A melanin-like-luteolin-copper metal composite nanomaterial, a preparation method and application thereof

Abstract

The application provides a kind of based on melanin-like-luteolin-copper composite nanomaterial and its preparation method and application. The composite nanomaterial uses melanin-like nanoparticle as carrier, and is loaded with luteolin and copper. In order to make up for the deficiency of single photothermal therapy, the application adopts the scheme of combining photothermal therapy with copper death to improve the effect of tumor treatment. The application creatively introduces Nrf2 inhibitor Luteolin in the composite nanomaterial, which can effectively inhibit Nrf2 in tumor cells, cause the content of ROS in tumor cells to increase, the content of GSH to decrease, reduce the chelation of copper ions, and further enhance the effect of copper death.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a melanin-like compound nanomaterial based on luteolin-copper and a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this BACKGROUND section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission or any kind of suggestion that this information forms the general prior art before the application.

[0003] Traditional cancer treatment strategies, such as surgery, chemotherapy and radiotherapy, usually have poor treatment effects because they can cause some side effects and lead to multi-drug resistance. New anti-cancer treatment strategies such as photothermal therapy have been widely studied. Photothermal therapy based on near-infrared light can burn tumors by local high temperature, and has controllability, simple operation and reliable biological safety. However, single photothermal therapy generally has problems such as insufficient photothermal conversion efficiency, thermal damage to normal tissues around the tumor, and limitations of single therapy, which makes photothermal therapy face great challenges in the process of clinical transformation.

[0004] Metals are essential elements for normal physiological activities of life, but their content in the body is very small. Common trace elements include iron (Fe), manganese (Mn), copper (Cu) and zinc (Zn), etc. Metal elements are regulated and controlled by the body's balance mechanism to maintain normal levels and metal homeostasis. When the homeostasis is destroyed, various pathological damages to the body will occur. For example, when the intracellular iron ion content is too high, it will lead to cell iron death; at the same time, it has been reported that copper death is an independent form of cell death, which mainly combines extracellular copper with copper ion carriers and transports it into cells, interferes with normal cell physiological processes, and induces cell death. Copper ions delivered to tumor sites can enter cells to directly kill tumor cells and inhibit tumor growth on the one hand; on the other hand, they can also induce immunogenic cell death (ICD), thereby activating an anti-tumor immune response. However, the anti-tumor immune response activated by ICD alone is not enough to inhibit tumor growth well. Therefore, other treatment methods are often combined to further activate and amplify the anti-tumor immune response to achieve the desired combined treatment effect. Copper death is strongly dependent on the sustained accumulation of intracellular copper ions, and the intracellular copper ions are strongly limited by glutathione chelation, copper ion transporters and metabolic protein efflux, resulting in a steady state of intracellular copper content, and free copper is difficult to cause tumor cell copper death. In addition, free copper ions also enter normal tissue cells, causing damage. Therefore, improving the accumulation of copper ions in tumor cells is of great significance for anti-tumor treatment based on copper death. SUMMARY

[0005] In order to overcome the above problems, the application provides a melanin-like-luteolin-copper composite nanomaterial and a preparation method and application thereof.

[0006] In order to achieve the above technical purposes, the application adopts the following technical solutions.

[0007] In a first aspect, the application provides a melanin-like-luteolin-copper composite nanomaterial, which takes melanin-like nanoparticles as a carrier, and is loaded with luteolin and copper.

[0008] In one or more embodiments, the melanin-like nanoparticles are obtained by self-assembly of 1,8-dihydroxynaphthalene (1,8-DHN) oxidation.

[0009] In one or more embodiments, the melanin-like composite nanomaterial has a hydration particle size of about 100-300 nm.

[0010] In a second aspect, the application provides a preparation method of the melanin-like-luteolin-copper composite nanomaterial of the first aspect, which comprises the following steps.

[0011] (1) oxidizing 1,8-dihydroxynaphthalene (1,8-DHN) with NaIO4 to obtain melanin-like nanoparticles by self-assembly;

[0012] (2) mixing the melanin-like nanoparticles with a luteolin solution to obtain an intermediate;

[0013] (3) mixing the intermediate with a copper salt solution to obtain the melanin-like-luteolin-copper composite nanomaterial.

[0014] In a third aspect, the application provides an application of the melanin-like-luteolin-copper composite nanomaterial of the first aspect and / or the melanin-like-luteolin-copper composite nanomaterial prepared by the preparation method of the second aspect in the preparation of an antitumor drug.

[0015] In a fourth aspect, the application provides an antitumor drug composition comprising the melanin-like-luteolin-copper composite nanomaterial of the first aspect and / or the melanin-like-luteolin-copper composite nanomaterial prepared by the preparation method of the second aspect.

[0016] The application has the following beneficial effects.

[0017] (1) The application provides a kind of based on melanin-like-luteolin-copper metal composite nanomaterial and its preparation method and application.The composite nanomaterial uses melanin-like nanoparticle as carrier, and is loaded with luteolin and metal copper.In order to make up for the deficiency of single photothermal therapy, the application adopts the scheme of combining photothermal therapy and copper death to improve the effect of tumor treatment.Melanin-like nanoparticle is the aggregate of polyphenol, and its structure contains a large number of voids, which can be used as a nanocarrier to carry drugs for transportation, and can also generate a large amount of heat under the irradiation of near-infrared light, thereby playing the role of photothermal therapy.Using melanin-like particles as light-heat agent as the carrier of metal copper ions, it can efficiently deliver copper ions into tumor cells, induce cell copper death and enhance the killing effect on tumor cells.

[0018] (2) The application creatively introduces Nrf2 inhibitor Luteolin in the composite nanomaterial, which can effectively inhibit Nrf2 in tumor cells, increase the content of ROS in tumor cells, reduce the content of GSH, reduce the chelation of copper ions, and further enhance the effect of copper death. BRIEF DESCRIPTION OF DRAWINGS

[0019] The drawings accompanying the specification of the application form part of the disclosure of the application and serve to further understand the application, the illustrative embodiments of the application and their description serve to explain the application and do not constitute an improper limitation of the application.

[0020] Figure 1 Transmission electron microscope picture of ACL nanoparticles prepared by the application;

[0021] Figure 2 Particle size distribution of ACL nanoparticles prepared by the application;

[0022] Figure 3 Zeta potential of AMNP prepared in example 1, ACu prepared in comparative example 1, ALut prepared in comparative example 2 and ACL prepared in example 2 in the application;

[0023] Figure 4 Temperature change of nanoparticles prepared by the application under irradiation of laser with different intensities;

[0024] Figure 5 Temperature change of nanoparticles prepared by the application under irradiation of laser with different concentrations;

[0025] Figure 6 Luteolin and Cu released by nanoparticles prepared by the application under acidic conditions 2+ ;

[0026] Figure 7 Cell viability chart of nanoparticles prepared by the application;

[0027] Figure 8 The nanoparticles prepared by the present application induce calreticulin transfer;

[0028] Figure 9 The nanoparticles prepared by the present application induce high mobility group protein B1 release;

[0029] Figure 10 The nanoparticles prepared by the present application cause mouse tumor copper death analysis;

[0030] Figure 11 The nanoparticles prepared by the present application affect the expression of CD80, a dendritic cell maturation marker, in the draining lymph nodes of mice;

[0031] Figure 12 The nanoparticles prepared by the present application affect the expression of CD86, a dendritic cell maturation marker, in the draining lymph nodes of mice. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0033] It is also important to note that the terms used herein are not intended to limit the particular embodiments of the present application to the preferred embodiments described. Rather, the terms are used only to describe specific embodiments of the present application. As used herein, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" are intended to include the plural forms as well, i.e., the term "a" is meant to include the forms "at least one" and "one or more." Furthermore, it is to be understood that the terms "comprising" and / or "including," when used herein, specify the presence of stated features, steps, operations, devices, components, and / or combinations thereof, but do not preclude the presence or addition of one or more other features, steps, operations, devices, components, and / or combinations thereof.

[0034] A first exemplary embodiment of the present application provides a melanin-like-luteolin-copper-based composite nanomaterial, which uses melanin-like nanoparticles as a carrier and is loaded with luteolin and copper.

[0035] In one or more embodiments, the melanin-like nanoparticles are obtained by oxidation of 1,8-dihydroxynaphthalene from a self-contained kit.

[0036] In one or more embodiments, the melanin-like composite nanomaterial has a hydration particle size of about 100-300 nm.

[0037] A second exemplary embodiment of the present application provides a preparation method of the melanin-like-luteolin-copper-based composite nanomaterial of the first aspect, which comprises the following steps:

[0038] (1) using NaIO4 to oxidize 1,8-dihydroxynaphthalene (1,8-DHN) to obtain melanin-like nanoparticles by self-assembly;

[0039] (2) mixing the melanin-like nanoparticles with a luteolin solution to obtain an intermediate;

[0040] (3) mixing the intermediate with a copper salt solution to obtain a melanin-like-luteolin-copper composite nanomaterial.

[0041] In one or more embodiments, the melanin-like-luteolin-copper composite nanomaterial is modified using 8-arm-PEG-NH2 and 8-arm-PEG-NHS solutions to obtain a modified melanin-like-luteolin-copper composite nanomaterial.

[0042] Preferably, the melanin-like-luteolin-copper composite nanomaterial obtained by mixing the intermediate with the copper salt solution is sequentially immersed in 8-arm-PEG-NH2 and 8-arm-PEG-NHS solutions to obtain a modified melanin-like-luteolin-copper composite nanomaterial.

[0043] Further preferably, the concentration of the 8-arm-PEG-NH2 solution is 0.8-1.2 mg / mL, preferably 1 mg / mL; and the time of immersion in the 8-arm-PEG-NH2 solution is 3-5 h, preferably 4 h.

[0044] Further preferably, the concentration of the 8-arm-PEG-NHS solution is 0.8-1.2 mg / mL, preferably 1 mg / mL; and the time of immersion in the 8-arm-PEG-NHS solution is 10-15 h, preferably 12 h.

[0045] In one or more embodiments, in step (1), the method for obtaining melanin-like nanoparticles by self-assembly using NaIO4 to oxidize 1,8-dihydroxynaphthalene (1,8-DHN) comprises:

[0046] NaIO4 solution is added to the 1,8-dihydroxynaphthalene solution, the reaction is stirred, and then the solid is collected to obtain melanin-like nanoparticles using methanol as a precipitant.

[0047] Preferably, the solvent in the 1,8-dihydroxynaphthalene solution is a mixed solution of acetonitrile and water, and the volume ratio of acetonitrile to water is 1:(18-22), preferably 1:19; and the concentration of the 1,8-dihydroxynaphthalene solution is 0.9-1.2 g / mL, preferably 1.0 g / mL.

[0048] Preferably, the concentration of the NaIO4 solution is 0.8-1.2 mol / L, preferably 1 mol / L.

[0049] Preferably, the molar ratio of 1,8-dihydroxynaphthalene to NaIO4 is (1.8-2.4):1, preferably 2:1.

[0050] In one or more embodiments, in step (2), the mass ratio of melanin-like nanoparticles to luteolin is (2-3):(7-8), preferably 2.5:8.

[0051] In one or more embodiments, in step (3), the copper salt is CuCl2.

[0052] Preferably, the mass ratio of melanin-like nanoparticles to CuCl2 is (2-3):4, preferably 2.5:4.

[0053] In a third typical embodiment of the present application, there is provided a use of the melanin-like-luteolin-copper-based composite nanomaterial provided in the first aspect and / or prepared by the method of the second aspect in the preparation of an anti-tumor drug.

[0054] In a fourth typical embodiment of the present application, there is provided an anti-tumor drug composition comprising the melanin-like-luteolin-copper-based composite nanomaterial provided in the first aspect and / or prepared by the method of the second aspect.

[0055] In one or more embodiments, the anti-tumor drug composition further comprises a tumor treatment-related drug.

[0056] Preferably, the tumor treatment-related drug is an anti-tumor drug, an immune adjuvant, a checkpoint inhibitor, or an antigenic protein.

[0057] In one or more embodiments, the tumor comprises a benign tumor and / or a malignant tumor; the malignant tumor comprises a solid tumor and a blood tumor.

[0058] It should be noted that the tumor is used in the present application as known by those skilled in the art, which includes a benign tumor and / or a malignant tumor. The benign tumor is defined as an excessive proliferation of cells that cannot form an aggressive, metastatic tumor in the body. Conversely, the malignant tumor is defined as a cell with various cellular abnormalities and biochemical abnormalities that can form a systemic disease (e.g., tumor metastasis in a distant organ).

[0059] solid tumors such as tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (e.g. thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, kidney, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, renal, small intestine, soft tissue, testis, stomach, skin (e.g. melanoma), ureter, vagina and vulva. Malignomas include hereditary cancers such as retinoblastoma and Wilm's tumor. Furthermore, malignomas include primary tumors in the mentioned organs and corresponding secondary tumors in distant organs (metastases). Hematological tumors such as leukemias and lymphomas in aggressive and indolent forms, i.e. non-Hodgkin's disease, chronic and acute myeloid leukemia (CML / AML), acute lymphoblastic leukemia (ALL), Hodgkin's disease, multiple myeloma and T-cell type lymphomas. Also included are myelodysplastic syndrome, plasmacytoma, paraneoplastic syndrome and cancer of unknown primary site and AIDS-related malignomas.

[0060] In order to enable a person skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples.

[0061] Example 1

[0062] Preparation of melanin-like nanoparticles AMNP:

[0063] Weigh 1,8-DHN (20 mg) into 20 mL 5% acetonitrile solution, place it on a magnetic stirrer and stir for 5 min to make it fully dissolved. Quickly add 62.5 μL of NaIO4 solution (1 M) to it, and the color of the reaction system quickly turns yellow, then gray. Stir the reaction for 20 h, then centrifuge at high speed (11000 r / min, 10 min), wash with water for 3 times, and finally disperse in water and store at room temperature.

[0064] After 24 h, centrifuge to remove water (11000 r / min, 10 min), add methanol to make the concentration 0.5 mg / mL, mix by vortexing (stirring speed 4000 rpm) for 30 s, and place on a shaker (90 r / min). After 6 days, dialysis with a 10 kDa dialysis bag in pure water for 2 days, and if necessary, concentrate by centrifugation (11000 r / min, 10 min).

[0065] Example 2

[0066] Preparation of melanin-like-luteolin-copper metal composite nanomaterial ACL:

[0067] Take AMNP solution (2.5 mg / mL) 1 mL and luteolin ethanol solution (4 mg / mL) 2 mL mixed vortex (stirring speed 4000 rpm) 10 min, then rotary evaporation to remove the solvent. Add CuCl2solution (4 mg / mL) 1 mL, mixed vortex (stirring speed 4000 rpm) 10 min, then high speed centrifugation (14000 r / min, 10 min), washed with water 3 times. Add 8-arm-PEG-NH2(1 mg / mL) 2.5 mL, magnetic stirring 4 h, then high speed centrifugation (14000 r / min, 10 min), washed with cold water 3 times. Add 8-arm-PEG-NHS solution (1 mg / mL) 2.5 mL, magnetic stirring 12 h, centrifugation (14000 r / min) water washing 3 times, finally dispersed into PBS and stored at room temperature.

[0068] The morphology of the composite nanomaterial ACL based on melanin-like-luteolin-metal copper obtained in this example is shown in Figure 1 , which shows that the composite material based on melanin-like-luteolin-metal copper is a nanoparticle.

[0069] The particle size distribution of the composite nanomaterial based on melanin-like-luteolin-metal copper is shown in Figure 2 , which shows that the average hydrated particle size is about 200 nm.

[0070] Comparative Example 1

[0071] Preparation of melanin-like nanoparticle ACu loaded with CuCl2:

[0072] Take AMNP solution (2.5 mg / mL) 1 mL and CuCl2solution (4 mg / mL) 1 mL mixed vortex (stirring speed 4000 rpm) 10 min, then high speed centrifugation (14000 r / min, 10 min), washed with cold water 3 times. Add 8-arm-PEG-NH2(1 mg / mL) 2.5 mL, magnetic stirring 4 h, centrifugation (14000 r / min) water washing 3 times. Add 8-arm-PEG-NHS solution (1 mg / mL) 2.5 mL, magnetic stirring 12 h, then high speed centrifugation (14000 r / min, 10 min), washed with cold water 3 times, finally dispersed into water and stored at room temperature.

[0073] Comparative Example 2

[0074] Preparation of melanin-like nanoparticle ALut loaded with luteolin:

[0075] AMNP solution (2.5 mg / mL) 1 mL and Luteolin ethanol solution (4 mg / mL) 2 mL were mixed by vortex (stirring speed 4000 rpm) for 10 min, and then the solvent was removed by rotary evaporation. 8-arm-PEG-NH2 solution (1 mg / mL) 2.5 mL was added thereto, and magnetic stirring was performed for 4 h, followed by high-speed centrifugation (14000 r / min, 10 min) and washing with cold water 3 times. 8-arm-PEG-NHS solution (1 mg / mL) 2.5 mL was added thereto, and magnetic stirring was performed for 12 h, followed by high-speed centrifugation (14000 r / min, 10 min) and washing with cold water 3 times, and finally dispersed in PBS and stored at room temperature.

[0076] The zeta potentials of AMNP prepared in Example 1, ACu prepared in Comparative Example 1, ALut prepared in Comparative Example 2, and ACL prepared in Example 2 are shown in Figure 3 , indicating that the zeta potential can be changed after adsorbing copper ions, and further changed under the action of Luteolin to change the potential of the nanoparticles. Since the AMNP particles themselves are negatively charged and have a pore structure, they will be electrostatically adsorbed with the cation Cu 2+ , resulting in the loading of Cu 2+ . In addition, Cu 2+ can complex with Luteolin to form a metal polyphenol complex loaded on the particles.

[0077] The photothermal performance analysis of AMNP prepared in Example 1 and ACL prepared in Example 2 is shown in Figure 4 , Figure 5 , indicating that under 808 nm laser irradiation, the temperature of the nanoparticles can be significantly increased with the increase of the concentration of the nanoparticles, the passage of irradiation time, and the increase of the laser intensity.

[0078] In order to verify that ACL prepared in Example 2 can be decomposed to release the loaded material under acidic conditions, and then decomposed and applied in tumor cells, part of the ACL was placed in solutions with pH = 5.0 and pH = 7.4, respectively, the supernatant was collected and the content of Cu 2+ , Luteolin in the solution was quantitatively analyzed. The results are shown in Figure 6 , indicating that under the slightly acidic conditions of the tumor, ACL can be decomposed to release Cu 2 + , Luteolin, while it is relatively stable under physiological pH.

[0079] Example 3

[0080] Culture of 4T1 (mouse breast cancer cells): 1640 medium was preheated in 37°C water bath, 5 mL preheated medium was taken into 15 mL centrifuge tube in the biological safety cabinet. 4T1 cells were taken from liquid nitrogen tank and quickly put into 37°C water bath, gently shaken to make it evenly heated until thawing. The thawed cell suspension was transferred into the pre-divided medium in the biological safety cabinet, centrifuged, and the supernatant was discarded. Add medium to resuspend the cells and transfer to the culture bottle, shake well and place in the incubator for culture. When the cells grow to 80%, trypsinize and transfer to a centrifuge tube, centrifuge, and discard the supernatant. Add 1 mL of medium to resuspend the cells, take 250 μL of cell suspension into the culture bottle, add the required medium, shake well, and place in the incubator for culture.

[0081] Cell viability detection: the newly recovered cells were subcultured for the third time, and according to the cell count results, the cells were inoculated into a 96-well plate at a density of 8 x 10 3 cells / well in 100 μL of medium per well. After plating, shake well and place the plate in the incubator overnight. The next day, prepare AMNP, ACu, ALut, and ACL nanomaterials in medium at concentrations of 25, 50, and 100 μg / mL. Remove the medium from the original 96-well plate and add the new medium containing different concentrations of nanomaterials. Set 6 drug groups at each concentration, including AMNP light group, ACL light group, AMNP group, ACu group, ALut group, and ACL group. The non-drug group was used as a negative control, with 5 replicate wells per group. After drug addition, place in the incubator for 24 h, and the light groups were irradiated with 808 nm laser (1 W / cm 2 , 10 min) after 12 h of incubation after drug addition, and then placed in the incubator for 24 h. After the time, remove the supernatant, and wash each well with PBS three times. Add 100 μL of prepared MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazolium bromide) solution (0.5 mg / mL) under dark conditions, and incubate in the incubator for 4 h. Remove the supernatant under dark conditions, add 100 μL of DMSO to each well, and shake on a shaker for 10 min. Use a multifunctional enzyme marker to detect the absorbance at 490 nm. Calculate the cell viability by the formula. The results are shown in Figure 7 ACL particles prepared can significantly kill tumor cells.

[0082] Example 4

[0083] Laser confocal analysis of CRT: 4T1 cells (1 x 10 5Cells were cultured at 37°C for 12 hours. Five treatment groups were set up, including AMNP light-treated group, ACL light-treated group, AMNP group, ACu group, ALut group, and ACL group (100 μg / mL) for 24 hours. The untreated group served as a negative control. After treatment, the cells were incubated in an incubator for 24 hours. The light-treated group was incubated in an incubator for 12 hours after treatment and then treated with 808 nm laser irradiation (1 W / cm²). 2 The cells were incubated for 10 min, then placed in an incubator for 24 h. They were washed three times with PBS. The PBS was removed, and the cells were fixed with 1 mL of 4% paraformaldehyde at room temperature in the dark for 20 min, followed by three washes with PBS. The cells were blocked with PBS containing 5% BSA for 30 min, and washed three times with PBS. 100 μL of anti-mouse CRT primary antibody was added, and the cells were incubated at room temperature in the dark for 2 h, followed by three washes with PBS. 100 μL of secondary antibody was added, and the cells were incubated at room temperature in the dark for 1 h, followed by three washes with PBS. The nuclei were stained with Hoechst dye (500 μL, 37℃) for 20 min in the dark, followed by three washes with PBS. The cells were then observed under a laser confocal microscope.

[0084] Quantitative release of HMGB1 using an ELISA kit: 4 T1 cells (1 × 10⁻⁶) were seeded in 24-well plates. 5 Cells were cultured at 37°C for 12 hours. Five treatment groups were set up, including AMNP light-treated group, ACL light-treated group, AMNP group, ACu group, ALut group, and ACL group (100 μg / mL) for 24 hours. The untreated group served as a negative control. After treatment, the cells were incubated in an incubator for 24 hours. The light-treated group was incubated in an incubator for 12 hours after treatment and then treated with 808 nm laser irradiation (1 W / cm²). 2, 10 min), and then placed in the incubator for 24 h. The culture medium was collected and centrifuged (13000 rpm, 10 min). The blank hole (blank control hole without sample and enzyme label reagent, the same as the following steps), standard hole, and sample hole were set. The standard sample was accurately added to the enzyme label coating plate, 50 μL. The sample hole was added with sample diluent, 40 μL, and then the sample to be detected, 10 μL, and then gently shaken to mix. After sealing the plate with a sealing film, it was incubated at 37°C for 30 min. The 30-fold concentrated washing solution was diluted with distilled water 30 times for standby use. The sealing film was carefully removed, and the liquid was discarded. After drying, the washing solution was added to each hole, and after standing for 30 s, it was discarded. The above steps were repeated 5 times, and then dried. Enzyme label reagent, 50 μL, was added to each hole except the blank hole. After sealing the plate with a sealing film, it was incubated at 37°C for 30 min. The sealing film was carefully removed, and the liquid was discarded. After drying, the washing solution was added to each hole, and after standing for 30 s, it was discarded. The above steps were repeated 5 times, and then dried. Color reagent A, 50 μL, and color reagent B, 50 μL, were mixed, and then 100 μL of the mixed solution was added to each hole. After gently shaking, it was incubated at 37°C for 10 min. 50 μL of stop solution was added to each hole to stop the reaction. The blank hole was zeroed, and the absorbance of each hole was measured at 450 nm.

[0085] Copper ions and Luteolin can induce immunogenic cell death (ICD) while killing tumor cells, thereby activating the anti-tumor immune response. During the ICD process, the cell membrane surface specifically expresses CRT protein. The anti-CRT-FITC antibody was used to label the CRT on the cell surface, and the results showed that the CRT expression in the assembled nanoparticle group was significantly higher than that in the free drug group. Figure 8 The confocal results showed that the assembled nanoparticles and the light group showed obvious green fluorescence signals, while the PBS group and the free drug group did not detect obvious fluorescence signals. During the ICD process, high mobility group box 1 (HMGB1) was also released, so the released HMGB1 was quantitatively determined by using an ELISA kit. As shown in FIG. 6B, there was a significant difference between the assembled nanoparticles and the free drug group, and it was further enhanced after light. The results of CRT and HMGB1 showed that the treatment group of the assembled nanoparticles could effectively cause the ICD phenomenon, which provided a good foundation for the subsequent specific immunotherapy. Figure 9

[0086] Example 5

[0087] Analysis of composite nanoparticles inducing copper death of mouse tumor cells:

[0088] Female Balb / C mice were subcutaneously injected with 4T1 cells (1 × 10 6 ​) A mouse breast cancer model was established, and the groups were set up as AMNP light irradiation group, ACL light irradiation group, ACu group, ALut group, ACL group and PBS group. The drug administration group was injected with 100 μL of drug (6.25 mg / kg) through the tail vein, and the PBS group was used as a negative control. The light irradiation group was irradiated with 808 nm laser (1 W / cm 2 , 10 min) 12 h after the end of drug administration. The tail vein was administered on the 9th, 13th, 17th and 21st days, and the mice were treated on the 23rd day. The tumor tissue was paraffin-embedded for sectioning, and the accumulation of copper death marker DLAT was detected by immunofluorescence staining. As shown in Figure 10 , the nanoparticles significantly induced copper death of tumor cells.

[0089] Effect of composite nanoparticles on activation of dendritic cells (BMDCs) in the draining lymph nodes of mice:

[0090] The tumor-side lymph nodes of the mice in the above experimental groups were removed and ground to extract cells. According to different experimental groups, the cells were divided into 1.5 mL centrifuge tubes, centrifuged at 1500 r / min and washed with PBS for 3 times. The PBS solution containing APC-CD11c antibody, PE-Cy7-CD86 antibody and FITC-CD80 antibody was prepared in advance, and 100 μL of the antibody-containing PBS was added to each centrifuge tube, with blank and single-stained samples reserved. Incubate at 4°C in the dark, and after 30 min, wash the cells with 1 mL of PBS. Finally, resuspend the cells in PBS and perform flow cytometry analysis. The fluorescence intensity and percentage of the flow cytometer were used to analyze the activation of dendritic cells in the draining lymph nodes of mice by nanoparticles. The maturation of BMDCs is a key step in initiating an immune response, accompanied by up-regulation of co-stimulatory markers (CD80, CD86). As shown in Figure 11 and Figure 12 , compared with the PBS group, although free drugs (ACu, ALut) and photothermal groups can up-regulate the expression of CD80 and CD86 to a certain extent, the expression level is still lower than that of ACL promoted by BMDCs under light irradiation, which can be attributed to less cell uptake of free drugs, lower immunogenicity of copper ions, etc. The prepared nanoparticles improved the uptake of tumor-associated antigens and significantly increased the expression levels of mature marker CD86 and CD80 on the surface of BMDCs, demonstrating the superiority of the prepared nanoparticles in promoting the maturation of BMDCs.

[0091] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. The application of a composite nanomaterial based on melanin-luteolin-metallic copper in the preparation of antitumor drugs, characterized in that, The composite nanomaterial based on melanin-luteolin-copper uses melanin-like nanoparticles as a carrier and is loaded with luteolin and copper. The melanin-like nanoparticles were obtained by oxidation of 1,8-dihydroxynaphthalene; the mass ratio of the melanin-like nanoparticles to luteolin was (2~3):(7~8). The hydrated particle size of the melanin-based composite nanomaterial is 100~300 nm. The preparation method of the composite nanomaterial based on melanin-luteolin-metallic copper includes the following steps: (1) 1,8-dihydroxynaphthalene was oxidized by NaIO4 and self-assembled to obtain melanin-like nanoparticles; (2) The intermediate was obtained by mixing melanin-like nanoparticles with luteolin solution; (3) After mixing the intermediate with the copper salt solution, a melanin-luteolin-metallic copper composite nanomaterial was obtained; In step (3), the copper salt is CuCl2, and the mass ratio of melanin-like nanoparticles to CuCl2 is (2~3):4; The melanin-luteolin-copper composite nanomaterial obtained by mixing the intermediate with a copper salt solution was then sequentially immersed in 8-arm-PEG-NH2 and 8-arm-PEG-NHS solutions to obtain the modified melanin-luteolin-copper composite nanomaterial.

2. The application as described in claim 1, characterized in that, The concentration of the 8-arm-PEG-NH2 solution was 0.8~1.2 mg / mL; the soaking time in the 8-arm-PEG-NHS solution was 10~15 h.

3. The application as described in claim 1, characterized in that, The concentration of the 8-arm-PEG-NH2 solution was 1 mg / mL; the soaking time in the 8-arm-PEG-NHS solution was 12 h.

4. The application as described in claim 1, characterized in that, In step (1), the method for obtaining melanin-like nanoparticles by oxidizing 1,8-dihydroxynaphthalene with NaIO4 and self-assembling includes: NaIO4 solution was added to a 1,8-dihydroxynaphthalene solution, the mixture was stirred and reacted, the solid was collected, and methanol was used as a precipitant to obtain melanin-like nanoparticles. The solvent in the 1,8-dihydroxynaphthalene solution is a mixture of acetonitrile and water, with a volume ratio of acetonitrile to water of 1:(18~22); the concentration of the 1,8-dihydroxynaphthalene solution is 0.9~1.2 g / mL; the concentration of the NaIO4 solution is 0.8~1.2 mol / L; and the molar ratio of 1,8-dihydroxynaphthalene to NaIO4 is (1.8~2.4):

1.

5. The application as described in claim 4, characterized in that, The volume ratio of acetonitrile to water is 1:

19.

6. The application as described in claim 4, characterized in that, The concentration of the 1,8-dihydroxynaphthalene solution was 1.0 g / mL.

7. The application as described in claim 4, characterized in that, The concentration of the NaIO4 solution is 1 mol / L.

8. The application as described in claim 4, characterized in that, The molar ratio of 1,8-dihydroxynaphthalene to NaIO4 is 2:

1.

9. The application as described in claim 1, characterized in that, The mass ratio of melanin-like nanoparticles to luteolin is 2.5:

8.

10. The application as described in claim 1, characterized in that, The mass ratio of melanin-like nanoparticles to CuCl2 is 2.5:4.

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