Intelligent responsive copper-death nanomedicine, preparation and anti-tumor application

Abstract

The present invention discloses an intelligent responsive copper-death nanomedicine, its preparation and anti-tumor application. The intelligent responsive copper-death nanomedicine is obtained by loading a copper ion carrier in an intelligent responsive nanocarrier. The intelligent responsive nanocarrier uses copper nanoparticles as the core, and its outer layer is wrapped with a shell of organic mesoporous silica containing X-ray responsive double bonds, and the shell is modified with an acid-responsive polymer. The intelligent responsive nanocarrier has multiple responsive stimulation effects, which can change the charge carried by the drug-carrying system and increase the amount of drug administered into the cell; and has a drug delivery system with good targeting and spatiotemporal controlled release, which can reduce the toxic and side effects of drug molecules on other normal tissues and achieve precise treatment of tumors. The intelligent responsive copper-death nanomedicine can combine the metabolic therapy controlled by copper death with immunotherapy, which will effectively improve tumor resistance, reduce the side effects of radiotherapy on the whole body, and improve the survival rate of patients.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an intelligent responsive nanocarrier and a preparation method thereof, and also relates to an intelligent responsive copper-death nanomedicine obtained based on the intelligent responsive nanocarrier and its anti-tumor application. Background Art

[0002] Treatment for malignant tumors primarily relies on surgical resection, radiotherapy, and chemotherapy. Current clinical cancer treatment relies on tailoring treatment modalities to the specific stage of tumor progression, combined with their respective advantages and disadvantages. However, these therapies all have drawbacks and limitations, hindering optimal therapeutic outcomes. For example, surgery is one of the most commonly used treatments in clinical practice. However, it is invasive and risky, unable to remove even small lesions or metastases, and can easily lead to metastasis and recurrence. Compared to surgery, chemotherapy and radiotherapy have a wider range of applications and are suitable for treating tumors at all stages. However, they also have limitations: most chemotherapy and radiotherapy methods lack specificity and cannot precisely target the lesion, resulting in severe systemic side effects. Repeated treatments can lead to multidrug resistance in tumor cells, significantly reducing treatment efficacy. Therefore, developing new therapeutic approaches that modulate tumor metabolic pathways and promote programmed cell death (PD) could effectively reduce the toxic side effects of traditional treatments, alleviate and reverse drug resistance, and prolong patient survival.

[0003] Mitochondria regulate respiratory chain transport, oxidative phosphorylation, the tricarboxylic acid cycle, and apoptosis, playing a crucial role in tumor development and progression. A deeper understanding of the mechanisms regulating these key pathways and the reversal of mitochondrial dysfunction by drug molecules will be crucial for the subsequent development of targeted drug delivery within mitochondria for tumor therapy, minimizing off-target effects, reversing tumor resistance, and maximizing drug anti-tumor efficacy. Studies have shown that ferredoxin 1 (FDX1) is a mitochondrial reductase that participates in the formation of iron-sulfur (Fe-S) clusters, a process crucial for mitochondrial electron transport and oxidative phosphorylation. The specific mechanism is that FDX1 utilizes the redox properties of its [2Fe-2S] cofactor to reduce Cu(II) to Cu(I) within mitochondria, inhibiting Fe-S cluster synthesis and, in turn, affecting the production of Fe-S cluster proteins. Furthermore, FDX1 can reduce mitochondrial cytochrome P450 enzymes and confer catalytic activity, for example, in the conversion of cholesterol to pregnenolone, aldosterone, and cortisol. In these biochemical pathways, electrons are transferred from nicotinamide adenine dinucleotide phosphate (NADPH) to FDX1 via ferredoxin reductase (FDXR). Therefore, intervening in the stability of FDX1 protein in mitochondria is extremely important for resolving tumor resistance.

[0004] Copper, a trace metal essential for life, is an essential cofactor in enzymes that regulate cellular function in the human body. It mediates a range of important cellular functions, including mitochondrial respiration, antioxidant defense, and the biosynthesis of hormones, neurotransmitters, and pigments. However, dysregulation of copper reserves can lead to oxidative stress and cytotoxicity. Within mitochondria, FDX1 is involved in regulating protein lipidation. FDX1 reduces Cu(II) to Cu(I), inhibiting Fe-S cluster synthesis and, in turn, impairing the production of Fe-S cluster proteins. FDX1 deficiency also inhibits the fatty acylation of dihydrolipoamide S-acetyltransferase (DLAT). The resulting Cu(I) can directly bind to the acylated DLAT, inducing abnormal oligomerization of the acylated DLAT. Increased levels of insoluble DLAT lead to cellular proteotoxic stress and, subsequently, cell death. Cu ion accumulation within cells can also interfere with the synthesis of iron-sulfur cluster proteins within the respiratory chain complex, triggering a proteotoxic stress response. Therefore, disrupting copper homeostasis in tumor cells to achieve copper death is crucial for cancer treatment.

[0005] However, long-term use of compounds including copper ionophores and copper chelators can disrupt essential metal homeostasis and may cause serious side effects in treated patients. Summary of the Invention

[0006] In light of this, the primary objective of the present invention is to provide a smart, responsive nanocarrier that can alter the charge of the drug-carrying system, increasing the intracellular delivery volume. This nanocarrier also offers a drug delivery system with excellent targeting and spatiotemporal controllable release. Crucially, during drug delivery, the nanocarrier maintains drug toxicity "inactivation" while circulating in the body, but upon entering tumor tissue and / or cells, exhibits a specific spatiotemporal response, "activating" the drug's toxicity, thereby reducing the drug's toxic side effects on other normal tissues and achieving precise tumor treatment.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention first provides an intelligent responsive nanocarrier, which uses copper nanoparticles as the core. The copper nanoparticles are wrapped with a shell layer, which is organic mesoporous silica containing X-ray responsive double bonds, and the shell layer is modified with an acid-responsive polymer.

[0009] In a further embodiment, the copper nanoparticles are copper-containing compounds, and the copper-containing compound is CuCl2, Cu(NO3)2, CuSO4, CuO, CuS or Cu3BiS3.

[0010] In a further embodiment, the X-ray responsive double bond is a diselenide bond, a ditellurium bond or a disulfide bond.

[0011] In a further embodiment, the X-ray responsive double bond is a diselenide bond.

[0012] In a further embodiment, the acid-responsive polymer is a block polymer of polymer X modified with maleic anhydride, and the polymer X is any one of polyetherimide, chitosan, or poly-(2-ethyl-2-oxazoline).

[0013] The present invention further provides a method for preparing the aforementioned smart responsive nanocarrier, comprising the following steps:

[0014] obtaining copper nanoparticles;

[0015] The outer layer of the copper nanoparticles is coated with organic mesoporous silica containing X-ray responsive double bonds to form a shell layer;

[0016] A shell acid-responsive polymer is modified on the shell layer.

[0017] In a further embodiment, the copper nanoparticles are Cu3BiS3, and their preparation comprises the following steps:

[0018] Dispersing bismuth salt and copper salt into oleylamine solution, degassing and stirring to mix evenly;

[0019] A sulfur source is quickly injected into the reaction mixture and stirred to obtain Cu3BiS3 nanoparticles.

[0020] In a further embodiment, the method for forming organic mesoporous silica containing X-ray responsive double bonds in the shell layer comprises the following steps:

[0021] dissolving copper nanoparticles, a surfactant, and an alkaline catalyst in deionized water and mixing them uniformly to obtain a mixed solution;

[0022] Adding the silica precursor and the compound containing an X-ray responsive double bond into the mixed solution in multiple portions, and stirring to react;

[0023] The surfactant in the product is removed, and organic mesoporous silica containing X-ray responsive double bonds is formed on the surface of the copper nanoparticles.

[0024] In a further embodiment, the method for modifying the acid-responsive polymer on the shell layer comprises the following steps:

[0025] Dispersing copper nanoparticles coated with organic mesoporous silica containing X-ray responsive double bonds in an alcohol solvent to form a dispersion;

[0026] A crosslinking agent is injected into the dispersion, and after stirring, a solution of polymer X is introduced into the reaction solution, and stirred for reaction to obtain Cu@X nanoparticles, wherein polymer X is polyetherimide, chitosan, or poly-(2-ethyl-2-oxazoline);

[0027] Cu@X nanoparticles are dissolved in an alkaline buffer solution and stirred at low temperature. Subsequently, 3 to 20 mol equivalents of maleic anhydride relative to polymer X are added, stirred for reaction, and dried to obtain Cu@X-DA.

[0028] In a further embodiment, the cross-linking agent is (3-glycidyloxypropyl)trimethoxysilane or (3-triethoxysilyl)propylsuccinic anhydride after carboxyl activation.

[0029] In a further embodiment, the alkaline buffer solution is a NaHCO 3 buffer solution or a Na 2 HPO 4 buffer solution with a pH of 8 to 10.

[0030] The present invention further provides an intelligent responsive nanomedicine comprising the intelligent responsive nanocarrier as described above, wherein the intelligent responsive nanocarrier is loaded with a copper ion carrier.

[0031] In a further embodiment, the copper ion carrier is ilisimol or disulfiram.

[0032] The present invention further provides the use of the aforementioned smart responsive nanomedicine in the preparation of a drug for treating tumors.

[0033] Beneficial effects of the present invention:

[0034] The intelligent responsive nanocarriers described in this invention utilize copper nanoparticles as their core. An organic mesoporous silica carrier containing an X-ray-responsive structure is constructed around the copper nanoparticles, and the shell is modified with an acid-responsive polymer. By forming an X-ray- and acid-responsive shell around the copper nanoparticles, the invention addresses the serious toxic side effects of existing nanoparticles, such as premature drug release after entering the bloodstream, difficulty in tumor accumulation and uptake, and low drug release efficiency after entering tumor cells. This allows for more precise and controlled killing of tumor cells.

[0035] Specifically, mesoporous silica nanoparticles have the characteristics of good biocompatibility, excellent stability in physiological environment, modifiable outer surface, large specific surface area and high drug loading rate. Using them as shell can improve the bioavailability and efficacy of drugs. Furthermore, in order to improve the controlled release of drugs in deep tumor tissues, the present invention constructs organic mesoporous silica containing X-ray responsive double bonds. When excited by exogenous X-rays and endogenous reactive oxygen species, the silica shell containing X-ray responsive double bonds will rapidly rupture, allowing the core copper nanoparticles and the loaded copper ion carriers in the shell pores to react in situ to form CuET complexes, leading to cell apoptosis. While the CuET complex is formed, Cu 2+ Converted to Cu + , oligomerizes with mitochondrial proteins in the tricarboxylic acid cycle and causes copper death in tumor cells.

[0036] This shell is modified with an acid-responsive polymer to create a nanocarrier with charge reversal properties. By modifying the acid-responsive polymer within a mesoporous silica shell coated with X-ray-responsive double bonds, the constructed smart responsive nanocarrier exhibits a negative charge in the blood circulation. Upon entering the tumor microenvironment through body fluid circulation, the modified polymer falls off the surface, exposing the positively charged nanoparticles. This effectively promotes the uptake of the nanodrug by tumor cells, thereby achieving both circulatory stability and good uptake efficiency.

[0037] In summary, the intelligent, responsive nanomedicine constructed in this invention exhibits excellent spatiotemporal controllable release properties, effectively addressing the issues of premature drug release in the bloodstream, difficulty in tumor cell uptake, and low drug release efficiency after entry into tumor cells. Combining copper-mediated metabolic therapy with immunotherapy will effectively improve tumor drug resistance, reduce systemic side effects of radiotherapy, and improve patient survival. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the structure of Cu@Cs-DA prepared in Example 1 of the present invention and Cu@DSF prepared in Example 2.

[0039] Figure 2 Transmission electron microscopy (TEM) characterization of Cu3BiS3 in Example 1 of the present invention and the particle size distribution results of Cu3BiS3.

[0040] Figure 3 Transmission electron microscopy (TEM) characterization of Cu@SeSiO2 in Example 1 of the present invention.

[0041] Figure 4 This is the X-ray photoelectron spectrum (XPS) of selenium element in Cu@SeSiO2 in Example 1 of the present invention before and after X-ray stimulation.

[0042] Figure 5 Zeta potential of Cu3BiS3, Cu@SeSiO2, Cu@Cs-DA in Example 1 and Cu@DSF in Example 2 in PBS.

[0043] Figure 6 Transmission electron microscopy characterization of the Cu@DSF morphology structure in Example 2 of the present invention.

[0044] Figure 7 Zeta potential of Cu@Cs-DA at different pH values ​​in Example 1 of the present invention.

[0045] Figure 8 Uptake of Cu@SeSiO2-FITC and Cu@CS-DA-FITC by 4T1 at different pH values.

[0046] Figure 9 Cell viability of 4T1 cells after treatment with the same concentration of Cu@SeSiO2-DSF and Cu@DSF.

[0047] Figure 10 Cell viability of 4T1 cells treated with the same concentration of Cu@DSF with or without irradiation.

[0048] Figure 11 Cell viability of 4T1 cells treated with different methods.

[0049] Figure 12 The live-dead staining results of 4T1 cells treated in different ways.

[0050] Figure 13 The data of tumor volume changes over time in mice in different treatment groups are shown in Figure 2.

[0051] Figure 14 Transmission electron microscopy characterization of the morphology of Cu@SSiO2 before and after irradiation in Example 4 of the present invention.

[0052] Figure 15 This is the Zeta potential of Cu@PEI-DA at different pH values ​​in Example 5 of the present invention. DETAILED DESCRIPTION

[0053] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0055] The first aspect of the present invention discloses an intelligent responsive nanocarrier, which has a copper nanoparticle as a core, and a shell layer wrapped around the outer layer of the copper nanoparticle. The shell layer is organic mesoporous silica containing X-ray responsive double bonds, and the shell layer is modified with an acid-responsive polymer.

[0056] The shell of this intelligent responsive nanocarrier has multiple activation properties, which can effectively promote the uptake of the loaded nanodrugs by tumor cells and the intracellular drug release through the combination of X-ray stimulation response and acid response, thereby improving the therapeutic effect.

[0057] Specifically, the nanocarrier stimulates charge reversal through acid response and carrier rupture through X-ray stimulation, giving it good targeting and spatiotemporal controllable release, thereby increasing the amount of drug delivered into the cell. Most importantly, during the drug delivery process, the loaded drug can remain toxic and "inactivated" during circulation in the body, and after entering the tumor tissue / cell, it will show a specific spatiotemporal response to "activate" the drug's toxicity, thereby reducing the toxic side effects of the drug molecules on other normal tissues and achieving precise tumor treatment.

[0058] The copper nanoparticles described herein refer to copper-containing compounds with a particle size of approximately 10 to 100 nm. Specific types may include copper oxides, sulfides, and the like, or inorganic or organic salts containing copper ions. Specific examples include, but are not limited to, CuCl2, Cu(NO3)2, CuSO4, CuO, CuS, and Cu3BiS3. Preferably, in some specific embodiments of the present invention, the copper nanoparticles are Cu3BiS3.

[0059] The X-ray responsive double bond described herein refers to a double bond that can be oxidized or broken under X-ray activation, and specific examples that can be mentioned include a diselenide bond (Se-Se), a ditellurium bond (Te-Te) or a disulfide bond (SS). Preferably, the X-ray responsive double bond is a diselenide bond (Se-Se). Compared with other types of X-ray responsive double bonds, while having the effects that other X-ray responsive double bonds can bring, silicon dioxide containing a diselenide bond can also be oxidized to selenious acid, which can promote cell apoptosis and reduce the expression of human leukocyte antigen E (HLA-E) in different cancer cells, thereby promoting cancer cells to be recognized and eliminated by NK cells. In addition, some selenium elements can be released into the blood circulation system, and by being converted into selenoenzymes or selenoproteins, the body's immunity is enhanced throughout the body and the side effects of radiotherapy on the body are reduced.

[0060] The acid-responsive polymer described in this article refers to a block polymer containing a chemical group (such as imidazole, amino, etc.) that can be protonated as the pH value of the environment decreases. Specific examples that can be mentioned can be one of the block polymers of maleic anhydride-modified polyetherimide (PEI), chitosan (CS) or poly-(2-ethyl-2-oxazoline) (PEOz).

[0061] The second aspect of the present invention provides a method for preparing the smart responsive nanocarrier according to the first aspect of the present invention, the main steps of which are as follows:

[0062] S1. Obtaining copper nanoparticles

[0063] The copper nanoparticles herein can be prepared or obtained using methods known in the art. Using the preparation of Cu3BiS3 as an example, a bismuth salt and a copper salt are dispersed in an oleylamine solution, degassed, and stirred to mix thoroughly. Subsequently, a sulfur source is rapidly injected into the reaction mixture and stirred to produce Cu3BiS3 nanoparticles.

[0064] The bismuth salt can be Bi(NO3)3·5H2O, Bi(OAc)3 or bismuth octanoate, the copper salt can be Cu(CH3COO)2, Cu(NO3)2·3H2O or CuCl2·2H2O; and the sulfur source can be thioacetamide, elemental sulfur, Na2S·9H2O or sodium diethyldithiocarbamate.

[0065] The molar ratio of the bismuth salt, the copper salt and the sulfur source is 1:2:3 to 1:6:12, preferably 1:3:3.

[0066] The temperature for uniform stirring and mixing is 60-160° C., the stirring speed is 500-2000 rpm, and the time can be adjusted as needed; the preferred temperature is 100° C., and the stirring speed is 1500 rpm.

[0067] Before injecting the sulfur source into the reaction mixture, the temperature of the reaction mixture needs to be rapidly raised to 160-500°C and maintained for 10 minutes. Preferably, the temperature is rapidly raised to 300°C.

[0068] S2, wrapping the outer layer of the copper nanoparticles with an X-ray responsive double-bond organic mesoporous silica to form Shell The specific steps are as follows:

[0069] S21. Dissolve the copper nanoparticles, surfactant, and alkaline catalyst in deionized water and mix them evenly to obtain a mixed solution.

[0070] The surfactant serves as a cationic template for the mesoporous silica. Specific examples include cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride, or cetyltrimethylammonium p-toluenesulfonate. Cetyltrimethylammonium bromide is preferred. Preferably, the mass ratio of surfactant to copper nanoparticles is 5:1.

[0071] The base catalyst is used to subsequently catalyze the hydrolysis of the silica precursor to generate mesoporous silica. Specific examples include ammonia water or triethylamine (TEA). The specific amount used can be determined according to the amount of silica precursor used; preferably, triethylamine is used.

[0072] Furthermore, in this step, the mixing and stirring temperature is 50-95°C, preferably 65°C.

[0073] S22, adding the silica precursor and the compound containing an X-ray responsive double bond into the mixed solution in multiple times, and stirring to react.

[0074] The silicon dioxide precursor may be tetraethyl orthosilicate (TEOS) or methyltrimethoxysilane (MTMS), but is not limited thereto; preferably, it is tetraethyl orthosilicate (TEOS).

[0075] The compound containing an X-ray responsive double bond is selected according to the type of double bond, and specifically can be bis[3-(triethoxysilyl)propyl]diselenide (BTESePD), bis-[3-(triethoxysilyl)propyl]-disulfide, bis-[3-(triethoxysilyl)propyl]-ditelluride; preferably, it is BTESePD.

[0076] Specifically, the mass ratio of the added silicon oxide precursor to the compound containing an X-ray responsive double bond is 4:1 to 1:1, preferably 2: 1. Furthermore, in this step, the reaction is maintained at 50 to 95°C, preferably 65°C.

[0077] S23. Removing the surfactant from the product, and forming organic mesoporous silica containing X-ray responsive double bonds on the surface of the copper nanoparticles.

[0078] The method for removing the surfactant may specifically be to remove it by refluxing in ethanol containing 10% HCl at 70° C. for 24 hours.

[0079] S3, modifying the shell acid responsive polymer on the shell layer

[0080] The specific steps are as follows:

[0081] S31. Dispersing copper nanoparticles coated with organic mesoporous silica containing X-ray responsive double bonds in an alcohol solvent to form a dispersion.

[0082] S32, injecting a crosslinking agent into the dispersion, stirring, introducing a solution of polymer X into the reaction solution, stirring and reacting, to obtain Cu@X nanoparticles.

[0083] Specifically, the pH of the system is adjusted to between 3.5 and 4.5, and then a silane coupling agent, such as (3-glycidyloxypropyl)trimethoxysilane or carboxyl-activated (3-triethoxysilyl)propyl succinic anhydride, is injected into the dispersion. The silane coupling agent utilizes the CO bond cleavage of the epoxy functional group on (3-glycidyloxypropyl)trimethoxysilane to undergo a nucleophilic addition reaction with the NH2 on the polymer X; alternatively, the carboxyl-activated (3-triethoxysilyl)propyl succinic anhydride undergoes an amidation reaction with the NH2 on the polymer X.

[0084] The carboxyl-activated (3-triethoxysilyl)propyl succinic anhydride is prepared by adding EDC and NHS and stirring for 1 to 12 hours to activate the carboxyl group.

[0085] S33. Dissolve the Cu@X nanoparticles in an alkaline buffer solution, gently stir at low temperature (such as 4° C.), then add 3-20 mol equivalents of maleic anhydride relative to polymer X, stir to react, and dry to obtain Cu@X-DA.

[0086] The alkaline buffer described herein refers to a buffer with a pH between 8 and 10. Specific examples include NaHCO3 buffer or Na2HPO4. The specific concentration can be adjusted as needed. In some specific embodiments of the present invention, the concentration of the alkaline buffer is 0.5M.

[0087] The polymer X is polyetherimide (PEI), chitosan (CS) or poly-(2-ethyl-2-oxazoline) (PEOz), and the final products are Cu@PEI-DA, Cu@CS-DA, and Cu@PEOz-DA, respectively.

[0088] The low temperature and alkaline buffer solution are used to prevent maleic anhydride from reacting violently after dissolving in water. At the same time, the alkaline condition also promotes the amidation reaction between maleic anhydride and the amino group on polymer X.

[0089] The third aspect of the present invention provides an intelligent responsive nanomedicine comprising the intelligent responsive nanocarrier as described in the first aspect of the present invention, wherein the intelligent responsive nanocarrier is loaded with a copper ion carrier.

[0090] The copper ion carrier refers to a compound known in the art that can combine with copper ions to induce copper cell death in tumor cells or a pharmaceutically acceptable salt thereof, and specific examples include ilisimol and disulfiram.

[0091] The specific method of loading the copper ion carrier into the smart responsive nanocarrier can be carried out by a method known in the art, for example, dispersing the nanocarrier in a solution containing the copper ion carrier and stirring the solution to obtain the nanocarrier.

[0092] The fourth aspect of the present invention provides the use of the smart responsive nanomedicine as described in the third aspect of the present invention in the preparation of a drug for treating tumors.

[0093] The technical solution of the present invention will be more clearly described below with reference to specific embodiments. In addition, unless otherwise specified, methods without specific conditions or steps are conventional methods, and the reagents and materials used can be obtained from commercial sources.

[0094] Example 1 Preparation of Intelligent Responsive Nanocarriers

[0095] This embodiment provides a smart responsive nanocarrier and a preparation method thereof. The structure of the smart responsive nanocarrier is as follows: Figure 1 As shown in , it uses copper nanoparticles as the core, its surface is coated with a mesoporous silica shell containing diselenide bonds, and the shell is modified with a chitosan block polymer modified with maleic anhydride. The specific steps of this smart responsive nanocarrier are as follows:

[0096] S1. Preparation of copper nanoparticles (Cu3BiS3)

[0097] Bi(NO3)3·5H2O (1 mmol) and Cu(CH3COO)2 (1 mmol) were placed in a 50 mL three-necked flask containing 12 mL of OM (oleylamine) and degassed for 5 min. The temperature was raised to 100°C and the mixture was gently stirred for 1 h. Subsequently, the temperature of the reaction mixture was rapidly raised to 300°C and maintained for 10 min.

[0098] TAA (thioacetamide) was dispersed in OM (oleylamine) (final concentration of 1.2 mM), and then 2.5 ml of TAA solution was quickly injected into the above reaction mixture; after 30 minutes, the reaction system was cooled to 70°C and 20 mL of cyclohexane was added; the product was collected by centrifugation and then washed three times with ethanol; finally, the prepared Cu3BiS3 nanoparticles were dispersed in 10 mL of cyclohexane to avoid agglomeration of the Cu3BiS3 nanoparticles.

[0099] S2. Preparation of copper nanoparticles coated with organic mesoporous silica containing diselenide bonds (Cu@SeSiO2)

[0100] Dissolve cetyltrimethylammonium bromide (CTAB) (0.5 g), triethylamine (TEA) (0.2 g), and the copper nanoparticles (Cu3BiS3) prepared in step S1 (100 mg) in 35 mL of deionized water, heat to 65 °C, and stir slowly for 30 min;

[0101] Tetraethyl orthosilicate (TEOS) (1.2 ml) was dissolved in cyclohexane (20 ml), added to the above reaction system and maintained at 65°C for 30 min; the mixed solution was further heated and maintained at 65°C for 1 h, and then a mixture of 0.5 ml of tetraethyl orthosilicate (TEOS) and 0.5 ml of bis[3-(triethoxysilyl)propyl] diselenide (BTESePD) was slowly injected and stirred for 12 h. The product was washed three times with ethanol and placed in ethanol;

[0102] The solid CTAB-stabilized product was added to excess ethanol and deionized water and centrifuged. Finally, the precipitate was refluxed in ethanol containing 10% (v / v) HCl at 70°C for 24 h to remove the surfactant CTAB.

[0103] The mixture was centrifuged at 12000r / min for 5min and washed with water and ethanol several times. After the precipitate was dried, copper nanoparticles wrapped with organic mesoporous silica containing diselenide bonds, namely Cu@SeSiO2, were obtained.

[0104] In addition, ordinary organic mesoporous silica-coated copper nanoparticles (Cu@SiO2) were prepared in the same manner as step S2, with the only difference being that 0.5 mL of bis[3-(triethoxysilyl)propyl] diselenide (BTESePD) was replaced with an equal volume of tetraethyl orthosilicate (TEOS), and the other steps were the same as step S2.

[0105] S3. Preparation of chitosan and maleic anhydride modified organic mesoporous silica coated copper nanoparticles (Cu@Cs-DA)

[0106] Chitosan (1.0 g) was dissolved in 200 ml of glacial acetic acid aqueous solution (3 wt%) and stirred at room temperature for 24 h.

[0107] The Cu@SeSiO2 (10 mg) prepared in step S2 was dispersed in 5 mL of anhydrous ethanol and ultrasonically treated for 15 min. Then, glacial acetic acid was added to adjust the pH value to 3.5-4.5 to obtain a Cu@SeSiO2 dispersion.

[0108] (3-Oxypropyl)trimethoxysilane (GPTMS) (0.1 g) was quickly injected into the Cu@SeSiO2 dispersion and stirred at room temperature for 12 h. Then, 5 ml of pre-acidified chitosan solution (0.5% w / v) was added and stirred at room temperature for 24 h. The final product was collected by centrifugation (7000 rpm), washed with excess deionized water and ethanol multiple times, and then freeze-dried to obtain Cu@chitosan nanoparticles (Cu@CS).

[0109] The above-mentioned Cu@CS was dissolved in NaHCO3 buffer (0.5M, pH 9.0) and gently stirred at 4°C, followed by the slow addition of excess maleic anhydride (DMMA) (10 mol equivalent relative to chitosan); the reaction mixture was stirred at pH 9.0 for 24 h; the resulting nanoparticles were centrifuged and washed with distilled water, and freeze-dried to obtain chitosan and maleic anhydride-modified organic mesoporous silica-encapsulated copper nanoparticles, namely Cu@Cs-DA.

[0110] Example 2 Preparation of Intelligent Responsive Copper Death Nanomedicine

[0111] This embodiment provides a smart responsive copper death nanomedicine (Cu@DSF) obtained based on the smart responsive nanocarrier prepared in Example 1, and its structure is as follows: Figure 1 As shown in FIG, DSF is loaded into the smart responsive nanocarrier Cu@Cs-DA. The specific steps for preparing the smart responsive copper-death nanomedicine are as follows:

[0112] 10 mg of Cu@CS-DA was dispersed in 2 mL of ethanol solution under ultrasonic oscillation for 1 h, and then the mixture was vigorously stirred overnight. The ethanol solution contained 50 mg of DSF (disulfiram).

[0113] After subsequent centrifugation and washing with ethanol three times, the copper-death nanomedicine, namely Cu@DSF, was obtained.

[0114] In addition, for comparison, this article provides a copper-death nanodrug (Cu@SiO2-DSF) obtained based on copper nanoparticles coated with ordinary organic mesoporous silica (Cu@SiO2) and a copper-death nanodrug (Cu@SeSiO2-DSF) obtained based on copper nanoparticles coated with organic mesoporous silica containing diselenide bonds (Cu@SeSiO2). The preparation of these nanodrugs is the same as in Example 2, except that Cu@CS-DA is replaced with an equal mass of Cu@SiO2 or Cu@SeSiO2. All other steps and parameters are the same as in Example 2.

[0115] Results and Analysis

[0116] 1. Test Characterization

[0117] 1. Characterization and testing of copper nanoparticles Cu3BiS3

[0118] Transmission electron microscopy was used to characterize the particle size and morphology of the copper nanoparticles Cu3BiS3 prepared in Example 1 ( Figure 2 ), it can be seen that Cu3BiS3 is a spherical particle with uniform particle size; in addition, the particle size of the synthesized Cu3BiS3 was statistically analyzed, and the results showed that the average particle size of Cu3BiS3 was about 15.3±4.6nm.

[0119] 2. Characterization test of Cu@SeSiO2

[0120] (1) Transmission electron microscopy was used to characterize the particle size and morphology of Cu@SeSiO2 constructed in Example 1. The organic mesoporous silica shell of Cu@SeSiO2 showed obvious dendritic shape, and the pore size formed was about 5 to 10 nm ( Figure 3 ).

[0121] (2) Subsequently, in order to detect whether the constructed Cu@SeSiO2 has X-ray response characteristics, the X-ray photoelectron spectrum of Cu@SeSiO2 was characterized using an X-ray photoelectron spectrometer. The increase in Se 3d5 binding energy from 56eV to 60eV confirmed the formation of selenious acid ( Figure 4 ), which proves that irradiation can promote the breakage and oxidation of diselenide bonds, indicating that the Cu@SeSiO2 prepared in Example 1 has X-ray response characteristics.

[0122] 3. Characterization and testing of Cu@Cs-DA and Cu@DSF

[0123] (1) The Zeta potential of Cu3BiS3, Cu@SeSiO2, Cu@CS, Cu@CS-DA prepared in Example 1 and Cu@DSF prepared in Example 2 were tested by Zeta potential instrument. The results are shown in Figure 5As can be seen, Cu3BiS3 exhibits a significant negative charge. When modified with a silica shell, the charge shifts from positive to negative. Subsequently, after chitosan and maleic anhydride are added, the zeta potential shifts from negative to positive and then back to negative. This change in zeta potential indicates that chitosan and maleic anhydride are successfully modified on the Cu@SeSiO2 surface.

[0124] (2) To further prove that the constructed Cu@Cs-DA has the characteristics of charge reversal, the potential of Cu@Cs-DA at different pH was tested using a Zeta potential meter. The results are shown in Figure 7 It can be seen that the surface potential of Cu@Cs-DA in a solution with a pH of 7.4 is lower than -3.74 mV. As the pH decreases, the maleic anhydride on the surface of Cu@Cs-DA is hydrolyzed and Cs is exposed on the surface of the nanoparticles, which causes the charge on the surface of Cu@Cs-DA to change from negative to positive. Under weakly acidic conditions of pH 5.5, the potential is about 2.76 mV.

[0125] (3) To further prove whether the Cu@DSF was successfully synthesized, the particle size and morphology of the Cu@DSF constructed in Example 1 were characterized using transmission electron microscopy. The size of the Cu@DSF was about 150 nm. At the same time, it was observed that the core of the organic silica carrier had obvious granular substances, and the mesoporous structure was completely filled ( Figure 6 ).

[0126] 2. In vitro cell assay

[0127] 1. Mouse breast cancer cells 4T1 (Wuhan Pricella Biotechnology Co, Ltd., CL-0007) were cultured in 6-well plates, with 10 4 Each well contained 2 mL of R1640 culture medium. After the cells were cultured in the incubator for 24 h and attached to the wall, the original culture medium was aspirated. The experimental grouping and specific operations were as follows:

[0128] R1640 growth medium: RPMI-1640 (PM150110) + 10% FBS (164210-50) + 1% P / S (PB180120).

[0129] Culture conditions: Gas phase: air, 95%; CO2, 5%; Temperature: 37°C.

[0130] Group 1: 2 ml of 100 μg / ml Cu@SeSiO2-FITC was added and cultured for 12 h at pH 7.4 and pH 6.5, respectively.

[0131] Group 2: 2 ml of 100 μg / ml Cu@Cs-DA-FITC was added and co-cultured for 12 h at pH 7.4 and pH 6.5, respectively.

[0132] The above experimental groups were observed by laser confocal microscopy, and the results were as follows: Figure 8 shown.

[0133] pass Figure 8 It can be seen that the Cu@Cs-DA-FITC modified with the charge-flipping material is uptaken by tumor cells more frequently in a weakly acidic environment of pH 6.5. However, the uptake of Cu@SeSiO2-FITC at pH 6.5 and pH 7.4 is relatively low, showing no increase in uptake efficiency with changes in acidic environment. This suggests that nanomaterials modified with charge-flipping materials can significantly enhance their ability to be taken up by tumor cells, thereby improving therapeutic efficacy.

[0134] 2. Mouse breast cancer cells 4T1 were cultured in 96-well plates, with 10 4 Each well contained 100 μL of R1640 culture medium. After the cells were cultured in the incubator for 24 hours and attached to the wall, the original culture medium was aspirated. The experimental grouping and specific operations were as follows:

[0135] Group 1: add 100 μL of fresh R1640 medium containing 0, 0.625, 1.25, 2.5, and 5 μg / ml Cu@SeSiO2-DSF (as described above) and incubate for 12 h;

[0136] Group 2: Add 100 μL of fresh R1640 medium containing 0, 0.625, 1.25, 2.5, and 5 μg / ml of Cu@DSF and incubate for 12 h.

[0137] The cell viability of the experimental group was detected by CCK-8. The experimental results are as follows Figure 9 .

[0138] according to Figure 9 It can be seen that compared with Cu@SeSiO2-DSF without Cs-DA modification, the cell viability of Cu@DSF is significantly reduced, further indicating that after the nanomaterial is modified with a material with charge reversal, its ability to be taken up by tumor cells can be significantly improved, thereby improving the therapeutic effect.

[0139] 3. Mouse breast cancer cells 4T1 were cultured in 96-well plates, with 10 4 Each well contained 100 μL of R1640 culture medium. After the cells were cultured in the incubator for 24 hours and attached to the wall, the original culture medium was aspirated. The experimental grouping and specific operations were as follows:

[0140] Group 1: add 100 μL of fresh R1640 medium containing 0, 0.625, 1.25, 2.5, and 5 μg / ml Cu@SiO2-DSF and incubate for 12 h;

[0141] Group 2: Add 100 μL of fresh R1640 medium containing 0, 0.625, 1.25, 2.5, and 5 μg / ml Cu@SiO2-DSF+X (pretreated with X-rays, irradiation dose of 1 Gy) and incubate for 12 h;

[0142] Group 3: add 100 μL of fresh R1640 medium containing 0, 0.625, 1.25, 2.5, and 5 μg / ml Cu@SeSiO2-DSF and incubate for 12 h;

[0143] Group 4: 100 μL of fresh R1640 medium containing 0, 0.625, 1.25, 2.5, and 5 μg / ml Cu@SeSiO2-DSF+X (pretreated with X-rays, 1 Gy) was added and incubated for 12 h.

[0144] The cell viability of the above experimental groups was detected by CCK-8, and the results are shown in Figure 10 .

[0145] pass Figure 10 It can be seen that Cu@SiO2-DSF, which does not contain diselenide bonds, did not significantly reduce cell viability before and after X-ray irradiation. However, the cytotoxicity of Cu@SeSiO2-DSF increased significantly after X-ray irradiation, showing better killing efficiency than the Cu@SiO2-DSF group. These results indicate that organic silica supports containing diselenide bonds have good X-ray response characteristics. After irradiation stimulation, the silica supports with diselenide bonds are destroyed, releasing the loaded Cu3BiS3 and DSF, generating the more toxic CuET in situ, further improving the therapeutic effect.

[0146] 4. Mouse breast cancer cells 4T1 were cultured in 96-well plates, with 10 cells per well. 4 Each well contained 100 μL of R1640 culture medium. After the cells were cultured in the incubator for 24 hours and attached to the wall, the original culture medium was aspirated. The experimental grouping and specific operations were as follows:

[0147] Group 1: add 100 μL of fresh R1640 medium containing 5 μg / ml Cu3BiS3 and incubate for 12 h;

[0148] Group 2: add 100 μL of fresh R1640 medium containing 5 μg / ml DSF and incubate for 12 h;

[0149] Group 3: add 100 μL of fresh R1640 medium containing 5 μg / ml Cu3BiS3+DSF and incubate for 12 h;

[0150] Group 4: add 100 μL of fresh R1640 medium containing 5 μg / ml Cu@CS-DA and incubate for 12 h;

[0151] Group 5: add 100 μL of fresh R1640 medium containing 5 μg / ml Cu@DSF and incubate for 12 h;

[0152] Group 6: Add 100 μL of fresh R1640 medium containing 5 μg / ml Cu@DSF+X (pretreated with X-rays, irradiation dose of 1 Gy) and incubate for 12 h;

[0153] The cell viability of the above experimental groups was detected by CCK-8 (Yeasen Biotechnology (Shanghai) Co., Ltd., 40203ES80) and AM / PI kit (Yeasen Biotechnology (Shanghai) Co., Ltd., 40747ES76). The color depth of CCK-8 is proportional to the proliferation of cells and inversely proportional to the cytotoxicity. The OD value was measured at a wavelength of 450 nm using a microplate reader, which indirectly reflects the number of viable cells ( Figure 11 ). For the AM / PI kit, Calcein-AM (which does not emit fluorescence itself) is cleaved by intracellular esterases to form the membrane-impermeable polar molecule Calcein. Since dead cells lack esterases, Calcein-AM is retained in the cells and emits strong green fluorescence only in living cells. Propidium iodide (PI) cannot pass through the cell membrane of living cells, but can only pass through the disordered regions of the dead cell membrane to reach the cell nucleus and embed into the cell's DNA double helix to produce red fluorescence (Ex = 535nm, Em = 617nm). Therefore, PI only stains dead cells ( Figure 12 ).

[0154] As can be seen, the toxicity of Cu3BiS3, DSF, and Cu@CS-DA alone to 4T1 cells was negligible, indicating the excellent cytocompatibility of Cu3BiS3, DSF, and Cu@CS-DA. However, when Cu3BiS3 and DSF were directly mixed, significant cell death occurred, indicating that DSF deprived Cu3BiS3 of its stable copper ions, converting them into the more toxic CuET. To mitigate the toxic side effects of direct contact between Cu3BiS3 and DSF, DSF was loaded into Cu@CS-DA to obtain Cu@DSF. This exhibited significantly reduced toxicity to 4T1 cells, exhibiting significant toxic activation only after X-ray excitation. This is likely attributed to X-ray-induced rapid degradation of the Cu@DSF shell, allowing for the release of large amounts of Cu3BiS3 and DSF, enabling direct contact and enhanced synergistic copper cell death / radiotherapy. These results demonstrate that, compared to DSF alone or in combination with CuET, X-ray control can transform Cu@DSF from a "non-toxic" to a "toxic" state, significantly inhibiting the proliferation of 4T1 tumor cells.

[0155] 3. Animal Model Experiment

[0156] Establishment of tumor-bearing mouse model: 1×10 7 Cells / ml 4T1 cell suspension was used to establish a tumor-bearing Balb / c model. 42 tumor-bearing female BALB / c mice were randomly divided into 7 groups (n=6). The specific groups are as follows:

[0157] (a) Control group: normal saline; (b) Cu3BiS3 treatment group; (c) DSF treatment group; (d) Cu@Cs-DA treatment group; (e) Cu@DSF treatment group; (f) normal saline + X-ray treatment group; (g) Cu@DSF + X-ray treatment group.

[0158] The drug was injected into the tail vein on days 1, 4, 7, 10, 13, and 16 (n=6). The concentration of the drug injected in each group was 10 mg / kg, and the radiotherapy condition was 1 Gy of X-ray irradiation 24 hours after the tail vein injection of the drug. The length (a, mm) and width (b, mm) of the tumor were measured every 2 days using a digital vernier caliper. The tumor volume (V, mm) was calculated using the following equation 3 ):

[0159] V=(ab 2 ) / 2

[0160] Figure 13Figure 3 shows the change in tumor volume over time in different treatment groups. The results show that Cu@DSF alone and radiotherapy inhibited tumor growth, while irradiation with Cu@DSF+X significantly outperformed both the Cu@DSF and radiotherapy groups. This demonstrates that X-rays can effectively stimulate the drug toxicity of Cu@DSF, significantly improving the therapeutic efficacy in tumor-bearing mice.

[0161] Other parallel implementation plans

[0162] Example 3

[0163] This example provides another method for preparing smart responsive nanocarriers, using the same method as in Example 1, except that the copper nanoparticles used are CuO. Other process steps and conditions are the same as in Example 1.

[0164] CuO was prepared by placing 1 mmol of copper acetate and 18 ml of glacial acetic acid into a 250 ml three-necked flask and degassing for 5 minutes. The temperature was raised to 100°C, allowing the solution to fully boil and maintaining this temperature for 1 hour. Subsequently, 3.50 ml of a 0.08 g / mL NaOH solution was quickly added, immediately forming a large amount of black precipitate. The precipitate was centrifuged, washed three times with anhydrous ethanol, and then freeze-dried to obtain copper nanoparticles (CuO).

[0165] Example 4

[0166] This example provides another method for preparing smart responsive nanocarriers, using the same implementation as in Example 1, except that the compound containing an X-ray responsive double bond used is bis[3(triethoxysilyl)propyl]-disulfide (BTEPDS), and the X-ray responsive double bond formed is a disulfide bond, thereby obtaining organic mesoporous silica-coated copper nanoparticles (Cu@SSiO2) containing a disulfide bond.

[0167] Figure 14 The transmission electron microscopy characterization results of Cu@SSiO2 prepared in Example 4 and Cu@SSiO2+X after X-ray irradiation treatment (irradiation dose 1 Gy) are shown. It can be seen that irradiation treatment can achieve the breakage and oxidation of disulfide bonds, indicating that the Cu@SSiO2 prepared in Example 4 has X-ray response characteristics.

[0168] Example 5

[0169] This example provides another method for preparing smart responsive nanocarriers, using the same method as in Example 1, except that the acid-responsive polymer formed is a maleic anhydride-modified polyetherimide. All other steps and conditions are the same as in Example 1.

[0170] The modification steps of the acid-responsive polymer are as follows:

[0171] Dissolve polyetherimide (PEI, 25KD) (100 mg) in 100 ml of glacial acetic acid aqueous solution (3 wt%) and stir at room temperature for 24 h.

[0172] The Cu@SeSiO2 (10 mg) prepared in step S2 of Example 1 was dispersed in 5 mL of anhydrous ethanol and ultrasonically treated for 15 min. Then, glacial acetic acid was added to adjust the pH value to 3.5-4.5 to obtain a Cu@SeSiO2 dispersion.

[0173] (3-Oxypropyl)trimethoxysilane (GPTMS) (0.1 g) was quickly injected into the Cu@SeSiO2 dispersion and stirred at room temperature for 12 h. Then, 5 ml of the pre-acidified polyetherimide solution was added and stirred at room temperature for 24 h. The final product was collected by centrifugation (7000 rpm), washed with excess deionized water and ethanol multiple times, and then freeze-dried to obtain Cu@polyetherimide nanoparticles (Cu@PEI).

[0174] The above-mentioned Cu@PEI was dissolved in NaHCO3 buffer (0.5 M, pH 9.0) and gently stirred at 4°C, followed by the slow addition of excess maleic anhydride (DMMA) (10 mol equivalent relative to the shell PEI); the reaction mixture was stirred at pH 9.0 for 24 h; the resulting nanoparticles were centrifuged and washed with distilled water, and freeze-dried to obtain copper nanoparticles coated with polyetherimide and maleic anhydride-modified organic mesoporous silica (Cu@PEI-DA).

[0175] Figure 15 The potential test results of Cu@PEI-DA in Example 5 are shown. It can be seen that the surface potential of Cu@PEI-DA in a solution with a pH of 7.4 is lower than -4.17 mV. As the pH decreases, the maleic anhydride on the surface of Cu@PEI-DA is hydrolyzed, and PEI is exposed on the surface of the nanoparticles, which causes the charge on the surface of Cu@PEI-DA to change from negative to positive. Under weakly acidic conditions of pH 5.5, the potential is approximately 3.02 mV.

[0176] Example 6

[0177] This example provides another method for preparing nanomedicines, which uses the same implementation as Example 2, except that the loaded drug is ilisimol.

[0178] The constructed acid-stimuli-responsive copper nanoparticles (Cu@PEI-DA, Cu@CS-DA, or Cu@PEOz-DA) were dispersed in 2 mL of ethanol solution under ultrasonic oscillation for 1 h, and then the mixture was vigorously stirred overnight. The ethanol solution contained 50 mg of ilisimol (Es).

[0179] After subsequent centrifugation, the mixture was washed with ethanol three times and freeze-dried to obtain copper nanoparticles coated with ilisimol-loaded organic mesoporous silica, namely Cu@Es.

[0180] In general, the present invention uses copper nanoparticles as the core, and the outer layer is wrapped with an organic silica shell containing X-ray responsive double bonds. The shell surface is modified with an acid-responsive block polymer with charge reversal characteristics to improve the biocompatibility of the carrier and the uptake efficiency in tumor tissue, and the copper ion carrier is loaded into the silica mesoporous channel. When the carrier enters the weakly acidic microenvironment of the tumor, the polymer modified on the shell surface is hydrolyzed, resulting in the exposure of the cationic polymer on the shell surface. This promotes the uptake of tumor cells. Subsequently, through external X-ray remote triggering, the outer shell of the carrier is promoted to rupture, resulting in the rapid release of copper nanoparticles and copper ion carriers, and the in situ generation of CuET, leading to cell apoptosis. In particular, the formation of CuET is accompanied by the formation of Cu 2+ Converted to Cu + , oligomerizes with mitochondrial proteins in the tricarboxylic acid cycle, and causes copper death in tumor cells. This solves the problems of existing nanoparticles, such as the serious side effects caused by premature release of drugs after entering the blood circulation, the difficulty in accumulation and uptake at the tumor site, and the low efficiency of drug release after entering the tumor cells, thereby achieving more precise and controllable killing of tumor cells.

[0181] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0182] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A smart responsive nanocarrier, characterized in that: The core of the invention is a copper nanoparticle, which is coated with a shell layer. The shell layer is organic mesoporous silica containing X-ray responsive double bonds, and the shell layer is modified with an acid-responsive polymer. Wherein, the copper nanoparticles are copper-containing compounds with a particle size of 10 to 100 nm, and the copper-containing compound is CuCl2, Cu(NO3)2, CuSO4, CuO, CuS or Cu3BiS3; The X-ray responsive double bond is a diselenide bond, a ditellurium bond or a disulfide bond; The acid-responsive polymer is a block polymer of polymer X modified with maleic anhydride, wherein polymer X is any one of polyetherimide, chitosan, or poly-(2-ethyl-2-oxazoline); The modification of the acid-responsive polymer on the shell is achieved by treating the copper nanoparticles coated with the shell with a crosslinker, and the crosslinker reacts with NH2 on the polymer X. The crosslinker is (3-glycidyloxypropyl)trimethoxysilane or (3-triethoxysilyl)propylsuccinic anhydride after carboxyl activation.

2. The smart responsive nanocarrier according to claim 1, wherein The X-ray responsive double bond is a diselenide bond.

3. A method for preparing the smart responsive nanocarrier according to any one of claims 1 to 2, characterized in that: The following steps are involved: obtaining copper nanoparticles; The outer layer of the copper nanoparticles is coated with organic mesoporous silica containing X-ray responsive double bonds to form a shell layer; An acid-responsive polymer is modified on the shell layer.

4. The preparation method according to claim 3, wherein The copper nanoparticles are Cu3BiS3, and their preparation includes the following steps: Dispersing bismuth salt and copper salt into oleylamine solution, degassing and stirring to mix evenly; A sulfur source is quickly injected into the reaction mixture and stirred to obtain Cu3BiS3 nanoparticles.

5. The preparation method according to claim 3, wherein The method for forming organic mesoporous silica containing X-ray responsive double bonds in the shell layer comprises the following steps: dissolving copper nanoparticles, a surfactant, and an alkaline catalyst in deionized water and mixing them uniformly to obtain a mixed solution; Adding the silica precursor and the compound containing an X-ray responsive double bond into the mixed solution in multiple portions, and stirring to react; The surfactant in the product is removed, and organic mesoporous silica containing X-ray responsive double bonds is formed on the surface of the copper nanoparticles.

6. The preparation method according to claim 3, wherein The method for modifying the acid-responsive polymer on the shell layer comprises the following steps: Dispersing copper nanoparticles coated with organic mesoporous silica containing X-ray responsive double bonds in an alcohol solvent to form a dispersion; A crosslinking agent is injected into the dispersion, and after stirring, a solution of polymer X is introduced into the reaction solution, and stirred for reaction to prepare Cu@X nanoparticles, where polymer X is polyetherimide, chitosan, or poly-(2-ethyl-2-oxazoline); Cu@X nanoparticles were dissolved in alkaline buffer and stirred at low temperature. Then, 3 to 20 mol equivalents of maleic anhydride equivalent to polymer X were added, stirred for reaction, and dried to obtain Cu@X-DA.

7. The preparation method according to claim 6, wherein The cross-linking agent is (3-glycidyloxypropyl)trimethoxysilane or (3-triethoxysilyl)propylsuccinic anhydride after carboxyl activation.

8. The preparation method according to claim 6, wherein The alkaline buffer solution is a NaHCO3 buffer solution or a Na2HPO4 buffer solution with a pH of 8 to 10.

9. An intelligent responsive copper-death nanomedicine, characterized in that: The invention relates to a smart responsive nanocarrier comprising the smart responsive nanocarrier according to claim 1 or 2 or a smart responsive nanocarrier prepared by the preparation method according to any one of claims 3 to 8, wherein the smart responsive copper death nanocarrier is loaded with a copper ion carrier, and the copper ion carrier is ilisimol or disulfiram.

10. Use of the smart responsive copper-death nanomedicine according to claim 9 in the preparation of a drug for treating breast cancer.

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