Drug delivery system loaded with radionuclide labeled natural polyphenol compound and preparation method and application thereof

Abstract

The invention belongs to the technical field of tumor diagnosis and treatment, and particularly relates to a drug delivery system loaded with a radionuclide-labeled natural polyphenol compound as well as a preparation method and application of the drug delivery system. The drug delivery system provided by the invention comprises a responsive liposome, a radionuclide-labeled natural polyphenol compound encapsulated in the responsive liposome, an unlabeled natural polyphenol compound and artificial metalloenzyme. The drug delivery system of the natural polyphenol compound loaded with the radionuclide label provided by the invention realizes specific delivery of radionuclide (131I), reduces non-specific distribution of the radionuclide, and prolongs the residence time of tumors, so that the growth of the tumors can be effectively inhibited; the drug delivery system of the natural polyphenol compound loaded with the radionuclide label can also be used for PET imaging, and tumor targeting, tumor treatment effect and drug in-vivo tissue distribution can be effectively monitored.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor diagnosis and treatment, and specifically relates to a drug delivery system of a natural polyphenol compound labeled with a radionuclide, and a preparation method and application thereof. Background Art

[0002] Radiotherapy is the mainstay of treatment for solid tumors. Clinically, approximately 50-60% of cancer patients receive radiotherapy alone or in combination with other treatment modalities, such as surgery, chemotherapy, targeted therapy, and immunotherapy. Generally, radiotherapy is divided into two categories: external beam radiotherapy and internal radioisotope therapy. Growing evidence suggests that radiotherapy can cause irreparable DNA damage, directly killing tumor cells.

[0003] However, for external beam radiotherapy, only a small portion of the radiation energy is absorbed by the tumor, so high-intensity radiation beams are usually required to effectively kill the tumor, which will cause serious damage to adjacent normal tissues in the radiation beam path (such as X-rays). Several clinical trials have found that when a single dose exceeds 15Gy, a large number of patients begin to experience grade 3 toxicity, including bleeding and intestinal perforation. In contrast, internal radioisotope therapy is the introduction of radioisotopes into the tumor site. The transported radioisotopes emit energy from the cell nucleus, generating ionized atoms and ROS, causing DNA single-strand breaks, thereby inducing cell apoptosis and effectively killing cancer cells.

[0004] Studies have shown that clinical 131 With each injection of 60 mCi of I-iodized oil, the cumulative radiation dose absorbed by the tumor was 248 ± 176 Gy, and the threshold of absorbed dose for effective tumor response was 280 Gy. 131 I-iodized oil has poor tumor retention, resulting in a short effective half-life in tumors (4.3 to 5.5 days). 131 Excessive accumulation of I-iodized oil in the lungs can cause adverse reactions such as interstitial pneumonia. 131 Systemic distribution of I-iodized oil requires patients to take non-radioactive iodine before treatment to block the thyroid gland from 131 I-Iodized oil intake. 131 I-iodized oil has low tumor specificity, and patients receive a radioactive dose of up to 2.22 GBq (60 mCi) to reach the effective absorbed dose threshold.

[0005] In summary, current internal radioisotope therapy or PET imaging has the disadvantages of poor radionuclide specificity and short tumor retention time. Summary of the Invention

[0006] The purpose of the present invention is to provide a drug delivery system of a natural polyphenol compound loaded with radionuclide labeling, a preparation method thereof and an application thereof. The drug delivery system of a natural polyphenol compound loaded with radionuclide labeling provided by the present invention realizes the radionuclide ( 131 I) specific delivery, reducing the nonspecific distribution of radionuclides, and prolonging tumor retention time, thereby effectively inhibiting tumor growth; the drug delivery system of the natural polyphenol compound labeled with radionuclides provided by the present invention can also be used for PET imaging to effectively monitor tumor targeting, tumor treatment effects and drug tissue distribution in vivo.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a drug delivery system for a natural polyphenol compound loaded with radionuclide labeling, comprising a responsive liposome, a natural polyphenol compound labeled with radionuclide, an unlabeled natural polyphenol compound and an artificial metalloenzyme encapsulated in the responsive liposome, wherein the natural polyphenol compound labeled with radionuclide comprises 131 I-labeled natural polyphenol compounds and / or 124 I-labeled natural polyphenolic compounds.

[0009] Preferably, the responsive liposomes include one or more of pH responsive liposomes, cathepsin B enzyme responsive liposomes and matrix metalloproteinase-9 enzyme responsive liposomes; the pH responsive liposomes are composed of dioleoylphosphatidylethanolamine, cis-9-octadecenoic acid and distearoylphosphatidylethanolamine-polyethylene glycol 2000 in a molar ratio of (5-9): (1-5): 1; the cathepsin B enzyme responsive liposomes are composed of 1-palmitoyl-2-oleoylphosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol 2000. Phosphatidylethanolamine-polyethylene glycol 2000 and cathepsin B cleavage peptide are composed of a molar ratio of (5-9):(1-5):(0.5-1):(0.5-1); the matrix metalloproteinase-9 enzyme-responsive liposomes are composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine, matrix metalloproteinase-9 cleavage peptide, distearoylphosphatidylethanolamine-polyethylene glycol 2000 and cholesterol in a molar ratio of (5-9):(1-5):(0.5-1):(0.1-0.5).

[0010] Preferably, the natural polyphenol compound in the radionuclide-labeled natural polyphenol compound is epigallocatechin gallate;

[0011] The unlabeled natural polyphenol compound is epigallocatechin gallate.

[0012] Preferably, the artificial metalloenzyme has a core-shell structure, the core is a copper cluster, and the shell material includes proteins and / or peptides, the proteins include one or more of naturally derived bovine serum albumin, naturally derived human serum albumin, naturally derived mouse serum albumin, naturally derived transferrin and other water-soluble proteins; the peptides include naturally derived peptides and / or artificially synthesized peptides.

[0013] Preferably, the particle size of the artificial metalloenzyme is 1 to 10 nm, and the core of the artificial metalloenzyme contains 1 to 100 metal atoms.

[0014] Preferably, the radioactivity of the drug delivery system loaded with radionuclide-labeled natural polyphenol compounds is 150 to 750 μCi.

[0015] The present invention provides a method for preparing a drug delivery system of a natural polyphenol compound labeled with a radionuclide as described in the above technical solution, comprising the following steps:

[0016] dissolving a radionuclide-labeled natural polyphenol compound, an unlabeled natural polyphenol compound, and an artificial metalloenzyme in a buffer solution to obtain a drug solution;

[0017] mixing the drug solution and the responsive liposome solution to obtain a liposome mixed solution;

[0018] The liposome mixed solution is sequentially subjected to membrane filtration, dialysis purification and concentration to obtain the drug delivery system loaded with the natural polyphenol compound labeled with radioactive nuclides.

[0019] Preferably, the radioactive nuclide-labeled natural polyphenol compound is prepared by using a radioactive nuclide salt solution and a natural polyphenol compound as raw materials and adopting an Iodogen direct labeling method;

[0020] The liposome solution is prepared by a thin film hydration method.

[0021] The present invention provides the use of the drug delivery system of the natural polyphenol compound labeled with a radionuclide as described in the above technical solution or the drug delivery system of the natural polyphenol compound labeled with a radionuclide prepared by the preparation method described in the above technical solution in the preparation of tumor diagnosis and treatment reagents, wherein the tumor diagnosis and treatment reagents include anti-tumor drugs and / or tumor positron emission tomography imaging reagents.

[0022] Preferably, the anti-tumor drug is a drug for treating solid tumors.

[0023] The present invention provides a drug delivery system for a natural polyphenol compound loaded with radionuclide labeling, comprising a responsive liposome, a natural polyphenol compound labeled with radionuclide, an unlabeled natural polyphenol compound and an artificial metalloenzyme encapsulated in the responsive liposome, wherein the natural polyphenol compound labeled with radionuclide comprises 131 I-labeled natural polyphenol compounds and / or 124 I-labeled natural polyphenol compounds. The present invention uses responsive liposome encapsulation 131 I-labeled natural polyphenol compounds, unlabeled natural polyphenol compounds and artificial metalloenzymes are used as drug formulations for inhibiting tumor proliferation; responsive liposome encapsulation 124 I-labeled natural polyphenol compounds, unlabeled natural polyphenol compounds and artificial metalloenzymes are pharmaceutical dosage forms for tumor positron emission tomography (PET imaging). Specifically, natural polyphenol compounds are labeled with radioactive nuclides, and unlabeled natural polyphenol compounds are used as bioactive substrates of artificial metalloenzymes. The drug delivery system provided by the present invention is stable under physiological conditions, but demulsifies in the tumor microenvironment to release artificial metalloenzymes, unlabeled natural polyphenol compounds and radioactive nuclides-labeled natural polyphenol compounds. Specific delivery of radionuclides is achieved, and nonspecific distribution of radionuclides is reduced. Among them, the loaded 131 After the responsive liposomes containing the labeled natural polyphenolic compound, the unlabeled natural polyphenolic compound and the artificial metalloenzyme arrive at the tumor site, the artificial metalloenzyme accelerates the oxidative polymerization of the unlabeled polyphenolic compound in the environment around the tumor tissue and within the tumor cells to form a hydrogel self-assembly. 131 I is stably present in tumor tissue and prolongs tumor retention time. 131 The continuous β-radiation provided by I causes direct DNA damage in tumor cells and can effectively inhibit tumor growth. 124 Liposomes containing I-labeled natural polyphenolic compounds, unlabeled natural polyphenolic compounds, and artificial metalloenzymes can be used for PET imaging to monitor tumor targeting, tumor therapeutic efficacy, and drug tissue distribution in vivo. In summary, the drug delivery system provided by the present invention, which carries radionuclide-labeled natural polyphenolic compounds, can specifically deliver radioisotopes to tumor sites, increase their bioavailability, and minimize their toxicity to normal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The radioactive thin layer chromatogram of EGCG labeled with radionuclide is shown in Figure 2. Figure 1 a and b are radioactive thin-layer chromatograms of the product before purification; Figure 1 Figures c and d are radioactive thin-layer chromatograms of the purified products;

[0025] Figure 2 This is a morphological characterization diagram of the artificial metalloenzyme. Figure 2 a in the figure is the morphology of BSA-CuNCs observed by transmission electron microscopy (TEM). Figure 2 b in the figure shows the morphology of GSH-CuNCs observed by transmission electron microscopy (TEM). Figure 2 c in the figure shows the morphology of TGA-CuNCs observed by transmission electron microscopy (TEM);

[0026] Figure 3 This is the morphological analysis diagram of responsive liposome demulsification. Figure 3 The a in 127 The morphology of I-EGCG-BSA-CuNCs-Lp, Figure 3 b in 127 The morphology of I-EGCG-GSH-CuNCs-Lp, Figure 3 The c in 127 Morphology of I-EGCG-TGA-CuNCs-Lp;

[0027] Figure 4 This is an optical image of the oxidative polymerization of EGCG catalyzed by artificial metalloenzymes under conditions simulating tumor microenvironment in vitro;

[0028] Figure 5 Loading for tail vein administration 131 The inhibitory effects of I-EGCG and EGCG-artificial metalloenzyme responsive liposomes on orthotopic breast cancer xenografts in mice;

[0029] Figure 6 For load 124 PET imaging of mouse tumors responsive to I-EGCG, EGCG, and artificial metalloenzyme liposomes. DETAILED DESCRIPTION

[0030] The present invention provides a drug delivery system for a natural polyphenol compound loaded with radionuclide labeling, comprising a responsive liposome, a natural polyphenol compound labeled with radionuclide, an unlabeled natural polyphenol compound and an artificial metalloenzyme encapsulated in the responsive liposome, wherein the natural polyphenol compound labeled with radionuclide comprises 131 I-labeled natural polyphenol compounds and / or 124 I-labeled natural polyphenolic compounds.

[0031] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0032] The drug delivery system for a radionuclide-labeled natural polyphenol compound provided by the present invention includes responsive liposomes. In the present invention, the responsive liposomes preferably include one or more of pH-responsive liposomes, cathepsin B (CTSB) enzyme-responsive liposomes, and matrix metalloproteinase-9 (MMP-9) enzyme-responsive liposomes.

[0033] In the present invention, the pH-responsive liposomes are preferably composed of dioleoylphosphatidylethanolamine (DOPE), cis-9-octadecenoic acid (OA), and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) in a molar ratio of (5-9):(1-5):1, more preferably (6-8):(2-4):1, and even more preferably 7:3:1. In the embodiment, the ratio can be 5:5:1.

[0034] In the present invention, the CTSB enzyme-responsive liposomes are preferably composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), cholesterol (Ch), DSPE-PEG2000, and CTSB cleavage peptide in a molar ratio of (5-9):(1-5):(0.5-1):(0.5-1), more preferably (6-8):(2-4):1:1, further preferably (6-7):(2-3):1:1, and in embodiments, may be 6.36:2.45:1:1. The CTSB cleavage peptide is preferably SMAC-P-FRRG. The amino acid sequence of SMAC-P-FRRG is shown in SEQ ID NO. 1, which is: Ala-Val-Pro-Iso-Ala-Glu-Phe-Arg-Arg-Gly.

[0035] In the present invention, the MMP-9 enzyme-responsive liposomes are preferably composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), MMP-9 cleavage peptide, DSPE-PEG2000 and Ch in a molar ratio of (5-9):(1-5):(0.5-1):(0.1-0.5), more preferably (6-8):(2-4):(0.5-0.8):(0.2-0.4), further preferably (6-8):(3-4):(0.5-0.6):(0.3-0.4), and in the embodiment, it can be 7:3.5:0.58:0.35. The MMP-9 cleavage peptide is preferably PLGLWA-SA. The PLGLWA-SA is preferably Pro-Leu-Gly-Leu-Trp-Ala-CH3(CH2) 16 The PLGLWA-SA preferably consists of the amino acid sequence shown in SEQ ID NO.2 and -CH3(CH2) linked to the Ala of the amino acid sequence shown in SEQ ID NO.2.16 The SEQ ID NO. 2 is Pro-Leu-Gly-Leu-Trp-Ala.

[0036] The drug delivery system of the radionuclide-labeled natural polyphenol compound provided by the present invention comprises the radionuclide-labeled natural polyphenol compound, the unlabeled natural polyphenol compound and the artificial metalloenzyme encapsulated in the responsive liposome. In the present invention, the radionuclide-labeled natural polyphenol compound comprises 131 I-labeled natural polyphenol compounds and / or 124 I-labeled natural polyphenolic compounds.

[0037] In the present invention, the natural polyphenol compound in the radionuclide-labeled natural polyphenol compound is preferably epigallocatechin gallate (EGCG).

[0038] In the present invention, the unlabeled natural polyphenol compound is preferably EGCG.

[0039] In the present invention, the artificial metalloenzyme preferably has a core-shell structure, the core is preferably a copper cluster, and the material of the shell preferably includes proteins and / or peptides. The protein preferably includes one or more of bovine serum albumin (BSA) of natural origin, human serum albumin (HSA) of natural origin, mouse serum albumin (MSA) of natural origin, transferrin (TRF) of natural origin and other water-soluble proteins. The peptides preferably include peptides of natural origin and / or artificially synthesized peptides, more preferably glutathione (GSH) and / or thioglycolic acid (TGA). The material of the shell more preferably includes one or more of BSA, glutathione (GSH) and thioglycolic acid (TGA).

[0040] In the present invention, the artificial metalloenzyme more preferably includes one or more of BSA-coated copper clusters (BSA-CuNCs), GSH-coated copper clusters (GSH-CuNCs) and TGA-coated copper clusters (TGA-CuNCs).

[0041] In the present invention, the particle size of the artificial metalloenzyme is preferably 1 to 10 nm. The core of the artificial metalloenzyme preferably contains 1 to 100 metal atoms.

[0042] The drug delivery system of the natural polyphenol compound loaded with radionuclide labeling provided by the present invention includes a drug dosage form for inhibiting tumor growth and a drug dosage form for tumor PET imaging.

[0043] In the present invention, the pharmaceutical dosage form for inhibiting tumor growth preferably includes encapsulated 131pH-responsive liposomes of I-EGCG, EGCG and BSA-CuNCs ( 131 I-EGCG-BSA-CuNCs-Lp), encapsulation 131 CTSB enzyme-responsive liposomes of I-EGCG, EGCG and GSH-CuNCs ( 131 I-EGCG-GSH-CuNCs-Lp) or encapsulation 131 MMP-9 enzyme-responsive liposomes of I-EGCG, EGCG and TGA-CuNCs ( 131 I-EGCG-TGA-CuNCs-Lp).

[0044] In the present invention, the pharmaceutical dosage form for tumor PET imaging preferably includes encapsulated 124 pH-responsive liposomes of I-EGCG, EGCG and BSA-CuNCs ( 124 I-EGCG-BSA-CuNCs-Lp), encapsulation 124 CTSB enzyme-responsive liposomes of I-EGCG, EGCG and GSH-CuNCs ( 124 I-EGCG-GSH-CuNCs-Lp) or encapsulation 124 MMP-9 enzyme-responsive liposomes of I-EGCG, EGCG and TGA-CuNCs ( 124 I-EGCG-TGA-CuNCs-Lp).

[0045] The radioactivity of the drug delivery system loaded with radionuclide-labeled natural polyphenol compounds provided by the present invention is preferably 150 to 750 μCi.

[0046] The present invention provides a method for preparing a drug delivery system of a natural polyphenol compound labeled with a radionuclide as described in the above technical solution, comprising the following steps:

[0047] dissolving a radionuclide-labeled natural polyphenol compound, an unlabeled natural polyphenol compound, and an artificial metalloenzyme in a buffer solution to obtain a drug solution;

[0048] mixing the drug solution and the responsive liposome solution to obtain a liposome mixed solution;

[0049] The liposome mixed solution is sequentially subjected to membrane filtration, dialysis purification and concentration to obtain the drug delivery system loaded with the natural polyphenol compound labeled with radioactive nuclides.

[0050] The invention dissolves a radionuclide-labeled natural polyphenol compound, an unlabeled natural polyphenol compound and an artificial metal enzyme in a buffer solution to obtain a drug solution.

[0051] In the present application, the radionuclide-labeled natural polyphenol compound includes 131 I-labeled natural polyphenol compound 131 I-EGCG) and / or 124 I-labeled natural polyphenol compound 124 I-EGCG). The radionuclide-labeled natural polyphenol compound is preferably prepared using Iodogen direct labeling method with a radionuclide salt solution and a natural polyphenol compound as raw materials.

[0052] The preparation method of the radionuclide-labeled natural polyphenol compound is preferably Iodogen (1,3,4,6-tetrachloro-3α,6α-diphenylglycouril) direct labeling method. The Iodogen direct labeling method preferably uses Iodogen as an oxidizing agent to radionuclide-label the natural polyphenol compound by direct electrophilic substitution.

[0053] In the present application, the preparation method of the radionuclide-labeled natural polyphenol compound preferably includes the following steps:

[0054] The Iodogen is dissolved in an organic solvent to obtain an Iodogen solution, and the Iodogen solution is placed in a reaction container to obtain a reaction container with Iodogen after removing the solvent. The organic solvent for dissolving the Iodogen is preferably dichloromethane. The reaction container is preferably an Ep tube. The method for removing the solvent is preferably cold air blowing. The mass ratio of the Iodogen to the volume of the organic solvent is preferably 1-5 mg: 1 mL.

[0055] The natural polyphenol compound solution and the radionuclide salt solution are stirred and mixed to obtain a mixed solution. In the present application, the natural polyphenol compound solution preferably includes a natural polyphenol compound and PBS. The mass ratio of the natural polyphenol compound in the natural polyphenol compound solution to the volume of the PBS is preferably 2 mg: (600-800) μL. The radionuclide salt solution is preferably an aqueous radionuclide salt solution. The radionuclide salt preferably includes Na 131 I and / or Na 124 I. The radionuclide salt solution is preferably an aqueous Na 131 I solution or an aqueous Na 124 I solution. The radioactivity of the radionuclide salt solution is 1-5 mCi. In the present application, the ratio of the radioactivity of the radionuclide salt solution to the mass of the natural polyphenol compound in the natural polyphenol compound solution is preferably (1-5) mCi: (2-10) mg. The volume ratio of the natural polyphenol compound solution to the radionuclide salt solution is preferably (3-4): (1-2).

[0056] In the present invention, the mass ratio of the natural polyphenol compound in the natural polyphenol compound solution to the lodogen in the lodogen solution is preferably 1:1.

[0057] After obtaining the mixed solution, the present invention places the mixed solution in a reaction vessel containing an iodogen and reacts in the dark. After the reaction is completed, a Na2S2O3 solution in PBS is added to terminate the reaction, thereby obtaining a crude solution of a radionuclide-labeled natural polyphenol compound. In the present invention, the reaction temperature is preferably 35-37°C. Gently mix the mixture at regular intervals during the reaction. The reaction time is preferably 12-24 hours. After the reaction is completed, the present invention preferably transfers the resulting reaction solution to a new vessel and then adds a Na2S2O3 solution in PBS to terminate the reaction.

[0058] After preparing a crude solution of a radionuclide-labeled natural polyphenol compound using the direct labeling method, the present invention preferably further comprises purifying the crude solution of the radionuclide-labeled natural polyphenol compound. The purification is preferably performed by column chromatography. The purification is preferably performed using a C18 column, preferably a Sep-Pak C18 Plus Short Cartridge, 360 mg Sorbentper Cartridge. The C18 column is preferably washed before use, and the solvents used for washing are preferably methanol and then ultrapure water, respectively. The crude solution of the radionuclide-labeled natural polyphenol compound is preferably diluted with ultrapure water before loading, and the elution preferably comprises rinsing with ultrapure water and then eluting with anhydrous ethanol. After obtaining a pure solution of the radionuclide-labeled natural polyphenol compound through purification, the present invention preferably removes the solvent to obtain a pure radionuclide-labeled natural polyphenol compound. The solvent removal is preferably performed by blowing away the solvent with nitrogen. The present invention preferably dissolves the pure radionuclide-labeled natural polyphenol compound in ultrapure water.

[0059] In the present invention, the preparation method of the artificial metalloenzyme preferably comprises the following steps:

[0060] The metal salt solution, the shell raw material solution and the sodium hydroxide solution are mixed to carry out a reduction reaction, and the obtained reduction reaction solution is dialyzed and concentrated to obtain an artificial metal enzyme. In the present application, the metal salt solution is preferably a copper sulfate solution. The molar concentration of the metal salt solution is preferably 1-200 mM, and more preferably 50-150 mM. The shell raw material solution is preferably a protein solution or a peptide solution; the concentration of the shell raw material solution is preferably 1-100 mg / mL, and more preferably 20-80 mg / L. The concentration of the sodium hydroxide solution is preferably 0.1-1 M, and more preferably 0.3-0.6 M; the solvents of the metal salt solution, the shell raw material solution and the sodium hydroxide solution are all preferably water; the volume ratio of the shell raw material solution to the metal salt solution is preferably (0.2-2):(0.1-1), and preferably 1:(0.3-1); the volume ratio of the shell raw material solution to the sodium hydroxide solution is preferably (0.2-2):(0.04-0.4), and more preferably 1:0.1-0.35. In the present application, the reduction reaction preferably further includes a reducing agent; the reducing agent preferably includes one or more of sodium borohydride, ascorbic acid and hydrazine, and more preferably sodium borohydride; the reducing agent is preferably used in the form of a reducing agent solution, and the molar concentration of the reducing agent solution is preferably 1-200 mM; in specific embodiments of the present application, the protein or peptide itself has a certain reducing property; if the reducing property of the protein or peptide is sufficient to reduce the metal compound to a metal atom, no reducing agent needs to be added; when the reducing property of the protein or peptide is insufficient to reduce the metal compound to a metal atom, a reducing agent is added to promote the reduction; in specific embodiments of the present application, when bovine serum albumin is used to reduce copper sulfate, no reducing agent is preferably added.

[0061] In the present application, the temperature of the reduction reaction is preferably 20-55℃, and more preferably 25-45℃; the time of the reduction reaction is preferably 1-12 h, and more preferably 2-10 h. In specific embodiments of the present application, the metal salt solution and the shell raw material solution are first mixed, stirred at room temperature for 2-5 min, then the sodium hydroxide solution is added, and a reducing agent solution is added or not added as needed, and then the obtained mixed solution is subjected to a reduction reaction at the above-mentioned temperature. After the reduction reaction is completed, the obtained reduction reaction solution is dialyzed and concentrated to obtain an artificial metal enzyme. In the present application, the molecular weight cut-off of the dialysis bag used for dialysis is preferably 3-10 kDa, and the time of dialysis is preferably 0.5-2 h; the dialysis is used to remove unreacted proteins or peptides and free metal ions; the concentration is preferably carried out by using an ultrafiltration tube; the molecular weight cut-off of the ultrafiltration tube is preferably 10-100 kDa. The obtained artificial metal enzyme after concentration is in a solution state, has a fluorescence characteristic, and has high stability in physiological saline, cell culture medium and fetal bovine serum (FBS)

[0062] The present invention preferably mixes the radionuclide-labeled natural polyphenol compound, the unlabeled natural polyphenol compound, and the artificial metalloenzyme to obtain a mixture; and then dissolves the mixture in a buffer solution. In the present invention, the mixing preferably includes: premixing the radionuclide-labeled natural polyphenol compound and the unlabeled natural polyphenol compound to obtain a premix; and mixing the premix with the artificial metalloenzyme. In the present invention, the mass ratio of the natural polyphenol compound to the unlabeled natural polyphenol compound in the radionuclide-labeled natural polyphenol compound is preferably 1:4. When the radionuclide-labeled natural polyphenol compound and the unlabeled natural polyphenol compound are mixed, the radioactivity of the radionuclide-labeled natural polyphenol compound is preferably 1 to 5 mCi.

[0063] In the present invention, the molar ratio of the unlabeled natural polyphenol compound to the metal atom in the artificial metalloenzyme is preferably (1-5): 1. The buffer solution is preferably PBS (pH 7.4).

[0064] After obtaining the drug solution, the present invention mixes the drug solution with a responsive liposome solution to obtain a liposome mixed solution. In the present invention, the liposome solution is preferably prepared using a thin film hydration method. The thin film hydration method preferably comprises: dissolving the raw materials for preparing the liposomes in an organic solvent to obtain an organic reaction solution; evaporating the organic reaction solution to remove the solvent to obtain a liposome film; and hydrating the liposome film with water to obtain the responsive liposome liposome solution. The liposomes are preferably prepared by determining the type and molar ratio of the raw materials based on the composition of the responsive liposomes described above. The evaporation is preferably performed under reduced pressure. The mass ratio of the liposomes in the liposome solution to the natural polyphenol compounds in the drug solution is preferably (5-50):(1-10), with the mass of the liposomes being based on the total mass of the raw materials. The mixing is preferably performed under ultrasonic conditions, and the ultrasonication time is preferably 1-2 hours. During the ultrasonication process, the liposomes self-assemble and encapsulate the radionuclide-labeled natural polyphenol compound, the unlabeled natural polyphenol compound, and the artificial metalloenzyme.

[0065] After obtaining the liposome mixed solution, the liposome mixed solution is sequentially subjected to membrane filtration, dialysis purification and concentration to obtain the drug delivery system of the radionuclide-labeled natural polyphenol compound. In the present application, the pore size of the filter membrane used for membrane filtration is preferably 0.22 μm; the large liposome vesicles are repeatedly extruded by membrane filtration to filter out particles with a particularly large particle size, and the large particles can be extruded to reduce the particle size, thereby obtaining the drug delivery system of the radionuclide-labeled natural polyphenol compound with a particle size of 100-200 nm. In the present application, the molecular weight cut-off (MWCO) of the dialysis bag used for dialysis purification is preferably 1000 kDa, and the dialysis time is preferably 0.5-2 h; the dialysis purification is used to remove unreacted substances. The concentration is preferably concentrated by using an ultrafiltration tube; the molecular weight cut-off (MWCO) of the ultrafiltration tube is preferably 100 kDa; the rotation speed of the ultrafiltration tube is preferably 5000 r / min, the single concentration time is preferably 10 min, and the concentration times are preferably 3-5 times; the impurities are removed by centrifugation of the ultrafiltration tube to obtain the drug delivery system of the radionuclide-labeled natural polyphenol compound.

[0066] In the present application, the concentrated solution of the drug delivery system of the radionuclide-labeled natural polyphenol compound obtained after concentration preferably further comprises purifying the concentrated solution of the drug delivery system of the radionuclide-labeled natural polyphenol compound, and the purification is preferably performed by using a Sep-Pak C18 column. The purification preferably removes free radionuclides and other impurities.

[0067] The present application provides the use of the drug delivery system of the radionuclide-labeled natural polyphenol compound or the drug delivery system of the radionuclide-labeled natural polyphenol compound prepared by the preparation method in the preparation of a tumor diagnosis and treatment reagent, and the tumor diagnosis and treatment reagent comprises an anti-tumor drug and / or a tumor positron emission computed tomography reagent.

[0068] In the present application, the anti-tumor drug is a drug for treating solid tumors, and in the examples, it can be a breast cancer tumor.

[0069] The drug delivery system of the radionuclide-labeled natural polyphenol compound provided by the present application is particularly a drug dosage form for inhibiting tumor proliferation, and the drug dosage form comprises a responsive liposome encapsulating 131 I-labeled natural polyphenol compound, a natural polyphenol compound and an artificial metalloenzyme. 124I-labeled natural polyphenol compounds, natural polyphenol compounds and artificial metalloenzymes are pharmaceutical dosage forms for tumor PET imaging. The present invention uses radioactive nuclides to label natural polyphenol compounds, uses natural polyphenol compounds as biologically active substrates of artificial metalloenzymes, and uses metal clusters as artificial metalloenzymes. The resulting drug delivery system is stable under physiological conditions, but demulsifies in the tumor microenvironment to release artificial metalloenzymes, natural polyphenol compounds and radioactive nuclides-labeled natural polyphenol compounds. Specific delivery of radionuclides is achieved, and nonspecific distribution of radionuclides is reduced. 131 After the responsive liposomes of natural polyphenolic compounds labeled with I and natural polyphenolic compounds and artificial metalloenzymes reach the tumor site, the artificial metalloenzymes accelerate the oxidative polymerization of polyphenolic compounds in the environment around the tumor tissue and within the tumor cells to form a hydrogel self-assembly. 131 I is stably present in tumor tissue and prolongs tumor retention time. 131 The continuous β-radiation provided by I causes direct DNA damage in tumor cells and can effectively inhibit tumor growth. 124 I-labeled natural polyphenolic compounds, natural polyphenolic compounds and artificial metalloenzyme-responsive liposomes can be used for PET imaging to monitor tumor targeting, tumor treatment efficacy and drug tissue distribution in vivo.

[0070] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0071] In the following embodiments, the load 131 Test method for inhibiting tumor growth by responsive liposome drug delivery system of I-EGCG, EGCG and artificial metalloenzyme:

[0072] (1) Establish an orthotopic breast cancer xenograft model in mice. Select female Balb / c nude mice aged 4-6 weeks, weighing approximately 16-18 g. Take 5×10 MDA-MB-231 cells in the logarithmic growth phase. 6 Each was suspended in 100 μL PBS and injected subcutaneously into the fourth mammary fat pad on the right side of female Balb / c nude mice.

[0073] (2) 5 to 7 days after injection, when the tumor volume reaches about 50 to 100 mm 3 The mice were randomly divided into 3 groups, 5 in each group. The 3 groups of mice were treated with drugs, namely: 1. saline group, 2. load group 131 3. Loading of I-EGCG into Responsive Liposomes 131 I-EGCG, EGCG and artificial metalloenzyme responsive liposome group. Among them, 131The radioactivity of I-EGCG was 50 μCi, and the EGCG concentration was 5–50 mg kg -1 The concentration of artificial metalloenzyme (measured by copper atomic concentration) is 0.5-10 mg / kg -1 The administration method was tail vein injection (200 μL / mouse), the administration frequency was once every 7 days, and the treatment cycle was 21 days. During the treatment period, the mice were weighed every other day, and the tumor diameter was measured and recorded. Tumor volume calculation formula: V = 1 / 2 (L × W 2 ), where L and W represent the long and short diameters of the tumor, respectively.

[0074] In the following embodiments, the load 124 Testing methods for PET imaging of mouse tumors using drug delivery systems of I-EGCG, EGCG, and artificial metalloenzymes:

[0075] (1) Establish a mouse breast cancer xenograft model. Select female Balb / c nude mice aged 4 to 6 weeks, weighing approximately 16 to 18 g. Take 5 × 10 MDA-MB-231 cells in the logarithmic growth phase. 6 Each was suspended in 100 μL PBS and injected subcutaneously into the fourth mammary fat pad on the right side of female Balb / c nude mice.

[0076] (2) On the 10th day after injection, when the tumor volume reached about 100 mm 3 When injected via tail vein 124 I-EGCG, EGCG, and artificial metalloenzyme-responsive liposomes (150 μCi, 150 μL). 6 h after injection, tumor-bearing mice were imaged using a small animal PET scanner. The tumor-bearing mice were anesthetized with 2% isoflurane, and the signal intensity at the tumor site was measured using PET imaging software.

[0077] All solutions in the following examples, unless otherwise specified, are aqueous solutions prepared from ultrapure water.

[0078] Example 1:

[0079] (one) 131 I-labeled EGCG:

[0080] (1) Iodogen direct labeling method: Iodogen is used as an oxidant to radiolabel EGCG through direct electrophilic substitution.

[0081] a. Dissolve 2 mg of lodogen in 1 mL of dichloromethane in a 4 mL Eppendorf tube. Blow dry the liquid in the tube with cold air to obtain a reaction tube for later use.

[0082] b. Take another tube and add 600 μL EGCG solution (2 mg EGCG dissolved in 600 μL PBS solution), add 400 μL Na 131 I(Na 131 Add the radioactive substance I (with a radioactivity of 1 mCi) in water and mix thoroughly. After mixing, transfer the liquid to a reaction tube.

[0083] c. Incubate the reaction at 37°C in the dark overnight, gently mixing every once in a while. After overnight, transfer the liquid in the tube to a new Eppendorf tube and add 100 μL of 32 mg mL -1 The reaction was terminated by adding Na2S2O3 solution in PBS.

[0084] (2) 131 Purification of I-EGCG:

[0085] a. Activate the C18 column in advance. First, rinse the column with 10 mL of methanol and empty the syringe to remove the residual liquid in the column. Then rinse the column with 10 mL of ultrapure water and empty the syringe to remove the residual liquid in the column.

[0086] b. Mix 1 mL of the above reaction solution with 4 mL of ultrapure water and pass it through a C18 column. Then rinse the C18 with 10 mL of ultrapure water. Then, elute the product remaining in the C18 column with 1 mL of anhydrous ethanol and collect it in an Eppendorf tube.

[0087] c. The product dispersed in ethanol was dried with nitrogen to remove the solvent, leaving 50 μL of product, to which 500 μL of ultrapure water was added for use in the next step.

[0088] (two) 124 I-labeled EGCG:

[0089] (1) Iodogen direct labeling method: Iodogen is used as an oxidant to radiolabel EGCG through direct electrophilic substitution.

[0090] a. Dissolve 2 mg of lodogen in 1 mL of dichloromethane in a 4 mL Eppendorf tube. Blow dry the liquid in the tube with cold air to obtain a reaction tube for later use.

[0091] b. Take another tube and add 600 μL EGCG solution (2 mg EGCG dissolved in 600 μL PBS solution), add 400 μL Na 124 I solution (radioactivity is 1 mCi), mix thoroughly while adding. After mixing, transfer the above liquid to the reaction tube.

[0092] c. Incubate the reaction at 37°C in the dark overnight, gently mixing every once in a while. After overnight, transfer the liquid in the tube to a new Eppendorf tube and add 100 μL of 32 mg mL-1 The reaction was terminated by adding Na2S2O3 solution in PBS.

[0093] (2) 124 Purification of I-EGCG:

[0094] a. Activate the C18 column in advance. First, rinse the column with 10 mL of methanol and empty the syringe to remove the residual liquid in the column. Then rinse the column with 10 mL of ultrapure water and empty the syringe to remove the residual liquid in the column.

[0095] b. Mix 1 mL of the above reaction solution with 4 mL of ultrapure water and pass it through a C18 column. Then rinse the C18 with 10 mL of ultrapure water. Then, elute the product remaining in the C18 column with 1 mL of anhydrous ethanol and collect it in an Eppendorf tube.

[0096] c. The product dispersed in ethanol was dried with nitrogen to remove the solvent, leaving 50 μL of product, to which 500 μL of ultrapure water was added for use in the next step.

[0097] (III) Preparation of BSA-coated copper clusters (BSA-CuNCs):

[0098] (1) Mix 1 mL of 40 mg / mL BSA solution with 0.4 mL of 20 mM CuSO₄ aqueous solution and stir at room temperature for 5 min. Slowly add 0.1 mL of 0.5 M NaOH solution to the mixed solution and continue stirring the resulting solution at 55°C for 8 h.

[0099] (2) The copper clusters synthesized above were dialyzed using a dialysis bag with a molecular weight cutoff of 100 kDa for 0.5 h to remove unreacted BSA and free metal ions.

[0100] (3) The copper clusters were concentrated using an ultrafiltration tube with a molecular weight cut-off of 30 kDa.

[0101] (4) Load 124 I-EGCG / 131 Preparation of pH-responsive liposomes of I-EGCG, EGCG and BSA-CuNCs:

[0102] (1) 7 μmol DOPE, 7 μmol OA, and 1.4 μmol DSPE-PEG2000 were dissolved and mixed in 1 mL of chloroform. The solution was then transferred to a 20 mL round flask and the solvent was evaporated under reduced pressure using a rotary evaporator at 52°C to obtain a lipid film.

[0103] (2) The obtained liposome membrane was hydrated in a round flask for 2 h.

[0104] (3) The purified 124I-EGCG or 131 I-EGCG was mixed with 8mg EGCG (where 124 I-EGCG or 131 2 mg of EGCG was used in the preparation of I-EGCG, which was then mixed with 8 mg of free EGCG, resulting in a total of 10 mg of EGCG. 124 I-EGCG or 131 I-EGCG (mixed with EGCG raw material, with a radioactivity of 1-5 mCi) was added to 800 μL of ultrapure water, and then added to the flask from step (2) along with 1 mL of a 10 mM BSA-CuNCs solution. The liposome mixture was then sonicated for 60 min to obtain a liposome mixture.

[0105] (4) The liposome mixture solution was then extruded and filtered through a membrane with a pore size of 0.22 μm.

[0106] (5) The liposome mixed solution obtained after membrane filtration in step (4) was dialyzed for 30 min using a dialysis bag with a molecular weight cutoff (MWCO) of 1000 kDa to remove unreacted substances. The liposome mixed solution was then centrifuged 5 times at 5000 r / min, 10 min / time using an ultrafiltration tube with a MWCO of 100 kDa to concentrate and remove impurities. The synthesized pH-responsive liposomes are represented as 124 I-EGCG-BSA-CuNCs-Lp / 131 I-EGCG-BSA-CuNCs-Lp (radioactivity is 150 μCi).

[0107] This example uses thin layer chromatography to detect 124 I-EGCG-BSA-CuNCs-Lp / 131 I-EGCG-BSA-CuNCs-Lp 124 I-EGCG, 131 The labeling rate of I-EGCG was determined by using a mixed solution of chloroform and acetonitrile (volume ratio of 1:1) as a developing solvent. The obtained product solution was spotted on iTLC chromatography paper for thin layer chromatography, and a radioactive thin layer scanner was used for detection and analysis. Figure 1 The results show that the product solution contains the product 124 In addition to I-EGCG, it also contains some free 124 I - Calculation shows that Na 124 The labeling rate of I-labeled EGCG was 76.5%. The product solution was then purified using a Sep-Pak C18 column to remove free 124 I -Then, the purified product solution was spotted on iTLC chromatography paper for thin layer chromatography and analyzed using a radioactive thin layer scanner. Figure 1 As shown in b. The results showed that after purification, the free 124 I - was successfully removed, and the product solution 124 The radiochemical purity of I-EGGC is almost 100%, which meets the requirements for application in liposome synthesis. 131 The labeling rate of I-EGCG, such as Figure 1 As shown in c, before purification 131 The labeling rate of I-EGCG was 83.8%, which was close to 100% after purification. Figure 1 As shown in d.

[0108] Next, in this example, transmission electron microscopy (TEM) was used to determine the morphology and particle size of BSA-coated copper clusters (BSA-CuNCs). Figure 2 As shown in a, TEM shows that the synthesized BSA-CuNCs have good dispersion and uniform particle size. 127 I instead of Na 124 I / Na 131 I prepared 127 I-EGCG-BSA-CuNCs-Lp, pH-responsive liposomes confirmed by TEM 127 Structural changes of I-EGCG-BSA-CuNCs-Lp. Figure 3 As shown in a, TEM shows that in pH 6.5 buffer, the liposomes broke and released BSA-CuNCs, EGCG and 127 I-EGCG. Under the action of H2O2, BSA-CuNCs catalyzes the oxidative polymerization of EGCG to form a hydrogel self-assembly. Figure 4 As shown in Figure a, 8 μM BSA-CuNCs, 100 μM H2O2, and 1 mg / mL EGCG were dissolved in a pH 6.5 buffer solution and incubated at 37°C for 24 h. A hydrogel was formed at the bottom of the test tube.

[0109] Figure 2 a in the figure is the morphological characterization diagram of the artificial metalloenzyme (BSA-CuNCs) prepared in Example 1. Figure 3 This is the morphological analysis diagram of responsive liposome demulsification. Figure 3 The a in 127 I-EGCG-BSA-CuNCs-Lp was dispersed in pH 6.5 buffer and incubated at 37°C for 24 h. The morphology was observed using cryo-transmission electron microscopy. Figure 3 b in 127I-EGCG-GSH-CuNCs-Lp was dispersed in pH 6.5 buffer and incubated at 37°C for 24 h. The morphology was observed using cryo-transmission electron microscopy. Figure 3 The c in 127 I-EGCG-TGA-CuNCs-Lp was dispersed in pH 6.5 buffer and incubated at 37°C for 24 h. The morphology was observed using cryo-transmission electron microscopy.

[0110] Figure 4 This is an optical image of the oxidative polymerization of EGCG catalyzed by artificial metalloenzymes under conditions simulating tumor microenvironment in vitro. Figure 4 a in the figure represents the formation of hydrogel self-assembly by oxidative polymerization of EGCG catalyzed by artificial metalloenzyme in a buffer solution of pH 6.5 in the presence of 100 μM H2O2 to simulate the tumor microenvironment. The reaction conditions are: 8 μM BSA-CuNCs, 100 μM H2O2 and 1 mg / mL EGCG. The reaction mixture was incubated at 37°C for 24 h. Figure 4 b in the figure shows that in a buffer solution of pH 6.5, in the presence of 100 μM H2O2 to simulate the tumor microenvironment, the artificial metalloenzyme catalyzed the oxidative polymerization of EGCG to form a hydrogel self-assembly. The reaction conditions were: 8 μM GSH-CuNCs, 100 μM H2O2 and 1 mg / mL EGCG. The reaction mixture was incubated at 37°C for 24 h. Figure 4 c in the figure represents the formation of hydrogel self-assembly by oxidative polymerization of EGCG catalyzed by artificial metalloenzyme in a buffer solution of pH 6.5 in the presence of 100 μM H2O2 to simulate the tumor microenvironment. The reaction conditions are: 8 μM TGA-CuNCs, 100 μM H2O2 and 1 mg / mL EGCG. The reaction mixture was incubated at 37°C for 24 h.

[0111] Next, 131 I-EGCG-BSA-CuNCs-Lp was used to evaluate the inhibition of tumor growth in vivo. An orthotopic breast cancer xenograft model was established in mice. When the tumor volume reached about 50 mm 3 The mice were randomly divided into 3 groups, 5 in each group. Every 7 days, saline was injected into the tail vein. 131 I-EGCG-Lp, 131 I-EGCG-BSA-CuNCs-Lp (EGCG concentration of 11 mg / kg, Cu concentration of 0.75 mg / kg) was treated for 21 consecutive days. During the treatment period, the mice were weighed and the tumor diameter was measured every other day. 131I-EGCG-BSA-CuNCs-Lp breaks the emulsion in the acidic microenvironment of the tumor, and the released BSA-CuNCs catalyzes the oxidative polymerization of EGCG to form a hydrogel self-assembly under the action of H2O2 in the tumor microenvironment. 131 I is stably present in tumor tissue and prolongs tumor retention time. 131 The β-radiation continuously provided by I causes direct DNA damage in tumor cells and has a significant inhibitory effect on the proliferation of in situ tumor cells.

[0112] like Figure 5 As shown in a, after 21 days of treatment, 131 The average tumor volume of the I-EGCG-BSA-CuNCs-Lp treatment group was 110.86 ± 5.86 mm 3 , which was significantly smaller than the other control groups. Compared with the control group injected with saline, 131 The tumor volume of the I-EGCG-BSA-CuNCs-Lp treatment group decreased by 89.4%.

[0113] Figure 5 Tail vein administration load 131 Inhibitory effects of I-EGCG, EGCG and artificial metalloenzyme-responsive liposomes on orthotopic breast cancer xenografts in mice, Figure 5 The a in it stands for saline, 131 I-EGCG-Lp, 131 I-EGCG-BSA-CuNCs-Lp; Figure 5 The b in it stands for saline. 131 I-EGCG-Lp, 131 I-EGCG-GSH-CuNCs-Lp; Figure 5 The c in it stands for saline. 131 I-EGCG-Lp, 131 I-EGCG-TGA-CuNCs-Lp (n=5; * P < 0.5; ** P < 0.1; *** P<0.01).

[0114] Finally 124 In vivo PET imaging of breast cancer subcutaneous tumor-bearing mice was performed using I-EGCG-BSA-CuNCs-Lp as a contrast agent to analyze the tumor targeting ability of liposomes. 124 Six hours after the injection of I-EGCG-BSA-CuNCs-Lp via the tail vein, PET imaging was performed using a small animal PET scanner, as shown in Figure 6 As shown in middle a, the PET signal intensity of the tumor site in the yellow circle is obvious. Figure 6 For load 124PET imaging of mouse tumors in responsive liposomes of I-EGCG, EGCG, and artificial metalloenzymes. 124 I-EGCG, EGCG and artificial metalloenzyme responsive liposomes, PET imaging was performed 6 hours later, Figure 6 The artificial metalloenzymes in 124 I-EGCG-BSA-CuNCs-Lp( Figure 6 a) in the above sentence; b) 124 I-EGCG-GSH-CuNCs-Lp( Figure 6 b) in the above; 124 I-EGCG-TGA-CuNCs-Lp( Figure 6 c) in the above example.

[0115] Example 2:

[0116] (one) 131 I. Labeling of EGCG: The same labeling method as in Example 1 was used.

[0117] (two) 124 I. Labeling of EGCG: The same labeling method as in Example 1 was used.

[0118] (III) Preparation of GSH-coated copper clusters:

[0119] (1) Mix 1 mL of 40 mg / mL GSH solution with 0.4 mL of 20 mM CuSO4 solution and stir at room temperature for 5 min. Slowly add 0.1 mL of 0.5 M NaOH solution to the mixed solution and continue stirring the resulting solution at 55°C for 8 h.

[0120] (2) The GSH-CuNCs synthesized above were dialyzed for 0.5 h using a dialysis bag with a molecular weight cutoff of 10 kDa to remove unreacted GSH and free copper ions.

[0121] (3) The GSH-CuNCs were concentrated using an ultrafiltration tube with a molecular weight cut-off of 30 kDa.

[0122] (4) Load 124 I-EGCG / 131 Preparation of CTSB enzyme-responsive liposomes of I-EGCG, EGCG, and GSH-CuNCs:

[0123] (1) 7 μmol POPC, 2.7 μmol Ch, 1.1 μmol DSPE-PEG2000, and 1.1 μmol SMAC-P-FRRG were dissolved and mixed in 1 mL of chloroform. The solution was then transferred to a 20 mL round flask and the solvent was evaporated under reduced pressure using a rotary evaporator at 52°C to obtain a lipid film.

[0124] (2) The obtained liposome film was hydrated in a round flask for 2 h.

[0125] (3) The purified 124 I-EGCG / 131 I-EGCG and 8mg EGCG (of which 124 I-EGCG or 131 2 mg of EGCG was used in the preparation of I-EGCG, which was then mixed with 8 mg of free EGCG, resulting in a total of 10 mg of EGCG. 124 I-EGCG or 131 I-EGCG (mixed with EGCG raw material at a radioactivity of 1-5 mCi) was added to 800 μL of ultrapure water, and then added to a flask along with 1 mL of a 10 mM GSH-CuNCs solution. The flask was then sonicated for 60 minutes to obtain a liposome solution.

[0126] (4) The liposomes were then extruded and filtered through a membrane with a pore size of 0.22 μm.

[0127] (5) The liposome solution was dialyzed for 30 min using a dialysis bag with a molecular weight cutoff (MWCO) of 1000 kDa to remove unreacted substances. The liposome solution was then centrifuged 3 to 5 times using an ultrafiltration tube with a MWCO of 100 kDa at 5000 r / min for 10 min / time to concentrate and remove impurities. The synthesized CTSB enzyme-responsive liposomes are represented as 124 I-EGCG-GSH-CuNCs-Lp or 131 I-EGCG-GSH-CuNCs-Lp (radioactivity is 150 μCi).

[0128] Subsequently, transmission electron microscopy (TEM) was used to determine the morphology and particle size of GSH-CuNCs. Figure 2 As shown in b, TEM shows that the synthesized GSH-CuNCs are well dispersed and have uniform particle size. 127 I instead of Na 124 I / Na 131 I prepared 127 I-EGCG-GSH-CuNCs-Lp, CTSB enzyme-responsive liposomes confirmed by TEM 127 Structural changes of I-EGCG-GSH-CuNCs-Lp. Figure 3 As shown in b, TEM shows that in the presence of CTSB enzyme, the liposomes were demulsified and GSH-CuNCs, EGCG and 127I-EGCG. Under the action of H2O2, GSH-CuNCs catalyzes the oxidative polymerization of EGCG to form a hydrogel self-assembly. Figure 4 As shown in (b), 8 μM GSH-CuNCs, 100 μM H2O2, and 1 mg / mL EGCG were dissolved in a buffer solution containing CTSB enzyme. After incubation at 37°C for 24 h, a hydrogel was formed at the bottom of the test tube.

[0129] Next, 131 I-EGCG-GSH-CuNCs-Lp was used to evaluate the inhibition of tumor growth in vivo. An orthotopic breast cancer xenograft model was established in mice. When the tumor volume reached about 50 mm 3 The mice were randomly divided into 3 groups, 5 in each group. Saline was injected into the tail vein every 7 days. 131 I-EGCG-Lp, 131 I-EGCG-GSH-CuNCs-Lp (EGCG concentration was 11 mg / kg, Cu concentration was 0.75 mg / kg) was treated for 21 consecutive days. During the treatment, the mice were weighed and the tumor diameter was measured every other day. In the tumor microenvironment, 131 I-EGCG-GSH-CuNCs-Lp breaks the emulsion under the catalysis of CTSB enzyme, and the released GSH-CuNCs catalyzes the oxidative polymerization of EGCG to form a hydrogel self-assembly under the action of H2O2 in the tumor microenvironment. 131 I is stably present in tumor tissue and prolongs tumor retention time. 131 The continuous β-radiation provided by I causes direct DNA damage in tumor cells and significantly inhibits the proliferation of in situ tumor cells. Figure 5 As shown in b, after 21 days of treatment, 131 The average tumor volume of the I-EGCG-GSH-CuNCs-Lp treatment group was 142.26±18.16 mm 3 , which was significantly smaller than the other control groups. Compared with the control group injected with saline, 131 The tumor volume of the I-EGCG-GSH-CuNCs-Lp treatment group decreased by 86%.

[0130] The last pair 124 I-EGCG-GSH-CuNCs-Lp was used for in vivo PET imaging in tumor-bearing mice to analyze the tumor targeting ability of liposomes. 124 Six hours after I-EGCG-GSH-CuNCs-Lp was injected into the tail vein, PET imaging was performed using a small animal PET scanner, as shown in Figure 6 As shown in b, the signal intensity of the tumor site in the yellow circle is obvious.

[0131] Example 3

[0132] (one) 131 I. Labeling of EGCG: The same labeling method as in Example 1 was used.

[0133] (two) 124 I. Labeling of EGCG: The same labeling method as in Example 1 was used.

[0134] (III) Preparation of TGA-coated copper clusters:

[0135] (1) Mix 1 mL of 40 mg / mL TGA solution with 0.4 mL of 20 mM CuSO4 solution and stir at room temperature for 5 min. Slowly add 0.1 mL of 0.5 M NaOH solution to the mixed solution and continue stirring the resulting solution at 55°C for 8 h.

[0136] (2) The TGA-CuNCs synthesized above were dialyzed for 0.5 h using a dialysis bag with a molecular weight cutoff of 10 kDa to remove unreacted TGA and free copper ions.

[0137] (3) The TGA-CuNCs copper clusters were concentrated using an ultrafiltration tube with a molecular weight cutoff of 30 kDa.

[0138] (4) Load 124 I-EGCG / 131 Preparation of MMP-9 enzyme-responsive liposomes of I-EGCG, EGCG, and TGA-CuNCs:

[0139] (1) 7 μmol DOPC, 3.5 μmol PLGLWA-SA, 0.58 μmol DSPE-PEG2000, and 0.35 μmol Ch were dissolved and mixed in 1 mL of chloroform. The solution was then transferred to a 20 mL round flask and the solvent was evaporated under reduced pressure using a rotary evaporator at 52°C to obtain a lipid film.

[0140] (2) The obtained liposome membrane was hydrated for 2 h.

[0141] (3) The purified 124 I-EGCG / 131 I-EGCG was mixed with 8mg EGCG (where 124 I-EGCG or 131 2 mg of EGCG was used in the preparation of I-EGCG, which was then mixed with 8 mg of free EGCG, resulting in a total of 10 mg of EGCG. 124 I-EGCG or 131I-EGCG (mixed with EGCG raw material at a radioactivity of 1-5 mCi) was added to 800 μL of ultrapure water and then added to a flask along with 1 mL of a 10 mM TGA-CuNCs solution. The liposome solution was then sonicated for 60 min to obtain a liposome solution.

[0142] (4) The liposomes were then extruded and filtered through a membrane with a pore size of 0.22 μm.

[0143] (5) The liposome solution was dialyzed for 30 min using a dialysis bag with a molecular weight cutoff (MWCO) of 1000 kDa to remove unreacted substances. The liposome solution was then centrifuged 3 to 5 times using an ultrafiltration tube with a MWCO of 100 kDa at 5000 r / min for 10 min / time to concentrate and remove impurities. The synthesized MMP-9 enzyme-responsive liposomes are represented as 131 I-EGCG-TGA-CuNCs-Lp (radioactivity is 150 μCi).

[0144] Next, transmission electron microscopy (TEM) was used to determine the morphology and particle size of TGA-CuNCs. Figure 2 As shown in Figure c, TEM shows that the synthesized TGA-CuNCs have good dispersion and uniform particle size. 127 I instead of Na 124 I / Na 131 I-EGCG-TGA-CuNCs-Lp was prepared and the structural changes of MMP-9 enzyme-responsive liposomes I-EGCG-TGA-CuNCs-Lp were determined by TEM. Figure 3 As shown in Figure c, TEM shows that in the presence of MMP-9, the liposomes were demulsified, releasing TGA-CuNCs, EGCG and I-EGCG. Under the action of H2O2, TGA-CuNCs catalyzed the oxidative polymerization of EGCG to form a hydrogel self-assembly. Figure 4 As shown in Figure c, 8 μM TGA-CuNCs, 100 μM H2O2, and 1 mg / mL EGCG were dissolved in a buffer solution in the presence of MMP-9 enzyme. After incubation at 37°C for 24 h, a hydrogel was formed at the bottom of the test tube.

[0145] Next, 131 I-EGCG-TGA-CuNCs-Lp was evaluated for its ability to inhibit tumor growth in vivo. An orthotopic breast cancer xenograft model was established in mice. When the tumor volume reached approximately 50 mm 3 The mice were randomly divided into 3 groups, 5 in each group. Saline was injected into the tail vein every 7 days. 131 I-EGCG-Lp, 131I-EGCG-TGA-CuNCs-Lp (EGCG concentration of 11 mg / kg, Cu concentration of 0.75 mg / kg) was treated for 21 consecutive days. During the treatment period, the mice were weighed and the tumor diameter was measured every other day. 131 I-EGCG-TGA-CuNCs-Lp breaks the emulsion under the catalysis of MMP-9 in the tumor microenvironment, and the released TGA-CuNCs catalyzes the oxidative polymerization of EGCG to form a hydrogel self-assembly under the action of H2O2 in the tumor microenvironment. 131 I is stably present in tumor tissue and prolongs tumor retention time. 131 The continuous β-radiation provided by I causes direct DNA damage in tumor cells and significantly inhibits the proliferation of in situ tumor cells. Figure 5 As shown in c, after 21 days of treatment, 131 The average tumor volume of the I-EGCG-TGA-CuNCs-Lp treatment group was 153.86 ± 19.9 mm 3 , which was significantly smaller than the other control groups. Compared with the control group injected with saline, 131 The tumor volume of the I-EGCG-TGA-CuNCs-Lp treatment group decreased by 85.3%.

[0146] The last pair 124 I-EGCG-TGA-CuNCs-Lp was used for in vivo PET imaging in tumor-bearing mice to analyze the tumor targeting ability of liposomes. 124 Six hours after I-EGCG-TGA-CuNCs-Lp was injected into the tail vein, PET imaging was performed using a small animal PET scanner, as shown in Figure 6 As shown in c, the signal intensity of the tumor site in the yellow circle is obvious.

[0147] From the above examples, it can be seen that the present invention provides a drug delivery system for natural polyphenol compounds labeled with radioactive nuclides. 131 I-labeled natural polyphenol compounds, natural polyphenol compounds and artificial metalloenzymes are pharmaceutical formulations for inhibiting tumor proliferation; or responsive liposome encapsulation 124 I-labeled natural polyphenol compounds, natural polyphenol compounds and artificial metalloenzymes are pharmaceutical dosage forms for tumor positron emission tomography imaging. Specifically, natural polyphenol compounds are used as substrates for artificial metalloenzymes, and metal clusters are used as artificial metalloenzymes. The prepared drug delivery system of natural polyphenol compounds loaded with radioactive nuclides is stable under physiological conditions, but demulsifies in the tumor microenvironment to release artificial metalloenzymes, natural polyphenol compounds and natural polyphenol compounds labeled with radioactive nuclides. The present invention achieves specific delivery of radionuclides and reduces the nonspecific distribution of radionuclides in the body. 131After the responsive liposomes of natural polyphenolic compounds labeled with I and natural polyphenolic compounds and artificial metalloenzymes reach the tumor site, the artificial metalloenzymes accelerate the oxidative polymerization of polyphenolic compounds in the environment around the tumor tissue and within the tumor cells to form a hydrogel self-assembly. 131 I is stably present in tumor tissue and prolongs tumor retention time. 131 Continuous β-irradiation causes direct DNA damage in tumor cells and can effectively inhibit tumor growth. 124 I-labeled natural polyphenolic compounds, natural polyphenolic compounds, and artificial metalloenzyme-responsive liposomes can be used for PET imaging to monitor tumor targeting, tumor treatment efficacy, and drug tissue distribution in vivo.

[0148] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A drug delivery system loaded with a radionuclide-labeled natural polyphenol compound, characterized in that: The invention comprises a responsive liposome, a natural polyphenol compound labeled with radionuclide, an unlabeled natural polyphenol compound and an artificial metalloenzyme encapsulated in the responsive liposome, wherein the natural polyphenol compound labeled with radionuclide comprises 131 I-labeled natural polyphenol compounds and / or 124 I-labeled natural polyphenolic compounds.

2. The drug delivery system of the natural polyphenol compound loaded with radionuclide labeling according to claim 1, characterized in that: The responsive liposomes include one or more of pH-responsive liposomes, cathepsin B enzyme-responsive liposomes, and matrix metalloproteinase-9 enzyme-responsive liposomes; the pH-responsive liposomes are composed of dioleoylphosphatidylethanolamine, cis-9-octadecenoic acid, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 in a molar ratio of (5-9): (1-5): 1; the cathepsin B enzyme-responsive liposomes are composed of 1-palmitoyl-2-oleoylphosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine, and -Polyethylene glycol 2000 and cathepsin B cleavage peptide are composed of a molar ratio of (5-9):(1-5):(0.5-1):(0.5-1); the matrix metalloproteinase-9 (MMP-9) enzyme-responsive liposomes are composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine, matrix metalloproteinase-9 cleavage peptide, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and cholesterol in a molar ratio of (5-9):(1-5):(0.5-1):(0.1-0.5).

3. The drug delivery system of the natural polyphenol compound loaded with radionuclide labeling according to claim 1, characterized in that: The natural polyphenol compound in the radionuclide-labeled natural polyphenol compound is epigallocatechin gallate; The unlabeled natural polyphenol compound is epigallocatechin gallate.

4. The drug delivery system of the natural polyphenol compound loaded with radionuclide labeling according to claim 1, characterized in that: The artificial metalloenzyme has a core-shell structure, wherein the core is a copper cluster, and the shell material includes proteins and / or peptides. The proteins include one or more of naturally derived bovine serum albumin, naturally derived human serum albumin, naturally derived mouse serum albumin, naturally derived transferrin, and other water-soluble proteins; and the peptides include naturally derived peptides and / or artificially synthesized peptides.

5. The drug delivery system of the natural polyphenol compound loaded with radionuclide labeling according to claim 4, characterized in that: The particle size of the artificial metalloenzyme is 1 to 10 nm, and the core of the artificial metalloenzyme contains 1 to 100 metal atoms.

6. The drug delivery system of the natural polyphenol compound loaded with radionuclide labeling according to claim 1, characterized in that: The radioactivity of the drug delivery system loaded with the natural polyphenol compound labeled with radioactive nuclides is 150 to 750 μCi.

7. The method for preparing the drug delivery system of the natural polyphenol compound loaded with radionuclide labeling according to any one of claims 1 to 6, characterized in that: The following steps are involved: dissolving a radionuclide-labeled natural polyphenol compound, an unlabeled natural polyphenol compound, and an artificial metalloenzyme in a buffer solution to obtain a drug solution; mixing the drug solution and the responsive liposome solution to obtain a liposome mixed solution; The liposome mixed solution is sequentially subjected to membrane filtration, dialysis purification and concentration to obtain the drug delivery system loaded with the natural polyphenol compound labeled with radioactive nuclides.

8. The preparation method according to claim 7, characterized in that The radioactive nuclide-labeled natural polyphenol compound is prepared by using a radioactive nuclide salt solution and a natural polyphenol compound as raw materials and adopting an Iodogen direct labeling method; The liposome solution is prepared by a thin film hydration method.

9. Use of the drug delivery system of a natural polyphenol compound labeled with a radionuclide as described in any one of claims 1 to 6 or the drug delivery system of a natural polyphenol compound labeled with a radionuclide prepared by the preparation method according to any one of claims 7 or 8 in the preparation of tumor diagnostic and therapeutic reagents, wherein the tumor diagnostic and therapeutic reagents include anti-tumor drugs and / or tumor positron emission tomography imaging reagents.

10. The use according to claim 9, characterized in that The anti-tumor drug is a drug for treating solid tumors.

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