Nano-drug for promoting liver cancer cell copper death and enhancing disulfiram chemotherapy, preparation method and application

Abstract

The invention relates to a preparation method and application of a nano-drug for promoting liver cancer cell copper death and synergistic disulfiram (DSF) chemotherapy. At present, DSF in drugs for liver cancer cell copper death has huge potential, but the drugs are insufficient to cause liver cancer cell copper death. In order to overcome the defects, the invention provides a novel nano-drug which comprises inorganic nano-material hollow copper sulfide nano-particles (HCuS NPs) with near-infrared light (NIR) response, Au-Pt nano-enzyme with bimetallic catalytic activity, a photosensitizer indocyanine green (ICG), a diethyl dithiocarbamic acid-copper conjugate (Cu (DTC) 2) and peptide anchored with liver cancer targeting. The nano-drug HCuS / Au-Pt (at) DSF / ICG (at) 9R-P (HAPDI9P) capable of promoting liver cancer cell copper death and enhancing DSF chemotherapy is obtained, the nano-drug has an excellent combined treatment effect of chemotherapy, photothermal therapy, photodynamic therapy and chemical dynamic therapy, liver cancer cell immunogenicity death is induced, the immune response of liver cancer cells is enhanced, and the application prospect is wide. The important significance is realized on promoting liver cancer cell copper death.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and health technology, and specifically relates to a nano drug that promotes copper death of liver cancer cells and enhances disulfiram (DSF) chemotherapy, a preparation method and an application thereof. Background Art

[0002] Copper (Cu) is one of the essential trace elements for the body and plays an important role in physiological processes including mitochondrial respiration, antioxidant reactions, and biomacromolecule synthesis. Both copper deficiency and copper overload can cause damage to cells. In recent years, a newly reported copper death (Cuproptosis) is a new form of death caused by excessive copper. Intracellular copper stimulates the lipoylation aggregation process of mitochondrial-related proteins, promotes the degradation of Fe-S cluster proteins and protein toxicity reactions, and ultimately leads to cell death. This major discovery undoubtedly provides a new perspective for the treatment of copper metabolism imbalance, especially copper overload-related diseases. In addition, disulfiram, an FDA-approved drug for the treatment of chronic alcoholism, also shows obvious advantages in anti-tumor effects, and its anti-tumor effect has been shown to have a unique copper-dependent mode. Under physiological conditions, disulfiram can be rapidly metabolized into diethyldithiocarbamate (DTC), which reacts with Cu 2+ The reaction forms a complex of diethyl dithiocarbamate and copper (II) Cu(DTC) 2 , that is, in situ conversion from “non-toxic to highly toxic” for enhanced chemotherapy of liver cancer. At the same time, the bimetallic Au / Pt nanozyme catalyzes H 2 O 2 The generated OH and O 2 It can solve TME hypoxia and cooperate with DTC and Cu 2+ The chelated Cu+ generates ·OH through Fenton-like reaction, which increases the generation of ROS and induces cell apoptosis. Therefore, the nanodrug designed to promote copper death of liver cancer cells and enhance disulfiram (DSF) chemotherapy has excellent combined therapeutic effects of chemotherapy, photothermal therapy, photodynamic therapy and chemodynamic therapy, and induces immunogenic death of liver cancer cells, enhances the immune response of liver cancer cells, and is of great significance to promote copper death of liver cancer cells.

[0003] Although the nanomaterial that promotes copper death of liver cancer cells and enhances disulfiram (DSF) chemotherapy is a highly promising carrier, its application in the preparation of tumor therapeutic drugs still faces many challenges, such as: hypoxic tumor microenvironment, low tumor cell targeting, etc., which makes it difficult to achieve targeted drug transport and high-efficiency, low-toxic combination chemotherapy. Therefore, how to prepare a liver cancer targeting peptide-modified nano drug carrier that promotes copper death of liver cancer cells and enhances disulfiram (DSF) chemotherapy to achieve its application in the preparation of tumor therapeutic drugs is a technical problem that needs to be seriously solved. Based on this, this application was developed. Summary of the invention

[0004] The main purpose of the present invention is to overcome the defects of the prior art and provide a method for preparing a nano drug carrier that promotes copper death of liver cancer cells and enhances disulfiram (DSF) chemotherapy. The liver cancer targeting peptide modified nano drug carrier that promotes copper death of liver cancer cells and enhances disulfiram (DSF) chemotherapy prepared by this method has good hydrophilicity, high safety, small size and specific targeting of tumor sites.

[0005] Another object of the present invention is to provide a use of the nano drug carrier for promoting copper death of liver cancer cells and enhancing disulfiram (DSF) chemotherapy in drug loading.

[0006] The present invention also provides a method for preparing nano-drugs by using the nano-drug carrier.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: A method for preparing a nano drug carrier for promoting copper death of liver cancer cells and enhancing disulfiram (DSF) chemotherapy comprises the following steps: Step 1, at room temperature, dispersing cupric chloride dihydrate solution and polyvinyl pyrrolidone (PVP-K30) in water (stirring for 2-10 min) to obtain a mixed solution A; wherein the cupric chloride dihydrate solution preferably has a concentration range of 0.4-0.6 M; and the polyvinyl pyrrolidone preferably has a concentration range of 8-12 mg / mL; Step 2: Add 45-55% (mass percentage) hydrazine hydrate solution for reducing effect, sodium hydroxide solution with alkaline condition of pH=8-10 and 310-330 mg / mL sodium sulfide nonahydrate solution to the mixed solution A, and stir for 2-10 minutes to obtain a mixed solution B; further, in step 2, the volume ratio of the cupric chloride dihydrate solution to the hydrazine hydrate solution, the sodium hydroxide solution and the sodium sulfide nonahydrate solution is 1:0.05-0.07:200-300:1-3; Step 3, under room temperature, first centrifuge the mixed solution B (11000-13000 rpm for 10-15 min, the same below), wash (wash with ultrapure water for 2-4 times, the same below), and freeze-dry (freeze-dry under low temperature and high vacuum conditions for 20-30 hours, the same below) to obtain a dark green solid; then disperse the dark green solid in ultrapure water to obtain a product C suspension (HCuS); wherein the solid-liquid ratio of the product C suspension is 1:4000-6000 g / mL (the unit of the solid-liquid ratio is g / mL, the same below); Step 4: at room temperature, dispersing 0.5-1.5 mL of the product C suspension in 1.0-1.4 mL of ethanol to obtain a mixed solution D; preferably, the volume ratio of the product C suspension to ethanol is 1:1.0-1.5; Step 5, at room temperature, first add polyvinyl pyrrolidone to the mixed solution D, stir, then add chloroauric acid aqueous solution, stir, then add sodium borohydride aqueous solution, stir, and finally add potassium chloroplatinate and L-ascorbic acid, stir in an oil bath at 70-90 ° C for 20-40 min to obtain a mixed solution E; Step 6: Under room temperature, centrifuge the mixed solution E, wash it, and freeze-dry it to obtain a black solid; then disperse the black solid in ultrapure water to obtain a product F suspension (i.e., a nano drug carrier, HCuS / Au-Pt). The solid-liquid ratio of the product F suspension is 1:1000-3000 g / mL.

[0008] Specifically, in step 1, the mass ratio of cupric chloride dihydrate to polyvinyl pyrrolidone may be 1:20-30. The molecular weight of polyvinyl pyrrolidone (PVP-K30) may be 50000-60000 Da.

[0009] Further, in step 5, 5-15 mg of polyvinyl pyrrolidone can be first added to the mixed solution D containing 0.1-0.3 mg of dark green solid, stirred for 20-40 min, then 5-7 mL of 0.2-0.4 mM chloroauric acid aqueous solution is added, stirred for 5-15 min, then 0.4-0.6 mL of 2-4 mM sodium borohydride aqueous solution is added, stirred for 20-40 min, and finally 50-150 mL of 0.05-0.15 mM potassium chloroplatinate and 4-6 mL of 4-6 mM L-ascorbic acid are added.

[0010] The present invention provides a nano drug carrier prepared by the above method and capable of promoting copper death of liver cancer cells and enhancing disulfiram (DSF) chemotherapy.

[0011] The present invention provides the application of the nano drug carrier for promoting copper death of liver cancer cells and enhancing disulfiram (DSF) chemotherapy in drug loading. For example, the nano drug carrier can be used for preparing drugs, especially anti-tumor drugs.

[0012] The present invention also provides a method for preparing a liver cancer targeting peptide-modified disulfiram (DSF) chemotherapy nanodrug that promotes copper death of liver cancer cells by using the above nano drug carrier, which comprises the following steps: Step a, at room temperature, subjecting the product F suspension nano drug carrier to ultrasonic treatment for 20-30 min, adding the chemotherapy drug disulfiram and the photosensitizer indocyanine green, stirring for 2-48 h in the dark, centrifuging, washing, and freeze drying (freeze drying for 20-30 h under low temperature and high vacuum conditions, the same below) to obtain a black solid; then dispersing the black solid in a pH 7-9 phosphate buffer to obtain a product G suspension (HCuS / Au-Pt@DSF / ICG, abbreviated as HAPDI); Step b: at room temperature, the product G suspension is sonicated for 20-30 min, then a thiol-containing coating is added, stirred for 2-48 h in the dark, centrifuged, washed, and freeze-dried to obtain a black solid; the black solid is then dispersed in ultrapure water to obtain a product H suspension (HCuS / Au-Pt@DSF / ICG-NH2, abbreviated as HAPDI-NH 2 ), Step c, placing the liver cancer targeting peptide in a phosphate buffer at pH 7-9 at room temperature, adding carbodiimide hydrochloride and N-hydroxysuccinimide, and stirring for 20-40 min to obtain an activated peptide solution I; Step d: at room temperature, the product H suspension and the activated peptide solution I are mixed, stirred for 12-48 h in the dark, centrifuged, washed, and freeze-dried to obtain a black solid, thereby obtaining nanodrug J (HCuS / Au-Pt@DSF / ICG@9R-P, abbreviated as HAPDI9P).

[0013] Specifically, in step a, the mass ratio of the chemotherapy drug disulfiram, the photosensitizer indocyanine green and the black solid in the product F suspension can be 1-3: 1-3: 1. In step a, the disulfiram can be converted from "non-toxic to highly toxic" in situ for enhanced chemotherapy of liver cancer.

[0014] Specifically, in step b, the thiol-containing coating includes β-mercaptoethylamine, mercaptopropionic acid or thioglycolic acid, etc., which is used as a bridge to connect HCuS / Au-Pt@DSF / ICG and liver cancer targeting peptide. The mass ratio of the thiol-containing coating to the black solid in the product G suspension can be 1-3:1.

[0015] Specifically, liver cancer targeting peptides include 9R-P peptide, A54 peptide, SP94 peptide, AM-2 peptide, T7 peptide, BP9 peptide, X1 peptide, L5 peptide, etc. Further, in step c, the sequence of the liver cancer targeting peptide is: rrrrrrrrrGSGSWHLDYPSMWYLD.

[0016] Specifically, in step c, the mass ratio of the liver cancer targeting peptide to carbodiimide hydrochloride and N-hydroxysuccinimide can be 1:0.3-0.5:0.6-0.8. In step d, the mass ratio of the liver cancer targeting peptide to the black solid in the product H suspension can be 0.4-0.6:1.

[0017] The present invention also provides a liver cancer targeting peptide modified disulfiram (DSF) chemotherapy nano-drug that promotes copper death of liver cancer cells and is prepared by the above method. The nano-drug can be used in chemotherapy, hyperthermia, photodynamic, chemodynamic and immunotherapy. That is, the present invention provides a drug with chemotherapy-hyperthermia-photodynamic-chemodynamic-immunotherapy, which comprises the disulfiram (DSF) chemotherapy nano-drug carrier that promotes copper death of liver cancer cells. The present invention introduces the chemotherapy drug disulfiram and the photosensitizer indocyanine green into the disulfiram (DSF) chemotherapy nano-drug carrier that promotes copper death of liver cancer cells and can be applied to the combination treatment of cancer chemotherapy, photodynamic therapy, chemodynamic therapy, photothermal therapy and immunotherapy.

[0018] The present invention provides a method for preparing a nano drug carrier for promoting copper death of liver cancer cells and enhancing disulfiram (DSF) chemotherapy: firstly, a near-infrared light (NIR) responsive inorganic nanomaterial hollow copper sulfide nanoparticle (HCuSNPs) is synthesized, and its unique cage structure can be used as a carrier of photosensitizer indocyanine green (ICG) and chemotherapy drug disulfiram (DSF), and Au-Pt nanozyme with bimetallic catalytic activity can be grown on the surface, so that it has excellent catalytic effect, and finally a liver cancer targeting peptide is anchored to obtain a nano drug HCuS / Au-Pt@DSF / ICG@9R-P (abbreviated as HAPDI9P) with the function of promoting copper death of liver cancer cells and enhancing disulfiram (DSF) chemotherapy, and finally the nano drug has excellent chemotherapy, photothermal therapy, photodynamic therapy and chemodynamic therapy combined therapeutic effect, and induces immunogenic death of liver cancer cells, enhances the immune response of liver cancer cells, and is of great significance to promoting copper death of liver cancer cells.

[0019] The present invention introduces a liver cancer targeting peptide into the surface of the nano drug carrier for promoting copper death of liver cancer cells and enhancing disulfiram (DSF) chemotherapy, which can be applied to the specific targeting of liver cancer. Compared with the prior art, the nano drug carrier for promoting copper death of liver cancer cells and enhancing disulfiram (DSF) chemotherapy has the following advantages: 1) Using copper chloride dihydrate, polyvinyl pyrrolidone (PVP-K30), sodium hydroxide, hydrazine hydrate, chloroauric acid, sodium borohydride, potassium chloroplatinate, L-ascorbic acid and sodium sulfide nonahydrate as raw materials, the prepared black solid is nanoparticles that promote copper death of liver cancer cells and enhance disulfiram (DSF) chemotherapy; then using chemotherapy drug disulfiram, photosensitizer indocyanine green, β-mercaptoethylamine, liver cancer targeting peptide and nanoparticles that promote copper death of liver cancer cells and enhance disulfiram (DSF) chemotherapy as raw materials to obtain liver cancer targeting peptide-modified nanomedicine that promotes copper death of liver cancer cells and enhances disulfiram (DSF) chemotherapy. The raw materials used in the present invention have low toxicity and high safety.

[0020] 2) Au / Pt nanozymes were obtained by taking advantage of their high catalytic activity, good stability, low cost and easy scale-up preparation. In addition, non-toxic and easily available polyvinyl pyrrolidone (PVP-K30) was used as an active agent and stabilizer to synthesize nanoparticles that promote copper death of liver cancer cells and enhance disulfiram (DSF) chemotherapy at room temperature. The operation is simple, the reaction conditions are mild and controllable, and the cost is low. Liver cancer targeting peptides were modified on the surface to further improve the hydrophilicity of the prepared liver cancer targeting peptide-modified nanodrug carrier that promotes copper death of liver cancer cells and enhances disulfiram (DSF) chemotherapy in aqueous solution and its specific targeting effect on liver cancer.

[0021] 3) The surface potential of the nano drug carrier for promoting copper death of liver cancer cells and enhancing disulfiram (DSF) chemotherapy of the present invention is positive, and the positive potential is conducive to the targeting of the nano drug carrier to cancer cells, higher membrane penetration efficiency and stronger stability. The size is 8-120 nm, the size distribution is uniform and the dispersion is even, which is conducive to circulation in the organism.

[0022] 4) The present invention can effectively combine the chemokinetic and photothermal therapeutic effects of nanomaterials that promote copper death of liver cancer cells and enhance disulfiram (DSF) chemotherapy with the chemotherapy and photodynamic effects of the chemotherapy drug disulfiram and the photosensitizer indocyanine green, thereby enhancing the anti-tumor effect; the prepared liver cancer targeting peptide-modified nanomedicine that promotes copper death of liver cancer cells and enhances disulfiram (DSF) chemotherapy has little toxicity to normal liver cells and is highly toxic to liver cancer tumor cells, thereby achieving photothermal / photodynamic / chemotherapy / chemokinetic / immunotherapy combined treatment of tumors.

[0023] 5) The present invention has the characteristics of simple operation, high safety, mild reaction conditions, controllable reaction process and low cost. The prepared nano drug carrier for promoting copper death of liver cancer cells and enhancing disulfiram (DSF) chemotherapy has good hydrophilicity, small size, specific targeting, is conducive to circulation in the body, and the reagent toxicity is low during the preparation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1Transmission electron microscope (TEM) images of product C (HCuS) (a), product F (HCuS / Au-Pt) (b), product G (HAPDI) (c), product J (HAPDI9P) (d) in Example 1, and a mapping image of S, Cu, Au, Pt, and Merge of product F (HCuS / Au-Pt); Figure 2 The UV absorption graphs of product C (HCuS), product F (HCuS / Au-Pt), product G (HAPDI), product J (HAPDI9P), DSF and ICG in Example 1; Figure 3 Zeta potential diagrams of product C (HCuS), product F (HCuS / Au-Pt), product G (HAPDI), product H (HAPDI-NH2), and product J (HAPDI9P) in Example 1; Figure 4 The infrared spectra of product F (HCuS / Au-Pt) and DSF@HCuS / Au-Pt in Example 1; Figure 5 is the EDS image of product F (HCuS / Au-Pt) in Example 1; Figure 6 This is the experimental graph of the hemolysis rate of product J (HAPDI9P) in Example 1; Figure 7 The product J (HAPDI9P) in Example 1 was irradiated with 808 nm laser (1.0 W / cm 2 ) for 10 min under different concentrations (a) and the temperature change diagram of product J (HAPDI9P) 100 μg / mL under 808 nm laser irradiation for 10 min under different powers (b); Figure 8 The product J (HAPDI9P) in Example 1 was irradiated with 808 nm laser (1.0 W / cm 2 ) under irradiation of 100 μg / mL during 5 laser on / off cycles; Fig. 9 This is a graph showing the survival rates of product J (HAPDI9P) in Example 1 on HepG2 cells, SNU-739 cells, HeLa cells and HL-7702 cells; Fig.10The following are (a) graphs showing changes in mouse body weight, (b) graphs showing tumor growth curves, (c) digital images of tumor tissues, (d) graphs showing changes in tumor weight, and (e) graphs showing changes in tumor inhibition rate at the end of treatment of H22 tumor-bearing mice with product F (HCuS / Au-Pt), product F (HCuS / Au-Pt) + NIR, product G (HAPDI), product J (HAPDI9P), product J (HAPDI9P) + NIR, DSF, and ICG + NIR of the present invention. DETAILED DESCRIPTION

[0025] In order to further understand the present invention, the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention rather than limiting the scope of protection of the present invention.

[0026] Experimental instrument name and model: American Perkin-Elmer Lambda-850 UV spectrophotometer; Japan JEOL JEM-200CX transmission electron microscope; BioTek multifunctional microplate reader from the United States; German LEICA TCS SP8+STED laser confocal microscope; BD Facsverse flow cytometer, USA; LABCONCO-FreeZone 6L freeze drying system from the United States; Cytiva AI800flour ultra-sensitive multifunctional imager from the United States; In the examples, the required raw materials are all common commercial products that can be directly purchased or can be prepared according to conventional methods in the art.

[0027] Unless otherwise specified, room temperature refers to 25±5℃.

[0028] Example 1 A method for preparing a nano drug carrier that promotes copper death of liver cancer cells and enhances disulfiram (DSF) chemotherapy. The steps of the preparation method described in this embodiment are as follows: Step 1: Disperse 100 μL of cupric chloride dihydrate solution (0.5 M) and 240.0 mg of polyvinyl pyrrolidone (9.6 mg / mL) (PVP-K30, molecular weight 50,000-60,000 Da) in 25 mL of water at room temperature and stir for 5 min to obtain solution A.

[0029] Step 2: Add 6.4 μL of 50% hydrazine hydrate solution, 25 mL of sodium hydroxide solution with a pH of 9, and 200 μL of 320 mg / mL sodium sulfide nonahydrate solution to the solution A, and stir for 5 min to obtain a mixed solution B.

[0030] Step 3. Under room temperature, the mixed solution B was first centrifuged at 12000 rpm for 10 min, then washed three times with ultrapure water, and freeze-dried for 24 h under low temperature (-80 ° C) and high vacuum (1 Pa) conditions to obtain 4 mg of dark green solid; then the dark green solid was dispersed in 20 mL of ultrapure water to obtain a product C suspension (HCuS).

[0031] Step 4: At room temperature, 1.0 mL of product C suspension was dispersed in 1.2 mL of ethanol to obtain a mixed solution D.

[0032] Step 5. At room temperature, add 10 mg of polyvinyl pyrrolidone (PVP-K30, molecular weight 50000-60000 Da) to the mixed solution D and stir for 30 min. Then add 6.4 mL (0.3 mM) of chloroauric acid aqueous solution and stir for 10 min. Then add 0.5 mL (3 mM) of sodium borohydride aqueous solution and stir for 30 min. Finally, add 100 mL of 0.1 mM potassium chloroplatinate and 5 mL of 5 mM L-ascorbic acid and stir in an 80 °C oil bath for 30 min to obtain a mixed solution E.

[0033] Step 6. Under room temperature, the mixed solution E was first centrifuged at 12000 rpm for 10 min, then washed three times with ultrapure water, and freeze-dried for 24 h under low temperature (-80 ° C) and high vacuum (1 Pa) conditions to obtain 0.5 mg of black solid; then the black solid was dispersed in 1 mL of ultrapure water to obtain a product F suspension (HCuS / Au-Pt), which is a nano drug carrier.

[0034] The present invention also provides a method for preparing a liver cancer targeting peptide-modified disulfiram (DSF) chemotherapy nanodrug that promotes copper death of liver cancer cells by using the above nano drug carrier, which comprises the following steps: Step a, at room temperature, ultrasonically treat the 1 mL product F suspension for 25 min, then add 1 mg of the chemotherapy drug disulfiram (DSF) and 1 mg of the photosensitizer indocyanine green (ICG), stir at 1000 rpm in the dark for 24 h, centrifuge at 12000 rpm for 10 min, wash three times with ultrapure water, freeze-dry at low temperature (-80 ° C) and high vacuum (1 Pa) for 24 h to obtain 1 mg of black solid; then disperse the black solid in 1.0 mL of pH 7.4 phosphate buffer to obtain product G suspension (HCuS / Au-Pt@DSF / ICG, abbreviated as HAPDI). At the same time, for comparison and verification, when only the chemotherapy drug disulfiram (DSF) was added without the photosensitizer indocyanine green (ICG), the product DSF@HCuS / Au-Pt was prepared by referring to the operation of step a.

[0035] Step b, at room temperature, the 1.0 mL product G suspension was ultrasonicated for 25 min, and then 2.0 mg β-mercaptoethylamine was added, stirred at 1000 rpm in the dark for 24 h, centrifuged at 12000 rpm for 10 min, washed three times with ultrapure water, and freeze-dried at low temperature (-80 ° C) and high vacuum (1 Pa) for 24 h to obtain 2 mg of black solid; the black solid was then dispersed in 2.0 mL of ultrapure water to obtain a product H suspension (HCuS / Au-Pt@DSF / ICG-NH 2 , abbreviated as HAPDI-NH 2 ).

[0036] Step c: At room temperature, 1.0 mg of liver cancer targeting peptide (manufacturer: Shanghai Qiangyao Biotechnology Co., Ltd., number: 04010020031) was placed in 1.0 mL of pH 7.4 phosphate buffer, 0.4 mg of carbodiimide hydrochloride and 0.7 mg of N-hydroxysuccinimide were added, and the mixture was stirred for 30 min for activation to obtain an activated peptide solution L. The sequence of the liver cancer targeting peptide is: rrrrrrrrrGSGSWHLDYPSMWYLD.

[0037] Step d, at room temperature, the 2.0 mL product H suspension and the activated peptide solution L were mixed, stirred at 1000 rpm in the dark for 24 h, centrifuged at 12000 rpm for 10 min, washed three times with ultrapure water, and freeze-dried at low temperature (-80 ° C) and high vacuum (1 Pa) for 24 h to obtain 2.5 mg of black solid, which is nanodrug J (HCuS / Au-Pt@DSF / ICG@9R-P, abbreviated as HAPDI9P).

[0038] The size and dispersion of the hollow copper sulfide and the composite were characterized by transmission electron microscopy (TEM). Figure 1 .from Figure 1 It can be observed that: the prepared hollow copper sulfide (a) HCuS has an obvious hollow structure, small size, uniform morphology, and a size of 100-140 nm. (b) HCuS / Au-Pt can see that the surface Au-Pt nanoparticles are successfully attached to the surface of the hollow copper sulfide. (c) HAPDI can see that the hollow structure is no longer obvious after loading DSF and ICG. (d) HAPDI9P can see that the outline of the nanoparticles becomes clearer after connecting to the liver cancer targeting peptide, and the mapping of HCuS / Au-Pt S, Cu, Au, Pt element mapping can see that the signal evenly passes through the selected area of ​​the sample, further confirming the synthesis of HCuS / Au-Pt.

[0039] The nanoparticles were scanned at full wavelength using UV absorption spectroscopy. Figure 2 . Figure 2 It is the ultraviolet absorption diagram of product C (HCuS), product F (HCuS / Au-Pt), product G (HAPDI), product J (HAPDI9P), DSF and ICG of the present invention. Figure 2 It can be seen that the synthesis of each product can be further proved by the UV spectra of different products; the UV absorption of product G (HAPDI) and product J (HAPDI9P) in the near-infrared region is almost the same, and the modification of the targeting peptide has almost no effect on the UV absorption, which further shows that product J (HAPDI9P) has strong penetrating ability.

[0040] The Zeta potential of each component of the nanoparticles was studied using a Zeta potential instrument. The results are shown in Figure 3 . Figure 3 The product C (HCuS), product F (HCuS / Au-Pt), product G (HAPDI), product H (HAPDI-NH 2 ), and Zeta potential diagram of product J (HAPDI9P). Figure 3 It can be seen that after the addition of Au-Pt, the potential value of product F (HCuS / Au-Pt) becomes -25 mV; after the loading of DSF and ICG, the potential value of product G (HAPDI) becomes -31.3 mV; after the modification of amino groups and the attachment of liver cancer targeting peptides, the potential value of product J (HAPDI9P) becomes 30.4 mV.

[0041] The products F (HCuS / Au-Pt) and DSF@HCuS / Au-Pt were characterized by infrared spectroscopy. Figure 4 . Figure 4The results show that DSF@HCuS / Au-Pt has a -1 Nearby and 700 cm -1 The characteristic peaks of CN and CS appeared nearby, proving the successful loading of DSF.

[0042] The energy spectrum elements of the product F (HCuS / Au-Pt) were analyzed by transmission electron microscopy. The results are shown in Figure 5 . Figure 5 It can be seen that the main elements detected in the sample are Cu, S, Pt and Au, which provides a basis for the synthesis of product F (HCuS / Au-Pt).

[0043] Figure 6 This is a safety test for the hemolysis rate of product J (HAPDI9P). Figure 6 It can be seen that the hemolysis rate of product J (HAPDI9P) NPs (NPs refers to nanoparticles) at 500 μg / mL is still less than 5%, indicating that the safety of the entire system is relatively high.

[0044] The obtained product J (HAPDI9P) was prepared into solutions of different concentrations with water, and then the product was analyzed by 808 nm, 1.0 W / cm 2 The temperature change of the solution was recorded by near-infrared light irradiation with a FLIR thermal imager to obtain the temperature change curve of the product J (HAPDI9P) in the aqueous solution. Figure 7 .from Figure 7 It can be observed that the concentration change diagram (a) shows that when the product J (HAPDI9P) is 100 μg / mL, it is highly concentrated under 808 nm laser (1.0 W / cm 2 ) irradiated with an increase in concentration, the temperature gradually increased, and reached about 45.6 °C in 10 min. The power variation diagram of (b) shows that when the product J (HAPDI9P) was 100 μg / mL, it was heated to 45.6 °C under 808 nm laser (1.0 W / cm 2 ) Under irradiation, the temperature gradually increases with the increase of power, and the temperature can rise to about 45.8 ℃ in 10 minutes; it can kill tumor cells, which corresponds to the concentration change diagram. Therefore, the photothermal conversion properties of product J (HAPDI9P) prove that it can be used as an excellent photothermal material for tumor photothermal therapy.

[0045] Figure 8 To evaluate the photothermal stability of nanodrug product J (HAPDI9P) by multiple laser on / off treatments. In each cycle, the nanodrug solution was irradiated with laser for 10 min, and then cooled to room temperature naturally after the laser was turned off. The on / off cycles were repeated 5 times, and the temperature change curves during these cycles were recorded by plotting the temperature change curves as a function of irradiation time. Figure 8The experimental results show that product J (HAPDI9P) has good photothermal stability.

[0046] In vitro cell inhibition The in vitro tumor activity of product J (HAPDI9P) was detected by MTT method. HepG2 liver cancer cells, SNU-739 liver cancer cells, HeLa cervical cancer cells and HL-7702 normal liver cells in good culture state were spread on the bottom of the culture dish, digested with trypsin, centrifuged, and the supernatant was aspirated and 1 mL of culture medium was added (the composition of the culture medium: 1 L triple distilled water + 13.4 g / pkg DMEM powder + 3.7 g NaHCO 3 +10 mL penicillin-streptomycin dual antibody (penicillin 10000 U / mL, streptomycin 10 mg / mL)) was blown to homogenize the cells, and an appropriate amount of the cell suspension was diluted with culture medium so that the number of cells per well was about 4500 and inoculated in a 96-well plate and cultured for 24 hours. Product J (HAPDI9P) was diluted with culture medium to different concentrations and placed in test tubes for use. Each concentration contained 4 sub-wells, and a blank group without inoculated cells and a negative group without drug were set up for 48 hours. 50 µL 1 mg / mL MTT solution was added to each well for 4 hours, the plate was shaken, 100 µL DMSO test solution was added to each well, and the shaker was shaken for 10 min. The absorbance value A at 570 nm was measured with a multifunctional microplate reader; the inhibition rate of each concentration of the sample on the cells was calculated by the formula: cell inhibition rate × 100% = (average OD value of the negative group - average OD value of the experimental group) / (average OD value of the negative group - average OD value of the blank group), and the corresponding IC was calculated using software 50 The results are shown in Fig. 9 .

[0047] from Fig. 9 It can be seen that the results of the proliferation rates of HepG2 cells, SNU-739 cells, HeLa cells and HL-7702 cells show that product J (HAPDI9P) has the greatest toxicity to HepG2 cells (human liver cancer), HeLa cells (human cervical cancer) and SNU-739 cells (human liver cancer), and has little toxicity to HL-7702 cells (human normal liver cells). It is speculated that the modification of the liver cancer targeting peptide binds to the FOXM1 receptor on the surface of HepG2 cells, which can specifically target HepG2 cells and has little toxicity to HL-7702 cells.

[0048] In vivo tumor therapy To further investigate the in vivo antitumor effects of nanodrug product J (HAPDI9P) and free ICG that promote copper death-enhancing disulfiram (DSF) chemotherapy in liver cancer cells, we constructed a subcutaneous transplant tumor model in BALB / c mice using H22 liver cancer cells and studied the antitumor effects of the nanodrug by tail vein injection (dose: 5 mg / kg) combined with laser control.

[0049] The mice were randomly divided into 7 groups, 5 in each group. A subcutaneous transplant tumor model of BALB / c mice was established using H22 cells, and the anti-tumor effect of the nanomedicine was studied by tail vein injection (dose: 5 mg / kg) combined with laser control. The tumor size of the mice was observed every day, and the tumor volume was about 100 mm 3 Seven groups of mice were dosed. They were ① control group (normal saline), ② HCuS / Au-Pt group, ③ HCuS / Au-Pt+NIR group, ④ positive drug DSF group, ⑤ ICG+NIR group, ⑥ HAPDI group, ⑦ HAPDI9P NPs group, and ⑧ HAPDI9P NPs+NIR group. The mice were dosed every other day for 3 times. The body weight and tumor volume of the mice were measured at the same time. The mice were irradiated with 808nm laser (1.0 W / cm 2 , 5 min) to irradiate the tumor area. The tumor volume of mice is calculated by V=W 2 ×L / 2 (V represents tumor volume, W represents tumor short diameter, and L represents tumor long diameter).

[0050] After the treatment, the experimental results are shown in Fig.10 .like Fig.10 As shown in the figure, the weight gain of mice in all groups was similar, indicating that the selected materials and the entire system were safe. Compared with the control group, the HAPDI9P NPs group and the HAPDI9P NPs+NIR group had a more obvious inhibitory effect on tumor growth, among which the HAPDI9P NPs+NIR group had the best inhibitory effect. The tumor weight of the HAPDI9PNPs+NIR group was only 0.035 g, which was much lower than the control group (1.083 g) and the DSF group (0.816 g). The tumor volume of mice in the HAPDI9P NPs+NIR group was only 26.08 mm 3 , which was much lower than the control group (625.42 mm3) and the DSF group (516.85 mm3). 3 The tumor inhibition rate reached 96.9%, indicating that the nano drug HAPDI9P of the present invention can better inhibit tumor growth under laser irradiation conditions.

[0051] In summary, the nano drug carrier prepared by the present invention for promoting copper death of liver cancer cells and enhancing disulfiram (DSF) chemotherapy has good hydrophilicity, high safety, small size and liver cancer-specific targeting effect. The present application provides a novel nano drug, which includes hollow copper sulfide nanoparticles (HCuSNPs) of inorganic nanomaterials with near-infrared light (NIR) response, bimetallic catalytic active Au-Pt nanozymes, photosensitizer indocyanine green (ICG), diethyldithiocarbamate-copper conjugate (Cu(DTC) 2 ) and a peptide targeting liver cancer were anchored to obtain the nanodrug HCuS / Au-Pt@DSF / ICG@9R-P (abbreviated as HAPDI9P) with the ability to promote copper death of liver cancer cells and enhance DSF chemotherapy. The nanodrug finally achieved the combined therapeutic effect of excellent chemotherapy, photothermal therapy, photodynamic therapy and chemodynamic therapy, induced immunogenic death of liver cancer cells, enhanced the immune response of liver cancer cells, and was of great significance for promoting copper death of liver cancer cells. In in vitro and in vivo studies, the nanodrug HAPDI9P actively targeted HepG2 cells through endocytosis mediated by specific receptors on the surface of liver cancer cells, and then triggered the release of the drug in the tumor microenvironment (TME). Under the irradiation of near-infrared (NIR) light, HAPDI9P can not only effectively convert near-infrared light into the heat required for photothermal therapy, but also generate high levels of reactive oxygen species (ROS) for photodynamic therapy. In the tumor-bearing mouse experiment, under the near-infrared light (808 nm, 1.0 W / cm 2 The tumor-bearing mice treated with HAPDI9P+NIR group under irradiation of 100 μg / mL for 5 min showed a tumor inhibition rate of about 96.9%. Therefore, the nanodrug designed to promote copper death of liver cancer cells and enhance disulfiram (DSF) chemotherapy has excellent combined therapeutic effects of chemotherapy, photothermal therapy, photodynamic therapy and chemodynamic therapy, and induces immunogenic death of liver cancer cells, enhances the immune response of liver cancer cells, and is of great significance to promote copper death of liver cancer cells.

Claims

1. A method for preparing a nano drug carrier that promotes copper death of liver cancer cells and enhances disulfiram chemotherapy, characterized in that: The steps include: Step 1, at room temperature, dispersing cupric chloride dihydrate solution and polyvinyl pyrrolidone in water to obtain a mixed solution A; Step 2, adding 45-55% hydrazine hydrate solution, sodium hydroxide solution with pH=8-10 and 310-330 mg / mL sodium sulfide nonahydrate solution to the mixed solution A, stirring to obtain a mixed solution B; Step 3: at room temperature, centrifuge, wash, and freeze-dry the mixed solution B to obtain a dark green solid; then disperse the dark green solid in ultrapure water to obtain a product C suspension; Step 4: Dispersing the product C suspension in ethanol at room temperature to obtain a mixed solution D; Step 5, at room temperature, first add polyvinyl pyrrolidone to the mixed solution D, stir, then add chloroauric acid aqueous solution, stir, then add sodium borohydride aqueous solution, stir, and finally add potassium chloroplatinate and L-ascorbic acid, stir in an oil bath at 70-90 ° C for 20-40 min to obtain a mixed solution E; Step 6: Under room temperature, centrifuge, wash, and freeze-dry the mixed solution E to obtain a black solid; then disperse the black solid in ultrapure water to obtain a product F suspension.

2. The method for preparing the nano drug carrier for promoting copper death of liver cancer cells and enhancing disulfiram chemotherapy according to claim 1, characterized in that: In step 1, the mass ratio of cupric chloride dihydrate to polyvinyl pyrrolidone is 1:20-30.

3. The method for preparing the nano drug carrier for promoting copper death of liver cancer cells and enhancing disulfiram chemotherapy according to claim 1, characterized in that: In step 5, first add 5-15 mg of polyvinyl pyrrolidone to the mixed solution D containing 0.1-0.3 mg of dark green solid, stir for 20-40 min, then add 5-7 mL of 0.2-0.4 mM chloroauric acid aqueous solution, stir for 5-15 min, then add 0.4-0.6 mL of 2-4 mM sodium borohydride aqueous solution, stir for 20-40 min, and finally add 50-150 mL of 0.05-0.15 mM potassium chloroplatinate and 4-6 mL of 4-6 mM L-ascorbic acid.

4. A nano drug carrier for promoting copper death of liver cancer cells and enhancing disulfiram chemotherapy, prepared by the method according to any one of claims 1 to 3.

5. Use of the nano drug carrier for promoting copper death of liver cancer cells and enhancing disulfiram chemotherapy as claimed in claim 4 in drug delivery.

6. A method for preparing a liver cancer targeting peptide-modified disulfiram chemotherapy nanodrug that promotes copper death of liver cancer cells using the nano drug carrier according to claim 4, characterized in that: The steps include: Step a, at room temperature, sonicating the product F suspension nano drug carrier, then adding the chemotherapy drug disulfiram and the photosensitizer indocyanine green, stirring for 2-48 h in the dark, centrifuging, washing, and freeze-drying to obtain a black solid; then dispersing the black solid in a pH 7-9 phosphate buffer to obtain a product G suspension; Step b, at room temperature, subjecting the product G suspension to ultrasonic treatment, adding a thiol-containing coating, stirring for 2-48 h in the dark, centrifuging, washing, and freeze-drying to obtain a black solid; and then dispersing the black solid in ultrapure water to obtain a product H suspension. Step c, placing the liver cancer targeting peptide in a phosphate buffer at pH 7-9 at room temperature, adding carbodiimide hydrochloride and N-hydroxysuccinimide, and stirring to obtain an activated peptide solution I; Step d: at room temperature, the product H suspension and the activated peptide solution I are mixed, stirred for 12-48 h in the dark, centrifuged, washed, and freeze-dried to obtain a black solid.

7. The method for preparing a liver cancer targeting peptide modified disulfiram chemotherapy nanodrug that promotes copper death of liver cancer cells according to claim 6, characterized in that: In step a, the mass ratio of the chemotherapy drug disulfiram, the photosensitizer indocyanine green and the black solid in the product F suspension is 1-3:1-3:1; in step b, the mass ratio of the thiol-containing coating and the black solid in the product G suspension is 1-3:

1.

8. The method for preparing a liver cancer targeting peptide modified disulfiram chemotherapy nanodrug that promotes copper death of liver cancer cells according to claim 6, characterized in that: In step c, the sequence of the liver cancer targeting peptide is: rrrrrrrrrGSGSWHLDYPSMWYLD.

9. The method for preparing a liver cancer targeting peptide modified disulfiram chemotherapy nanodrug that promotes copper death of liver cancer cells according to claim 6, characterized in that: In step c, the mass ratio of the liver cancer targeting peptide to carbodiimide hydrochloride and N-hydroxysuccinimide is 1:0.3-0.5:0.6-0.8; in step d, the mass ratio of the liver cancer targeting peptide to the black solid in the product H suspension is 0.4-0.6:

1.

10. A liver cancer targeting peptide-modified disulfiram chemotherapy nanodrug that promotes copper death of liver cancer cells and is prepared by the method of any one of claims 6 to 9.

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