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

Abstract

The invention discloses preparation of supramolecular nanoparticles self-assembled by polypeptide coupling drugs with stimulation responsiveness and application of the supramolecular nanoparticles in brain tumors. The supramolecular nanoparticles comprise a requinomod analogue and a camptothecin analogue. A requinomod analogue and a camptothecin analogue are connected to a brain targeting peptide through a sensitive peptide, and are further self-assembled to form supramolecular nanoparticles, so that the brain targeting ability and intracerebral retention efficiency of the drug are improved. The polypeptide coupled drug supramolecular nanoparticles provided by the invention show excellent capabilities of killing tumor cells and polarizing anti-inflammatory macrophages in vivo and in vitro, have a strong inhibition effect on brain tumors, and have huge clinical application value.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the preparation of a class of polypeptide-coupled drugs and the application of self-assembled nanoparticles thereof in brain tumors. Background Art

[0002] Brain tumors are a type of global tumor disease that occurs in the brain and seriously endangers human health, and has placed a serious burden on global health. Due to the particularity and importance of its location, as well as its strong invasiveness, the incidence (21 people / 100,000 people) and mortality of brain tumors remain high. Among them, the incidence of more malignant gliomas accounts for about one-third of all brain tumors, and the most malignant glioblastoma (5-year survival rate 9.8%) among gliomas accounts for more than half of gliomas. At present, the first-line treatment for brain tumors in clinical practice is maximum surgical resection combined with postoperative radiotherapy and chemotherapy, but the treatment effect is not ideal. The main reason is that in addition to the pathological characteristics of brain tumors themselves, the existence of the blood-brain barrier also greatly limits the therapeutic drugs from entering the brain tissue to exert their therapeutic effects. The development of anti-tumor drugs, prodrugs, and drug carriers that have the ability to penetrate the blood-brain barrier can effectively increase the concentration of drugs in the brain and enhance the therapeutic effect of brain tumors.

[0003] There are many ways to cross the blood-brain barrier. The method of using ligand-receptor-mediated transcytosis to deliver drugs to brain tissue has been widely studied and is currently the most recognized. After the ligand binds to the corresponding receptor on the luminal membrane of brain microvascular / capillary endothelial cells, clathrin or caveolin will aggregate on the luminal membrane of the binding site, causing the membrane to indent, and then further indent to form vesicles. The vesicles containing the ligand can directly fuse with the cell membrane of the endothelial cell near the brain side, release the ligand into the brain tissue, and complete the blood-brain barrier transport process of the ligand. Ligands that have been widely used in the field of drug delivery for brain diseases include low-density lipoprotein receptor-related ligands, transferrin receptor-related ligands, insulin / insulin-like growth factor 1 receptor-related ligands, integrin-related ligands, etc. However, some of the above ligands, such as low-density lipoprotein receptor-related protein 1, are expressed simultaneously on the luminal membrane and the proximal cell membrane of endothelial cells, resulting in the possibility that the ligand that has entered the brain tissue will bind to the receptor on the proximal cell membrane again and be excreted back into the blood. The same problem exists in drug delivery systems that modify these ligands, which will reduce the accumulation of drugs in brain tissue, and the efficacy will be reduced or even lost. Using specific components in the brain tumor microenvironment, such as matrix metalloproteinases, cathepsin B, granzyme B, etc., to cause ligand shedding and thus block this efflux phenomenon is a promising means to further improve the efficiency of this type of drug delivery system.

[0004] In addition, brain tumors are typical "cold tumors" and their immune microenvironment is in an inhibitory state. It is difficult for cytotoxic T cells and helper T cells to infiltrate into the tumor; the differentiation of suppressive T cells is significantly enhanced; macrophages and microglia show an anti-inflammatory phenotype, which not only makes it difficult for emerging immunotherapies to produce effective immune responses in brain tumors, but also seriously weakens the direct killing effect of chemotherapy drugs on brain tumor cells. Among them, anti-inflammatory macrophages and microglia account for about 30-50% in the brain tumor microenvironment, and play an important role in the construction of an immunosuppressive microenvironment. Polarizing these highly inflammatory macrophages and microglia into pro-inflammatory phenotype cells can effectively relieve the inhibitory immune microenvironment of brain tumors, activate the body's own specific immune response, and at the same time re-sensitize brain tumors to chemotherapy drugs, giving full play to the function of chemotherapy drugs.

[0005] Camptothecin analogs are alkaloids (camptothecin) and their derivatives isolated from extracts of Camptotheca acuminata with antitumor activity. They can selectively inhibit topoisomerase I (TopoⅠ), bind to the complex formed by TopoⅠ-DNA, stabilize the complex, and prevent the broken DNA chains from rejoining, thereby preventing DNA replication and RNA synthesis, and achieving the effect of preventing cell proliferation. They have become the first-line chemotherapy drugs for many tumors. Resiquimod analogs are a class of immunomodulators that can activate Toll-like receptors 7 / 8 expressed on monocytes and macrophages, leading to the induction and release of proinflammatory cytokines, thereby polarizing anti-inflammatory macrophages into proinflammatory macrophages. Resiquimod analogs have been approved for the treatment of various tumor diseases such as cutaneous T-cell lymphoma. In recent years, they have also been developed as adjuvants for various vaccines to enhance immune effects. Summary of the invention

[0006] The existing brain tumor drug treatment system has problems such as insufficient drug accumulation concentration in the brain and the drug efficacy being suppressed by the tumor suppressive immune microenvironment. The present invention connects the therapeutic drug to the corresponding ligand of the receptor on the luminal membrane of brain microvessels / capillary endothelial cells through the sensitive sequence of the tumor microenvironment specific components, constructs a class of polypeptide-coupled drugs, and further self-assembles to form supramolecular nanoparticles. The system has the functions of brain targeting, inhibiting drug efflux, exerting drug synergistic effects, etc., and can effectively inhibit the development of brain tumors.

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

[0008] Another object of the present invention is to provide the preparation method of the following general formula (I) and (II) and their use in treating brain tumors.

[0009] Another object of the present invention is to provide supramolecular nanoparticles self-assembled from the following general formulae (I) and (II), a preparation method thereof, and application thereof 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: IEFD, IEPD; apoptosis enzyme sensitive sequence: DEVD;

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

[0015]

[0016] Among them, R 1 is selected from hydrogen and halogen;

[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-C 1-3 -alkyl, alkoxy-C 1-12 -alkyl-hydroxy, alkoxy-C 1-12 -Alkylcarboxyl, alkoxy-C 1-3 -alkyl-carbonylpiperidine, acyloxy-C 1-12 -alkyl, acyloxy-C 1-12 -alkylcarboxyl, acyloxy-amino-C 1-3 -alkylcarboxyl, acyloxy-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 phosphoate;

[0018] R 3 is 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, ethynyl, 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, 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 is selected from hydrogen, methylene hydroxyl, methylene tetrahydropyrrole, R 5 and R 6 It cannot be methylene tetrahydropyrrole 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: IEFD, IEPD; apoptosis enzyme sensitive sequence: DEVD;

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

[0023] Furthermore, in the polypeptide conjugate of the resiquimod analogue of the present 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 sequence, and Pep2 is selected from low-density lipoprotein receptor-related ligand.

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

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

[0026]

[0027] The compounds of the present invention can be prepared by the following route:

[0028] Solution 1

[0029]

[0030] Step 1, dissolving dithiodipyridine in methanol at 0-4°C, adding mercaptoethanol dropwise thereto to obtain a mixed solution, continuing to stir for 2-6 hours, and purifying by silica gel column chromatography to obtain a compound of formula AA;

[0031] Step 2, dissolving the compound of formula AA and 4-nitrophenyl chloroformate in dichloromethane at 0-4° C., adding a base thereto for catalysis, continuing stirring for 2-6 hours, and purifying by silica gel column chromatography to obtain a compound of formula AB;

[0032] Step 3, dissolving the compound of formula AB and the compound of formula AC in dichloromethane at 30-55° C., adding a base thereto for catalysis, continuing stirring for 12-24 hours, and purifying by silica gel column chromatography to obtain a compound of formula AD;

[0033] Step 4, dissolving the compound of formula AD and the sensitive peptide-brain targeting peptide conjugate in dimethyl sulfoxide at 15-35° C., continuing stirring for 12-24 hours, dialyzing and freeze-drying to obtain the compound of formula 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 defined in claim 2; the base is as defined in claim 4.

[0035] Solution 2

[0036]

[0037] Step 1, dissolve dithiodipyridine in methanol at 0-4°C, add mercaptoethanol dropwise thereto to obtain a mixed solution, continue stirring for 2-6 hours, and purify by silica gel column chromatography to obtain a compound of formula AA.

[0038] Step 2, at 0-4° C., dissolve the compound of formula BA and 4-nitrophenyl chloroformate in dichloromethane, add a base for catalysis, continue stirring for 2-6 hours, and purify by silica gel column chromatography to obtain a compound of formula BB.

[0039] Step 3, at 35-60° C., dissolve the compound of formula AA and the compound of formula BB in dichloromethane, add a base thereto for catalysis, continue stirring for 12-24 hours, and purify by silica gel column chromatography to obtain a compound of formula BC.

[0040] Step 4, dissolving the compound of formula BC and the sensitive peptide-brain targeting peptide conjugate in dimethyl sulfoxide at 15-35° C., continuing stirring for 12-24 hours, dialyzing and freeze-drying to obtain the compound of formula 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 defined in claim 2; the base is as defined in claim 4.

[0042] Among them, 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 = tetrahydrothiazolidine-2-carboxylic acid, Pen = 3-mercaptovaline.

[0043] Furthermore, the present invention also provides supramolecular nanoparticles formed by self-assembly of the above-mentioned polypeptide conjugate of resiquimod analog and the polypeptide conjugate of camptothecin analog, comprising the following steps:

[0044] Step 1, dissolving the polypeptide conjugate of the resiquimod analog and the polypeptide conjugate of the camptothecin analog in solvent I at a ratio of 5:1-1:1;

[0045] Step 2, adding the solution obtained in step 1 dropwise into solvent II at a ratio of 1:20-1:100, and stirring for 1-5 minutes;

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

[0047] Step 4, resuspending and collecting the upper nanoparticles with solvent II to obtain supramolecular nanoparticles.

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

[0049] The present invention also relates to supramolecular nanoparticles having general formula (I) and (II) and self-assembled therefrom, which are used as therapeutic active substances. Specifically, they are used to treat brain tumor diseases, including glioblastoma, oligodendroglioma, astrocytoma, oligoastrocytoma, ependymoma, glioneuronal tumor, choroid plexus tumor, medulloblastoma, meningioma, pituitary tumor, and schwannoma.

[0050] Beneficial Effects

[0051] The present invention synthesizes a completely new compound, which has not been reported in any literature or patent.

[0052] 1) The present invention is based on the ligand-receptor transport function of brain microvessel / capillary endothelial cells, and modifies the therapeutic drug with ligands to give it the ability to penetrate the blood-brain barrier and increase the drug content entering the brain tissue;

[0053] 2) The present invention further provides a sensitive bond connection between the therapeutic drug and the ligand with a specific component in the tumor microenvironment, so that the outer layer of the ligand can be cut off after entering the brain tissue, preventing the ligand from binding to the receptors on the inner side of the brain microvessel / capillary endothelial cells and being excreted back into the blood, thereby effectively improving the accumulation efficiency of the drug in the brain tissue;

[0054] 3) The supramolecular nanoparticles prepared by the present invention, which are self-assembled by the polypeptide conjugates of the resiquimod analogs and the polypeptide conjugates of the camptothecin analogs, can polarize the anti-inflammatory macrophages present in large quantities 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 the tumor to chemotherapeutic drugs, play a synergistic role in drugs, and effectively inhibit tumor development.

[0055] The supramolecular nanoparticles involved in the present invention are easy to prepare, simple in composition, free of adjuvant incorporation, high in targeted delivery efficiency, and have drug synergistic effects, thus providing powerful conditions for the development of preparations. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0059] Figure 4 The polarization of underlying microglia by supramolecular nanoparticles in an in vitro blood-brain barrier model;

[0060] Figure 5 for the distribution of supramolecular nanoparticles in the brain of tumor-bearing mice;

[0061] Figure 6 The effects of supramolecular nanoparticles on the body weight and survival rate of tumor-bearing mice. DETAILED DESCRIPTION

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

[0063] Example 1: Synthesis of 2-dithiopyridineethanol

[0064]

[0065] Under ice bath conditions, disulfide dipyridine (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, and the solvent was removed by rotary evaporation. The mixture was purified by silica gel column chromatography (0% to 20% ethyl acetate in petroleum ether) and concentrated to dryness to obtain the title compound (2.01 g, 64%) as a yellow oily liquid. 1 H NMR (400 MHz, CDCl 3 )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) acetate

[0067]

[0068] Under ice bath conditions, 2-dithiopyridineethanol (781 mg, 4.18 mmol) was dissolved in 15 mL of dichloromethane, and then 4-nitrophenyl chloroformate (1.68 g, 8.35 mmol) dissolved in 10 mL of dichloromethane was added dropwise, and then diisopropylethylamine (1.46 mL, 8.34 mmol) was added. The mixture was stirred in an ice bath for 2 h, and the solvent was removed by rotary evaporation. The mixture was purified by silica gel column chromatography (3% to 10% ethyl acetate in petroleum ether) and concentrated to dryness 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 Resiquimod-(2-dithiopyridine)aminoacetate

[0070]

[0071] Resiquimod (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, followed by the addition of diisopropylethylamine (1.40 mL, 8.00 mmol), refluxed at 35 degrees with stirring for 16 h, and the solvent was removed by rotary evaporation, and then purified by silica gel column chromatography (0% to 5% methanol in dichloromethane), and concentrated to dryness to obtain the title compound (605 mg, 61%) as a light 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: Resiquimod-C 2 Synthesis of K-GPLGLAG-TFFYGGSRGKRNNFKTEEY(Ⅰ-a)

[0073]

[0074] Resiquimod-(2-dithiopyridine) aminoacetate (13 mg, 0.025 mmol) and C 2 K-GPLGLAG-TFFYGGSRGKRNNFKTEEY (20 mg, 0.006 mmol) was dissolved in 2 mL of anhydrous dimethyl sulfoxide, replaced with nitrogen and protected with nitrogen, stirred overnight at room temperature, dialyzed in pure water with MWCO 2000 for 24 h, and freeze-dried to obtain 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) formate

[0076]

[0077] Camptothecin (104 mg, 0.298 mmol) and 4-nitrophenyl chloroformate (157 mg, 0.772 mmol) were dissolved in dichloromethane under ice bath conditions, followed by the addition of dimethylaminopyridine (206 mg, 1.69 mmol). The mixture was stirred in an ice bath for 3 h, and the solvent was removed by rotary evaporation. The mixture was purified by silica gel column chromatography (0% to 75% ethyl acetate in dichloromethane) and concentrated to dryness to give the title compound (109 mg, 71%) as a light yellow solid. 1 H NMR (400 MHz, CDCl 3 )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(d q, 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)carboxylate

[0079]

[0080] 4-Nitrophenyl-(camptothecin) formate (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-dithiopyridineethanol (545 mg, 2.92 mmol), refluxed at 45 degrees with stirring for 12 h, the solvent was removed by rotary evaporation, and then purified by silica gel column chromatography (0% to 100% ethyl acetate in dichloromethane), and concentrated to dryness to give the title compound (324 mg, 60%) as a light yellow solid. 1 HNMR (400MHz, CDCl 3)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: Camptothecin-C 2 Synthesis of K-GPLGLAG-TFFYGGSRGKRNNFKTEEY(Ⅱ-a)

[0082]

[0083] Camptothecin-(2-dithiopyridine)carboxylate (13.5 mg, 0.024 mmol) and C 2 K-GPLGLAG-TFFYGGSRGKRNNFKTEEY (20 mg, 0.006 mmol) was dissolved in 2 mL of anhydrous dimethyl sulfoxide, replaced with nitrogen and protected with nitrogen, stirred overnight at room temperature, dialyzed in pure water with MWCO 2000 for 24 h, and freeze-dried to obtain 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 Resiquimod-C 2 K-GPLGLAG-TFFYGGSRGKRNNFKTEEY (7.5 mg) and II-a camptothecin-C 2K-GPLGLAG-TFFYGGSRGKRNNFKTEEY (1.5 mg) was dissolved in 1 mL of dimethyl sulfoxide; then 100 μL of the solution was added dropwise to 5 mL of phosphate buffer and stirred at 150 rpm for 2 minutes; then, an ultrafiltration tube with a molecular weight cutoff of 10 kDa was used for centrifugation at 8000×g for 10 minutes, and the upper layer of supramolecular nanoparticles was collected with 1 mL of phosphate buffer to obtain the target sample.

[0086] The particle size distribution of supramolecular nanoparticles was determined by dynamic light scattering. Figure 1 The results showed that the prepared supramolecular nanoparticles had a particle size of about 95 nm and a polydispersity coefficient of 0.145, with a smaller particle size and a narrower 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 in 6-well Transwell chambers at 150,000 cells / well and cultured at 37 degrees and 5% carbon dioxide for 5 days; mouse glioma cells GL261 cells were seeded in 6-well plates at 150,000 cells / well and cultured at 37 degrees and 5% carbon dioxide for 24 hours. Then the Transwell chamber seeded with bEnd.3 cells was placed in a 6-well plate seeded with GL261 cells, and free drugs (resiquimod + camptothecin) or supramolecular nanoparticles were added to the Transwell chamber. bEnd.3 cells and GL261 cells were collected 4 and 12 hours after drug addition, washed with phosphate-buffered saline, and the fluorescence intensity of camptothecin in the cells was analyzed by flow cytometry.

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

[0090] Example 10: Cytotoxicity of supramolecular nanoparticles

[0091] Cells were inoculated and cultured as described in Example 9, and then phosphate buffered saline, free drugs (resiquimod + camptothecin) or supramolecular nanoparticles were added to the Transwell chamber. GL261 cells were collected 36 hours after drug addition and stained with Annexin-FITC / PT according to the operating instructions. After completion, cell apoptosis was analyzed using flow cytometry.

[0092] Figure 3It is the apoptosis of GL261 cells, indicating that the killing ability of supramolecular nanoparticles on GL261 cells is much stronger than that of free drugs.

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

[0094] Mouse brain microvascular / capillary endothelial cells bEnd.3 cells were inoculated into 6-well Transwell chambers at 150,000 cells / well and cultured at 37 degrees and 5% carbon dioxide for 5 days; mouse primary microglia (which can be regarded as macrophages) were inoculated into 6-well plates at 150,000 cells / well, and 10 ng / mL of IL-10 and 20 ng / mL of IL-4 were added, and cultured at 37 degrees and 5% carbon dioxide for 24 hours to polarize them into anti-inflammatory microglia. Then the Transwell chamber inoculated with bEnd.3 cells was placed in a 6-well plate inoculated with microglia, and phosphate buffered saline, free drugs (resiquimod + camptothecin) or supramolecular nanoparticles were added to the Transwell chamber. Microglia were collected 24 hours after drug addition and stained with CD86 (a pro-inflammatory macrophage marker) and CD206 (an anti-inflammatory macrophage marker) antibodies. After completion, microglia polarization was analyzed using flow cytometry.

[0095] Figure 4 is the polarization of microglia, indicating 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 Ten days after inoculation, free fluorescent dye DiD or DiD-labeled supramolecular nanoparticles (DiD 1.5 mg / kg) were administered through the tail vein. 48 hours later, the mice were killed, and the brain tissues were collected. The distribution of fluorescent dye signals in the brain tissues was observed and quantified using a small animal in vivo imager.

[0098] Figure 5 It is the quantification and ratio of fluorescence intensity in normal brain tissue area and brain tumor area. The overall fluorescence signal of the supramolecular nanoparticle group is stronger, and the fluorescence intensity ratio between the brain tumor area and the normal area is significantly higher than that of the free dye group.

[0099] Example 13: Evaluation of the anti-tumor ability of supramolecular nanoparticles

[0100] GL261 cells (3×10 6After 10 days, normal saline, free drugs (resiquimod + camptothecin) or supramolecular nanoparticles (resiquimod 0.9 μmol / kg, camptothecin 4.4 μmol / kg) were administered via the tail vein, and the normal saline group was given an equal amount of normal saline. The body weight and survival of the mice were recorded.

[0101] Figure 6 The weight changes and survival rates of mice in each group were analyzed. The supramolecular nanoparticle group reversed the weight loss caused by brain tumor, and the survival time of mice was significantly prolonged.

[0102] It should be noted that the technical means disclosed in the present 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 on the basis of the present invention, and these modifications are obvious to those skilled in the art, therefore, these modifications all belong to the protection scope of the present invention.

Claims

1. A brain-targeted peptide-drug conjugate self-assembled supramolecular nanoparticle with stimulus responsiveness, characterized in that: It is formed by the self-assembly of a polypeptide conjugate of a resiquimod analog and a polypeptide conjugate of a camptothecin analog.

2. According to claim 1, the polypeptide conjugate and pharmaceutically acceptable salt of the resiquimod analogue have the following general structural formula: in 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; 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; 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: IEFD, IEPD; apoptosis enzyme sensitive sequence: DEVD; Pep2 is a brain-targeting peptide selected from low-density lipoprotein receptor-related ligands: TFFYGGSRGKRNNFKTEEY, LRKLRKRLLLRKLRKRLL, SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS, TEELRVRLASHLRKLRKRLLRDR, cyl[CMPRLRGC], cyl[CM'Thz'RLRG'Pen']; transferrin receptor-related ligands: HAIYPRH, THRPPMWSPVWP, GYR; integrin-related ligand: RGD.

3. A method for preparing the compound according to claim 2, characterized in that: The steps include: Step 1, dissolving dithiodipyridine in methanol at 0-4°C, adding mercaptoethanol dropwise thereto to obtain a mixed solution, continuing to stir for 2-6 hours, and purifying by silica gel column chromatography to obtain a compound of formula AA; Step 2, at 0-4° C., dissolve the compound of formula AA and 4-nitrophenyl chloroformate in dichloromethane, add a base for catalysis, continue stirring for 2-6 hours, and purify by silica gel column chromatography to obtain a compound of formula AB. Step 3, at 30-55° C., dissolve the compound of formula AB and the compound of formula AC in dichloromethane, add a base thereto for catalysis, continue stirring for 12-24 hours, and purify by silica gel column chromatography to obtain a compound of formula AD. Step 4, dissolving the compound of formula AD and the sensitive peptide-brain targeting peptide conjugate in dimethyl sulfoxide at 15-35° C., continuing stirring for 12-24 hours, dialyzing and freeze-drying to obtain the compound of formula I.

4. The method for preparing the compound according to claim 3, characterized in that: In the compound R 1 , R 2 As defined in claim 2; Its characteristics also include that the sensitive peptide-brain targeting peptide conjugate is selected from different combinations of Pep1 and Pep2 defined in claim 2; The invention is also characterized in that the base is selected from one or a combination of the following: dimethylaminopyridine, triethylamine, diisopropylethylamine, pyridine, hydroxybenzotriazole, and carbodiimide.

5. The polypeptide conjugate of camptothecin analog and the pharmaceutically acceptable salt thereof according to claim 1, wherein the general structural formula is as follows: in R 1 is selected from hydrogen and halogen; R 2 Selected from hydrogen, hydroxyl, halogen, C 1-6 -alkyl, alkoxy-C 1-6 -alkyl, alkoxy-tert-butyl, alkoxy-C 1-3 -alkyl-ester-tert-butyl, alkoxy-C 1-3 -alkyl-ester-C 1-3 -alkyl, alkoxy-C 1-12 -alkyl-hydroxy, alkoxy-C 1-12 -Alkylcarboxyl, alkoxy-C 1-3 -alkyl-carbonylpiperidine, acyloxy-C 1-12 -alkyl, acyloxy-C 1-12 -alkylcarboxyl, acyloxy-amino-C 1-3 -alkylcarboxyl, acyloxy-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 phosphoate; R 3 is selected from hydrogen, hydroxy, amino, nitro, aldehyde, carbonylamino, carbonylamino-C 1-6 -alkyl, C 2-6 -Alkenyl, C 1-6 -alkyl-dimethylamino; R 4 Selected from hydrogen, hydroxyl, halogen, aldehyde, carboxyl, ethynyl, 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, 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; R 5 , R 6 is selected from hydrogen, methylene hydroxyl, methylene tetrahydropyrrole, R 5 and R 6 It cannot be methylene tetrahydropyrrole at the same time. Pep1 is a sensitive peptide as defined in claim 2; Pep2 is a brain targeting peptide as defined in claim 2.

6. A method for preparing the compound according to claim 5, characterized in that: The steps include: Step 1, dissolve dithiodipyridine in methanol at 0-4°C, add mercaptoethanol dropwise thereto to obtain a mixed solution, continue stirring for 2-6 hours, and purify by silica gel column chromatography to obtain a compound of formula AA. Step 2, at 0-4° C., dissolve the compound of formula BA and 4-nitrophenyl chloroformate in dichloromethane, add a base for catalysis, continue stirring for 2-6 hours, and purify by silica gel column chromatography to obtain a compound of formula BB. Step 3, at 35-60° C., dissolve the compound of formula AA and the compound of formula BB in dichloromethane, add a base thereto for catalysis, continue stirring for 12-24 hours, and purify by silica gel column chromatography to obtain a compound of formula BC. Step 4, dissolving the compound of formula BC and the sensitive peptide-brain targeting peptide conjugate in dimethyl sulfoxide at 15-35° C., continuing stirring for 12-24 hours, dialyzing and freeze-drying to obtain the compound of formula II.

7. The method for preparing the compound according to claim 6, characterized in that: In the compound of formula VII, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 As defined in claim 5; Its characteristics also include that the sensitive peptide-brain targeting peptide conjugate is selected from different combinations of Pep1 and Pep2 defined in claim 2; its characteristics also include that the base is defined in claim 4.

8. The supramolecular nanoparticle according to claim 1, characterized in that: The preparation method also includes the generation of self-assembled supramolecular nanoparticles, including the following steps: Step 1, dissolving the compound defined in claim 2 and claim 5 in solvent I at a ratio of 5:1-1:

1. Step 2: slowly add the solution obtained in step 1 to solvent II at a ratio of 1:20-1:100 and stir for 1-5 minutes. Step 3: centrifuge the system obtained in step 2 at 6000-15000 rpm using an ultrafiltration tube Step 4, resuspending and collecting the upper nanoparticles with solvent II, which are the supramolecular nanoparticles self-assembled by brain-targeted peptide-drug conjugates with stimulus responsiveness.

9. The method for preparing self-assembled supramolecular nanoparticles according to claim 8, characterized in that: The solvent I in step 1 is selected from one or more of dimethyl sulfoxide, N,N-dimethylformamide, dioxane, acetonitrile, acetone, dimethyl carbonate, and tetrahydrofuran; the solvent II in steps 2 and 4 is selected from ultrapure water, physiological saline, phosphate buffer, and Hanks balanced salt buffer.

10. Use of the self-assembled supramolecular nanoparticles according to claim 1 or the self-assembled supramolecular nanoparticles prepared by the preparation method according to any one of claims 2 to 9 in inhibiting the growth, metastasis and recurrence of brain tumors. Characterized in that: The inhibition of brain tumor growth, metastasis and recurrence includes directly killing tumor cells; polarizing anti-inflammatory macrophages into pro-inflammatory macrophages to improve the tumor immune microenvironment; and promoting the infiltration of lymphocytes in the tumor microenvironment. It is also characterized in that the brain tumors include glioblastoma, oligodendroglioma, astrocytoma, oligoastrocytoma, ependymoma, glial neuron tumor, choroid plexus tumor, medulloblastoma, meningioma, pituitary tumor, and schwannoma.

Images