Tumor mitochondria targeted dual copper depletion nano sponge as well as preparation method and application thereof

Abstract

The invention discloses a tumor mitochondria targeted dual copper depletion nano sponge and a preparation method and application thereof, and relates to the technical field of biological medicines. The nanometer sponge comprises inner core nanometer particles with double copper adsorption characteristics, mitochondrial targeting molecules and cancer cell membranes, wherein the mitochondrial targeting molecules are sequentially grafted on the surfaces of the inner core nanometer particles, and the cancer cell membranes are coated on the surfaces of the inner core nanometer particles. The inner core nano-particles comprise polydopamine nano-particles and silicon dioxide nano-particles grafted on the surfaces of the polydopamine nano-particles. The inherent homing effect of a cancer cell membrane in the nano sponge and the natural mitochondrial targeting capability of TPP are utilized to compete with a compound IV (COX) on tumor mitochondria and efficiently grab copper ions in situ, so that oxidative phosphorylation is inhibited, oxidative stress is increased, and breast cancer cell energy consumption is accompanied.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a tumor mitochondria-targeted dual copper depletion nanosponge, a preparation method thereof, and an application thereof. Background Art

[0002] Triple-negative breast cancer (TNBC), accounting for about 15% - 20% of all diagnosed breast cancer cases, is characterized by high malignancy, strong invasiveness, and poor prognosis, which is a highly unmet medical need area. Therefore, improving the treatment plan for TNBC remains a current public health issue. Research has confirmed that triple-negative breast cancer cells often highly rely on oxidative phosphorylation as their energy source. Therefore, oxidative phosphorylation is an ideal broad-spectrum target for triple-negative breast cancer treatment. However, limited by compensatory glycolytic metabolism, only inhibiting oxidative phosphorylation is not enough. Since triple-negative breast cancer is less sensitive to anti-glycolytic therapies represented by blocking glucose transport, anti-angiogenic alternative therapies have attracted great attention by cutting off nutrient supply and directly relieving hypoxia. Copper ions are widely considered to be involved in the energy supply, angiogenesis, metastasis, and immune escape of cancer cells. Among them, complex IV in the electron transport in the mitochondrial respiratory chain, namely cytochrome C oxidase (COX), has a high activity dependence on copper ions. In addition, tumors and endothelial cells require copper to secrete several angiogenic factors and thus participate in proliferation and migration. Therefore, copper ions play a crucial role in angiogenesis.

[0003] Traditional chelating agents, such as tetrathiomolybdate, have the disadvantages of low copper adsorption selectivity and serious side effects caused by off-target effects. Therefore, there is an urgent need to develop a new nano-chelator-based therapeutic platform with high targeting characteristics. Summary of the Invention

[0004] Aiming at the deficiencies existing in the above background art, the present invention provides a tumor mitochondria-targeted dual copper depletion nanosponge, a preparation method thereof, and an application thereof. This nanosponge can precisely target tumor mitochondria and highly selectively adsorb Cu 2+ , enabling it to competitively plunder Cu in situ on tumor cell mitochondria 2+ , inhibit the activity of COX, a key copper-containing enzyme in oxidative phosphorylation metabolism, block the energy metabolism dependence of triple-negative breast cancer, and mediate tumor starvation therapy. At the same time, it can also effectively inhibit tumor angiogenesis, further block the delivery / metabolism of nutrients and close the tumor metastasis channel from the root, thereby achieving a safe and precise tumor dual starvation therapy effect.

[0005] The first object of the present invention is to provide a tumor mitochondria-targeted dual copper depletion nanosponge, which includes a core nanoparticle with dual copper adsorption characteristics, and a mitochondria-targeting molecule grafted in sequence on the surface of the core nanoparticle and a triple-negative breast cancer cell membrane coated thereon; The core nanoparticles include polydopamine nanoparticles and silica nanoparticles grafted onto the surface of the polydopamine nanoparticles; wherein, the silica nanoparticles are imidazole-modified organic silica nanoparticles.

[0006] Preferably, the core nanoparticles are prepared by Michael addition reaction of polydopamine nanoparticles and silica nanoparticles.

[0007] Preferably, the silica nanoparticles are prepared according to the following steps: Dissolve trisodium citrate in an aqueous solvent, add 3-mercaptopropyltriethoxysilane and N-[3-(triethoxysilyl)propyl]-4,5-dihydroimidazole, mix evenly, transfer the mixture to an autoclave, and carry out hydrothermal reaction at 180-200 °C for 12-24 h to obtain silica nanoparticles.

[0008] Preferably, the polydopamine nanoparticles are prepared according to the following steps: Disperse Igepal CO-520 in anhydrous cyclohexane, add ammonium hydroxide, mix evenly, inject an aqueous solution of DA·HCl into the reaction mixture, stir at 20 °C for 12-36 h, and precipitate PDA nanoparticles with ethanol to obtain polydopamine nanoparticles.

[0009] Preferably, the mass percentage of the polydopamine nanoparticles to the silica nanoparticles is 9:1.

[0010] Preferably, the mass ratio of the core nanoparticles to the cancer cell membrane is 2:1; The mitochondrial targeting molecules include triphenylphosphine, Szeto-Schiller peptide, mitochondrial penetrating peptide, rhodamine, cyanine or pyridinium ion; the cancer cell membrane is triple-negative breast cancer cell membrane or 4T1 tumor cell membrane.

[0011] Preferably, the nanosponge is spherical with a particle size of 80-90 nm.

[0012] The second object of the present invention is to provide a preparation method of a tumor mitochondrial targeting dual copper depletion nanosponge, comprising the following steps: Prepare core nanoparticles and formulate a core nanoparticle solution; Add an aqueous solution of mitochondrial targeting molecules to the core nanoparticle solution, and react at room temperature for 12-24 h to obtain core nanoparticles grafted with mitochondrial targeting molecules on the surface; Resuspend the core nanoparticles grafted with mitochondrial targeting molecules on the surface in an aqueous solvent, add a cancer cell membrane, and perform ultrasonic treatment to obtain a tumor mitochondrial targeting dual copper depletion nanosponge.

[0013] Preferably, the cancer cell membrane is a triple-negative breast cancer cell membrane, and the triple-negative breast cancer cell membrane is prepared according to the following steps: After MDA-MB-231 cells reach confluence, collect the cells, then wash them with pre-cooled PBS. Add a membrane protein extraction reagent containing 1% phenylmethanesulfonyl fluoride to the above solution and lyse it on ice for 15-20 min. Freeze-thaw the cell suspension with liquid nitrogen for multiple cycles, and then centrifuge. Collect the supernatant and centrifuge again to obtain the triple-negative breast cancer cell membrane.

[0014] The third object of the present invention is to provide an application of a tumor mitochondria-targeted dual copper depletion nanosponge in the preparation of a drug for dual inhibition of tumor-dependent oxidative phosphorylation and angiogenesis. Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a tumor mitochondria-targeted dual copper depletion nanosponge and its preparation method and application. The present invention uses a one-step hydrothermal method to prepare ultra-small organosilicon nanoparticles (IMS) modified with imidazole groups on the surface; subsequently, a reverse microemulsion method is used to synthesize polydopamine particles (PDA) rich in catechol groups; finally, a large number of small-sized IMS are grafted onto the surface of PDA by Michael addition reaction, and functionalization means such as TPP and tumor cell membrane are coupled on its surface to construct a nanosponge system CCM@PDA-IMS-TPP (abbreviated as CCM@PIT) with dual targeting to tumors and high-selective Cu 2+ chelating properties.

[0015] Based on the natural homing effect of tumor cell membrane and TPP and the mitochondrial targeting advantage, this Cu 2+ depletion-type nanosponge can efficiently and specifically accumulate in tumor cell mitochondria, improving the safety and effectiveness of delivery.

[0016] Based on the high-selective chelating properties of imidazole / catechol functional groups for Cu 2+ , this nanosponge can competitively and efficiently adsorb Cu on tumor cell mitochondria 2+ and inhibit the activity of the key Cu 2+ enzyme COX in oxidative phosphorylation metabolism, blocking the breast cancer-dependent energy metabolism and inducing tumor starvation damage. The competitive depletion of Cu by the nanosponge provided by the present invention 2+ will also interfere with the activity of Cu 2+ enzymes closely related to the angiogenesis pathway, blocking tumor angiogenesis and the delivery and compensatory metabolism of nutrients such as glucose, cascading and amplifying the tumor starvation treatment effect and inhibiting its metastasis.

[0017] The strategy provided by the present invention can not only overcome the off-target effects on tumor cells and mitochondria, improve the safety and effectiveness of delivery, but also competitively scavenge Cu on mitochondria 2+ , reduce the activities of key Cu 2+ -containing enzymes in oxidative phosphorylation metabolism and angiogenesis pathways, and broadly inhibit tumor-dependent energy metabolism and angiogenesis, thereby achieving effective dual tumor starvation and metastasis inhibition. It is worth mentioning that healthy cells have a lower demand for Cu 2+ . With the introduction of tumor cell membranes and TPP targeting molecules, normal cells hardly uptake this nanosponge. Therefore, this system ensures the unity of excellent biosafety and effective tumor-targeted killing. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 shows the characterization of PDA-IMS in the present invention. Among them, A is the TEM image of PDA; B is the TEM image of IMS; C is the TEM image of PDA-IMS; D is the EDS spectrum of PDA-IMS; E is the XPS spectrum of PDA-IMS; F is the thermogravimetry of PDA and PDA-IMS.

[0019] Figure 2 shows the characterization of CCM@PIT and its intermediates in the present invention. Among them, A is the Zeta potential of PDA, IMS, PDA-IMS, PIT, CCM and CCM@PIT; B is the TEM image of CCM@PIT; C is the SDS-PAGE analysis of MDA-MB-231 cell lysates, cell membranes and CCM@PIT; C is the Zeta potential of PDA, IMS, PDA-IMS, PIT, CCM and CCM@PIT; D is the DLS graph of IMS, PDA, PDA-IMS and CCM@PIT.

[0020] Figure 3 shows the verification of the copper adsorption effect in the present invention. A is the competitive metal ion adsorption of PDA, IMS and PDA-IMS; B and C are the fluorescence imaging and corresponding quantitative statistics of RhB co-incubated with different doses of PDA-IMS for 24 hours; D is the viability of MDA-MB-231 cells after being treated with different doses of metal ions and CCM@PIT for 24 hours.

[0021] Figure 4 shows the cytotoxicity of the products in the present invention. Among them, A is the cytotoxicity of PIT and CCM@PIT on MDA-MB-231 at different concentrations; B is the viability of MDA-MB-231 cells treated with different groups for 24 hours.

[0022] Figure 5Tumor cell uptake efficiency diagram of the copper-depleted nanosponge of the present invention: Among them, A is the confocal microscope image of MDA-MB-231 cells after co-incubation with PIT and CCM@PIT for 4 h and 12 h, and the cell nucleus and cytoskeleton are stained with DAPI (blue) and ActinRed TM 555 (red), respectively; B is the flow cytometry analysis diagram under the above treatment conditions; Scale bar of A: 50 µm.

[0023] Figure 6 Lysosomal escape ( Figure 6 A) and mitochondrial targeting ( Figure 6 B) CLSM diagrams of the copper-depleted nanosponge CCM@PIT of the present invention. Figure 6 The scale bar is 20 µm.

[0024] Figure 7 Apoptosis and mitochondrial damage result diagrams of the copper-depleted nanosponge of the present invention: Among them, A is the flow cytometry analysis diagram of cell apoptosis after treating MDA-MB-231 cells with different groups using AnnexinV-FITC / PI staining; B is the confocal microscope image after treating MDA-MB-231 cells with different groups using a mitochondrial membrane potential detection kit; C is the western blot diagram of apoptosis-related proteins in MDA-MB-231 cells induced by different group treatments; Scale bar of B: 30 µm.

[0025] Figure 8 Mechanism study on copper depletion-induced mitochondrial damage activated by the copper-depleted nanosponge CCM@PIT of the present invention: Figure 8 A, Figure 8 B, and Figure 8 C are the intracellular COX levels ( Figure 8 A), ATP levels ( Figure 8 B), and SOD activities ( Figure 8 C) of MDA-MB-231 cells after treatment with different groups, respectively; Figure 8 D is the confocal microscope image of intracellular ROS after treatment with different groups; Scale bar of D: 40 µm.

[0026] Figure 9 Migration ( Figure 9 A), invasion ( Figure 9 B), and tube formation ( Figure 9 C) results of the supernatant separated after co-incubating the copper-depleted nanosponge in the present invention with MDA-MB-231 for 24 h on HUVEC cells; Scale bar of A: 500 µm; Scale bar of A: 150 µm; Scale bar of A: 200 µm.

[0027] Figure 10Anti-angiogenic mechanism of the copper-depleted nanosponge of the present invention: Among them, A is the levels of pro-angiogenic factors IL-1α, IL-8, VEGF, and MMP-2 in HUVEC cells detected by an ELISA kit after the above administration; B and C are the NF-κB expressions in the nucleus and cytoplasm of HUVEC cells after the above administration observed by confocal microscopy and Western blotting, respectively.

[0028] Figure 11 Oxidative phosphorylation ( Figure 11 A) and glycolytic metabolism ( Figure 11 B) of MDA-MB-231 cells in vivo by the copper-depleted nanosponge CCM@PIT of the present invention, as analyzed by Seahorse.

[0029] Figure 12 Metabolomics analysis of MDA-MB-231 cells in vivo by the copper-depleted nanosponge CCM@PIT of the present invention: A is a bubble plot of significantly upregulated / downregulated metabolic pathways in MDA-MB-231 cells treated with CCM@PIT; B is the result of a hierarchical clustering heat map of differential metabolites between the control group and CCM@PIT.

[0030] Figure 13 In vivo biosafety of the copper-depleted nanosponge CCM@PIT of the present invention: Among them, A is the H&E staining of various organs of tumor-bearing mice; B is a graph of the body weight change of tumor-bearing mice; C is the evaluation of blood routine and liver and kidney functions of tumor-bearing mice in different treatment groups; Scale bar of A: 50 µm.

[0031] Figure 14 Evaluation of the in vivo anti-tumor effect of the copper-depleted nanosponge CCM@PIT of the present invention. Among them, A are the tumor images of different treatment groups at 0 days, 7 days, and 18 days; B and C are the survival rate and tumor volume of tumor-bearing mice.

[0032] Figure 15 H&E, TUNEL, Ki67, and CD31 immunofluorescence staining images of tumor sections of the copper-depleted nanosponge CCM@PIT of the present invention. Scale bar: 100 µm.

[0033] Figure 16 Evaluation of the in vivo anti-vascular effect of the copper-depleted nanosponge CCM@PIT of the present invention. Among them, A is the optical imaging of blood vessels in tumor-bearing mice after different treatments; B is the optical imaging of blood vessels in tumor-bearing mice treated with CCM@PIT at 0 h, 12 h, and 24 h.

[0034] Figure 17 Schematic diagram of the construction of the copper-depleted nanosponge CCM@PIT of the present invention and its mechanism of inhibiting metabolism and angiogenesis in triple-negative breast cancer. Detailed implementation manners

[0035] To enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention will be further described below in conjunction with specific embodiments and the accompanying drawings. However, the embodiments cited do not limit the present invention.

[0036] The present invention constructs a dual copper-depleting nanosponge modified with tumor cell membrane and mitochondrial targeting molecules. This nanosponge can effectively compete with COX, scavenge copper ions in situ, thereby causing oxidative phosphorylation-dependent inhibition. In addition, the copper deficiency caused by the CCM@PIT nanosponge can also significantly inhibit key copper-dependent enzymes related to angiogenesis, resulting in the blockage of internal tumor blood vessels and the normalization of peripheral blood vessels, further blocking the supply of glucose or other nutrients from the root, inhibiting energy compensation, and thus achieving effective dual starvation of tumors.

[0037] To achieve the above object, the first aspect of the present invention provides a tumor mitochondrial-targeted dual copper-depleting nanosponge, which includes a core nanoparticle with dual copper adsorption properties, and a mitochondrial targeting molecule and a coated cancer cell membrane sequentially grafted on the surface of the core nanoparticle; The core nanoparticle includes a polydopamine nanoparticle, and an organosilica nanoparticle grafted on the surface of the polydopamine nanoparticle; wherein, the organosilica nanoparticle is an imidazole-modified organosilica nanoparticle.

[0038] The nanosponge provided by the present invention is a nanosystem prepared by coupling a mitochondrial targeting molecule TPP and a triple-negative breast cancer cell membrane on the surface of a core nanoparticle containing catechol / imidazole groups with dual copper adsorption properties.

[0039] The present invention utilizes the inherent homing effect of the cancer cell membrane in the nanosponge and the natural mitochondrial targeting ability of TPP to compete with complex IV (COX) on tumor mitochondria, scavenge copper ions efficiently in situ, thereby inhibiting oxidative phosphorylation, increasing oxidative stress, and accompanying energy consumption in breast cancer cells. More importantly, the copper deficiency caused by the nanosponge can also significantly inhibit the activity of key copper-dependent enzymes related to angiogenesis, resulting in the blockage of internal tumor blood vessels and the normalization of peripheral blood vessels, further blocking the supply of glucose or other nutrients from the root, inhibiting the energy compensation involved in glycolysis, and thus achieving dual starvation therapy for tumors.

[0040] Among them, the core nanoparticle is prepared by Michael addition reaction of polydopamine nanoparticles and organosilica nanoparticles.

[0041] The silica nanoparticles are prepared according to the following steps: Dissolve trisodium citrate in an aqueous solvent, add 3-mercaptopropyltriethoxysilane and N-[3-(triethoxysilyl)propyl]-4,5-dihydroimidazole. After mixing evenly, transfer the mixture to an autoclave and carry out a hydrothermal reaction at 180-200 °C for 12-24 h to obtain the silica nanoparticles.

[0042] The polydopamine nanoparticles are prepared according to the following steps: Disperse Igepal CO-520 in anhydrous cyclohexane, then add ammonium hydroxide. After mixing evenly, inject an aqueous solution of DA·HCl into the reaction mixture and stir at 20 °C for 12-36 h. Then precipitate the PDA nanoparticles with ethanol to obtain the polydopamine nanoparticles.

[0043] The mass percentage of the polydopamine nanoparticles to the silica nanoparticles is 9:1.

[0044] The mass ratio of the core nanoparticles to the cancer cell membrane is 2:1; The mitochondrial targeting molecules include triphenylphosphine, Szeto-Schiller peptide, mitochondrial penetrating peptide, rhodamine, cyanine or pyridinium ion; The cancer cell membrane is a triple-negative breast cancer cell membrane or a 4T1 tumor cell membrane.

[0045] The nanosponge is spherical and has a particle size of 85 nm.

[0046] The second aspect of the present invention provides a method for preparing a tumor mitochondrial-targeted dual copper-depleting nanosponge, comprising the following steps: Prepare core nanoparticles and formulate a core nanoparticle solution; Add an aqueous solution of mitochondrial targeting molecules to the core nanoparticle solution and react at room temperature for 12-24 h to obtain core nanoparticles grafted with mitochondrial targeting molecules on the surface; Resuspend the core nanoparticles grafted with mitochondrial targeting molecules on the surface in an aqueous solvent, add the cancer cell membrane, and perform ultrasonic treatment to obtain the tumor mitochondrial-targeted dual copper-depleting nanosponge.

[0047] Among them, the concentration of the aqueous solution of mitochondrial targeting molecules is 10 mg / mL; The concentration of the core nanoparticle solution is 5 mg / mL; The volume ratio of the aqueous solution of mitochondrial targeting molecules to the core nanoparticle solution is 1:2; The mass ratio of the core nanoparticles grafted with mitochondrial targeting molecules on the surface to the cancer cell membrane is 2:1.

[0048] The cancer cell membrane is a triple-negative breast cancer cell membrane, and the triple-negative breast cancer cell membrane is prepared according to the following steps: After MDA-MB-231 cells reach confluence, the cells are collected, then washed with pre-cooled PBS. A membrane protein extraction reagent containing 1% phenylmethanesulfonyl fluoride is added to the above solution, and the mixture is lysed on ice for 15 - 20 min. The cell suspension is frozen and thawed with liquid nitrogen for multiple cycles, and then centrifuged. The supernatant is collected and centrifuged again to obtain the triple-negative breast cancer cell membrane.

[0049] Exemplarily, a preparation method of a tumor mitochondria-targeted dual copper depletion nanosponge includes: (1) Preparation of nanoparticles with copper depletion ability, i.e., polydopamine particles (PDA) and imidazole-modified organosilica nanoparticles (IMS); (2) Synthesis of core nanoparticles with dual copper adsorption (PDA-IMS); (3) Grafting of the mitochondria-targeting molecule TPP; (4) Preparation of a tumor mitochondria-targeted copper depletion nanosponge CCM@PIT.

[0050] Step (1) includes: 11) Synthesis of ultra-small-sized imidazole-modified organosilica copper chelating nanoparticles (IMS) by hydrothermal reaction: Dissolve 0.368 g of trisodium citrate hydrate in 8 mL of double-distilled water. Then, add 0.5 mL of 3-mercaptopropyltriethoxysilane and 1 - 2 mL of N-[3-(triethoxysilyl)propyl]-4,5-dihydroimidazole (TESDI) to the above solution under stirring for 30 min to obtain a pale yellow transparent solution. Then, transfer the mixture to an autoclave and carry out hydrothermal reaction at 180 °C for another 20 h. Subsequently, dialyze the obtained solution with a dialysis bag (MWCO = 2000) for 3 days, then filter it using a 0.22 μm filter and store it at 4 °C for future use.

[0051] 12) Synthesis of nanoparticles with copper adsorption ability, i.e., polydopamine (PDA): Inject 0.65 mL of Igepal CO-520 into 10 mL of anhydrous cyclohexane and stir for 10 minutes. Then, add ammonium hydroxide (80 μL, 28 wt%) to the above mixture, and then sonicate for 20 minutes. After stirring for another 30 minutes, inject an aqueous solution of DA·HCl (25 wt%, 7.5 - 12.5 μL) into the reaction mixture. After stirring at 20 °C for 24 hours, precipitate the PDA nanoparticles with ethanol, collect them by centrifugation (10000 rpm, 10 minutes), and wash them twice with ethanol and water. Finally, store the PDA in anhydrous ethanol.

[0052] Step (2) is specifically: Synthesis of dual copper-adsorbing nano-core PDA-IMS: Dissolve 2 mg of PDA and 6 - 8 mg of IMS in 10 mL of double-distilled water, and adjust the pH value to 9.0 with NaOH. Next, ultrasonicate the solution for 10 min, and then stir it at room temperature for 12 h. Collect the resulting product PDA-IMS by centrifugation (10 min, 10000 rpm), and wash it with double-distilled water.

[0053] Step (3) is specifically as follows: Under stirring, add 1 mL of TPP-NH2 aqueous solution (10 mg / mL) to the PDA-IMS solution (5 mg / mL, 2 mL), and react at room temperature for 24 h. Then centrifuge the mixture (12000 rpm, 10 minutes), and wash the resulting precipitate 3 times with double-distilled water, denoted as PIT.

[0054] Step (4) is specifically as follows: 41) Preparation of MDA-MB-231 tumor cell membranes: Collect cells after MDA-MB-231 reaches confluence, and then wash them twice with pre-cooled PBS. Add a membrane protein extraction reagent containing 1% phenylmethylsulfonyl fluoride (PMSF) to the above solution, and lyse it on ice for 15 minutes. Freeze-thaw the cell suspension 5 cycles with liquid nitrogen. After centrifugation at 700 g for 10 min, collect the supernatant and centrifuge it at 100000 g for 30 min to obtain tumor cell membranes.

[0055] 42) Synthesis of nano-sponge CCM@PIT: Use the ultrasonic treatment method to coat the obtained cell membranes on the surface of PIT. Briefly, resuspend the PIT obtained in step (3) in water, and then add MDA-MB-231 cancer cell membranes. Ultrasonicate the mixture on ice for 10 minutes to obtain CCM@PIT, and finally store it at -80 °C; wherein, the mass ratio of the core nanoparticles (PIT) grafted with mitochondrial targeting molecules on the surface to MDA-MB-231 cancer cell membranes is 2:1.

[0056] The present invention uses a one-step hydrothermal method to prepare ultra-small organosilica nanoparticles (IMS) modified with imidazole groups on the surface; subsequently, uses the reverse microemulsion method to synthesize polydopamine particles (PDA) rich in catechol groups; finally, uses the Michael addition reaction to graft a large number of small-sized IMS onto the surface of PDA, and couples functionalization means such as TPP and tumor cell membranes on its surface to construct a nano-sponge system CCM@PDA-IMS-TPP (abbreviated as CCM@PIT) with dual targeting of tumors and high-selectivity Cu 2+ chelating properties.

[0057] The third aspect of the present invention provides an application of a tumor mitochondrial-targeted dual copper-depleting nano-sponge in the preparation of drugs for dual inhibition of tumor-dependent oxidative phosphorylation and angiogenesis.

[0058] It should be noted that, unless otherwise specified, the experimental methods used in the present invention are all conventional methods; the reagents and materials used, unless otherwise specified, can all be purchased on the market.

[0059] Example 1 A tumor mitochondrial-targeted dual copper-depleting nanosponge, the nanosponge comprising a core nanoparticle with dual copper adsorption properties, and a mitochondrial targeting molecule grafted in sequence on the surface of the core nanoparticle and a triple-negative breast cancer cell membrane coated thereon; The core nanoparticle comprises a polydopamine nanoparticle, and an organosilica nanoparticle grafted on the surface of the polydopamine nanoparticle; wherein, the organosilica nanoparticle is an imidazole-modified organosilica nanoparticle.

[0060] The preparation process of the tumor mitochondrial-targeted dual copper-depleting nanosponge CCM@PIT comprises the following steps: (1) Preparation of nanoparticles with copper-depleting ability, polydopamine particles (PDA) and imidazole-modified organosilica nanoparticles (IMS): 11) Synthesis of ultra-small-sized imidazole-modified organosilica copper chelating nanoparticles (IMS) by hydrothermal reaction: Dissolve 0.368 g of trisodium citrate hydrate in 8 mL of double-distilled water. Then, add 0.5 mL of 3-mercaptopropyltriethoxysilane and 2 mL of N-[3-(triethoxysilyl)propyl]-4,5-dihydroimidazole (TESDI) to the above solution under stirring for 30 min to obtain a light yellow transparent solution, and then transfer the mixture to an autoclave and carry out hydrothermal reaction at 180 °C for another 20 h. Subsequently, dialyze the obtained solution with a dialysis bag (MWCO = 2000) for 3 days, then filter it using a 0.22 μm filter and store it at 4 °C for future use.

[0061] 12) Synthesis of nanoparticles with copper adsorption ability, polydopamine (PDA): Inject 0.65 mL of Igepal CO-520 into 10 mL of anhydrous cyclohexane and stir for 10 minutes, then add ammonium hydroxide (80 μL, 28 wt%) to the above mixture, and then sonicate for 20 minutes. After stirring for another 30 minutes, inject an aqueous solution of DA·HCl (25 wt%, 7.5 μL) into the reaction mixture. After stirring at 20 °C for 24 hours, precipitate the PDA nanoparticles with ethanol, collect them by centrifugation (10000 rpm, 10 minutes), and wash them twice with ethanol and water. Finally, store the PDA in anhydrous ethanol.

[0062] (2) Synthesis of dual copper adsorption nanocore PDA-IMS: Dissolve 2 mg of PDA and 6 mg of IMS in 10 mL of double-distilled water, and adjust the pH value to 9.0 with NaOH. Next, ultrasonicate the solution for 10 min, and then stir it at room temperature for 12 h. Collect the resulting product PDA-IMS by centrifugation (10000 rpm, 10 min), and wash it with double-distilled water.

[0063] (3)Grafting of mitochondrial targeting molecule TPP: Add 1 mL of TPP-NH2 aqueous solution (10 mg / mL) to the PDA-IMS solution (5 mg / mL, 2 mL) under stirring, and react at room temperature for 24 h. Then centrifuge the mixture (12000 rpm, 10 minutes), and wash the resulting precipitate 3 times with double-distilled water, denoted as PIT.

[0064] (4)Preparation of tumor mitochondrial targeting copper depletion nanosponge CCM@PIT: 41)Preparation of MDA-MB-231 tumor cell membrane: Collect cells after MDA-MB-231 reaches confluence, and then wash them twice with pre-cooled PBS. Add the membrane protein extraction reagent containing 1% phenylmethylsulfonyl fluoride (PMSF) to the above solution, and lyse it on ice for 15 minutes. Freeze-thaw the cell suspension 5 cycles with liquid nitrogen. After centrifugation at 700 g for 10 min, collect the supernatant and centrifuge it at 100000 g for 30 min to obtain the tumor cell membrane.

[0065] 42)Synthesis of nanosponge CCM@PIT: Use the ultrasonic treatment method to coat the obtained cell membrane on the surface of PIT. Briefly, resuspend the PIT obtained in step (3) in water, and then add the MDA-MB-231 cancer cell membrane. Ultrasonicate the mixture on ice for 10 minutes to obtain CCM@PIT, and finally store it at -80 °C; wherein, the mass ratio of PIT to MDA-MB-231 cancer cell membrane is 2:1.

[0066] In the nanosponge prepared in this example, the mass percentage of polydopamine nanoparticles to the silica nanoparticles is 9:1.

[0067] Use modern nano-test and analysis techniques such as transmission electron microscopy (TEM), EDS, XPS, thermogravimetry, DLS and Zeta potential analyzer, SDS-PAGE gel electrophoresis, etc. to systematically study the morphology and composition of the products obtained in each step of Example 1, and the results are as follows: The present invention proves the successful construction of PDA-IMS by TEM, EDS, XPS, and thermogravimetry.

[0068] See Figure 1As shown in A and B, the TEM results show that they are PDA and IMS respectively, with uniform spherical structures and particle sizes. The particle sizes are approximately 45 nm and 5 nm respectively.

[0069] See Figure 1 As shown in C, for PDA-IMS obtained after grafting IMS on the surface of PDA, its size significantly increases to approximately 58 nm, demonstrating the successful introduction of IMS.

[0070] See Figure 1 As shown in D and E, detected by energy dispersive spectroscopy and X-ray photoelectron spectroscopy respectively, the unique Si and S elements of IMS are also shown on the surface of PDA-IMS, confirming the successful synthesis of PDA-IMS.

[0071] See Figure 1 As shown in F, according to the thermogravimetric curve analysis of PDA and PDA-IMS, the grafting efficiency of IMS in PDA-IMS is calculated to be approximately 8.86%.

[0072] The CCM@PIT obtained in the examples was characterized by TEM, DLS and Zeta potential analyzer, and SDS-PAGE gel electrophoresis in the present invention.

[0073] See Figure 2 As shown in A, the mitochondrial targeting molecule TPP was grafted onto the above-mentioned PDA-IMS by Michael addition reaction and denoted as PIT. The Zeta potential results show that the surface charge of PIT increases from 24.96 mV to 31.74 mV, demonstrating the successful grafting of TPP.

[0074] See Figure 2 As shown in B, the cell membrane of MDA-MB-231 was extracted and covered on the surface of PIT to form the final copper-depleting nanosponge CCM@PIT. The TEM results show an obvious thin film on the surface of CCM@PIT, and the size of TEM increases to about 85 nm, which is Figure 2 consistent with the DLS data shown in C, demonstrating the successful coating of the cell membrane. In addition, the surface potential of CCM@PIT becomes negatively charged like that of the cell membrane, attributed to the successful coating of the tumor cell membrane. See Figure 2 As shown in D, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) determination shows that the protein expression of CCM@PIT is similar to that of MDA-MB-231 cell lysates and cell membranes, once again demonstrating the successful encapsulation of the cell membrane. The above results all demonstrate the successful preparation of the copper-depleting nanosponge CCM@PIT.

[0075] Experimental Example 2 Evaluation of the in vitro copper adsorption effect of the tumor mitochondrial-targeted dual copper depletion nanosponge CCM@PIT provided in Example 1.

[0076] In the present invention, the excellent dual copper adsorption effect of the core PDA-IMS is demonstrated by metal ion competitive adsorption and copper-specific probe RhB fluorescence quantification, and the in vitro copper chelation characteristics of the nanosponge are detected by metal ion supplementation experiments. The specific results are as follows: 1) Evaluation of the copper adsorption effect of the copper depletion nanosponge core PDA-IMS To study the excellent dual copper adsorption effect of the nanosponge core PDA-IMS, the selective chelation behavior of the nanosponge towards copper ions was first studied by adding various essential elements in HEPES buffer (pH = 7.4). The results are as Figure 3 shown in Figure A, PDA, IMS, and PDA-IMS can selectively absorb copper more than other metal ions. In addition, the copper adsorption effect of PDA-IMS is significantly greater than that of PDA and IMS alone, demonstrating the stronger copper chelation ability of PDA-IMS.

[0077] Secondly, the copper 2+ specific fluorescent probe RhB was used to further demonstrate the copper chelation ability of the nanosponge. Cu 2+ can bind to RhB and exhibit obvious fluorescence. The results are as Figure 3 shown in Figures B and C, under the same conditions, PDA-IMS significantly reduced the fluorescence intensity, which is attributed to the copper ion competitive chelation induced by PDA-IMS, further confirming the copper adsorption characteristics of PDA-IMS.

[0078] 2) In vitro copper chelation effect of the copper depletion nanosponge CCM@PIT The "metal supplementation" experiment was used to co-incubate MDA-MB-231 cells with CCM@PIT with or without various metal ions to demonstrate the key effect of copper depletion on cell viability. The results show that CCM@PIT can effectively kill tumor cells. When copper ions are provided additionally, the cell viability can be restored in a dose-dependent experiment, while the supplementation of other metal ions has no obvious effect, indicating the key role of Cu 2+ in cell proliferation, and the Cu 2+ chelation achieved by the nanosponge CCM@PIT alleviates the cytotoxicity of tumors. The above results together reveal that the CCM@PIT nanosponge has excellent copper chelation ability, which is beneficial to achieving good anti-tumor effects.

[0079] Experimental Example 3 In vitro biological evaluation of the tumor mitochondrial-targeted dual copper depletion nanosponge CCM@PIT provided in Example 1.

[0080] (1) Cell culture Cell culture: Human breast cancer cells (MDA-MB-231) and human umbilical vein endothelial cells (HUVEC) provided by the Institute of Zoology, Chinese Academy of Sciences were selected in this invention. MDA-MB-231 and HUVEC cells were cultured in high-glucose DMEM medium containing 10% serum and 1% double antibiotics (100 μg / mL penicillin and 100 μg / mL streptomycin) in a cell incubator at 37 °C and 5% CO2.

[0081] (2) Cell viability detection To evaluate the in vitro tumor cell cytotoxicity of nano sponge CCM@PIT, the CCK-8 method was used in this invention to detect the effects of PDA, IMS, PDA-IMS, PIT, and CCM@PIT on the viability of MDA-MB-231 cells, respectively. First, MDA-MB-231 cells were seeded on 48-well plates. When the cell confluence reached about 60-70%, MDA-MB-231 cells were co-cultured with the above drugs for 24 h. Subsequently, the cells were washed and incubated with fresh medium containing CCK-8 for 1 h, and the ultraviolet absorbance at a wavelength of 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The results are shown in Figure 4 Figure A of Figure 4 As shown in Figure B of

[0082] (3) In vitro tumor cell uptake To further investigate the cell uptake efficiency, MDA-MB-231 cells were seeded in confocal dishes. When the cell confluence reached 60-70%, TPP and CCM@PIT were labeled with FITC and co-cultured with them for 4 and 12 h, and the endocytosis of CCM@PIT was visualized using confocal laser scanning microscopy (CLSM) and flow cytometry (FCM). The results are shown in Figure 5 Figure A of Figure 5 As shown, MDA-MB-231 cells showed time-dependent uptake behavior towards PIT and CCM@PIT nano sponges, which benefited from the strong positive charge of PIT and the introduction of tumor cell membranes. Meanwhile, the uptake amount of CCM@PIT by tumor cells was significantly higher than that of PIT, demonstrating the first-order targeting effect achieved by tumor cell membranes due to the homing effect. In addition, as shown in

[0083] (4)Lysosomal escape and mitochondrial targeting effect To study the subcellular distribution of the nanosponge, MDA-MB-231 cells seeded on confocal microscope dishes were allowed to grow to 60-70%. Subsequently, FITC-labeled CCM@PIT and the lysosomal fluorescent dye LysotrackerRed or the mitochondrial fluorescent dye MitotrackerRed were added to the above mixture. After co-incubation at 37 °C for different time intervals, the cells were washed and imaged by CLSM. The results are as Figure 6 shown in Figure A. After co-incubation for 1 hour, most of the green fluorescent-labeled CCM@PIT was distributed at the edges of MDA-MB-231 cells, indicating that the cells were about to endocytose; the green fluorescence of CCM@PIT actually overlapped with the red-labeled lysosomes, and when the incubation time was extended to 4 h, the green fluorescence of CCM@PIT overlapped with the red-labeled lysosomes, showing obvious yellow fluorescence, indicating that the lysosomes successfully captured CCM@PIT; when it was 8 hours, CCM@PIT successfully escaped from the lysosomes, manifested as the separation of red fluorescence and green fluorescence, indicating that CCM@PIT could successfully escape from the lysosomes. As Figure 6 shown in Figure B, after co-incubation for 1 hour, most of the FITC-labeled CCM@PIT was distributed around the cells; partial and complete co-localization of the green fluorescent CCM@PIT and the red fluorescent mitochondria were achieved at 4 h and 8 h of co-incubation, respectively, demonstrating the good mitochondrial targeting ability of TPP.

[0084] (5)In vitro tumor cell apoptosis To study the in vitro tumor killing effect of the dual copper-depleting nanosponge CCM@PIT with tumor mitochondrial targeting, the apoptosis levels of MDA-MB-231 cells induced by CCM@PIT were analyzed by flow cytometry using the AnnexinV-FITC / PI apoptosis kit, the mitochondrial membrane potential kit, and Western blotting protein immunoblotting method. Specifically as follows: 1) Detection by AnnexinV-FITC / PI flow cytometer To investigate the apoptosis level of tumor cells, the apoptosis ratio of MDA-MB-231 cells induced by different experimental groups was quantitatively detected using the AnnexinV-FITC / PI staining kit and FCM. The specific experimental procedure was as follows: MDA-MB-231 cells were seeded in 6-well plates and cultured in PBS, PDA, IMS, PDA-IMS, PIT, and CCM@PIT for 24 hours. Subsequently, the apoptosis degree of the cells was detected using the AnnexinV-FITC / PI cell apoptosis detection kit according to the manufacturer's instructions and detected by FCM. The results are as Figure 7As shown in Figure A, compared with the control, PDA-IMS showed higher cytotoxicity than free PDA and IMS, which was attributed to the excellent dual copper adsorption ability of PDA-IMS. In addition, due to the primary and secondary targeting of tumor cell membranes and TPP, PIT and CCM@PIT induced stronger apoptosis, and showed the highest apoptosis rate on CCM@PIT, which demonstrated the excellent in vitro anti-tumor efficiency of the nanosponge.

[0085] 2) Detection of mitochondrial membrane potential To further explore the activation of the mitochondrial damage pathway triggered by copper depletion, the membrane-permeable JC-1 probe was used to measure the mitochondrial damage level and membrane potential changes: MDA-MB-231 cells seeded on confocal microscope dishes were co-incubated with PBS, PDA, IMS, PDA-IMS, PIT and CCM@PIT for 12 hours, the cells were washed with PBS and treated with a mitochondrial membrane potential kit. Finally, the above cell samples were detected by CLSM. The ratio of red / green fluorescence was used to detect the changes in mitochondrial membrane potential and damage status. The results are as Figure 7 shown in Figure B. After 12 hours of co-incubation, the red fluorescence of different treatment groups gradually decreased and the green fluorescence gradually increased relative to the control, indicating depolarization of the mitochondrial membrane potential. Among them, the red / green fluorescence ratio induced by the CCM@PIT nanosponge was the weakest, indicating that CCM@PIT induced the most severe mitochondrial damage.

[0086] 3) Western blotting The expression of Bcl-2 family-related genes plays an important role in the tumor cell apoptosis signal transduction pathway. Among them, the expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax was used to characterize the apoptosis status of tumor cells. To further study the apoptosis mechanism mediated by mitochondrial damage, MDA-MB-231 cells were co-incubated with PBS, IMS, PDA, PDA-IMS, PIT and CCM@PIT for 24 h, and then the cells were lysed and protein samples were collected by centrifugation. Western blotting was used to detect and analyze the expression of Bax, Bcl-2, Cyt-C and Caspase-3. The results are as Figure 7 shown in Figure C. Compared with the control group, different treatment groups induced an upregulation of Bax / Bcl-2, Cyt-C, and cleaved Caspase-3 in the order of PDA or IMS < PDA-IMS < PIT < CCM@PIT, indicating the successful activation of the apoptosis pathway caused by mitochondrial damage. Due to the modification of the mitochondrial targeting molecule TPP and the cancer cell membrane, CCM@PIT induced the highest level of tumor cell apoptosis. These results indicate that the CCM@PIT nanosponge can significantly activate the apoptosis pathway mediated by mitochondrial damage in vitro.

[0087] (6)Mitochondrial damage mechanism of tumor cells The present invention demonstrates the mitochondrial damage mechanism induced by copper depletion of CCM@PIT by measuring the activities of COX enzyme, ATP and SOD enzyme and intracellular ROS detection in MDA-MB-231 cells. The specific results are as follows: First, MDA-MB-231 in a 6-well plate with a density of 60%-70% was incubated with PBS, IMS, PDA, PDA-IMS, PIT and CCM@PIT for 24 h, then centrifuged and collected, washed 3 times with PBS, and the cells were treated with a mitochondrial isolation kit to obtain mitochondria. After protein quantification, 5 μg of mitochondrial protein was used to measure COX activity. The enzyme activity was calculated by detecting the decrease in absorbance of cytochrome C at 550 nm. The results are as Figure 8 shown in Figure A. Compared with other treatment groups, CCM@PIT had the strongest effect on reducing COX activity, indicating that CCM@PIT nanosponges can effectively induce copper depletion-activated mitochondrial damage by regulating the upstream COX in the oxidative phosphorylation metabolic pathway.

[0088] Second, MDA-MB-231 cells were seeded on a 6-well plate, cultured with the above-mentioned drugs for 24 h, the cells were lysed and centrifuged to separate, the protein concentration of the supernatant was measured, and then the intracellular ATP content was detected according to the protocol of the instruction manual. The results are as Figure 8 shown in Figure B. The copper depletion nanosponge CCM@PIT reduced the content of ATP in the cells.

[0089] The typical copper-dependent enzyme superoxide dismutase (SOD) plays a key role in maintaining intracellular oxidative stress, which is another key regulator upstream of the mitochondrial damage pathway. To further detect the activity of intracellular SOD enzyme, MDA-MB-231 cells seeded on a 6-well plate were cultured with the above-mentioned drugs for 24 hours. After centrifuging and collecting the cells, SOD sample preparation solution was added to the above mixture to lyse the cells. After lysis, the samples were centrifuged and the supernatant was measured using a BCA protein concentration assay kit, and the SOD enzyme activity was measured and calculated using an SOD activity detection kit. The results are as Figure 8 shown in Figure C. The activity of SOD enzyme in MDA-MB-231 cells in all experimental groups decreased, and the CCM@PIT group showed the strongest inhibitory effect, confirming the inhibition of copper depletion on SOD enzyme activity.

[0090] To further verify the disruption of redox homeostasis caused by copper depletion, MDA-MB-231 cells inoculated on confocal microscope culture dishes were grown to 60 - 70% and treated with the above-mentioned drug administration for 12 hours. After co-incubating the cells with the DCFH-DA probe for 30 minutes according to the instructions, the cells were washed and fluorescence was imaged by CLSM. The results are as Figure 8 shown in Panel D of Figure 8 . The green fluorescence intensity of the DCFH-DA probe method gradually increased in MDA-MB-231 cells, demonstrating that the inactivation of SOD enzyme also disrupted the balance of redox homeostasis and increased the ROS level in tumor cells, which further strengthened the mitochondrial damage pathway.

[0091] (7) Anti-angiogenic mechanism in vitro The present invention evaluated the anti-angiogenic effect of nano-sponge CCM@PIT using wound healing, invasion, angiogenesis, ELISA and nuclear translocation experiments of NF-κB. The specific results are as follows: HUVEC cells (8×10 5 cells / well) inoculated in 6-well plates were incubated for 24 hours. When the cells grew into a monolayer, a linear wound was created in the middle of the culture well through the tip of a p200 pipette. Subsequently, the supernatants of MDA-MB-231 cells treated with PBS, IMS, PDA, PDA-IMS, PIT and CCM@PIT for 24 hours were co-incubated with HUVEC cells for 24 h, the cells were washed, and imaged by microscope. The results are as Figure 9 shown in Panel A of Figure 9 . The cell scratch in the control group almost disappeared, indicating that tumor cells inherently have strong healing ability. In contrast, the CCM@PIT group showed the highest anti-healing ability, attributed to the fact that copper deficiency affects the motility of HUVEC cells.

[0092] For the invasion experiment, a suspension of HUVEC cells with a density of 10 5 cells was added to the upper chamber of the invasion chamber containing Matrigel in a pre-hydrated 24-well plate, and then the supernatant medium obtained from the above-mentioned drug administration was added for co-incubation. At the same time, a medium containing 10% serum was added as a chemoattractant to the lower chamber. After incubating for 24 hours, non-invasive cells were removed by scrubbing with a cotton swab, and invasive cells were stained with crystal violet. Finally, the number of cells was counted under a microscope, with at least 4 views per well. The results are as Figure 9 shown in Panel B of Figure 9 . The invasion experiment also showed a similar trend to the above-mentioned scratch healing experiment. Among them, the CCM@PIT group had the strongest inhibitory effect on the invasion of HUVEC cells. The results showed that copper depletion caused by nano-sponge CCM@PIT could effectively inhibit the metastasis of HUVEC cells.

[0093] To investigate the anti-angiogenic effect of copper-depleted nanosponge CCM@PIT, Matrigel was added to 24-well plates and incubated overnight at 4 °C and then solidified at 37 °C for 1 hour. Next, HUVEC (1×10 4 cells / well) cells were cultured on the matrix with 500 μL of supernatant medium obtained from the above administration, and after further incubation for 6 hours, the cell tubes were imaged using a microscope. The results are as shown in Figure 9 Panel C. In the control group, HUVEC cells formed a unique tubular network in the plate, indicating their natural tube-forming ability. However, in the CCM@PIT group, most of the tubes remained open, significantly inhibiting the tube-forming ability, further indicating that copper chelation can effectively inhibit angiogenesis.

[0094] Since the Cu 2+ -binding protein IL-1α and the downstream factor NF-κB play important roles in angiogenesis and invasion by regulating the expression of pro-angiogenic factors (including VEGF, IL-8, and MMP-2). To further clarify the mechanism of the nanosponge in inhibiting angiogenesis, HUVEC cells seeded on 6-well plates were cultured with the supernatant medium containing 10% serum obtained from the above administration. After incubation for 24 hours, the medium was extracted, centrifuged to obtain the supernatant, and ELISA kits were used to measure the levels of interleukin-1α (IL-1α), vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and matrix metalloproteinase-2 (MMP-2). The results are as shown in Figure 10 Panel A. The expressions of the pro-angiogenic factors IL-1α, VEGF, IL-8, and MMP-2 in HUVEC cells were greatly reduced, demonstrating that copper depletion induced by the nanosponge CCM@PIT can inhibit angiogenesis.

[0095] To demonstrate the nuclear translocation of NF-κB in HUVEC cells, HUVEC cells were seeded in confocal microscope dishes and allowed to grow to 60 - 70% and then the cells were treated with the above administration for 24 hours. Subsequently, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% TritonX-100, and blocked with 5% BSA, and then the cells were incubated with an antibody against NF-κB (P65) and stained with Coralite-594 secondary antibody. Finally, the cells were incubated with DAPI to stain the nuclei and imaged by confocal microscopy. The results are as shown in Figure 10 Panel B. NF-κB in the treatment group translocated from the cytoplasm to the nucleus, demonstrating a decrease in the expression of the upstream factor IL-1α induced by copper chelation, and again demonstrating that copper chelation can inhibit angiogenesis-related factors to normalize the blood vessels around the tumor.

[0096] To quantitatively investigate the nuclear translocation of HUVEC cells, nuclear and cytoplasmic protein extraction kits were used to extract the nuclear and cytoplasmic proteins in HUVEC, and the expression level of NF-κB protein was detected by a Western blotting system. Histone-3 and β-actin were used to normalize the results of nuclear and cytoplasmic proteins, respectively. The results are as Figure 10 shown in Figure C of Figure 10 , which is consistent with the confocal results of NF-κB. The above results indicate that copper deficiency induced by CCM@PIT nanosponges can actually induce anti-angiogenesis closely related to tumor growth by regulating IL-1α and NF-κB nuclear translocation and the expression of VEGF, IL-8, and MMP-2, thereby enhancing the anti-tumor effect of the nanosponge CCM@PIT.

[0097] Experimental Example 4 In vivo Seahorse and metabolomics analysis of the tumor mitochondria-targeted dual copper depletion nanosponge CCM@PIT provided in Example 1.

[0098] The present invention uses Seahorse and metabolomics to detect the effect of the nanosponge CCM@PIT on in vivo metabolism. The specific results are as follows: (1) Construction of a tumor-bearing mouse model Tumor-bearing MDA-MB-231 mice were established, and 100 μL of normal saline, IMS, IMS, PDA, PDA-IMS, PIT, and CCM@PIT with a concentration of 50 mg / kg PDA-IMS equivalent were intravenously injected. The treatment was administered once a day for 3 days. Next, MDA-MB-231 cells were extracted from the tumor-bearing MDA-MB-231 mice for subsequent experiments.

[0099] (2) In vivo Seahorse analysis The present invention used a Seahorse 24XFe extracellular flux analyzer to measure the real-time changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Primary MDA-MB-231 cells were seeded in a standard XF24 plate, and the cells were cultured in XF basal medium at 37 °C and incubated for 1 hour under CO2-free conditions. For OCR measurement, oligomycin (1 μM), FCCP (1 μM), and rotenone / antimycin A (0.5 μM) were injected at the designated time points, respectively. For ECAR detection, glucose (0.1 μM), oligomycin (10 μM), and 2-DG (0.5 μM) were added, respectively. For all analyses, the results were normalized using a BCA protein assay. The results are as Figure 11As shown in Figure A, compared with the control, the OCR values of PDA-IMS, PIT, and CCM@PIT decreased, demonstrating effective inhibition of oxidative phosphorylation metabolism, which can be attributed to copper depletion reducing COX activity and thus inhibiting the electron transfer respiratory chain in oxidative phosphorylation metabolism. Among all groups, CCM@PIT showed the highest degree of oxidative phosphorylation inhibition and energy blockade. As Figure 11 shown in Figure B, the glycolysis levels of the treatment groups decreased significantly in the order of IMS or PDA < PDA-IMS < PIT < CCM@PIT, demonstrating a reduction in glycolysis and energy cut-off. The glycolysis inhibition of CCM@PIT is attributed to competitive copper depletion, which can inhibit angiogenesis around tumors by regulating the IL-1α and NF-κB-related pathways.

[0100] (3) In vivo metabolomics analysis In this experiment, targeted metabolomics analysis was used to determine the effects of nanosponges on tumor cell metabolic pathways and metabolites in vivo. After extracting MDA-MB-231 cells from MDA-MB-231 tumor-bearing mice, they were treated with PBS and CCM@PIT for 24 h. Subsequently, the cells were washed with pre-cooled PBS, collected with a cell scraper, and frozen in liquid nitrogen. Finally, metabolomics experiments were conducted by Beijing Biotree Co., Ltd. The results are as Figure 12 shown in Figure A, oxidative phosphorylation metabolism in the CCM@PIT group was effectively inhibited, which is attributed to CCM@PIT causing chelation of copper in COX, resulting in interruption of the electron transfer respiratory chain and blockade of oxidative phosphorylation. In addition, enrichment analysis of metabolic pathways also demonstrated that CCM@PIT significantly inhibited glycolytic metabolism and its downstream tricarboxylic acid cycle, which is attributed to the anti-angiogenic effect caused by copper depletion in CCM@PIT hindering glucose delivery and thus blocking subsequent energy metabolism. As Figure 12 shown in Figure B, compared with the control group, the contents of metabolites related to oxidative phosphorylation (such as NAD and NADH), glycolytic products (such as phosphoenolpyruvate and dihydroxyacetone phosphate), and key intermediates of the tricarboxylic acid cycle (such as malate and succinate) in the CCM@PIT group decreased, further demonstrating that copper depletion caused by CCM@PIT inhibited the electron transfer respiratory chain and angiogenesis, thus inhibiting oxidative phosphorylation, glycolysis, and tricarboxylic acid cycle metabolism. The above in vivo metabolomics analysis clearly shows that with the help of mitochondrial-targeted precise copper consumption, CCM@PIT can be efficiently targeted to mitochondria, effectively inhibit the metabolism of oxidative phosphorylation by reducing COX activity, while blocking angiogenesis and cutting off nutrient delivery at the root to inhibit glycolysis, achieving the effect of dual starvation therapy for tumors.

[0101] Experimental Example 5 Evaluation of the in vivo anti-tumor and anti-angiogenic effects of the tumor mitochondrial-targeted dual copper depletion nanosponge CCM@PIT provided in Example 1.

[0102] 1) Construction of a tumor-bearing mouse model for in vivo anti-tumor effect All animal experiments in this invention were conducted in accordance with the relevant regulations of the Laboratory Animal Management and Use Committee Work Manual. Female Balb / c nude mice aged 5 - 6 weeks and weighing about 20 g were purchased from the Beijing Institute for Drug Control, China. In this invention, a 100 μL cell suspension was prepared using MDA-MB-231 cells in the exponential growth phase, and the suspension was subcutaneously inoculated under the right groin of each mouse to establish a tumor-bearing mouse model. When the average tumor size reached about 50 mm 3 , the mice were randomly divided into six groups according to weight and injected intravenously with normal saline, IMS, PDA, PDA-IMS, PIT, and CCM@PIT, twice a week for 18 days. The health status and behavior of the mice were observed daily, and the body weight and tumor volume of the tumor-bearing mice were recorded every 2 days. The tumor volume (V) was calculated according to the following formula: V = L × S 2 / 2 (L, the long diameter of the tumor; S, the short diameter of the tumor). After the administration ended, the mice were euthanized. The hearts, livers, spleens, lungs, kidneys, and tumors of the mice were collected for histopathological analysis by H&E, TUNEL, Ki-67, CD31, and HIF-1α staining.

[0103] 2) Biological safety evaluation As shown in Figure A of Figure 13 , CCM@PIT did not cause obvious damage to the main organs of the mice. At the same time, as shown in Figure B of Figure 13 , there was no significant difference in the body weight of the mice in all treatment groups compared with that of the control group during the administration period, further confirming the good biological safety of CCM@PIT.

[0104] In addition, normal saline and CCM@PIT were intravenously injected into non-tumor-bearing mice. After treatment, mouse blood was collected from the orbital socket, and the whole blood samples of the mice were collected into anticoagulation tubes and subjected to routine blood tests using a blood test analyzer. In addition, after the whole blood was incubated at room temperature for 4 h, serum was extracted from the whole blood by centrifugation at 1000 g for 15 min to measure the levels of liver / kidney function indicators. The results are shown in Figure 13 Figure C of

[0105] 3) Anti-tumor effect After the mice were sacrificed, the main organs and tumor tissues were collected. The results are shown in Figure 14As shown in Figure A, the PDA-IMS group and the PIT group showed slight and moderate inhibitory effects on tumor growth, respectively. Among all treatment groups, the CCM@PIT group induced the most significant inhibitory effect on tumor growth, indicating that the copper depletion therapy induced by CCM@PIT can efficiently kill tumors. As Figure 14 shown in Figure B, the statistical results of tumor volume confirmed the same trend of tumor growth inhibition. In addition, the results Figure 14 shown in Figure C, compared with other treatment groups, the CCM@PIT group significantly prolonged the survival time of tumor-bearing mice, which was significantly higher than that of other treatment groups, indicating that CCM@PIT has excellent anti-tumor effects.

[0106] 4) Histopathological analysis To reveal the good anti-tumor effects of CCM@PIT at the tissue and molecular levels, tumor tissues were sectioned and immunofluorescently stained. The results Figure 15 are shown in the figure. In the H&E results, the CCM@PIT group induced the largest number of apoptotic tissues, showing nuclear shrinkage and darker color. In addition, the CCM@PIT group had the largest number of pink fluorescent dots formed by the co-localization of Cy3-labeled fragmented DNA and DAPI-labeled cell nuclei, and the smallest number of proliferation-related antigen ki67 labeled with green fluorescence, which again confirmed the strongest anti-tumor effect of CCM@PIT in vivo, attributed to the tumor starvation damage induced by dual copper depletion resulting in blocked energy metabolism and inhibited angiogenesis. In addition, the in vivo angiogenesis inhibitory effect was studied. The immunofluorescence intensity of CD31 in the control group was the strongest, indicating tumor angiogenesis, while the blood vessels of the treated mice became sparse and narrower. It is worth noting that CCM@PIT induced the most obvious angiogenesis inhibitory effect, which was due to superior tumor targeting and dual copper chelation. The fluorescence intensity labeled by the HIF-1α probe in the CCM@PIT group was the weakest, demonstrating that CCM@PIT-mediated vascular normalization can effectively block the glucose supply of tumors and reverse the hypoxic state around tumors.

[0107] 5) Anti-angiogenesis effect in vivo In the present invention, a photoacoustic imaging system was used to image the tumors and their surrounding blood vessels in mice. After the average size of the tumors in tumor-bearing mice inoculated with MDA-MB-231 cells on the back reached about 50 mm 3 , the drug was administered once a day for 7 consecutive days, and then the blood vessel density around the tumors was detected using a photoacoustic microscope. In addition, for the acute effect, after the average size of the tumors reached about 50 mm 3 , CCM@PIT was injected every 6 h for treatment, and the blood vessel density was observed at 0 h, 12 h, and 24 h. The results Figure 16As shown in Figure A, subcutaneous blood vessel imaging of mice verified the high inhibition of tumor angiogenesis by the nanosponge CCM@PIT, manifested as a significant decrease in blood vessel density. As Figure 16 As shown in Figure B, the results of the time-dependent experiment further confirmed the acute anti-angiogenic effect of CCM@PIT. The above results indicate that the dual copper depletion induced by CCM@PIT caused significant damage to angiogenesis, which in turn led to the inhibition of tumor-dependent energy metabolism and had excellent anti-tumor effects in vivo.

[0108] Figure 17 It is a schematic diagram of the construction of the copper-depleted nanosponge CCM@PIT of the present invention and its inhibition of metabolism and angiogenesis in triple-negative breast cancer. As shown in the figure, the present invention constructs a nanosponge with dual copper chelating effects, and couples the mitochondrial targeting molecule TPP and breast cancer cell membrane on the surface, so that it can accurately target the mitochondria of breast cancer cells to deplete copper, thereby blocking oxidative phosphorylation metabolism and angiogenesis, starving and killing breast cancer doubly and inhibiting its metastasis.

[0109] Example 5 Same as Example 1, except that the nanosponge prepared from 4T1 tumor cell membrane was used.

[0110] Preparation of 4T1 tumor cell membrane: Collect cells after 4T1 is confluent, and then wash twice with pre-cooled PBS. Add a membrane protein extraction reagent containing 1% phenylmethylsulfonyl fluoride (PMSF) to the above solution and lyse on ice for 15 minutes. The cell suspension was frozen and thawed 5 cycles with liquid nitrogen. After centrifugation at 700g for 10 min, the supernatant was collected and centrifuged at 100,000g for 30 min to obtain tumor cell membrane.

[0111] Synthesis of 4T1 cell membrane-coated nanosponge CCM-B@PIT: The obtained cell membrane was coated on the surface of PIT by sonication. Briefly, PIT was resuspended in water, and then 4T1 cancer cell membrane was added. The mixture was sonicated on ice for 10 minutes to obtain CCM-B@PIT, and finally stored at -80 °C.

[0112] In summary, the present invention developed a nanosponge CCM@PIT with dual copper chelating ability and coated with mitochondrial targeting molecules and tumor cell membranes, and verified its therapeutic effect against triple-negative breast cancer. This nanosponge can accurately target the mitochondria of triple-negative breast cancer cells, efficiently chelate copper ions like leeches, directly inhibit oxidative phosphorylation metabolism by robbing the copper of COX on the electron transfer respiratory chain, activate the mitochondrial damage pathway, and induce tumor starvation by amplifying oxidative stress. Importantly, competitive copper chelation can also effectively block root angiogenesis and compensatory glycolytic metabolism by directly cutting off the delivery of nutrients, thereby achieving a dual starvation treatment effect.

[0113] This application solves the following problems: (1) Solving the problem of low adsorption capacity of traditional copper chelators: The copper adsorption core PDA-IMS of the nanosponge combines catechol groups derived from PDA and a large number of imidazole groups derived from IMS, achieving dual copper absorption characteristics, and thus chelating copper ions selectively and efficiently like leeches.

[0114] (2) Solving the problems of insufficient targeting and poor safety in in vivo delivery of chelators: Triphenylphosphine (TPP) is a well-known lipophilic cation that can achieve intracellular mitochondrial targeting effects; the autologous tumor cell membrane coating can achieve tumor targeting through the natural homing effect. The nanosponge CCM@PIT is surface-coupled with TPP and autologous tumor cell membranes, and by virtue of the homing effect and mitochondrial targeting advantages, it specifically accumulates in the mitochondria of tumor cells, thereby enhancing the therapeutic effect and reducing side effects, and improving the safety and effectiveness of delivery.

[0115] (3) Solving the compensatory metabolism caused by the inhibition of oxidative phosphorylation: The nanosponge CCM@PIT can compete with COX, efficiently scavenging copper ions in situ, thereby causing oxidative phosphorylation-dependent inhibition, activation of the mitochondrial damage pathway caused by increased oxidative stress, and concomitant energy consumption of breast cancer cells. However, due to the limitation of compensatory glycolytic metabolism, single oxidative phosphorylation inhibition is insufficient. Moreover, the copper deficiency caused by the CCM@PIT nanosponge can also significantly inhibit angiogenesis-related factors, resulting in the occlusion of internal tumor blood vessels and the normalization of peripheral blood vessels, thereby further blocking the supply of glucose or other nutrients from the root, and inhibiting the energy compensation involved in glycolysis.

[0116] This invention describes the preferred embodiments and their effects. However, those skilled in the art can make additional changes and modifications to these embodiments once they learn the basic creative concepts. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of this invention.

[0117] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A tumor mitochondrial-targeted dual copper-depleted nanosponge, characterized in that: The nanosponge includes a core nanoparticle with dual copper adsorption characteristics, and a mitochondrial targeting molecule and a coated cancer cell membrane sequentially grafted onto the surface of the core nanoparticle; The core nanoparticles include polydopamine nanoparticles and silicon dioxide nanoparticles grafted on the surface of the polydopamine nanoparticles; wherein the silicon dioxide nanoparticles are imidazole-modified organic silicon dioxide nanoparticles.

2. The tumor mitochondria-targeted dual copper-depleted nanosponge according to claim 1, characterized in that: The core nanoparticles are prepared by Michael addition reaction of polydopamine nanoparticles and silicon dioxide nanoparticles.

3. The tumor mitochondria-targeted dual copper-depleted nanosponge according to claim 2, characterized in that: The silicon dioxide nanoparticles are prepared according to the following steps: dissolving trisodium citrate in a water solvent, adding 3-mercaptopropyltriethoxysilane and N-[3-(triethoxysilyl)propyl]-4,5-dihydroimidazole, mixing evenly, transferring the mixture to an autoclave, and performing a hydrothermal reaction at 180-200° C. for 12-24 hours to obtain silicon dioxide nanoparticles.

4. The tumor mitochondria-targeted dual copper-depleted nanosponge according to claim 2, characterized in that: The polydopamine nanoparticles are prepared according to the following steps: Igepal CO-520 was dispersed in anhydrous cyclohexane, and then ammonium hydroxide was added. After mixing evenly, a DA·HCl aqueous solution was injected into the reaction mixture. After stirring at 20°C for 12 to 36 hours, PDA nanoparticles were precipitated with ethanol to obtain polydopamine nanoparticles.

5. The tumor mitochondria-targeted dual copper-depleted nanosponge according to claim 1, characterized in that: The mass ratio of the polydopamine nanoparticles to the silicon dioxide nanoparticles is 9:

1.

6. The tumor mitochondria-targeted dual copper-depleted nanosponge according to claim 1, characterized in that: Said The mass ratio of the core nanoparticles to the cancer cell membrane is 2:1; The mitochondrial targeting molecule includes triphenylphosphine, Szeto-Schiller peptide, mitochondrial penetrating peptide, rhodamine, cyanine or pyridinium ion; The cancer cell membrane is a triple-negative breast cancer cell membrane or a 4T1 tumor cell membrane.

7. The tumor mitochondria-targeted dual copper-depleted nanosponge according to claim 1, characterized in that: The nano sponge is spherical and has a particle size of 80-90 nm.

8. A method for preparing the tumor mitochondria-targeted dual copper-depleted nanosponge according to any one of claims 1 to 7, characterized in that: The following steps are involved: preparing core nanoparticles and preparing a core nanoparticle solution; The mitochondrial targeting molecule aqueous solution is added to the core nanoparticle solution, and the reaction is carried out at room temperature for 12 to 24 hours to obtain the core nanoparticles with the mitochondrial targeting molecule grafted on the surface; The inner core nanoparticles with mitochondrial targeting molecules grafted on the surface are resuspended in an aqueous solvent, cancer cell membranes are added, and ultrasonic treatment is performed to obtain a tumor mitochondrial targeted dual copper depletion nanosponge.

9. The method for preparing the tumor mitochondria-targeted dual copper-depleted nanosponge according to claim 8, characterized in that: The cancer cell membrane is a triple-negative breast cancer cell membrane, and the triple-negative breast cancer cell membrane is prepared according to the following steps: After MDA-MB-231 cells have grown to full size, the cells are collected and then washed with pre-cooled PBS. A membrane protein extraction reagent containing 1% phenylmethylsulfonyl fluoride is added to the above solution and lysed on ice for 15-20 minutes. The cell suspension is frozen and thawed with liquid nitrogen for multiple cycles, and after centrifugation, the supernatant is collected and centrifuged again to obtain triple-negative breast cancer cell membranes.

10. Use of the tumor mitochondria-targeted dual copper-depleted nanosponge according to any one of claims 1 to 7 in the preparation of a drug for dual inhibition of tumor-dependent oxidative phosphorylation and angiogenesis.

Images