mixed to obtain R848-SS-COOH; R848-SS-COOH, N-hydroxysuccinimide, N,N-dicyclohexylcarbodiimide and N,N-diisopropylethylamine are mixed to obtain R848-SS-CONSH (N-hydroxysuccinimide ester of R848-SS-COOH); human serum albumin and R848-SS-CONSH are stirred and mixed, dialyzed and freeze-dried to obtain the protein-coupled imidazole-quinoline compound;

[0013] The structure of R848 is The structure of the 2,2'-dithiodiethanol is , R is S.

[0014] Furthermore, the preparation step of R848-SS-OH includes: dissolving R848 and N,N-carbonyldiimidazole in anhydrous tetrahydrofuran, adding 2,2'-dithiodiethanol after sufficient stirring to continue the reaction, pressurizing the solvent to dryness, and purifying by column chromatography to obtain R848-SS-OH.

[0015] Furthermore, the preparation step of R848-SS-COOH includes: dissolving R848-SS-OH, succinic anhydride, N,N-diisopropylethylamine, and 4-dimethylaminopyridine in 50 mL of dichloromethane, stirring the reaction sufficiently, extracting with ethyl acetate, combining the organic phases, drying over anhydrous sodium sulfate, and decompressing the solvent to obtain R848-SS-COOH.

[0016] Furthermore, the molar ratio of R848-SS-OH, succinic anhydride, N,N-diisopropylethylamine, and 4-dimethylaminopyridine is 4:4~20:4~20:1.

[0017] Furthermore, the molar ratio of R848, N,N-carbonyldiimidazole and 2,2'-dithiodiethanol is 1:1-3:2-4.

[0018] Furthermore, the molar ratio of R848-SS-COOH to human serum albumin is 0.5~5:1.

[0019] The present invention also provides application of the above protein-coupled imidazole-quinoline compound in preparing a drug carrier.

[0020] The present invention also provides the use of the above-mentioned protein-coupled imidazole-quinoline compound in the preparation of anti-tumor drugs.

[0021] Furthermore, the drug is a glutathione-responsive drug.

[0022] Beneficial Effects

[0023] According to the difference that the concentration of glutathione (GSH) in tumor cells is much higher than that in normal cells, the active amino group of imidazoquinoline agonists and the carboxyl group on albumin are connected by GSH-responsive linkers to prepare GSH-responsive R848-coupled albumin. Due to the occupation of the active amino group of imidazoquinoline agonists, their activity is significantly reduced, thereby reducing toxic side effects. Through the tumor targeting function of albumin, it accumulates in the tumor site, and the imidazoquinoline agonists are responsively broken and released to restore their activity by the excessive GSH in the tumor site, thereby improving the immunosuppressive microenvironment and activating anti-tumor immunity. In addition, this modification has little effect on the drug binding site and electronegativity of albumin, so it is not easy to affect the interaction between albumin and other drugs. It can be predicted that the modified albumin can still load some drugs such as paclitaxel, amphiphilic molecules (such as amphiphilic peptide ipep), etc., or form a higher-level structure (such as nanometer and micrometer level) with them. Therefore, the agonist-albumin conjugate can be used as a therapeutic drug and a carrier for the transportation of other drugs.

[0024] The present invention reduces the systemic toxicity and side effects of the imidazoquinoline agonist by covalently bonding the GSH-responsive linker to the active amino group of the imidazoquinoline agonist; utilizes albumin to enhance the tumor targeting of the imidazoquinoline agonist and enhances the accumulation in the tumor site; utilizes the tumor microenvironment-responsive release of the GSH linker to restore the activity of the imidazoquinoline agonist, reduce the toxicity and side effects, and enhance the efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the technical roadmap of the present invention;

[0026] Figure 2 For R848-SS-OH 1 H spectrum;

[0027] Figure 3 For R848-CC-OH 1 H spectrum;

[0028] Figure 4 is the mass spectrum of R848-SS-COOH;

[0029] Figure 5 is the mass spectrum of R848-CC-COOH;

[0030] Figure 6 is the mass spectrum of HSA;

[0031] Figure 7is the mass spectrum of RS-HSA (R848-SS-COOH:HSA=0.6:1, n / n);

[0032] Figure 8 is the mass spectrum of RS-HSA (R848-SS-COOH:HSA=1.6:1, n / n);

[0033] Fig. 9 is the mass spectrum of RS-HSA (R848-SS-COOH:HSA=1.9:1, n / n);

[0034] Fig.10 is the mass spectrum of RC-HSA (R848-CC-COOH:HSA=2.9:1, n / n);

[0035] Fig.11 is the mass spectrum of BSA;

[0036] Fig.12 is the mass spectrum of RS-BSA (R848-SS-COOH:BSA=1.6:1, n / n);

[0037] Fig.13 In vitro GSH-responsive release of RS-HSA and free R848;

[0038] Fig.14 The trend of body weight changes after tail vein injection of mice with PBS, R848, RS-HSA (R848-SS-COOH:HSA=1.6:1, n / n), and RS-HSA (R848-SS-COOH:HSA=2.9:1, n / n);

[0039] Fig.15 The water content and particle size and transmission electron microscopy images of RS-HSA nanoparticles (iP-RS NP) loaded with amphiphilic peptide iPep;

[0040] Fig.16 is the water and particle size of paclitaxel RC-HSA nanoparticles;

[0041] Fig.17 Comparison of the difference in R848 release inside and outside the cells of RS / RC-HSA nanoparticles (iP-RS / RC NPs) loaded with the amphiphilic peptide iPep;

[0042] Fig.18 This is a comparison of the maturation of mouse bone marrow-derived macrophages (BMDCs) promoted by PBS, RC-HSA, RS-HSA, and lipopolysaccharide (LPS). The LPS incubation group was used as a positive control, *** p < 0.001;

[0043] Fig.19Comparison of the polarization of mouse bone marrow-derived macrophages (BMDMs) promoted by PBS, R848, RC-HSA, and RS-HSA, *** p < 0.001;

[0044] Fig. 20 The hemolysis of iPep, iP-RC NP, and iP-RS NP in Example 14, *** p < 0.001;

[0045] Fig.21 is the chemical structure of the amphiphilic polypeptide iPep;

[0046] Fig. 22 Tissue distribution of amphiphilic peptide iPep and amphiphilic peptide nanoparticles coated with RC-HSA in mice after tail vein injection for 24 hours, *** p < 0.001;

[0047] Fig.23 The pharmacokinetics of iPep, an amphiphilic peptide nanoparticle coated with RC-HSA / RS-HSA, injected via tail vein in mice;

[0048] Fig.24 The maturation of dendritic cells (DCs) in the lymph nodes of mice was promoted by the control group, R848, RC-HSA, and RS-HSA 24 hours after tail vein injection, ** p < 0.01;

[0049] Fig.25 PBS, R848, RC-HSA, and RS-HSA were injected into the tail vein for 4 consecutive days. The ratio of M1 and M2 macrophages in the tumor site 3 days later was *** p < 0.001;

[0050] Fig.26 The graph is a comparison of the growth curves of breast cancer orthotopic tumors in PBS and RS-HSA mice, ***p<0.001. DETAILED DESCRIPTION

[0051] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0052] Example 1

[0053] The technical route of the present invention is as follows Figure 1 As shown, R1=CH2 is a non-GSH responsive functional group, and R2=S is a GSH responsive functional group;

[0054] The structural formulas of R848 (Resiquimod) and R837 (Imiquimod) are as follows:

[0055]

[0056] Synthesis of R848-SS-OH

[0057] R848-SS-OH synthesis steps: Take a 100 mL flask, dissolve R848 (4.78 mmol) and ‌N,N-carbonyldiimidazole (7.17 mmol) in 50 mL of anhydrous tetrahydrofuran, and stir at room temperature for 8 h. Add 2,2'-dithiodiethanol (9.56 mmol) and continue the reaction for 24 h. The solvent is pressurized and dried, and purified by column chromatography (ethyl acetate) to obtain yellow solid R848-SS-OH (yield, 78%). 1 H spectrum Figure 2 shown.

[0058] Example 2

[0059] Synthesis of R848-CC-OH

[0060] R848-CC-OH synthesis steps: Take a 100 mL flask, dissolve R848 (4.78 mmol) and N,N-carbonyldiimidazole (7.17 mmol) in 50 mL of anhydrous tetrahydrofuran, and stir at room temperature for 8 h. Add 1,6-hexanediol (9.56 mmol) and continue the reaction for 24 h. The solvent is pressurized and dried, and purified by column chromatography (ethyl acetate) to obtain a yellow solid R848-CC-OH (yield, 80%). 1 H spectrum Figure 3 shown.

[0061] Example 3

[0062] Synthesis of R848-SS-COOH

[0063] R848-SS-COOH synthesis steps: Take a 100 mL flask, dissolve R848-CC-OH (4 mmol), succinic anhydride (16 mmol), N,N-diisopropylethylamine (20 mmol), and 4-dimethylaminopyridine (1 mmol) in 50 mL of dichloromethane, and stir at room temperature for 12 h. Extract with ethyl acetate three times (50 mLx3), combine the organic phases, dry over anhydrous sodium sulfate, and decompress the solvent to obtain a yellow solid R848-CC-COOH (yield, 69%). The mass spectrum of R848-SS-COOH is shown in Figure 4 shown.

[0064] Example 4

[0065] Synthesis of R848-CC-COOH

[0066] R848-CC-COOH synthesis steps: Take a 100 mL flask, dissolve R848-CC-OH (4 mmol), succinic anhydride (16 mmol), N,N-diisopropylethylamine (20 mmol), and 4-dimethylaminopyridine (1 mmol) in 50 mL of dichloromethane, and stir at room temperature for 12 h. Extract with ethyl acetate three times (50 mLx3), combine the organic phases, dry over anhydrous sodium sulfate, and decompress the solvent to obtain a yellow solid R848-CC-COOH (yield, 70%). The mass spectrum of R848-CC-COOH is shown in Figure 5 shown.

[0067] Example 5

[0068] Synthesis of R848-SS-HSA (hereinafter referred to as RS-HSA)

[0069] RS-HSA synthesis steps: Take a 100 mL flask, dissolve R848-SS-COOH (4 mmol), N-hydroxysuccinimide (12 mmol), N,N-dicyclohexylcarbodiimide (6 mmol), N,N-diisopropylethylamine (16 mmol) in 20 mL dichloromethane, and stir at room temperature for 24 h. Filter through a baffle funnel to remove the white solid, and the solvent is dried under reduced pressure to obtain a yellow solid R848-SS-CONSH.

[0070] Weigh HSA (human serum albumin, 10 mmol), dissolve it in ultrapure water, add R848-SS-CONSH (10, 20, 30, 40, 50, 100, 200 mmol), stir at room temperature for 16 h, put it into a dialysis bag (cut-off molecular weight 14 KDa), change the liquid at 4, 6, 8, 12, 16, and 24 h, put it into a 50 mL centrifuge tube, put it at -80℃ for 12 hours, and freeze-dry it to obtain R848-SS-HSA. The mass spectrum of human serum albumin (HSA) is shown in Figure 6 The mass spectrum of RS-HSA is shown in Figures 7-9 shown.

[0071] Example 6

[0072] Synthesis of R848-CC-HSA (hereinafter referred to as RC-HSA)

[0073] RC-HSA synthesis steps: Take a 100 mL flask, dissolve R848-CC-COOH (4 mmol), N-hydroxysuccinimide (12 mmol), N,N-dicyclohexylcarbodiimide (6 mmol), N,N-diisopropylethylamine (16 mmol) in 20 mL dichloromethane, and stir at room temperature for 24 h. Filter through a baffle funnel to remove the white solid, and the solvent is dried under reduced pressure to obtain a yellow solid R848-CC-CONSH.

[0074] HSA (10 mmol) was weighed and dissolved in ultrapure water. R848-CC-CONSH (10, 20, 30, 40, 50, 100, 200 mmol) was added and stirred at room temperature for 16 h. The mixture was placed in a dialysis bag (molecular weight cutoff 14 KDa), and the solution was changed every 4, 6, 8, 12, 16, and 24 h. The mixture was placed in a 50 mL centrifuge tube and placed at -80 °C for 12 h. The tube was freeze-dried to obtain R848-CC-HSA. Fig.10 This is the mass spectrum of RC-HSA (R848-CC-COOH:HSA=2.9:1, n / n).

[0075] Example 7

[0076] Synthesis of R848-SS-BSA (hereinafter referred to as RS-BSA)

[0077] RS-BSA synthesis steps: Take a 100 mL flask, dissolve R848-SS-COOH (4 mmol), N-hydroxysuccinimide (4-12 mmol), N,N-dicyclohexylcarbodiimide (4-10 mmol), N,N-diisopropylethylamine (4-16 mmol) in 20 mL of dichloromethane, and stir at room temperature for 24 h. Filter through a baffle funnel to remove the white solid, and the solvent is dried under reduced pressure to obtain a yellow solid R848-SS-CONSH. BSA (bovine serum albumin, 10 mmol) was weighed and dissolved in ultrapure water. R848-SS-CONSH (10, 20, 30, 40, 50, 100, 200 mmol) was added and stirred at room temperature for 16 h. The mixture was placed in a dialysis bag (molecular weight cutoff 14 KDa), and the solution was changed every 4, 6, 8, 12, 16, and 24 h. The mixture was placed in a 50 mL centrifuge tube and placed at -80 °C for 12 h for freeze drying. Fig.11 is the mass spectrum of bovine serum albumin (BSA); Fig.12 This is the mass spectrum of RS-BSA (R848-SS-COOH:BSA=1.6:1, n / n).

[0078] Example 8

[0079] GSH-responsive release of RS-HSA in vitro

[0080] Prepare 10 mM pH 6.5 phosphate buffered saline solution, add GSH to prepare 0, 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 5.0, 10.0 mM receiving solution, draw 0.5 mL of nanoparticles (containing R848 1.0 mg) into a dialysis bag with a molecular weight cutoff of 3 KDa, add 5 mL of receiving solution, place in a shaker at 37°C, 180 rpm, and duplicate 3 samples in each group. Draw 1 mL of receiving solution at 0, 1, 2, 4, 6, 8, 12, 16, 24, 48 h, and then add 1 mL of new receiving solution, and measure by liquid phase. At the same time, set up an R848 free control group, prepare R848 treated with dimethyl sulfoxide solution, and treat with 40% PEG 300 Dilute to 2.0 mg / mL, draw 0.5 mL into a dialysis bag with a molecular weight cutoff of 3 KDa, add 5 mL of receiving solution (pH 6.5 phosphate buffered saline solution, containing GSH, 0.2, 0.3, 0.4 mM), place in a shaker at 37°C, 180 rpm, and duplicate 3 samples in each group. Draw 1 mL of receiving solution at 0, 1, 2, 4, 6, 8, 12, 16, 24, and 48 h, and measure by liquid chromatography. The chromatographic conditions are as follows: chromatographic column, InertSustain ® C18 (4.6 mm×150 mm, 5μm); column temperature, 30℃; flow rate, 1.0 mL / min; injection volume, 10 μL; diode array detector detection wavelength, 254 nm; run time, 40 min; diluent, acetonitrile: water (V:V) = 60:40; needle washing solution, 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%; 35min, 90%; 40 min, 90%. %B, 0 min, 10%; 5 min, 10%; 25 min, 90%; 33 min, 90%; 35 min, 10%; 40min, 10%.). The in vitro GSH responsive release of RS-HSA and free R848 is shown in the figure. Fig.13 As shown in the figure, the release rate of R848 gradually increased with the increase of GSH concentration, and only 0.5 mM GSH could release about 60% of R848, indicating that the synthesized RS-HSA has good and sensitive GSH-responsive drug release behavior.

[0081] Example 9

[0082] Biosafety of RS-HSA and R848

[0083] BalB / c mice aged 6-8 weeks and 16-18 g were injected with 500 μM R848, RS-HSA (R848:HSA=1.6:1, n / n), and RS-HSA (R848:HSA=2.9:1, n / n) 100 μL each through the tail vein for 4 consecutive days, and their body weight changes were measured. The weight change trend of mice after tail vein injection of each sample is shown in the figure. Fig.14 As shown, RS-HSA significantly reduced the in vivo toxicity of R848 compared with free R848.

[0084] Example 10

[0085] RS-HSA as a drug carrier

[0086] 500 mM RS-HSA was mixed with equal concentrations of iridium-coordinated oligoarginine amphiphilic peptides (iPep, chemical structure: Fig.21 The samples were mixed at a volume ratio of 1:1 at room temperature for at least 2 h to obtain RS-HSA nanoparticles (iP-RS NPs) loaded with amphiphilic polypeptides. The nanoparticles were diluted with pure water to 10 mM, and the hydrated particle size of the nanoparticles was detected by dynamic light scattering.

[0087] Paclitaxel was dissolved in acetonitrile to prepare a 1 mg / mL solution, and then 10 mL of 10 mg / mL RC-HSA aqueous solution was taken, and 500 mL of paclitaxel solution was slowly added to the aqueous solution under stirring at room temperature, and stirring was continued for at least 2 h to obtain paclitaxel RC-HSA nanoparticles. Take 1 mL of the sample, centrifuge at 4000 rpm for 10 min, and then use a dynamic light scattering instrument to detect the hydrated particle size of the nanoparticles. The hydrated particle size of the iP-RS NP loaded with the amphiphilic peptide iPep and the transmission electron microscopy are shown in the figure. Fig.15 As shown, the water content and particle size of RC-HSA nanoparticles loaded with hydrophobic compound paclitaxel are as follows Fig.16 As shown, both formed nanoparticles with a particle size of 100-200 nm, and the particle size dispersion index was less than 0.3, with good uniformity, indicating that RS-HSA / RC-HSA can be used as a drug carrier to load a variety of different types of drugs.

[0088] Embodiment 11

[0089] Intracellular GSH-responsive release of iP-RS NPs

[0090] 14 μM iP-RS / RC NP (RS / RC-HSA nanoparticles loaded with amphiphilic peptides) was mixed with 10 64T1 cells were co-incubated for 4 h, the culture supernatant and cells were taken separately, 1 mL of ultrapure water was added to the cells and then ultrasonically disrupted, then 4 mL of chloroform was added to the supernatant and the cell disruption solution respectively, the liquid was placed in a separatory funnel for extraction, the chloroform layer was taken, and then an equal volume of new chloroform was added again, the extraction process was repeated three times, the collected chloroform layer was evaporated to dry the organic solution, and then 200 mL of dimethyl sulfoxide was added to re-dissolve, centrifuged at 10000 g for 10 min, the upper supernatant was taken, and the content of R848 was detected by ultra-performance liquid chromatography. The chromatographic conditions were as follows: chromatographic 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; needle washing solution, 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 differences in the responsive release of intracellular and extracellular GSH from the two nanoparticles are shown in Figure 2. Fig.17 As shown, the release of intracellular R848 of GSH-responsive iP-RS NP was about 40 times different from that of non-responsive iP-RC NP, and the release of extracellular R848 was about 4 times different, indicating that after entering the cells, RS-HSA can respond well to the intracellular GSH to release R848, while the non-responsive nanoparticles have a poor ability to release R848.

[0091] Example 12

[0092] RS-HSA promotes the maturation of mouse bone marrow-derived dendritic cells (BMDCs)

[0093] 8 mM RS-HSA / RC-HSA, LPS (1 μg / mL) and 5x10 5Mouse bone marrow-derived dendritic cells (BMDCs) were co-incubated for 24 h, and BMDCs were collected. Dead / live staining was first performed, and the cells were blocked after washing with PBS three times. After blocking, they were washed with PBS twice, and 200 μL of cell staining buffer was added, followed by 1 μL of anti-mouse CD11c-FITC, anti-mouse CD80-PE, and anti-mouse CD86-APC. In addition, single staining tubes for the three dyes and blank cell tubes were set up. After the cells and dyes were mixed evenly, they were stained on ice in the dark for 15 min, centrifuged at 1500 rpm for 5 min to discard the dyes, and then washed three times with 1 mL of cell staining buffer. Finally, the cells were resuspended with 500 μL of cell staining buffer and detected by flow cytometry. The maturation of BMDCs promoted by PBS, RC-HSA, RS-HSA, and LPS is shown in the following figure. Fig.18 As shown, RS-HSA significantly increased the maturation effect of BMDCs by more than 10% compared with the non-responsive RC-HSA, indicating that RS-HSA can release R848, promote the maturation of dendritic cells in vitro, and has the potential to activate immunity.

[0094] Example 13

[0095] RS-HSA promotes BMDMs polarization

[0096] Mouse bone marrow-derived macrophages (BMDMs) were induced to polarize into M2 type by IL-4 (20 ng / mL), and the percentage of M0 macrophages polarized into M2 type was detected by flow cytometry using anti-mouse-CD206-PE antibody. If the induction was successful, the next step was performed. 8 mM RS-HSA / RC-HSA, LPS (1 μg / mL) and 5x10 5 M2 BMDMs were co-incubated for 24 h, and BMDMs were collected. Dead / live staining was first performed, and the cells were blocked after washing 3 times with PBS. After blocking, they were washed twice with PBS, and 200 μL of cell staining buffer was added, followed by 1 μL of anti-mouse CD11b-FITC, anti-mouse F4 / 80-APC, anti-mouse CD80-PE (M1) or anti-mouse CD206-PE (M2). In addition, single staining tubes of the three dyes and blank cell tubes were set up. After the cells and dyes were mixed evenly, they were stained on ice in the dark for 15 min, centrifuged at 1500 rpm for 5 min to discard the dyes, and then washed 3 times with 1 mL of cell staining buffer. Finally, the cells were resuspended with 500 μL of cell staining buffer and detected by flow cytometry. The polarization of BMDMs promoted by PBS, R848, RC-HSA, and RS-HSA is shown in the figure. Fig.19As shown, RS-HSA has a similar macrophage polarization ability to R848, with an M1 / M2 ratio of about 20, while RC-HSA has a poor polarization ability due to its inability to release R848, indicating that RS-HSA can promote the differentiation of macrophages into the pro-inflammatory M1 type and reduce the anti-inflammatory M2 type, thereby having the ability to improve the tumor immunosuppressive microenvironment.

[0097] Embodiment 14

[0098] Hemolytic toxicity of RS / RC-HSA

[0099] The blood of the mice was collected in a blood collection tube containing EDTA and mixed evenly. The blood cells were washed and diluted to 4 × 10 per ml with 1 × PBS. 8 Cells. Set up blank control group, negative control group, positive control group and sample group. Blank control: add 140 mL pH7.4 1×PBS to each well. Negative control: add 70 mL cell suspension and 70 mL pH7.4 1×PBS to each well. Positive control: take 2 mL of cell suspension, centrifuge to remove PBS, then add 2 mL of ultrapure water to obtain red blood cell lysis solution, add 70 mL red blood cell lysis solution and 70 mL ultrapure water to each well. Sample group: add 70 mL cell suspension and 70 mL sample to each well. The final concentrations of the sample group are 8, 16, 32, 64, 128, and 256 mM. The liquids were transferred to U-shaped 96-well plates respectively, and shaken at 37°C and 87rpm for 2 h. Then, the 96-well plate was centrifuged at 4300 g for 5 min, and 90 μL of supernatant was transferred to a flat-bottom 96-well plate. The absorbance of each group at 405 nm was measured by an enzyme reader, and the hemolysis rate = (OD experiment-OD negative) / (OD positive-OD negative). Fig. 20 The figure shows the hemolysis of iPep, iP-RC NP (RC-HSA nanoparticles loaded with amphiphilic peptides), and iP-RS NP (RS-HSA nanoparticles loaded with amphiphilic peptides). The hemolysis rate of iPep alone reached 86.4% at 256 μM, while after being encapsulated by RS / RC-HSA to make nanoparticles, the hemolysis rate was less than 10% at the highest concentration, indicating that the hemolytic toxicity of iPep with strong hemolytic toxicity can be significantly reduced after being loaded with RS / RC-HSA.

[0100] Embodiment 15

[0101] Tumor targeting of iP-RS NP

[0102] Mice were injected with 500 μM iPep and 100 μL iP-RS NP through the tail vein. The heart, liver, spleen, lung, kidney, and tumor of the mice were taken out 24 hours later, weighed, cut into small pieces, and homogenized by ultrasonic wall breaking. 1 mL of perchloric acid and 4 mL of aqua regia (nitric acid: hydrochloric acid = 1:3, v / v) were added, and heated at 260°C to digest until only about 1 mL of liquid remained. Pure water was added to the total volume of 10 mL. At the same time, 0.1, 0.5, 1, 5, 10, 50, and 100 ppb standard solutions were prepared with iridium standard solution to establish a standard curve. ICP-MASS was used to detect the iridium content of each group. The tissue distribution of amphiphilic peptide iPep and amphiphilic peptide nanoparticles coated with RS-HSA in mice 24 hours after tail vein injection is shown in the figure. Fig. 22 As shown in the figure, the accumulation of nanoparticles in the tumor site at 24 h was about 4 times that of the amphiphilic peptide iPep, indicating that RS-HSA, as a drug carrier, can significantly improve the tumor targeting of drugs.

[0103] Example 16

[0104] Pharmacokinetics of iP-RS NPs

[0105] Mice were injected with 500 μM iPep, iP-RC NP, and iP-RS NP, 100 μL each, and blood was collected at 0.017, 0.5, 2, 4, 8, 24, and 48 h. The blood was centrifuged at 10,000 rpm for 10 min, and 100 μL of serum was collected. 1 mL of perchloric acid and 4 mL of aqua regia (nitric acid: hydrochloric acid = 1:3, v / v) were added, and the solution was heated at 260°C until only about 1 mL of liquid remained. Pure water was added to the total volume of 10 mL. At the same time, 0.1, 0.5, 1, 5, 10, 50, and 100 ppb standard solutions were prepared with iridium standard solution to establish a standard curve. The iridium content of each group was detected by inductively coupled plasma mass spectrometry. The pharmacokinetics of amphiphilic peptide iPep injected with tail vein and amphiphilic peptide nanoparticles coated with RC-HSA / RS-HSA in mice were shown in Figure 2. Fig.23 As shown, for biologically unstable amphiphilic peptides, their half-life in vivo is less than half an hour, but after being encapsulated into nanoparticles, their half-life in vivo exceeds 15 h, indicating that RC-HSA / RS-HSA encapsulated drugs can significantly prolong the circulation time of drugs in the body, which is especially beneficial for biologically unstable drugs in the body.

[0106] Embodiment 17

[0107] Effect of RS-HSA on maturation of lymph node DCs

[0108] The mice were injected with 500 μM PBS, R848, RC-HSA, and RS-HSA, 100 μL each, through the tail vein. After 24 hours, the inguinal lymph nodes of the mice were collected, cut into small pieces, and then digested with collagenase IV and DNase I at 37°C for 30 min. The digested tissue blocks were gently blown to disperse them into single cells. Then they were washed with PBS three times, and after dead-live staining and CD16 / 32 blocking, the cells were resuspended in 200 μL of cell staining buffer and 10 6 1 μL of anti-mouse CD11c-FITC, anti-mouse CD80-PE, and anti-mouse CD86-APC were added to the cells and stained on ice in the dark for 15 min. Unbound dyes were then washed off with cell staining buffer, and the maturation of DCs in the inguinal lymph nodes of tumor-bearing mice was detected by flow cytometry. The maturation of DCs in the lymph nodes of mice was promoted 24 h after the tail vein injection of the control group, R848, RC-HSA, and RS-HSA samples. Fig.24 As shown, RS-HSA and R848 have similar abilities to promote the maturation of lymph node DCs. The DCs maturation rate in the RS-HSA group was significantly increased by about 20% compared with the non-responsive RC-HSA group, indicating that RS-HSA can release R848 in vivo to promote DCs maturation and activate anti-tumor immunity.

[0109] Embodiment 18

[0110] RS-HSA improves tumor microenvironment

[0111] The mice were injected with 500 μM PBS, R848, RC-HSA, and RS-HSA, 100 μL each, for 4 consecutive days. After another 3 days, the subcutaneous tumors of the mice were collected, cut into small pieces, and digested with collagenase IV and DNase I at 37°C for 30 min. The digested tissue pieces were gently blown to disperse them into single cells. Then they were washed with PBS 3 times, and after dead-live staining and CD16 / 32 blocking, the cells were resuspended with 200 μL of cell staining buffer and 10 6 1 μL of anti-mouse CD11b-FITC, anti-mouse F4 / 80-APC, anti-mouse CD80-PE or anti-mouse CD206-PE was added to the cells and stained on ice in the dark for 15 min. Unbound dyes were then washed away with cell staining buffer, and the polarization state of macrophages in the tumor site was detected by flow cytometry. PBS, R848, RC-HSA, and RS-HSA samples were injected into the tail vein for 4 consecutive days. After 3 days, the ratio of M1 and M2 macrophages in the tumor site was as follows. Fig.25As shown, RS-HSA has the most significant pro-inflammatory macrophage polarization ability in vivo, and the M1 / M2 ratio is 1.5 times that of R848 and 5 times that of RC-HSA, indicating that RS-HSA can release R848 in vivo to promote macrophage polarization to the pro-inflammatory M1 type and improve the tumor immunosuppressive microenvironment.

[0112] Embodiment 19

[0113] RS-HSA inhibits the growth of breast cancer orthotopic tumors in mice

[0114] Mouse breast cancer cell line 4T1 cells were inoculated into the right mammary gland of mice for 10 6 When it grows to 80 mm 3 Around 24 hours after the injection, mice were injected with 100 μL of 500 μM RS-HSA via the tail vein, and the same volume of PBS was used as a control for 4 consecutive days. The tumor size was measured from the day of administration. The growth curves of breast cancer in situ tumors in mice with PBS and RS-HSA are shown in the figure below. Fig.26 As shown, RS-HSA significantly inhibited the growth of 4T1 orthotopic tumors compared with PBS. At the end of the experiment, the average tumor volume of the PBS group was about twice that of the RS-HSA group, indicating that RS-HSA can play a good role in inhibiting tumor growth while reducing the in vivo toxicity of R848.

Claims

1. A protein-coupled imidazole-quinoline compound, characterized in that: The structure of the protein-coupled imidazole-quinoline compound is shown in formula (I): ； Formula (I) In formula (I), R is S, n is an integer between 1 and 20, and A is albumin.

2. The method for synthesizing protein-coupled imidazole-quinoline compounds according to claim 1, characterized in that: R848, ‌N,N-carbonyldiimidazole and 2,2'-dithiodiethanol are reacted to obtain R848-SS-OH; R848-SS-OH, succinic anhydride, N,N-diisopropylethylamine and 4-dimethylaminopyridine are mixed to obtain R848-SS-COOH; R848-SS-COOH, N-hydroxysuccinimide, N,N-dicyclohexylcarbodiimide and N,N-diisopropylethylamine are mixed to obtain R848-SS-CONSH, human serum albumin and R848-SS-CONSH are stirred and mixed, dialyzed and freeze-dried to obtain protein-coupled imidazole-quinoline compounds; The structure of R848 is The structure of the 2,2'-dithiodiethanol is , R is S.

3. The method for synthesizing protein-coupled imidazole-quinoline compounds according to claim 2, characterized in that: The preparation steps of R848-SS-OH include: dissolving R848 and N,N-carbonyldiimidazole in anhydrous tetrahydrofuran, adding 2,2'-dithiodiethanol after sufficient stirring to continue the reaction, pressurizing the solvent to dryness, and purifying by column chromatography to obtain R848-SS-OH.

4. The method for synthesizing protein-coupled imidazole-quinoline compounds according to claim 2, characterized in that: The preparation steps of R848-SS-COOH include: dissolving R848-SS-OH, succinic anhydride, N,N-diisopropylethylamine and 4-dimethylaminopyridine in 50 mL of dichloromethane, stirring the mixture to react, extracting the mixture with ethyl acetate, combining the organic phases, drying the mixture with anhydrous sodium sulfate, and drying the solvent under reduced pressure to obtain R848-SS-COOH.

5. The method for synthesizing protein-coupled imidazole-quinoline compounds according to claim 2, characterized in that: The molar ratio of R848-SS-OH, succinic anhydride, N,N-diisopropylethylamine and 4-dimethylaminopyridine is 4:4~20:4~20:

1.

6. The method for synthesizing protein-coupled imidazole-quinoline compounds according to claim 2, characterized in that: The molar ratio of R848, N,N-carbonyldiimidazole and 2,2'-dithiodiethanol is 1:1~3:2~4.

7. The method for synthesizing protein-coupled imidazole-quinoline compounds according to claim 2, characterized in that: The molar ratio of R848-SS-COOH to human serum albumin is 0.5~5:

1.

8. Use of the protein-coupled imidazoquinoline compound according to claim 1 in the preparation of a drug carrier.

9. Use of the protein-coupled imidazoquinoline compound according to claim 1 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The drug is a glutathione responsive drug.

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