Nano-drug for inducing cupric death of tumor cells and preparation method of nano-drug

Abstract

The invention relates to the field of anti-tumor drugs, in particular to a nano-drug for inducing tumor cell copper death and a preparation method of the nano-drug. The nano-drug is of a core-shell structure, the core is Cu < 2 + >-doped zeolite imidazole skeleton-8 and a composite drug, and the shell is hyaluronic acid; wherein the composite drug comprises hemoglobin and dichloroacetic acid containing a mitochondrial targeting group. According to the nano-drug, the concentration of Cu < 2 + > in tumor cells is increased and the respiratory metabolism mode of the tumor cells is changed through three strategies of consuming GSH, destroying the steady state of copper metabolism and regulating and controlling the respiratory metabolism mode, so that the cascade enhancement reaction is activated, the sensitivity of the tumor cells to copper death is improved, and the immunotherapy is enhanced; the sustainable development of copper death driven immunotherapy is greatly promoted.

Description

Technical Field

[0001] The present application relates to the field of anti-tumor drugs, and in particular to a nano drug that induces copper death of tumor cells and a preparation method thereof. Background Art

[0002] Existing treatments for breast cancer mainly include surgery, radiotherapy and chemotherapy, but these methods all have problems such as incomplete treatment and easy recurrence. According to statistics, about 90% of breast cancer-related deaths are caused by tumor metastasis. Immunotherapy is based on natural immune response to fight tumor metastasis. Although immunotherapy has revolutionized traditional cancer treatment in recent years, it is not effective for all patients, which is mainly limited by insufficient immune activation and tumor immunosuppressive microenvironment. Immunogenic cell death (ICD) refers to the release of damaging molecular patterns such as calreticulin (CRT), high mobility group protein B1 (HMGB1) and adenosine triphosphate (ATP) by dead tumor cells to promote the maturation of dendritic cells (DCs) and improve the infiltration of cytotoxic T lymphocytes in the tumor, thereby effectively enhancing the sensitivity of immunotherapy. However, because the immunosuppressive tumor microenvironment significantly limits the effect of immunotherapy, the immunosuppressive tumor microenvironment is also a major challenge for immunotherapy. Tumor cells usually metabolize energy through aerobic glycolysis, and the lactic acid produced by tumor cell glycolysis will accumulate in the tumor microenvironment and form an acidic environment, which will lead to immune tolerance and accelerate tumor growth. At present, the acidic microenvironment of tumors can be improved by calcium carbonate neutralization reaction and lactate oxidase catalytic reaction, but these methods have a short duration of action and the drugs used are prone to lose activity. Therefore, the development of innovative strategies that can accurately induce immunogenic cell death (ICD) and effectively regulate lactic acid metabolism is of vital importance for enhancing the immunogenicity of tumors, reversing the immunosuppressive tumor microenvironment, and thus improving the effect of immunotherapy.

[0003] As a new type of programmed cell death, copper death is characterized by copper-dependent mitochondrial dysfunction; copper death is mainly manifested by excessive intracellular copper, esterified mitochondrial protein aggregation, and degradation of iron-sulfur cluster proteins, which ultimately leads to proteotoxic stress and cell death. Existing studies have shown that copper death can activate tumor cells to release damage molecular patterns and tumor-associated antigens, thereby inducing ICD, thereby transforming immunosuppressive "cold" tumors into immune-responsive "hot" tumors. Although copper death has potential, its therapeutic effect is significantly limited by the inherent respiratory metabolic pathways and copper homeostasis mechanisms in tumor cells. Copper death is closely related to mitochondrial respiration, and tumor cells mainly produce ATP through aerobic glycolysis to meet their high energy needs, which significantly reduces their sensitivity to copper death. In addition, the copper level in tumor cells is strictly regulated, including the lack of copper influx transporters and the overexpression of copper export proteins based on Cu-ATPase. In addition, excess endogenous glutathione (GSH) rapidly chelates free copper ions to regulate the copper level in tumor cells, which is another important factor in limiting copper death. Therefore, it becomes extremely important to develop a copper carrier that can modulate respiratory metabolism and disrupt copper homeostasis to promote the significant induction of copper death. Summary of the invention

[0004] The present application provides a nano drug that induces copper death in tumor cells and a preparation method thereof to solve the following technical problems: how to increase the copper content in tumor cells 2+ concentration while changing the respiratory metabolism of tumor cells.

[0005] In the first aspect, the present application provides a nano drug that induces copper death of tumor cells, wherein the nano drug is a core-shell structure, the core is Cu 2+ The doped zeolite imidazole framework-8 and the composite drug have a shell of hyaluronic acid; wherein the composite drug comprises hemoglobin and dichloroacetic acid containing a mitochondrial targeting group.

[0006] Optionally, the weight m1 of the hemoglobin and the weight m2 of the dichloroacetic acid containing the mitochondrial targeting group satisfy the relationship: m1:m2≥2:1.

[0007] Optionally, the total weight m3 of the composite drug and the total weight m4 of the nano drug satisfy the relationship: m3:m4=(3.6-4.4):(21.2-25.6).

[0008] Optionally, the thickness of the hyaluronic acid is 8.6 nm to 12.8 nm.

[0009] Optionally, the Cu 2+The weight m5 of the doped zeolite imidazole framework-8 and the total weight m4 of the nano drug satisfy the relationship: m5:m4=(15.5-18.5):(21.2-25.6); and / or

[0010] The Cu of the zeolite imidazole framework-8 2+ The doping amount is 0.33mg~0.41mg.

[0011] Optionally, the average particle size of the nanomedicine is 150nm to 200nm.

[0012] In a second aspect, the present application provides a method for preparing the nanomedicine described in the first aspect, the method comprising:

[0013] Dissolving 2-methylimidazole in deionized water to obtain a 2-methylimidazole solution;

[0014] Copper acetate, zinc acetate, hemoglobin and dichloroacetic acid with a mitochondrial targeting group are mixed, and deionized water is added to the mixture to obtain a raw material mixed solution;

[0015] The raw material mixture and the 2-methylimidazole solution are mixed to allow the copper acetate, the zinc acetate, the hemoglobin, the dichloroacetic acid and the 2-methylimidazole solution to undergo an encapsulation reaction to obtain a nano drug core;

[0016] The nano drug core is dispersed in an aqueous solution of hyaluronic acid so that the hyaluronic acid covers the surface of the nano drug core to obtain the nano drug.

[0017] Optionally, the temperature of the packaging reaction is 10° C. to 40° C., and the time of the packaging reaction is ≥ 6 hours.

[0018] Optionally, the dispersion method includes ultrasonic dispersion, the frequency of the ultrasonic dispersion is 15kHz to 25kHz, and the time of the ultrasonic dispersion is ≥15min.

[0019] In a third aspect, the present application provides a tumor killing and immune activation drug, which includes the nanomedicine described in the first aspect.

[0020] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0021] The present invention provides a nano drug that induces copper death of tumor cells. The nano drug is Cu 2+The doped zeolite imidazole skeleton-8 was used as a carrier, and then a composite drug including hemoglobin (Oxy-Hb) and dichloroacetic acid (Mito-DCA) containing a mitochondrial targeting group was encapsulated. Finally, hyaluronic acid (HA) was used as the shell. The interaction between HA and CD44 on the cell membrane surface was used to mediate the selective endocytosis of nanomedicines by tumor cells, so that the nanomedicines could be disintegrated in a pH-responsive manner in tumor cells. At this time, the Cu 2+ Doped zeolite imidazolate framework-8 releases Cu 2+ , and the released Cu 2+ In the transformation to Cu + At the same time, it depletes the endogenous glutathione (GSH) of tumor cells, effectively catalyzing the Fenton-like reaction process of hydrogen peroxide in tumor cells and converting it into hydroxyl radicals (·OH), thereby hindering the copper efflux mediated by copper-ATPase, so as to overload the copper level in tumor cells; in addition, the Oxy-Hb of the nanodrug also transports oxygen, and in combination with Mito-DCA, it can promote the metabolic mode of tumor cells from aerobic glycolysis to mitochondrial oxidative phosphorylation (OXPHOS), thereby inhibiting the function of pyruvate dehydrogenase kinase (PDK) of tumor cells; this nanodrug can The above three strategies of GSH depletion, copper homeostasis disruption and respiratory metabolic mode change can lead to oligomerization of dihydrolipoic acid S-acetyltransferase (DLAT) and loss of lipoyl synthase (LIAS) in tumor cells, which ultimately increases the sensitivity of tumor cells to copper death. Tumor cells with increased copper death sensitivity will activate immune cell death (ICD), triggering a cascade immune response based on the release of tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs), thereby promoting the maturation of dendritic cells (DCs) and CD8 + Infiltration of T cells; in addition, the conversion of glycolysis to OXPHOS in tumor cells will effectively reduce the lactate level of tumor cells to reprogram the lactate-rich immunosuppressive tumor microenvironment (TME), by promoting the transformation of tumor-associated macrophages (TAMs) from the immunosuppressive M2 phenotype to the immunostimulatory M1 phenotype; in addition, while tumor cells die from copper, they also interfere with the metabolic process of lactate to enhance the immunogenicity of tumor cells and reverse their immunosuppressive TME, thereby effectively activating the body's systemic immunity against tumor development and metastasis. Therefore, this nanomedicine increases Cu in tumor cells and inhibits the growth of tumor cells. 2+ concentration and changing the respiratory metabolism of tumor cells to activate cascade enhancement reactions to increase the sensitivity of tumor cells to copper death and enhance immunotherapy, which has greatly promoted the continued development of copper death-driven immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0024] Figure 1 The morphological characteristics result diagram of the nano drug HMCZH provided in the embodiment of the present application, wherein a is the TEM spectrum of the nano drug HMCZH, and b is the SEM spectrum of the nano drug HMCZH;

[0025] Figure 2 A schematic flow chart of a method for preparing nanomedicine provided in an embodiment of the present application;

[0026] Figure 3 The Zeta potential and infrared spectrum result diagram of each material in the process of preparing nanomedicine provided in the embodiment of the present application, wherein a is the Zeta potential of each material in the process of preparing nanomedicine, and b is the infrared spectrum result diagram of each material in the process of preparing nanomedicine;

[0027] Figure 4 The cell-targeted uptake behavior result graph of the nanodrug HMCZH provided in the embodiment of the present application, wherein a is a confocal microscope result graph of the nanodrug HMCZH, b is a graph showing the uptake effect of the nanodrug HMCZH on 4T1 cells during flow cytometry analysis, and c is a statistical result graph showing the relevant average fluorescence intensity value of the nanodrug HMCZH on 4T1 cells during flow cytometry analysis;

[0028] Figure 5 The nano drug HMCZH provided in the embodiment of the present application has Cu in different cells 2+ The performance characterization results of metabolic homeostasis disruption, where a is the change of Cu-ATPase activity of nanomedicine HMCZH in different cells, and b is the change of Cu in nanomedicine HMCZH in different cells 2+ Content change graph;

[0029] Figure 6 The evaluation results of the killing effect of different mass concentrations and different types of nano drugs on tumor cells provided in this application are shown in Figure 1, wherein a is a staining result diagram of 4T1 cells treated with different types of nano drugs at the same mass concentration, and b is a statistical result diagram of cell survival rate of 4T1 cells treated with different types of nano drugs at the same mass concentration;

[0030] Figure 7 The in vivo anti-tumor effect diagram of the nano drug HMCZH in 4T1 tumor-bearing mice provided in the examples of the present application, wherein a is a diagram showing the change in tumor volume of 4T1 tumor-bearing mice treated with nano drug HMCZH within 15 days, and b is a diagram showing the statistical results of the tumor inhibition rate of 4T1 tumor-bearing mice treated with nano drug HMCZH after 15 days;

[0031] Figure 8 The graphs are result graphs of the nanodrug HMCZH inducing phenotypic transformation of tumor-associated macrophages in vivo provided in the embodiments of the present application, wherein a is an immunofluorescence image of the nanodrug HMCZH inducing tumor cell-associated CD86 in vivo, b is an immunofluorescence quantitative analysis result graph of the nanodrug HMCZH inducing tumor cell-associated CD86 in vivo, c is an immunofluorescence image of the nanodrug HMCZH inducing tumor cell-associated CD206 in vivo, and d is an immunofluorescence quantitative analysis result graph of the nanodrug HMCZH inducing tumor cell-associated CD206 in vivo. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0033] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited number (fractional or integer) within the indicated range.

[0034] In this article, the terms including "include" and the like mean "including but not limited to". "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone; wherein A and B can be singular or plural. "At least one" means one or more, and "plurality" means two or more; "at least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single or plural items; for example, "at least one of a, b, or c", or "at least one of a, b, and c", can all mean: a, b, c, ab (i.e. a and b), ac, bc, or abc, wherein a, b, c can be single or multiple, respectively. "Parts" such as parts by weight, parts by mass, etc. indicate the proportional relationship between the components. In the proportional relationship involved in this article, the parameters that need to be described by proportion should be understood as the first term of the proportional formula according to the order of description, and the proportional numbers should be understood as the second term of the proportional formula. For example, the mass ratio of substance A, substance B and substance C is 1:2:3, then substance A, substance B and substance C should correspond one by one to the proportional numbers in the proportional formula according to the order of description, that is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

[0035] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this article can be purchased from the market or prepared by existing methods.

[0036] Figure 1 The morphological characteristics result diagram of the nano drug HMCZH provided in the examples of the present application is shown exemplarily;

[0037] like Figure 1 As shown, the present invention provides a nano drug that induces copper cell death in tumor cells. The nano drug has a core-shell structure, and the core is Cu 2+ The doped zeolite imidazole framework-8 and the composite drug have a shell of hyaluronic acid; wherein the composite drug comprises hemoglobin and dichloroacetic acid containing a mitochondrial targeting group.

[0038] It should be noted that the preparation method of the dichloroacetic acid containing a mitochondrial targeting group can refer to the relevant content recorded in "Mito-DCA: A Mitochondria Targeted Molecular Scaffold for Efficacious Delivery of Metabolic Modulator Dichloroacetate".

[0039] In some optional embodiments, the weight m1 of the hemoglobin and the weight m2 of the dichloroacetic acid containing the mitochondrial targeting group satisfy the relationship: m1:m2≥2:1;

[0040] In these embodiments, the weight m1 of hemoglobin and the weight m2 of dichloroacetic acid containing mitochondrial targeting groups can satisfy the relationship: m1:m2≥2:1, so that the composite drug can have sufficient hemoglobin and dichloroacetic acid containing mitochondrial targeting groups. Sufficient hemoglobin can increase the intracellular oxygen concentration, and combined with sufficient dichloroacetic acid containing mitochondrial targeting groups, it can promote the metabolic mode of tumor cells from anaerobic glycolysis to mitochondrial oxidative phosphorylation, thereby inhibiting the function of pyruvate dehydrogenase kinase of tumor cells to affect the respiratory metabolic homeostasis of tumor cells; in addition, the conversion of glycolysis to mitochondrial oxidative phosphorylation in tumor cells can effectively reduce the lactate level of tumor cells, so as to reprogram the lactate-rich immunosuppressive tumor microenvironment, and promote the transformation of tumor-associated macrophages from the M2 phenotype of immunosuppressive state to the M1 phenotype of immunostimulation, so as to activate the body's systemic immunity against tumor development and metastasis.

[0041] In some optional embodiments, the total weight m3 of the composite drug and the total weight m4 of the nano drug satisfy the relationship: m3:m4=(3.6-4.4):(21.2-25.6);

[0042] In these embodiments, the total weight m3 of the composite drug and the total weight m4 of the nano drug can satisfy the relationship: m3:m4=(3.6-4.4):(21.2-25.6), so that the nano drug contains a sufficient amount of composite drug, and the sufficient amount of composite drug can affect the respiratory metabolic homeostasis of tumor cells through the synergistic effect between hemoglobin and dichloroacetic acid containing mitochondrial targeting groups, and at the same time promote the transformation of tumor-associated macrophages from the M2 phenotype of the immunosuppressive state to the M1 phenotype of the immunostimulatory state, so as to activate the body's systemic immunity against tumor development and metastasis.

[0043] The total weight m3 of the composite drug can be 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3 or 4.4; the total weight m4 of the nano drug can be 21.2, 21.6, 22.0, 22.4, 22.8, 23.2, 23.6, 23.0, 23.4, 23.8, 25.2 or 25.6.

[0044] In some optional embodiments, the thickness of the hyaluronic acid is 8.6 nm to 12.8 nm;

[0045] In these embodiments, the thickness of the hyaluronic acid can be 8.6nm to 12.8nm, so that the hyaluronic acid shell has a sufficient thickness. Hyaluronic acid of sufficient thickness can better interact with the CD44 of tumor cells to mediate the selective internalization of the nanodrug, thereby delivering the core containing the composite drug into the tumor cells to enable the composite drug to better exert its efficacy.

[0046] The thickness of the hyaluronic acid may be 8.6 nm, 9.2 nm, 9.8 nm, 10.4 nm, 11.0 nm, 11.6 nm, 12.2 nm or 12.8 nm.

[0047] In some optional embodiments, the Cu 2+ The weight m5 of the doped zeolite imidazole framework-8 and the total weight m4 of the nano drug satisfy the relationship: m5:m4=(15.5-18.5):(21.2-25.6); and / or

[0048] The Cu of the zeolite imidazole framework-8 2+ The doping amount is 0.33mg~0.41mg;

[0049] In these embodiments, Cu 2+ The weight m5 of the doped zeolite imidazole framework-8 and the total weight m4 of the nanodrug can satisfy the relationship: m5:m4=(15.5-18.5):(21.2-25.6), and the Cu of the zeolite imidazole framework-8 2+ The doping amount can be 0.33mg~0.41mg, which makes the nano drug have sufficient amount of Cu 2+ Doped zeolite imidazole framework-8, sufficient amount of Cu 2+ The doped zeolite imidazole framework-8 can effectively encapsulate a sufficient amount of composite drugs, and a sufficient amount of Cu 2+ The doped zeolite imidazole framework-8 also releases sufficient Cu 2+ , and sufficient Cu 2+ Will transform into Cu + At the same time, it depletes the endogenous glutathione (GSH) of tumor cells to effectively catalyze the Fenton-like reaction process of hydrogen peroxide in tumor cells and convert it into hydroxyl radicals (·OH), thereby hindering the copper efflux mediated by copper-ATPase and overloading the copper level in tumor cells.

[0050] The Cu 2+The weight m5 of the doped zeolite imidazole framework-8 can be 15.5, 16.0, 16.5, 17.0, 17.5, 18.0 or 18.5; the total weight m4 of the nano drug can be 21.2, 21.6, 22.0, 22.4, 22.8, 23.2, 23.6, 23.0, 23.4, 23.8, 25.2 or 25.6

[0051] The Cu of the zeolite imidazolyl framework-8 2+ The doping amount can be 0.33 mg, 0.34 mg, 0.35 mg, 0.36 mg, 0.37 mg, 0.38 mg, 0.39 mg, 0.40 mg or 0.41 mg.

[0052] In some optional embodiments, the average particle size of the nanomedicine is 150nm to 200nm;

[0053] In these embodiments, the average particle size of the nanodrug can be 150nm to 200nm, so that the nanodrug has a sufficiently small particle size to facilitate the nanodrug to quickly enter the tumor cells and enable the composite drug to better exert its efficacy.

[0054] The average particle size of the nano drug is 150nm, 160nm, 170nm, 180nm, 190nm or 200nm.

[0055] Figure 2 A schematic diagram of a method for preparing nanomedicine provided in an embodiment of the present application is shown exemplarily;

[0056] Based on a general inventive concept, such as Figure 2 As shown, the present application embodiment provides a method for preparing the nano drug, the method comprising:

[0057] S1. dissolving 2-methylimidazole in deionized water to obtain a 2-methylimidazole solution;

[0058] S2. mixing copper acetate, zinc acetate, hemoglobin and dichloroacetic acid with a mitochondrial targeting group, and then adding deionized water to the mixture to obtain a raw material mixture;

[0059] S3. mixing the raw material mixture and the 2-methylimidazole solution to allow the copper acetate, the zinc acetate, the hemoglobin, the dichloroacetic acid and the 2-methylimidazole solution to undergo an encapsulation reaction to obtain a nano drug core;

[0060] S4. Dispersing the nano drug core in an aqueous solution of hyaluronic acid so that the hyaluronic acid is coated on the surface of the nano drug core to obtain a nano drug.

[0061] This method is a method for preparing the above-mentioned nanomedicine. The specific structure of the nanomedicine can refer to the above-mentioned embodiment. Since this method adopts part or all of the technical solutions of the above-mentioned embodiment, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiment, which will not be repeated here one by one.

[0062] It should be noted that the raw material mixture and the 2-methylimidazole solution can be mixed by gradually adding the raw material mixture to the 2-methylimidazole solution; in addition, after the encapsulation reaction, the nano drug core precipitate can be collected by centrifugation, washed three times with deionized water, and then vacuum dried to obtain the nano drug core.

[0063] It should be noted that the dispersion can be first performed by ultrasonic dispersion and then by mechanical stirring in a dark environment; in addition, after the dispersion treatment, the mixture needs to be centrifuged to obtain a precipitate, and then the precipitate is washed with deionized water to remove free hyaluronic acid, and finally a pure nano drug is obtained.

[0064] It should be noted that this method first encapsulates the composite drug into Cu 2+ The hyaluronic acid is doped in the zeolite imidazole framework-8, and then through electrostatic interaction, the hyaluronic acid is covered on the surface of the nano-drug core.

[0065] In some optional embodiments, the temperature of the encapsulation reaction is 10° C. to 40° C., and the time of the encapsulation reaction is ≥ 6 h;

[0066] In these embodiments, the encapsulation reaction temperature can be 10° C. to 40° C., and the encapsulation reaction time can be ≥ 6 hours, so as to promote sufficient reaction between copper acetate, zinc acetate, hemoglobin, dichloroacetic acid and 2-methylimidazole solution, so as to promote the effective encapsulation of the composite drug in the Cu 2+ The nano drug core is obtained within the doped zeolite imidazole framework-8.

[0067] The temperature of the encapsulation reaction may be 10°C, 20°C, 30°C or 40°C.

[0068] In some optional embodiments, the dispersion method includes ultrasonic dispersion, the frequency of the ultrasonic dispersion is 15kHz to 25kHz, and the time of the ultrasonic dispersion is ≥15min;

[0069] In these embodiments, the dispersion method may include ultrasonic dispersion, and the frequency of ultrasonic dispersion may be 15kHz to 25kHz, and the time of ultrasonic dispersion may be ≥15min. The nano drug core may be fully dispersed into the aqueous solution of hyaluronic acid by ultrasonic dispersion, so that the hyaluronic acid fully wraps the nano drug core, thereby finally obtaining a nano drug with sufficient hyaluronic acid thickness.

[0070] The frequency of the ultrasonic dispersion may be 15 kHz, 16 kHz, 17 kHz, 18 kHz, 19 kHz, 20 kHz, 21 kHz, 22 kHz, 23 kHz, 24 kHz, or 25 kHz.

[0071] Based on a general inventive concept, an embodiment of the present application provides a tumor killing and immune activation drug, which includes the nano drug.

[0072] The drug is realized based on the above-mentioned nanomedicine. The specific structure of the nanomedicine can refer to the above-mentioned embodiment. Since the drug adopts part or all of the technical solutions of the above-mentioned embodiment, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiment, which will not be described one by one here.

[0073] It should be noted that, in addition to nanomedicines, the drug may also contain various adjuvants to enhance the drug's ability to target tumor cells and the therapeutic effect of the nanomedicine.

[0074] The present application is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually measured in accordance with national standards / industry standards; if there are no corresponding national standards / industry standards, they are measured in accordance with common international standards, conventional conditions or conditions recommended by the manufacturer.

[0075] Example 1

[0076] like Figure 2 As shown, a method for preparing nanomedicine comprises:

[0077] S1. Dissolve 492.6 mg of 2-methylimidazole in 3.0 mL of deionized water to obtain a 2-methylimidazole solution;

[0078] S2. 5.449 mg of copper acetate, 37.317 mg of zinc acetate, 4 mg of hemoglobin (Hb) and 2 mg of dichloroacetic acid with a mitochondrial targeting group (Mito-DCA) were mixed, and deionized water was added to the mixture to obtain a raw material mixture;

[0079] S3. The raw material mixture and the 2-methylimidazole solution are mixed to allow copper acetate, zinc acetate, hemoglobin, dichloroacetic acid and 2-methylimidazole solution to undergo encapsulation reaction to obtain the nanodrug core Hb / Mito-DCA@Cu / ZIF-8 (HMCZ);

[0080] S4. Disperse the nanodrug core HMCZ in an aqueous solution of hyaluronic acid (mass concentration is 10 mg / mL) so that the hyaluronic acid is coated on the surface of the nanodrug core to obtain the nanodrug HMCZH.

[0081] The temperature of the encapsulation reaction is 25° C., and the time of the encapsulation reaction is 6 h.

[0082] The dispersion method includes ultrasonic dispersion, the frequency of ultrasonic dispersion is 20kHz, the time of ultrasonic dispersion is 15 minutes, and stirring is required in the dark for 24 hours after ultrasonic dispersion.

[0083] like Figure 1 As shown, a nanomedicine that triggers copper cell death in tumor cells has a core-shell structure, and the core is Cu 2+ The doped zeolite imidazole framework-8 (Cu / ZIF-8) and the composite drug have a shell of hyaluronic acid; wherein the composite drug includes hemoglobin (Hb) and dichloroacetic acid (Mito-DCA) containing a mitochondrial targeting group.

[0084] The weight m1 of hemoglobin and the weight m2 of dichloroacetic acid containing a mitochondrial targeting group satisfy the relationship: m1:m2=2:1.

[0085] The total weight m3 of the composite drug and the total weight m4 of the nano drug satisfy the relationship: m3:m4=4:23.4.

[0086] The thickness of hyaluronic acid is 8.6nm to 12.8nm.

[0087] Cu 2+ The weight m5 of the doped zeolite imidazole framework-8 and the total weight m4 of the nano-drug satisfy the relationship: m5:m4=17:23.4;

[0088] Cu of zeolite imidazolate framework-8 2+ The doping amount is 0.35 mg.

[0089] Example 2

[0090] Based on the contents disclosed in Example 1, the following modifications are further made:

[0091] The total weight m3 of the composite drug and the total weight m4 of the nano drug satisfy the relationship: m3:m4=3.6:25.6.

[0092] Cu 2+ The weight m5 of the doped zeolite imidazole framework-8 and the total weight m4 of the nano-drug satisfy the relationship: m5:m4=15.5:25.6;

[0093] Cu of zeolite imidazolate framework-8 2+The doping amount is 0.33 mg.

[0094] Example 3

[0095] Based on the contents disclosed in Example 1, the following modifications are further made:

[0096] The total weight m3 of the composite drug and the total weight m4 of the nano drug satisfy the relationship: m3:m4=4.4:21.2.

[0097] Cu 2+ The weight m5 of the doped zeolite imidazole framework-8 and the total weight m4 of the nano-drug satisfy the relationship: m5:m4=18.5:21.2;

[0098] Cu of zeolite imidazolate framework-8 2+ The doping amount is 0.40 mg.

[0099] Comparative Example 1

[0100] Based on the contents disclosed in Example 1, the following modifications are further made:

[0101] Without adding the composite drug, the nanodrug Cu / ZIF-8@HA (CZH) was obtained.

[0102] Comparative Example 2

[0103] Based on the contents disclosed in Example 1, the following modifications are further made:

[0104] Without adding the hemoglobin of the composite drug, only dichloroacetic acid containing the mitochondrial targeting group was added to obtain the nanodrug Mito-DCA@Cu / ZIF-8@HA(MCZH).

[0105] Comparative Example 3

[0106] Based on the contents disclosed in Example 1, the following modifications are further made:

[0107] Without adding dichloroacetic acid containing mitochondrial targeting groups as a composite drug, only hemoglobin was added to obtain the nanodrug Hb@Cu / ZIF-8@HA (HCZH).

[0108] Related experiments and effect data:

[0109] 1. Morphological characterization of HMCZH:

[0110] The nanoparticles of HMCZH were characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Figure 1As shown, the average particle size of the synthesized nanomedicine HMCZH is about 180nm, and the surface presents a regular dodecahedral structure.

[0111] 2. Potential and infrared characterization of HMCZH:

[0112] The ZIF-8, Cu / ZIF-8, nanomedicine core HMCZ, nanomedicine HMCZH and nanocore HMCZ of Example 1 were tested using a Zeta potential analyzer and an infrared spectrometer. The results are as follows: Figure 3 It can be seen that the Zeta potential of some nanomedicine core HMCZ is positive (+1.8mV). After HA is modified on its surface, the Zeta potential of nanomedicine HMCZH becomes negative (-11.3mV), which indicates that HA is successfully modified on HMCZ nanoparticles. At the same time, the infrared spectrum results also show that HA is successfully modified on HMCZ nanoparticles.

[0113] 3. Verification of HMCZH's targeting performance on cancer cells (4T1)

[0114] In order to observe the cell-targeted uptake behavior of the nanodrug HMCZH in Example 1, rhodamine B (Rhm B) fluorescent dye was loaded into the nanodrug HMCZH. The specific loading method was that 5.449 mg of copper acetate, 37.317 mg of zinc acetate, 4 mg of hemoglobin (Hb), 2 mg of dichloroacetic acid with mitochondrial targeting groups (Mito-DCA) and 2 mg of Rhm B were mixed in the process of preparing the nanodrug core by one-pot method, and then deionized water was added to the mixture to obtain a raw material mixture, and then the nanodrug HMCZRH was prepared according to the method of Example 1. 4T1 cells were co-incubated with the nanodrug HMCZRH, and the cells were analyzed by laser confocal microscopy and flow cytometry analysis after 0 h, 2 h, 4 h and 6 h of co-culture, respectively. The results are shown in FIG. Figure 4 As shown. Among them, Figure 4 Figure a shows that after the nanodrug HMCZRH was co-incubated with 4T1 cells for 4 hours, the 4T1 cells showed a strong fluorescence intensity. Figure 4 Figures b and c show that the nanodrug HMCZRH has fully entered the 4T1 cells after being co-incubated with 4T1 cells for 4 hours. These results indicate that HMCZH can target and enter 4T1 cells within 4 hours.

[0115] 4. Characterization of the performance of nanomedicine HMCZH on the disruption of Cu2+ metabolic homeostasis in cancer cells:

[0116] The nanomedicine HMCZH of Study Example 1 affects Cu in tumor cells at the cellular level. 2+ Effects on metabolic homeostasis.

[0117] Based on the steps of Experiment 3, 4T1 cells were co-incubated with nanodrug HMCZH. Normal cells L929 and normal cells HUVECs were used as controls. The intracellular Cu 2+ The changes in concentration, such as Figure 5 shown. Figure 5 The results showed that the nanodrug HMCZH significantly inhibited the Cu-ATPase activity of tumor cells (4T1), while the Cu in normal cells HUVECs and L929 cells 2+ The concentration did not change significantly, indicating that these normal cells have intact Cu 2+ Metabolic homeostasis, which can effectively excrete excess Cu in cells 2+ , maintaining intracellular Cu 2+ Balance. Cu in 4T1 cells 2+ The concentration increased by only 37% within 4 hours and did not change significantly within 12 hours. The above results show that the nanodrug HMCZH can specifically destroy the Cu 2+ Metabolic homeostasis to enhance intracellular Cu 2+ concentration, thereby inducing copper death of tumor cells.

[0118] 5. In vitro verification of the killing effect of nanodrug HMCZH on cancer cells

[0119] The nanodrug HMCZH of Example 1, the nanodrug CZH of Comparative Example 1, the nanodrug MCZH of Comparative Example 2, and the nanodrug HCZH of Comparative Example 3 were co-incubated with 4T1 cells, and then the killing effects of nanodrugs of different concentrations and types on cancer cells were evaluated by laser confocal microscopy and CCK-8 method. The results are as follows: Figure 6 As shown. After 24 hours of treatment with different types of nanodrugs at the same mass concentration, the toxicity of 4T1 cells was significantly enhanced. When the nanoparticle concentration was 80 μg / mL, the nanodrug HMCZH could kill more than 87% of 4T1 cells after 24 hours of treatment. However, it was difficult for nanodrugs CZH, nanodrugs MCZH and nanodrugs HCZH to reach this level (nanodrug MCZH killed about 75% of 4T1 cells, nanodrug HCZH killed about 40% of 4T1 cells, and nanodrug CZH killed about 30% of 4T1 cells), which indicates that there is a strong affinity between Hb in nanodrug HMCZH and Mito-DCA for Cu in tumor cells. 2+ The effects of these two drugs have a synergistic effect and have a significant killing effect on tumor cells.

[0120] 6. Investigation of anti-tumor effects in vivo

[0121] The in vivo antitumor effects of different nanomedicines were evaluated using 4T1 tumor-bearing mice. 3 Under the condition of Cu, 4T1 tumor-bearing mice were randomly divided into 6 groups for different treatments, namely: Control group, ZH group (only Cu 2+ doped zeolite imidazole framework-8), CZH group, HCZH group, MCZH group and HMCZH group were treated with different nanomedicines. The results are shown in Figure 7 As shown. The tumor volume of the control group increased dramatically during the treatment, reaching more than 12 times the original volume on the 15th day. In contrast, the tumor growth of the CZH group and the HCZH group was inhibited to a certain extent, and the final tumor volumes of these two groups were 5.5 times and 4.9 times the initial tumor volumes, respectively. It is worth noting that after the introduction of Mito-DCA, the anti-tumor effects of the MCZH group and the HMCZH group were significantly enhanced, which is consistent with the above in vitro experimental results. Figure 7 It can be seen that the tumor inhibition rate of the HMCZH group was the highest at 88.85%.

[0122] The above results confirmed that the HMCZH group had the strongest anti-tumor effect, further indicating that the interaction between Hb in the nanomedicine HMCZH and Mito-DCA on Cu in tumor cells 2+ The effects of these two drugs have a synergistic effect and have a significant killing effect on tumor cells.

[0123] 7. Investigation of in vivo immunotherapy effects

[0124] Immunofluorescence staining was used to evaluate the phenotypic transformation of tumor-associated macrophages induced by HMCZH in 4T1 tumor-bearing mice. Figure 8 As shown. Figure 8 As shown in Figures a and b, the positive expression rate of CD86 in the Control group increased significantly from 5.1% to 23.8% in the HMCZH group. Figure 8 As shown in Figures c and d, the positive expression rate of CD206 in the control group decreased from 22.6% to 3.6% in the HMCZH group. This indicates that the HMCZH group can significantly increase the proportion of M1 macrophages and reduce the proportion of M2 macrophages, thereby enhancing the anti-tumor immune effect; this shows that the nanodrug HMCZH shows great clinical therapeutic potential.

[0125] In summary, the present invention provides a nano drug that induces copper death in tumor cells. The nano drug is based on a composite drug including hemoglobin and dichloroacetic acid containing a mitochondrial targeting group. The synergistic effect of the two can effectively affect the Cu in tumor cells. 2+ metabolic homeostasis, thereby achieving increased intracellular Cu 2+The purpose is to increase the concentration while changing the metabolic mode of tumor cells.

[0126] In addition, the nanomedicine that induces copper death of tumor cells provided in the embodiment of the present application can promote the nanomedicine to reverse the tumor respiratory metabolism mode through the combined action of hemoglobin of the composite drug and dichloroacetic acid containing mitochondrial targeting groups, so as to destroy the Cu 2+ The metabolic homeostasis of the tumor cells is altered, thereby inducing copper death of tumor cells, thereby effectively killing the in situ tumor and enhancing the immune response.

[0127] In addition, the present invention provides a nano drug that induces copper death of tumor cells. The nano drug can increase the amount of Cu in cancer cells. 2+ By increasing the concentration and changing the respiratory metabolism of tumor cells, it is possible to not only achieve copper cell death treatment for tumor cells, but also reverse the immunosuppressive tumor microenvironment to enhance the immunogenicity of tumor cells. This provides a new idea for the development of multifunctional nanomedicines that have both tumor killing and immune activation, which has important medical significance and application potential.

[0128] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. It will be apparent to those skilled in the art that various modifications to these embodiments are possible, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown in the present application, but will conform to the widest range consistent with the principles and novel features applied for by the present application.

Claims

1. A nano drug that induces copper death of tumor cells, wherein the nano drug has a core-shell structure, and the core is Cu 2+ The doped zeolite imidazole framework-8 and the composite drug have a shell of hyaluronic acid; wherein, The compound drug comprises hemoglobin and dichloroacetic acid containing a mitochondrial targeting group.

2. The nanomedicine according to claim 1, wherein the weight m1 of the hemoglobin and the weight m2 of the dichloroacetic acid containing the mitochondrial targeting group satisfy the relationship: m1:m2≥2:

1.

3. According to the nanomedicine according to claim 1, the total weight m3 of the composite drug and the total weight m4 of the nanomedicine satisfy the relationship: m3:m4=(3.6-4.4):(21.2-25.6).

4. The nanomedicine according to claim 1, wherein the thickness of the hyaluronic acid is 8.6 nm to 12.8 nm.

5. The nano drug according to claim 1, wherein the Cu 2+ The weight m5 of the doped zeolite imidazole framework-8 and the total weight m4 of the nano drug satisfy the relationship: m5:m4=(15.5-18.5):(21.2-25.6); and / or The Cu of the zeolite imidazole framework-8 2+ The doping amount is 0.33mg~0.41mg. The nanomedicine according to claim 1 , wherein the average particle size of the nanomedicine is 150 nm to 200 nm.

7. A method for preparing the nanodrug according to any one of claims 1 to 6, the method comprising: Dissolving 2-methylimidazole in deionized water to obtain a 2-methylimidazole solution; Copper acetate, zinc acetate, hemoglobin and dichloroacetic acid with a mitochondrial targeting group are mixed, and deionized water is added to the mixture to obtain a raw material mixed solution; The raw material mixture and the 2-methylimidazole solution are mixed to allow the copper acetate, the zinc acetate, the hemoglobin, the dichloroacetic acid and the 2-methylimidazole solution to undergo an encapsulation reaction to obtain a nano drug core; The nano drug core is dispersed in an aqueous solution of hyaluronic acid so that the hyaluronic acid covers the surface of the nano drug core to obtain the nano drug. 8 . The method according to claim 7 , wherein the temperature of the encapsulation reaction is 10° C. to 40° C., and the time of the encapsulation reaction is ≥ 6 hours. 9 . The method according to claim 7 , wherein the dispersing method comprises ultrasonic dispersion, the frequency of the ultrasonic dispersion is 15 kHz to 25 kHz, and the time of the ultrasonic dispersion is ≥15 min.

10. A drug for tumor killing and immune activation, comprising the nano drug according to any one of claims 1 to 6.

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