Adriamycin-tetravalent platinum drug-norcantharidin three-drug combined nano-drug as well as preparation method and application thereof

Abstract

The invention discloses an adriamycin-tetravalent platinum drug-norcantharidin three-drug combined nano-drug as well as a preparation method and application thereof, and belongs to the technical field of medical materials. A tetravalent platinum drug modified by demethylated cantharidin and adriamycin are used as raw materials, a novel nano-drug is constructed through Cu < 2 + > coordination, and then the nano-drug combining adriamycin, the tetravalent platinum drug and demethylated cantharidin is prepared by polymerizing polydopamine with a mild photo-thermal effect. The polymer has the capacity of controllably releasing drugs under acidity and reduction, can realize photo-thermal conversion under laser irradiation to generate a mild photo-thermal effect, has a better tumor cell killing effect, has the capacity of consuming glutathione and generating hydroxyl radicals, can enter cancer cells in an endocytosis mode and release DOX, and can be used for killing tumor cells. And a new thought is provided for multi-drug combination and chemotherapy-chemokinetics therapy-photothermal therapy multi-mode combined therapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, and specifically relates to a doxorubicin-quadrivalent platinum-norcanthracen triple-drug combination nanomedicine and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] As we all know, doxorubicin is an antitumor antibiotic with a wide antitumor spectrum and is effective against a variety of tumors. It is a non-specific drug for the cycle and has a killing effect on tumor cells in various growth cycles. However, it also has serious side effects: cardiotoxicity, heart failure and bone marrow suppression in severe cases. Scientists have done a lot of research on doxorubicin and are also gradually overcoming its own toxicity in the hope of making it play a greater anti-cancer role. Cisplatin is a platinum (II)-based chemotherapy drug used in the first-line anti-tumor treatment of many cancers. For example, breast cancer, cervical cancer, lung cancer and liver cancer. However, it also has a non-specific distribution throughout the body, which seriously affects its clinical use and causes a series of serious consequences. Therefore, it is urgently needed to modify it to maximize its anti-cancer effect. Norcantharidin is a demethylated analogue of cantharidin, which has the characteristics of inhibiting cell proliferation, autophagy, migration, metastasis, inducing cell apoptosis and enhancing anti-cancer immunity. It is easier to synthesize and has fewer side effects. It only causes mild gastrointestinal side effects and no adverse reactions to the urinary system.

[0004] Chemotherapy has been widely used in clinical anti-tumor treatment, but the results of chemotherapy alone cannot achieve the expected effect. The main reason is that chemotherapy drugs are not specific in the process of delivery to tumor tissues and the content reaching the tumor site is very small. At the same time, long-term use of chemotherapy drugs can cause drug resistance in tumor cells, which can lead to failure, recurrence or metastasis of cancer treatment. Patent CN110051858A discloses a class of polysaccharide drug composite nanoparticles, which use Ganoderma lucidum polysaccharide as the main chain and covalently bond with norcantharidin, doxorubicin and (Z)-diamminedichloroplatinum to prepare low-toxicity composite nanoparticles, which can make the two drugs have a significant inhibitory effect on tumor cells at low levels, overcome the problems of drug resistance and toxic side effects of anticancer drugs, but when it is used to treat cancer, it is still a single treatment mode, and the treatment effect needs to be further improved. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a doxorubicin-quadrivalent platinum-norcanthracen triple-drug combination nanomedicine and its preparation method and application. The nanomedicine provided by the present invention has multiple treatment modes and can greatly improve the anti-cancer effect.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] The first aspect of the present invention provides a nano drug, which includes a nano particle core and polydopamine wrapped on the surface of the core, wherein the nano particle is self-assembled by a tetravalent platinum drug modified with norcantharidin and doxorubicin under the coordination of metal ions.

[0008] The second aspect of the present invention provides a method for preparing the above-mentioned nanomedicine, comprising:

[0009] Adding a tetravalent platinum aqueous solution modified with norcantharidin to a metal salt aqueous solution, and then dropping an adriamycin hydrochloride aqueous solution, reacting at room temperature in the dark, to obtain nanoparticles;

[0010] The nanoparticles are dispersed in water, dopamine hydrochloride is added, stirred, and then ammonia water is added, stirred in the dark to react, thereby obtaining a nanomedicine.

[0011] The third aspect of the present invention provides a use of the above-mentioned nanomedicine or the nanomedicine prepared by the above-mentioned preparation method in the preparation of anti-tumor drugs.

[0012] The fourth aspect of the present invention provides an anti-tumor drug, comprising the above-mentioned nanodrug or the nanodrug prepared by the above-mentioned preparation method.

[0013] The beneficial effects of the present invention are:

[0014] The present invention uses demethylcantharidin-modified tetravalent platinum drug (D-Pt-D) and small molecule doxorubicin (DOX) as raw materials, constructs a new type of nano drug DDC NPs through metal coordination, and then polymerizes polydopamine with mild photothermal effect to prepare nano drug PDDC NPs for the combination of DOX, tetravalent platinum drug and demethylcantharidin. PDDC NPs have an ultra-high drug loading capacity. The results of dynamic light scattering show that PDDC NPs are stable under pH 7.4 conditions, while under acidic and reducing conditions, the particle size and particle size distribution change sharply, indicating that the prepared nano drug has pH and reduction responsiveness. The results of in vitro drug release also show that PDDC NPs have the ability to controllably release drugs under acidic and reducing conditions. In vitro photothermal experiments show that PDDC NPs can achieve photothermal conversion and produce mild photothermal effects under 880nm laser irradiation. PDDC NPs have the ability to consume glutathione and generate hydroxyl free radicals. The results of cell experiments showed that PDDC NPs can enter cancer cells through endocytosis and release DOX; compared with small molecule DOX and small molecule D-Pt-D, PDDC NPs have the best tumor cell killing effect under 880nm light conditions. In summary, PDDC NPs is a promising multifunctional nanomedicine, which provides a new idea for multi-drug combination therapy, chemotherapy-chemodynamic therapy-photothermal therapy multi-modal combined therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0016] Figure 1 Schematic diagram of the preparation and action mechanism of the nano drug PDDC NPs provided by the present invention;

[0017] Figure 2 The NMR and IR spectra of OH-Pt-OH and D-Pt-D prepared in Example 1 of the present invention, wherein A is the 1 H-NMR spectrum; B is D-Pt-D 1 H-NMR spectrum; C is the FT-IR spectrum of OH-Pt-OH and D-Pt-D;

[0018] Figure 3Spectra of nanoparticles, nanomedicines, etc. prepared in Example 1 of the present invention, wherein A is a UV-visible absorption spectrum of DOX, D-Pt-D, CuCl2·2H2O, DDC NPs, and PDDC NPs in DMSO; B is a UV-visible absorption spectrum of DOX, D-Pt-D, CuCl2·2H2O, DDC NPs, and PDDC NPs in deionized water; C is a fluorescence spectrum of DOX, D-Pt-D, CuCl2·2H2O, DDC NPs, and PDDC NPs in DMSO; D is a fluorescence spectrum of DOX, D-Pt-D, CuCl2·2H2O, DDC NPs, and PDDC NPs in deionized water;

[0019] Figure 4 This is the XPS spectrum of the nanoparticles DDC NPs prepared in Example 1 of the present invention;

[0020] Figure 5 The Tyndall effect diagrams of DDC NPs and PDDC NPs prepared in Example 1 of the present invention; wherein A is the Tyndall effect diagram of DDC NPs in DMSO; B is the Tyndall effect diagram of DDC NPs in deionized water; C is the Tyndall effect diagram of PDDC NPs in DMSO; D is the Tyndall effect diagram of PDDC NPs in deionized water;

[0021] Figure 6 The morphology characterization diagrams of DDC NPs and PDDC NPs prepared in Example 1 of the present invention; wherein A is a particle size distribution diagram of DDC NPs; B is a particle size distribution diagram of PDDC NPs; C is a TEM diagram of DDC NPs; and D is a TEM diagram of PDDC NPs.

[0022] Figure 7 The stability and responsiveness characterization diagram of DDC NPs prepared in Example 1 of the present invention; wherein A is a diagram showing the change in average particle size of DDC NPs in different solutions; B is a diagram showing the change in dispersibility of DDC NPs in different solutions;

[0023] Figure 8 The stability and responsiveness characterization diagram of PDDC NPs prepared in Example 1 of the present invention; wherein A is a diagram showing the change in average particle size of PDDC NPs in different solutions; B is a diagram showing the change in dispersibility of PDDC NPs in different solutions;

[0024] Fig. 9 The photothermal effect diagram of PDDC NPs prepared in Example 1 of the present invention; wherein A is the photothermal effect of PDDC NPs under laser irradiation (808nm, 1.5W / cm 2) photothermal effect diagram; B is the photothermal effect diagram of PDDC NPs irradiated with different laser intensities (0.5 mg / mL); C is the photothermal effect diagram of PDDC NPs (0.5 mg / mL) irradiated with 808 nm laser (1.5 W / cm 2 ) photothermal stability curve under irradiation; D is the relationship between the negative natural logarithm of -ln(θ) and the cooling time of PDDC NPs;

[0025] Fig.10 808nm (1.5W / cm 2 )’s thermal infrared images;

[0026] Fig.11 The graphs are the experimental results of GSH and H2O2 consumption of PDDC NPs prepared in Example 1 of the present invention; wherein A is the GSH consumption curve of PDDC NPs (0.1 mg / mL); B is the GSH consumption curve of PDDC NPs (0.2 mg / mL); C is the H2O2 consumption curve of PDDC NPs with different concentrations; and D is the H2O2 consumption curve of PDDC NPs (0.1 mg / mL);

[0027] Fig.12 The in vitro drug release curve of PDDC NPs prepared in Example 1 of the present invention; wherein A is the release curve of DOX, B is the release curve of Pt, and C is the release curve of DMC;

[0028] Fig.13 It is a diagram of the in vitro cell endocytosis of PDDC NPs prepared in Example 1 of the present invention; wherein, A is a CLSM image of HeLa cells incubated with small molecule DOX and PDDC NPs for 1, 3 and 6 hours (scale: 25 μm); B is a flow cytometric analysis of HeLa cells incubated with small molecule DOX and PDDC NPs for 1, 3 and 6 hours; C is a CLSM image of MCF-7 cells incubated with small molecule DOX and PDDC NPs for 1, 3 and 6 hours (scale: 25 μm); D is a flow cytometric analysis of MCF-7 cells incubated with small molecule DOX and PDDC NPs for 1, 3 and 6 hours;

[0029] Fig.14 Cell viability of HeLa cells incubated with different concentrations of DOX+D-Pt-D and PDDC NPs in the dark or under laser irradiation for 24 h (A) and 48 h (B);

[0030] Fig.15 DLS image (A) and TEM image (B) of the iron-coordinated nanoparticles DDC NPs obtained in Example 8 of the present invention;

[0031] Fig.16is the standard curve of DOX at 484 nm;

[0032] Fig.17 This is the drug loading amount of PDDC NPs obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0033] Chemotherapy has been widely used in clinical anti-tumor treatment, but the results of chemotherapy alone cannot achieve the expected results. The main reason is that chemotherapy drugs are not specific during delivery to tumor tissues and the content reaching the tumor site is very small. At the same time, long-term use of chemotherapy drugs can cause drug resistance in tumor cells, which can lead to failure of cancer treatment, recurrence or metastasis. Therefore, chemodynamic therapy (CDT) has gradually come into the field of vision of researchers. CDT is a new method for treating tumors by activating nanodrugs in the tumor microenvironment to produce Fenton (Fenton-like) reactions and generate strong oxidizing hydroxyl radicals. Among them, the use of exogenous metal ions to induce chemodynamic therapy has been widely studied. Copper is a common metal and a transition metal element with redox activity. Under conventional chemical reactions and physiological conditions, oxidized Cu 2+ Can be converted into reduced Cu + . Copper ions participate in a variety of biochemical reactions by providing or accepting electrons. Among them, copper mediates the Fenton reaction to catalyze the synthesis of the most active hydroxyl radicals, which leads to an increase in the concentration of ROS in tumor cells and kills tumor cells. In addition, copper can oxidize reduced glutathione into oxidized glutathione disulfide, leading to the consumption of the antioxidant glutathione, thereby interfering with the glutathione-related antioxidant defense system. Once the system is disturbed, its scavenging effect on highly active hydroxyl radicals will be weakened, leading to tumor cell apoptosis.

[0034] As the research on light-mediated photothermal therapy (PTT) and photodynamic therapy (PDT) becomes more and more in-depth, they are increasingly valued in tumor treatment. PTT can convert near-infrared (NIR) light energy into heat energy through photothermal agents, and can quickly and effectively kill tumor cells in the presence of photosensitizers and laser irradiation. However, due to the uneven distribution of photosensitizers, the efficiency of PTT will be significantly reduced, and the large amount of heat generated by PTT may damage normal cells and tissues. Therefore, it is necessary to choose a suitable photosensitizer. Dopamine has strong absorption properties and has high absorption peaks in the visible light and near-infrared spectrum regions. This makes it a high-quality optical material. Under light, the energy absorbed in dopamine can be converted into heat energy, thereby increasing the temperature to achieve a mild photothermal effect. In addition, dopamine has good biocompatibility, which expands its application in tumor treatment.

[0035] Nowadays, a single treatment method can no longer meet people's needs, and long-term use of chemotherapy drugs will cause cancer patients' bodies to develop a certain drug resistance, which will greatly reduce the anti-cancer effect of chemotherapy drugs.

[0036] Therefore, the present invention has developed a new type of nano drug, which can greatly improve the anti-cancer effect by combining multiple treatment methods. The present invention first uses small molecule DOX and tetravalent platinum drugs modified by norcantharidin as raw materials, and self-assembles to prepare carrier-free nano drug DDC NPs under the coordination of metal ions, and then polymerizes polydopamine on the surface to construct a new type of nano drug PDDCNPs to achieve multi-mode synergistic anti-tumor effects of chemotherapy, chemodynamic therapy and photothermal therapy (such as Figure 1 As described). First, dopamine exerts its mild photothermal effect under the irradiation of near-infrared light and releases the drug it encapsulates. Secondly, the copper ions in the drug can perform chemodynamic therapy, reduce the content of glutathione in tumor cells, increase the content of hydroxyl free radicals, and accelerate the death of tumor cells. Finally, chemotherapy drugs such as doxorubicin, platinum (IV) drugs and norcantharidin can further kill tumor cells and enhance the effect of tumor treatment. The nanomedicine provided by the present invention can significantly improve the anti-tumor effect through chemotherapy, chemodynamic therapy and photothermal therapy.

[0037] The innovative features of the nano drug PDDC NPs provided by the present invention are:

[0038] ①Preparation of carrier-free nanomedicines by small molecule co-assembly method, which is simple to prepare;

[0039] ② Co-delivery of three chemotherapy drugs, DOX, platinum drugs, and demethylcantharidin, through a simple, integrated nano-drug delivery system;

[0040] ③Metal ions (Fe 3+ , Gd 3+ , Mn 2+ 、Co 2+ 、Ni 2+ and Cu 2+ etc.) can drive self-assembly through metal coordination and also have the effect of chemodynamic therapy;

[0041] ④PDDC NPs have synergistic anti-tumor effects of chemotherapy, chemodynamic therapy, and photothermal therapy.

[0042] Specifically, the present invention provides a nanomedicine for the combination of doxorubicin, a tetravalent platinum drug and norcantharidin, wherein the nanomedicine comprises nanoparticles and polydopamine coated on the surface of the nanoparticles, wherein the nanoparticles are self-assembled by tetravalent platinum drug modified with norcantharidin (DMC) and doxorubicin (DOX) under the coordination of metal ions.

[0043] According to the present invention, the structure of the tetravalent platinum drug modified with norcantharidin is as follows:

[0044]

[0045] It should be noted that the nano drug provided by the present invention has an ultra-high drug loading capacity. The present invention has no special requirements for the amount of doxorubicin in the nano drug, and those skilled in the art can select a suitable loading amount according to actual needs. The present invention has no special requirements for the thickness of the outer shell polydopamine, as long as it can completely cover the inner core.

[0046] In some embodiments of the present invention, the metal ion is selected from Fe 3+ , Gd 3+ , Mn 2+ 、Co 2+ 、Ni 2+ and Cu 2+ One of them.

[0047] In some embodiments of the present invention, the nanoparticles are spherical and have a particle size of 140-180 nm.

[0048] In some embodiments of the present invention, the nanomedicine is spherical and has a particle size of 200-250 nm.

[0049] The present invention also provides a method for preparing the above-mentioned nanomedicine, comprising:

[0050] Adding a tetravalent platinum aqueous solution modified with norcantharidin to a metal salt aqueous solution, and then dropping an adriamycin hydrochloride aqueous solution, reacting at room temperature in the dark, to obtain nanoparticles;

[0051] The nanoparticles are dispersed in water, dopamine hydrochloride is added, stirred, and then ammonia water is added, stirred in the dark to react, thereby obtaining a nanomedicine.

[0052] In some embodiments of the present invention, the mass ratio of doxorubicin hydrochloride (DOX), tetravalent platinum drug modified with norcantharidin (D-Pt-D) and metal salt is 1:1:0.5-1, specifically 1:1:0.5, 1:1:0.6, 1:1:0.7, 1:1:0.8, 1:1:0.9, 1:1:1, etc., preferably 1:1:0.6.

[0053] It should be noted that the metal salt includes one of an iron salt, a copper salt, a gadolinium salt, a manganese salt, a cobalt salt and a nickel salt. The iron salt includes but is not limited to iron chloride, iron sulfate or iron nitrate. The copper salt includes but is not limited to one or more of copper chloride, copper sulfate or copper nitrate, preferably copper chloride or its hydrate. The gadolinium salt includes but is not limited to gadolinium chloride or gadolinium nitrate. The manganese salt includes but is not limited to manganese sulfate. The cobalt salt includes but is not limited to cobalt chloride, cobalt sulfate and cobalt nitrate. The nickel salt includes but is not limited to nickel chloride, nickel sulfate and nickel nitrate.

[0054] In some embodiments of the present invention, the mass ratio of the nanoparticles to dopamine hydrochloride is 3-5:1, specifically 3:1, 4:1, 5:1, etc., preferably 4:1.

[0055] In some embodiments of the present invention, after the reaction at room temperature in the dark or the reaction with stirring in the dark is completed, the reaction solution is dialyzed and freeze-dried to obtain the target product;

[0056] The molecular weight cut-off of the dialysis is 900-1100D, preferably 1000D.

[0057] In some embodiments of the present invention, the synthesis route of the tetravalent platinum drug modified with norcantharidin is as follows:

[0058]

[0059]

[0060] The preparation method comprises the following steps:

[0061] Cisplatin, water and hydrogen peroxide are mixed and reacted at room temperature in the dark to obtain a hydroxyl-modified tetravalent platinum drug;

[0062] Under nitrogen protection and 65-75° C., the hydroxyl-modified tetravalent platinum drug and norcantharidin react in the dark to obtain the norcantharidin-modified tetravalent platinum drug.

[0063] According to the present invention, at room temperature, cisplatin, water and hydrogen peroxide are mixed and reacted in the dark. After the reaction is completed, the mixture is filtered under reduced pressure, and the filter residue is rinsed with an organic solvent and then dried in a vacuum and in the dark to obtain a hydroxyl-modified tetravalent platinum drug. The organic solvent is preferably one or more of acetone, ether, chloroform and ethanol, preferably acetone and ether. The temperature of vacuum and light-proof drying is preferably 30-50°C, and more preferably 40°C.

[0064] In some embodiments of the present invention, the usage ratio of cisplatin, water and hydrogen peroxide is preferably 1 g:5-10 mL:8-12 mL, and more preferably 1 g:8 mL:10 mL.

[0065] According to the present invention, a hydroxyl-modified tetravalent platinum drug, norcantharidin and an organic solvent are mixed, and then reacted in the dark at 65-75°C under nitrogen protection. After the reaction is completed, the mixture is filtered under reduced pressure, then precipitated with ether, washed with methanol and acetone, then precipitated with ether, and finally dried in a vacuum and in the dark to obtain a tetravalent platinum drug modified with norcantharidin. The organic solvent is preferably one or more of N,N-dimethylformamide, acetone and chloroform, preferably N,N-dimethylformamide.

[0066] In some embodiments of the present invention, the mass ratio of the hydroxyl-modified tetravalent platinum drug to norcantharidin is preferably 1:1.8-2.2.

[0067] The present invention also provides a use of the nano drug or the nano drug prepared by the above preparation method in the preparation of anti-tumor drugs.

[0068] According to the present invention, the tumor includes but is not limited to cervical cancer and breast cancer.

[0069] The present invention also provides an anti-tumor drug, comprising the above-mentioned nano drug or the nano drug prepared by the above-mentioned preparation method.

[0070] In some embodiments of the present invention, the anti-tumor drug further comprises pharmaceutically acceptable excipients, including but not limited to lactose, glucose, sorbitol, mannitol, xylitol, maltitol, starch, gum arabic, gelatin, calcium phosphate, cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, and mineral oil, etc., which are not specifically limited here.

[0071] According to the present invention, the tumor includes but is not limited to cervical cancer and breast cancer.

[0072] The present invention uses tetravalent platinum drug (D-Pt-D) modified with demethylcantharidin and small molecule doxorubicin (DOX) as raw materials, constructs a new type of nano drug DDC NPs by metal ion coordination, and then polymerizes polydopamine with mild photothermal effect to prepare nano drug PDDC NPs for the combination of DOX, tetravalent platinum drug and demethylcantharidin. PDDC NPs have an ultra-high drug loading capacity. The results of dynamic light scattering show that PDDC NPs are stable under pH 7.4 conditions, while under acidic and reducing conditions, the particle size and particle size distribution change sharply, indicating that the prepared nano drug has pH and reduction responsiveness. The results of in vitro drug release also show that PDDC NPs have the ability to controllably release drugs under acidic and reducing conditions. In vitro photothermal experiments show that PDDC NPs can achieve photothermal conversion and produce mild photothermal effects under 880nm laser irradiation. PDDC NPs have the ability to consume glutathione and generate hydroxyl free radicals. The results of cell experiments showed that PDDC NPs can enter cancer cells through endocytosis and release DOX; compared with small molecule DOX and small molecule D-Pt-D, PDDC NPs have the best tumor cell killing effect under 880nm light conditions. In summary, PDDC NPs is a promising multifunctional nanomedicine, which provides a new idea for multi-drug combination therapy, chemotherapy-chemodynamic therapy-photothermal therapy multi-modal combined therapy.

[0073] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0074] Example 1

[0075] A method for preparing a nano drug comprises the following steps:

[0076] 1. Synthesis of OH-Pt-OH and D-Pt-D

[0077] At room temperature, 1 g of cisplatin was weighed and added with 8 mL of H2O and 10 mL of H2O2. After the reaction was allowed to react in the dark for 24 h, the mixture was filtered under reduced pressure. The filter residue was rinsed with acetone and ether respectively, and then dried in a vacuum drying oven at 40 °C in the dark for 24 h to obtain a yellow powdery solid product (i.e., a hydroxyl-modified tetravalent platinum drug), which was named OH-Pt-OH.

[0078] Weigh OH-Pt-OH (0.6 g, 1.8 mmol) and norcantharidin (DMC) (1.21 g, 7.19 mmol) into a round-bottom flask, add DMF (17 mL), and then react at 70 ° C in the dark for 10 h under nitrogen protection. Finally, the reaction solution was filtered under reduced pressure, precipitated with ether, washed with methanol and acetone, and then precipitated with ether. Finally, it was dried in a vacuum oven at 37 ° C in the dark to obtain a light white powder solid product (i.e., a tetravalent platinum drug modified with norcantharidin), named D-Pt-D.

[0079] 2. Synthesis of DDC NPs

[0080] Take doxorubicin hydrochloride (DOX) (20 mg, 17.24 mM), D-Pt-D (20 mg, 14.92 mM), and CuCl2·2H2O (12 mg, 3.519 mM) and add water to dissolve them respectively, then add D-Pt-D solution to CuCl2·2H2O solution under ultrasound, then add DOX solution dropwise under stirring, and then react at room temperature in the dark for 5 hours. The reaction solution is dialyzed (MWCO: 1000D) for 24 hours, and finally freeze-dried to obtain a solid (i.e., nanoparticles), which is named DDC NPs.

[0081] Note: Taking doxorubicin hydrochloride as an example, "20 mg, 17.24 mM" means that 20 mg of doxorubicin hydrochloride is dissolved in water to obtain a doxorubicin hydrochloride solution with a concentration of 17.24 mM. Therefore, the mass ratio between doxorubicin hydrochloride, D-Pt-D and CuCl2·2H2O is clear.

[0082] 3. Synthesis of PDDC NPs

[0083] DDC NPs (40 mg) were dispersed in ultrapure water, and 2 mL of dopamine hydrochloride solution (10 mg, 26.37 mM) was added after 5 h. After stirring for 30 min, 1.03 mL of ammonia water (1.556 M) was added, and then stirred for 10 h in the dark. The reaction solution was dialyzed (MWCO: 1000D) for 24 h, and finally freeze-dried to obtain a solid (i.e., nanodrug), which was named PDDC NPs.

[0084] Experimental Example 1

[0085] The OH-Pt-OH, D-Pt-D, DDC NPs, and PDDC NPs used in this experimental example were all prepared in Example 1.

[0086] 1. Structural characterization

[0087] 1.1 Structural characterization of OH-Pt-OH and D-Pt-D

[0088] OH-Pt-OH and D-Pt-D were synthesized according to the synthetic route design and 1 H-NMR and FT-IR confirmed its successful synthesis ( Figure 2 ).Depend on Figure 2 As shown in (B), the signal peak of δ=6.17ppm belongs to the characteristic peak of -NH3. The signal peak of δ=4.6~4.8ppm belongs to the characteristic peak of the methine (-CH) on the six-membered ring of DMC connected to oxygen. The signal peak of δ=2.9~3.0ppm belongs to the characteristic peak of the methine (-CH) on the six-membered ring of DMC connected to platinum and carboxyl. Figure 2 The FT-IR spectrum in (C) proves the successful synthesis of OH-Pt-OH and D-Pt-D.

[0089] 1.2 Characterization of DDC NPs and PDDC NPs

[0090] DOX, D-Pt-D and CuCl2·2H2O were synthesized through metal coordination and non-covalent interaction to obtain DDCNPs, and then a layer of polydopamine was coated on its surface to obtain PDDC NPs. The successful preparation of DDC NPs and PDDC NPs was confirmed by UV-visible spectrophotometer, fluorescence spectrophotometer, DLS and TEM, and their particle size and microstructure were characterized.

[0091] First, if Figure 3 As shown in the UV-visible absorption spectra of (A) and (B), the characteristic absorption peaks of DOX appeared in the solutions of DDC NPs and PDDC NPs, and there was an obvious red shift phenomenon, which was consistent with the fluorescence spectrum ( Figure 3 Combined with (C) and (D) in the figure, the successful coordination of DDC NPs and PDDC NPs and the successful loading of DOX were preliminarily proved. Combined with the XPS of DDCNPs ( Figure 4 ) analysis shows that D-Pt-D and CuCl2·2H2O were also successfully assembled. Figure 5 The Tyndall effect further revealed the successful assembly of DDC NPs and PDDC NPs.

[0092] like Figure 6 As shown in (A) and (B), the particle sizes of DDC NPs and PDDC NPs are 159.9 nm and 225.7 nm, respectively. Figure 6(C) and (D) are TEM images of DDC NPs and PDDC NPs, showing that both DDC NPs and PDDC NPs are spherical in shape. The appropriate particle size and morphology make DDC NPs and PDDC NPs have better EPR effect. The above results also show the successful preparation of DDC NPs and PDDC NPs.

[0093] 2. Drug loading

[0094] Using DMSO as solvent, DOX solutions of different concentrations were prepared, and the UV-visible absorption spectra of each solution were measured. The standard curve of DOX was drawn based on the absorbance value at the maximum absorption peak ( Fig.16 PDDC NPs (2 mg) were added to 15 mL of DMSO and dissolved by ultrasound. The absorbance at the maximum absorption peak of the solution was measured by UV-visible spectrophotometry ( Fig.17 ) to calculate the drug loading capacity (DLC) of PDDC NPs.

[0095] The Pt loading was determined by ICP.

[0096] The drug loading of demethylcantharidin was characterized by HPLC.

[0097]

[0098] 3. Stability and responsiveness tests of DDC NPs and PDDC NPs

[0099] In order to study the stability and responsiveness of DDC NPs and PDDC NPs in the blood circulation and tumor microenvironment in vivo, the changes in the particle size of DDC NPs and PDDC NPs over time under different conditions were determined by DLS and UV-Vis. Taking DDCNPs as an example, (1) DDC NPs were dispersed in PBS at pH = 7.4, and the particle size of the sample was measured at 0, 1, 2, 4, 6, 12 and 24h; (2) DDC NPs were dispersed in PBS at pH = 5.0, and the particle size of the sample was measured at 0, 1, 2, 4, 6, 12 and 24h; (3) DDC NPs were dispersed in PBS at pH = 7.4 with 20mM GSH added, and the particle size of the sample was measured at 0, 1, 2, 4, 6, 12 and 24h; (4) DDC NPs were dispersed in PBS at pH = 5.0 with 20mM GSH added, and the particle size of the sample was measured at 0, 1, 2, 4, 6, 12 and 24h. PDDC NPs were also measured using the same method. In each test group, the concentration of DDC NPs and PDDC NPs was 1mg / mL.

[0100] Experimental results:

[0101] The stability of nanomedicine helps it maintain its structure and activity in the body, thereby improving the therapeutic effect of the drug, and can prolong the circulation time of the drug in the body and reduce the loss of the drug during the clearance process; the responsiveness of nanomedicine can achieve precise treatment, reduce toxic side effects on normal tissues, and improve the therapeutic effect of the drug. Figure 8 As shown in the figure, PDDC NPs were dispersed in PBS at pH = 7.4. As stirring continued, the changes in particle size measured at different time points were negligible, proving that PDDC NPs have good stability. However, PDDC NPs underwent significant changes immediately after being added to the three solutions for 1 hour. The reason may be that the Cu in PDDC NPs 2+ The reaction between Pt(Ⅳ) and GSH in high concentrations caused the PDDC NPs to crack and thus destroyed the nanoparticle structure.

[0102] At the same time, the stability and responsiveness of DDC NPs were characterized by the same method as PDDC NPs, and similar results were obtained. Figure 7 As shown, DDC NPs also exhibited good stability and responsiveness.

[0103] 3. In vitro photothermal performance test of PDDC NPs

[0104] 3.1 Concentration-dependent and laser-dependent photothermal properties of PDDC NPs

[0105] Different concentrations of PDDC NPs solutions (0, 0.2, 0.4, 0.6, and 0.8 mg / mL) were prepared, and 200 μL of each solution was placed in a 96-well plate. The 808 nm laser (1.5 W / cm 2 ) was used to irradiate the solution for 10 min, and the temperature change was recorded every 20 s. Secondly, an infrared thermal imager was used to record the thermal imaging of PDDC NPs solution (0.2, 0.4, 0.6, 0.8 mg / mL) at 0, 2, 4, 6, 8, and 10 min, respectively. Finally, the PDDC NPs solution was prepared with a concentration of 0.5 mg / mL and different powers (0.4, 0.8, 1.2, 1.6 W / cm 2 ) were used to irradiate the solutions with 808 nm laser for 10 min, and the changes in solution temperature were recorded every 20 s.

[0106] 3.2 Photothermal stability and photothermal conversion efficiency of PDDC NPs

[0107] Prepare PDDC NPs solution (0.5 mg / mL), take 200 μL of the solution and place it in a 96-well plate. 2The solution was irradiated with an 808nm laser for 10 minutes, and then the light source was turned off and the temperature was naturally cooled for 10 minutes. This heating and cooling cycle was repeated 5 times. The temperature change was recorded every 20 seconds during the whole process to study the photothermal stability of the sample.

[0108] The photothermal conversion efficiency of PDDC NPs was calculated according to the reported formula:

[0109]

[0110] Among them, T max and T sur are the maximum temperature of the solution and the ambient temperature, Q s is the heat dissipated by the solvent used, I is the power of the laser used, A 808 nm is the absorbance of the PDDC NPs solution at 808 nm, h and A are the heat transfer coefficient and the surface area of ​​the sample cell, respectively. The calculation formula of hA is as follows:

[0111]

[0112] Among them, m D and C D is the mass and specific heat of the solvent used (4.2 J / g for water), τ s is the time constant of heat transfer in the system, τ s The calculation formula is as follows:

[0113]

[0114] Where t represents time, θ is the unit driving force, dimensionless, and is defined as follows:

[0115]

[0116] Where T represents the temperature at a certain moment.

[0117] Q s Obtained by the following formula:

[0118]

[0119] Test results:

[0120] As a photothermal agent, polydopamine is widely used in photothermal therapy due to its excellent photothermal conversion ability and biocompatibility. Fig. 9 As shown in (A), compared with the deionized water control group, with the increase of PDDC NPs concentration, at the same power of 1.5 W / cm 2The solution temperature increased significantly after 808nm laser irradiation. In the PDDC NPs solution with a concentration of 0.8mg / mL, the temperature increased (ΔT) to 30.7℃ after 10min of laser irradiation. Fig. 9 As shown in (B), the temperature of PDDC NPs also changes with the increase of power. 2 When the concentration of PDDC NPs was 0.5 mg / mL, the temperature increased (ΔT) to 35.2°C after laser irradiation for 10 min. This fully illustrates the power dependence of PDDC NPs.

[0121] In addition, the photostability and photothermal conversion efficiency of PDDC NPs were also studied. Fig. 9 As shown in (C), under the 808nm laser (1.5W / cm 2 ) After five repeated irradiation / off cycles, the maximum temperature of the PDDC NPs solution remained basically unchanged, showing good photostability. According to the first heat generation-dissipation curve, substituting Fig. 9 From the relationship equation between the negative logarithm of -ln(θ) and the cooling time of PDDC NPs shown in (D), the photothermal conversion efficiency of PDDC NPs was obtained to be 6.67%.

[0122] Finally, the photothermal imaging of PDDC NPs at different concentrations was further studied. As shown in Figure 10, the color change trend and photothermal temperature curve are basically consistent.

[0123] The above photothermal results show that PDDC NPs have excellent photothermal properties, with significant concentration and laser power dependence, and also have good photostability and photothermal imaging capabilities.

[0124] 4. GSH and H2O2 consumption experiments of PDDC NPs

[0125] 4.1 GSH consumption experiment of PDDC NPs

[0126] First, prepare the 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) standard solution (10mM): take 39.6mg of DTNB, dissolve it with Na2HPO4·12H2O (50mM), and make up to volume in a 10mL volumetric flask; Tris-HCl buffer solution (0.25mM): take 0.3581g of tris(hydroxymethyl)aminomethane, dissolve it with deionized water, adjust its pH to 8 with concentrated hydrochloric acid, and finally make up to 100mL with deionized water; DTNB analytical solution (0.1mM): take 1mL of DTNB standard solution (10mM) into a volumetric flask, and then make up to 100mL with Tris-HCl buffer solution (0.25mM).

[0127] Take PDDC NPs freeze-dried powder (4 mg) and add HAc-NaAc buffer solution (4 mL pH = 5.5) to prepare PDDCNPs (1 mg / mL) solution. Take 1 mL of the above solution and add 9 mL HAc-NaAc buffer solution (pH = 5.5), then add glutathione (GSH) (10 mM, 0.6 mL) in a 37 ° C water bath, take 0.5 mL of the reaction solution at 0 and 30 min, add DTNB analysis solution (0.1 mM) pre-temperatured at 25 ° C, react for 5 min, and measure the absorbance at 300-800 nm using a UV spectrophotometer. At the same time, take 2 mL of PDDC NPs (1 mg / mL) solution and add 8 mL HAc-NaAc buffer solution (pH = 5.5), then add glutathione (GSH) (10 mM, 0.6 mL) in a 37 ° C water bath and repeat the above experiment.

[0128] 4.2 H2O2 consumption experiment of PDDC NPs

[0129] First, prepare 3,3',5,5'-tetramethylbenzidine (TMB) ethanol solution (10mM, 10mL) and H2O2 (1M, 10mL). Secondly, take PDDC NPs freeze-dried powder (4mg) and add HAc-NaAc buffer solution (4mL pH=5.5) to prepare PDDC NPs (1mg / mL) solution. Then, use HAc-NaAc buffer solution (pH=5.5) to prepare PDDCNPs (1mg / mL) solution with concentrations of 0, 0.02, 0.04, 0.06, 0.08 and 0.1mg / mL, respectively, add H2O2 (1M, 1mL) and TMB ethanol solution (10mM, 100μL), and use UV-visible spectrophotometer to detect the absorbance at 400-800nm ​​after 10min. At the same time, H2O2 (1 M, 1 mL) and TMB ethanol solution (10 mM, 100 μL) were added to the 0.1 mg / mL PDDCNPs solution, and the absorbance at 400-800 nm was detected using a UV-visible spectrophotometer after 0, 2, 4, 6, 8, and 10 min of reaction, respectively.

[0130] Experimental results:

[0131] like Fig.11As shown in (A) and (B), PDDC NPs are cleaved under acidic conditions, and the released copper ions can react with GSH and consume GSH. When GSH is not added to the solution, the absorption peak of DTNB can be seen at 320nm; when DTNB is mixed with GSH, TNB is generated, and a corresponding absorption peak is generated at 412nm, and the absorption peak at 320nm decreases. When PDDC NPs solution is added to the mixed solution of DTNB and GSH, it can be seen that the absorption peak at 412nm decreases with time, and the absorption peak at 320nm increases, which indicates that the copper ions released after PDDC NPs are cleaved under acidic conditions can react with GSH to reduce the content of GSH.

[0132] like Fig.11 As shown in (C), with the increase of PDDC NPs concentration, the absorption peak at 652nm also increases, indicating that PDDC NPs react with H2O2 to generate ·OH. At the same time, when the PDDC NPs concentration is 0.1mg / mL ( Fig.11 In (D), the content of ·OH generated by the reaction of PDDC NPs with H2O2 increases with time, but its content does not change much after 6 minutes.

[0133] In summary, PDDC NPs possess chemodynamic therapeutic properties.

[0134] 5. In vitro drug release experiment of PDDC NPs

[0135] After 5 mg of PDDC NPs freeze-dried powder was dispersed in 5 mL of PBS (pH = 7.4), PBS (pH = 5.0), PBS (pH = 7.4, 20 mM GSH) and PBS (pH = 5.0, 20 mM GSH), the solution was placed in a dialysis bag (MWCO: 1000D) and then placed in a glass bottle containing 45 mL of the corresponding buffer solution. The solution was shaken in a constant temperature water bath oscillator at 37 ° C, and then 3 mL of the dialysate was taken out at the specified time points (1, 2, 4, 6, 8, 10, 14, 24, 32, 36 and 48 h), and the same volume of new buffer solution was added. The UV-visible absorption spectra of the solutions were measured, and the cumulative release of DOX at each time point was calculated according to the standard curve; the cumulative release of Pt at each time point was calculated according to the standard curve of Pt element measured by ICP; the cumulative release of DMC at each time point was calculated according to the standard curve of DMC measured by HPLC.

[0136] Experimental results:

[0137] The drug release behavior of PDDC NPs was evaluated by dialysis method. Fig.12As shown in (A), when pH = 7.4, the release of DOX was only 13.65% at 48h, further proving that PDDC NPs can maintain stability under normal conditions. However, when the pH is acidic, the release of DOX increased significantly, reaching 38.86% at 48h. At the same time, 20mM GSH was added to PBS at pH = 7.4 to verify the sensitivity of PDDC NPs to a reducing environment. The results were consistent with expectations. When GSH was added to PBS at pH = 7.4, the release of DOX reached 52.37%, while after adding GSH in an acidic environment, the release of DOX reached 80.45%. It can be seen that the effective release of DOX in PDDC NPs can be effectively promoted in the tumor microenvironment to play a chemotherapeutic role. In addition, as Fig.12 As shown in (B) and (C), the release behavior of Pt and DMC was also studied. When pH = 7.4, the release amounts of Pt and DMC were 15.23% and 14.84%, respectively. This also proves the stability of PDDC NPs under normal conditions. However, in two sets of acidic environments, the release amounts of Pt were 40.56% (pH = 5.0) and 84.65% (pH = 5.0 + 20mM GSH); the release amounts of DMC were 39.89% (pH = 5.0) and 83.55% (pH = 5.0 + 20mM GSH). In summary, it is shown that PDDC NPs can release drugs normally to achieve therapeutic effects.

[0138] 6. Cellular studies of PDDC NPs

[0139] 6.1 Cell culture

[0140] Human cervical cancer cells (HeLa) and human breast cancer cells (MCF-7) used in the experiment were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were inoculated in cell culture dishes containing DMEM and cultured in a sterile incubator at 37°C, 90% relative humidity, and 5% CO2.

[0141] 6.2 In vitro endocytosis and intracellular distribution

[0142] To study the uptake of PDDC NPs by cancer cells and the distribution of drugs in cancer cells, HeLa cells were cultured at 4×10 5The cells were inoculated at a density of 4×10 / well in a six-well plate covered with a coverslip. After incubation, culture medium containing small molecule DOX or PDDC NPs was added for treatment at different time points (1, 3 and 6h). After treatment, the cells were first washed three times with PBS, and then treated with 4% paraformaldehyde solution for 20 minutes to fix the cells. Subsequently, they were washed three times with PBS, and then the cells were sealed and stained with anti-fluorescence quenching sealing solution containing DAPI. Finally, laser confocal microscopy (CLSM) was used to observe the uptake mode and distribution of small molecule DOX and PDDC NPs in HeLa cells. At the same time, flow cytometry was used to quantitatively analyze the uptake of PDDC NPs by cancer cells. A density of 4×10 5 HeLa cells per well were inoculated in a six-well plate, and the cells were treated with small molecule DOX or PDDCNPs at different time points (1, 3 and 6h) after incubation until the cells adhered. After incubation, the culture medium was removed and washed three times with PBS buffer. The cells were digested with trypsin, collected by centrifugation and redispersed in PBS buffer to obtain a cell suspension, and the fluorescence intensity of DOX in cells treated at different times was detected by flow cytometry. At the same time, MCF-7 cells were also experimentally verified according to the above operation.

[0143] 6.3 In vitro cytotoxicity

[0144] The in vitro cytotoxicity of PDDC NPs was evaluated using the MTT assay. HeLa cells were cultured at a density of 1 × 10 4 The cells were seeded at a density of 100 cells / well in a 96-well plate and incubated for 24 hours. After the cells adhered to the wall, DMEM medium containing small molecule DOX plus D-Pt-D and PDDCNPs was added for treatment. The experiment set up the following 5 different treatment methods: (1) control group, supplemented with medium without drugs; (2) medium containing small molecule DOX and D-Pt-D was added; (3) medium containing small molecule DOX and D-Pt-D was added and irradiated with 808nm laser; (4) medium containing PDDC NPs was added; (5) medium containing PDDC NPs was added and irradiated with 808nm laser; laser irradiation was given 6 hours after administration, with 808nm (1.5W / cm 2 ) laser irradiation. After the treatment is completed and incubated for 24h and 48h, MTT solution is added to each well. The culture is continued for 4h, the culture medium in the well is discarded, and DMSO is added to dissolve the formazan formed in the cells. The absorbance of each well at 490nm is measured using a multifunctional microplate reader. The cell survival rate can be calculated by the following formula:

[0145]

[0146] Experimental results:

[0147] Effective cellular endocytosis is an important prerequisite for the anticancer nanomedicine to work. Taking HeLa cells as an example, first, the in vitro cell performance of PDDC NPs was studied by laser confocal fluorescence microscopy (CLSM). PDDCNPs were added to HeLa cells at 1, 3, and 6 hours respectively. Small molecule DOX was used as the control group. Fig.13 As shown in (A), after 1 hour of incubation, the fluorescence of HeLa cells treated with small molecule DOX was basically located in the cell nucleus. For HeLa cells treated with PDDC NPs, most of the DOX fluorescence was located in the cytoplasm after 1 hour; after 6 hours of incubation, DOX was distributed in both the cell nucleus and cytoplasm, indicating that the prepared nanomedicine can effectively release DOX. The results show that PDDC NPs can be absorbed by HeLa cells through endocytosis, while small molecule DOX enters the cells through passive diffusion. At the same time, flow cytometry (FCM) was used to quantitatively verify the cellular absorption of small molecule DOX and PDDC NPs. The FCM results confirmed that cellular absorption is related to time, and the results are consistent with the CLSM analysis ( Fig.13 Meanwhile, the CLSM and FCM results of MCF-7 cells were similar to those of HeLa cells ( Fig.13 (C) and (D) ), both demonstrated that PDDC NPs could be effectively internalized by cells and successfully release DOX.

[0148] The in vitro cytotoxicity of DOX, D-Pt-C, and PDDC NPs on HeLa cells with or without 808 nm laser irradiation was determined by MTT assay. Fig.14 As shown in the figure, with the increase of concentration, all drugs showed gradually enhanced cell killing ability. In the absence of laser irradiation, small molecule DOX has a strong killing effect on cells. After 808nm laser irradiation, PDDC NPs significantly reduced the survival rate of cells, demonstrating the excellent PTT effect. The MTT experiment showed that PDDC NPs may be a potential drug for chemotherapy-synergistic PTT treatment.

[0149] In summary, DDC NPs were obtained by coordinating copper ions with DOX and D-Pt-D, and a layer of dopamine was coated on its surface to further self-assemble PDDC NPs. PDDC NPs have suitable particle size and particle size distribution, spherical morphology, significant photothermal conversion ability, pH and GSH responsiveness, corresponding controllable drug release curves, and chemodynamic therapy effects. In vitro cell experiments showed that PDDC NPs can be effectively internalized by cancer cells and show obvious cytotoxicity under the action of 808nm laser. Therefore, PDDC NPs is a potential nano anticancer drug for synergistic chemotherapy-PTT-chemodynamic therapy.

[0150] Example 2

[0151] A method for preparing a nano drug, which differs from Example 1 in that: at room temperature, 1 g of cisplatin is weighed and added to 5 mL of H2O and 8 mL of H2O2, and the remaining steps are consistent with those of Example 1.

[0152] Example 3

[0153] A method for preparing a nano drug, which differs from Example 1 in that: at room temperature, 1 g of cisplatin is weighed and added to 10 mL of H2O and 12 mL of H2O2, and the remaining steps are consistent with those of Example 1.

[0154] Example 4

[0155] A method for preparing a nano drug, which differs from Example 1 in that: OH-Pt-OH (0.6 g) and norcantharidin (DMC) (1.08 g) are weighed and poured into a round-bottom flask, and the remaining steps are consistent with those of Example 1.

[0156] Example 5

[0157] A method for preparing a nano drug, which differs from Example 1 in that: OH-Pt-OH (0.6 g) and norcantharidin (DMC) (1.32 g) are weighed and poured into a round-bottom flask, and the remaining steps are consistent with those of Example 1.

[0158] Example 6

[0159] A method for preparing a nano drug, which differs from Example 1 in that: DDC NPs (30 mg) are dispersed in ultrapure water, and 2 mL of dopamine hydrochloride solution (10 mg, 26.37 mM) is added thereto after 5 hours, and the remaining steps are consistent with those of Example 1.

[0160] Example 7

[0161] A method for preparing a nano drug, which differs from Example 1 in that: DDC NPs (50 mg) are dispersed in ultrapure water, and 2 mL of dopamine hydrochloride solution (10 mg, 26.37 mM) is added thereto after 5 hours, and the remaining steps are consistent with those of Example 1.

[0162] Example 8

[0163] A method for preparing a nano drug, which differs from Example 1 in that FeCl3 is used instead of CuCl2·2H2O, the concentration of FeCl3 in the solution is still 3.519 mM, and the remaining steps are consistent with Example 1. The particle size of the iron-coordinated nanoparticles DDC NPs obtained in this example is 176.3 nm, and PDI=0.209 ( Fig.15(A).

[0164] After testing, the performance of the nanodrugs obtained in Examples 2-8 is similar to that of the nanodrug obtained in Example 1, and they also have the ability to controllably release drugs under acidity and reduction, can achieve photothermal conversion under laser irradiation to produce a mild photothermal effect, have a tumor cell killing effect, have the ability to consume glutathione and produce hydroxyl free radicals, can enter cancer cells through endocytosis, and release DOX, and can significantly improve the anti-tumor effect through chemotherapy, chemodynamic therapy and photothermal therapy.

[0165] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nano drug, characterized in that: The invention comprises nanoparticles and polydopamine wrapped on the surface of the nanoparticles. The nanoparticles are self-assembled by tetravalent platinum medicine modified by norcantharidin and doxorubicin under the coordination of metal ions.

2. The nano drug according to claim 1, characterized in that The structure of the tetravalent platinum drug modified by norcantharidin is as follows:

3. The nano drug according to claim 1, characterized in that The metal ion is selected from Fe 3+ , Gd 3+ , Mn 2+ 、Co 2+ 、Ni 2+ and Cu 2+ One of them.

4. The nano drug according to claim 1, characterized in that The nanoparticles are spherical and have a particle size of 140-180 nm; Preferably, the nano drug is spherical and has a particle size of 200-250 nm.

5. A method for preparing the nano drug according to any one of claims 1 to 4, characterized in that: include: Adding a tetravalent platinum aqueous solution modified with norcantharidin to a metal salt aqueous solution, and then dropping an adriamycin hydrochloride aqueous solution, reacting at room temperature in the dark to obtain nanoparticles; The nanoparticles are dispersed in water, dopamine hydrochloride is added, stirred, and then ammonia water is added, stirred in the dark to react, thereby obtaining a nanomedicine.

6. The preparation method according to claim 5, characterized in that: The preparation method of the tetravalent platinum drug modified by norcantharidin comprises the following steps: Cisplatin, water and hydrogen peroxide are mixed and reacted at room temperature in the dark to obtain a hydroxyl-modified tetravalent platinum drug; Under nitrogen protection and 65-75°C, the hydroxyl-modified tetravalent platinum drug and norcantharidin react in the dark to obtain the norcantharidin-modified tetravalent platinum drug; Preferably, the dosage ratio of cisplatin, water and hydrogen peroxide is 1 g: 5-10 mL: 8-12 mL; Preferably, the mass ratio of the hydroxyl-modified tetravalent platinum drug to norcantharidin is 1:1.8-2.2; Preferably, the structure of the hydroxyl-modified tetravalent platinum drug is:

7. The preparation method according to claim 5, characterized in that: The mass ratio of doxorubicin hydrochloride, tetravalent platinum drug modified with norcantharidin and metal salt is 1:1:0.5-1; Preferably, the mass ratio of the nanoparticles to dopamine hydrochloride is 3-5:1, preferably 4:

1.

8. The preparation method according to claim 5, characterized in that: After the reaction is completed at room temperature and away from light or with stirring in the dark, the reaction solution is dialyzed and freeze-dried to obtain the target product; The molecular weight cut-off of the dialysis is 900-1100D.

9. Use of the nano drug according to any one of claims 1 to 4 or the nano drug prepared by the preparation method according to any one of claims 5 to 8 in the preparation of anti-tumor drugs; Preferably, the tumor includes cervical cancer and breast cancer.

10. An anti-tumor drug, characterized in that: A nanodrug comprising any one of claims 1 to 4 or a nanodrug prepared by the preparation method of any one of claims 5 to 8; Preferably, it also includes pharmaceutically acceptable excipients; Preferably, the tumor includes cervical cancer and breast cancer.

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