PH / GSH dual-response copper silicate-based nano system for synergistically treating breast cancer

Abstract

The invention belongs to the technical field of tumor treatment, and particularly discloses a pH / GSH dual-response copper silicate-based nano system for synergistic treatment of breast cancer, the pH / GSH dual-response copper silicate-based nano system comprises a CuSiO3 nano carrier and molybdenum-based polyoxometallate, the molybdenum-based polyoxometallate is loaded on the CuSiO3 nano carrier, the loading concentration of the molybdenum-based polyoxometallate is 0.2-1.0 mg / mL, and the loading concentration of the molybdenum-based polyoxometallate is 0.2-1.0 mg / mL. And adding molybdenum-based polyoxometallate into the dispersion liquid of the CuSiO3 nano carrier, stirring at room temperature, and centrifuging to obtain the copper silicate-based nano system modified by the molybdenum-based polyoxometallate. The pH / GSH dual-response copper silicate-based nano system for synergistically treating the breast cancer shows a synergistic effect in the aspects of photothermal therapy and induction of copper death and iron death, effectively inhibits tumor cells and improves the treatment efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, and particularly to a pH / GSH dual-responsive copper silicate-based nanosystem for synergistic treatment of breast cancer. Background Art

[0002] The tumor microenvironment (TME) has some unique physicochemical characteristics different from normal tissues, mainly manifested in: (1) Weak acidity. The pH of the internal environment of normal cells is 7.2 - 7.4, while the pH of most TME (tumor microenvironment) is 5.5 - 6.5. This is mainly due to abnormal metabolism of tumor cells, resulting in lactic acid accumulation. (2) Overproduction of hydrogen peroxide (H2O2). The level of H2O2 in tumor cells (5 μM - 1 mM) is significantly higher than that in normal cells (less than 0.7 μM). And H2O2 plays an important role in tumorigenesis and growth. (3) Excessive glutathione (GSH). Usually, the concentration of GSH in cancer cells is 2 - 10 mM, much higher than that in normal cells. As an important antioxidant in cells, GSH can effectively neutralize excessive reactive oxygen species (ROS) and protect tumor cells from damage caused by oxidative stress. Therefore, the unique properties of the tumor microenvironment pose new challenges for tumor treatment, and also provide opportunities for new treatment strategies.

[0003] Although copper silicate nanomaterials have shown good effects in tumor treatment, the treatment mechanism relying solely on copper silicate nanomaterials may not be able to meet the complex and variable nature of the tumor microenvironment. When further exploring strategies to improve the tumor treatment effect, the scheme of integrating a copper silicate nanoplatform with nanomaterials responsive to the tumor microenvironment into the same nanosystem shows excellent potential. Among them, molybdenum-based polyoxometalates (POM) as a typical acid-responsive material can form large aggregates in acidic TME through proton-induced hydrogen bonds, which is beneficial for its long-term retention in tumor tissues through the enhanced permeability and retention effect (EPR effect). In addition, the high level of GSH in TME triggers the conversion of Mo 6+ to Mo 5+ . Due to the charge transfer between valence states, the reduced Mo 5+ significantly enhances the near-infrared absorption of POM, making the POM clusters a promising photothermal conversion agent under the tumor microenvironment. In addition, Mo 5+ can also react with H2O2 through the Russel mechanism to generate cytotoxic 1 O2. Based on the above, it is speculated that the TME-responsive nanosystem integrating POM and copper silicate is expected to overcome the limitations of single copper silicate nanomaterials and play the synergistic role of multiple mechanisms, so as to achieve more comprehensive and efficient tumor treatment.

[0004] Therefore, a pH / GSH dual-responsive POM-modified CuSiO3 intelligent nanosystem (CuSiO3@POM) was designed to explore the synergistic therapeutic effect mediated by CuSiO3@POM. Summary of the Invention

[0005] The object of the present invention is to provide a pH / GSH dual-responsive copper silicate-based nanosystem for the synergistic treatment of breast cancer, which demonstrates a synergistic effect in photothermal therapy, as well as inducing cuproptosis and ferroptosis, effectively inhibiting tumor cells and improving the treatment efficiency.

[0006] To achieve the above object, the present invention provides a pH / GSH dual-responsive copper silicate-based nanosystem for the synergistic treatment of breast cancer, which includes a CuSiO3 nanocarrier and a molybdenum-based polyoxometalate. The molybdenum-based polyoxometalate is loaded on the CuSiO3 nanocarrier, and the loading concentration of the molybdenum-based polyoxometalate is 0.2 - 1.0 mg / mL. The molybdenum-based polyoxometalate is added to the dispersion of the CuSiO3 nanocarrier, stirred at room temperature, and the molybdenum-based polyoxometalate-modified copper silicate-based nanosystem is obtained by centrifugation.

[0007] Preferably, the loading concentration of the molybdenum-based polyoxometalate is 0.2 mg / mL.

[0008] Preferably, the preparation method of the CuSiO3 nanocarrier is as follows:

[0009] A. Synthesis of Cu2O: Dissolve Cu(NO3)2 and NH4NO3 in deionized water, add the NaOH solution at a rate of 1.8 mL / min, continue stirring, add the ascorbic acid solution at a rate of 0.6 mL / min, stir again, centrifuge to collect the product Cu2O, wash it with ethanol, and disperse it in ethanol;

[0010] B. Synthesis of Cu2O@mSiO2: Dilute the above Cu2O dispersion in a mixed solution of ethanol and deionized water, sequentially add a 25 wt% aqueous solution of cetyltrimethylammonium chloride and triethylamine diluted 10 times with ethanol, add tetraethyl orthosilicate, stir the mixture at room temperature, collect the solid Cu2O@mSiO2 by centrifugation and washing, and redisperse it in ethanol;

[0011] C. Synthesis of CuSiO3: Add 2-methylimidazole and ethanol to the Cu2O@mSiO2 dispersion, stir at room temperature, centrifuge and wash twice with ethanol to obtain CuSiO3, and redisperse the obtained CuSiO3 in ethanol for use.

[0012] Preferably, in step C, the concentration of 2-methylimidazole is 0.2 g / mL.

[0013] Preferably, in step C, CuSiO3 is a layered CuSiO3 nanoflower support.

[0014] Preferably, the preparation method of the molybdenum-based polyoxometalate is as follows:

[0015] Add (NH4)6Mo7O 24 ·4H2O to ultrapure water, stir at room temperature for 1 h, then quickly add NaH2PO4, stir, and finally add ethanol, centrifuge, wash, and dry to obtain the molybdenum-based polyoxometalate.

[0016] Preferably, the molybdenum-based polyoxometalate has a spherical structure in a neutral solution with a pH of 7.4, and aggregates into clusters under acidic conditions with a pH of 5.5, showing pH responsiveness.

[0017] The advantages and beneficial effects of the pH / GSH dual-responsive copper silicate-based nanosystem for synergistically treating breast cancer in the present invention are as follows:

[0018] 1. CuSiO3@POMs designed in the present invention, as a tumor microenvironment-responsive nanosystem, demonstrates a synergistic effect in photothermal therapy, as well as inducing cuproptosis and ferroptosis, effectively inhibiting tumor cells and improving the treatment efficiency.

[0019] 2. Under 808 nm laser irradiation, CuSiO3@POM in the present invention exhibits good photothermal conversion performance under weakly acidic and GSH conditions, and the photothermal conversion efficiency is 38%. The detection results of in vitro reactive oxygen species and glutathione consumption show that after glutathione reduction, CuSiO3@POM generates ·OH and + O2 through the Cu 5+ -mediated Fenton-like reaction and the Mo 1 -catalyzed Russel mechanism respectively, significantly increasing the level of reactive oxygen species and effectively consuming glutathione, and the consumption is positively correlated with the reaction time and concentration.

[0020] 3. In vivo treatment in the present invention shows that CuSiO3@POM injected into MCF-7 tumor-bearing nude mice effectively accumulates in the tumor region, has biosafety, and the CuSiO3@POM + Laser treatment shows a significant anti-tumor effect, with a tumor inhibition rate of 72.9%.

[0021] 4. In the present invention, Cu2O is used as a copper source, and an amorphous mesoporous SiO2 layer is coated on the outside to form a Cu2O@mSiO2 core-shell structure as a sacrificial template. The concentration of 2-methylimidazole is determined to be 0.2 g / mL, and layered CuSiO3 nanoflowers are prepared. The synthesized CuSiO3 nanoflowers have a relatively high specific surface area of 1370 m 2 / g.

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Brief Description of the Drawings

[0023] Figure 1 It is the TEM image and particle size distribution statistical chart of POM at different pH values of the present invention. Among them, (a) is the TEM image and particle size distribution statistics of POM at pH = 7.4, (b) is the TEM image and particle size distribution statistics of POM at pH = 5.5, (c) is the ultraviolet absorption spectrum, and (d) is the photothermal heating curve under 808 nm laser irradiation;

[0024] Figure 2 It is the TEM images of CuSiO3@POM at different pH values (pH = 7.4 and 5.5) of the present invention. Among them, (a) is the TEM image of CuSiO3@POM at pH = 7.4, (b) is the TEM image of CuSiO3@POM at pH = 5.5, (c) is the dynamic light scattering (DLS) distribution map, and (d) is the EDX mapping map of CuSiO3@POM;

[0025] Figure 3 It is the photothermal performance test diagram of the present invention. Among them, (a) is the picture of the CuSiO3@POM dispersion liquid with or without the addition of GSH at different pH values, (b) is the photothermal heating curve (808 nm, 1W cm -1 ), (c) is the temperature change curve of heating and natural cooling, (d) is the graph and linear fitting of the cooling time and -ln(θ), (e) is the heating-cooling cycle curve, and (f) is the infrared thermal imaging diagram of the CuSiO3@POM dispersion liquid changing with time;

[0026] Figure 4 It is the in vitro ROS and GSH consumption detection diagram of the present invention. Among them, (a) is the mechanism diagram of CuSiO3@POM generating ROS, (b) is the XPS photoelectron energy spectrum diagram of Mo 3d, (c) is the XPS photoelectron energy spectrum diagram of Cu 2p, (d) is the time-dependent DPBF absorption curve of the reaction between CuSiO3@POM and H2O2, (e) is the corresponding relative content of DPBF, (f) is the SOSG fluorescence detection of the generated 1 O2, (g) is the ESR spectrum detection of TEMP capturing 1 O2, (h) is the UV-Vis spectrum of the MB solution under different treatments, and (i) is the ESR spectrum verification of ·OH generation;

[0027] Figure 5This is the cytotoxicity verification detection diagram of the present invention. Among them, (a) is the determination of GSH consumption ability, (b) is the corresponding quantitative analysis, (c) is the evaluation of the viability of MCF-7 cells after incubation with CuSiO3@POM by the CCK-8 method, (d) is the CLSM image of MCF-7 cells stained with Calcein-AM and PI under different treatments, (e) is the analysis of apoptosis of MCF-7 cells under different treatments, (f) is the CLSM image of cells stained with DCFH-DA dye after different treatments, and (g) is the CLSM image of cells stained with SOSG dye after different treatments;

[0028] Figure 6 This is the diagram of CuSiO3@POM of the present invention conjugated with FITC green fluorescent probe for tracking cell uptake. Among them, (a) is the cell uptake after incubation for 2, 6, and 12 h, and (b) is the DNA damage in MCF-7 after different treatments;

[0029] Figure 7 This is the immunostaining result and CLSM image of CuSiO3@POM of the present invention. Among them, (a) is the oligomerization of DLAT protein in MCF-7 cells after different treatments, (b) is the CLSM image of MCF-7 cells stained with C11-BODIPY581 / 591 dye, (c) is the protein expression of MCF-7 cells after different treatments, and (d) is the change of mitochondrial membrane potential of MCF-7 cells after different treatments;

[0030] Figure 8 This is the diagram of different differential genes of the control group and the CuSiO3@POM+Laser group obtained by RNA-seq analysis of the present invention. Among them, (a) is the heat map, (b) is the volcano map, (c) is the GO enrichment analysis of different genes after CuSiO3@POMs+Laser treatment, (d) is the up-regulated genes, and (e) is the down-regulated genes;

[0031] Figure 9 This is the diagram for evaluating the in vivo therapeutic effect and biosafety of CuSiO3@POM of the present invention. Among them, (a) is the establishment of the MCF-7 tumor model and the anti-tumor treatment plan, (b) is the in vivo fluorescence imaging of MCF-7 tumor-bearing mice after intravenous injection of CuSiO3@POM loaded with Cy5 (the blue dotted area indicates the tumor site) and the in vitro fluorescence imaging 24 h after injection, (c) is the infrared thermal imaging of tumor-bearing mice injected with PBS and CuSiO3@POM respectively under 808 nm laser irradiation, (d) is the tumor photos of different groups after 14 days of treatment, (e) is the change of tumor volume of different groups during treatment, (f) is the change of body weight of mice in different groups during 14 days of treatment, and (g) is the H&E staining, TUNEL staining, and GPX4 immunohistochemical analysis of tumor tissues after different treatments;

[0032] Figure 10 These are the staining diagrams of tumor tissues and major organs collected from different groups of mice in the present invention. Among them, (a) is the H&E staining of major organs, and (b) is the DCFH staining of tumor sections of each group. Detailed implementation mode

[0033] The technical solution of the present invention will be further described below with reference to the drawings and examples.

[0034] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the field to which the present invention belongs.

[0035] Unless otherwise defined, the reagents, equipment and other materials used in the present invention are all obtained from conventional commercial sources.

[0036] Example 1

[0037] The pH / GSH dual-responsive copper silicate-based nanosystem for the synergistic treatment of breast cancer demonstrates a synergistic effect in photothermal therapy, as well as inducing cuproptosis and ferroptosis, effectively inhibiting tumor cells and improving the treatment efficiency. It includes a CuSiO3 nanocarrier and a molybdenum-based polyoxometalate (POM). The molybdenum-based polyoxometalate is loaded on the CuSiO3 nanocarrier, and the loading concentration of the molybdenum-based polyoxometalate is 0.2 - 1.0 mg / mL. The molybdenum-based polyoxometalate is added to the dispersion of the CuSiO3 nanocarrier, stirred at room temperature, and the molybdenum-based polyoxometalate-modified copper silicate-based nanosystem (CuSiO3@POM) is obtained by centrifugation.

[0038] The loading concentration of the molybdenum-based polyoxometalate (POM) is 0.2 mg / mL.

[0039] The preparation method of the CuSiO3 nanocarrier is as follows:

[0040] A. Synthesis of Cu2O: Dissolve 0.1 mmol of Cu(NO3)2 and 0.12 mmol of NH4NO3 in 40 mL of deionized water. Then, add 2.0 mL of NaOH (0.2 M) solution at a specific speed of 1.8 mL / min. After continuing to stir for 10 min, add 3.5 mL of ascorbic acid (0.1 M) solution at a specific speed of 0.6 mL / min, and stir for another 10 min. After the reaction is completed, collect the product Cu2O by centrifugation, wash it with ethanol, and disperse it in 10 mL of ethanol. -1 Add 3.5 mL of ascorbic acid (0.1 M) solution at a specific speed of 0.6 mL / min. Stir for another 10 min. After the reaction is completed, collect the product Cu2O by centrifugation, wash it with ethanol, and disperse it in 10 mL of ethanol.

[0041] B. Synthesis of Cu2O@mSiO2: Dilute the above-mentioned Cu2O dispersion in a mixture of 20 mL of ethanol and 10 mL of deionized water. Subsequently, add 0.16 mL of an aqueous solution of 25 wt% cetyltrimethylammonium chloride (CTAC) and 0.15 mL of triethylamine (TEA) diluted 10 times with ethanol in sequence. After adding 50 μL of tetraethyl orthosilicate (TEOS), stir the mixture at room temperature for 14 h. Collect the solid Cu2O@mSiO2 by centrifugation and washing, and redisperse it in 10 mL of ethanol.

[0042] C. Synthesis of CuSiO3: Add 2 mL of 2-methylimidazole (2-Melm) with a concentration of 0.2 g / mL and 4.5 mL of ethanol to 1 mL of the Cu2O@mSiO2 dispersion. Stir at room temperature for 14 h. After centrifugation and washing twice with ethanol, redisperse the obtained CuSiO3 in 10 mL of ethanol for use.

[0043] CuSiO3 is a layered CuSiO3 nanoflower carrier.

[0044] Cu2O nanospheres were synthesized by the room-temperature ascorbic acid reduction method. During the synthesis process, NH4NO3 was added to inhibit the rapid growth of the Cu(OH)2 intermediate phase and enable the Cu2O crystals to grow in a size-controlled manner. By uniformly growing a mesoporous SiO2 shell on the Cu2O core, a Cu2O@mSiO2 core-shell structure was formed, where the thickness of the mesoporous SiO2 shell was approximately 22 nm. Subsequently, the effect of the amount of 2-MeIm on the morphological evolution of Cu2O@mSiO2 was studied. As the concentration of 2-MeIm increased (0.02 - 0.04 g / mL), the Cu2O core was gradually etched, and the Cu2O@mSiO2 core-shell structure transformed into a hollow sphere. When the amount of 2-MeIm continued to increase to 0.08 g / mL, the nanoflower structure began to appear. When the amount of 2-MeIm reached 0.2 g / mL, the Cu2O@mSiO2 with a core-shell structure was completely transformed into a uniform nanoflower-like structure (i.e., CuSiO3), indicating that 2-MeIm plays a key role in regulating the phase transition from the core-shell structured Cu2O@mSiO2 to the hollow-structured CuSiO3.

[0045] The preparation method of the molybdenum-based polyoxometalate is as follows:

[0046] Add 4 mmol of (NH4)6Mo7O 24 ·4H2O to 20 mL of ultrapure water, stir at room temperature for 1 h, then quickly add 10 mL of NaH2PO4 with a concentration of 2.34 mmol, stir for 15 min, and finally add 80 mL of ethanol, centrifuge, wash, and dry to obtain the molybdenum-based polyoxometalate (POM).

[0047] The molybdenum-based polyoxometalate exhibits a spherical structure in a neutral solution with a pH of 7.4, and aggregates into clusters under acidic conditions with a pH of 5.5, showing pH responsiveness.

[0048] Example 2

[0049] Perform performance tests on the synthesized CuSiO3@POM.

[0050] 1. Synthesis and characterization of CuSiO3@POM.

[0051] First, synthesize POM clusters according to a simple method. Transmission electron microscope (TEM) images show ( Figure 1 in (a) and (b)), POM exhibits a spherical structure, with uniform size in a neutral solution (pH = 7.4), and an ultra-small diameter of 2.7 ± 0.6 nm. When the pH is reduced to 5.5, the POM clusters aggregate into large clusters (with a diameter of 4.5 ± 0.8 nm). This phenomenon demonstrates the pH-responsive assembly of POM, attributed to acid-induced protonation of POM. In addition, POM in the UV-vis spectrum shows a near-infrared absorption ability with enhanced reducibility and acidity in the NIR-I (700 - 900 nm) window ( Figure 1 in (c)), endowing it with the potential for photothermal therapy. Correspondingly, the reduced POM exhibits excellent photothermal properties. Under 808 nm laser irradiation for 10 min, the temperature of the POM aqueous solution increases by approximately 28 °C and 19 °C at pH = 7.4 and 5.5, respectively ( Figure 1 in (d)).

[0052] As shown in Figure 2 (a) and (d), the synthesized CuSiO3@POM shows high dispersibility in a neutral solution. Elemental mapping shows the uniform distribution of POM in the CuSiO3 nanoflower structure. This high-density distribution is conducive to the acid-induced aggregation of the entire nanosystem. In the simulated weakly acidic tumor environment (pH = 5.5), TEM images show that CuSiO3@POM undergoes severe aggregation, and the hydrodynamic particle size distribution also increases from a center of 534.5 nm to 1206.4 nm ( Figure 2 in (b) and (c)). This aggregation of CuSiO3@POM is conducive to its long-term retention in the tumor, thus enhancing the EPR effect. Subsequently, the optical properties of CuSiO3@POM under physiological conditions were studied. The UV-vis spectrum shows that similar to individual POM, CuSiO3@POM exhibits enhanced near-infrared absorption with GSH reduction and acid response.

[0053] 2. Photothermal performance test.

[0054] Figure 3As shown in (a), when the CuSiO3@POM solution was incubated with GSH overnight, the color gradually changed to blue, and the blue color was deeper in an acidic environment than in a neutral environment. This phenomenon can be attributed to the more frequent redox reactions between Mo ions caused by the acid-triggered aggregation of CuSiO3@POM. Next, the photothermal conversion performance of CuSiO3@POM was systematically studied. As Figure 3 shown in (b)-(e), after 10 min of irradiation with an 808 nm laser, the temperature of the reduced acidic CuSiO3@POM solution increased by about 22 °C, which was higher than 17 °C under neutral conditions. As for the unreduced CuSiO3@POM solution, the temperature change was basically the same in acidic and neutral solutions, increasing by about 11 °C. This is because the CuSiO3 nanocarrier itself also has a certain near-infrared absorption. Then, the photothermal conversion efficiency η of CuSiO3@POM was calculated to be 38% based on the temperature change curves of heating and natural cooling under 808 nm laser irradiation. In addition, CuSiO3@POM after reaction with GSH under the condition of pH = 5.5 showed excellent photostability even after 5 laser irradiations. The thermal imaging diagram in the figure also provided direct evidence ( Figure 3 in (f)), proving that the designed CuSiO3@POM can be used for pH / GSH dual-responsive photothermal therapy (PTT) treatment.

[0055] 3. Detection of in vitro ROS and GSH consumption.

[0056] In the XPS spectrum of the reduced CuSiO3@POM (ReCuSiO3@POM), the increase in Cu + and the appearance of Mo 5+ ( Figure 4 in (b) and (c)) confirmed the redox properties of CuSiO3@POM. The cyclic voltammogram also confirmed that CuSiO3@POM had reversible oxidation / reduction peaks, which enabled CuSiO3@POM to respond well to H2O2 or GSH. Next, the ability of CuSiO3@POM to generate ROS was explored. As Figure 4 shown in (a), the mechanism of ROS generation by CuSiO3@POM can be explained as follows: After being taken up by cancer cells, the acidic intracellular environment gradually degrades the aggregated CuSiO3@POM structure, releasing Cu 2+ and POM. The reducing intracellular environment reduces Mo 6+ and Cu 2+ to Mo 5+ and Cu + respectively, where Mo 5+ catalyzes the decomposition of H2O2 to generate 1 O2 through the Russell mechanism driven by the POM structure, and Cu +Then, the Fenton-like reaction catalyzes the generation of hydroxyl radicals (·OH) from H2O2. The generation of singlet oxygen ( 1 O2) and hydroxyl radicals (·OH) rapidly increases the ROS level.

[0057] First, DPBF and the singlet oxygen fluorescence probe (SOSG) were used to explore the 1 O2 generation respectively. Due to the 1 oxidation of DPBF by 1 O2, the ultraviolet absorption peak at 415 nm will decrease. Therefore, the level of Figure 4 O2 can be verified by the change in the absorbance of DPBF. As shown in (d) of Figure 4 , when ReCuSiO3@POM reacts with H2O2, the absorbance of DPBF gradually decreases with the prolongation of the reaction time. At 240 min, the content of DPBF has dropped to 37% of the original ( Figure 4 (e)), proving that 1 O2 is continuously generated during the reaction. 1 The generation of 1 O2 is specifically explained by the Russell mechanism involving the tetraoxide intermediate (ROOOOR): POM-Mo in CuSiO3@POM 5+ will react with H2O2 to form this tetraoxide, which further decomposes to produce 1 O2. The singlet oxygen sensor green (SOSG) fluorescence probe is also often used to detect 1 O2 due to its high selectivity for 1 O2. In the presence of 1 O2, SOSG can emit green fluorescence. As shown in (f) of Figure 4 , the increase in the SOSG fluorescence signal at 525 nm clearly indicates that ReCuSiO3@POM reacts with H2O2 to generate 1 O2, and with the prolongation of the reaction time, the level of 1 O2 increases.

[0058] To confirm the Cu + -mediated Fenton-like reaction, MB was selected as an indicator, and the generated ·OH was determined based on the degradation of MB. As shown in (h) of Figure 4 , in GSH and HCO 3-In the presence of CuSiO3@POM, after reacting with the MB-H2O2 solution for 24 h, the absorbance of MB decreased significantly. After irradiation with an 808 nm laser, the generation efficiency of ·OH increased significantly, and the absorbance decreased to nearly 0, which was due to the enhanced generation efficiency of ROS caused by the increase in temperature induced by photothermal effect. In addition, it was found that the degradation of MB showed time dependence. As the reaction time extended, the absorbance of MB gradually decreased until it almost completely disappeared after 24 h of reaction, indicating that CuSiO3@POM could promote the generation of ·OH and had excellent Fenton-like activity. Further verification by ESR 1 confirmed the generation of Figure 4 O2 and ·OH ( 1 in (g) and (i)), using TEMP and DMPO as spin trapping reagents respectively. During the reaction of ReCuSiO3@POM with H2O2, 6+ -Mo 5+ the generation of 1 O2 corresponded to the triplet feature in the ESR spectrum with a relative intensity of 1:1:1. The presence of ·OH corresponded to the characteristic quartet with a relative intensity of 1:2:2:1. The above results indicated that due to the redox reaction between GSH and CuSiO3@POM and the

[0059] reversible transformation of -1 Mo Figure 5 as shown in (a) and (b) of 2+ Cu 6+ , the content of GSH decreased with the increase of reaction time. At 60 min of reaction, about 79.2% of GSH had been lost. The above results showed that the consumption of GSH was positively correlated with the reaction time and the concentration of CuSiO3@POM.

[0060] 4. Cytotoxicity detection.

[0061] The standard CCK-8 method was used to study the in vitro cytotoxicity of CuSiO3@POM. Human cerebral microvascular endothelial cells (hCMEC / D3) were incubated with different concentrations of CuSiO3@POM for 24 h. Even when the concentration of CuSiO3@POM was as high as 200 μg / mL, the cell viability of hCMEC / D3 cells remained above 80%, indicating that CuSiO3@POM has good biocompatibility. It is worth noting that the CuSiO3@POM+Laser treatment had very little toxicity to hCMEC / D3, and the cell viability only decreased by about 10%. On the one hand, it may be because the temperature induced by CuSiO3@POM+Laser was not sufficient to kill normal cells. On the other hand, normal cells are less sensitive to the heat caused by laser compared to cancer cells. However, when human breast cancer cells (MCF-7) were incubated with different concentrations of CuSiO3@POM for 24 h, the cell viability decreased significantly ( Figure 5 in (c)). At a concentration of 200 μg / mL, the cell survival rate decreased to less than 40%. The obvious toxicity of CuSiO3@POM to MCF-7 cells can be attributed to the fact that in the reducing environment and high H2O2 conditions of cancer cells, CuSiO3@POM triggered the Cu + -mediated Fenton-like reaction and the Russell mechanism reaction participated by Mo 5+ , generating ·OH and 1 O2 respectively, which caused a ROS storm. At the same time, the toxicity of MCF-7 cells incubated with CuSiO3@POM was further enhanced under the irradiation of 808 nm laser (1 W cm -2 ), indicating that the photothermal effect of CuSiO3@POM further enhanced the killing ability to MCF-7 cells. In addition, the cell viability under different treatments was also evaluated by double staining with a live / dead cell double staining kit (Calcein-AM / PI) and imaging with a laser confocal scanning microscope (CLSM) ( Figure 5 in (d)). Strong red fluorescence emitted by PI in MCF-7 was observed in the CuSiO3@POM+Laser group, indicating that almost all cells were dead, which was consistent with the cytotoxicity results measured by the CCK-8 method. Flow cytometry analysis of cell death was performed on MCF-7 cells treated differently using an apoptosis detection kit (annexinV-FITC) and PI staining ( Figure 5(e)). Annexin V is a protein that binds to phosphatidylserine (PS). In the early stage of apoptosis, PS flips from the inner side of the cell membrane to the outer side, and AnnexinV-FITC can bind to the exposed PS on the outer side, showing green fluorescence. PI is a dye that cannot penetrate the intact cell membrane of living cells or early apoptotic cells, but can enter late apoptotic and necrotic cells with damaged membranes, bind to DNA and emit red fluorescence. After CuSiO3@POM + Laser treatment, nearly 64.2% of MCF-7 cells underwent apoptosis. The above results together illustrate that the strategy of CuSiO3@POM + Laser can cause satisfactory anti-cancer effects in MCF-7 cells.

[0062] 5. Detection of intracellular ROS.

[0063] The fluorescence probe (DCFH-DA) was used as the ROS fluorescence probe to measure the intracellular ROS levels of MCF-7 cells treated differently. As Figure 5 shown in (f), the green fluorescence signal in the CuSiO3@POM group was significant, indicating a significant increase in ROS levels. 808 nm laser irradiation (1 W cm -2 , 10 min) could further promote ROS generation, as shown by the strongest green fluorescence in the CuSiO3@POM + Laser group, which was attributed to the accelerated reaction rate of ROS production due to the increased temperature. In addition, the obvious green fluorescence of SOSG in the cells also supported the 1 O2 generation mediated by CuSiO3@POM ( Figure 5 shown in (g)). CuSiO3@POM generated 5+ Mo in the reduction reaction with intracellular GSH, and thus reacted with intracellular H2O2 through the Russell mechanism to produce effective 1 O2. The cell uptake experiment can evaluate the enrichment of nanodrugs in cells, thus providing a reference for optimizing the tumor treatment effect. CuSiO3@POM was conjugated with the FITC green fluorescence probe to track cell uptake. As Figure 6 shown in (a), compared with the control group, obvious green fluorescence was observed in the cytoplasm of MCF-7 cells after 2 h of incubation, proving that CuSiO3@POM was successfully taken up by the cells. And the uptake behavior of MCF-7 cells showed time dependence. As the incubation time was extended to 12 h, the green fluorescence signal around the blue cell nucleus stained with DAPI solution was significantly enhanced. Since the ROS storm oxidizes proteins and lipids in cancer cells and damages DNA, γ-H2AX immunofluorescence staining was used to measure the DNA damage levels of different treatment groups ( Figure 6 shown in (b)). Obviously, the cells treated with CuSiO3@POM plus 808 nm laser showed the most serious DNA damage.

[0064] 6. Copper death / ferroptosis synergistic cell death mechanism.

[0065] Inspired by the excellent in vitro anti-tumor efficacy of CuSiO3@POM, its mechanism of action was attempted to be elucidated. Copper death is a new and regulated form of cell death, characterized by the excessive accumulation of copper ions in the body, the aggregation of lipoylated dihydrolipoamide S-acetyltransferase (DLAT), and the loss of Fe-S cluster proteins. Usually, excessive Cu2+ ions in cells are reduced to Cu+ by ferredoxin 1 (FDX1), and then Cu+ binds to lipoylated DLAT, leading to its aggregation. At the same time, Cu+ also causes the loss of Fe-S cluster proteins. These abnormal phenomena together trigger the toxic stress of proteins, ultimately leading to cell death. In addition, in the context of copper death, GSH can bind to Cu+ to form a stable chelate, hindering the occurrence of copper death. Therefore, it can be inferred that the increase in intracellular Cu + concentration and the consumption of endogenous GSH may trigger copper death in cells. In view of this, it was further evaluated whether CuSiO3@POM could effectively induce copper death in MCF-7 cells. The results of immunofluorescence staining showed ( Figure 7 in (a) below), no obvious DLAT foci were seen in the control group and the Laser group, while obvious DLAT foci were observed in MCF-7 cells after treatment with CuSiO3@POM, indicating the obvious oligomerization of DLAT. In addition, the results of immunoblotting experiments also showed obvious DLAT oligomers in cells treated with CuSiO3@POM, and at the same time, the expression of FDX1, the upstream regulator of DLAT lipoylation, was also inhibited. The FDX1 level in the CuSiO3@POM+Laser group was 0.19 times that of the control group ( Figure 7 in (c) below). The above results indicate that CuSiO3@POM can effectively induce copper death in cells.

[0066] It has been previously demonstrated that intracellular CuSiO3@POM significantly induces a ROS storm, and the burst of ROS promotes lipid peroxidation (LPO), which is one of the typical characteristics of ferroptosis. Therefore, the ferroptosis performance was further studied. LPO can be detected by C11-BODIPY 581 / 591 fluorescent probe because it has high sensitivity to LPO in cell membranes. C11-BODIPY 581 / 591 shows red fluorescence under normal conditions, and when LPO occurs, the fluorescence of C11-BODIPY 581 / 591 changes from red to green. The CLSM images of C11-BODIPY 581 / 591 cells after incubation with CuSiO3@POM are as Figure 7As shown in (b). Obviously, the cells incubated with CuSiO3@POM showed bright green fluorescence and dim red fluorescence, indicating a high level of LPO. Notably, after laser irradiation, the green fluorescence was enhanced in the CuSiO3@POM+Laser group, which was due to the increased ROS level promoted by photothermal effect deepening the degree of LPO. In addition, the expression of glutathione peroxidase 4 (GPX4) was significantly downregulated in the cells treated with CuSiO3@POM+Laser, being 0.39 times that of the control group ( Figure 7 as shown in (c)), which is another typical feature of ferroptosis. GPX4 is an important antioxidant enzyme that can catalyze the reduction of highly toxic LOOH to non-toxic hydroxy compounds (LOH) in the presence of GSH. The reaction of CuSiO3@POM with intracellular GSH depleted GSH, resulting in a decrease in GPX4 activity, thus leading to the accumulation of cytotoxic lipid peroxides (LOOH), and further causing ferroptosis of cells. In addition, it was also found that apoptosis was also involved in CuSiO3@POM-induced cell death, because the expression level of the key apoptotic protein caspase-3 was significantly upregulated in the CuSiO3@POM / CuSiO3@POM+Laser groups, and the expression of caspase-3 increased by about 9 times compared with the control group.

[0067] These two unique forms of cell death, cuproptosis and ferroptosis, although with different triggering factors, often are accompanied by a significant decline in mitochondrial function, leading to insufficient energy supply and increased oxidative stress. Next, it was explored whether the mitochondrial function in cells was damaged after CuSiO3@POM treatment. The decrease or loss of mitochondrial membrane potential (MMP) is one of the most critical markers of mitochondrial damage. A mitochondrial membrane potential detection kit (JC-1) is usually used to identify changes in mitochondrial membrane potential. Under normal mitochondrial membrane potential, JC-1 forms aggregates and presents red fluorescence. However, when the mitochondrial membrane potential is lost, JC-1 cannot aggregate and presents green fluorescence. The situation of mitochondrial membrane potential in MCF-7 cells after different treatments is as Figure 7 shown in (d). Compared with the control group, the JC-1 fluorescence signal in MCF-7 cells changed from red to green after incubation with CuSiO3@POM, indicating the loss of mitochondrial membrane potential, which may be due to the combined effect of a large amount of ROS generation induced by CuSiO3@POM and cuproptosis and ferroptosis of cells, resulting in severe damage to mitochondrial function. After 808nm laser irradiation, the green fluorescence intensity of JC-1 was further enhanced, which was attributed to the damage of mitochondria caused by photothermal-enhanced ROS generation. In summary, CuSiO3@POM induced effective ferroptosis by jointly causing LPO through GSH depletion, ROS burst, and GPX4 inactivation. Excessive intracellular Cu 2+It can also induce DLAT aggregation in mitochondria to induce cell copper death. In addition, intracellular GSH-reduced CuSiO3@POM exhibits good photothermal effect under near-infrared laser irradiation. This photothermal-enhanced mechanism of synergistic ferroptosis and copper death is conducive to maximizing cancer cell death.

[0068] 7. RNA sequencing.

[0069] In order to further explore the potential mechanism of the anti-tumor effect of CuSiO3@POM+Laser treatment, further attempts were made to reveal its mechanism of action through RNA sequencing analysis. After treatment with phosphate buffered saline (PBS) and CuSiO3@POM+Laser, the total RNA of MCF-7 cells was extracted and compared with the control group for comprehensive analysis. A total of 13,927 genes of tumor cells were analyzed, and differentially expressed genes (DEGs) were screened according to Pvalue<0.05 and |log2(foldchange)|≥1. The heat map and volcano map showed that there were 476 significant DEGs between the groups ( Figure 8 (a) and (b)). Compared with the control group, 207 genes were down-regulated and 269 genes were up-regulated in the CuSiO3@POM+Laser group. The bubble chart of the top 20 biological process entries enriched in Gene Ontology (GO) is shown in Figure 2. Figure 8 As shown in (c). The color of the bubbles in the figure usually represents the range of p-value. The smaller the p-value, the more significant the enrichment result. The size of the bubble represents the number of enriched genes. The larger the bubble, the more genes are contained in the relevant entry. The biological processes with high GO enrichment and a large number of enriched genes mainly involve oxidative stress response, cellular heat stress response, drug response, Rho-dependent serine / threonine kinase activity regulation, etc. This proves that CuSiO3@POM plus near-infrared light irradiation can significantly increase the intracellular temperature and induce cell apoptosis. In addition, the massive generation of ROS causes excessive oxidative stress in cells, prompting cell death. In addition, the negative regulatory changes in Rho-dependent serine / threonine kinase activity may suggest that the CuSiO3@POM+Laser group plays a role in inhibiting the invasion and metastasis of cancer cells. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that ( Figure 8In (d) and (e)), compared with the control group, the differentially expressed genes in the CuSiO3@POM+Laser group were mainly enriched in the "ferroptosis" pathway and the "D-glutamine and D-glutamate metabolism" pathway. Among them, D-glutamine and D-glutamate metabolism was inhibited, indicating that the treatment with CuSiO3@POM plus Laser weakened the metabolic function related to GSH synthesis in cells. In addition, the estrogen signaling pathway and the protein processing pathway in the endoplasmic reticulum pathway were activated, which might be due to the cells' activation of survival signaling pathways to cope with the stress response induced by the CuSiO3@POM nanodrug. The above results confirmed that the CuSiO3@POM+Laser treatment caused oxidative stress in MCF-7 cells and the possibility of inducing ferroptosis and cuproptosis.

[0070] 8. In vivo therapeutic effect and biosafety evaluation.

[0071] Inspired by the enhanced photothermal synergistic therapy effect of cuproptosis / ferroptosis, the anti-tumor effect of CuSiO3@POM was then confirmed in MCF-7 tumor-bearing nude mice. First, the biocompatibility of CuSiO3@POM was evaluated. Phosphate buffer solution (PBS) and CuSiO3@POM were intravenously injected into normal nude mice respectively. The serum biochemical and blood routine indexes of normal nude mice showed normal, proving that CuSiO3@POM could be used for further safe in vivo tumor nanodrug therapy. To determine whether CuSiO3@POM could effectively accumulate in the tumor region, an in vivo fluorescence imaging experiment was carried out by intravenously injecting Cy5-labeled CuSiO3@POM into MCF-7 tumor-bearing mice ( Figure 9 in (b)). The results showed that the fluorescence signal intensity at the tumor site reached the peak at 24 h and then began to decline. Ex vivo imaging of the tumor 24 h after injection also showed that a large amount of CuSiO3@POM accumulated in the tumor. This result determined that the 808 nm laser irradiation started 24 h after the intravenous injection of CuSiO3@POM. At the same time, ex vivo imaging of the main organs showed that CuSiO3@POM was mainly distributed in the liver, kidney and spleen because these organs are usually responsible for the metabolism and filtration of nanodrugs.

[0072] Subsequently, the in vivo anti-tumor efficiency of CuSiO3@POM was evaluated. MCF-7 tumor-bearing nude mice were randomly divided into four groups (n = 4): control group (PBS buffer solution), Laser group, CuSiO3@POM group, CuSiO3@POM+Laser group. As Figure 9 shown in (a), CuSiO3@POM (20 mg / kg) was intravenously injected into the tail vein on day 0 and day 7, and 808 nm laser (1 W / cm -1 , 10 min) was irradiated on day 1 and day 8. The temperature change was monitored by an infrared thermal imager during the laser irradiation (Figure 9 In (c)). For the control group, the surface temperature of the tumor hardly changed after laser irradiation, only increasing by about 1 °C, while the temperature in the CuSiO3@POM+Laser group increased by about 21 °C. The results showed that CuSiO3@POM could effectively convert near-infrared light into heat energy in vivo. After 14 days of treatment, the tumor growth of each group of mice showed significantly different trends, indicating that CuSiO3@POM had an effective anti-tumor effect( Figure 9 In (d) and (e)). The tumor sizes of the control group and the Laser group increased rapidly, while the tumor growth was significantly inhibited after CuSiO3@POM treatment. Especially in the CuSiO3@POM+Laser group, the tumor growth was the slowest, and its tumor inhibition rate was 72.9%. In addition, the body weights of all mice during the treatment did not decrease significantly( Figure 9 In (f)), confirming that the negative impacts of these treatments on the health of mice could be ignored.

[0073] In addition, the tumor tissues and major organs collected from mice in different groups after treatment were subjected to histological analysis using H&E staining, as Figure 9 shown in (g). The treatment with CuSiO3@POM plus Laser resulted in the most obvious tumor tissue damage. No histopathological abnormalities were observed in the major organs (heart, liver, spleen, lung, and kidney) of mice in different treatment groups( Figure 10 In (a)), indicating that CuSiO3@POM had good biocompatibility in vivo. More significant tumor cell apoptosis was observed in the CuSiO3@POM+Laser group through the TUNEL assay. At the same time, the activity of GPX4 was also affected by the treatment with CuSiO3@POM plus Laser, and the immunohistochemical expression decreased to a certain extent, further illustrating the effectiveness of CuSiO3@POM in inducing ferroptosis of cancer cells. In addition, bright green fluorescence of ROS was shown in the tumor tissues of the CuSiO3@POM+Laser group, which was significantly different from that of the control group( Figure 10 In (b)), indicating that the level of ROS in the tumor tissues increased after CuSiO3@POM treatment, which was beneficial to the co-activation of cuproptosis and ferroptosis. Overall, CuSiO3@POM had satisfactory biosafety and excellent therapeutic effects, with potential application value in vivo.

[0074] The designed CuSiO3@POMs of the present invention, as a TME-responsive nanosystem, demonstrate a synergistic effect in photothermal therapy, as well as inducing cuproptosis and ferroptosis, effectively inhibiting tumor cells and improving the treatment efficiency. First, POM clusters were successfully synthesized, and TEM showed that they had a spherical structure in neutral solution with a diameter of about 2.7 nm. Under acidic conditions (pH = 5.5), the POM clusters aggregated into larger clusters (diameter about 4.5 nm), showing pH responsiveness. X-ray photoelectron spectroscopy (XPS) and ultraviolet-visible (UV-vis) spectroscopy indicated that POM had the potential for photothermal therapy. Then, POM was loaded onto the CuSiO3 nanocarrier through electrostatic interaction. Characterizations such as TEM, XRD, XPS, and Zeta potential confirmed the successful loading of POM and optimized the POM loading concentration (0.2 mg mL -1 ). Under 808 nm laser irradiation, CuSiO3@POM showed good photothermal conversion performance under weakly acidic and GSH conditions, and the photothermal conversion efficiency was 38%. The in vitro ROS and GSH consumption detection results showed that after GSH reduction, CuSiO3@POM generated ·OH and + O2 through the Cu 5+ -mediated Fenton-like reaction and the Russell mechanism catalyzed by Mo 1 respectively, significantly increasing the ROS level and effectively consuming GSH, and the consumption was positively correlated with the reaction time and concentration.

[0075] Cytotoxicity verification showed that CuSiO3@POM showed good biocompatibility to normal cells (hCMEC / D3), but had significant toxicity to MCF-7. Under laser irradiation, the cell survival rate decreased to less than 20% at a concentration of 200 μg mL -1 . DCFH-DA and SOSG probe experiments confirmed that CuSiO3@POM significantly increased the ROS level in cells. Further exploration of the cell death mechanism showed that CuSiO3@POM induced ferroptosis through GSH consumption, ROS burst, and GPX4 inactivation, and at the same time induced cuproptosis through excessive accumulation of Cu 2+ , manifested as the oligomerization of DLAT protein and the decrease in the expression of FDX1 protein. RNA-seq analysis further revealed that the CuSiO3@POM+Laser treatment significantly affected the gene expression related to oxidative stress, heat stress, and ferroptosis pathways in MCF-7 cells. In vivo treatment showed that the injected CuSiO3@POM in MCF-7 tumor-bearing nude mice effectively accumulated in the tumor area, had biosafety, and the CuSiO3@POM+Laser treatment showed a significant anti-tumor effect with a tumor inhibition rate of 72.9%.

[0076] The design of CuSiO3@POMs shows that combining TME responsiveness and multimodal therapy is one of the effective ways to improve the therapeutic effect. This rationally designed multifunctional nanosystem provides a unique example of a synergistic therapy strategy, which can achieve efficient tumor treatment and has ideal prospects for clinical translation.

[0077] Therefore, the present invention uses the above-mentioned pH / GSH dual-responsive copper silicate-based nanosystem for synergistic treatment of breast cancer, demonstrating a synergistic effect in photothermal therapy, as well as inducing cuproptosis and ferroptosis, effectively inhibiting tumor cells and improving the treatment efficiency.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A pH / GSH dual-responsive copper silicate-based nanosystem for the co-treatment of breast cancer, characterized in that: It includes a CuSiO3 nanocarrier and a molybdenum-based polyoxometalate. The molybdenum-based polyoxometalate is loaded on the CuSiO3 nanocarrier, and the loading concentration of the molybdenum-based polyoxometalate is 0.2 - 1.0 mg / mL. The molybdenum-based polyoxometalate is added to the dispersion of the CuSiO3 nanocarrier, stirred at room temperature, and the molybdenum-based polyoxometalate-modified copper silicate-based nanosystem is obtained by centrifugation.

2. The pH / GSH dual-responsive copper silicate-based nanosystem for the co-treatment of breast cancer according to claim 1, wherein: The loading concentration of the molybdenum-based polyoxometalate is 0.2 mg / mL.

3. The pH / GSH dual-responsive copper silicate-based nanosystem for the synergistic treatment of breast cancer according to claim 1, wherein, The preparation method of the CuSiO3 nanocarrier is as follows: A. Synthesis of Cu2O: Dissolve Cu(NO3)2 and NH4NO3 in deionized water, add the NaOH solution at a rate of 1.8 mL / min, continue stirring, add the ascorbic acid solution at a rate of 0.6 mL / min, stir again, centrifuge to collect the product Cu2O, wash it with ethanol, and disperse it in ethanol; B. Synthesis of Cu2O@mSiO2: Dilute the above Cu2O dispersion in a mixed solution of ethanol and deionized water, successively add a 25 wt% aqueous solution of cetyltrimethylammonium chloride and triethylamine diluted 10 times with ethanol. After adding tetraethyl orthosilicate, stir the mixture at room temperature, collect the solid Cu2O@mSiO2 by centrifugation and washing, and redisperse it in ethanol; C. Synthesis of CuSiO3: Add 2-methylimidazole and ethanol to the Cu2O@mSiO2 dispersion, stir at room temperature, centrifuge and wash twice with ethanol to obtain CuSiO3, and redisperse the obtained CuSiO3 in ethanol for use.

4. The pH / GSH dual-responsive copper silicate-based nanosystem for the co-treatment of breast cancer according to claim 3, wherein: In step C, the concentration of 2-methylimidazole is 0.2 g / mL.

5. The pH / GSH dual-responsive copper silicate-based nanosystem for the synergistic treatment of breast cancer according to claim 3, wherein: In step C, CuSiO3 is a layered CuSiO3 nanoflower carrier.

6. The pH / GSH dual-responsive copper silicate-based nanosystem for the synergistic treatment of breast cancer according to claim 1, wherein The preparation method of the molybdenum-based polyoxometalate is as follows: Add (NH4)6Mo7O 24 ·4H2O to ultrapure water, stir at room temperature for 1 h, then quickly add NaH2PO4, stir, and finally add ethanol, centrifuge, wash, and dry to obtain molybdenum-based polyoxometalates.

7. The pH / GSH dual-responsive copper silicate-based nanosystem for the co-treatment of breast cancer according to claim 1, wherein: The molybdenum-based polyoxometalate has a spherical structure in a neutral solution with a pH of 7.4, and aggregates into clusters under acidic conditions with a pH of 5.5, showing pH responsiveness.

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