A copper death-selective inducing nanoparticle targeting tumor mitochondria

Abstract

The application belongs to the technical field of nanomedicine, tumor treatment and immunotherapy, and specifically provides a copper death selective induction nanoparticle targeting tumor mitochondria, which is prepared by the following preparation method: S1, TPY is specifically coupled with MHI through a condensation reaction to obtain MTPY; S2, MTPY is complexed with copper ions to obtain an MTPY-Cu complex; and S3, the MTPY-Cu complex is self-assembled with albumin to form an MTPY-Cu@Alb nanoparticle. The copper dose of the application is only 1 / 1000 of that of free Cu 2+ , and the equivalent immune regulation and killing effects can be achieved; the primary tumor and distant tumor foci can be inhibited, and lung metastasis can be reduced; and the application can induce immune memory and prevent recurrence; the application is combined with cisplatin to significantly enhance the therapeutic effect and reverse the induced immunosuppression; and the application has high biological safety, and has no obvious hepatorenal toxicity or hemolytic reaction.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomedicine, tumor treatment and immunotherapy, and particularly relates to a copper death selective induction nanoparticle targeting tumor mitochondria. BACKGROUND

[0002] Copper death is a form of mitochondria-dependent programmed cell death discovered in recent years, which is triggered by the direct combination of copper ions with lipidated proteins in the TCA cycle, leading to protein aggregation, iron-sulfur cluster protein loss and metabolic disaster. Studies have shown that copper death can activate the immune system by releasing tumor-associated antigens, ROS and DAMPs, but existing research has failed to reveal its systematic regulation of immune checkpoints.

[0003] The prior art mainly has the following deficiencies:

[0004] 1. Lack of tumor-selective delivery system: Common copper carriers (such as Disulfiram, Elesclomol, 8-HQ, TPY, etc.) lack tumor selectivity and are easily enriched in normal tissues, causing systemic toxicity.

[0005] 2. Unable to achieve mitochondrial-targeted delivery: Existing nanometer copper carriers are mostly located in the cytoplasm and are difficult to directly act on the mitochondrial "copper pool", resulting in low copper death efficiency.

[0006] 3. Single immune regulation: Existing immunotherapy is mostly directed at a single checkpoint (such as PD-1 / PD-L1, CTLA-4, CD47, etc.), which is difficult to reverse both innate and adaptive immune escape.

[0007] 4. Resistance to conventional treatment: Traditional treatments such as radiotherapy and chemotherapy easily induce upregulation of CD276 and CD47, weakening the immune killing effect.

[0008] 5. High toxicity: Accumulation of copper ions in non-target tissues can cause liver and kidney damage, hematopoietic suppression and other side effects.

[0009] Therefore, it is necessary to design a copper death selective induction nanoparticle targeting tumor mitochondria. SUMMARY

[0010] The purpose of the present application is to provide a copper death selective induction nanoparticle targeting tumor mitochondria to solve the problems of poor tumor selectivity, high systemic toxicity, and inability to achieve mitochondrial-specific delivery of existing copper ion carriers, resulting in low copper death induction efficiency and difficulty in effectively regulating immune checkpoint expression, which cannot simultaneously reverse innate immune escape (CD47-mediated) and adaptive immune tolerance (CD276-mediated).

[0011] To achieve the above object, the application provides a copper death selective induction nanoparticle targeting tumor mitochondria, which is prepared by the following preparation method:

[0012] S1, TPY is specifically coupled with MHI through a condensation reaction to obtain MTPY;

[0013] S2, MTPY is complexed with copper ions to obtain MTPY-Cu complex;

[0014] S3, MTPY-Cu complex is self-assembled with albumin to form MTPY-Cu@Alb nanoparticles.

[0015] In a specific embodiment, the MHI is prepared by the following preparation method:

[0016] N-[(3-(anilinemethylene)-2-chloro-1-cyclohexen-1-yl)methylene] aniline hydrochloride, 1-ethyl-2,3,3-trimethyl-3H-indolium iodide and 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indolium iodide are dissolved in anhydrous ethanol, sodium acetate is added, and the reaction is heated to reflux in an oil bath; after the reaction is completed, it is cooled to room temperature, the reaction solution is concentrated under reduced pressure, an appropriate amount of ethyl acetate is added for crystallization, the solid product is collected, and high-performance preparative liquid chromatography is used for purification to obtain MHI.

[0017] In a specific embodiment, in step S1, the condensation reaction is that MHI and TPY are dissolved in a mixed solvent, a condensing agent is added, and the reaction is stirred at room temperature, and the whole process is carried out under nitrogen protection.

[0018] In a specific embodiment, the condensing agent is DMTMM.

[0019] In a specific embodiment, the mixed solvent is a mixed solvent obtained by mixing anhydrous methanol and anhydrous dichloromethane at a volume ratio of 1:1.

[0020] In a specific embodiment, after the condensation reaction is completed, the reaction solution is concentrated under reduced pressure, ether is added to precipitate, the solid is collected and crystallized with an ethanol-water mixture, and the crystallized product is further purified by high-performance preparative liquid chromatography to obtain MTPY.

[0021] In a specific embodiment, in step S2, MTPY is first dissolved in DMSO, and then an aqueous solution of CuCl2·2H2O with an equal molar amount is added, and the reaction is stirred at room temperature to obtain MTPY-Cu complex.

[0022] In a specific embodiment, in step S3, albumin is weighed and dissolved in ultrapure water, and the obtained MTPY-Cu complex solution is slowly added dropwise into the albumin solution, and magnetic stirring is performed for a period of time, to obtain MTPY-Cu@Alb nanoparticles.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] The present application provides a nano-carrier system that can efficiently and selectively enrich copper ions in the mitochondria of tumor cells, thereby achieving efficient induction of copper death.

[0025] The present application realizes bidirectional down-regulation of immune checkpoints CD276 and CD47, and synergistically activates T cell killing and macrophage phagocytosis, thereby simultaneously reversing innate and adaptive immune tolerance.

[0026] The present application overcomes the immune tolerance induced by chemotherapy, improves the comprehensive anti-tumor effect, and induces immune memory, thereby reducing tumor recurrence and metastasis.

[0027] The present application reduces systemic toxic side effects while maintaining anti-tumor activity, thereby improving biological safety and clinical convertibility.

[0028] The copper dose of the present application is only 1 / 1000 of free Cu 2+ , and can achieve equivalent immune regulation and killing effects; can also inhibit primary tumors and distant tumor foci, reduce lung metastasis; and can induce immune memory to prevent recurrence; the present application, in combination with cisplatin, significantly enhances the therapeutic effect and reverses the immune suppression induced by cisplatin; the present application has high biological safety and no obvious hepatotoxicity, nephrotoxicity or hemolytic reaction.

[0029] In addition to the objects, features, and advantages described above, the present application has other objects, features, and advantages. The present application will be further described in detail below. BRIEF DESCRIPTION OF DRAWINGS

[0030] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application, illustrate the preferred embodiments of the application, and assist in the explanation of the application. In the drawings:

[0031] Figure 1 is a synthesis diagram of MHI and MTPY of an embodiment of the present application.

[0032] Figure 2 is a synthesis diagram of MTPY-Cu@Alb of an embodiment of the present application.

[0033] Figure 3 is a hemolysis test diagram for biological safety evaluation of MTPY-Cu@Alb of an embodiment of the present application, wherein,Figure 3 a is the macroscopic result diagram of MTPY-Cu@Alb hemolysis test, Figure 3 b is the hemolysis rate diagram of MTPY-Cu@Alb under different concentrations.

[0034] Figure 4 is the ALT detection result diagram of the biosafety evaluation of MTPY-Cu@Alb of an embodiment of the application;

[0035] Figure 5 is the AST detection result diagram of the biosafety evaluation of MTPY-Cu@Alb of an embodiment of the application;

[0036] Figure 6 is the CR detection result diagram of the biosafety evaluation of MTPY-Cu@Alb of an embodiment of the application;

[0037] Figure 7 is the BUN detection result diagram of the biosafety evaluation of MTPY-Cu@Alb of an embodiment of the application.

[0038] Figure 8 is the organ HE staining diagram of the biosafety evaluation of MTPY-Cu@Alb of an embodiment of the application.

[0039] Figure 9 is the in vivo tumor targeting diagram of MTPY-Cu@Alb of an embodiment of the application, wherein, Figure 9 a is the distribution diagram of MTPY-Cu@Alb at different time points in the body of a mouse; Figure 9 b is the distribution diagram of MTPY-Cu@Alb in main organs and tumors in vivo.

[0040] Figure 10 is the in situ anti-tumor model treatment diagram of MTPY-Cu@Alb of an embodiment of the application;

[0041] Figure 11 is the tumor volume and weight statistical diagram of different treatment groups of MTPY-Cu@Alb in situ anti-tumor model of an embodiment of the application, wherein, Figure 11 a is the statistical diagram of tumor volume change with time of different treatment groups of mice and the representative picture of tumor at the termination time point; Figure 11 b is the statistical diagram of tumor weight of different treatment groups of mice at the termination time point; group 1 of the grouping is the PBS control group, group 2 is the TPY-Cu group, group 3 is the MHI@Alb group, and group 4 is the MTPY-Cu@Alb group.

[0042] Figure 12 is the immunohistochemical representative diagram of the detection of T cell infiltration of the tumor of a mouse in different treatment groups of an embodiment of the application.

[0043] Figure 13 Figure 1 is a graph of WB detection of the anti-tumor ability and activation ability of T cells and macrophages of different treatment groups of mice with tumors according to an embodiment of the present application, wherein, Figure 13 a is WB detection of the anti-tumor proliferation and immune checkpoint inhibition ability of MTPY-Cu@Alb; Figure 13 b is WB detection of the T cell activation ability of MTPY-Cu@Alb; Figure 13 c is WB detection of the apoptosis promotion and tumor migration inhibition ability of MTPY-Cu@Alb; Figure 13 d is WB detection of the macrophage activation ability of MTPY-Cu@Alb.

[0044] Figure 14 Figure 2 is a graph of flow cytometry detection of T cell infiltration of different treatment groups of mice with tumors according to an embodiment of the present application, wherein, Figure 14 a is a total T cell number statistical graph; Figure 14 b is a helper T cell number statistical graph; Figure 14 c is a cytotoxic T cell number statistical graph.

[0045] Figure 15 Figure 3 is a graph of statistics and immunohistochemical detection of lung metastases of different treatment groups of mice according to an embodiment of the present application, wherein, Figure 15 a is a photograph of lung metastases and a representative HE staining graph of different groups; Figure 15 b is a lung metastasis number statistical graph of different groups; Figure 15 c is a mouse body weight statistical graph of different groups over time.

[0046] Figure 16 Figure 4 is a schematic diagram of an immune memory model of MTPY-Cu@Alb according to an embodiment of the present application;

[0047] Figure 17 Figure 5 is a tumor volume and weight statistical graph of different treatment groups of mice with tumors in an immune memory model of MTPY-Cu@Alb according to an embodiment of the present application, wherein, Figure 17 a is a statistical graph of tumor volume change over time and a representative picture of tumors at the termination time point of different treatment groups of mice; Figure 17 b is a tumor weight statistical graph of different treatment groups of mice at the termination time point, wherein group 1 is a PBS control group, group 2 is a TPY-Cu group, group 3 is an MHI@Alb group, and group 4 is an MTPY-Cu@Alb group.

[0048] Figure 18 Figure 6 is a schematic diagram of a model of MTPY-Cu@Alb combined with cisplatin according to an embodiment of the present application;

[0049] Figure 19Figure 2 is a tumor volume and weight statistical chart of different treatment groups of the MTPY-Cu@Alb combined with cisplatin model according to an embodiment of the present application, wherein, Figure 19 Figure 2a is a tumor volume statistical chart of different treatment groups of mice and representative pictures of tumors at the termination time point; Figure 19 Figure 2b is a tumor weight statistical chart of different treatment groups of mice at the termination time point, wherein group 1 is the PBS control group, group 2 is the cisplatin group, group 3 is the MTPY-Cu@Alb group, and group 4 is the cisplatin+MTPY-Cu@Alb combined group.

[0050] Figure 20 Figure 3 is an immunohistochemical chart of tumors of different treatment groups of the MTPY-Cu@Alb combined with cisplatin model according to an embodiment of the present application.

[0051] Figure 21 Figure 4 is a flow cytometry detection chart of tumors of different treatment groups of the MTPY-Cu@Alb combined with cisplatin model according to an embodiment of the present application. DETAILED DESCRIPTION

[0052] The embodiments of the present application are described in detail below, and the specific embodiments described herein are only used to explain the present application and not to limit the present application.

[0053] The present application provides a copper death selective induction nanoparticle targeting tumor mitochondria, named MTPY-Cu@Alb, which comprises a copper ion chelating group and a mitochondria targeting group.

[0054] Copper ion chelating group:

[0055] 4-([2,2':6',2"-Terpyridin]-4'-yl)aniline (abbreviated as TPY), which has a high-affinity terpyridine coordination skeleton and can stably complex with Cu 2+ ions in a 1:1 molar ratio, thereby achieving efficient capture and delivery of copper ions.

[0056] Mitochondria targeting group:

[0057] heptamethine cyanine (abbreviated as MHI), which is a typical heptamethine cyanine dye, has a positive charge and a carboxyl side chain in its molecular structure, has mitochondria targeting property and near-infrared fluorescence characteristics, can realize accurate positioning of mitochondria and has the function of in vivo imaging.

[0058] Construction of MTPY:

[0059] TPY and MHI are specifically coupled through condensation reaction to obtain a bifunctional small molecule MTPY which has both copper ion chelating ability and mitochondria targeting performance.

[0060] MTPY and Cu 2+ After complexation, MTPY-Cu@Alb nanoparticles are self-assembled with albumin (Alb), which not only ensures the stability and injectability of the drug, but also enhances the biological safety of in vivo distribution.

[0061] The preparation method of MTPY-Cu@Alb nanoparticles is to first synthesize MTPY, then complex MTPY with CuCl2 in a solution to generate MTPY-Cu; and then self-assemble with an albumin solution to form nanoparticles with a particle size of about 114 nm and a zeta potential of about -23 mV; and verify the good storage stability by freeze-drying and reconstitution.

[0062] Example 1

[0063] Synthesis of MHI and MTPY:

[0064] 1. Synthesis of MHI.

[0065] 1.1. Preparation of raw materials:

[0066] N-[(3-(anilinemethylene)-2-chloro-1-cyclohexen-1-yl)methylene] aniline hydrochloride (708 mg);

[0067] 1-ethyl-2,3,3-trimethyl-3H-indolium iodide (602 mg);

[0068] 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indolium iodide (774 mg);

[0069] Sodium acetate (975 mg, 11.9 mmol);

[0070] Anhydrous ethanol (150 mL).

[0071] 1.2. Reaction conditions:

[0072] Dissolve the above three dye precursors in anhydrous ethanol, add sodium acetate, and heat to reflux in an oil bath for 3 h. After the reaction is completed, cool to room temperature, and concentrate the reaction solution under reduced pressure.

[0073] 1.3. Purification of product:

[0074] Add an appropriate amount of ethyl acetate to crystallize, collect the solid product, and purify by high-performance preparative liquid chromatography (prep-HPLC) to obtain MHI dark solid (631 mg, yield 47.2%, purity about 95%).

[0075] 1.4. Structural characterization:

[0076] The molecular structure of MHI was characterized by nuclear magnetic resonance (^1H NMR, ^13C NMR) and high resolution mass spectrometry (HRMS), and its structure and targeting performance were confirmed.

[0077] 2. Synthesis of MTPY.

[0078] 2.1, Preparation of raw materials:

[0079] MHI (669 mg, from the synthesis of the previous step);

[0080] 4'- (4-aminophenyl) -2, 2': 6', 2 "-terpyridine (TPY, 486 mg);

[0081] Condensing agent DMTMM (828 mg);

[0082] Solvent: anhydrous methanol (50 mL) and anhydrous dichloromethane (50 mL).

[0083] 2.2, Reaction conditions:

[0084] Dissolve MHI and TPY in the mixed solvent, add DMTMM, and stir at room temperature for 24 h. The whole process is carried out under nitrogen protection.

[0085] 2.3, Purification of product:

[0086] After the reaction, concentrate under reduced pressure to 20 mL, add 50 mL of ether to precipitate the precipitate. Collect the solid and crystallize with ethanol-water mixture. The final product is purified by prep-HPLC to obtain dark green solid MTPY (508 mg, yield 52.1%, purity about 95%).

[0087] 2.4, Structure characterization:

[0088] MTPY is verified by ^1H NMR, ^13C NMR and HRMS, confirming that it is successfully coupled by TPY and MHI, and has the dual functional characteristics of copper ion chelation and mitochondrial targeting.

[0089] Example 2

[0090] Preparation of MTPY-Cu@Alb nanoparticles:

[0091] 1, MTPY-Cu complex formation.

[0092] Dissolve MTPY in DMSO with a final concentration of 10 mM; add an equimolar amount of CuCl2·2H2O aqueous solution (pH 7.0) to complex it at a 1:1 molar ratio; stir at room temperature for 2 hours to obtain stable MTPY-Cu complex.

[0093] 2, Self-assembly with albumin.

[0094] Bovine serum albumin (BSA, Sigma-Aldrich, 300 mg) was weighed and dissolved in 60 mL ultrapure water; the above MTPY-Cu solution was slowly added dropwise into the BSA solution, and magnetic stirring was performed for 4 hours; and the self-assembled nanoparticles MTPY-Cu@Alb were obtained.

[0095] 3. Purification and stabilization.

[0096] Free DMSO, Cu 2+ and unbound MTPY were removed by repeated filtration with 30 kDa ultrafiltration centrifuge tubes; the solution was concentrated to 10 mL; and further heating was performed at 70°C for 10 minutes to enhance the structural stability of the nanoparticles, followed by cooling to room temperature.

[0097] 4. Particle size and performance characterization.

[0098] The dynamic light scattering (DLS) detection results showed that the average hydrated particle size of the obtained MTPY-Cu@Alb was 110-120 nm, and the surface potential was -23 mV; fluorescence spectroscopy and electron microscope characterization showed that the nanoparticles had good stability and dispersibility.

[0099] Example 3

[0100] Safety evaluation of MTPY-Cu@Alb:

[0101] 1. Hemolysis experiment.

[0102] In one embodiment, a fresh isolated mouse red blood cell suspension (40 μL) was mixed with different concentrations of MTPY-Cu@Alb solution (0, 10, 20, 40, 80 μM, based on the content of MTPY-Cu). Deionized water treatment was used as a positive control, and PBS treatment was used as a negative control. After incubation at 37°C for 2 hours, each group was centrifuged, and the supernatant was collected for detection of absorbance at 540 nm. The results showed that there was no obvious release of hemoglobin in the MTPY-Cu@Alb group compared with the PBS control group, indicating that it had good hemolytic compatibility. Figure 3 .

[0103] 2. Serological detection.

[0104] In another embodiment, C57BL / 6 mice were randomly divided into groups, and MTPY-Cu@Alb (dose 5 mg / kg) or an equal volume of PBS was injected via the tail vein. After 24 hours, the orbital blood was taken to separate the serum, and the serum biochemical indicators were detected, including creatinine (CR), blood urea nitrogen (BUN), glutamic-pyruvic transaminase (ALT), and glutamic-oxalacetic transaminase (AST). The detection results were all within the normal reference range, suggesting that there was no obvious hepatorenal toxicity. Figures 4-7 .

[0105] 3. Histological examination.

[0106] In yet another embodiment, after the end of the experiment, the animals are sacrificed, and the heart, liver, spleen, lung, kidney and other major organs are taken, fixed with 10% neutral formaldehyde, routinely paraffin-embedded, sectioned, and stained with hematoxylin-eosin (H&E). No inflammation, necrosis or tissue structure destruction is observed under optical microscope, further proving that MTPY-Cu@Alb has good in vivo biological safety. Figure 8

[0107] Example 4

[0108] In vivo fluorescence localization of MTPY-Cu@Alb:

[0109] 1. Establishment of experimental animal model.

[0110] In one embodiment, C57BL / 6 mice are taken, and MB49 bladder cancer cells (5 x 10 5 6 per mouse, 100 μL of PBS suspension) are subcutaneously inoculated to establish a tumor xenograft model in the right axillary. When the tumor volume is about 100 mm 3 3, the subsequent treatment is started.

[0111] 2. Drug administration and in vivo imaging.

[0112] MTPY-Cu@Alb solution (5 mg / kg, tail vein injection) is injected into tumor-bearing mice. In vivo near-infrared fluorescence imaging (IVIS Lumina imaging system) is performed at 0, 1, 6, 12, 24 and 48 hours after administration to observe the distribution of nanoparticles in vivo.

[0113] 3. Results analysis.

[0114] The imaging results show that MTPY-Cu@Alb gradually accumulates at the tumor site, and reaches a fluorescence signal peak at 24 hours after administration, and then gradually decays. The animals are sacrificed at 48 hours, and the heart, liver, spleen, lung, kidney and tumor are taken for ex vivo fluorescence imaging. The results show that the drug is mainly distributed in the tumor tissue, with a small amount in the liver and kidney, showing good tumor targeting and in vivo imaging performance.

[0115] Example 5

[0116] Evaluation of in situ anti-tumor effect of MTPY-Cu@Alb:

[0117] 1. Establishment of animal model.

[0118] In one embodiment, C57BL / 6 mice are taken, and MB49 bladder cancer cells (5 x 10​5 tumor volume is about 50-100mm 3 , the drug administration is started.

[0119] 2. Grouping and drug administration.

[0120] The mice were randomly divided into PBS control group, TPY-Cu group (2mg / kg), MHI@Alb group (5mg / kg), and MTPY-Cu@Alb group (5mg / kg). Each group was injected through the tail vein, and the drug was administered once on days 0, 3, 6, and 9.

[0121] 3. Detection method.

[0122] Tumor volume was measured every two days during the experiment, and the formula was: volume = length x width 2 / 2. Body weight changes were also monitored. At the end of the experiment, the animals were sacrificed, and tumor tissue was taken for immunohistochemistry (IHC), Western blotting (WB), and flow cytometry analysis of tumor infiltrating lymphocytes.

[0123] 4. Results.

[0124] The results showed that the tumor volume and weight of the MTPY-Cu@Alb group were significantly lower than those of the control group and other control drug groups. WB results suggested that MTPY-Cu@Alb could inhibit tumor proliferation, migration, and activate T and macrophages. Immunohistochemistry and flow cytometry results showed that CD3 + , CD8 + T cell infiltration was significantly increased, indicating that MTPY-Cu@Alb had significant in situ anti-tumor activity.

[0125] Example 6

[0126] Lung metastasis inhibition effect of MTPY-Cu@Alb:

[0127] 1. Animal model establishment.

[0128] In another embodiment, BALB / c mice were subcutaneously inoculated with 4T1 breast cancer cells (5x10 5 cells per mouse, 100μL of PBS suspension) in the right axillary to establish a breast cancer orthotopic tumor model.

[0129] 2. Grouping and drug administration.

[0130] The mice were randomly divided into PBS control group, TPY-Cu group, MHI@Alb group, and MTPY-Cu@Alb group (dose same as Example 5). Each group was injected through the tail vein on days 0, 3, 6, and 9.

[0131] 3. Detection method.

[0132] On day 14, the animals were sacrificed, and the lung tissues were fixed, sectioned, and stained with H&E for counting the number of lung metastatic nodules. The body weight and general state of the animals were monitored synchronously.

[0133] 4. Results.

[0134] The results showed that the lung metastatic nodules were significantly reduced in the MTPY-Cu@Alb group, the lung tissue structure was well preserved, and the animal body weight remained stable, indicating that the nano-drug had obvious advantages in inhibiting lung metastasis.

[0135] Example 7

[0136] Evaluation of the immune memory effect of MTPY-Cu@Alb:

[0137] 1. Establishment of animal models.

[0138] In another embodiment, BALB / c mice were inoculated with 4T1 breast cancer cells (8 x 10 5 mm in volume, and were randomly divided and administered (grouping was the same as in Example 5). The administration period was day 0, 3, 6, and 9. 3

[0139] 2. Resection of primary tumors and secondary attack.

[0140] On day 14, the primary tumors of the mice in each group were surgically removed. Four days after the operation (day 18), the same number of 4T1 cells were again inoculated in the left groin to simulate recurrence / secondary attack.

[0141] 3. Detection method.

[0142] Tumor growth was monitored every two days during the experiment, and the animals were sacrificed on day 32, and the tumor tissue at the secondary inoculation site was weighed.

[0143] 4. Results.

[0144] The results showed that the growth of the tumors inoculated in the MTPY-Cu@Alb treatment group was significantly inhibited, indicating that it induced a persistent anti-tumor immune memory.

[0145] Example 8

[0146] Chemotherapy sensitization effect of MTPY-Cu@Alb combined with cisplatin:

[0147] 1. Establishment of animal models.

[0148] In one embodiment, C57BL / 6 mice were inoculated with MB49 bladder cancer cells (5 x 10 5 ​Each mouse was injected with 100 μL of PBS suspension of tumor cells to establish orthotopic tumor model. When the tumor volume reached about 50 mm 3 Then, drug treatment was started.

[0149] 2. Grouping and drug administration

[0150] The mice were randomly divided into four groups:

[0151] PBS control group;

[0152] Cisplatin group (2 mg / kg, intraperitoneal injection, once every other day);

[0153] MTPY-Cu@Alb group (5 mg / kg, tail vein injection, administered on days 0, 3, 6, and 9);

[0154] Cisplatin + MTPY-Cu@Alb combination group (administered in the same way as above).

[0155] 3. Detection method

[0156] The tumor volume and body weight were measured every two days during the drug administration period. After the experiment, the animals were sacrificed, and the tumor tissue was taken for immunohistochemistry (IHC) and flow cytometry analysis.

[0157] 4. Experimental results

[0158] Tumor inhibition effect: the tumor volume and weight of the combination group were significantly lower than those of the cisplatin or MTPY-Cu@Alb groups alone (P < 0.05). Figures 18-19 ).

[0159] Molecular level detection: the cisplatin + MTPY-Cu@Alb combination group could promote tumor apoptosis (P < 0.05). Figure 20 ).

[0160] Immune activation: flow cytometry detection showed that the CD8 + T cell activation level was the highest in the combination group, and the proportion of tumor-infiltrating immune cells was significantly increased (P < 0.05). Figure 21 ).

[0161] The above content is a further detailed description of the present application in combination with specific preferred embodiments, and cannot be considered as limiting the specific implementation of the present application to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, without departing from the concept of the present application, a number of simple deductions and substitutions can be made, which should be considered as falling within the protection scope of the present application.

Claims

1. A copper death-selective inducing nanoparticle targeting tumor mitochondria, characterized in that, The preparation method is as follows: S1, coupling TPY with MHI through a condensation reaction to obtain MTPY; The structural formula of TPY is: ; MHI has the structural formula: ; The structural formula of MTPY is: ; S2, complexing MTPY with copper ions to obtain MTPY-Cu complex; S3, self-assembling MTPY-Cu complex with albumin to form MTPY-Cu@Alb nanoparticles.

2. The tumor mitochondria-targeted copper death-selective inducing nanoparticle according to claim 1, characterized in that, The MHI is prepared by the following preparation method: Dissolving N-[(3-(anilinemethylene)-2-chloro-1-cyclohexen-1-yl)methylene] aniline hydrochloride, 1-ethyl-2,3,3-trimethyl-3H-indolium iodide and 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indolium iodide in anhydrous ethanol, adding sodium acetate, and heating to reflux in an oil bath; after the reaction is completed, cooling to room temperature, concentrating the reaction solution under reduced pressure, adding an appropriate amount of ethyl acetate for crystallization, collecting the solid product, and purifying by high-performance preparative liquid chromatography to obtain MHI.

3. The tumor mitochondria-targeted copper death-selective inducing nanoparticle according to claim 1, characterized in that, In step S1, the condensation reaction is to dissolve MHI and TPY in a mixed solvent, add a condensing agent, and stir the reaction at room temperature, and the whole process is carried out under nitrogen protection.

4. The tumor mitochondria-targeted copper death-selective inducing nanoparticle according to claim 3, characterized in that, The condensing agent is DMTMM.

5. The tumor mitochondria-targeted copper death-selective induction nanoparticle of claim 3, wherein, The mixed solvent is a mixed solvent obtained by mixing anhydrous methanol and anhydrous dichloromethane at a volume ratio of 1:

1.

6. The tumor mitochondria-targeted copper death-selective induction nanoparticle of claim 3, wherein, After the condensation reaction is completed, concentrate under reduced pressure, add ether to precipitate, collect the solid and crystallize with an ethanol-water mixture, and then purify the crystallized product by high-performance preparative liquid chromatography to obtain MTPY.

7. The tumor mitochondria-targeted copper death-selective induction nanoparticle of claim 1, wherein, In step S2, first dissolve MTPY in DMSO, then add an equimolar amount of CuCl2·2H2O aqueous solution, and stir the reaction at room temperature to obtain MTPY-Cu complex.

8. The tumor mitochondria-targeted copper death-selective induction nanoparticle of claim 1, wherein, In step S3, weigh albumin and dissolve it in ultrapure water, slowly add the obtained MTPY-Cu complex solution to the albumin solution, and magnetically stir for a period of time to obtain MTPY-Cu@Alb nanoparticles.

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