CN109806252B - Ternary composite nano system and preparation method and application thereof - Google Patents

Ternary composite nano system and preparation method and application thereof Download PDF

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CN109806252B
CN109806252B CN201910084885.0A CN201910084885A CN109806252B CN 109806252 B CN109806252 B CN 109806252B CN 201910084885 A CN201910084885 A CN 201910084885A CN 109806252 B CN109806252 B CN 109806252B
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polyphenol
iron
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composite nano
ternary composite
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CN109806252A (en
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姚静
熊慧
蒋志杰
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China Pharmaceutical University
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Abstract

The invention discloses a ternary composite nano system and a preparation method and application thereof, wherein the system comprises an iron compound, a micromolecular anti-tumor active compound containing a benzene ring and a polyphenol compound, and the weight ratio of the iron compound to the micromolecular anti-tumor active compound containing a benzene ring to the polyphenol compound is 1-4:2-10: 5-20. Compared with the prior art, different micromolecular compounds or medicines can be stably assembled only by a physical assembly means, the formed composite nano-medicine not only has the anti-tumor treatment effect of micromolecular anti-tumor active compounds, but also has the iron death treatment effect mediated by iron compounds and polyphenol compounds based on intracellular Fenton reaction.

Description

Ternary composite nano system and preparation method and application thereof
Technical Field
The invention belongs to the field of pharmaceutics, and relates to a novel anti-tumor pharmaceutical preparation, in particular to a ternary composite nano system based on an iron compound, a small molecular anti-tumor active compound containing a benzene ring and a polyphenol compound, and a preparation method and application thereof.
Background
Cancer is one of the main diseases threatening human health at present, and the traditional chemotherapy drugs as the main body of the current clinical cancer treatment can not obtain satisfactory treatment effect due to the defects of low bioavailability, easy generation of multi-drug resistance and the like, so the clinical combined drug administration mode is usually adopted. The combined use of the chemotherapeutic drugs can generate a synergistic effect and improve the treatment effect; and the chemotherapy drugs have different mechanisms of action, so that the generation of multidrug resistance can be prevented to a certain extent. However, chemotherapy combination therapy cannot solve the inherent defects of traditional chemotherapeutic drugs, such as poor specificity, unexpected pharmacokinetics, biodistribution and the like, which all cause the chemotherapeutic drugs to cause serious damage to normal tissues when killing tumor cells; and the combined treatment often has enhanced toxic and side effects, and poor patient compliance and tolerance, thus greatly limiting the clinical application of the combined chemotherapy.
"Iron Death" (ferroptosis) is a novel mode of Cell Death proposed by Dxion in 2012, which can ultimately induce Cell Death by mediating the accumulation of intracellular Lipid Peroxides (LPO) to the level of ferritin Death, and is essentially a novel mode of oxidative Cell Death (Dixon S, LembergK, LamprchM, et al. Ferroptosis: An Iron-Dependent Form of nonapoptotic Cell Death [ J ]. Cell,2012(5), 149.). The use of iron death to mediate tumor cell death bypasses the apoptotic pathway of tumor cells, thus avoiding resistance caused by apoptosis-mediated cell death in other tumor therapies such as chemotherapy and photothermal therapy (V.S. Viswanathan, M.J.Ryan, H.D.Dhruv, et al.dependency of a thermal-resistant state of cancer cells a lipid peroxidase pathway [ J ] Nature,2017 (7664): 453-. The combined application of iron death therapy and other tumor treatment modes such as chemotherapy, photothermal therapy and photodynamic therapy can relieve the limitations of treatment resistance easily caused by monotherapy. Taking the iron death inducer Erastin as an example, Erastin can inhibit cystine/glutamic acid reverse transport protein from absorbing cystine so as to inhibit the synthesis of intracellular Glutathione (GSH), further reduce intracellular Glutathione Peroxidase (GPXs) and reduce the antioxidant capacity of cells. While intracellular polyunsaturated fatty acids (PUFAs) can be continuously oxidized and LPO is produced in a stereospecific manner. In the presence of iron, LPO further forms toxic lipid radicals, such as alkoxy radicals, which ultimately induce iron death. In addition, these generated lipid radicals can also transfer protons adjacent to PUFAs, subsequently initiating a new round of lipid oxidation and further transmitting oxidative damage, producing a circularly amplified Cell killing effect (Stockwell B R, Jos bee Pehydro, Angeli F, et al.Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease [ J ] Cell,2017(2): 273) 285.). In addition, iron compounds can also mediate iron death efficiently by participating in intracellular fenton reactions, producing large amounts of Reactive Oxygen Species (ROS) and inserting into lipid membranes to form LPO (Hofmans S, Berghe T V, Devischer L, et al. novel dehydration inhibitors with improved reactivity [ J ]. Journal of Medicinal Chemistry,2015(5): 2041-53). However, the iron compound is used in a limited amount because it requires a large dose for tumor treatment and has a high risk of toxic and side effects.
The micromolecule antitumor active compound containing benzene ring often has serious toxic and side effects, and in addition, other inorganic nano photothermal materials have the defect of being incapable of being degraded, thereby bringing unpredictable potential toxicity.
Disclosure of Invention
The technical problem to be solved is as follows: in order to overcome the defects of the prior art, solve the defect that the antitumor active small molecular compound is easy to induce drug resistance in single treatment and improve the technical bottleneck of applying the iron compound to tumor treatment drugs, the invention provides a ternary composite nano system based on the iron compound, the benzene ring-containing small molecular antitumor active compound and the polyphenol compound, and a preparation method and application thereof. The specific objectives are as follows:
purpose 1: to obtain a ternary composite nano system based on an iron compound, a small molecular anti-tumor active compound containing a benzene ring and a polyphenol compound and a preparation method thereof.
Purpose 2: provides the application of the ternary composite nano system in preparing anti-tumor medicaments, and realizes the combined treatment of iron death treatment, photo-thermal treatment and chemotherapy or photodynamic treatment.
Purpose 3: broadens the therapeutic window of the micromolecule antitumor active compound containing benzene ring in the ternary composite nano system.
Purpose 4: the barriers of drug resistance of the micromolecule antitumor active compound containing benzene ring in the ternary composite nano system, deep delivery of the antitumor nano-drug and the like are broken.
Purpose 5: based on the ternary composite nano system, a strategy for improving toxic and side effects of triple combination therapy of iron death therapy, photothermal therapy, chemotherapy or photodynamic therapy is provided. The ternary composite nano system of the micromolecule antitumor active compound containing the benzene ring, the polyphenol compound and the iron compound can reduce the existing or potential toxic and side effects through the following mechanisms: firstly, the tumor targeted distribution of each active component is improved, and the non-selective systemic distribution of the active components is reduced, so that the system toxicity is reduced; secondly, all assembly units of the nano system are degradable and metabolizable active ingredients, so that toxic and side effects caused by incapability of degrading inorganic photo-thermal materials such as gold nanoparticles and the like are avoided; and thirdly, the fenton reaction of the polyphenol compound and iron ions in cells can greatly improve the reaction efficiency of pure iron ion mediated iron death by promoting the generation of intracellular ROS and LPO, thereby improving the utilization rate of the iron agent, reducing the dosage of the iron agent and achieving the effect of attenuation.
The technical scheme is as follows: the ternary composite nano system comprises an iron compound, a small molecular anti-tumor active compound containing a benzene ring and a polyphenol compound, wherein the weight ratio of the iron compound to the small molecular anti-tumor active compound containing the benzene ring to the polyphenol compound is 1-4:2-10: 5-20.
The particle size of the ternary composite nano system is 10-1000 nm.
Preferably, the iron compound is at least one of ferric ammonium citrate, ferric citrate, ferrous sulfate, ferric ammonium citrate, ferrous fumarate, ferrous chloride and ferric chloride hexahydrate.
Preferably, the small-molecule antitumor active compound containing a benzene ring is at least one of doxorubicin hydrochloride, idarubicin hydrochloride, daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride, epirubicin hydrochloride, mitoxantrone mesylate, melphalan, ondansetron, indocyanine green, methotrexate, irinotecan hydrochloride and paclitaxel.
Preferably, the polyphenol compound is at least one of tannic acid, catechin, epicatechin, bisabolol catechin, epigallophenol catechin, tea polyphenol, apple polyphenol, eriodictyol, grape polyphenol and resveratrol.
The preparation method of any one of the three-element composite nano-systems comprises the step of self-assembling an iron compound, a small-molecular anti-tumor active compound containing a benzene ring and a polyphenol compound in a solution environment to form the three-element composite nano-system.
Preferably, the method is that the iron compound and the polyphenol compound are firstly assembled through coordination chemistry to form a pre-assembly element, and then the pre-assembly element and the small molecular anti-tumor active compound containing a benzene ring form a ternary composite nano system through pi-pi conjugation.
Further, the method comprises the following specific steps:
(1) respectively weighing an iron compound, a micromolecular antitumor active compound containing a benzene ring and a polyphenol compound, and preparing into an aqueous solution;
(2) mixing the iron compound and the polyphenol compound solution, and adding an organic solvent in the mixing process;
(3) adding a small-molecule anti-tumor active compound solution containing benzene rings into the mixed solution in the step (2), uniformly mixing, adding a precipitation solvent, centrifuging, redissolving, and carrying out ultrasonic treatment to collect a product.
Preferably, the organic solvent in step (2) is at least one of ethanol, ethanol water, methanol water, isopropanol, and isopropanol water.
Preferably, the precipitation solvent in step (3) is at least one of ammonia water, sodium carbonate, urea, ammonium bicarbonate, hexamethylenetetramine, disodium ethylenediamine tetraacetate and sodium hydroxide solution.
Preferably, the adding amount of the organic solvent in the step (2) is 2-100 times of the total volume of the iron compound and the aqueous solution of the polyphenol compound in the step (1), and the adding amount of the precipitation solvent in the step (3) is 0.2-50 times of the total volume of the iron compound and the aqueous solution of the polyphenol compound in the step (1).
The application of any one of the ternary composite nano systems in preparing antitumor drugs.
The principle of the ternary composite nano system is as follows: after the ternary composite nano system is depolymerized in cells, free micromolecule antitumor active compounds containing benzene rings exert corresponding chemotherapy or photodynamic therapy effects in the cells to inhibit proliferation of tumor cells, and free iron ions and polyphenol compounds generate Fenton reaction in the cells to generate a large amount of ROS to be accumulated on lipid membranes, so that intracellular LPO is further induced to generate, and iron death is induced. In addition, the nanoparticle composite system has a good photothermal effect, so that the intracellular ROS can be further promoted to be generated, the deep delivery of the nanoparticles in the tumor can be promoted, the interstitial delivery obstacle to the nano-drug is broken, and the combined treatment of iron death treatment, photothermal treatment and chemotherapy or photodynamic treatment is finally realized. The ternary composite nano system can keep higher tumor cell killing activity under the condition of low concentration of the small molecular antitumor active compound containing the benzene ring, and remarkably improves the high tolerance of the treatment effect of the small molecular antitumor active compound containing the benzene ring to the concentration of the medicine. In addition, the accumulation and detention of the small-molecule antitumor active compound containing the benzene ring in the tumor cells are improved, the small-molecule antitumor active compound containing the benzene ring keeps effective cell killing capability in a wider concentration floating interval, the adverse effect of large fluctuation of treatment effect caused by drug concentration change in the cell material balance process is obviously counteracted, the treatment effect of the small-molecule antitumor active compound containing the benzene ring is improved, the toxicity of the small-molecule antitumor active compound containing the benzene ring is reduced, and the treatment window of the small-molecule antitumor active compound containing the benzene ring is effectively widened.
Has the advantages that: (1) the ternary composite nano system of the micromolecule antitumor active compound containing benzene ring, the polyphenol compound and the iron compound, which is constructed by the invention, only needs physical assembly without changing the chemical structure of the medicine, and is different from the change of the structure-activity relationship possibly caused by chemical modification means; the development and application limitations caused by factors such as the complex synthesis process, the change of the chemical structure of the drug micromolecule due to chemical modification, the uncertain structure-activity relationship and the like of the polymer prodrug micelle, the dendrimer prodrug nanoparticle and the like constructed by the traditional conjugated modification means are broken through.
(2) The ternary composite nano system of the micromolecule antitumor active compound containing the benzene ring, the polyphenol compound and the iron compound has the advantages of simple preparation method, mild preparation conditions, avoidance of the problems of poor quality uniformity and the like caused by a complex preparation method, good reproducibility and simple industrial conversion technology.
(3) The ternary composite nano system of the micromolecule antitumor active compound containing benzene ring, the polyphenol compound and the iron compound is also a degradable novel photo-thermal composite nano material.
(4) The ternary composite nano system of the micromolecule antitumor active compound containing the benzene ring, the polyphenol compound and the iron compound, which is constructed by the invention, conveniently and efficiently realizes the multi-bit integrated combined treatment of the iron death treatment, the photothermal treatment and the chemotherapy or the photodynamic therapy, the treatment functions are highly synergistic, and the potential toxicity is mutually decomposed; the ternary composite nano system provided by the invention not only realizes the combined treatment of tumor iron death treatment, photothermal treatment and chemotherapy or photodynamic therapy, but also can reduce the toxic and side effects of the multi-element combined treatment scheme and improve the treatment practicability of the multi-bit integrated combined system by improving the tumor targeting precision by off-target distribution of fewer drugs and promoting the intracellular reaction of micromolecule antitumor active compounds, polyphenol compounds and iron compounds of which the main unit components contain benzene rings.
(5) The ternary composite nano system of the micromolecule anti-tumor active compound containing the benzene ring, the polyphenol compound and the iron compound can widen the treatment window of the micromolecule anti-tumor active compound containing the benzene ring.
(6) The ternary composite nano system of the micromolecule antitumor active compound containing the benzene ring, the polyphenol compound and the iron compound, which is constructed by the invention, also widens the application range of the iron preparation in the field of tumor treatment, greatly improves the efficiency of mediating the tumor cell iron death, and pertinently solves the application dilemma of large dosage and high toxicity when the iron preparation is simply applied to tumor treatment.
(7) The ternary composite nano system of the micromolecule anti-tumor active compound containing the benzene ring, the polyphenol compound and the iron compound has excellent tumor targeting property, can selectively concentrate the loaded active compound on tumor cells to improve the targeting distribution efficiency of the tumor cells, can protect the loaded compound from being metabolized into a compound with reduced or inactivated activity in blood circulation, and keeps the stability of the loaded compound.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1: preparation of ferric chloride/tannic acid/doxorubicin hydrochloride ternary composite nano system
Accurately weighing ferric chloride, tannic acid and doxorubicin hydrochloride according to a weight ratio of 4:2:5, respectively dissolving in purified water, uniformly mixing by vortex, mixing and stirring the prepared ferric chloride solution and the tannic acid solution, injecting absolute ethyl alcohol with a volume 5 times of the total volume of the ferric chloride and tannic acid mixed solution under a stirring condition, and continuously stirring for 1-60 min. And adding the doxorubicin hydrochloride solution into the stirred system, continuously stirring for 1-60 min, then adding ammonia water with the volume of 0.2 times of the total volume of the mixed solution of ferric chloride and tannic acid, centrifuging, adding water to dissolve the precipitate again, and performing ultrasonic treatment on the precipitate for 8-30 min to obtain the ternary composite nano system.
Example 2: preparation of ferric citrate/tea polyphenol/daunorubicin hydrochloride ternary composite nano system
Precisely weighing ferric citrate, tea polyphenol and daunorubicin hydrochloride according to the weight ratio of 4:6:9, respectively dissolving in purified water, uniformly mixing by vortex, mixing and stirring the prepared ferric citrate solution and the prepared tea polyphenol solution, injecting anhydrous methanol with the volume 10 times of the total volume of the ferric citrate and tea polyphenol mixed solution under the stirring condition, and continuously stirring for 1-60 min. And adding the daunorubicin hydrochloride solution into the stirred system, continuously stirring for 1-60 min, then adding urea with the volume 2 times of the total volume of the ferric citrate and tea polyphenol mixed solution, centrifuging, adding water into the precipitate for redissolution, and carrying out water bath ultrasound for 8-30 min to obtain the ternary composite nano system.
Example 3: preparation of ferric citrate/tea polyphenol/doxorubicin hydrochloride ternary composite nano system
Precisely weighing ferric citrate, tea polyphenol and doxorubicin hydrochloride according to the weight ratio of 4:6:9, respectively dissolving in purified water, uniformly mixing in a vortex manner, mixing and stirring the prepared ferric citrate solution and the prepared tea polyphenol solution, injecting anhydrous isopropanol with the volume being 30 times of the total volume of the ferric citrate and tea polyphenol mixed solution under the stirring condition, and continuously stirring for 1-60 min. And adding the doxorubicin hydrochloride solution into the stirred system, continuously stirring for 1-60 min, then adding urea with the volume of 0.4 times of the total volume of the ferric citrate and tea polyphenol mixed solution, centrifuging, adding water to dissolve the precipitate again, and performing ultrasonic treatment on a probe for 8-30 min to obtain the ternary composite nano system.
Example 4: preparation of ferrous sulfate/catechin/epirubicin hydrochloride ternary composite nano system
Precisely weighing ferrous sulfate, catechin and epirubicin hydrochloride according to the weight ratio of 2:4:6, respectively dissolving the ferrous sulfate, the catechin and the epirubicin hydrochloride in purified water, uniformly mixing by vortex, mixing and stirring the prepared ferrous sulfate solution and the catechin solution, injecting an ethanol-water mixture with the volume being 20 times of the total volume of the ferrous sulfate and catechin mixed solution under the stirring condition, and continuously stirring for 1-60 min. And adding epirubicin hydrochloride solution into the stirred system, continuously stirring for 1-60 min, then adding sodium bicarbonate with the volume 50 times of the total volume of the ferrous sulfate and catechin mixed solution, centrifuging, adding water into the precipitate for redissolution, and performing ultrasonic treatment on the probe for 8-30 min to obtain the ternary composite nano system.
Example 5: preparation of ferrous sulfate/epicatechin/epirubicin hydrochloride ternary composite nano system
Precisely weighing ferrous sulfate, epicatechin and epirubicin hydrochloride according to the weight ratio of 2:4:6, respectively dissolving the ferrous sulfate, the epicatechin and the epirubicin hydrochloride in purified water, uniformly mixing by vortex, mixing and stirring the prepared ferrous sulfate solution and the epicatechin solution, injecting a methanol-water mixture with the volume 40 times of the total volume of the ferrous sulfate and epicatechin mixed solution under the stirring condition, and continuously stirring for 1-60 min. And adding epirubicin hydrochloride solution into the stirred system, continuously stirring for 1-60 min, then adding sodium bicarbonate with the volume which is 42 times of the total volume of the ferrous sulfate and epicatechin mixed solution, centrifuging, adding water into the precipitate for redissolution, and performing water bath ultrasound for 8-30 min to obtain the ternary composite nano system.
Example 6: preparation of ferric ammonium citrate/resveratrol/mitoxantrone hydrochloride ternary composite nano system
Accurately weighing ferric ammonium citrate, resveratrol and mitoxantrone hydrochloride according to the weight ratio of 1:5:10, respectively dissolving in purified water, uniformly mixing by vortex, mixing the prepared ferric ammonium citrate solution and the resveratrol solution, stirring, injecting an isopropanol water mixture with the volume being 60 times of the total volume of the ferric ammonium citrate and resveratrol mixed solution under the stirring condition, and continuously stirring for 1-60 min. Adding the mitoxantrone hydrochloride solution into the stirred system, continuously stirring for 1-60 min, then adding hexamethylenetetramine with the volume being 34 times of the total volume of the mixed solution of ferric ammonium citrate and resveratrol, centrifuging, adding water into the precipitate for redissolution, and performing ultrasonic treatment on the precipitate for 8-30 min to obtain the ternary composite nano system.
Example 7: preparation of ferric ammonium citrate/resveratrol/epirubicin hydrochloride ternary composite nano system
Accurately weighing ferric ammonium citrate, resveratrol and epirubicin hydrochloride according to the weight ratio of 1:5:10, respectively dissolving in purified water, uniformly mixing in a vortex manner, mixing the prepared ferric ammonium citrate solution and resveratrol solution, stirring, adding anhydrous methanol with the volume 50 times of the total volume of the ferric ammonium citrate and resveratrol mixed solution under the stirring condition, and continuously stirring for 1-60 min. And adding epirubicin hydrochloride solution into the stirred system, continuously stirring for 1-60 min, then adding hexamethylene tetramine with the volume being 26 times of the total volume of the mixed solution of ferric ammonium citrate and resveratrol, centrifuging, precipitating, adding water for redissolution, and performing ultrasonic treatment by using a probe for 8-30 min to obtain the ternary composite nano system.
Example 8: preparation of ferrous fumarate/apple polyphenol/idarubicin hydrochloride ternary composite nano system
Precisely weighing ferrous fumarate, apple polyphenol and idarubicin hydrochloride according to the weight ratio of 2:6:5, respectively dissolving in purified water, uniformly mixing in a vortex manner, mixing and stirring the prepared ferrous fumarate solution and the prepared apple polyphenol solution, adding absolute ethyl alcohol with the volume being 60 times of the total volume of the ferrous fumarate and apple polyphenol mixed solution under the stirring condition, and continuously stirring for 1-60 min. And adding the idarubicin hydrochloride solution into the stirred system, continuously stirring for 1-60 min, then adding ammonium bicarbonate with the volume 36 times of the total volume of the ferrous fumarate and apple polyphenol mixed solution, centrifuging, adding water into the precipitate for redissolution, and performing water bath ultrasound for 8-30 min to obtain the ternary composite nano system.
Example 9: preparation of ferrous fumarate/apple polyphenol/aclarubicin hydrochloride ternary composite nano system
Precisely weighing ferrous fumarate, apple polyphenol and aclarubicin hydrochloride according to the weight ratio of 2:6:5, respectively dissolving in purified water, uniformly mixing in a vortex manner, mixing and stirring the prepared ferrous fumarate solution and the prepared apple polyphenol solution, injecting an ethanol-water mixture with the volume being 80 times of the total volume of the ferrous fumarate and apple polyphenol mixed solution under the stirring condition, and continuously stirring for 1-60 min. And adding the aclarubicin hydrochloride solution into the stirred system, continuously stirring for 1-60 min, then adding ammonium bicarbonate with the volume 50 times of the total volume of the ferrous fumarate and apple polyphenol mixed solution, centrifuging, adding water into the precipitate for redissolution, and performing ultrasonic treatment by using a probe for 8-30 min to obtain the ternary composite nano system.
Example 10: preparation of ferrous chloride/tannic acid/indocyanine green ternary composite nano system
Accurately weighing ferric chloride, tannic acid and indocyanine green according to the weight ratio of 1:5:4, respectively dissolving the ferric chloride, the tannic acid and the indocyanine green in purified water, uniformly mixing the ferric chloride, the tannic acid and the indocyanine green in a vortex manner, mixing and stirring the prepared ferric chloride solution and tannic acid solution, injecting anhydrous isopropanol with the volume being 40 times of the total volume of the ferrous chloride and tannic acid mixed solution under the stirring condition, and continuously stirring for 1-60 min. And adding the indocyanine green solution into the stirred system, continuously stirring for 1-60 min, then adding disodium ethylene diamine tetraacetate with the volume being 34 times of the total volume of the ferrous chloride and tannic acid mixed solution, centrifuging, adding water into the precipitate for redissolution, and performing ultrasonic treatment by using a probe for 8-30 min to obtain the ternary composite nano system.
Example 11: preparation of ferrous chloride/eriodictyol/paclitaxel ternary composite nano system
Accurately weighing ferric chloride, eriodictyol and paclitaxel according to the weight ratio of 1:5:10, respectively dissolving in purified water, uniformly mixing by vortex, mixing and stirring the prepared ferric chloride solution and eriodictyol solution, injecting anhydrous methanol with the volume 50 times of the total volume of the ferric chloride and eriodictyol mixed solution under the stirring condition, and continuously stirring for 1-60 min. And adding the paclitaxel solution into the stirred system, continuously stirring for 1-60 min, then adding ammonia water with the volume 15 times of the total volume of the mixed solution of the ferric chloride and the eriodictyol, centrifuging, adding water into the precipitate for redissolution, and performing ultrasonic treatment on the precipitate for 8-30 min by using a probe to prepare the ternary composite nano system.
Example 12: preparation of ferrous chloride/eriodictyol/methotrexate ternary composite nano system
Accurately weighing ferric chloride, eriodictyol and methotrexate according to the weight ratio of 1:5:10, respectively dissolving the ferric chloride, the eriodictyol and the methotrexate in purified water, uniformly mixing by vortex, mixing and stirring the prepared ferric chloride solution and the eriodictyol solution, injecting an isopropanol water mixture with the volume being 70 times of the total volume of the ferric chloride and eriodictyol mixed solution under the stirring condition, and continuously stirring for 1-60 min. And adding the methotrexate solution into the stirred system, continuously stirring for 1-60 min, then adding ammonia water with the volume being 18 times of the total volume of the mixed solution of the ferric chloride and the eriodictyol, centrifuging, adding water into the precipitate for redissolution, and carrying out water bath ultrasound for 8-30 min to obtain the ternary composite nano system.
Example 13: preparation of ferric chloride/bisabolol catechin/melphalan ternary composite nano system
Accurately weighing ferric chloride, bisacodyl phenol catechin and melphalan according to the weight ratio of 3:6:9, respectively dissolving in purified water, uniformly mixing in a vortex manner, mixing and stirring the prepared ferric chloride solution and the bisacodyl phenol catechin solution, injecting anhydrous ethanol with the volume being 30 times of the total volume of the ferric chloride and bisacodyl phenol catechin mixed solution under the stirring condition, and continuously stirring for 1-60 min. And adding the melphalan solution into the stirred system, continuously stirring for 1-60 min, then adding 17 times of ammonia water by volume of the total volume of the mixed solution of ferric chloride and bisabolol catechin, centrifuging, adding water into the precipitate for redissolution, and performing ultrasonic treatment on the precipitate for 8-30 min to obtain the ternary composite nano system.
Example 14: preparation of ferric chloride/epigallophenol catechin/melphalan ternary composite nano system
Accurately weighing ferric chloride, epigallocatechin and melphalan according to the weight ratio of 3:6:9, respectively dissolving the ferric chloride, the epigallocatechin and the melphalan in purified water, uniformly mixing by vortex, mixing and stirring the prepared ferric chloride solution and the epigallocatechin solution, injecting anhydrous methanol with the volume being 45 times of the total volume of the ferric chloride and epigallocatechin mixed solution under the stirring condition, and continuously stirring for 1-60 min. And adding the melphalan solution into the stirred system, continuously stirring for 1-60 min, then adding ammonia water with the volume being 24 times of the total volume of the mixed solution of ferric chloride and epigallocatechin, centrifuging, adding water into the precipitate for redissolution, and carrying out ultrasonic treatment on the precipitate for 8-30 min to obtain the ternary composite nano system.
Example 15: preparation of ferric chloride/grape polyphenol/ondansetron ternary composite nano system
Accurately weighing ferric chloride, grape polyphenol and ondansetron according to the weight ratio of 5:6:8, respectively dissolving the ferric chloride, the grape polyphenol and the ondansetron in purified water, uniformly mixing by vortex, mixing and stirring the prepared ferric chloride solution and the prepared grape polyphenol solution, injecting absolute ethyl alcohol with the volume 40 times of the total volume of the ferric chloride and grape polyphenol mixed solution under the stirring condition, and continuously stirring for 1-60 min. Adding the ondansetron solution into the stirred system, continuously stirring for 1-60 min, then adding ammonia water with the volume being 27 times of the total volume of the mixed solution of ferric chloride and grape polyphenol, centrifuging, adding water to dissolve the precipitate again, and performing ultrasonic treatment on a probe for 8-30 min to obtain the ternary composite nano system.
Example 16: preparation of ferric chloride/grape polyphenol/irinotecan hydrochloride ternary composite nano system
Accurately weighing ferric chloride, grape polyphenol and irinotecan hydrochloride according to the weight ratio of 5:6:8, respectively dissolving in purified water, uniformly mixing by vortex, mixing and stirring the prepared ferric chloride solution and the prepared grape polyphenol solution, injecting an ethanol-water mixture with the volume being 100 times of the total volume of the ferric chloride and grape polyphenol mixed solution under the stirring condition, and continuously stirring for 1-60 min. And adding the irinotecan hydrochloride solution into the stirred system, continuously stirring for 1-60 min, then adding ammonia water with the volume being 48 times of the total volume of the mixed solution of ferric chloride and grape polyphenol, centrifuging, adding water into the precipitate for redissolution, and carrying out water bath ultrasound for 8-30 min to obtain the ternary composite nano system.
Example 17: particle size measurement of ternary composite nano system
The ternary complex nanosystems prepared in examples 1 to 16, 1mL diluted to 3mL with water, were measured with a particle size analyzer (malverm Instruments, Malvern, UK) and the results are shown in table 1. As can be seen from the table, the particle size of the ternary composite nano system prepared by the method reaches the nanometer level, and the particle size distribution is uniform.
TABLE 1 characterization of ternary composite nanosystems
Figure BDA0001961449350000091
Example 18: drug loading of ternary composite nanosystems
The drug loading detection method comprises the following steps: and drawing a standard curve of peak area and concentration of the therapeutic drug containing the conjugated structure at the maximum absorption wavelength by adopting an ultraviolet absorption photometry. And (3) measuring the peak area of the ternary composite nano system after carrying the medicine at the maximum absorption wavelength, calculating the concentration according to a standard curve, and calculating the medicine carrying amount according to a formula (1). As can be seen from Table 2, the drug loading of each system is more than 6.5%, and the system is proved to have higher drug loading efficiency, thereby being beneficial to improving the selective aggregation of the drug at the tumor part, improving the drug loading of the drug and reducing the side effect of the drug.
Figure BDA0001961449350000101
Note: m1(mg) is the input mass of the small-molecule antitumor active compound containing benzene ring, M2(mg) is the mass of the benzene ring-containing small-molecule antitumor active compound in the supernatant, M3(mg) mass of iron compound input, M4(mg) represents the charged mass of the polyphenol compound.
TABLE 2 drug loading of ternary composite nanosystems
Figure BDA0001961449350000102
Example 19: PI single staining method for detecting inhibition effect of different systems on MCF7 cells
MCF7 cells were collected at 5X 105Inoculating the mixture into a 6-well plate, incubating for 24h at 37 ℃, absorbing the culture solution, respectively adding 0.35 mu g/mL of free micromolecule antitumor active compound containing benzene ring and 2mL of ternary composite nano solution of micromolecule antitumor active compound containing benzene ring, polyphenol compound and iron compound, incubating for 48h at 37 ℃, adding 100 mu L of PI, gently mixing, and reacting for 15 min at room temperature in a dark place or for 30min at 4 ℃. Add 2mL of labeling buffer and machine (flow cytometer) detect immediately. Fluorescence intensity of each group was measured, and fluorescence intensity of the blank group and fluorescence intensity of the control group were measured in the same manner, where n was 6, and PI (%) value of the flow cytometer was recorded. It can be seen from table 3 that the cytotoxicity (PI%) of the ternary complex nanosystem is significantly higher than that of the free drug, and the preparation can be used in combination with photodynamic therapy, thus realizing the combined use of chemotherapeutic drugs and photodynamic therapy and having good anti-tumor effect.
TABLE 3 PI Single staining method for detecting the inhibition of MCF7 cells by different systems
Figure BDA0001961449350000111
Figure BDA0001961449350000121
Note: compared with the free drug group**P<0.01, compared with the ternary composite nano system group without laser irradiation##P<0.01。
Example 20: PI single staining method for detecting inhibition effect of different systems on human breast cancer multidrug resistance cell line MCF-7/ADR cells
Taking MCF7/ADR cells at 5X 105Inoculating the mixture into a 6-well plate, incubating for 24h at 37 ℃, absorbing the culture solution, respectively adding 0.35 mu g/mL of free micromolecule antitumor active compound containing benzene ring and 2mL of ternary composite nano solution of micromolecule antitumor active compound containing benzene ring, polyphenol compound and iron compound, incubating for 48h at 37 ℃, adding 100 mu L of PI, gently mixing, and reacting for 15 min at room temperature in a dark place or for 30min at 4 ℃. Add 2mL of labeling buffer and machine (flow cytometer) detect immediately. Fluorescence intensity of each group was measured, and fluorescence intensity of the blank group and fluorescence intensity of the control group (n-6) were measured in the same manner, and PI (%) values of the flow cytometer were recorded. It can be seen from table 4 that the therapeutic effect (PI%) of the ternary complex nano-system group on drug-resistant cell lines is significantly stronger than that of free drugs, and it is proved that the ternary complex nano-system also has a better therapeutic effect on drug-resistant tumor cells.
TABLE 4 PI Single staining method for detecting the inhibition of MCF7/ADR cells by different systems
Figure BDA0001961449350000131
Figure BDA0001961449350000141
Note: compared with the free drug group**P<0.01, compared with the ternary composite nano system group without laser irradiation##P<0.01。
Example 21 photothermal test
And recording the photo-thermal performance of the small-molecular anti-tumor active compound-polyphenol compound-iron compound ternary composite nano solution containing the benzene ring by using a thermal infrared imager so as to monitor the temperature. PBS dispersions containing different concentrations of nanoparticles (10. mu.g/mL) were exposed to laser light (808nm, 0.5W/cm)2) Irradiation was continued for 10min and the temperature change was recorded. From table 5, it can be seen that the ternary nano-system group has better photo-thermal conversion capability, and the ternary composite nano-system of the invention can be used for photo-thermal treatment, so that a novel photo-thermal material is developed.
TABLE 5 temperature Change of ternary composite nanosystems
Figure BDA0001961449350000151
Example 22 ROS detection assay
MCF7 cells were collected at 5X 105Inoculating the mixture into a 6-well plate, incubating for 24h at 37 ℃, absorbing the culture solution, respectively adding 0.35 mu g/mL of free micromolecule antitumor active compound containing benzene rings and 2mL of ternary composite nano solution of micromolecule antitumor active compound containing benzene rings, polyphenol compound and iron compound, incubating for 48h at 37 ℃, adding a DHE fluorescent probe, gently mixing uniformly, and incubating for 30min in an incubator. After incubation, the cells were washed three times with PBS, resuspended in 1mL ice-cold PBS, and immediately tested on the machine (flow cytometer). The fluorescence intensity of each group was measured, and the fluorescence intensity of the blank group and the fluorescence intensity of the control group were measured in the same manner, and the relative concentration of ROS was calculated according to the formula (2). It is known from table 6 that the ternary complex nanosystem group can generate a large amount of ROS, which indicates that an iron compound and a multi-class compound generate fenton reaction in cells to induce the generation of ROS, and a large amount of ROS is synergistically induced by combining laser irradiation with photodynamic therapy, so that the three-element complex nanosystem group has a better killing effect on cells.
Figure BDA0001961449350000161
TABLE 6 ROS detection experiment
Figure BDA0001961449350000162
Figure BDA0001961449350000171
Example 23 LPO detection experiment
MCF7 cells were collected at 5X 105The cells were inoculated in 6-well plates per well, incubated at 37 ℃ for 24h, the culture solution was aspirated, 0.35. mu.g/mL of each of a small molecule antitumor active compound free of benzene rings, a small molecule antitumor active compound containing benzene rings-a polyphenol compound-an iron compound ternary complex nano solution was added for 2mL, incubated at 37 ℃ for 48h, washed 3 times with PBS, cells were digested with 0.25% trypsin, centrifuged at 1000R for 5min, the cells were resuspended with 0.5mL of PBS and transferred to a glass test tube, an equal volume of Extract R saturated methanol was added to each tube and vortexed, then 1mL of cold chloroform was added to each tube, vortexed to mix well, and the mixture was centrifuged at 0 ℃ for 5min at 1,500 Xg, and the bottom chloroform layer was collected with a Pasteur pipette or syringe needle. Transferring the chloroform layer to another test tube, storing on ice, adding a certain amount of developing solution, and detecting light absorption value with a microplate reader at 500nm wavelength. The fluorescence intensity of each group was measured, and the fluorescence intensity of the blank group and the fluorescence intensity of the control group were measured in the same manner, and the relative concentration of LPO was calculated according to the formula (3). As can be seen from table 7, the ternary composite nanosystem can induce a large amount of LPO, and has significant difference from the free preparation group and the control group, and the promotion effect of the illumination group on LPO is more obvious, which indicates that the ternary nanosystem group can generate LPO to induce iron death, and can further promote the occurrence of iron death when combined with laser irradiation, thereby realizing the combined use of phototherapy and iron death, and the phototherapy has a promotion effect on iron death.
Figure BDA0001961449350000181
Table 7 LPO measurement of relative concentration of different systems to LPO in MCF7 cells
Figure BDA0001961449350000182
Figure BDA0001961449350000191
Example 24 pharmacodynamic evaluation
The tumor-bearing mice were divided into 22 groups, each group containing 3 mice each, negative Control group (Control), free adriamycin group, ferric chloride/tannic acid/adriamycin hydrochloride + Laser group, free indocyanine green group, ferric chloride/tannic acid/indocyanine green + Laser group, free paclitaxel group, ferric chloride/eriodictyol/paclitaxel group, free methotrexate group, ferric chloride/eriodictyol/methotrexate group, free melphalan group, ferric chloride/bisabolol catechin/melphalan group, and the like, Free ondansetron group, ferric chloride/grape polyphenol/ondansetron + Laser group, free irinotecan hydrochloride group, ferric chloride/grape polyphenol/irinotecan hydrochloride + Laser group, when the tumor volume of the mouse is 100mm3In cases of administration, free doxorubicin (5mg/kg), iron chloride/tannic acid/doxorubicin hydrochloride group (doxorubicin dose of 2mg/kg), iron chloride/tannic acid/doxorubicin hydrochloride + Laser group (doxorubicin dose of 2mg/kg), free indocyanine green group (350 μ g/kg), iron chloride/tannic acid/indocyanine green group (indocyanine green dose of 140 μ g/kg), iron chloride/tannic acid/indocyanine green + Laser group ((indocyanine green dose of 140 μ g/kg), free paclitaxel (10mg/kg), iron chloride/eriodictyol/paclitaxel group (paclitaxel dose of 4mg/kg), iron chloride/eriodictyol/paclitaxel + Laser group (paclitaxel dose of 4mg/kg) and the like were injected via the tail vein on days 1,3 and 5, Free methotrexate group (3mg/kg), iron chloride/eriodictyol/methotrexate group (methotrexate dose)1.2mg/kg), iron chloride/eriodictyol/methotrexate + Laser group (methotrexate dose of 1.2mg/kg), free melphalan group (7.5mg/kg), iron chloride/bisabolol catechin/melphalan group (melphalan dose of 3mg/kg), iron chloride/bisabolol catechin/melphalan + Laser group (melphalan dose of 3mg/kg), free ondansetron group (8mg/kg), iron chloride/grape polyphenol/ondansetron group (ondansetron dose of 3.2mg/kg), iron chloride/grape polyphenol/ondansetron + Laser group (ondansetron dose of 3.2mg/kg), free irinotecan hydrochloride group (2mg/kg), iron chloride/grape polyphenol/irinotecan hydrochloride group (irinotecan dose of 0.8mg/kg), Ferric chloride/grape polyphenol/irinotecan hydrochloride + Laser group (irinotecan dose 0.8mg/kg), for the combined Laser treatment experimental group, the tumor of the mouse was irradiated with a Laser at 808nm at 1W/cm 2 for 5 minutes. On day 21 of the experiment, mice were euthanized and mice were daily recorded for weight and tumor size for tumor volume and weight calculation and tumor inhibition efficiency calculation. It can be seen from table 8 that the ternary complex nanosystem group has a better therapeutic effect on tumor-bearing mice, because the well-formulated nanoparticles are aggregated at the tumor site by the EPR effect, the anti-tumor effect of the free drug is improved, and the toxic and side effects of the free drug on the mice are reduced.
TABLE 8 pharmacodynamic evaluation of different ternary complex formulations
Figure BDA0001961449350000201
Figure BDA0001961449350000211
Note: compared with Control group**P<0.01, compared to the free drug group##P<0.01, compared with the group without the laser preparation@@P<0.01
Example 25 toxicity test experiment
Dividing tumor-bearing mice into 22 groups, each group containing 3 mice is negative Control group (Control), free adriamycin group, ferric chloride/tannic acid/adriamycin hydrochloride group+ Laser group, free indocyanine green group, ferric chloride/tannic acid/indocyanine green + Laser group, free paclitaxel group, ferric chloride/eriodictyol/paclitaxel + Laser group, free methotrexate group, ferric chloride/eriodictyol/methotrexate group, free melphalan group, ferric chloride/eriodictyol/methotrexate group, ferric chloride/guaiacol/melphalan group, ferric chloride/guaiacol catechin/melphalan group, free ondansetron group, ferric chloride/grape polyphenol/ondansetron group, free irinotecan hydrochloride group, ferric chloride/grape polyphenol/irinotecan hydrochloride group, irinotecan group, Ferric chloride/grape polyphenol/irinotecan hydrochloride + Laser group, when the tumor volume of the mouse grows to 100mm3In cases of administration, free doxorubicin (5mg/kg), iron chloride/tannic acid/doxorubicin hydrochloride group (doxorubicin dose of 2mg/kg), iron chloride/tannic acid/doxorubicin hydrochloride + Laser group (doxorubicin dose of 2mg/kg), free indocyanine green group (350 μ g/kg), iron chloride/tannic acid/indocyanine green group (indocyanine green dose of 140 μ g/kg), iron chloride/tannic acid/indocyanine green + Laser group ((indocyanine green dose of 140 μ g/kg), free paclitaxel (10mg/kg), iron chloride/eriodictyol/paclitaxel group (paclitaxel dose of 4mg/kg), iron chloride/eriodictyol/paclitaxel + Laser group (paclitaxel dose of 4mg/kg) and the like were injected via the tail vein on days 1,3 and 5, Free methotrexate group (3mg/kg), iron chloride/eriodictyol/methotrexate group (methotrexate dose 1.2mg/kg), iron chloride/eriodictyol/methotrexate + Laser group (methotrexate dose 1.2mg/kg), free melphalan group (7.5mg/kg), iron chloride/bisabolol catechin/melphalan group (melphalan dose 3mg/kg), iron chloride/bisabolol catechin/melphalan + Laser group (melphalan dose 3mg/kg), free ondansetron group (8mg/kg), iron chloride/grape polyphenol/ondansetron group (ondansetron dose 3.2mg/kg), iron chloride/grape polyphenol/ondansetron + Laser group (ondansetron dose 3.2mg/kg), free irinotecan hydrochloride group (2mg/kg), Iron chloride/grape polyphenol/irinotecan hydrochloride group (irinotecan dose 0.8mg/kg), iron chloride/grape polyphenol/irinotecan hydrochloride + Laser group (irinotecan dose 0.8mg/kg), and for the experimental group combined with Laser treatment, 1W Laser was applied at 808nm/cm 2Tumors of mice were irradiated for 5 minutes. On day 21 of the experiment, mice were euthanized, and heart, liver, spleen, lung and kidney were taken to prepare pathological sections and examined for pathology. It can be known from table 9 that the free drug has high toxic and side effects on normal tissues, and the ternary complex nano system can effectively improve the toxic and side effects generated by the free drug, because the polyphenol compound is a good hydrogen donor, plays a role in reducing in cells, and reduces the damage of ROS to normal tissues. The preparation has good anti-tumor effect, good protection effect on normal tissues and wide use value.
TABLE 9 toxicity evaluation
Figure BDA0001961449350000221
Figure BDA0001961449350000231
Note: "-" no damage, "+" mild damage, "+ +" moderate damage.

Claims (4)

1. The ternary composite nano system for preparing the antitumor drugs is characterized by comprising an iron compound, a small molecular antitumor active compound containing a benzene ring and a polyphenol compound in a weight ratio of 1-4:2-10: 5-20; the ternary composite nano system is prepared by the following method: self-assembling an iron compound, a micromolecular antitumor active compound containing a benzene ring and a polyphenol compound in a single-phase solution environment to form a ternary composite nano system; the iron compound is at least one of ferric ammonium citrate, ferric citrate, ferrous sulfate, ferric ammonium citrate, ferrous fumarate, ferrous chloride and ferric chloride hexahydrate; the micromolecule antitumor active compound containing the benzene ring is at least one of adriamycin hydrochloride, idarubicin hydrochloride, daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride, epirubicin hydrochloride, mitoxantrone mesylate, melphalan, ondansetron, indocyanine green, methotrexate, irinotecan hydrochloride and paclitaxel; the polyphenol compound is at least one of tannic acid, catechin, epicatechin, bisabolol catechin, epigallophenol catechin, tea polyphenol, apple polyphenol, eriodictyol, grape polyphenol and resveratrol; the method comprises the following specific steps:
(1) respectively weighing an iron compound, a micromolecular antitumor active compound containing a benzene ring and a polyphenol compound, and preparing into an aqueous solution;
(2) mixing the iron compound and the polyphenol compound solution, and adding an organic solvent in the mixing process;
(3) adding a small-molecule anti-tumor active compound solution containing benzene rings into the mixed solution in the step (2), uniformly mixing, adding a precipitation solvent, centrifuging, redissolving, and carrying out ultrasonic treatment to collect a product.
2. The ternary complex nanosystem as claimed in claim 1, wherein the iron compound and the polyphenol compound are first assembled by coordination chemistry to form a pre-assembled element, and then conjugated with the small molecule antitumor active compound containing benzene ring by pi-pi to form the ternary complex nanosystem.
3. The ternary composite nanosystem for preparing antitumor drugs according to claim 1, wherein the organic solvent in step (2) is at least one of ethanol, ethanol water, methanol water, isopropanol, and isopropanol water.
4. The ternary composite nanosystem for preparing antitumor drugs according to claim 1, wherein the precipitation solvent in step (3) is at least one of ammonia, sodium carbonate, urea, ammonium bicarbonate, hexamethylenetetramine, disodium ethylenediaminetetraacetate, and sodium hydroxide solution.
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