A drug delivery system loaded with artificial metalloenzyme and polyphenol compound, and a preparation method and application thereof

Abstract

The present application relates to the technical field of antitumor drugs, and provides a drug delivery system loaded with artificial metalloenzyme and polyphenol compound as well as a preparation method and application thereof.The present application takes metal clusters fixed in a protein cavity as artificial metalloenzyme, takes abundant edible polyphenol compound as a biological active substrate of the artificial metalloenzyme, and simultaneously adopts pH-sensitive liposomes to encapsulate the artificial metalloenzyme and the polyphenol compound.The pH-sensitive liposomes are stable at physiological pH, but are broken in an acidic tumor microenvironment to release the artificial metalloenzyme and the polyphenol compound; the artificial metalloenzyme accelerates oxidation and polymerization of the polyphenol compound in an environment around tumor tissues and in tumor cells to form a hydrogel self-assembly, and the self-assembly can effectively inhibit growth and distant metastasis of an in-situ tumor.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antitumor drugs, and particularly relates to a drug delivery system loaded with artificial metalloenzyme and polyphenol compound as well as a preparation method and application thereof. BACKGROUND

[0002] At present, the survival rate of patients with solid primary tumors has been significantly improved, and distant metastasis has become the main cause of death of tumor patients. The treatment methods for cancer in the clinic are still surgery, chemotherapy, radiotherapy or combined treatment of the above therapies. Among them, systemic chemotherapy including neoadjuvant chemotherapy can effectively control the condition of early primary tumor patients, but in some cases, the curative effect on metastatic tumor patients is poor, especially the treatment effect on advanced tumor patients is not satisfactory. In addition, more and more preclinical evidences show that one of the side effects of traditional chemotherapy is to promote the metastasis of primary cancer cells to the distal end, especially to other organs.

[0003] In view of the potential link between traditional chemotherapy and higher risk of tumor metastasis, it is very urgent to develop a new drug dosage form based on systemic treatment, which can eliminate primary tumor cells and inhibit cancer cells peeled from primary tumors from entering the vascular system to inhibit distant metastasis. SUMMARY

[0004] Therefore, the present application provides a drug delivery system loaded with artificial metalloenzyme and polyphenol compound as well as a preparation method and application thereof. The drug delivery system loaded with artificial metalloenzyme and polyphenol compound provided by the present application can effectively inhibit the growth of primary tumors and distant metastasis, and has a broad application prospect in the field of antitumor.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The drug delivery system loaded with artificial metalloenzyme and polyphenol compound comprises a pH-sensitive liposome, an artificial metalloenzyme and a polyphenol compound; the artificial metalloenzyme and the polyphenol compound are encapsulated in the pH-sensitive liposome.

[0007] The pH-sensitive liposome comprises dioleoyl phosphatidylethanolamine, 1,2-dipalmitoyl-SN-glycero-3-succinic acid and distearoyl phosphatidylethanolamine-polyethylene glycol.

[0008] The molar ratio of the dioleoyl phosphatidylethanolamine, 1,2-dipalmitoyl-SN-glycero-3-succinic acid and distearoyl phosphatidylethanolamine-polyethylene glycol is (5-9):(1-5):1.

[0009] Preferably, the artificial metalloenzyme is a core-shell structure, the core is a metal cluster, and the shell is a protein or a peptide; the particle size of the artificial metalloenzyme is 1-10 nm, and the number of metal atoms is 1-100.

[0010] Preferably, the metal element of the metal cluster includes one or more of copper, gold, and platinum.

[0011] The protein is a water-soluble protein.

[0012] Preferably, the water-soluble protein includes serum albumin or transferrin.

[0013] Preferably, the polyphenol compound includes one or more of epigallocatechin gallate, epicatechin gallate, and epigallocatechin.

[0014] The application also provides a preparation method of the drug delivery system loaded with the artificial metalloenzyme and the polyphenol compound, comprising the following steps:

[0015] Dissolve dioleoylphosphatidylethanolamine, 1,2-dipalmitoyl-SN-glycero-3-succinate, and distearoylphosphatidylethanolamine-polyethylene glycol in an organic solvent, and then evaporate and remove the organic solvent to obtain a pH-sensitive liposome film;

[0016] Mix the artificial metalloenzyme, the polyphenol compound, a PBS solution, and the pH-sensitive liposome film, and then ultrasonicate to obtain a liposome solution loaded with the artificial metalloenzyme and the polyphenol compound;

[0017] Filter, dialyze, and concentrate the liposome solution loaded with the artificial metalloenzyme and the polyphenol compound in sequence to obtain the drug delivery system loaded with the artificial metalloenzyme and the polyphenol compound.

[0018] Preferably, the preparation method of the artificial metalloenzyme comprises the following steps:

[0019] Mix a metal compound solution, a shell raw material solution, and a sodium hydroxide solution to perform a reduction reaction, dialyze the obtained reduction reaction solution, and concentrate to obtain the artificial metalloenzyme; the concentration of the metal compound solution is 1-200 mM; the shell raw material solution is a protein solution or a peptide solution; the concentration of the shell raw material solution is 1-100 mg / mL; and the concentration of the sodium hydroxide solution is 0.1-1 M.

[0020] Preferably, the reduction reaction further includes a reducing agent; and the reducing agent includes one or more of sodium borohydride, ascorbic acid, and hydrazine.

[0021] Preferably, the molar ratio of the artificial metalloenzyme and the polyphenol compound is (1-5):1; the mass ratio of the polyphenol compound and the liposome film is (1-10):(5-50).

[0022] The application also provides the use of the drug delivery system loaded with the artificial metalloenzyme and the polyphenol compound described in the above scheme or prepared by the preparation method described in the above scheme in the preparation of an anti-tumor drug.

[0023] The application provides a drug delivery system loaded with an artificial metalloenzyme and a polyphenol compound, comprising a pH-sensitive liposome, an artificial metalloenzyme and a polyphenol compound; the artificial metalloenzyme and the polyphenol compound are encapsulated in the pH-sensitive liposome; the pH-sensitive liposome comprises dioleoylphosphatidylethanolamine, 1,2-dipalmitoyl-SN-glycero-3-succinate and distearoylphosphatidylethanolamine-polyethylene glycol; the molar ratio of the dioleoylphosphatidylethanolamine, 1,2-dipalmitoyl-SN-glycero-3-succinate and distearoylphosphatidylethanolamine-polyethylene glycol is (5-9):(1-5):1. The application uses a metal cluster fixed in a protein cavity as an artificial metalloenzyme, uses an edible polyphenol compound with abundant sources as a biological active substrate of the artificial metalloenzyme, and encapsulates the artificial metalloenzyme and the polyphenol compound by using a pH-sensitive liposome. The pH-sensitive liposome is stable at a physiological pH, but is broken in an acidic tumor microenvironment to release the artificial metalloenzyme and the polyphenol compound. The artificial metalloenzyme accelerates the oxidation and polymerization of the polyphenol compound to form a hydrogel-like self-assembly in the environment around tumor tissues and in tumor cells, and the self-assembly can effectively inhibit the growth and distant metastasis of an in-situ tumor. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 TEM images of artificial metalloenzymes, wherein: a) CuCs; b) AuCs; c) PtCs;

[0025] Figure 2 Morphology analysis images of the breakage of pH-sensitive liposomes under acidic conditions, wherein: a) EGCG-CuCs-Lp; b) ECG-AuCs-Lp; c) EGC-PtCs-Lp;

[0026] Figure 3 Optical images of the oxidation and polymerization of polyphenol compounds catalyzed by artificial metalloenzymes under in-vitro simulated tumor microenvironment conditions, the composition of the substances in the last test tube in each group is: a) 8 μM CuCs, 100 μM H2O2 and 1 mg / mL EGCG; b) 8 μM AuCs, 100 μM H2O2 and 1 mg / mL ECG; c) 8 μM PtCs, 100 μM H2O2 and 1 mg / mL EGC;

[0027] Figure 4 Inhibition effect of pH-sensitive liposomes loaded with artificial metalloenzyme and polyphenol compound on orthotopic breast cancer xenograft tumor in mice given tail vein administration, wherein: a) EGCG-CuCs-Lp vs saline, EGCG-Lp, CuCs-Lp; b) ECG-AuCs-Lp vs saline, ECG-Lp, AuCs-Lp; c) EGC-PtCs-Lp vs saline, EGC-Lp, PtCs-Lp (n = 5; * P < 0.5; ** P < 0.1; *** P < 0.01).

[0028] Figure 5 Inhibition effect of pH-sensitive liposomes loaded with artificial metalloenzyme and polyphenol compound on tumor lung metastasis, wherein: a), b), c) are H&E staining images of lung metastatic nodules of tumor-bearing mice after tail vein injection treatment for 21 days, the area in the black circle is the metastatic tumor nodules (scale = 1000 μm). d), e), f) are quantitative analysis images of lung metastatic nodule area of mice after receiving various treatments. DETAILED DESCRIPTION

[0029] The present application provides a drug delivery system loaded with artificial metalloenzyme and polyphenol compound, comprising pH-sensitive liposomes, artificial metalloenzyme and polyphenol compound; the artificial metalloenzyme and the polyphenol compound are encapsulated in the pH-sensitive liposomes.

[0030] In the present application, the pH-sensitive liposomes comprise dioleoylphosphatidylethanolamine (DOPE), 1,2-dipalmitoyl-SN-glycero-3-succinate (DPSG) and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG); the molar ratio of the dioleoylphosphatidylethanolamine, 1,2-dipalmitoyl-SN-glycero-3-succinate and distearoylphosphatidylethanolamine-polyethylene glycol is (5-9):(1-5):1, preferably 5-6:4-5:1, more preferably 5:5:1.

[0031] In the present application, the artificial metal enzyme preferably has a core-shell structure, the core is preferably a metal cluster, and the shell is preferably a protein or a peptide; the particle size of the artificial metal enzyme is preferably 1-10 nm, more preferably 2-6 nm, and the number of metal atoms is preferably 1-100, more preferably 10-60; the metal element type of the metal cluster preferably includes one or more of copper, gold and platinum; the protein is preferably a water-soluble protein; the water-soluble protein preferably includes serum albumin or transferrin (TRF); the serum albumin is preferably one or more of bovine serum albumin (BSA), human serum albumin (HSA) and mouse serum albumin (MSA); and the present application has no special requirements for the peptide, which can be a naturally occurring peptide or an artificially synthesized peptide.

[0032] In the present application, the polyphenol compound can be a natural polyphenol compound or an artificially synthesized polyphenol compound, and is preferably a natural polyphenol compound; specifically, the polyphenol compound preferably includes one or more of epigallocatechin gallate (EGCG), epicatechin gallate (ECG) and epigallocatechin (EGC).

[0033] The present application also provides a preparation method of the drug delivery system loaded with the artificial metal enzyme and the polyphenol compound as described in the above scheme, which comprises the following steps:

[0034] Dissolve dioleoylphosphatidylethanolamine, 1,2-dipalmitoyl-SN-glycero-3-succinate and distearoylphosphatidylethanolamine-polyethylene glycol in an organic solvent, and then evaporate and remove the organic solvent to obtain a pH-sensitive liposome film;

[0035] Mix the artificial metal enzyme, the polyphenol compound, the PBS solution and the pH-sensitive liposome film, and then ultrasonicate to obtain a liposome solution loaded with the artificial metal enzyme and the polyphenol compound;

[0036] Filter, dialyze and concentrate the liposome solution loaded with the artificial metal enzyme and the polyphenol compound in sequence to obtain a drug delivery system loaded with the artificial metal enzyme and the polyphenol compound.

[0037] First, the preparation method of the artificial metal enzyme is described.

[0038] In the present application, the preparation method of the artificial metal enzyme preferably comprises the following steps:

[0039] Mix the metal compound solution, the shell material solution and the sodium hydroxide solution to perform a reduction reaction, dialyze the obtained reduction reaction solution, and then concentrate to obtain the artificial metal enzyme.

[0040] In the present application, the concentration of the metal compound solution is preferably 1-200 mM, more preferably 50-150 mM; the metal compound is preferably copper sulfate (CuSO4), chloroauric acid (HAuCl4) or chloroplatinic acid (H2PtCl6); the shell layer raw material solution is preferably a protein solution or a peptide solution; the concentration of the shell layer raw material solution is preferably 1-100 mg / mL, more preferably 20-80 mg / L; the concentration of the sodium hydroxide solution is preferably 0.1-1 M, more preferably 0.3-0.6 M; the solvent of the metal compound solution, the shell layer raw material solution and the sodium hydroxide solution is preferably water; the volume ratio of the shell layer raw material solution to the metal compound solution is preferably (0.2-2):(0.1-1), preferably 1:(0.3-1); the volume ratio of the shell layer raw material solution to the sodium hydroxide solution is preferably (0.2-2):(0.04-0.4), more preferably 1:0.1-0.35.

[0041] In the present application, the raw material of the reduction reaction preferably further comprises a reducing agent; the reducing agent preferably comprises one or more of sodium borohydride, ascorbic acid and hydrazine, more preferably sodium borohydride; the reducing agent is preferably used in the form of a reducing agent solution, and the 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, and if the reducing property of the protein or peptide is sufficient to reduce the metal compound into 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 into a metal atom, the addition of a reducing agent promotes the reduction; in specific embodiments of the present application, when bovine serum albumin is used to reduce copper sulfate or chloroauric acid, no reducing agent is preferably added; when bovine serum albumin is used to reduce chloroplatinic acid, a reducing agent is preferably added; the volume ratio of the shell layer raw material solution to the reducing agent solution is preferably 1:(0.3-0.5).

[0042] In the present application, the temperature of the reduction reaction is preferably 20-55°C, more preferably 25-45°C; the time of the reduction reaction is preferably 1-12 h, more preferably 2-10 h. In specific embodiments of the present application, the metal compound solution and the shell layer 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 according to the need, and then the obtained mixed solution is subjected to the reduction reaction at the above-mentioned temperature.

[0043] After the reduction reaction is completed, the present application concentrates the obtained reduction reaction liquid after dialysis 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 dialysis time is preferably 0.5-2 h; the present application removes unreacted proteins or peptides and free metal ions by dialysis; the concentration is preferably concentrated 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 and has a fluorescence characteristic, and has high stability in physiological saline, cell culture medium, and fetal bovine serum (FBS).

[0044] The preparation method of the drug delivery system loaded with artificial metal enzyme and polyphenol compound is described in detail as follows:

[0045] The present application dissolves dioleoyl phosphatidyl ethanolamine, 1,2-dipalmitoyl-SN-glycero-3-succinic acid and distearoyl phosphatidyl ethanolamine-polyethylene glycol in an organic solvent, and then evaporates and removes the organic solvent to obtain a pH-sensitive liposome film. In the present application, the organic solvent is preferably one or more of chloroform, diethyl ether, n-hexane and ethanol, and more preferably chloroform; the present application does not have special requirements for the amount of the organic solvent, and the above lipids can be dissolved. After the dissolution is completed, the present application preferably transfers the obtained lipid solution to a flask, evaporates the solvent under reduced pressure using a rotary evaporator, and forms a pH-sensitive liposome film on the inner wall of the flask.

[0046] After obtaining the pH-sensitive liposome film, the present application mixes the artificial metal enzyme, the polyphenol compound, the PBS solution and the pH-sensitive liposome film and ultrasonicates to obtain a liposome solution loaded with artificial metal enzyme and polyphenol compound. In the present application, the molar ratio of the artificial metal enzyme and the polyphenol compound is preferably (1-5):1, and more preferably (2-4):1; the mass ratio of the pH-sensitive liposome and the polyphenol compound is (5-50):(1-10), and the mass of the pH-sensitive liposome is based on the total mass of DOPE, DPSG and DSPE-PEG. The pH value of the PBS solution is preferably 7.4; the amount ratio of the polyphenol compound and the PBS solution is preferably 10 mg / 1.5 mL; in a specific embodiment of the present application, the polyphenol compound is dissolved in the PBS solution, and the obtained polyphenol compound solution and the artificial metal enzyme solution (prepared by the above method) are added to the flask for preparing the pH-sensitive liposome film; after the polyphenol compound solution and the artificial metal enzyme solution are added to the flask, the pH-sensitive liposome film on the inner wall of the flask is hydrated. In the present application, the ultrasonic time is preferably 1-2 h, and during the ultrasonic process, the pH-sensitive liposome self-assembles and coats the artificial metal enzyme and the polyphenol compound inside.

[0047] After obtaining the liposome solution loaded with artificial metalloenzyme and polyphenol compound, the liposome solution loaded with artificial metalloenzyme and polyphenol compound is sequentially filtered, dialyzed and concentrated to obtain the drug delivery system loaded with artificial metalloenzyme and polyphenol compound. In the present application, the pore size of the filter membrane used for the filtration is preferably 0.22 μm; the liposomes with a particle size of 100-200 nm are obtained by repeatedly extruding the large liposome vesicles through the filtration; in the present application, the molecular weight cut-off of the dialysis bag used for the dialysis is preferably 1000 kDa, and the time for the dialysis is preferably 0.5-2 h; the unreacted substances are removed through the dialysis; the concentration is preferably carried out by using an ultrafiltration tube; the molecular weight cut-off of the ultrafiltration tube is preferably 100 kDa; the rotation speed of the ultrafiltration tube is preferably 5000 r / min, the time for a single centrifugation is preferably 10 min, and the number of centrifugations is preferably 3-5 times; the impurities are removed through the centrifugation by using the ultrafiltration tube to concentrate, and thus the drug delivery system loaded with artificial metalloenzyme and polyphenol compound (also referred to as the pH-sensitive liposome loaded with artificial metalloenzyme and polyphenol compound) is obtained.

[0048] The present application also provides the use of the drug delivery system loaded with artificial metalloenzyme and polyphenol compound described in the above scheme or prepared by the preparation method described in the above scheme in the preparation of an anti-tumor drug; in the present application, the anti-tumor drug is preferably a drug for inhibiting the growth of tumors in situ and the distant metastasis; the tumor is preferably breast cancer.

[0049] In the specific embodiments of the present application, the effect of inhibiting the growth of tumors in situ and the distant metastasis by using the drug delivery system is preferably verified through in vivo animal experiments, and the specific experimental steps are as follows:

[0050] (1) A mouse orthotopic breast cancer xenograft tumor model is established. Female Balb / c nude mice aged 4-6 weeks and weighing about 16-18 g are selected. 5×10 6 The MDA-MB-231 cells in the logarithmic growth phase are suspended in 100 μL PBS and subcutaneously injected into the right fourth mammary fat pad of the female Balb / c nude mice.

[0051] (2) 5-7 days after the injection, when the tumor volume reaches about 50-100 mm 3The mice were randomly divided into 4 groups, 5 mice in each group. Every 3 days, physiological saline, pH-sensitive liposomes loaded with polyphenol compounds, pH-sensitive liposomes loaded with artificial metalloenzyme and pH-sensitive liposomes loaded with artificial metalloenzyme and polyphenol compounds were injected into the tail vein of the mice respectively. The injection concentration of the polyphenol compounds was 5-50 mg / kg, the injection concentration of the artificial metalloenzyme (measured according to the metal atom concentration) was 0.5-10 mg / kg, and the continuous treatment lasted for 21 days. During the treatment, the body weight of the mice was measured every other day, the tumor diameter was measured and recorded. The tumor volume calculation formula: V = 1 / 2 (L x W 2 ), wherein L and W represent the long diameter and short diameter of the tumor respectively.

[0052] (3) After 21 days of treatment by tail vein injection, the mice were euthanized, the lung tissues were taken out by dissection, and the lung metastatic nodules were observed by H&E staining of the sections.

[0053] The technical solutions in the present application will be clearly and completely described below in combination with the embodiments in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0054] Example 1

[0055] (I) Preparation of copper-containing artificial metalloenzyme:

[0056] (1) 1 mL of 40 mg / mL BSA solution was mixed with 0.4 mL of 20 mM CuSO4 solution, stirred at room temperature for 5 min, and 0.1 mL of 0.5 M NaOH solution was slowly added to the mixed solution. The formed solution was continuously stirred at 55℃ for 8 h.

[0057] (2) The above reaction solution was dialyzed in a dialysis bag with a molecular weight cut-off of 10 kDa for 0.5 h to remove unreacted BSA and free metal ions, and then concentrated by an ultrafiltration tube with a molecular weight cut-off of 30 kDa to obtain the copper-containing artificial metalloenzyme (denoted as CuCs).

[0058] (II) Preparation of pH-sensitive liposomes loaded with copper-containing artificial metalloenzyme and polyphenol compounds:

[0059] (1) DOPE, DPSG and DSPE-PEG were dissolved and mixed in 1 mL of chloroform at a molar ratio of 5:5:1 (wherein the amount of DOPE was 5 mg), and then the solution was transferred to a 20 mL round flask. The solvent was evaporated under reduced pressure using a rotary evaporator at 52℃ to obtain a pH-sensitive liposome film.

[0060] (2) 10 mg EGCG was dissolved in 1.5 mL PBS (pH = 7.4) and added to a flask with 1 mL of 10 mM CuCs solution, the obtained pH-sensitive liposome film was hydrated and then ultrasonicated for 60 min to obtain a liposome solution loaded with artificial metalloenzyme and polyphenol compound.

[0061] (3) The liposome solution loaded with artificial metalloenzyme and polyphenol compound was then extruded through a membrane with a pore size of 0.22 μm, dialyzed with a dialysis bag with a molecular weight cut-off of 1000 kDa, and then centrifuged at 5000 r / min for 3-5 times with an ultrafiltration tube with a molecular weight cut-off of 100 kDa, each time for 10 min, to concentrate and remove impurities, to obtain pH-sensitive liposomes loaded with copper-containing artificial metalloenzyme and EGCG, denoted as EGCG-CuCs-Lp.

[0062] Performance test:

[0063] 1. The copper-containing artificial metalloenzyme (CuCs) prepared in Example 1 was subjected to transmission electron microscopy test, and the results are shown in FIG. a of Figure 1 ; the TEM results show that the copper-containing artificial metalloenzyme synthesized in the present application has good dispersibility and uniform particle size.

[0064] 2. The structural changes of EGCG-CuCs-Lp under acidic conditions were determined by TEM: EGCG-CuCs-Lp was dispersed in a buffer solution with a pH of 6.5, and placed at 37°C for 24 h, and then observed by cryogenic transmission electron microscopy. The results are shown in FIG. a of Figure 2 ; the TEM results show that in the buffer solution with pH = 6.5, the EGCG-CuCs-Lp has been demulsified, releasing artificial metalloenzyme (CuCs) and polyphenol compound (EGCG).

[0065] 3. The artificial metalloenzyme catalyzed polyphenol compound oxidation was carried out in a buffer solution with pH = 6.5 to simulate the tumor microenvironment:

[0066] Four different test tubes were taken, and a buffer solution with a pH of 6.5 was added, and then EGCG (concentration of 1 mg / mL), EGCG + H2O2 (EGCG concentration of 1 mg / mL, H2O2 concentration of 100 μM), EGCG + CuCs (EGCG concentration of 1 mg / mL, CuCs concentration of 8 μM), and EGCG + CuCs + H2O2 (EGCG concentration of 1 mg / mL, CuCs concentration of 8 μM, H2O2 concentration of 100 μM) were added respectively; the above reaction mixtures were incubated at 37°C for 24 h, and the state of the reactants in the test tubes was observed, and the results are shown in FIG. a of Figure 3 ; according to Figure 3As can be seen in a, in the experimental group of adding EGCG+CuCs+H2O2, hydrogel was formed at the bottom of the test tube, while no hydrogel was observed in the control groups of EGCG, EGCG+H2O2 and EGCG+CuCs; the above experimental results show that the artificial metalloenzyme can catalyze the oxidative polymerization of polyphenol compounds under the action of tumor micro-acidic environment and H2O2.

[0067] 4. The performance of the drug delivery system in inhibiting the growth of tumors in situ and remote metastasis is evaluated by in vivo animal experiments, and the in vivo animal experiment method comprises the following steps:

[0068] (1) A mouse orthotopic breast cancer xenograft tumor model is established. Female Balb / c nude mice of 4-6 weeks old with a body weight of about 16-18 g are selected. 5×10 6 MDA-MB-231 cells in the logarithmic growth phase are suspended in 100 μL PBS and subcutaneously injected into the right fourth mammary fat pad of female Balb / c nude mice.

[0069] (2) 5-7 days after injection, when the tumor volume reaches about 50 mm 3 , the mice are randomly divided into 4 groups, 5 mice in each group. Saline, EGCG-Lp, CuCs-Lp and EGCG-CuCs-Lp are respectively injected into the tail vein of the mice every 3 days, wherein the injection concentration of EGCG in the control EGCG-Lp is 11 mg / kg, the injection concentration of Cu in the CuCs-Lp group is 0.75 mg / kg, and the injection concentration of EGCG-CuCs-Lp is EGCG 11 mg / kg+Cu 0.75 mg / kg, and the treatment is continuously performed for 21 days; during the treatment, the body weight of the mice is weighed every other day, the tumor diameter is measured and recorded. The tumor volume calculation formula: V=1 / 2(L×W 2 ), wherein L and W respectively represent the long diameter and the short diameter of the tumor.

[0070] (3) After the tumor-bearing mice are injected into the tail vein for 21 days, the mice are euthanized, the lung tissue is taken out by dissection, and the lung metastasis nodules are observed by H&E staining of the section.

[0071] Among them: the preparation method of EGCG-Lp and EGCG-CuCs-Lp is only different in that the addition of artificial metalloenzyme (CuCs) is omitted; the preparation method of CuCs-Lp and EGCG-CuCs-Lp is only different in that the addition of EGCG is omitted.

[0072] The results are as shown in a and Figure 4 b. Figure 5As shown in a and d, EGCG-CuCs-Lp undergoes demulsification in the acidic tumor microenvironment. The released CuCs, under the action of H2O2 in the tumor microenvironment, catalyze the oxidative polymerization of EGCG to form a hydrogel self-assembly, which significantly inhibits the proliferation of in situ tumor cells. Figure 4 As shown in a, after 21 days of treatment, the tumor volume in the EGCG-CuCs-Lp treatment group was 202.9 ± 42.06 mm. 3 The tumor volume was significantly smaller than that of other control groups. Compared with the saline control group, the tumor volume in the EGCG-CuCs-Lp treatment group was reduced by 80.2%. Simultaneously, EGCG-CuCs-Lp also significantly inhibited lung metastasis of tumors, such as... Figure 5 As shown in a, compared with the control groups, no obvious tumor metastatic nodules appeared in the lungs of the EGCG-CuCs-Lp treatment group. Figure 5 As shown in d, compared with the control group injected with saline, the EGCG-CuCs-Lp treatment group achieved a 100% inhibition rate of tumor metastasis.

[0073] Example 2

[0074] (I) Preparation of gold-containing artificial metalloenzymes:

[0075] (1) Mix 5 mL of 50 mg / mL BSA solution with 5 mL of 10 mM HAuCl4 solution and stir at 37°C for 5 min. Slowly add 0.5 mL of 1 M NaOH to the mixed solution. The color of the solution changes. Continue to stir the resulting solution at 37°C for 12 h.

[0076] (2) The above reaction solution was dialyzed for 0.5 h using a dialysis bag with a molecular weight cutoff of 10 kDa to remove unreacted BSA and free metal ions. Then it was concentrated using an ultrafiltration tube with a molecular weight cutoff of 30 kDa to obtain gold-containing artificial metalloenzymes (denoted as AuCs).

[0077] (II) Preparation of pH-sensitive liposomes loaded with gold-containing artificial metalloenzymes and polyphenolic compounds:

[0078] (1) Dissolve and mix DOPE, DPSG and DSPE-PEG in 1 mL of chloroform at a molar ratio of 5:5:1 (of which DOPE is 5 mg). Then transfer the solution to a 20 mL round flask and evaporate the solvent under reduced pressure using a rotary evaporator at 52 °C to obtain a pH-sensitive liposome membrane.

[0079] (2) Dissolve 10 mg ECG in 1.5 mL PBS (pH = 7.4) and add it to a flask along with 1 mL of 10 mM AuCs solution. Hydrate the resulting membrane and then sonicate for 1 h to obtain a liposome solution.

[0080] (3) Then the liposome solution was extruded through a membrane with a pore size of 0.22 μm, and then dialyzed with a dialysis bag (MWCO: 1000 kDa), and then the liposome solution was centrifuged 3-5 times with an ultrafiltration tube (MWCO: 100 kDa) at 5000 r / min, 10 min / time, to concentrate and remove impurities, to obtain the pH-sensitive liposome loaded with the gold-containing artificial metalloenzyme and ECG, denoted as ECG-AuCs-Lp.

[0081] Performance test:

[0082] 1. Transmission electron microscopy (TEM) was used to test the gold-containing artificial metalloenzyme (AuCs) prepared in Example 2, and the results are shown in FIG. 2b; the TEM results show that the gold-containing artificial metalloenzyme synthesized in the present application has good dispersibility and uniform particle size. Figure 1

[0083] 2. TEM was used to determine the structural changes of ECG-AuCs-Lp under acidic conditions: ECG-AuCs-Lp was dispersed in a buffer solution with a pH of 6.5, and then placed at 37°C for 24 h, and then observed with a cryogenic transmission electron microscope. The results are shown in FIG. 3b; the TEM results show that in the buffer solution with a pH of 6.5, the ECG-AuCs-Lp has demulsified, releasing the artificial metalloenzyme (AuCs) and the polyphenol compound (ECG). Figure 2

[0084] 3. The experiment of artificial metalloenzyme catalyzing the oxidative polymerization of polyphenol compounds was carried out using a buffer solution with a pH of 6.5 to simulate the tumor microenvironment:

[0085] Four different test tubes were taken, and a buffer solution with a pH of 6.5 was added, and then ECG (concentration of 1 mg / mL), ECG+H2O2 (concentration of ECG of 1 mg / mL, concentration of H2O2 of 100 μM), ECG+AuCs (concentration of ECG of 1 mg / mL, concentration of AuCs of 8 μM), and ECG+AuCs+H2O2 (concentration of ECG of 1 mg / mL, concentration of AuCs of 8 μM, concentration of H2O2 of 100 μM) were added respectively; the above-mentioned reaction mixtures were incubated at 37°C for 24 h, and the state of the reactants in the test tubes was observed, and the results are shown in FIG. 4b; as can be seen from FIG. 4b, in the experimental group of ECG+AuCs+H2O2, a hydrogel was formed at the bottom of the test tube, while no hydrogel was observed in the control groups of ECG, ECG+H2O2, and ECG+AuCs; the above experimental results show that the artificial metalloenzyme can catalyze the oxidative polymerization of polyphenol compounds under the action of the tumor micro-acidic environment and H2O2. Figure 3 Figure 3

[0086] ​​​​4. The in vivo animal experiment was used to evaluate the performance of the drug delivery system in inhibiting the growth of tumors in situ and remote metastasis. The in vivo animal experiment method was consistent with that in Example 1, and only when the tumor volume reached about 50mm 3 When the tumor volume reached about 50mm 3 , the mice were randomly divided into 4 groups, 5 mice in each group. Saline, ECG-Lp, AuCs-Lp and ECG-AuCs-Lp were injected into the tail vein of mice every 3 days, respectively. The injection concentration of ECG in ECG-Lp was 11.5mg / kg, the injection concentration of Au in AuCs-Lp was 2.5mg / kg, and the injection concentration of ECG-AuCs-Lp was ECG 11.5mg / kg+Au 2.5mg / kg. The treatment was continued for 21 days. Among them: the difference between the preparation methods of ECG-Lp and ECG-AuCs-Lp is only that the addition of artificial metal enzyme (AuCs) is omitted; the difference between the preparation methods of AuCs-Lp and ECG-AuCs-Lp is only that the addition of ECG is omitted.

[0087] The results are shown in b of Figure 4 and b, e of Figure 5 . ECG-AuCs-Lp breaks in the acidic microenvironment of the tumor, and the released AuCs catalyzes the oxidative polymerization of ECG to generate hydrogel self-assembly under the action of H2O2 in the tumor microenvironment, which can significantly inhibit the proliferation of in situ tumor cells. As shown in b of Figure 4 , after 21 days of treatment, the tumor volume of the ECG-AuCs-Lp treatment group was 213.86±50.5mm 3 , which was significantly smaller than that of the other control groups. Compared with the saline injection control group, the tumor volume of the ECG-AuCs-Lp treatment group was reduced by 79%. At the same time, ECG-AuCs-Lp can also significantly inhibit tumor lung metastasis. After 21 days of treatment by tail vein injection, the mice were euthanized, and lung tissue was removed for sectioning and H&E staining to observe lung metastatic nodules. As shown in b of Figure 5 , compared with the control groups, no obvious tumor metastatic nodules were found in the lungs of the ECG-AuCs-Lp treatment group; as shown in e of Figure 5 , compared with the saline injection control group, the tumor metastasis inhibition rate of the ECG-AuCs-Lp treatment group reached 100%.

[0088] Example 3

[0089] (I) Preparation of platinum-containing artificial metal enzyme:

[0090] (1) 1 mL, 86 mg / mL BSA solution was mixed with 0.33 mL, 50 mM H2PtCl6 solution, and stirred vigorously for 5 min. 0.34 mL, 0.5 M NaOH solution was slowly added to the mixed solution. Then 0.33 mL, 50 mM NaBH4 solution was added, and the color of the solution changed. The formed solution was continuously stirred at room temperature for 1 h.

[0091] (2) The above reaction solution was dialyzed with a dialysis bag with a molecular weight cut-off of 500 kDa for 3 times, 0.5 h each time, to remove unreacted H2PtCl6, NaOH and NaBH4, and then concentrated by an ultrafiltration tube with a molecular weight cut-off of 30 kDa to obtain a platinum-containing artificial metal enzyme (denoted as PtCs).

[0092] (B) Preparation of pH-sensitive liposomes loaded with platinum-containing artificial metal enzyme and polyphenol compound:

[0093] (1) The stock solution of DOPE, DPSG and DSPE-PEG (molar ratio of 5:5:1) was mixed in 1 mL of chloroform (wherein the amount of DOPE was 5 mg). Then the solution was transferred to a 20 mL round flask, and the solvent was evaporated under reduced pressure at 52°C using a rotary evaporator to obtain a pH-sensitive liposome film.

[0094] (2) 10 mg of EGC was dissolved in 1.5 mL of PBS (pH = 7.4) and added to the flask together with 1 mL of 10 mM PtCs solution. The obtained film was hydrated and then ultrasonicated for 1 h to obtain a liposome solution.

[0095] (3) Then the liposome was extruded through a membrane with a pore size of 0.22 μm, and then dialyzed with a dialysis bag (MWCO: 1000 kDa). Subsequently, the liposome solution was centrifuged 3-5 times with an ultrafiltration tube (MWCO: 100 kDa) at 5000 r / min, 10 min / time, and the impurities were removed by concentration to obtain pH-sensitive liposomes loaded with artificial metal enzyme containing gold and EGC, denoted as EGC-PtCs-Lp.

[0096] Performance test:

[0097] 1. The platinum-containing artificial metal enzyme (PtCs) prepared in Example 3 was subjected to transmission electron microscopy test, and the results are shown in Fig. 1c; the TEM results show that the platinum-containing artificial metal enzyme synthesized by the present application has good dispersibility and uniform particle size. Figure 1

[0098] 2. The structural changes of EGC-PtCs-Lp under acidic conditions were determined by TEM: EGC-PtCs-Lp was dispersed in a buffer solution with a pH value of 6.5, and placed at 37°C for 24 h, and then observed by cryogenic transmission electron microscopy. The results are shown in Fig. 2c. Figure 2 ​TEM results show that in the buffer solution with pH = 6.5, the EGc-PtCs-Lp is demulsified to release artificial metalloenzyme (PtCs) and polyphenol compound (EGC).

[0099] 3. The experiment of artificial metalloenzyme catalyzing polyphenol compound oxidative polymerization was carried out by using the buffer solution with pH 6.5 to simulate the tumor microenvironment:

[0100] Four different test tubes were taken, and the buffer solution with pH 6.5 was added, and then EGC (concentration of 1 mg / mL), EGC+H2O2 (concentration of EGC was 1 mg / mL, and concentration of H2O2 was 100 μM), EGC+PtCs (concentration of EGC was 1 mg / mL, and concentration of PtCs was 8 μM), and EGC+PtCs+H2O2 (concentration of EGC was 1 mg / mL, concentration of PtCs was 8 μM, and concentration of H2O2 was 100 μM) were added respectively into the test tubes. The above reaction mixtures were incubated at 37 °C for 24 h, and the state of the reactants in the test tubes was observed, and the results are shown in Figure 3 c of FIG. 6; according to Figure 3 It can be seen from c of FIG. 6 that the hydrogel was formed at the bottom of the test tube in the experimental group of EGC+PtCs+H2O2, while no hydrogel was observed in the control groups of EGC, EGC+H2O2, and EGC+PtCs. The above experimental results show that the artificial metalloenzyme can catalyze the oxidative polymerization of polyphenol compounds under the action of tumor micro-acidic environment and H2O2.

[0101] The in vivo animal experiment was used to evaluate the performance of the drug delivery system in inhibiting the growth of tumors in situ and remote metastasis. The in vivo animal experiment method was consistent with that in Example 1, and only when the tumor volume reached about 50 mm 3 When the tumor volume reached about 50 mm 3 , the mice were randomly divided into 4 groups, 5 mice in each group, and physiological saline (saline), EGC-Lp, PtCs-Lp, and EGC-PtCs-Lp were injected into the tail vein of the mice every 3 days, respectively. The injection concentration of EGC-Lp was 6 mg / kg, the injection concentration of Pt in the PtCs-Lp group was 2 mg / kg, and the injection concentration of EGC-PtCs-Lp was 6 mg / kg of EGC + 2 mg / kg of Pt. The continuous treatment lasted for 21 days. Among them: the difference between the preparation methods of EGC-Lp and EGC-PtCs-Lp is only that the addition of artificial metalloenzyme (PtCs) is omitted; the difference between the preparation methods of PtCs-Lp and EGC-PtCs-Lp is only that the addition of EGC is omitted.

[0102] The results are shown in c of FIG. 8 and Figure 4 c of FIG. 9. Figure 5EGC-PtCs-Lp in the acidic tumor microenvironment, the released PtCs catalyze the oxidative polymerization of EGC under the action of H2O2 in the tumor microenvironment to generate hydrogel self-assembly, which produces a significant inhibitory effect on tumor growth. As shown in Fig. 1c, after 21 days of treatment, the tumor volume of the EGC-PtCs-Lp treatment group was 229.9 ± 49.2 mm Figure 4 As shown in Fig. 1c, after 21 days of treatment, the tumor volume of the EGC-PtCs-Lp treatment group was 229.9 ± 49.2 mm 3 As shown in Fig. 1c, after 21 days of treatment, the tumor volume of the EGC-PtCs-Lp treatment group was 229.9 ± 49.2 mm 3 As shown in Fig. 1c, after 21 days of treatment, the tumor volume of the EGC-PtCs-Lp treatment group was 229.9 ± 49.2 mm Figure 5 As shown in Fig. 1c, after 21 days of treatment, the tumor volume of the EGC-PtCs-Lp treatment group was 229.9 ± 49.2 mm Figure 5 As shown in Fig. 1c, after 21 days of treatment, the tumor volume of the EGC-PtCs-Lp treatment group was 229.9 ± 49.2 mm

[0103] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A drug delivery system loaded with artificial metalloenzymes and polyphenolic compounds, characterized in that, It includes pH-sensitive liposomes, artificial metalloenzymes, and polyphenolic compounds; the artificial metalloenzymes and the polyphenolic compounds are encapsulated in the pH-sensitive liposomes. The pH-sensitive liposomes include dioleoylphosphatidylethanolamine, 1,2-dipalmitoyl-SN-glycerol-3-succinic acid, and distearate-phosphatidylethanolamine-polyethylene glycol; The molar ratio of dioleoylphosphatidylethanolamine, 1,2-dipalmitoyl-SN-glycerol-3-succinic acid and distearate phosphatidylethanolamine-polyethylene glycol is (5-9):(1-5):1; The artificial metalloenzyme has a core-shell structure, with a metal cluster as the core and a protein or peptide as the outer shell; the particle size of the artificial metalloenzyme is 1-10 nm, and the number of metal atoms is 1-100; the polyphenolic compound includes one or more of epigallocatechin gallate, epicatechin gallate, and epigallocatechin.

2. The drug delivery system according to claim 1, characterized in that, The metal clusters include one or more of the following metal elements: copper, gold, and platinum; The protein in question is a water-soluble protein.

3. The drug delivery system according to claim 2, characterized in that, The water-soluble proteins include serum albumin or transferrin.

4. A method for preparing the drug delivery system loaded with artificial metalloenzymes and polyphenolic compounds according to any one of claims 1 to 3, characterized in that, Includes the following steps: Dioleoylphosphatidylethanolamine, 1,2-dipalmitoyl-SN-glycerol-3-succinic acid and distearate-phosphatidylethanolamine-polyethylene glycol were dissolved in an organic solvent, and then the organic solvent was evaporated to remove it, to obtain a pH-sensitive liposome film. Artificial metalloenzymes, polyphenolic compounds, PBS solution and the pH-sensitive liposome membrane were mixed and sonicated to obtain a liposome solution loaded with artificial metalloenzymes and polyphenolic compounds. The liposome solution loaded with artificial metalloenzymes and polyphenolic compounds was sequentially filtered, dialyzed, and concentrated to obtain a drug delivery system loaded with artificial metalloenzymes and polyphenolic compounds.

5. The preparation method according to claim 4, characterized in that, The method for preparing the artificial metalloenzyme includes the following steps: A metal compound solution, a shell-forming raw material solution, and a sodium hydroxide solution are mixed and subjected to a reduction reaction. The resulting reduction reaction solution is dialyzed and concentrated to obtain an artificial metalloenzyme. The concentration of the metal compound solution is 1–200 mM. The shell-forming raw material solution is a protein solution or a peptide solution. The concentration of the shell-forming raw material solution is 1–100 mg / mL. The concentration of the sodium hydroxide solution is 0.1–1 M.

6. The preparation method according to claim 5, characterized in that, The raw materials used in the reduction reaction also include a reducing agent; the reducing agent includes one or more of sodium borohydride, ascorbic acid, and hydrazine.

7. The preparation method according to claim 5, characterized in that, The molar ratio of the artificial metalloenzyme to the polyphenol compound is (1-5):1; the mass ratio of the polyphenol compound to the liposome membrane is (1-10):(5-50).

8. The application of the drug delivery system loaded with artificial metalloenzymes and polyphenolic compounds according to any one of claims 1 to 3, or the drug delivery system loaded with artificial metalloenzymes and polyphenolic compounds prepared by the preparation method according to any one of claims 4 to 7, in the preparation of antitumor drugs.

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