A copper-based metabolic regulation nanomaterial and its preparation method and application

Abstract

The present invention discloses a copper-based metabolic regulation nanomaterial, its preparation method and application, and belongs to the technical field of tumor drug preparation. The copper-based metabolic regulation nanomaterial provided by the present invention has a ferroptosis / copper death inducer as the core and a liposome loaded with a glycolysis / NAMPT dual inhibitor as the shell; the glycolysis / NAMPT dual inhibitor is STF-31, and the ferroptosis / copper death inducer is a copper-tannic acid ligand, which can interfere with glycolysis and inhibit NAD + Metabolism, inducing ferroptosis / copper death and reversing the immunosuppressive tumor microenvironment, significantly inhibiting tumor growth and lung metastasis, providing a feasible treatment model for synergistic cancer treatment with good anti-tumor efficacy and practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor drug preparations, and in particular to a copper-based metabolism-regulating nanomaterial, a preparation method thereof, and applications thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance 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] Ferroptosis and copper death are newly discovered regulated cell death modes that are different from apoptosis and are powerful weapons for treating cancer. Due to the low level of intracellular copper, excess copper will be pumped out of the cell through ATP1A (copper efflux pump), making it difficult to maintain high levels of Cu in the cell to achieve effective ferroptosis and copper death induction. Based on this, copper ion carriers and nanodrug delivery systems have been developed to overcome the above difficulties. However, copper ion carriers lack tumor selectivity. Although ilisimol can selectively target mitochondria, its efficacy is poor. Nanocarriers can selectively accumulate at the tumor site, reduce unnecessary off-target, and thus effectively enhance intracellular Cu aggregation.

[0004] Studies have shown that tumor cells primarily metabolize through glycolysis (the Warburg effect), making them insensitive to ferroptosis and copper death. Therefore, overcoming the resistance mechanisms to ferroptosis and copper death within cells is a challenge that needs to be urgently addressed. Previous studies have shown that glycolysis intervention can effectively sensitize ferroptosis and also has a synergistic effect on copper death. However, whether glycolysis intervention can simultaneously sensitize ferroptosis and copper death has not yet been reported.

[0005] In addition, studies have found that when glycolysis is inhibited, tumor cells are in a glucose deprivation state, thereby compensating by upregulating the expression of nicotinamide phosphoribosyltransferase (NAMPT). + ) The rate-limiting enzyme for salvage synthesis, tumor cells use NAMPT to increase NAD + The accumulation of ferroptosis and copper apoptosis ultimately leads to an increase in the synthesis of reduced nicotinamide adenine dinucleotide phosphate (NADPH); NADPH may inhibit the efficacy of ferroptosis and copper apoptosis by mediating the synthesis and regeneration of glutathione (GSH).

[0006] Therefore, how to provide a nanomaterial that can simultaneously sensitize tumor cell ferroptosis and copper death to achieve simultaneous improvement in anti-tumor effect and tumor selectivity is an urgent problem to be solved. Summary of the Invention

[0007] In view of this, the present invention provides a copper-based metabolic regulation nanomaterial and its preparation method and application. The copper-based metabolic regulation nanomaterial provided by the present invention converts glycolysis / NAD + The combination of metabolic intervention and ferroptosis / copper death induction has excellent anti-tumor effects.

[0008] In the first aspect, the present invention provides a copper-based metabolic-regulating nanomaterial, which has a ferroptosis / copper death inducer as a core and a liposome loaded with a glycolysis / NAMPT dual inhibitor as an outer shell; the glycolysis / NAMPT dual inhibitor is STF-31, and the ferroptosis / copper death inducer is a copper-tannic acid ligand.

[0009] Preferably, the liposomes are composed of dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine, cholesterol hemisuccinate (CHEMS) and phosphatidylethanolamine-polyethylene glycol (DSPE-PEG).

[0010] Preferably, the mass ratio of the glycolysis / NAMPT dual inhibitor, the ferroptosis / copper death inducer and the liposome is 1:(5-20):(5-20).

[0011] In a second aspect, the present invention provides a method for preparing a copper-based metabolism-regulating nanomaterial, comprising the following steps:

[0012] adding a copper salt aqueous solution dropwise to a tannic acid aqueous solution, stirring for reaction, centrifuging, collecting the precipitate, and washing with water to obtain a copper-tannic acid complex; dispersing the copper-tannic acid complex in water, and ultrasonically preparing a copper-tannic acid aqueous dispersion;

[0013] Dilinoleylphosphatidylethanolamine, lecithin, cholesterol hemisuccinate, phosphatidylethanolamine-polyethylene glycol and STF-31 are dissolved in an organic solvent, and the organic solvent is removed by rotary evaporation to obtain a lipid film, and the copper-tannic acid aqueous dispersion is added, and the film is ultrasonically hydrated and extruded to obtain the film.

[0014] Preferably, the copper salt is selected from copper acetate, copper sulfate, copper chloride or copper nitrate.

[0015] Preferably, the mass ratio of the copper salt to tannic acid is 1:(1-8).

[0016] Preferably, the stirring reaction time is 5 to 20 hours, and the stirring speed is 400 to 1200 rpm.

[0017] Preferably, the molar ratio of dilinoleylphosphatidylethanolamine, lecithin, cholesterol hemisuccinate, phosphatidylethanolamine-polyethylene glycol and STF-31 is 1:(0.9-1.1):(0.9-1.1):(0.1-0.15):(0.3-0.4).

[0018] Preferably, the organic solvent is a mixed solvent of chloroform and methanol, and the volume ratio of chloroform to methanol is (2-4):1.

[0019] Preferably, the mass ratio of the lipid film to the copper-tannic acid coordination compound is 1:(0.5-4); and the concentration of the copper-tannic acid aqueous dispersion is 2-10 mg / mL.

[0020] Preferably, the extrusion step is specifically as follows: placing the ultrasonically hydrated liquid at a temperature of 35 to 45° C. and stirring for 0.5 to 2 hours, and then extruding through a 200 nm polycarbonate porous membrane.

[0021] In a third aspect, the present invention provides the use of the copper-based metabolism-regulating nanomaterial described in the first aspect or the copper-based metabolism-regulating nanomaterial prepared by the preparation method described in the second aspect in the preparation of an anti-tumor drug, wherein the drug comprises at least one or more of the following uses:

[0022] 1) Inhibit glycolysis, reduce lactate, ATP levels and copper-ATPase activity;

[0023] 2) Inhibition of NAD + metabolism, reducing NADPH production and thus inhibiting GSH synthesis;

[0024] 3) sensitized ferroptosis / copper death;

[0025] 4) Reverse the immunosuppressive tumor microenvironment and improve anti-tumor efficacy.

[0026] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0027] The copper-based metabolic regulation nanomaterial provided by the present invention has a core of a ferroptosis / copper death inducer copper-tannic acid coordination agent and a shell of a liposome loaded with a glycolysis / NAMPT dual inhibitor STF-31, which can interfere with glycolysis and inhibit NAD + Metabolism, inducing ferroptosis / copper death and reversing the immunosuppressive tumor microenvironment, significantly inhibiting tumor growth and lung metastasis, providing a feasible treatment model for synergistic cancer treatment with good anti-tumor efficacy and practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings, which constitute part of the present invention, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute undue limitations thereon. It is obvious that one of ordinary skill in the art could derive other drawings based on these drawings without inventive effort.

[0029] Figure 1 This is a particle size distribution diagram of Cu-TA in Example 1 of the present invention;

[0030] Figure 2 This is a particle size distribution diagram of SCu / L in Example 2 of the present invention;

[0031] Figure 3 The UV-vis spectra of ·OH generated by SCu / L at different times in Example 4 of the present invention are shown;

[0032] Figure 4 This is a bar graph of the colony formation rate of B16F10 cells after different treatments in Example 5 of the present invention;

[0033] Figure 5 is the relative content of lactic acid after different treatments in Example 6 of the present invention;

[0034] Figure 6 is the relative content of ATP in B16F10 cells after different treatments in Example 7 of the present invention;

[0035] Figure 7 NAD in B16F10 cells after different treatments in Example 8 of the present invention + and the relative content of NADPH;

[0036] Figure 8 is the relative content of GSH in B16F10 cells after different treatments in Example 9 of the present invention;

[0037] Figure 9 Graph showing tumor weight and tumor inhibition rate in mice after injection of different preparations in Example 10 of the present invention. DETAILED DESCRIPTION

[0038] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0039] The present invention provides a copper-based metabolism-regulating nanomaterial, which comprises a ferroptosis / copper death inducer as a core and a liposome loaded with a glycolysis / NAMPT dual inhibitor as an outer shell; the glycolysis / NAMPT dual inhibitor is STF-31, and the ferroptosis / copper death inducer is a copper-tannic acid ligand.

[0040] The present invention proposes a method for regulating tumor cell glycolysis and NAD + The metabolically enhanced activation model of ferroptosis and copper death was studied. The nanomaterial (denoted as SCu / L) is based on a copper-tannic acid complex (Cu-TA) and is encapsulated by liposomes loaded with STF-31. It enters cells by endocytosis, enhances cellular uptake and promotes the accumulation of copper ions in mitochondria. The mixed valence Cu (Cu II and Cu I )-mediated Fenton-like reaction and GSH consumption can induce ferroptosis and copper death in tumor cells. At the same time, STF-31-mediated dual glycolysis and NAMPT inhibition can reduce intracellular glucose (Glu), NAD + , NADPH, and ATP levels, as well as inhibiting copper-ATPase (Cu-ATPase) activity, reducing intracellular glutathione (GSH) synthesis and inhibiting Cu efflux, thereby sensitizing ferroptosis and copper death. Furthermore, SCu / L not only induces immunogenic cell death (ICD) and triggers T cell activation, but also inhibits lactate (LA) efflux, reshaping the immunosuppressive tumor microenvironment (TME), reducing the proportion of M2 tumor-associated macrophages (TAMs) and restoring the activity of cytotoxic T lymphocytes (CTLs), thereby activating anti-tumor immune responses. Therefore, SCu / L can significantly inhibit tumor growth and lung metastasis.

[0041] This invention is the first to inhibit glycolysis and NAD by dual inhibition + Metabolism can synchronously enhance ferroptosis and copper death, providing a feasible idea for sensitization of ferroptosis / copper death from a metabolic perspective, activating anti-tumor immunity, helping to enhance the anti-tumor efficacy of Cu-based systems, and improving the application potential of ferroptosis / copper death in combined tumor treatment.

[0042] The liposomes described in the present invention are composed of dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine, cholesterol hemisuccinate (CHEMS), and phosphatidylethanolamine-polyethylene glycol (DSPE-PEG). These liposomes are acid-sensitive; because tumor cells are weakly acidic, the liposomes are endocytosed and enter lysosomes, where they rupture under acidic conditions, releasing the drug STF-31.

[0043] In the present invention, the mass ratio of the glycolysis / NAMPT dual inhibitor, the ferroptosis / copper death inducer and the liposome is 1:(5-20):(5-20).

[0044] The present invention also provides a method for preparing a copper-based metabolism-regulating nanomaterial, comprising the following steps:

[0045] adding a copper salt aqueous solution dropwise to a tannic acid aqueous solution, stirring for reaction, centrifuging, collecting the precipitate, and washing with water to obtain a copper-tannic acid complex; dispersing the copper-tannic acid complex in water, and ultrasonically preparing a copper-tannic acid aqueous dispersion;

[0046] Dilinoleylphosphatidylethanolamine, lecithin, cholesterol hemisuccinate, phosphatidylethanolamine-polyethylene glycol and STF-31 are dissolved in an organic solvent, and the organic solvent is removed by rotary evaporation to obtain a lipid film, and the copper-tannic acid aqueous dispersion is added, and the film is ultrasonically hydrated and extruded to obtain the film.

[0047] In the present invention, the copper salt is selected from copper acetate, copper sulfate, copper chloride or copper nitrate. The copper salt has good solubility in aqueous solution, thereby providing copper ions, which can coordinate with tannic acid to form a precipitate.

[0048] In the present invention, the mass ratio of the copper salt to tannic acid is preferably 1:(1-8), more preferably 1:(1-3).

[0049] In the present invention, the stirring reaction time is 5 to 20 hours, more preferably 8 to 15 hours; the stirring speed is 400 to 1200 rpm, more preferably 600 to 1000 rpm, and further preferably 700 to 900 rpm.

[0050] The present invention imposes no particular restrictions on the centrifugation process, as long as solid-liquid separation can be achieved to obtain a precipitate. Preferred centrifugation conditions are: 8,000-12,000 rpm for 8-12 minutes. The present invention also imposes no particular restrictions on the water washing process. Washing can be performed by adding water for dispersion followed by re-centrifugation, with the washing process performed 2-4 times to remove unreacted raw materials and prevent impact on subsequent applications.

[0051] In the present invention, the molar ratio of dilinoleylphosphatidylethanolamine, lecithin, cholesterol hemisuccinate, phosphatidylethanolamine-polyethylene glycol and STF-31 is 1:(0.9-1.1):(0.9-1.1):(0.1-0.15):(0.3-0.4).

[0052] In the present invention, the organic solvent is a mixed solvent of chloroform and methanol, and the volume ratio of chloroform to methanol is (2-4):1.

[0053] The present invention does not impose any special restrictions on the specific process of the rotary evaporation, as long as the organic solvent can be removed and a lipid film can be formed. The preferred rotary evaporation conditions of the present invention are: a temperature of 35-60° C. and a rotation speed of 40-100 rpm.

[0054] In the present invention, the mass ratio of the lipid film to the copper-tannic acid ligand is 1:(0.5-4), more preferably 1:1. The mass of the lipid film is the total mass of dioleoylphosphatidylethanolamine, phosphatidylcholine, cholesterol hemisuccinate, phosphatidylethanolamine-polyethylene glycol, and STF-31. The concentration of the copper-tannic acid aqueous dispersion of the present invention is 2-10 mg / mL, more preferably 4-6 mg / mL.

[0055] The present invention does not impose any particular limitation on the ultrasonic hydration method, and any ultrasonic hydration method commonly used in the art may be used.

[0056] In the present invention, the extrusion step is specifically as follows: placing the ultrasonically hydrated liquid at a temperature of 35 to 45° C. and stirring for 0.5 to 2 hours, and then extruding it through a 200 nm polycarbonate porous membrane.

[0057] The present invention also provides the use of the copper-based metabolism-regulating nanomaterial described in the first aspect or the copper-based metabolism-regulating nanomaterial prepared by the preparation method described in the second aspect in the preparation of an anti-tumor drug, wherein the drug comprises at least one or more of the following uses:

[0058] 1) Inhibit glycolysis, reduce lactate, ATP levels and copper-ATPase activity;

[0059] 2) Inhibition of NAD + metabolism, reducing NADPH production and thus inhibiting GSH synthesis;

[0060] 3) sensitized ferroptosis / copper death;

[0061] 4) Reverse the immunosuppressive tumor microenvironment and improve anti-tumor efficacy.

[0062] The technical solution of the present invention is further described below with reference to specific embodiments.

[0063] In the following examples, the culture medium of the control group was RPMI 1640 medium (Gibco, USA); the culture medium containing STF-31, S / L, Cu / L, and SCu / L was obtained by adding STF-31, S / L, Cu / L, and SCu / L to the control group, respectively, and controlling the amount of Cu added to be 12 μg / mL and the amount of STF-31 added to be 7.5 μg / mL.

[0064] Example 1 Preparation of Ferroptosis / Copper Death Inducer Cu-TA

[0065] Dissolve 10 mg of copper acetate in water and slowly add it dropwise to 2 mL of a 10 mg / mL tannic acid aqueous solution. Stir for 12 hours at 800 rpm. Finally, centrifuge the reaction solution at 10,000 rpm for 10 minutes, collect the precipitate, and wash it three times with water to obtain a copper-tannic acid complex (Cu-TA). Disperse the Cu-TA in water and sonicate at 200 W for 5 minutes to obtain a 5 mg / mL Cu-TA aqueous dispersion.

[0066] Example 2 Preparation of SCu / L

[0067] DOPE (dioleoylphosphatidylethanolamine), lecithin, CHEMS (cholesterol hemisuccinate), DSPE-PEG (phosphatidylethanolamine-polyethylene glycol) and STF-31 were dissolved in 4 mL of a mixed solution of chloroform and methanol (3:1, v / v) at a molar ratio of 1:1:1:0.12:0.35. After thorough mixing, the organic solvent was removed by rotary evaporation at 80 rpm to obtain 20 mg of a uniform and transparent lipid film. Then, 4 mL of the preheated 5 mg / mL Cu-TA aqueous dispersion in Example 1 was added to the lipid film for ultrasonic hydration, and then placed in a 40°C water bath and stirred for 1 h. SCu / L was extruded through a 200 nm polycarbonate porous membrane.

[0068] Preparation of Comparative Example 1S / L

[0069] DOPE, lecithin, CHEMS, DSPE-PEG and STF-31 were dissolved in 4 mL of a mixed solution of chloroform and methanol (3:1, v / v) at a molar ratio of 1:1:1:0.12:0.35. After thorough mixing, the organic solvent was removed by rotary evaporation at 80 rpm to obtain 20 mg of a uniform and transparent lipid film. Then, 4 mL of preheated PBS was added to the lipid film for ultrasonic hydration, and then placed in a 40°C water bath and stirred for 1 h. S / L was obtained by extrusion through a 200 nm polycarbonate porous membrane.

[0070] Comparative Example 2 Preparation of Cu / L

[0071] DOPE, lecithin, CHEMS, and DSPE-PEG were dissolved in 4 mL of a mixed solution of chloroform and methanol (3:1, v / v) at a molar ratio of 1:1:1:0.12. After thorough mixing, the organic solvent was removed by rotary evaporation at 80 rpm to obtain 20 mg of a uniform and transparent lipid film. Then, 4 mL of the preheated 5 mg / mL Cu-TA aqueous dispersion in Example 1 was added to the lipid film for ultrasonic hydration, and then placed in a 40°C water bath and stirred for 1 h. Cu / L was extruded through a 200 nm polycarbonate porous membrane.

[0072] Example 3 SCu / L particle size detection

[0073] Take the Cu-TA in Example 1 and the SCu / L in Example 2 to measure the particle size. Figure 1 and Figure 2 As shown in the figure, the average particle size of Cu-TA is 116.2 nm, and the average particle size of SCu / L is 144.5 nm. Compared with Cu-TA, the particle size of SCu / L is larger.

[0074] Example 4 Evaluation of SCu / L Production of OH in Aqueous Solution

[0075] Methylene blue (MB) was used to evaluate the generation of OH in aqueous solution. The specific operation was as follows:

[0076] A 100 μg / mL SCu / L solution was mixed with a 10 mM H2O2 solution and a 20 μg / mL MB solution and incubated at 37°C for various times (0, 15, 30, 60, and 120 min). After incubation, the mixed solution was scanned with UV-visible light at wavelengths of 450-800 nm, and the spectra were recorded.

[0077] The results are as follows Figure 3 As shown in the figure, with the extension of SCu / L incubation time, the color of MB gradually faded and the absorbance of UV-vis spectrum decreased, indicating that SCu / L can consume MB to produce ·OH, and the generation of ·OH is time-dependent.

[0078] Example 5 Colony formation experiment

[0079] B16F10 cells were seeded at a density of 200 cells / well in a 6-well plate and incubated overnight. After the cells adhered, the old medium was discarded and fresh medium containing STF-31, S / L from Comparative Example 1, Cu / L from Comparative Example 2, and SCu / L from Example 2 was added to each well and culture continued. The old medium was discarded every other day and fresh medium was added for another week. After clear colonies formed, the cells were stained with 0.1% methylene blue, and the colony formation rate was calculated as shown below:

[0080]

[0081] The experimental results are as follows Figure 4 As shown in the results, compared with the control group, the STF-31 and S / L groups could inhibit the formation of colonies to a certain extent, while the Cu / L and SCu / L groups could significantly inhibit the formation of colonies. The colony formation rate of SCu / L was 12.5%, which was lower than that of Cu / L (27.0%), indicating that SCu / L could inhibit the proliferation of tumor cells and had an enhanced tumor killing effect.

[0082] Example 6 Determination of lactic acid content

[0083] B16F10 cells were cultured at 2.0×10 5 Cells were seeded at a density of 100 μg / well in 6-well plates. After cell attachment, the old medium was replaced with fresh medium, medium containing STF-31, S / L from Comparative Example 1, Cu / L from Comparative Example 2, or SCu / L from Example 2, respectively, and culture was continued for 24 h. Subsequently, the cell supernatant was collected and the LA level in the supernatant was measured using an LA kit according to the instructions in the manufacturer's manual.

[0084] The experimental results are as follows Figure 5 As shown in the data, compared with the control group, the STF-31, S / L and SCu / L groups could reduce the production of LA, among which the inhibitory effects of S / L and SCu / L were stronger, which may be attributed to the fact that STF-31 inhibited the uptake of Glu by downregulating the expression of GLUT1, thereby reducing the production and excretion of LA.

[0085] Example 7 ATP content determination

[0086] B16F10 cells were cultured at 2.0×10 5 Cells were seeded at a density of 100 μg / well in a 6-well plate. After cell attachment, the medium was replaced with fresh medium, medium containing STF-31, S / L from Comparative Example 1, Cu / L from Comparative Example 2, or SCu / L from Example 2, respectively, and culture was continued for 24 h. The cells were washed with PBS and lysed on ice by adding 200 μL of ATP lysis buffer. The cells were centrifuged at 12,000 g for 5 min at 4°C. The supernatant was collected and the ATP content was measured using an enhanced ATP content assay kit.

[0087] The experimental results are as follows Figure 6 As shown, compared with the control group, ATP content decreased by 70.8% after S / L treatment, which is attributed to STF-31-mediated inhibition of glycolysis. The ATP content in the Cu / L group decreased by 76.5%, which may be due to Cu generating ROS and inducing ferroptosis, thereby damaging mitochondria and reducing ATP synthesis. In addition, SCu / L had the strongest ATP inhibitory effect, reducing ATP to 57.3%, which was attributed to the synergistic effect of Cu and STF-31.

[0088] Example 8 NAD + and NADPH content determination

[0089] The NAD(H) and NADP(H) content detection kits were used to assess the intracellular NAD +First, B16F10 cells were seeded in a culture dish and placed in an incubator to culture until the cells adhered to the wall. The old culture medium was discarded, and fresh culture medium, culture medium containing STF-31, S / L of Comparative Example 1, Cu / L of Comparative Example 2, or SCu / L of Example 2 were added to each dish. After incubation overnight, the cells were washed with PBS, and appropriate amounts of acidic or alkaline extracts were added to extract NADPH according to the operating procedures in the kit instructions. + The absorbance of the supernatant was measured at 570 nm using a multi-mode microplate detection system to calculate the intracellular NAD + Or NADPH content.

[0090] The experimental results are as follows Figure 7 As shown in A, compared with the control group, cells treated with STF-31, S / L or SCu / L showed an increase in NAD + The contents of NAD were reduced to 71.1%, 37.3% or 42.2%, respectively, among which S / L and SCu / L inhibited NAD + The synthesis capacity is the strongest, which is attributed to the high uptake efficiency of the nanoformulation by cells, which is conducive to STF-31 downregulating the expression of NAMPT. The intracellular NADPH level ( Figure 7 B) in the figure, its changing trend is similar to that of NAD + Compared with Cu / L, SCu / L treatment reduced intracellular NADPH from 91.2% to 44.7% (P<0.01).

[0091] Example 9 GSH content determination

[0092] B16F10 cells were first seeded in a culture dish. When the cell density reached 70% or higher, the old culture medium was replaced with culture medium containing STF-31, S / L from Comparative Example 1, Cu / L from Comparative Example 2, or SCu / L from Example 2, respectively, and the cells were treated for another 24 hours. After incubation, the drug-containing culture medium was discarded, and the cells were collected by centrifugation. The cells were resuspended with Reagent 1 from the GSH content detection kit and repeatedly frozen and thawed two to three times in liquid nitrogen and then in a 37°C water bath. The supernatant was collected after centrifugation at 8000g for 10 minutes. The color was developed according to the GSH kit instructions, and the absorbance at 412 nm was measured to calculate the intracellular GSH content.

[0093] The experimental results are as follows Figure 8 As shown in the figure, compared with the control group, the GSH content of cells treated with STF-31 and S / L only decreased slightly, while the GSH content was significantly reduced after treatment with Cu / L and SCu / L, indicating that Cu IIIn addition, compared with Cu / L, the intracellular GSH content was lower after SCu / L treatment, decreasing from 53.9% to 41.2%, which may be due to the intervention of STF-31, which enhanced the GSH consumption capacity.

[0094] Example 10 Evaluation of the anti-tumor efficacy of SCu / L

[0095] Female C57BL / 6 mice were purchased from Beijing Weitonglihua Animal Technology Co., Ltd. All animal experiments were performed in strict accordance with the protocol approved by the Experimental Animal Center of Shandong University. To establish the tumor-bearing mouse model, 1×10 6 B16F10 cells (100 μL) were subcutaneously inoculated into the right armpit of the mouse. 3 The mice were randomly divided into five groups, each with 5 mice, and injected intratumorally with 100 μL of normal saline (NS), STF-31, S / L from Comparative Example 1, Cu / L from Comparative Example 2, and SCu / L from Example 2 (all at 6 mg / kg Cu ​​and 3.75 mg / kg STF-31 concentrations). The drugs were administered every three days for a total of five treatment cycles, and the tumor volume and body weight of the mice were recorded every other day. The tumor volume was calculated using the following formula:

[0096]

[0097] Where L represents the maximum length (mm) and W represents the minimum length (mm).

[0098] After the treatment, the mice were euthanized and the tumors were obtained by dissection. The tumor inhibition rate of tumor-bearing mice was calculated according to the following formula:

[0099]

[0100] Where W c : Average tumor weight of the saline group, W t : The average tumor weight of each treatment group.

[0101] The experimental results are as follows Figure 9 As shown in the results, compared with the control group, the tumor volume of the STF-31 group showed a rapid growth trend during the treatment cycle, which may be due to the high invasiveness of melanoma. In contrast, the tumor inhibition rate of S / L was enhanced, which was attributed to the fact that the nanoformulation S / L prolonged the retention time in the tumor, which was more conducive to the therapeutic effect of STF-31. Cu / L can significantly inhibit tumor growth with a tumor inhibition rate of 75.2%. After intervention with STF-31, the inhibition rate of SCu / L was as high as 85.8%, indicating that the excellent anti-tumor effect of SCu / L may be due to ferroptosis / copper death induction, glycolysis and NAD +The result of the synergistic effect of metabolic intervention and immunosuppressive microenvironment regulation.

[0102] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A copper-based metabolism-regulating nanomaterial, characterized in that: The copper-based metabolic regulation nanomaterial has a ferroptosis / copper death inducer as the core and a liposome loaded with a glycolysis / NAMPT dual inhibitor as the shell; the glycolysis / NAMPT dual inhibitor is STF-31, and the ferroptosis / copper death inducer is a copper-tannic acid ligand.

2. The copper-based metabolism-regulating nanomaterial according to claim 1, wherein The liposomes are composed of dilinoleylphosphatidylethanolamine, lecithin, cholesterol hemisuccinate and phosphatidylethanolamine-polyethylene glycol; the mass ratio of the glycolysis / NAMPT dual inhibitor, ferroptosis / copper death inducer and liposomes is 1:5~20:5~20.

3. A method for preparing a copper-based metabolism-regulating nanomaterial, characterized in that: The steps include: adding a copper salt aqueous solution dropwise to a tannic acid aqueous solution, stirring for reaction, centrifuging, collecting the precipitate, and washing with water to obtain a copper-tannic acid complex; dispersing the copper-tannic acid complex in water, and ultrasonically preparing a copper-tannic acid aqueous dispersion; Dilinoleylphosphatidylethanolamine, lecithin, cholesterol hemisuccinate, phosphatidylethanolamine-polyethylene glycol and STF-31 are dissolved in an organic solvent, and the organic solvent is removed by rotary evaporation to obtain a lipid film, and the copper-tannic acid aqueous dispersion is added, and the film is ultrasonically hydrated and extruded to obtain the film.

4. The preparation method according to claim 3, wherein The copper salt is selected from copper acetate, copper sulfate, copper chloride or copper nitrate; the mass ratio of the copper salt to tannic acid is 1:1-8.

5. The preparation method according to claim 3, wherein The stirring reaction time is 5 to 20 h, and the stirring speed is 400 to 1200 rpm.

6. The preparation method according to claim 3, wherein The molar ratio of dilinoleylphosphatidylethanolamine, lecithin, cholesterol hemisuccinate, phosphatidylethanolamine-polyethylene glycol and STF-31 is 1: 0.9~1.1: 0.9~1.1: 0.1~0.15: 0.3~0.

4.

7. The preparation method according to claim 3, wherein The organic solvent is a mixed solvent of chloroform and methanol, and the volume ratio of chloroform to methanol is 2-4:

1.

8. The preparation method according to claim 3, wherein The mass ratio of the lipid film to the copper-tannic acid coordination compound is 1:0.5-4; and the concentration of the copper-tannic acid coordination compound aqueous solution is 2-10 mg / mL.

9. The preparation method according to claim 3, wherein The extrusion step is specifically as follows: placing the ultrasonically hydrated liquid at a temperature of 35-45° C. and stirring for 0.5-2 h, and then extruding it through a 200 nm polycarbonate porous membrane.

10. Use of the copper-based metabolism-regulating nanomaterial according to any one of claims 1 to 2 or the copper-based metabolism-regulating nanomaterial prepared by the preparation method according to any one of claims 3 to 9 in the preparation of an anti-tumor drug, wherein the tumor is melanoma, characterized in that: The medicine includes at least one or more of the following uses: 1) Inhibit glycolysis, reduce lactate, ATP levels and copper-ATPase activity; 2) Inhibition of NAD + metabolism, reducing NADPH production and thus inhibiting GSH synthesis; 3) sensitized ferroptosis / copperoptosis; 4) Reverse the immunosuppressive tumor microenvironment and improve anti-tumor efficacy.

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