Curcumin derivative, preparation method of curcumin derivative, nano-composite, preparation method of nano-composite and application of nano-composite

Abstract

The invention relates to the technical field of medicines, and discloses a curcumin derivative and a preparation method thereof, and a nano-composite and a preparation method and application thereof. Wherein the curcumin derivative is (1E, 6E)-1, 7-bis (3, 4-dihydroxyphenyl)-1, 6-heptadiene-3, 5-diketone, and the preparation method of the curcumin derivative comprises the step of carrying out double demethylation treatment on curcumin. The nano-composite contains a chelate of a curcumin derivative and Cu (II). The nano-composite can specifically react with GSH in cancer cells, simultaneously trigger a Fenton reaction and a Michael addition reaction and inhibit GSH synthesis to induce cell apoptosis, consumption of a large amount of GSH further promotes lipid peroxidation (LPO) to trigger ferroptosis, mitochondria can be attacked to cause dysfunction to induce ferroptosis, and in addition, the nano-composite also has good biological safety and can be used for preparing a medicine for treating cancer cells. The compound has a wide prospect in the application of drugs for resisting the drug resistance of cancer cells.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a curcumin derivative and a preparation method thereof, a nanocomposite and a preparation method and application thereof. Background Art

[0002] Drug resistance has always been a major obstacle to the success of clinical cancer treatment. Long-term chemotherapy and targeted therapy can lead to drug resistance, which significantly reduces or loses the therapeutic effect. Drug efflux mediated by drug pumps (such as P-glycoprotein (P-gp)) and toxinization and excretion processes mediated by drug metabolizing enzymes (such as glutathione S-transferases (GSTs)) can cause drug tolerance. Many preclinical and clinical studies have focused on overcoming this tolerance. Common strategies include improving the efficiency of intracellular drug delivery or enhancing the cytotoxicity of drugs. However, continuous treatment will inevitably weaken the sensitivity of target cells or cancer cells to drugs and produce toxicity or side effects on normal tissues. Therefore, there is an urgent need to develop efficient and safe methods to resist tumor tolerance.

[0003] Ferroptosis is a new and promising cancer treatment method, especially for reversing resistance that occurs during cancer treatment. Certain resistant cancer cells, such as mesenchymal cells, dedifferentiated cancer cells, and cancer cells with persistent resistance to treatment are usually resistant to common chemotherapeutic drugs and difficult to apoptosis. However, these cells are highly sensitive to inducers of ferroptosis. Therefore, inducing ferroptosis can restore the sensitivity of certain cancer cells with intrinsic or acquired resistance to chemotherapy. Several compounds have been shown to induce ferroptosis in various tumor models, including reversing chemoresistance in hepatocellular carcinoma (HCC). The SLC7A11 / GSH / glutamate reductase 4 (GPX4) axis plays a very important role as a regulator of various antioxidant defense systems. Inhibition of SLC7A11 (Solute Carrier family 7 member 1), ferroptosis is a member of the cystine-glutamate antiporter system Xc -GPX4 is an important component of ferroptosis, which can be induced by disrupting cystine uptake, thereby limiting GSH synthesis. GSH is not only an important inhibitor but also a key substrate of GPX4. It can convert lipid hydroxyperoxides into corresponding alcohols to prevent the formation of toxic lipid reactive oxygen species (ROS) and LPO and further promote ferroptosis. Many studies have confirmed that the ferroptosis pathway initiated by depletion of glutathione (GSH) can successfully treat cancer. GSH consumption triggers the production of reactive oxygen species (ROS) and greatly reduces the activity of GPX4, which leads to the accumulation of lipid peroxidation products (LPO), thereby inducing ferroptosis. Therefore, disrupting the ferroptosis of drug-resistant cancer cells by strongly depleting GSH can be an effective strategy. However, high levels of GSH in the complex tumor microenvironment (TME) activate the activity of GPX4 and reduce the efficacy of ferroptosis. In addition, many studies have shown that high levels of intracellular GSH make resistant cancer cells more insensitive to chemotherapy. Although some drugs such as cisplatin (DDP) can induce ferroptosis, their efficacy is greatly limited due to the formation of GSH-Pt complexes (in the presence of GSTs) and is further pumped out of the cell by P-gp. Therefore, high levels of GSH in cancer cells are an important "line of defense" to hinder anti-cancer therapy mediated by ferroptosis, and reducing the level of GSH in resistant tumor cells to normal cell levels helps reduce drug resistance and enhance ferroptosis.

[0004] So far, researchers have proposed various promising strategies to achieve GSH depletion. Two common approaches to achieve GSH depletion include the use of metal ions (such as Fe 3+ , Cu 2+ ) to enhance GSH depletion reserves by oxidizing GSH, and to form GS-X conjugates with electronegative molecules or by inhibiting system Xc -to block GSH synthesis. Many metal-based nanomaterials or single compounds with GSH depletion ability have been shown to activate ferroptosis, which has attracted great interest among researchers. Some metal ions with variable valence states are often used for GSH depletion. For example, Cu(II) organic frameworks (MOFs) are designed for ferroptosis induction in cancer therapy. GSH depletion in MOFs is achieved through the Fenton reaction between Cu(II) and GSH. Fe(III)-based phenolic networks (MPNs) achieve GSH depletion through the Fenton reaction to achieve a powerful ferroptosis-based antitumor effect. Nie et al. proposed a metal nanocluster that depletes GSH by accelerating Ir(IV)-Ir(III) conversion, thereby inhibiting glutathione peroxidase 4 (GPX4) activity and enhancing ferroptosis effect. For single compounds that deplete GSH, Zhou et al. synthesized a lipid-like cinnamaldehyde dimer (CDC) to deplete GSH through Michael addition between cinnamaldehyde and GSH. Liu et al. developed a Fenton chemistry-based chemodynamic therapy (CDT) to deplete GSH via Michael addition between cinnamyl humulone and GSH. There are also studies on depleting GSH by inhibiting GSH synthesis with single molecules such as sorafenib (SRF) or by forming a GS-X complex.

[0005] However, most studies are limited to single-stage depletion of GSH through Fenton reaction, Michael addition reaction or inhibition of GSH synthesis to induce apoptosis, which is biologically toxic to healthy cells. Therefore, it is necessary to design a compound that can simultaneously trigger the three methods to combine and produce a cascade GHS depletion effect without damaging normal organs. Summary of the invention

[0006] The purpose of the present invention is to overcome the problem that the existing anticancer cell resistance drugs are limited to inducing cell apoptosis by consuming GSH in a single stage through Fenton reaction, Michael addition reaction or inhibiting GSH synthesis, and will damage normal organs. A curcumin derivative and a preparation method thereof, a nanocomposite and a preparation method and application thereof are provided. The nanocomposite can specifically react with GSH in cells, simultaneously trigger Fenton reaction, Michael addition reaction and inhibit GSH synthesis to induce cell apoptosis, induce a large amount of GSH consumption, significantly enhance the ferroptosis effect of drug-resistant cancer cells, and has better biosafety.

[0007] In order to achieve the above-mentioned object, the first aspect of the present invention provides a curcumin derivative, which is (1E, 6E)-1,7-bis(3,4-dihydroxyphenyl)-1,6-heptadiene-3,5-dione, and its structural formula is shown in formula (I).

[0008]

[0009] The second aspect of the present invention provides a method for preparing a curcumin derivative, the method comprising: subjecting curcumin to a double demethylation treatment to obtain a curcumin derivative having a structural formula as shown in formula (I);

[0010]

[0011] Preferably, the process of the double demethylation treatment comprises the following steps:

[0012] S1. Under anaerobic conditions, curcumin is reacted with a solution containing dichloromethane to obtain a reaction solution I, the reaction solution I is mixed with ice water with a pH value of ≥11, and then the dichloromethane is removed by phase separation to obtain an aqueous phase;

[0013] S2. Adjust the pH of the aqueous phase to 2-4 and filter to obtain a filtrate, dry the filtrate, and then dissolve and precipitate.

[0014] Preferably, the reaction conditions include: temperature of -85°C to -70°C, and time of 20-40 min.

[0015] Preferably, the weight ratio of the curcumin to the dichloromethane is 3-4:1.

[0016] Preferably, the solvent used for the dissolution is ethyl acetate, and the precipitant used for the precipitation is n-hexane.

[0017] The third aspect of the present invention provides a nanocomposite, which contains a chelate of a curcumin derivative and Cu(II), wherein the structural formula of the curcumin derivative is as shown in formula (I),

[0018]

[0019] Preferably, the nanocomposite has a lattice structure with a triangular plate-like morphology;

[0020] Further preferably, the spacing of the lattice structure is 0.2-0.35 nm.

[0021] Preferably, the weight ratio of the nanocomposite curcumin derivative to Cu(II) is 1.5-2.5:1.

[0022] Preferably, the solvent of the nanocomplex includes physiological buffer, PBS, Dulbecco's modified Eagle's medium (DMEM) and DMEM+10% PBS;

[0023] Further preferably, the solvent of the nanocomplex is PBS.

[0024] The fourth aspect of the present invention provides a method for preparing a nanocomposite, the method comprising: subjecting a curcumin derivative to a chelate reaction with a Cu(II) chelate; the curcumin derivative has a structural formula as shown in formula (I),

[0025]

[0026] Preferably, the conditions of the chelating reaction include at least: a temperature of 5-35° C. and a time of 30-50 min.

[0027] Preferably, the chelation reaction is carried out in Tris-HCl buffer.

[0028] The fifth aspect of the present invention provides the use of at least one of the aforementioned curcumin derivatives, the aforementioned method for preparing the curcumin derivatives, the aforementioned nanocomplexes, and the aforementioned method for preparing the nanocomplexes in the preparation of drugs for resisting cancer cell drug resistance.

[0029] Preferably, the drug for resisting cancer cell drug resistance is selected from at least one of a drug for consuming and / or inhibiting GSH, a drug for inducing and / or enhancing cell ferroptosis, and a drug for downregulating ferroptosis-related proteins and / or drug-resistant proteins;

[0030] Preferably, the ferroptosis-related protein is glutathione peroxidase 4 and / or transporter family 7 member 1, and the drug-resistant protein is P-glycoprotein and / or glutathione S-transferase.

[0031] Through the above technical scheme, the present invention designs and synthesizes a nanocomposite (represented by Cur-NCs), Cur-NCs can specifically react with GSH in cells, trigger Fenton reaction, Michael addition reaction and inhibit GSH synthesis to induce cell apoptosis, these processes cause a large amount of GSH consumption, weaken the resistance of drug-resistant cancer cells; Cur-NCs can be used as a "targeted attack" to destroy the "target" of drug-resistant cells, triggering ferroptosis dependent on glutathione (GSH) consumption, and a large amount of GSH consumption will not only induce toxic reactive oxygen (·OH), but also inhibit the activity of GPX4, further promote lipid peroxidation (LPO) and trigger ferroptosis; Cur-NCs can attack and cause mitochondrial ferroptosis and dysfunction, manifested as mitochondrial morphological damage, redox balance disorder (glutathione consumption in mitochondria and increased generation of reactive oxygen), etc. In addition, Cur-NCs has good biosafety and has broad prospects in the use of drugs to resist cancer cell resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The solubility and stability of Cur-NCs in different solvents in Example 3 are shown in FIG. Figure 1 A in the middle is an image of Cur-NCs dispersed in 0.9% sodium chloride buffer, PBS buffer, DMEM, and DMEM + 10% FBS buffer (7 days in total); Figure 1 Middle B is the particle size distribution diagram of Cur-NCs dispersed in 0.9% sodium chloride buffer and PBS buffer (7 days and 14 days in total);

[0033] Figure 2 is the HR-TEM image of Cur-NCs of Example 4, wherein: Figure 2 A in the middle is the HR-TEM image with a scale bar of 20 nm; Figure 2 Middle B is the HR-TEM image with a scale bar of 5 nm; Figure 2 Middle C is the HR-TEM magnified image with a scale bar of 5 nm;

[0034] Figure 3 This is an atomic force microscope (AFM) analysis image of Cur-NCs of Example 4;

[0035] Figure 4 is the XRD pattern of Cur-NCs of Example 4;

[0036] Figure 5 This is an X-ray photoelectron spectroscopy (XPS) observation and analysis diagram of Cur-NCs of Example 4;

[0037] Figure 6 FTIR spectra of Cur-NCs and Cur-C of Example 4;

[0038] Figure 7 is a performance diagram of Cur-NCs in Example 5 regarding GSH consumption, wherein: Figure 7 A in the middle is the UV-visible spectra of the reactions of Cur-NCs, Cur-C, GSH, and Cur-NCs+GSH to GSH; Figure 7 B is the UV-visible spectrum of Cur-NCs (10 μg / mL) reacting with different concentrations of GSH for 10 minutes; Figure 7 Middle C is the color change diagram of Cur-NCs solution under different concentrations of GSH treatment (0-30mM); Figure 7 D in the middle is the color change diagram when Cu(I) is detected by 10μg / mL Cur-NCs (a), 20μg / mL Cur-NCs (b), 10μg / mL Cur-NCs+GSH solution (c), and 20μg / mL Cur-NCs+GSH solution (d); Figure 7 E in the figure is the UV-visible spectrum of the concentration-dependent effect of Cur-C; Figure 7F in the middle is the UV-visible spectrum of Cur-C (20 μM) reacting with different concentrations of GSH for 10 minutes; Figure 7 G in the middle is the UV-visible spectrum of Cur-C (20 μM) reacting to 10 mM GSH at different time periods;

[0039] Figure 8 The figure is a graph showing the anti-HCC effect of Cur-NCs in Example 6 on drug-resistant and sensitive liver cancer cells in vitro, wherein: Figure 8 A in the middle is the cell viability diagram of HepG2 / DDP and HepG2 treated with different concentrations of Cur-NCs after 24 hours;

[0040] Figure 8 B is the cell viability diagram of HepG2 / DDP and HepG2 treated with different concentrations of Cur-C after 24 hours; Figure 8 Middle C is the cell viability diagram of HepG2 / DDP and HepG2 treated with different concentrations of Cu(Ⅱ) after 24 hours; Figure 8 D in the middle is the Live / dead staining CLSM images of HepG2 / DDP and HepG2 after 6 h of treatment with PBS, Cur-C, Cur-NCs 1, Cur-NCs 2, and DDP; Figure 8 Middle E is a flow cytometry analysis of apoptosis of HepG2 / DDP and HepG2 cells after 24 h of treatment with PBS, Cur-C, Cur-NCs 1, Cur-NCs 2, and DDP; (Cur-C = 20 μM, Cur-NCs 1 = 10 μg / mL, Cur-NCs 2 = 20 μg / mL, DDP = 5 μM);

[0041] Fig. 9 is a graph related to mitochondrial dysfunction caused by Cur-NCs in Example 7, wherein Fig. 9 A in the middle is the graph of GSH content detected in mitochondria of HepG2 / DDP cells treated with Cur-C, Cur-NCs 1, Cur-NCs 2, and DDP;

[0042] Fig. 9 Middle Ba is the detection graph of mitochondrial reactive oxygen species (Mito-ROS) levels in HepG2 cells after treatment with Cur-C, Cur-NCs 1, Cur-NCs 2, and DDP for 24 hours; Fig. 9 Middle Bb is the quantitative analysis of mitochondrial reactive oxygen species (Mito-ROS) in HepG2 cells after treatment with Cur-C, Cur-NCs 1, Cur-NCs 2, and DDP for 24 hours; Fig. 9Middle C is a biological transmission electron microscope observation of the mitochondrial morphological changes of HepG2 / DDP cells after being treated with Cur-NCs (10ug / mL, 24h); Fig. 9 D in the middle is a picture of mitochondrial membrane damage of HepG2 / DDP cells after 24 hours of treatment with Cur-C, Cur-NCs 1, Cur-NCs 2, and DDP after staining with Mito-Tracker RedCMXRos, observed by CLSM; (Cur-C = 20 μM, Cur-NCs 1 = 10 μg / mL, Cur-NCs 2 = 20 μg / mL, DDP = 5 μM);

[0043] Fig.10 The figure is a diagram showing the inhibition of the growth of HepG2 / DDP cells in vivo by Cur-NCs of Example 8, wherein: Fig.10 A in the middle shows the weight changes of mice 21 days after treatment with Cur-C, Cur-NCs 1, Cur-NCs 2, and DDP;

[0044] Fig.10 Middle B is the graph showing the changes in tumor volume of mice 21 days after treatment with Cur-C, Cur-NCs 1, Cur-NCs 2, and DDP; Fig.10 Middle C is the image of a single tumor (scale, 1 cm); Fig.10 Middle D is a comparison chart of the weight of a single tumor;

[0045] Fig.10 Middle E is the tumor inhibition rate (TIR) ​​graph of mice receiving Cur-C, Cur-NCs 1, Cur-NCs 2, and DDP treatment regimens; Fig.10 Middle F shows the H&E and TUNEL staining results of tumor tissues of mice receiving Cur-C, Cur-NCs 1, Cur-NCs 2, and DDP regimens; Fig.10 Figure Ga in the middle is the staining image of GPX4 in tumor tissues of mice treated with Cur-C, Cur-NCs 1, Cur-NCs 2, and DDP for 21 days. Fig.10 Gb in the middle is the staining image of SLC7A11 in tumor tissues of mice treated with Cur-C, Cur-NCs 1, Cur-NCs2, and DDP for 21 days. Fig.10 Gc in the middle is the staining image of P-gp in tumor tissues of mice treated with Cur-C, Cur-NCs 1, Cur-NCs 2, and DDP for 21 days. Fig.10 Gd in the middle is the staining image of GSTs in tumor tissues of mice treated with Cur-C, Cur-NCs 1, Cur-NCs 2, and DDP for 21 days; Fig.10Middle H is the detection diagram of glutathione (GSH) level in tumor tissue of mice treated with Cur-C, Cur-NCs 1, Cur-NCs2, and DDP for 21 days; (Cur-C = 20 μM, Cur-NCs 1 = 10 μg / mL, Cur-NCs 2 = 20 μg / mL, DDP = 5 μM);

[0046] Fig.11 is a correlation diagram of the biosafety of Cur-NCs in Example 9, wherein Fig.11 A in the middle is the hemolysis analysis diagram of Cur-NCs at different concentrations (4, 10, 20, 30, 40ug / mL); Fig.11 B is the coagulation analysis diagram of Cur-NCs at different concentrations (4, 10, 20, 30, 40ug / mL); Fig.11 Center C shows the H&E staining results of organs in different treatment groups of HepG2 / DDP mice. DETAILED DESCRIPTION

[0047] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0048] The first aspect of the present invention provides a curcumin derivative, which is (1E, 6E)-1,7-bis(3,4-dihydroxyphenyl)-1,6-heptadiene-3,5-dione, and its structural formula is shown in formula (I).

[0049]

[0050] According to the present invention, compared with curcumin, the number of phenolic hydroxyl groups of the curcumin derivative is increased from 2 to 4, the metal chelating ability is stronger, and the structure contains two α, β-unsaturated ketone groups, which can show good glutathione consumption ability through Michael reaction.

[0051] The second aspect of the present invention provides a method for preparing a curcumin derivative, the method comprising: subjecting curcumin to a double demethylation treatment to obtain a curcumin derivative having a structural formula as shown in formula (I);

[0052]

[0053] According to the present invention, preferably, the process of the double demethylation treatment comprises the following steps:

[0054] S1. Under anaerobic conditions, curcumin is reacted with a solution containing dichloromethane to obtain a reaction solution I, the reaction solution I is mixed with ice water with a pH value of ≥11, and then the dichloromethane is removed by phase separation to obtain an aqueous phase;

[0055] S2. Adjust the pH of the aqueous phase to 2-4 and filter to obtain a filtrate, dry the filtrate, and then dissolve and precipitate.

[0056] According to the present invention, preferably, the reaction conditions include: temperature of -85 to -70°C, and time of 20-40 min.

[0057] Preferably, the weight ratio of the curcumin to the dichloromethane is 3-4:1.

[0058] Preferably, the solvent used for the dissolution is ethyl acetate, and the precipitant used for the precipitation is n-hexane.

[0059] Exemplarily, the process of subjecting curcumin to a double demethylation treatment comprises:

[0060] (1) under anaerobic conditions, curcumin with a content of more than 95% is mixed with a boron tribromide solution containing 15-18vt% dichloromethane (DCM) in a reaction environment of -85°C to -70°C, wherein the weight ratio of the curcumin to the dichloromethane is 3-4:1, and after stirring for 20-40 minutes, the mixed solution of curcumin, dichloromethane and boron tribromide is gradually restored to room temperature;

[0061] (2) adding a saturated ice-water solution containing NaOH (pH ≥ 11) to the mixed solution described above to quench the reaction and separate the DCM phase;

[0062] (3) adding dilute hydrochloric acid dropwise to the aqueous phase to adjust the pH to 2-4, and then filtering the mixture;

[0063] (4) dissolving the obtained filter residue with methanol, filtering, and adding DCM to the obtained filtrate to extract the product;

[0064] (5) The obtained mixed filtrate was rotary dried and dissolved with ethyl acetate (EA), and then n-hexane was added to the mixture to obtain a precipitate, which was then filtered and dried to obtain an orange solid product, which was a curcumin derivative.

[0065] The third aspect of the present invention provides a nanocomposite (represented by Cur-NCs), wherein the nanocomposite contains a chelate of a curcumin derivative and Cu(II), wherein the structural formula of the curcumin derivative is as shown in formula (I),

[0066]

[0067] The Cur-NCs provided by the present invention can specifically react with GSH in cells, trigger Fenton reaction, Michael addition reaction and inhibit GSH synthesis to induce cell apoptosis, these processes cause a large amount of GSH consumption, weaken the resistance of drug-resistant cancer cells; Cur-NCs can be used as a "targeted attack" to destroy the "target" of drug-resistant cells, triggering ferroptosis that depends on glutathione (GSH) consumption, and a large amount of GSH consumption will not only induce toxic reactive oxygen (·OH), but also inhibit the activity of GPX4, further promote lipid peroxidation (LPO) and trigger ferroptosis; Cur-NCs can attack and cause mitochondrial ferroptosis and dysfunction, manifested as mitochondrial morphological damage, redox balance disorder (glutathione consumption in mitochondria and increased generation of reactive oxygen), etc. In addition, Cur-NCs have good biosafety and have broad prospects in the use of drugs against cancer cell resistance.

[0068] According to the present invention, preferably, the nanocomposite has a lattice structure of a triangular platelet-like morphology.

[0069] Further preferably, the spacing of the lattice structure is 0.2-0.35 nm.

[0070] In the present invention, Cu(II) refers to divalent copper ions, which can be provided by any salt containing divalent copper ions, such as Cu(ClO4)2.

[0071] According to the present invention, preferably, the weight ratio of the nanocomposite curcumin derivative to Cu(II) is 1.5-2.5:1.

[0072] According to the present invention, the nanocomplex can be compounded into a corresponding solution using a solvent to better contact with the organism and more efficiently exert the above-mentioned effects. Preferably, the solvent of the nanocomplex is selected from at least one of a physiological buffer, a PBS buffer, a Dulbecco's modified Eagle's medium (DMEM medium), and a Dulbecco's modified Eagle's medium (DMEM medium) + 10% PBS. Dulbecco's modified Eagle's medium (DMEM medium) + 10% PBS refers to a mixed solution obtained by adding a PBS buffer with a volume ratio of 10% to the DMEM medium.

[0073] Further preferably, the solvent of the nanocomplex is PBS buffer.

[0074] The structure of the nanocomposite can be analyzed using electron microscopy, and the nanocomposite is dissolved in different solvents to observe and compare its solubility and stability in different solvents. Exemplarily, the present invention uses a high-resolution transmission electron microscope (HR-TEM) image to display the lattice structure of the nanocomposite, and observes the lattice spacing of the nanocomposite after magnification; the present invention uses an atomic force microscope (AFM) to analyze the thickness of the nanocomposite; the present invention selects the best solvent for the nanocomposite as PBS buffer by comparing the solubility and stability of the nanocomposite in physiological buffer (0.9% NaCl solution), PBS buffer, Dulbecco's modified Eagle's medium (DMEM) and DMEM + 10% PBS.

[0075] The fourth aspect of the present invention provides a method for preparing a nanocomposite, the method comprising: subjecting a curcumin derivative to a chelate reaction with a Cu(II) chelate; the structural formula of the curcumin derivative is as shown in formula (I),

[0076]

[0077] Preferably, the conditions of the chelating reaction include at least: a temperature of 5-35° C. and a time of 30-50 min.

[0078] Preferably, the chelation reaction is carried out in Tris-HCl buffer.

[0079] The Tris-HCl buffer used in the present invention was purchased from GBCBIO Technologies Inc (Guangzhou, China).

[0080] Exemplarily, the method for preparing the nanocomposite of the present invention comprises:

[0081] (i) dissolving a curcumin derivative in DMSO to form a solution I with a concentration of 7-13 mM, and dissolving Cu(ClO4)2·6H2O in deionized water to form a solution II with a concentration of 7-13 mM;

[0082] (ii) adding solution I dropwise to solution II to form a mixed solution, and stirring slowly for 5-7 min;

[0083] (iii) adjusting the pH value of the mixture to 7-9 with Tris-HCl solution, then slowly stirring at 5-35° C. for 30-50 min, purifying the mixture, and vacuum drying the mixture.

[0084] The fifth aspect of the present invention provides the use of at least one of the aforementioned curcumin derivatives, the aforementioned method for preparing the curcumin derivatives, the aforementioned nanocomplexes, and the aforementioned method for preparing the nanocomplexes in the preparation of drugs for resisting cancer cell drug resistance.

[0085] According to the present invention, preferably, the drug against cancer cell drug resistance is selected from at least one of a drug that consumes and / or inhibits GSH, a drug that induces and / or enhances cell ferroptosis, and a drug that downregulates ferroptosis-related proteins and / or drug-resistant proteins;

[0086] According to the present invention, preferably, the ferroptosis-related protein is glutathione peroxidase 4 and / or transporter family 7 member 1, and the drug-resistant protein is P-glycoprotein and / or glutathione S-transferase.

[0087] The present invention illustrates the application effect of the nanocomplex (Cur-NCs) and curcumin derivatives (Cur-C) in the preparation of drugs for anti-cancer cell resistance through five studies on the ability of GSH consumption, the anti-HCC effect on resistant and sensitive liver cancer cells in vitro, the induced mitochondrial dysfunction, the inhibition of HepG2 / DDP cell growth in vivo, and biosafety.

[0088] The present invention will be described in detail below through examples.

[0089] In the following examples, Cu(ClO4)2·6H2O and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium blue bromide (MTT) and fetal bovine serum (FBS) were from Sigma-Aldrich (Merck, America), Dulbecco's modified Eagle's medium (DMEM) was purchased from Pricella (Wuhan, China), Tris-based reagents were from GBCBIO Technologies Inc. (Guangzhou, China), curcumin (Cur) was purchased from Shanghai Dibo Biotechnology Co., Ltd., cisplatin (DDP), glutathione (GSH), ferroptosis inducer and ferroptosis inhibitor-1 (Fer-1) were purchased from Aladdin Industries (Shanghai, China), and C11-BODIPY 581 / 591Mitochondrial membrane potential assay kit was purchased from MedChemExpress (MCE, America), Mito Tracker Red CMXRos and JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylimidazolium carbocyanine iodide) were purchased from Beyotime (Shanghai, China), MitoPeDPP and mitochondrial superoxide indicator (MitoSOX) were purchased from DOJINDO (Japan), mouse antibodies (GPX4, SLC7A11, P-gp, GSTs) were purchased from Proteintech Group (Wuhan, China) and Beyotime (Shanghai, China), respectively. Hematoxylin-eosin (H&E) and TdT-mediated dUTP nick end labeling (TUNEL) detection kit were purchased from Solarbio (Beijing, China), and the rest of the reagents and raw materials were conventional commercial products.

[0090] All cell experiments in the present invention were performed in a biological safety cabinet (AIRTECH).

[0091] Example 1

[0092] This example illustrates the preparation method of curcumin derivative (Cur-C)

[0093] The specific process includes the following steps:

[0094] (1) Take a clean, dry, 100 mL transparent, colorless three-necked reaction bottle, add 95% curcumin (2 g, 1.5 M), place the reaction bottle in a -78°C dry ice acetone bath, slowly add a 17vt% dichloromethane (DCM) boron tribromide solution (3.6 mL) into the reaction bottle, stir for 30 minutes under argon protection, then take the reaction bottle out of the ice acetone bath to allow the mixed solution of curcumin, dichloromethane and boron tribromide to gradually return to room temperature;

[0095] (2) adding a saturated ice-water solution containing NaOH (pH ≥ 11) to the mixed solution described above to quench the reaction and separate the DCM phase;

[0096] (3) adding dilute hydrochloric acid dropwise to the aqueous phase to adjust the pH to 3, and then filtering the mixture;

[0097] (4) dissolving the obtained filter residue with methanol and filtering it with diatomaceous earth, then subjecting the filtrate to rotary evaporation, dissolving the rotary evaporation liquid with a small amount of methanol, and then adding DCM to extract the product;

[0098] (5) The resulting mixed filtrate was spin-dried and dissolved with ethyl acetate (EA), and then n-hexane was added to the mixture to obtain a precipitate, which was then filtered and dried to obtain an orange solid product.

[0099] The orange solid obtained in this example is a curcumin derivative (Cur-C), and the Cur-C prepared in this example is used for research in subsequent examples.

[0100] Example 2

[0101] This example illustrates the preparation method of the nanocomposite (Cur-NCs) of the present invention.

[0102] The specific process includes the following steps:

[0103] (1) taking a curcumin derivative (Cur-C) and dissolving it in DMSO to form a solution I with a concentration of 10 mM, taking Cu(ClO4)2·6H2O and dissolving it in deionized water to form a solution II with a concentration of 10 mM, and the weight ratio of Cur-C to Cu(ClO4)2·6H2O is 2:1;

[0104] (2) Add solution I dropwise into solution II to form a mixed solution, and stir slowly for 5 min;

[0105] (3) The pH value of the mixture was adjusted to 7.4 with Tris-HCl solution (pH = 8.8), and then slowly stirred at room temperature (about 25°C) for 40 min;

[0106] (4) The resulting mixture was purified by dialysis (1 kD) and dried under vacuum to obtain Cur-NCs.

[0107] The mixture obtained in this example is a nano-mixture (Cur-NCs). The Cur-NCs prepared in this example are used for research in subsequent examples.

[0108] Example 3

[0109] In this example, the solubility and stability of Cur-NCs in physiological buffer (0.9% NaCl), PBS, Dulbecco's modified Eagle's medium (DMEM) and DMEM+10% PBS were compared to select the best solvent for Cur-NCs.

[0110] Four portions of Cur-NCs prepared in Example 2 with the same mass were dissolved in equal amounts of 0.9% NaCl solution, PBS, DMEM, and DMEM+10% PBS, respectively, and the solubility was observed and the stability was analyzed (the particle size distribution of Cur-NCs in the solution was observed on the seventh and fourteenth days). The results are as follows: Figure 1 As shown by Figure 1 As shown in Figure A, Cur-NCs have the highest solubility in 0.9% NaCl solution and PBS; Figure 1As shown in Figure B, Cur-NCs are more stable in PBS, so Cur-NCs were dissolved in PBS to prepare Cur-NCs solution for subsequent example studies.

[0111] Example 4

[0112] This example illustrates the characterization of the nanocomposite (Cur-NCs) of the present invention by morphological detection.

[0113] 1. The Cur-NCs solution prepared in Example 3 was observed and analyzed by high-resolution transmission electron microscopy (HR-TEM). The results are as follows: Figure 2 As shown by Figure 2 As can be seen in Figure A, Cur-NCs have a triangular flake-like morphology and a clearly discernible lattice structure. After zooming in, it can be seen that the lattice structure is very uniform. Figure 2 As shown in B and C, Figure 2 As can be seen in B and C, the lattice spacing is about 0.2879nm;

[0114] 2. The Cur-NCs solution prepared in Example 3 was observed and analyzed by atomic force microscopy (AFM). The results are as follows: Figure 3 As shown by Figure 3 It can be seen that Cur-NCs is a triangular layered structure.

[0115] 3. The Cur-NCs solution prepared in Example 3 was analyzed by XRD pattern analysis on Rigaku ULTIMA IV. The results are as follows: Figure 4 As shown by Figure 4 It can be seen that the XRD patterns of Cur-NCs correspond to the standard physical peaks in sequence, showing characteristic diffraction peaks located at 10°, 15°, 22.0°, 25°, 32°, and 40°.

[0116] 4. The Cur-NCs solution prepared in Example 3 was observed and analyzed by X-ray photoelectron spectroscopy (XPS). The results are as follows: Figure 5 As shown by Figure 5 It can be seen that C, O and Cu elements exist in the synthesized Cur-NCs and their structures.

[0117] 5. The Cur-NCs solution prepared in Example 3 and the Cur-C prepared in Example 1 were dissolved in PBS solution to obtain the Cur-C solutions respectively. The FTIR spectra were measured by FTIR spectrometer. The results are as follows: Figure 6 As shown in Figure 2, the FTIR spectrum of Cur-NCs is similar to that of Cur-C, in which the characteristic absorption peak of hydroxyl (OH) is located at 3300-3600 cm -1 Along with the carboxyl group (C=O), the CO stretching vibration peak is observed at 1750-1700cm-l and 1300-100cm -1 Between Figure 6 It can be seen that Cur-C skeleton exists in Cur-NCs.

[0118] The above results confirm that there is a lattice structure in the Cu-NCs of the present invention, and Cur-C skeleton precipitation exists in Cur-NCs.

[0119] Example 5

[0120] This example studies the performance of Cur-NCs with respect to GSH consumption. Figure 7 .

[0121] The Cur-C prepared in Example 2 and the Cur-NCs prepared in Example 3 were dissolved in PBS to prepare Cur-C solution (20 μM) and Cur-NCs solution (100 μg / mL). The Cur-NCs solution (100 μg / mL) was mixed with GSH (10 mM) to prepare Cur-NCs+GSH solution. Four groups of solutions (Cur-C group, Cur-NCs group, Cur-NCs+GSH group and GSH group) were set up to study the response of Cur-NCs to GSH using UV-visible spectroscopy. The results are shown in Figure 2. Figure 7 shown.

[0122] Depend on Figure 7 As shown in Figure A, after the reaction of Cur-NCs and GSH (Cur-NCs+GSH group), an absorption peak appeared at 430nm, which was consistent with the peak of the ligand Cur-C, while Cur-NCs or GSH alone had no characteristic peak at 430nm. The solution color of the Cur-NCs+GSH group was similar to that of Cur-C but lighter, which was different from the color of Cur-NCs or GSH alone. Therefore, Cur-NCs responded to GSH and released Cur-C, and the color of the Cur-NCs+GSH solution was lighter than that of Cur-C, which may be related to the reaction of the released Cur-C with GSH.

[0123] For the Cur-NCs+GSH group, under 10 mM GSH treatment, the results were as follows Figure 7 As shown in B, Figure 7 As shown in Figure B, the UV absorbance intensity at 430 nm increases positively with the increase of GSH concentration, while the absorbance intensity decreases gradually with the extension of reaction time. In addition, the color of Cur-NCs solution changes from brown to bright orange with different GSH concentrations (color change of Cur-NCs solution under different GSH concentrations (0-30 mM)). Figure 7As shown in Figure 5C, these results confirm that in the presence of GSH, Cur-NCs react with GSH and release Cur-C.

[0124] A sensitive reagent (neocuprotin) that reacts with Cu(I) to form a yellow complex was added, and the generation of Cu(I) can be determined by the color change. 100 μL of ethanol solution of neocuprotin (1 mg / mL) was mixed with 1000 μL of Cur-NCs (100 μg / mL), and after adding GSH aqueous solution (100 μL, 100 mM), the color change of the mixture solution was recorded. The results are shown in Figure 2. Figure 7 As shown in D, in the supernatant treated with Cur-NCs, the color changed from bright orange to yellow with almost no precipitation. Figure 7 As can be seen in Figure D, Cu(I) is converted from Cu(II) chelated in Cur-NCs.

[0125] Since Cur-C contains an α, β-unsaturated ketone structure that can react with GSH, the present invention further studied the response of Cur-C to GSH. The results are as follows: Figure 6 As shown in E, F and G, compared with Cur-C alone ( Figure 7 E), the intensity of Cur-C (20uM) treated with GSH at 430nm increases with the increase of GSH concentration ( Figure 7 F) and the extension of reaction time ( Figure 7 It was confirmed that Cur-C can also react with GSH.

[0126] From this example, it can be seen that Cur-NCs respond to GSH and release Cur-C, and Cur-C can also react with GSH, and Cu(I) is converted from Cu(II) chelated in Cur-NCs.

[0127] Example 6

[0128] This example illustrates the anti-HCC effect of Cur-NCs on drug-resistant and sensitive liver cancer cells in vitro

[0129] Normal human hepatocytes (HL-7702), human cisplatin-sensitive hepatoma cells (HepG2) and cisplatin-resistant hepatoma cells (HepG2 / DDP) were cultured in DMEM medium (10% fetal bovine serum + 100 U / mL / 1% streptomycin) and stored at 37°C, 5% CO2.

[0130] HepG2 / DDP and HepG2 cells were cultured at 6×10 3The cells were inoculated at a density of 10 cells / well in a 96-well plate and cultured for 24 h. Then, Cur-NCs (0, 1, 2, 3, 10, 20 μg / mL), Cu(Ⅱ) (0, 5, 10, 20, 50, 100, 200 μM) and Cur-C (0, 2.5, 5, 10, 15, 20 μM) were added and the cells were cultured for 24 h. The cell viability was detected by MTT method according to the product manual. The absorbance value of each well at 490 nm was measured by microplate reader with the ratio of the absorbance value of each well to that of the untreated control group, and the relative cell viability was calculated. The number of live cells in each group was counted under a light microscope. The results were as follows: Figure 8 A, B and C represent Figure 8 As shown in Figure A, the half inhibitory concentration (IC50) of Cur-NCs in HepG2 / DDP and HepG2 were 10.0±0.61ug / mL and 15.0±0.79ug / mL, respectively. At high concentrations (10-20ug / mL), the cytotoxicity of Cur-NCs to HepG2 / DDP was significantly stronger than that to HepG2. Figure 8 Middle A and Figure 7 As shown in Figure B, at the same concentration (0-20 μM), the cytotoxicity of Cur-C to cisplatin-resistant cells (HepG2 / DDP) was weaker than that to sensitive cells (HepG2). Figure 8 As shown in Figure C, Cu(II) did not show obvious cytotoxicity to HepG2 / DDP and HepG2 cells.

[0131] For live / dead cell staining analysis, cells treated with Cur-NCs, Cur-C, and DDP and untreated cells (control group) were stained with calcein AM and propidium iodide (PI), respectively, and then imaged using confocal laser scanning microscopy (CLSM). Figure 8 As shown in D, Figure 8 As can be seen in Figure D, the confocal laser scanning microscopy (CLSM) images showed that there was abundant red fluorescence in the dead cells of the Cur-NCs-treated group, while there was abundant green fluorescence in the living cells of the control group, Cur-C, and DDP groups, indicating that Cur-NCs had strong cytotoxicity in HepG2 / DDP and HepG2.

[0132] Apoptosis was assessed by flow cytometry. HepG2 / DDP and HepG2 cells were cultured in 6-well plates and incubated with fresh medium containing Cu-C (20 μM), Cur-NCs (10 μg / mL), Cur-NCs (20 μg / mL), and DDP (5 μM) for 24 h. Then, the cells were washed with 1× PBS, collected, and stained with Annexin V-FITC & PI according to the manufacturer's protocol. Flow cytometry data were analyzed using FlowJo software. The results are shown in Figure 8 As shown in Figure E, under the action of high concentration of Cur-NCs, the cell survival rate decreased from 85.1% to 55.4%, and the ability of Cur-NCs to induce apoptosis of HepG2 / DDP cells was stronger than that of HepG2 cells (apoptosis rates were 18.9% and 13.8%, respectively).

[0133] The above results fully confirmed the effectiveness of Cur-NCs in anti-HCC ability.

[0134] Example 7

[0135] This example demonstrates that Cur-NCs cause mitochondrial dysfunction

[0136] HepG2 cells were incubated with Cur-NCs (10 μg / mL), Cur-NCs (20 μg / mL), DDP (5 μM), Cur-C (20 μM), and PBS (control group) for 24 h, and then stained with Mito-Tracker Red probe to observe the colocalization of Cur-NCs and Cur-C. To further explore whether Cur-NCs can induce mitochondrial dysfunction, mitochondria were extracted using a cell mitochondrial isolation kit (Beyotime Biotechnology, Shanghai), and mitochondrial GSH levels were determined. The results are shown in Fig. 9 As shown in A, Fig. 9 As can be seen in Figure A, the lowest GSH level was detected in the mitochondria of HepG2 / DDP cells treated with Cur-NCs. The GSH level in the Cur-NCs (10ug / ml) group was reduced to 49.1%, and the GSH level in the Cur-NCs (20ug / ml) group was reduced to 55.4%, indicating that Cur-NCs had the strongest mitochondrial GSH consumption ability. The degree of decrease in GSH level in the Cur-C group was second only to that in the Cur-NCs group, but much higher than that in the DDP group, indicating that Cur-C had a slight mitochondrial GSH consumption ability.

[0137] After HepG2 cells were incubated with Cur-NCs (10 μg / mL), Cur-NCs (20 μg / mL), DDP (5 μM), Cur-C (20 μM), and PBS (control group) for 24 h, mitochondrial reactive oxygen species were detected by MitoSOX staining and flow cytometry. Fig. 9 As shown in B, Fig. 9As can be seen in Figure B, compared with the other three groups, Cur-NCs induced the most mitochondrial ROS content (2.5-2.8 times that of the control group), and the ROS content induced by Cur-NCs (20μg / mL) was significantly greater than that induced by Cur-NCs (10μg / mL), while Cur-C only induced a small amount of ROS production. There was no significant difference in ROS production between the DDP group and the control group. Therefore, Cur-NCs induced significantly more ROS at high concentrations.

[0138] HepG2 cells were incubated with Cur-NCs (10 μg / mL) and PBS (control group) for 24 h, and then the cells were collected. 6 ) were fixed with 2.5% glutaraldehyde overnight. The cells were washed three times with PBS, fixed in 1% osmium acid solution for 1 h, dehydrated with graded ethanol, and embedded in epoxy resin. Ultrathin sections were obtained using an ultramicrotome (Leica Microanalysis) and stained with lead citrate. Subsequently, the morphological changes of mitochondria in the cells were observed under TEM (Hitachi H-7650) to evaluate mitochondrial damage. The results are shown in Fig. 9 C in the middle means that Fig. 9 As shown in Figure C, compared with the PBS-treated group, the mitochondria treated with Cur-NCs showed the characteristics of mitochondrial dysfunction, namely, severe damage and swelling, significantly increased membrane density, decreased volume, and reduced cristae.

[0139] After HepG2 cells were incubated with Cur-NCs (10 μg / mL), Cur-NCs (20 μg / mL), DDP (5 μM), Cur-C (20 μM), and PBS (control group) for 24 h, the cells were collected and stained with 10 μM MitoPeDPP (this reagent is oxidized by mitochondrial lipid-soluble peroxides and emits green fluorescence) at 37 °C for 20 min to measure mitochondrial LPO. After that, the cells were resuspended three times in 500 μL fresh PBS and analyzed by confocal laser scanning microscopy (CLSM). The results are shown in Fig. 9 As shown in D, the Cur-NCs treatment group produced bright green fluorescence, the Cur-C treatment group produced moderate fluorescence, and the DDP treatment group produced very weak green fluorescence. Fig. 9 As shown in D, Cur-NCs can induce oxidized mitochondrial membrane and LPO.

[0140] The four groups of experiments in this example can prove that Cur-NCs can cause mitochondrial dysfunction in cells.

[0141] Example 8

[0142] This example illustrates that Cur-NCs inhibit the growth of HepG2 / DDP cells in vivo

[0143] Experimental animals: Female BALB / c nude mice (6 weeks, 18-20 g) were purchased from Slake Jingda Laboratory Animal Co., Ltd. (Changsha, China). All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources and approved by the Ethics Committee of Hunan University of Chinese Medicine with IACUC number LLBH-2024030155.

[0144] Experimental process:

[0145] 1. Add 100 μL containing 1×10 7 The PBS solution of HepG2 / DDP cells was injected into the subcutaneous tissue of mice. When the tumor size reached 80 mm 3 When chemotherapy is given;

[0146] 2. HepG2 / DDP tumor-bearing mice were randomly divided into 4 groups (5 mice in each group) and intravenously injected with PBS (5 mg / kg), Cur-C (5 mg / kg), Cur-NCs (5 mg / kg) and DDP containing 5 mg / kg equivalent Cur-NCs, respectively, once every two days. The tumor size and mouse body weight were recorded every 2 days. After 21 days of treatment, the mice were killed and the tumor tissues were obtained.

[0147] 3. Each tumor was weighed, and the heart, liver, spleen, lung, kidney and tumor sections of each mouse were collected for HE staining. The tumor tissue was stained with terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) to further verify the HE staining results. Subsequently, the tumor tissue collected after 21 days of treatment was repeatedly washed with PBS, homogenized and collected in buffer, and tissue proteins were extracted using a kit for Western blot processing to explore the mechanism of Cur-NCs anti-tumor resistance through ferroptosis, and the GSH level in the tumor tissue was further detected;

[0148] 4. Tumor tissues were tested by immunohistochemistry staining and Western blot to explore the mechanism by which Cur-NCs achieve anti-drug resistance through ferroptosis.

[0149] The experimental results are as follows Fig.10 As shown in (A, B, C, D, E, F, G, H).

[0150] Fig.10 As shown in the results in Figure A, compared with the control group (PBS group), the changes in the body weight of mice in the Cur-NCs group and Cur-C group during the treatment period were negligible, while the body weight of mice treated with DDP decreased significantly. Fig.10 As shown in Figure A, Cur-NCs have good biosafety;

[0151] Fig.10 As shown in the results of B and C, compared with the control group (PBS group), the growth of HepG2 / DDP tumors was effectively inhibited after treatment with Cur-NCs, while the inhibitory effect of Cur-C treatment on tumor volume was limited, and DDP did not inhibit tumor growth. Fig.10 As shown in Figures B and C, Cur-NCs have a good anticancer effect on drug-resistant HCC;

[0152] Fig.10 As shown in the results of D and E, compared with the control group (PBS group), the Cur-NCs group had the lowest tumor weight and the highest tumor inhibition rate (TIR) ​​at a given dose (5 mg / kg). In contrast, the Cur-C group only showed a mild tumor inhibition effect, and the DDP group only slightly reduced the tumor size and TIR. Fig.10 In D and E, it can be seen that the efficiency of Cur-C group and DDP group in treating drug-resistant HCC tumors is poor, while the Cur-NCs group has a good therapeutic effect;

[0153] Fig.10 As shown in the results in Figure F, the morphology of tumor cells in the Cur-NCs group was significantly destroyed, with a large number of missing cell nuclei and obvious nuclear condensation, while the Cur-C group only had a certain apoptosis induction effect. The morphology of tumor cells in the DDP group was basically normal. The results of H&E staining were further confirmed by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining of tumor tissues. The staining results showed that the DDP group showed almost no apoptosis, while the Cur-C group showed only limited apoptosis induction, and severe apoptosis and tumor growth inhibition were observed in the Cur-NCs group. These results indicate that the Cur-NCs group treatment produced an excellent anti-resistant tumor effect, while the DDP group and Cur-C group showed only limited anti-tumor effects.

[0154] Fig.10 The results of G(ad) immunohistochemistry showed that the expression levels of ferroptosis-related factors (GPX4 and SCL7A11) and drug resistance-related factors (P-gp and GSTs) in the Cur-NCs group were lower than those in the control group;

[0155] Fig.10 As shown in the results in Figure 3, when detecting the level of glutathione (GSH) in tumor tissue, the Cur-NCs group could greatly consume GSH in tumor tissue compared with the Cur-C group or DDP group.

[0156] In summary, this example effectively demonstrated that Cur-NCs effectively triggered ferroptosis through the consumption of GSH, thereby exhibiting excellent antioxidant capacity.

[0157] Example 9

[0158] This example illustrates the biosafety of Cur-NCs

[0159] Fresh blood from one mouse in the same batch of mice purchased was collected in an anticoagulation tube, and then washed with PBS under centrifugation (3500 rpm, 5 minutes) to collect red blood cells (RBC), which were then prepared into a 4% saline RBC solution. 0.8 mL of Cur-NCs at different concentrations (4, 10, 20, 30, 40 μg / mL) and RBC solution (0.2 mL) were mixed. After incubation for 6 hours, RBC was removed by centrifugation, and the absorption of the supernatant was measured at 540 nm. The red blood cell (RBC) hemolysis and coagulation test was performed, and the results were as follows Fig.11 As shown in A and B, no hemolysis and coagulation were observed, indicating that Cur-NCs have good blood compatibility.

[0160] Healthy female BALB / c mice were intravenously injected with PBS, Cur-C (5 mg / kg), Cur-NCs (5 mg / kg) and DDP (5 mg / kg) every 2 days for a total of 10 times, and the body weight was recorded every 2 days. The mice were killed 21 days after the first injection, and the main organs (heart, liver, spleen, lung, kidney) were taken for HE staining. The results are shown in Fig.11 As shown in middle C, HE staining of major organs (heart, liver, spleen, lung and kidney) showed no morphological abnormalities. Fig.11 As can be seen in center C, Cur-NCs did not affect animal growth or damage any major organs, indicating that Cur-NCs have good biocompatibility and almost no side effects.

[0161] The above examples demonstrate that Cur-NCs can effectively enhance the ferroptosis effect in cisplatin-resistant hepatocellular carcinoma (HCC). The Cur-NCs have the ability to simultaneously consume GSH and inhibit GSH synthesis, and can reduce the GSH level in resistant HCC cells (HepG2 / DDP), greatly reversing the drug resistance of cancer cells. In addition, mitochondrial-targeted Cur-NCs lead to mitochondrial lipid peroxidation, induce cell ferroptosis, and Cur-NCs exhibit excellent anti-HCC cisplatin resistance effects without damaging normal organs.

[0162] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A curcumin derivative, characterized in that The curcumin derivative is (1E, 6E)-1,7-bis(3,4-dihydroxyphenyl)-1,6-heptadiene-3,5-dione, and its structural formula is shown in formula (I).

2. A method for preparing a curcumin derivative, characterized in that: The method comprises: performing a double demethylation treatment on curcumin to obtain a curcumin derivative having a structural formula as shown in formula (I); 3. The preparation method according to claim 2, characterized in that: The process of described double demethylation treatment comprises the following steps: S1. Under anaerobic conditions, curcumin is reacted with a solution containing dichloromethane to obtain a reaction solution I, the reaction solution I is mixed with ice water with a pH value of ≥11, and then the dichloromethane is removed by phase separation to obtain an aqueous phase; S2, adjusting the pH of the aqueous phase to 2-4 and filtering to obtain a filtrate, drying the filtrate and then dissolving and precipitating; Preferably, the reaction conditions include: temperature of -85°C to -70°C, time of 20-40 min; Preferably, the weight ratio of the curcumin to the dichloromethane is 3-4:1; Preferably, the solvent used for the dissolution is ethyl acetate, and the precipitant used for the precipitation is n-hexane.

4. A nanocomposite, characterized in that: The nanocomposite contains a chelate of a curcumin derivative and Cu(II), wherein the structural formula of the curcumin derivative is shown in formula (I).

5. The nanocomposite according to claim 4, characterized in that: The nanocomposite has a lattice structure with a triangular plate-like morphology; Preferably, the spacing of the lattice structure is 0.2-0.35 nm.

6. The nanocomposite according to claim 4, characterized in that The weight ratio of the curcumin derivative to Cu(II) is 1.5-2.5:

1.

7. The nanocomposite according to any one of claims 4 to 6, characterized in that: The solvent of the nanocomplex is selected from at least one of physiological buffer, PBS buffer, Dulbecco's modified Eagle's medium, and Dulbecco's modified Eagle's medium + 10% PBS; Preferably, the solvent of the nanocomplex is PBS buffer.

8. A method for preparing a nanocomposite, characterized in that: The method comprises: subjecting a curcumin derivative to a chelate reaction with a Cu(II) chelate; the structural formula of the curcumin derivative is as shown in formula (I), 9. The preparation method according to claim 8, characterized in that: The conditions of the chelating reaction include at least: a temperature of 5-35°C and a time of 30-50 minutes; Preferably, the chelation reaction is carried out in Tris-HCl buffer.

10. Use of the nanocomposite according to any one of claims 4 to 7 and / or the nanocomposite prepared according to the method of claim 8 or 9 in the preparation of a drug for treating drug resistance of cancer cells; Preferably, the drug for resisting cancer cell drug resistance is selected from at least one of a drug for consuming and / or inhibiting GSH, a drug for inducing and / or enhancing cell ferroptosis, and a drug for downregulating ferroptosis-related proteins and / or drug-resistant proteins; Preferably, the ferroptosis-related protein is glutathione peroxidase 4 and / or transporter family 7 member 1, and the drug-resistant protein is P-glycoprotein and / or glutathione S-transferase.

Images