Preparation of stimulus-responsive polypeptide conjugated drug self-assembled supramolecular nanoparticles and application in brain tumor

Abstract

The application discloses preparation of a kind of supermolecular nanoparticle self-assembled by polypeptide conjugated drug with stimulation response and application in brain tumor: polypeptide conjugate comprising a class of quinoline derivative analogs and camptothecin analogs.Quinoline derivative analogs and camptothecin analogs are connected on brain targeting peptide through sensitive peptide, and are further self-assembled to form supermolecular nanoparticle, improve the brain targeting ability and brain retention efficiency of drug.The polypeptide conjugated drug supermolecular nanoparticle provided in the application shows excellent ability of killing tumor cells and polarizing anti-inflammatory macrophages in vivo and in vitro, has strong inhibition effect on brain tumor, and has great clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the preparation of a class of polypeptide-conjugated drugs and the application of their self-assembled nanoparticles in brain tumors. Background Technology

[0002] Brain tumors are a type of cancer that occurs in the brain and poses a serious global threat to human health, placing a heavy burden on global health efforts. Due to their unique and important location, as well as their highly invasive nature, the incidence (21 per 100,000 people) and mortality rate of brain tumors remain high. Gliomas, considered more malignant, account for approximately one-third of all brain tumors, and glioblastoma, the most malignant type (5-year survival rate 9.8%), accounts for more than half of all gliomas. Currently, the first-line treatment for brain tumors is maximum surgical resection combined with postoperative radiotherapy and chemotherapy, but the treatment effect is not ideal. Besides the pathological characteristics of brain tumors themselves, the presence of the blood-brain barrier significantly limits the entry of therapeutic drugs into brain tissue to exert their therapeutic effects. Developing anti-tumor drugs, prodrugs, and drug carriers with the ability to penetrate the blood-brain barrier can effectively increase drug concentrations in the brain and enhance the therapeutic effect of brain tumors.

[0003] There are various pathways for crossing the blood-brain barrier, and ligand-receptor-mediated transcytosis is the most widely studied and accepted method for delivering drugs to brain tissue. After a ligand binds to its corresponding receptor on the luminal membrane of brain microvascular / capillary endothelial cells, clathrin or caveolin accumulates at the binding site, causing membrane invagination, which further invaginates to form vesicles. These vesicles containing the ligand can directly fuse with the proximal endothelial cell membrane, releasing the ligand into the brain tissue, thus completing the blood-brain barrier transport process. Ligands widely used in drug delivery for brain diseases include low-density lipoprotein receptor-associated ligands (LDL-A1), transferrin receptor-associated ligands (TRAL-A1), insulin / insulin-like growth factor 1 (IGF-1) receptor-associated ligands (IL-1), and integrin-associated ligands. However, some of these ligands, such as LDL-A1, are expressed simultaneously on both the luminal membrane and the proximal endothelial cell membrane. This means that ligands already in the brain tissue may rebind to receptors on the proximal endothelial cell membrane and be re-expressed back into the bloodstream. Drug delivery systems modified with these ligands suffer from the same problem: reduced drug accumulation in brain tissue, leading to decreased or even lost efficacy. Utilizing specific components in the brain tumor microenvironment, such as matrix metalloproteinases, cathepsin B, and granzyme B, to induce ligand efflux and thus block this efflux is a promising approach to further improve the efficiency of such drug delivery systems.

[0004] Furthermore, brain tumors are typical "cold tumors," with their immune microenvironment exhibiting an inhibitory state. This is characterized by difficulties in infiltrating the tumor with cytotoxic T cells and helper T cells; significantly enhanced differentiation of suppressor T cells; and macrophages and microglia displaying anti-inflammatory phenotypes. This not only makes it difficult for emerging immunotherapies to generate effective immune responses in brain tumors but also severely weakens the direct killing effect of chemotherapy drugs on brain tumor cells. Anti-inflammatory macrophages and microglia account for approximately 30-50% of the brain tumor microenvironment and play a crucial role in constructing the immunosuppressive microenvironment. Polarizing these highly prevalent anti-inflammatory macrophages and microglia into pro-inflammatory phenotyped cells can effectively relieve the inhibitory immune microenvironment of brain tumors, activate the body's own specific immune response, and simultaneously resensitize brain tumors to chemotherapy drugs, allowing chemotherapy drugs to fully exert their function.

[0005] Camptothecin analogues are alkaloids (camptothecin) and their derivatives isolated from camptothecin extracts, possessing antitumor activity. They selectively inhibit topoisomerase I (TopoI), binding to the TopoI-DNA complex and stabilizing it, thus preventing the rejoining of broken DNA strands, inhibiting DNA replication and RNA synthesis, and thereby inhibiting cell proliferation. They have become first-line chemotherapy drugs for various tumors. Requimod analogues are immunomodulators that activate Toll-like receptors 7 / 8 expressed on monocytes and macrophages, leading to the induction and release of pro-inflammatory cytokines, thereby polarizing anti-inflammatory macrophages into pro-inflammatory macrophages. Requimod analogues have been approved for the treatment of various tumors, including cutaneous T-cell lymphoma, and in recent years have also been developed as adjuvants to various vaccines to enhance immune responses. Summary of the Invention

[0006] Existing brain tumor drug treatment systems suffer from problems such as insufficient drug accumulation concentration in the brain and suppression of drug efficacy by the tumor-suppressive immune microenvironment. This invention connects therapeutic drugs to corresponding ligands on receptors on the luminal membranes of brain microvascular / capillary endothelial cells via sensitive sequences specific to the tumor microenvironment, constructing a class of peptide-conjugated drugs that further self-assemble into supramolecular nanoparticles. This system possesses functions such as brain targeting, inhibition of drug efflux, and synergistic drug effects, effectively inhibiting brain tumor development.

[0007] The object of the present invention is to provide a class of polypeptide conjugates of retinomod analogues and polypeptide conjugates of camptothecin analogues as shown in general formulas (I) and (II) below, and pharmaceutically acceptable salts thereof.

[0008] Another object of the present invention is to provide a method for preparing the following general formulas (I) and (II) and their use in brain tumors.

[0009] Another object of the present invention is to provide supramolecular nanoparticles formed by self-assembly of the following general formulas (I) and (II), a method for preparing the same, and their application in brain tumors.

[0010]

[0011] Among them, R 1 Selected from hydrogen, C 2-6 -alkyl, C 1-3 -alkoxy-C 1-3 -alkyl, C 1-3 -alkylamino-C 1-3 -alkyl;

[0012] R 2 Selected from hydrogen, C 1-3 -alkyl-isopropyl, C 1-3 -alkyl-hydroxyisopropyl, C 1-3 -alkyl-aminoisopropyl, C 1-3 -alkyl-C 3-6 -cycloalkyl, C 1-3 -alkyl-phenyl;

[0013] Pep1 is a sensitive peptide selected from matrix metalloproteinase 2 sensitive sequences: PLGLAG, PVGLIG, PLGIAGQ, PLGVRGD; cathepsin B sensitive sequences: GFLG, GGFG, FRRG, ALAL, EV'Cit', V'Cit', FK; granzyme B sensitive sequences: IED, IEDD; and apoptosis enzyme sensitive sequence: DEVD.

[0014] Pep2 is a brain-targeting peptide selected from integrin-associated ligands: RGD; low-density lipoprotein receptor-associated ligands: SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS, TFFYGGSRGKRNNFKTEEY, LRKLRKRLLLRKLRKRLL, TEELRVRLASHLRKLRKRLLRDR, cyl[CMPRLRGC], cyl[CM'Thz'RLRG'Pen']; and transferrin receptor-associated ligands: HAIYPRH, THRPPMWSPVWP, GYR.

[0015]

[0016] Among them, R 1 Selected from hydrogen and halogens;

[0017] R 2 Selected from hydrogen, hydroxyl, halogen, C 1-6 -alkyl, alkoxy-C 1-6 -alkyl, alkoxy-tert-butyl, alkoxy-C1-3 -alkyl-ester-tert-butyl, alkoxy-C 1-3 -alkyl-ester group-C 1-3 -alkyl, alkoxy-C 1-12 -alkyl-hydroxy, alkoxy-C 1-12 -alkylcarboxyl group, alkoxy-C 1-3 -alkyl-carbonylpiperidine, acyloxy-C 1-12 -alkyl, acyloxy-C 1-12 -alkylcarboxyl group, acyl-amino-C 1-3 -alkylcarboxyl group, acyl-amino-C 1-3 -alkyl-ester benzyl, acyloxy-amino-C 1-3 -alkyl-ester-tert-butyl, acyloxy-amino-C 1-3 -alkyl-carbonylpiperidine, acyloxy-piperidine, acyloxy-piperidine-piperidine, dihydroxyboryl, methyl phosphate;

[0018] R 3 Selected from hydrogen, hydroxyl, amino, nitro, aldehyde, carbonylamino, C 1-6 -alkyl-dimethylamino, carbonylamino-C 1-6 -alkyl, C 2-6 -Alkenyl;

[0019] R 4 Selected from hydrogen, hydroxyl, halogen, aldehyde, carboxyl, acetylene, C 1-6 -alkyl, C 1-6 -alkyl-hydroxyl, C 1-6 -alkyl-halogen, C 1-3 -alkyl-amino-isopropyl, C 1-3 -alkyl-amino-C 1-3 -alkyl, C 1-3 -alkyl-amino-C 1-3 -Phenyl, C 1-3 -alkyl-piperazine, C 1-3 -alkyl-acyloxy-C 1-3 -alkylcarboxyl group, C 1-6 -alkyl-trimethylsilyl, carbonyl-C 1-6 -alkyl, carbonylamino-C 1-6 -alkyl, carbonylamino-C 1-3 -alkyl-phenyl, imine-alkoxy-tert-butyl;

[0020] R 5 R 6 Selected from hydrogen, methylene hydroxyl, methylenetetrahydropyrrole, R 5 and R 6 It cannot be methylenetetrahydropyrrole at the same time.

[0021] Pep1 is a sensitive peptide selected from matrix metalloproteinase 2 sensitive sequences: PLGLAG, PVGLIG, PLGIAGQ, PLGVRGD; cathepsin B sensitive sequences: GFLG, GGFG, FRRG, ALAL, EV'Cit', V'Cit', FK; granzyme B sensitive sequences: IED, IEDD; and apoptosis enzyme sensitive sequence: DEVD.

[0022] Pep2 is a brain-targeting peptide selected from integrin-associated ligands: RGD; low-density lipoprotein receptor-associated ligands: SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS, TFFYGGSRGKRNNFKTEEY, LRKLRKRLLLRKLRKRLL, TEELRVRLASHLRKLRKRLLRDR, cyl[CMPRLRGC], cyl[CM'Thz'RLRG'Pen']; and transferrin receptor-associated ligands: HAIYPRH, THRPPMWSPVWP, GYR.

[0023] Furthermore, in the polypeptide conjugate of the remiquimod analogue described in this invention, R... 1 Selected from C 1-3 -alkoxy-C 1-3 -alkyl, R 2 Selected from C 1-3 -alkyl-hydroxyisopropyl, Pep1 is selected from matrix metalloproteinase 2 sensitive sequences, and Pep2 is selected from low-density lipoprotein receptor-associated ligands.

[0024] Furthermore, in the polypeptide conjugate of the camptothecin analogue described in this invention, R... 1 Selected from hydrogen, R 2 Selected from hydrogen, hydroxyl, R 3 Selected from hydrogen, hydroxyl, R 4 Selected from hydrogen, R 5 Selected from hydrogen, R 6 Selected from hydrogen, Pep1 is selected from matrix metalloproteinase 2 sensitive sequence, and Pep2 is selected from low-density lipoprotein receptor-associated ligand.

[0025] In the most preferred experimental scheme of this invention, the polypeptide conjugate (Ⅰ-a) of the retinoic acid analog and the polypeptide conjugate (Ⅱ-a) of the camptothecin analog are the following compounds:

[0026]

[0027] The compounds involved in this invention can be prepared via the following route:

[0028] Option 1

[0029]

[0030] Step 1: Dissolve dithiopyridine in methanol at 0-4℃, add mercaptoethanol dropwise to obtain a mixture, continue stirring for 2-6 hours, and purify by silica gel column chromatography to obtain compound AA.

[0031] Step 2: At 0-4℃, compound AA and 4-nitrophenyl chloroformate are dissolved in dichloromethane, alkali is added to catalyze the reaction, stirring is continued for 2-6 hours, and compound AB is obtained by silica gel column chromatography.

[0032] Step 3: Dissolve compound AB and compound AC in dichloromethane at 30-55℃, add alkali to catalyze the reaction, continue stirring for 12-24 h, and purify by silica gel column chromatography to obtain compound AD.

[0033] Step 4: At 15-35℃, dissolve compound AD and the sensitive peptide-brain-targeting peptide conjugate in dimethyl sulfoxide, continue stirring for 12-24 h, and lyophilize by dialyzing to obtain compound I.

[0034] Among them, R 1 R 2 As defined in claim 2; the sensitive peptide-brain-targeting peptide conjugate is selected from different combinations of Pep1 and Pep2 as defined in claim 2; the base is as defined in claim 4.

[0035] Option 2

[0036]

[0037] Step 1: Dissolve dithiopyridine in methanol at 0-4℃, add mercaptoethanol dropwise to obtain a mixture, continue stirring for 2-6 hours, and purify by silica gel column chromatography to obtain compound AA.

[0038] Step 2: At 0-4℃, compound BA and 4-nitrophenyl chloroformate are dissolved in dichloromethane, and alkali is added to catalyze the reaction. Stirring is continued for 2-6 hours, and compound BB is obtained by silica gel column chromatography.

[0039] Step 3: Dissolve compound AA and compound BB in dichloromethane at 35-60℃, add alkali for catalysis, continue stirring for 12-24h, and purify by silica gel column chromatography to obtain compound BC.

[0040] Step 4: At 15-35℃, dissolve compound BC and the sensitive peptide-brain-targeting peptide conjugate in dimethyl sulfoxide, continue stirring for 12-24 h, and lyophilize by dialyzing to obtain compound II.

[0041] Among them, R 1 R 2 R 3 R4 R 5 R 6 As defined in claim 5; the sensitive peptide-brain-targeting peptide conjugate is selected from different combinations of Pep1 and Pep2 as defined in claim 2; the base is as defined in claim 4.

[0042] Wherein, A = alanine, R = arginine, N = aspartic acid, D = asparagine, C = cysteine, Q = glutamine, E = glutamic acid, G = glycine, H = histidine, I = isoleucine, L = leucine, K = lysine, M = methionine, F = phenylalanine, P = proline, S = serine, T = threonine, W = tryptophan, Y = tyrosine, V = valine, Cit = citrulline, Thz = tetrahydrothiazol-2-carboxylic acid, and Pen = 3-mercaptovaline.

[0043] Furthermore, the present invention also provides supramolecular nanoparticles formed by the self-assembly of peptide conjugates of the above-mentioned retinomod analogs and peptide conjugates of camptothecin analogs, comprising the following steps:

[0044] Step 1: Dissolve the polypeptide conjugate of remiquimod analogue and the polypeptide conjugate of camptothecin analogue in solvent I at a ratio of 5:1 to 1:1;

[0045] Step 2: Add the solution obtained in Step 1 dropwise to Solvent II at a ratio of 1:20 to 1:100, and stir for 1-5 minutes;

[0046] Step 3: Centrifuge the system obtained in Step 2 using an ultrafiltration tube at 6000-15000 rpm;

[0047] Step 4: Resuspend the upper nanoparticles in solvent II to obtain supramolecular nanoparticles.

[0048] In some embodiments of the present invention, solvent I in step 1 is selected from one or more of dimethyl sulfoxide, N,N-dimethylformamide, dioxane, acetonitrile, acetone, dimethyl carbonate, and tetrahydrofuran; solvent II in steps 2 and 4 is selected from ultrapure water, physiological saline, phosphate buffer, and Hanks balanced salt buffer.

[0049] This invention also relates to supramolecular nanoparticles having general formulas (I) and (II) and self-assembled therefrom, which are used as therapeutically active substances. Specifically, they are used to treat brain tumor diseases, including glioblastoma, oligodendroglioma, astrocytoma, oligodendroastroglioma, ependymoma, glial neuron tumors, choroid plexus tumors, medulloblastoma, meningioma, pituitary adenoma, and schwannoma.

[0050] Beneficial effects

[0051] This invention synthesizes a novel compound, which has not been reported in the literature or patents.

[0052] 1) Based on the ligand-receptor transport function of brain microvascular / capillary endothelial cells, this invention provides ligand-modified therapeutic drugs to give them the ability to penetrate the blood-brain barrier and increase the drug content entering brain tissue.

[0053] 2) The present invention further incorporates a sensitive bond between the therapeutic drug and the ligand, which is a specific component of the tumor microenvironment. This allows the drug to cleave the outer ligand after entering the brain tissue, preventing the ligand from binding to receptors on the inner side of brain microvascular / capillary endothelial cells and being expelled back into the bloodstream, thus effectively improving the drug's accumulation efficiency in the brain tissue.

[0054] 3) The supramolecular nanoparticles prepared in this invention, which are formed by the self-assembly of peptide conjugates of retinomod analogs and peptide conjugates of camptothecin analogs, can polarize the large number of anti-inflammatory macrophages in the tumor microenvironment into pro-inflammatory macrophages, reverse the immunosuppressive microenvironment, and stimulate the body's own specific immune response; at the same time, they enhance the sensitivity of tumors to chemotherapy drugs, play a synergistic role, and effectively inhibit tumor development.

[0055] The supramolecular nanoparticles involved in this invention are easy to prepare, have simple composition, require no excipients, have high targeted delivery efficiency, and exhibit drug synergistic effects, providing favorable conditions for formulation development. Attached Figure Description

[0056] Figure 1 This is a particle size distribution diagram of supramolecular nanoparticles;

[0057] Figure 2 To demonstrate the uptake of supramolecular nanoparticles by bEnd.3 and GL261 cells in an in vitro blood-brain barrier model;

[0058] Figure 3 To investigate the killing effect of supramolecular nanoparticles on lower GL261 cells in an in vitro blood-brain barrier model;

[0059] Figure 4 To illustrate the polarization of supramolecular nanoparticles on underlying microglia in an in vitro blood-brain barrier model;

[0060] Figure 5 Distribution of supramolecular nanoparticles in the brains of tumor-bearing mice;

[0061] Figure 6 The effects of supramolecular nanoparticles on body weight and survival rate in tumor-bearing mice. Detailed Implementation

[0062] The following embodiments are used to illustrate the purpose and technical solutions of the present invention in detail. It should be noted that the embodiments are only used to illustrate the invention in detail and are not intended to limit the scope of the invention.

[0063] Example 1: Synthesis of 2-Dithiopyridine Ethanol

[0064]

[0065] Under ice bath conditions, dithiopyridine (5.03 g, 22.9 mmol) was dissolved in 15 mL of anhydrous methanol, and then 2-mercaptoethanol (1.18 mL, 16.8 mmol) was added dropwise. The mixture was stirred in an ice bath for 3 h, the solvent was removed by rotary evaporation, and then purified by silica gel column chromatography (0% to 20% ethyl acetate in petroleum ether solution). The solution was concentrated and dried to give the title compound (2.01 g, 64%) as a yellow oily liquid. 1 H NMR(400MHz, CDCl3)δ8.57–8.45(m,1H),7.60(qd,J=7.7,1.8Hz,1H),7.40(dt, J=8.0,1.1Hz,1H),7.22–7.08(m,1H),3.88–3.73(m,2H),3.02–2.86(m,2H)ppm.

[0066] Example 2: Synthesis of 4-nitrophenyl-(2-dithiopyridine)acetic acid ester

[0067]

[0068] Under ice bath conditions, 2-dithiopyridine ethanol (781 mg, 4.18 mmol) was dissolved in 15 mL of dichloromethane, followed by dropwise addition of 4-nitrophenyl chloroformate (1.68 g, 8.35 mmol) dissolved in 10 mL of dichloromethane, and then diisopropylethylamine (1.46 mL, 8.34 mmol). The mixture was stirred in an ice bath for 2 h, the solvent was removed by rotary evaporation, and then purified by silica gel column chromatography (3% to 10% ethyl acetate solution in petroleum ether). The solution was concentrated and dried to give the title compound (718 mg, 51%) as a yellow oily liquid. 1 HNMR(400MHz,DMSO-d6)δ8.48(ddd,J=4.8,1.9,1.0Hz,1H),8.37–8.29(m,2H),7.85(td,J=7.7,1.8Hz,1H),7.79(dt,J= 8.1,1.1Hz,1H),7.61–7.52(m,2H),7.27(ddd,J=7.3,4.8,1.2Hz,1H),4.48(t,J=6.0Hz,2H),3.24(t,J=6.0Hz,2H)ppm.

[0069] Example 3: Synthesis of Requimod-(2-Dithiopyridine)aminoacetic acid ester

[0070]

[0071] Requimide (590 mg, 1.88 mmol), 4-nitrophenyl-(2-dithiopyridine) acetate (1.8 g, 5.31 mmol), and 1-hydroxybenzotriazole (750 mg, 5.55 mmol) were dissolved in 20 mL of dichloromethane. Then, diisopropylethylamine (1.40 mL, 8.00 mmol) was added, and the mixture was refluxed and stirred at 35°C for 16 h. The solvent was removed by rotary evaporation, and the mixture was then purified by silica gel column chromatography (0% to 5% methanol in dichloromethane). The solution was concentrated and dried to give the title compound (605 mg, 61%) as a pale yellow solid. 1 HNMR(400MHz,DMSO-d6)δ9.89(s,1H),8.55(d,J=8.3Hz,1H),8.46(ddd,J=4.8,1.8,1.0 Hz,1H),7.93(d,J=1.4Hz,1H),7.90–7.76(m,2H),7.63(ddd,J=8.3,6.9,1.3Hz,1H),7.5 9–7.51(m,1H),7.28–7.19(m,1H),6.61(d,J=63.9Hz,1H),4.95(s,1H),4.76(s,4H),4. 34(t,J=6.2Hz,2H), 3.53(q,J=7.0Hz,2H), 3.18(t,J=6.2Hz,2H), 1.25–1.12(m,9H)ppm. MALDI-Tof m / z 528.1(MH + ).

[0072] Example 4: Synthesis of Remiquimod-C2K-GPLGLAG-TFFYGGSRGKRNNFKTEEY(Ⅰ-a)

[0073]

[0074] Requimide-(2-dithiopyridine)aminoacetic acid ester (13 mg, 0.025 mmol) and C2K-GPLGLAG-TFFYGGSRGKRNNFKTEEY (20 mg, 0.006 mmol) were dissolved in 2 mL of anhydrous dimethyl sulfoxide. The mixture was purged with nitrogen and kept under nitrogen protection. The mixture was stirred overnight at room temperature, dialyzed against pure water at MWCO 2000 for 24 h, and freeze-dried to give the title compound (18.3 mg, 75.5%) as a white solid. MALDI-Tof m / z 4034.91 (MH) + ).

[0075] Example 5: Synthesis of 4-nitrophenyl-(camptothecin) carbamate

[0076]

[0077] Under ice bath conditions, camptothecin (104 mg, 0.298 mmol) and 4-nitrophenyl chloroformate (157 mg, 0.772 mmol) were dissolved in dichloromethane, followed by the addition of dimethylaminopyridine (206 mg, 1.69 mmol). The mixture was stirred in an ice bath for 3 h, the solvent was removed by rotary evaporation, and then purified by silica gel column chromatography (0% to 75% ethyl acetate in dichloromethane). The solution was concentrated and dried to give the title compound (109 mg, 71%) as a pale yellow solid. 1 H NMR (400MHz, CDCl3) δ8.45 (s, 1H), 8.25 (dd, J = 9.6, 7.7Hz, 3H), 8.02–7.95 (m, 1H), 7.89(ddd,J=8.5,6.9,1.5Hz,1H),7.72(ddd,J=8.1,6.9,1.2Hz,1H),7.46–7.38(m, 3H),5.74(d,J=17.3Hz,1H),5.45(d,J=17.3Hz,1H),5.34(dd,J=3.8,1.2Hz,2H),2. 40(dq,J=14.8,7.4Hz,1H), 2.27(dq,J=14.5,7.4Hz,1H), 1.09(t,J=7.5Hz,3H)ppm.

[0078] Example 6: Synthesis of Camptothecin-(2-dithiopyridine)carbamate

[0079]

[0080] 4-Nitrophenyl-(camptothecin) carbamate (498 mg, 0.967 mmol) and dimethylaminopyridine (315 mg, 2.56 mmol) were dissolved in 30 mL of dichloromethane, followed by slow addition of 2-dithiopyridine ethanol (545 mg, 2.92 mmol). The mixture was refluxed and stirred at 45°C for 12 h. The solvent was removed by rotary evaporation, and the mixture was purified by silica gel column chromatography (0% to 100% ethyl acetate in dichloromethane). The solution was concentrated and dried to give the title compound (324 mg, 60%) as a pale yellow solid. 1HNMR (400MHz, CDCl3) δ8.46–8.39(m,2H),8.23(dd,J=8.5,1.1Hz,1H),7.95(dd,J=8.2,1.4Hz,1H),7.84(ddd, J=8.5,6.9,1.5Hz,1H),7.68(ddd,J=8.2,6.9,1.3Hz,1H),7.67–7.59(m,2H),7.34(s,1H),7.09–6.99(m,1H), 5.70(d,J=17.2Hz,1H),5.40(d,J=17.2Hz,1H),5.30(dd,J=3.1,1.2Hz,2H),4.44–4.35(m,1H),4.38–4.29(m, 1H), 3.06 (t, J = 6.6Hz, 2H), 2.29 (dq, J = 14.9, 7.4Hz, 1H), 2.17 (hept, J = 7.3Hz, 1H), 1.00 (t, J = 7.5Hz, 3H) ppm. MALDI-Tof m / z562.32(MH + ).

[0081] Example 7: Synthesis of Camptothecin-C2K-GPLGLAG-TFFYGGSRGKRNNFKTEEY(Ⅱ-a)

[0082]

[0083] Camptothecin-(2-dithiopyridine)carboxylate (13.5 mg, 0.024 mmol) and C2K-GPLGLAG-TFFYGGSRGKRNNFKTEEY (20 mg, 0.006 mmol) were dissolved in 2 mL of anhydrous dimethyl sulfoxide, purged with nitrogen and protected under nitrogen atmosphere, stirred overnight at room temperature, dialyzed against pure water at MWCO 2000 for 24 h, and freeze-dried to give the title compound (16.6 mg, 76.8%) as a white solid. MALDI-Tof m / z 4103.13 (MH) + ).

[0084] Example 8: Preparation of supramolecular nanoparticles

[0085] I-a-Requimod-C2K-GPLGLAG-TFFYGGSRGKRNNFKTEEY (7.5 mg) and II-a-Camptothecin-C2K-GPLGLAG-TFFYGGSRGKRNNFKTEEY (1.5 mg) were dissolved in 1 mL of dimethyl sulfoxide; then 100 μL of this solution was added dropwise to 5 mL of phosphate buffer and stirred at 150 rpm for 2 minutes; then the mixture was centrifuged at 8000 × g for 10 minutes using an ultrafiltration tube with a molecular weight cutoff of 10 kDa, and the upper supramolecular nanoparticles were collected in 1 mL of phosphate buffer to obtain the target sample.

[0086] The particle size distribution of supramolecular nanoparticles was determined using dynamic light scattering. Figure 1 The results showed that the prepared supramolecular nanoparticles had a particle size of about 95 nm and a polydispersity index of 0.145, exhibiting a small particle size and a narrow particle size distribution.

[0087] Example 9: Determination of cellular uptake efficiency of supramolecular nanoparticles

[0088] Mouse brain microvascular / capillary endothelial cells (bEnd.3 cells) were seeded at 150,000 cells / well in 6-well Transwell chambers and cultured for 5 days at 37°C and 5% CO2. Mouse glioma cells (GL261 cells) were seeded at 150,000 cells / well in 6-well plates and cultured for 24 hours at 37°C and 5% CO2. The Transwell chambers seeded with bEnd.3 cells were then placed in 6-well plates seeded with GL261 cells, and free drug (requimod + camptothecin) or supramolecular nanoparticles were added to the Transwell chambers. bEnd.3 and GL261 cells were collected at 4 and 12 hours after drug addition, washed with phosphate-buffered saline (PBS), and the intracellular camptothecin fluorescence intensity was analyzed by flow cytometry.

[0089] Figure 2 a and Figure 2 b represents the quantitative fluorescence intensity of bEnd.3 and GL261 cells after taking up free drugs or supramolecular nanoparticles. It can be seen that the uptake of supramolecular nanoparticles by both bEnd.3 and GL261 cells is much higher than that by free drugs, indicating that supramolecular nanoparticles can effectively penetrate the biomimetic blood-brain barrier and remain in GL261 cells.

[0090] Example 10: Cytotoxicity of supramolecular nanoparticles

[0091] Cells were seeded and cultured as described in Example 9. Then, phosphate-buffered saline solution, free drug (requimod + camptothecin) or supramolecular nanoparticles were added to the Transwell chamber. GL261 cells were collected 36 hours after drug addition and Annexin-FITC / PT staining was performed according to the operating instructions. After completion, cell apoptosis was analyzed by flow cytometry.

[0092] Figure 3 The apoptosis of GL261 cells indicates that supramolecular nanoparticles have a much stronger killing effect on GL261 cells than free drugs.

[0093] Example 11: Polarization of macrophages by supramolecular nanoparticles

[0094] Mouse brain microvascular / capillary endothelial cells (bEnd.3 cells) were seeded at 150,000 cells / well in 6-well Transwell chambers and cultured for 5 days at 37°C and 5% CO2. Primary mouse microglia (which can be considered macrophages) were seeded at 150,000 cells / well in 6-well plates, with 10 ng / mL IL-10 and 20 ng / mL IL-4 added, and cultured at 37°C and 5% CO2 for 24 hours to polarize them into anti-inflammatory microglia. Then, the Transwell chambers seeded with bEnd.3 cells were placed in 6-well plates seeded with microglia, and phosphate-buffered saline solution, free drugs (requimod + camptothecin), or supramolecular nanoparticles were added to the Transwell chambers. Microglia were collected 24 hours after drug administration and stained with CD86 (a marker of pro-inflammatory macrophages) and CD206 (a marker of anti-inflammatory macrophages) antibodies. After the procedure, the polarization of microglia was analyzed by flow cytometry.

[0095] Figure 4 The polarization of microglia indicates that supramolecular nanoparticles can more effectively polarize anti-inflammatory microglia into pro-inflammatory microglia.

[0096] Example 12: Study on the brain tumor targeting ability of supramolecular nanoparticles

[0097] GL261 cells (3 × 10⁻⁶) were in situ injected into the brains of 18-22g C57 mice. 6 Mice were inoculated (10 mice per mouse). Ten days later, mice were given free fluorescent dye DiD or DiD-labeled supramolecular nanoparticles (DiD 1.5 mg / kg) via tail vein. The mice were sacrificed 48 hours later, and brain tissue was collected. The distribution of fluorescent dye signals in the brain tissue was observed and quantified using a small animal in vivo imaging system.

[0098] Figure 5It is a quantitative analysis of the fluorescence intensity and ratio between normal brain tissue and brain tumor regions. The supramolecular nanoparticle group has a stronger overall fluorescence signal, and the fluorescence intensity ratio between the brain tumor region and the normal region is significantly higher than that of the free dye group.

[0099] Example 13: Evaluation of the antitumor activity of supramolecular nanoparticles

[0100] GL261 cells (3 × 10⁻⁶) were in situ injected into the brains of 18-22g C57 mice. 6 Each mouse was administered saline, free drugs (requimod + camptothecin), or supramolecular nanoparticles (requimod 0.9 μmol / kg, camptothecin 4.4 μmol / kg) via tail vein after 10 days. The saline group received an equal volume of saline. The weight and survival status of the mice were recorded.

[0101] Figure 6 The study examined the changes in body weight and survival rate of mice in each group. The supramolecular nanoparticle group reversed the weight loss caused by brain tumors and significantly prolonged the survival time of the mice.

[0102] It should be noted that the technical means disclosed in this invention are not limited to the contents described in the above embodiments. For those skilled in the art, several non-essential modifications can be made based on this invention; these modifications are obvious to those skilled in the art, and therefore, all such modifications fall within the protection scope of this invention.

Claims

1. A supramolecular nanoparticle with stimulus-responsive brain-targeting peptide-conjugated drug self-assembly, characterized in that: It is formed by the self-assembly of a polypeptide conjugate of remiquintimod (I) and a polypeptide conjugate of camptothecin (II), and its general structural formula is as follows: in, Pep1 is a sensitive peptide selected from one of the following matrix metalloproteinase 2 sensitive sequences: PLGLAG, PVGLIG, PLGIAGQ, or PLGVRGD. Pep2 is a brain-targeting peptide, selected from the low-density lipoprotein receptor-associated ligand TFFYGGSRGKRNNFKTEEY.

2. A method for preparing supramolecular nanoparticles with stimulus-responsive brain-targeting peptide-conjugated drugs as described in claim 1, characterized in that, The synthetic steps of the polypeptide conjugate (I) including retinomod are as follows: Step 1: Dissolve dithiopyridine in methanol at 0-4℃, then add mercaptoethanol dropwise to obtain a mixture. Continue stirring for 2-6 h, and purify by silica gel column chromatography to obtain compound AA. Step 2: At 0-4°C, compound AA and 4-nitrophenyl chloroformate were dissolved in dichloromethane. A base was added for catalysis, and stirring was continued for 2-6 h. The mixture was then purified by silica gel column chromatography to obtain compound AB. Step 3: Dissolve compounds of formula AB and formula AC in dichloromethane at 30-55℃, add alkali for catalysis, continue stirring for 12-24 h, and purify by silica gel column chromatography to obtain compound AD. Step 4: At 15-35℃, dissolve compound AD and the sensitive peptide-brain-targeting peptide conjugate in dimethyl sulfoxide, continue stirring for 12-24 h, dialyze and lyophilize to obtain compound I. in, Pep1 is a sensitive peptide selected from one of the following matrix metalloproteinase 2 sensitive sequences: PLGLAG, PVGLIG, PLGIAGQ, or PLGVRGD. Pep2 is a brain-targeting peptide, selected from the low-density lipoprotein receptor-associated ligand TFFYGGSRGKRNNFKTEEY.

3. The preparation method according to claim 2, characterized in that, It also includes the synthetic steps of camptothecin polypeptide conjugate (II), as follows: Step 1: Dissolve dithiopyridine in methanol at 0-4℃, then add mercaptoethanol dropwise to obtain a mixture. Continue stirring for 2-6 h, and purify by silica gel column chromatography to obtain compound AA. Step 2: At 0-4°C, compound BA and 4-nitrophenyl chloroformate were dissolved in dichloromethane. A base was added for catalysis, and stirring was continued for 2-6 h. The mixture was then purified by silica gel column chromatography to obtain compound BB. Step 3: Dissolve compounds of formula AA and formula BB in dichloromethane at 35-60℃, add alkali for catalysis, continue stirring for 12-24 h, and purify by silica gel column chromatography to obtain compound of formula BC. Step 4: At 15-35℃, dissolve compound BC and the sensitive peptide-brain-targeting peptide conjugate in dimethyl sulfoxide, continue stirring for 12-24 h, dialyze and lyophilize to obtain compound II. in, Pep1 is a sensitive peptide selected from one of the following matrix metalloproteinase 2 sensitive sequences: PLGLAG, PVGLIG, PLGIAGQ, or PLGVRGD. Pep2 is a brain-targeting peptide, selected from the low-density lipoprotein receptor-associated ligand TFFYGGSRGKRNNFKTEEY.

4. The preparation method according to claim 3, characterized in that, It also includes the generation of self-assembled supramolecular nanoparticles, comprising the following steps: Step 1: Dissolve the polypeptide conjugate of remiquinoline and the polypeptide conjugate of camptothecin as described in claim 1 in solvent I at a ratio of 5:1 to 1:

1. Step 2: Slowly add the solution obtained in Step 1 to Solvent II at a ratio of 1:20 to 1:100, and stir for 1-5 minutes. Step 3: Centrifuge the system obtained in Step 2 using an ultrafiltration tube at 6000-15000 rpm. Step 4: Resuspend and collect the upper nanoparticles with solvent II; these are the supramolecular nanoparticles that are self-assembled with a stimulus-responsive brain-targeting peptide-conjugated drug. In step 1, solvent I is selected from dimethyl sulfoxide and N,N-dimethylformamide; in steps 2 and 4, solvent II is selected from ultrapure water, physiological saline and phosphate buffer.

5. The use of the stimulation-responsive brain-targeting peptide-conjugated drug-in-place supramolecular nanoparticles self-assembled according to claim 1, or the stimulation-responsive brain-targeting peptide-conjugated drug-in-place supramolecular nanoparticles self-assembled according to any one of claims 2-4, in the preparation of drugs for inhibiting the growth, metastasis, and recurrence of brain tumors, characterized in that... The inhibition of brain tumor growth, metastasis and recurrence means directly killing tumor cells, or polarizing anti-inflammatory macrophages into pro-inflammatory macrophages to improve the tumor immune microenvironment, or promoting the infiltration of lymphocytes in the tumor microenvironment; the brain tumor is glioblastoma, oligodendroglioma or oligoastrocytoma.

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