A visualized carbon-based nanocatalytic drug and its preparation method and application

Abstract

The present invention discloses a visualized carbon-based nanocatalytic drug, which includes a carbon nanodot substrate, a tumor targeting ligand, a metal ion chelator and a metal ion chelated therewith, wherein the metal ion chelator is modified on the surface of the carbon nanodot, and the tumor targeting ligand is coupled on the surface of the carbon nanodot. The preparation method includes the following steps: preparing a carbon nanodot substrate; preparing a metal ion chelator; preparing a tumor targeting ligand; preparing a bifunctional carbon nanodot; dispersing the bifunctional carbon nanodot in pure water, adding a metal ion solution and a tumor targeting ligand for reaction, and the product obtained after freeze-drying is a visualized carbon-based nanocatalytic drug. The drug of the present invention has the advantages of fluorescence emission function, uniform and stable structure, monodispersity, good water solubility, good catalytic efficiency, etc., and targets and transports $V ions to the inside of tumor cells, amplifies the oxidative stress of tumor cells by catalyzing a Fenton-like reaction, and thus efficiently kills tumor cells.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomaterials, biomedicine and nano-drugs, and in particular to a visualized carbon-based nano-catalytic drug and a preparation method and application thereof. Background Art

[0002] Regulating cellular oxidative stress is a novel and effective tumor treatment method. Chemodynamic therapy (CDT) utilizes Fenton or Fenton-like reactions mediated by iron and copper ions to induce intracellular oxidative stress by converting endogenous H2O2 into highly reactive OH, thereby killing tumor cells. CDT is rapidly becoming a novel treatment method, showing many advantages over traditional treatment methods. The development of new tumor catalytic drugs to improve the efficacy of tumor treatment has important clinical and economic significance.

[0003] Cu 2+ Nanocatalytic drugs are an emerging type of tumor therapeutic drugs. They can not only catalyze hydrogen peroxide to produce highly toxic superoxide anions in cells, but also reduce intracellular GSH, thereby amplifying the intracellular oxidative stress of tumor cells and improving the tumor treatment effect. Therefore, it has received extensive attention from the scientific research and medical communities.

[0004] However, currently research on Cu 2+ Nanocatalytic drugs have three disadvantages:

[0005] (1) Non-targeted;

[0006] (2)Cu 2+ Easy to leak;

[0007] (3) The treatment process is difficult to track.

[0008] Therefore, it is necessary to design a visualized carbon-based nanocatalytic drug and its preparation method and application. Summary of the invention

[0009] In order to overcome the defects in the prior art, a visualized carbon-based nanocatalytic drug and a preparation method and application thereof are provided.

[0010] The present invention is achieved through the following solutions:

[0011] A visualized carbon-based nanocatalytic drug comprises a carbon nanodot substrate, a tumor targeting ligand, a metal ion chelator and metal ions chelated therewith, wherein the metal ion chelator is modified on the surface of the carbon nanodots, and the tumor targeting ligand is coupled to the surface of the carbon nanodots.

[0012] The carbon nanodots are carbon nanodots with fluorescence emission performance.

[0013] The tumor targeting ligand includes a tumor targeting nucleic acid aptamer.

[0014] The nucleic acid aptamer targets a tumor membrane surface receptor protein, and the nucleic acid aptamer modifies DBCO.

[0015] The metal ion chelator and the amino azide ligand are covalently modified on the surface of the carbon nanodots.

[0016] The metal ion chelating agent is a Cu chelating agent, and the metal ion is Cu 2+ .

[0017] The Cu chelating agent is DTPA.

[0018] A method for preparing a visualized carbon-based nanocatalytic drug, the method comprising the following steps:

[0019] The first step is to prepare a carbon nanodot substrate;

[0020] The second step is to prepare a metal ion chelating agent;

[0021] Step 3: Prepare tumor targeting ligands;

[0022] Step 4: Preparation of bifunctional carbon nanodots: amino azide ligands and Cu chelator DTPA are covalently modified on the surface of carbon nanodots through amidation reaction to obtain bifunctional carbon nanodots;

[0023] Step 5. Preparation of visualized carbon-based nanocatalytic drugs: Disperse the bifunctional carbon nanodots in pure water, then add metal ion solution to the suspension, and then add tumor targeting ligands to continue the reaction. The product obtained after freeze-drying is the visualized carbon-based nanocatalytic drug.

[0024] The carbon nanodot substrate in the first step is prepared by a hydrothermal method, and the specific operation process is: adding polythiophene derivative powder to a NaOH solution, ultrasonicating for 10 minutes, adding it to a high-pressure reactor, reacting at 200°C for 8 hours, cooling to room temperature, filtering, dialysis, and freeze-drying to obtain a red solid product, which is a carbon nanodot substrate with fluorescent emission performance;

[0025] The specific operation process of the third step is: the tumor targeting ligand is a nucleic acid aptamer that can specifically target the receptor on the surface of the tumor cell membrane, the nucleic acid aptamer is prepared in an automated and batch manner by a solid phase synthesis method, and the nucleic acid aptamer is modified with DBCO for standby use;

[0026] The specific operation process of the fourth step is: dissolving the carbon nanodots in PBS and fully dispersing them, adding EDC, NHS, and TEA in sequence, reacting them at room temperature for 2 hours, adding amino azide ligands, reacting them at room temperature for more than 12 hours, dialyzing, and freeze-drying to obtain azide-functionalized carbon nanodots; using the azide-functionalized carbon nanodots as substrates, and reacting the Cu The chelating agent DTPA is modified on the surface of the azide-functionalized carbon nanodots to obtain dual-functionalized carbon nanodots;

[0027] The specific operation process of the fifth step is: dispersing the dual-functional carbon nanodots in pure water, then adding CuCl2 solution to the suspension, stirring at room temperature for 2 hours; ultrafiltration to remove the unchelated Cu 2+ After ionization, tumor targeting ligand was added and reacted at room temperature for 26 hours; unreacted nucleic acid aptamer was removed by ultrafiltration, and the red solid product obtained after freeze-drying was the visualized carbon-based nanocatalytic drug.

[0028] An application of a visualized carbon-based nanocatalytic drug, wherein the visualized carbon-based nanocatalytic drug is applied to tumor targeted therapy.

[0029] The beneficial effects of the present invention are:

[0030] The visualized carbon-based nanocatalytic drug of the present invention uses NIR fluorescent carbon quantum dots (carbon nanodots) with good biocompatibility as a substrate, and covalently modifies the surface of the carbon dots with DNA aptamer (tumor targeting ligand) and Cu complex (Cu chelator) through precise molecular design, thereby forming a visualized carbon-based nanocatalytic drug with fluorescence emission function, uniform and stable structure, monodispersity, good water solubility and good catalytic efficiency. 2+ Ions are transported into tumor cells in a targeted manner, and by catalyzing Fenton-like reactions, they amplify the oxidative stress of tumor cells, thereby effectively killing tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 .Atomic force microscopy images of carbon-based catalytic drugs for regulation of cellular oxidative stress.

[0032] Figure 2 .Carbon-based catalytic drug endocytosis in tumor cells

[0033] Figure 3 .Chart showing the killing effect of carbon-based catalytic drugs on tumor cells. DETAILED DESCRIPTION

[0034] The preferred embodiments of the present invention are further described below in conjunction with the accompanying drawings:

[0035] In this application, the meanings of some English abbreviations are as follows:

[0036] PBS: phosphate buffered saline;

[0037] EDC: 1-ethyl-(3-dimethylaminopropyl)carbodiimide;

[0038] NHS: N-hydroxysuccinimide;

[0039] TEA: triethylamine.

[0040] A visualized carbon-based nanocatalytic drug comprises a carbon nanodot substrate, a tumor targeting ligand, a metal ion chelator and metal ions chelated therewith, wherein the metal ion chelator is modified on the surface of the carbon nanodots, and the tumor targeting ligand is coupled to the surface of the carbon nanodots.

[0041] The carbon nanodots are carbon nanodots with fluorescence emission performance. The fluorescence of the carbon nanodots should include fluorescence ranges such as visible light, NIR I, and NIR II, and the carbon nanodots are NIR fluorescent carbon quantum dots with good biocompatibility.

[0042] The tumor targeting ligand includes a tumor targeting nucleic acid aptamer.

[0043] The nucleic acid aptamer targets a tumor membrane surface receptor protein, and the nucleic acid aptamer modifies DBCO.

[0044] The metal ion chelator and the amino azide ligand are covalently modified on the surface of the carbon nanodots.

[0045] The metal ion chelating agent is a Cu chelating agent, and the metal ion is Cu 2+ .

[0046] The Cu chelator is DTPA. In practical applications, the metal ion chelator also includes clinical metal ion chelators DOTA, NOTA and others.

[0047] A method for preparing a visualized carbon-based nanocatalytic drug, the method comprising the following steps:

[0048] The first step is to prepare a carbon nanodot substrate;

[0049] Step 2: Prepare a metal ion chelator; in this embodiment, the metal ion chelator is Cu chelator DTPA, and its preparation method is a well-known technology. For details, please refer to: Ji, Ding-Kun, et al. ACS Applied Nano Materials, 2021, 4(2): 1467-1477.

[0050] Step 3: Prepare tumor targeting ligands;

[0051] Step 4: Preparation of bifunctional carbon nanodots: amino azide ligands and Cu chelator DTPA are covalently modified on the surface of carbon nanodots through amidation reaction to obtain bifunctional carbon nanodots;

[0052] Step 5. Preparation of visualized carbon-based nanocatalytic drugs: Disperse the bifunctional carbon nanodots in pure water, then add metal ion solution to the suspension, and then add tumor targeting ligands to continue the reaction. The product obtained after freeze-drying is the visualized carbon-based nanocatalytic drug.

[0053] The carbon nanodot substrate in the first step is prepared by a hydrothermal method, and the specific operation process is: adding polythiophene derivative powder to a NaOH solution, ultrasonicating for 10 minutes, adding it to a high-pressure reactor, reacting at 200°C for 8 hours, cooling to room temperature, filtering, dialysis, and freeze-drying to obtain a red solid product, which is a carbon nanodot substrate with fluorescent emission performance;

[0054] The specific operation process of the third step is: the tumor targeting ligand is a nucleic acid aptamer that can specifically target the receptor on the surface of the tumor cell membrane, the nucleic acid aptamer is prepared in an automated and batch manner by a solid phase synthesis method, and the nucleic acid aptamer is modified with DBCO for standby use;

[0055] The specific operation process of the fourth step is: dissolving the carbon nanodots in PBS and fully dispersing them, adding EDC, NHS, and TEA in sequence, reacting at room temperature for 2 hours, adding amino azide ligands, reacting at room temperature for more than 12 hours, dialyzing, and freeze-drying to obtain azide-functionalized carbon nanodots; using azide-functionalized carbon nanodots as substrates, modifying the surface of the azide-functionalized carbon nanodots with Cu chelator DTPA through an amidation reaction to obtain dual-functionalized carbon nanodots;

[0056] The specific operation process of the fifth step is: dispersing the dual-functional carbon nanodots in pure water, then adding CuCl2 solution to the suspension, stirring at room temperature for 2 hours; ultrafiltration to remove the unchelated Cu 2+ After ionization, tumor targeting ligands were added and reacted at room temperature for 26 hours; unreacted nucleic acid aptamers were removed by ultrafiltration, and the red solid product obtained after freeze-drying was the visualized carbon-based nanocatalytic drug. The atomic force microscopy image of the visualized carbon-based nanocatalytic drug is shown in FIG. Figure 1 shown.

[0057] An application of a visualized carbon-based nanocatalytic drug, wherein the visualized carbon-based nanocatalytic drug is applied to tumor targeted therapy.

[0058] The visualized carbon-based nanocatalytic drug of the present application can be specifically internalized by colorectal cancer cells (HCT116) and show a stable near-infrared fluorescence emission signal in the cells. Specific experimental steps: HCT 116 (1×10 5 ) The cells were planted in a glass imaging dish and incubated for 24 hours. The cells were observed to be in good condition, and the nanocatalytic drugs were added and incubated for 8 hours. The unbound nanomedicines were washed away with PBS. The images were taken using a Leica laser confocal microscope. The results are detailed in Figure 2 .

[0059] The visualized carbon-based nanocatalytic drug of the present application can achieve specific killing of tumor cells. Specific experimental steps: HCT 116 (1×10 5 ) The cells were planted in a glass imaging dish and incubated for 24 hours. When the cells were observed to be in good condition, the nanocatalytic drugs were added and incubated for 24 hours. The unbound nanomedicines were washed away with PBS and stained with a dead cell detection kit for 30 minutes. The images were taken using a Leica laser confocal microscope. At the same time, the tumor cell survival rate was determined using CCK-8. The results are detailed in Figure 3 .

[0060] Although the technical solutions of the present invention have been described and listed in detail, it should be understood that it is obvious to those skilled in the art to make modifications to the above embodiments or adopt equivalent alternatives. These modifications or improvements made without departing from the spirit of the present invention are within the scope of protection required by the present invention.

Claims

1. A visualized carbon-based nanocatalytic drug, characterized in that: The drug comprises a carbon nanodot substrate, a tumor targeting ligand, a metal ion chelator and metal ions chelated therewith, wherein the metal ion chelator is modified on the surface of the carbon nanodot, and the tumor targeting ligand is coupled to the surface of the carbon nanodot; The tumor targeting ligand includes a tumor-targeting nucleic acid aptamer; The nucleic acid aptamer targets a tumor membrane surface receptor protein, and the nucleic acid aptamer modifies DBCO; The metal ion chelator and the aminoazide ligand are covalently modified on the surface of the carbon nanodots; The metal ion chelating agent is DTPA, and the metal ion is Cu 2+ ; The carbon nanodot substrate is prepared by a hydrothermal method using a polythiophene derivative.

2. A visualized carbon-based nanocatalytic drug according to claim 1, characterized in that: The carbon nanodots are carbon nanodots with fluorescence emission performance.

3. A method for preparing a visualized carbon-based nanocatalytic drug as claimed in any one of claims 1 to 2, characterized in that: The method comprises the following steps: The first step is to prepare a carbon nanodot substrate; The second step is to prepare a metal ion chelating agent; Step 3: Prepare tumor targeting ligands; Step 4: Preparation of bifunctional carbon nanodots: amino azide ligands and Cu chelator DTPA are covalently modified on the surface of carbon nanodots through amidation reaction to obtain bifunctional carbon nanodots; Step 5: Preparation of visualized carbon-based nanocatalytic drugs: Disperse the bifunctional carbon nanodots in pure water, then add metal ion solution to the suspension, and then add tumor targeting ligand to continue the reaction. The product obtained after freeze-drying is the visualized carbon-based nanocatalytic drug; The carbon nanodot substrate in the first step is prepared by a hydrothermal method, and the specific operation process is: adding polythiophene derivative powder to a NaOH solution, ultrasonicating for 10 minutes, adding it to a high-pressure reactor, reacting at 200°C for 8 hours, cooling to room temperature, filtering, dialysis, and freeze-drying to obtain a red solid product, which is a carbon nanodot substrate with fluorescent emission performance; The specific operation process of the third step is: the tumor targeting ligand is a nucleic acid aptamer that can specifically target the receptor on the surface of the tumor cell membrane, the nucleic acid aptamer is prepared in an automated and batch manner by a solid phase synthesis method, and the nucleic acid aptamer is modified with DBCO for standby use; The specific operation process of the fourth step is: dissolving the carbon nanodots in PBS and fully dispersing them, adding EDC, NHS, and TEA in sequence, reacting at room temperature for 2 hours, adding amino azide ligands, reacting at room temperature for more than 12 hours, dialyzing, and freeze-drying to obtain azide-functionalized carbon nanodots; using azide-functionalized carbon nanodots as substrates, modifying the surface of the azide-functionalized carbon nanodots with Cu chelator DTPA through an amidation reaction to obtain dual-functionalized carbon nanodots; The specific operation process of the fifth step is: dispersing the dual-functional carbon nanodots in pure water, then adding CuCl2 solution to the suspension, stirring at room temperature for 2 hours; ultrafiltration to remove the unchelated Cu 2+ After ionization, tumor targeting ligand was added and reacted at room temperature for 26 hours; unreacted nucleic acid aptamer was removed by ultrafiltration, and the red solid product obtained after freeze-drying was the visualized carbon-based nanocatalytic drug.

4. An application of a visualized carbon-based nanocatalytic drug as claimed in claim 3, characterized in that: The visualized carbon-based nanocatalytic drug is used for the preparation of tumor targeted therapeutic drugs or tumor imaging drugs.

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