A multifunctional anti-tumor nanodrug delivery system based on copper death and its preparation method and application

Abstract

The present invention discloses a multifunctional anti-tumor nano drug delivery system based on copper death, and its preparation method and application. The nano drug delivery system comprises: an amino diselenide bond-doped mesoporous silica loaded with chemotherapy drugs, and a copper-based shell coated on the surface of the amino diselenide bond-doped mesoporous silica loaded with chemotherapy drugs, and a liposome membrane loaded with small molecule inhibitors coated on the surface of the copper-based shell; the copper-based shell is formed by self-assembly of copper ions and ligand 3,3'-dithiobis(propionylhydrazine). In the nano drug delivery system, the diselenide bond-doped mesoporous silica has GSH-responsive matrix degradation behavior, exhibits good biosafety, and can realize on-demand release of chemotherapy drugs; the nano drug delivery system can realize acidic and GSH dual-responsive degradation and gradually release small molecule inhibitors, Cu 2+ The nanoparticles, which are known to be chemotherapeutic agents, exhibit excellent Fenton reaction activity, effectively blocking the energy supply of tumor cells and significantly depleting GSH, creating conditions for ferroptosis and copper proliferation. In vitro and in vivo experiments have demonstrated that the combination of ferroptosis and copper proliferation, combined with chemotherapy, can exhibit significant anti-tumor efficacy. The preparation method of this nanoparticle drug delivery system is simple and easy to operate.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterial technology and biomedicine, and particularly relates to a multifunctional anti-tumor nanomedicine delivery system based on copper ion and a preparation method and application thereof. Background Art

[0002] Cancer, with its high incidence and mortality rates, is a major threat to human health today. However, traditional cancer treatments are often limited. To safeguard human health and safety, more effective and safer cancer treatment strategies are urgently needed. In recent years, mesoporous silica nanoparticles (MSNs) have attracted widespread attention in drug delivery due to their adjustable pore size, morphology, and surface functionalization. However, the slow biodegradability of their purely inorganic frameworks limits their application in the context of a preferred matrix degradation-controlled release mechanism. Current studies have shown that integrating functional groups into the mesoporous silica framework at the molecular level can address this shortcoming. The diselenide bond has a relatively low bond energy and is easily oxidized to form selenious acid or reduced to form selenol, exhibiting good redox biodegradability.

[0003] Pemetrexed (Pem) is an antifolate agent with a pyrrolopyrimidine core structure. It inhibits tumor replication, growth, and proliferation by disrupting normal folate-dependent metabolic processes within tumor cells. It is a first-line chemotherapy drug for various cancers. However, Pem can only be internalized by cells via a folate carrier, making it difficult to accumulate at the tumor site, resulting in unsatisfactory therapeutic effects. Furthermore, single chemotherapy strategies are also ineffective in eliminating tumors. Combining multiple treatment approaches can overcome the limitations of single treatment strategies and achieve more comprehensive and thorough therapeutic effects.

[0004] Ferroptosis is a non-apoptotic cell death method with unique advantages over apoptosis. It primarily relies on lipid peroxidation caused by intracellular iron overload and imbalance in the antioxidant system to kill tumors. However, the expression levels of antioxidants in tumor cell redox homeostasis are significantly upregulated compared to normal cells, severely limiting the generation of reactive oxygen species (ROS). Glutathione (GSH) not only directly scavenges ROS but also serves as an important substrate for glutathione peroxidase 4 (GPX4). GPX4 has the ability to reduce harmful lipid peroxides to harmless lipid alcohols, protecting cells from oxidative stress and effectively inhibiting the occurrence of ferroptosis. Therefore, in order to induce ferroptosis with a powerful therapeutic effect on tumors, it is of great significance to develop a strategy that can both achieve a large accumulation of ROS and regulate the antioxidant microenvironment of tumor cells.

[0005] Copper overload-induced cell death pathway - copper death is a promising strategy for tumor treatment. Excessive copper induces cell death by promoting abnormal oligomerization of lipoacylated proteins (DLAT) in the tricarboxylic acid cycle and reducing the level of iron-sulfur (Fe-S) cluster proteins. It is fundamentally different from the widely studied programmed cell death modes such as apoptosis and necrosis. Therefore, it has great prospects in overcoming the shortcomings of current tumor chemotherapy. Copper ion carrier is a kind of 2+ Ligands that transport copper into cells are mostly small molecules with short blood half-lives, making it difficult to deliver sufficient copper to cancer cells. To overcome these obstacles, copper ion carriers and nanoplatforms have been combined with Cu 2+ Precise targeted delivery. However, under the cellular homeostasis mechanism, excess copper is usually pumped out of the cell by ATP7A and ATP7B (the main exporters controlling copper efflux). In addition, GSH can also act as a copper chelator to inhibit copper proliferation, and the copper in the cell is maintained at a very low level. Therefore, how to prepare a nano-delivery system with a controllable structure in a simple way, break through the various limitations of this therapeutic strategy, and ultimately achieve cellular copper deposition remains a daunting challenge. Summary of the Invention

[0006] The purpose of the present invention is to address the deficiencies in the existing technology and provide a multifunctional anti-tumor nano drug delivery system based on copper death, as well as its preparation method and application. The nano drug delivery system uses amino diselenide bond-doped mesoporous silica (SMON) as a carrier, loaded with chemotherapy drugs, and electrostatically adsorbed copper ions and ligand 3,3'-dithiobis(propionylhydrazine) (TPH) self-assembled to form a copper-based shell (CuT). The outermost layer is also coated with an acid-responsive liposome membrane (Chol-SPC / DOPE) and carries a small molecule inhibitor with high selectivity for glucose transporter (Glut1). The nano drug delivery system responds to the acidic pH and high concentration of GSH in the tumor microenvironment to gradually release small molecule inhibitors, Cu 2+ , TPH (with SS structure) and chemotherapy drugs, thereby consuming a large amount of GSH and reducing GPX4 activity, leading to an imbalance in the tumor antioxidant system (ferroptosis), Cu 2+ Reduced to Cu + The three aspects of synergistic treatment of tumors are to destroy the mitochondrial tricarboxylic acid cycle (copper death) and to inhibit tumor replication, growth and proliferation (chemotherapy) by chemotherapy drugs.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The multifunctional anti-tumor nano-drug delivery system based on copper apoptosis provided by the present invention comprises: aminated diselenide bond-doped mesoporous silica loaded with chemotherapy drugs, a copper-based shell coated on the surface of the aminated diselenide bond-doped mesoporous silica loaded with chemotherapy drugs, and a liposome membrane loaded with small molecule inhibitors coated on the surface of the copper-based shell; the copper-based shell is formed by self-assembly of copper ions and the ligand 3,3'-dithiobis(propionylhydrazine).

[0009] Preferably, the doping amount of selenium is 15-25 wt % based on the total mass of the amino diselenide bond-doped mesoporous silica.

[0010] Preferably, the mass ratio of the amino diselenide bond doped mesoporous silica to the chemotherapy drug is (28-32): (19-21); the amino diselenide bond doped mesoporous silica loaded with chemotherapy drug and Cu 2+ , 3,3'-dithiobis(propionylhydrazide) is in a mass ratio of (18-22):(7-9):(7-9); the mass ratio of aminodiselenide bond-doped mesoporous silica coated with a copper-based shell and loaded with chemotherapy drugs to small molecule inhibitors is in a mass ratio of (4-6):(2-4).

[0011] Preferably, the average particle size of the amino diselenide bond-doped mesoporous silica is 80 to 90 nm, and the specific surface area is 400 to 450 m 2 / g, pore volume is 0.2~0.4cm 3 / g, pore size is 2~4nm.

[0012] Preferably, the chemotherapy drug includes pemetrexed (Pem).

[0013] Preferably, the small molecule inhibitor comprises diclofenac (DC).

[0014] Preferably, the liposome membrane is made of four raw materials: phospholipid (PC), cholesterol (Chol), dioleoylphosphatidylethanolamine (DOPE), and polyethylene glycol; the mass ratio of the phospholipid, cholesterol, dioleoylphosphatidylethanolamine, and polyethylene glycol is (3-5): (2-4): (7-9): (4-6).

[0015] More preferably, the average molecular weight of the polyethylene glycol is 1900-2100.

[0016] In some specific embodiments, based on the mass of the finally synthesized multifunctional anti-tumor nano drug delivery system, the loading rate of pemetrexed is 10-15wt%, Cu 2+ The loading rate of acetaminophen is 7-9wt%, and the loading rate of diclofenac is 4-7wt%.

[0017] The present invention also provides a method for preparing the multifunctional anti-tumor nano-drug delivery system based on copper death, comprising the following steps:

[0018] 1) Adding template agents hexadecyltrimethylammonium p-toluenesulfonate (CTAT) and triethanolamine (TEAH3) to deionized water, followed by heating and stirring, followed by dropwise addition of tetraethyl orthosilicate (TEOs) and bis[3-(triethoxysilyl)propyl] diselenide (BTESePD), and continuing the reaction by stirring. After the reaction is completed, centrifugation is performed to obtain a precipitate; the precipitate is then dissolved in solvent 1 and stirred under reflux to remove the template agent, thereby obtaining diselenide bond-doped mesoporous silica (SeMSN);

[0019] 2) performing an amination treatment on the diselenide bond-doped mesoporous silica obtained in step 1) to obtain amination-diselenide bond-doped mesoporous silica (SMON);

[0020] 3) dispersing the amino diselenide bond-doped mesoporous silica obtained in step 2) in solvent 2, then adding the chemotherapy drug solution, stirring at room temperature for a set time, opening the lid and stirring to evaporate half of the solvent, and then centrifuging to collect the product to obtain the amino diselenide bond-doped mesoporous silica loaded with the chemotherapy drug;

[0021] 4) The amine diselenide bond-doped mesoporous silica loaded with chemotherapy drugs obtained in step 3) is dispersed in solvent 3, and then Cu is added. 2+ aqueous solution and 3,3'-dithiobis(propionylhydrazide) solution, stirring for reaction, and collecting the product by centrifugation after the reaction is completed to obtain an amino diselenide bond-doped mesoporous silica coated with a copper-based shell and loaded with chemotherapy drugs;

[0022] 5) mixing phospholipids, cholesterol, dioleoylphosphatidylethanolamine, polyethylene glycol and a small molecule inhibitor solution, rotary evaporating under vacuum conditions to form a thin film, adding amino diselenide bond-doped mesoporous silica with a surface coated with a copper-based shell loaded with chemotherapy drugs dispersed in water, hydrating the lipid film to form a nanoparticle coating layer, and then centrifuging to obtain the nano drug delivery system.

[0023] Preferably, in step 1), the ratio of hexadecyltrimethylammonium p-toluenesulfonate, triethanolamine, and deionized water is (70-80) mg: (14-18) μL: (4-6) mL; and the volume ratio of ethyl orthosilicate and bis[3-(triethoxysilyl)propyl] diselenide is (400-600): (100-200).

[0024] Further preferably, in step 1), the ratio of hexadecyltrimethylammonium p-toluenesulfonate, triethanolamine, and deionized water is 75 mg:16.74 μL:5 mL; and the volume ratio of ethyl orthosilicate and bis[3-(triethoxysilyl)propyl] diselenide is 533:100.

[0025] Preferably, in step 1), the reaction temperature is 75-85° C., and the reaction time is 3-5 h; the temperature for removing the template by stirring and reflux is 65-75° C., and the stirring and reflux time is 24-48 h.

[0026] Further preferably, in step 1), the reaction temperature is 80° C. and the reaction time is 4 h; the temperature for removing the template by stirring and reflux is 70° C. and the stirring and reflux time is 24 h.

[0027] Preferably, in step 1), the solvent 1 is a mixture of ethanol and hydrochloric acid.

[0028] In step 2), in order to facilitate subsequent drug loading and shell membrane coating, the diselenide bond-doped mesoporous silica is subjected to an amination treatment. The specific operation is: a mixed solution of diselenide bond-doped mesoporous silica, toluene and (3-aminopropyl)triethoxysilane (APTES) is stirred and refluxed. After the reflux is completed, the product is collected by centrifugation, washed, and vacuum-dried to obtain the amination diselenide bond-doped mesoporous silica.

[0029] Preferably, the ratio of the diselenide bond-doped mesoporous silica, toluene and (3-aminopropyl)triethoxysilane is (200-300) mg: (32-48) mL: (0.8-1.2) mL.

[0030] Further preferably, the ratio of the diselenide bond-doped mesoporous silica, toluene and (3-aminopropyl)triethoxysilane is 250 mg:40 mL:1 mL.

[0031] Preferably, in the amination treatment in step 2), the stirring and reflux temperature is 105-115° C., the stirring and reflux time is 23-25 ​​h, and the vacuum drying time is 8-12 h.

[0032] Further preferably, in the amination treatment in step 2), the temperature for stirring and reflux is 110° C., the time for stirring and reflux is 24 h, and the time for vacuum drying is 10 h.

[0033] Preferably, in step 3), the solvent 2 comprises methanol.

[0034] Preferably, in step 3), the mass ratio of amino diselenide bond-doped mesoporous silica and the chemotherapy drug is (28-32): (19-21); the concentration of amino diselenide bond-doped mesoporous silica dispersed in solvent 2 is 5-7 mg / mL, and the concentration of the chemotherapy drug solution is 3-5 mg / mL; the total stirring time is 22-26 h, of which the volatilization stirring time is 11-13 h.

[0035] Further preferably, in step 3), the mass ratio of amino diselenide bond-doped mesoporous silica and chemotherapy drugs is 3:2; the concentration of amino diselenide bond-doped mesoporous silica dispersed in solvent 2 is 6 mg / mL, and the concentration of chemotherapy drug solution is 4 mg / mL; the stirring time is 24 h, and the volatilization stirring time is 12 h.

[0036] Preferably, in step 3), the chemotherapy drug includes pemetrexed.

[0037] Preferably, in step 4), the solvent 3 comprises ethanol; the amino diselenide bond-doped mesoporous silica loaded with chemotherapy drugs, Cu 2+ , 3,3'-dithiobis(propionylhydrazide) mass ratio is (18-22): (7-9): (7-9); the concentration of the amino diselenide bond doped mesoporous silica loaded with chemotherapy drugs dispersed in solvent 3 is 9-11 mg / mL, Cu 2+ The concentration of the aqueous solution is 32-33 mg / mL, and the concentration of the 3,3'-dithiobis(propionylhydrazide) solution is 0.5-1 mg / mL; the stirring time is 18-20 h, and the stirring temperature is 30-37° C.

[0038] Further preferably, in step 4), the amino diselenide bond-doped mesoporous silica, Cu 2+ The mass ratio of 3,3'-dithiobis(propionylhydrazide) is 20:7.8:8; the concentration of the amino diselenide bond-doped mesoporous silica loaded with chemotherapy drugs dispersed in solvent 3 is 10 mg / mL, Cu 2+ The concentration of the aqueous solution was 32.56 mg / mL, and the concentration of the 3,3'-dithiobis(propionylhydrazide) solution was 0.8 mg / mL; the stirring time was 19 h, and the stirring temperature was 37°C.

[0039] Preferably, in step 5), the mass ratio of phospholipid, cholesterol, dioleoylphosphatidylethanolamine, polyethylene glycol and small molecule inhibitor is (3-5): (2-4): (7-9): (4-6): (2-4); the concentration of phospholipid solution is 9-11 mg / mL, the concentration of cholesterol solution is 1-3 mg / mL, the concentration of dioleoylphosphatidylethanolamine solution is 3-5 mg / mL, the concentration of polyethylene glycol solution is 2-3 mg / mL, and the concentration of small molecule inhibitor solution is 2-4 mg / mL. g / mL; the rotary evaporation time is 10 to 20 min; the concentration of amino-diselenide-doped mesoporous silica loaded with chemotherapy drugs and coated with a copper-based shell dispersed in water is 0.8 to 1.2 mg / mL; the mass ratio of amino-diselenide-doped mesoporous silica loaded with chemotherapy drugs and coated with a copper-based shell, liposome membrane and small molecule inhibitor is (4 to 6): (16 to 24): (2 to 4); the hydration time is 25 to 35 min, and the hydration temperature is 36 to 38°C.

[0040] Further preferably, in step 5), the mass ratio of phospholipid, cholesterol, dioleoylphosphatidylethanolamine, polyethylene glycol and small molecule inhibitor is 4:3:8:5:3; the concentration of the phospholipid solution is 10 mg / mL, the concentration of the cholesterol solution is 2 mg / mL, the concentration of the dioleoylphosphatidylethanolamine solution is 4 mg / mL, the concentration of the polyethylene glycol solution is 2.5 mg / mL, and the concentration of the small molecule inhibitor solution is 3 mg / mL; the rotary evaporation time is 15 min; the concentration of the amino-diselenide-doped mesoporous silica loaded with chemotherapy drugs coated with a copper-based shell dispersed in water is 1 mg / mL; the hydration time is 30 min, and the hydration temperature is 37°C.

[0041] Preferably, in step 5), the small molecule inhibitor includes diclofenac.

[0042] The present invention also provides an application of the multifunctional anti-tumor nano-drug delivery system based on copper apoptosis in the preparation of tumor-targeted therapeutic drugs.

[0043] Preferably, the tumor is breast cancer.

[0044] Beneficial effects of the present invention: The present invention provides a multifunctional anti-tumor nano-drug delivery system, which can accumulate and penetrate into tumor cells through the EPR effect (high permeability and long retention), and the outermost liposome membrane responds to cleavage under the acidic microenvironment of the tumor, releasing DC to inhibit glucose transport, thereby cutting off the energy source (ATP) of ATP7A and ATP7B to reduce their dependence on Cu. 2+ The exposed copper-based shell is cracked by the reaction of SS in TPH with GSH, consuming a large amount of GSH and releasing Cu 2+ , in order to realize Cu2+ Overload of GSH creates conditions. Excessive clearance of GSH will lead to reduced GPX4 activity and weakened cell antioxidant capacity, thus inducing ferroptosis. 2+ can be reduced by GSH and FDX1 to the more toxic Cu + , destroying the mitochondrial tricarboxylic acid cycle (TCA) and causing the aggregation of DLAT and the loss of Fe-S cluster proteins, triggering protein toxicity stress and causing copper death in cells. In addition, SMON can also release the chemotherapy drug Pem in response to the redox environment through the diselenide bond, inhibiting the replication, growth and proliferation of tumors. The present invention effectively improves the sensitivity of cancer cells to copper hyperplasia, and kills tumors through multi-level, multi-directional and multi-angle ferroptosis, copper death and chemotherapy, which is conducive to overcoming the drug resistance of tumor cells to conventional chemotherapy. In addition, the multifunctional anti-tumor nano drug delivery system provided by the present invention has no toxic side effects on normal cells and has good biocompatibility and degradability. And the preparation method of the nano drug delivery system is simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 TEM image of the amino diselenide bond-doped mesoporous silica nanocarrier prepared in Example 1 (A), TEM image after copper shell coating (B), and TEM and mapping images of the SMON-P@CuT / LipD nanodrug delivery system (C);

[0046] Figure 2 The nitrogen adsorption-desorption curve and pore size distribution of the amino diselenide bond-doped mesoporous silica nanocarrier prepared in Example 1;

[0047] Figure 3 GSH consumption capacity of the Pem-loaded amino diselenide bond-doped mesoporous silica nanocarrier (SMON-P), the Pem-loaded amino diselenide bond-doped mesoporous silica with a copper-based shell (SMON-P@CuT), and SMON-P@CuT / LipD prepared in Example 1;

[0048] Figure 4 Infrared spectra of Pem, TPH, DC, the amino diselenide bond-doped mesoporous silica nanocarrier prepared in Example 1, the amino diselenide bond-doped mesoporous silica loaded with Pem, the amino diselenide bond-doped mesoporous silica with a copper-based shell and loaded with Pem, and the SMON-P@CuT / LipD nanodrug delivery system;

[0049] Figure 5 This is the DC release of the SMON-P@CuT / LipD nanodrug delivery system prepared in Example 1 under acidic conditions;

[0050] Figure 6The SMON-P@CuT / LipD nano drug delivery system prepared in Example 1 was Cu under acidic and high-level GSH conditions. 2+ and Pem release;

[0051] Figure 7 UV absorption graphs of the SMON-P@CuT / LipD nanodrug delivery system prepared in Example 1 after incubation with TMB and H2O2 at different time and different pH values;

[0052] Figure 8 This is a bar graph of cell survival of normal cells (MOVAS) treated with different concentrations of SMON and SMON-P@CuT / LipD nanodrug delivery systems prepared in Example 1 for 48 h;

[0053] Figure 9 This is a histogram of cell survival after treating mouse breast cancer cells (4T1) with different concentrations of SMON and SMON-P@CuT / LipD nanodrug delivery systems prepared in Example 1 for 48 h;

[0054] Figure 10 Graph showing changes in tumor volume and weight over time in mice in different experimental groups in Example 1. DETAILED DESCRIPTION

[0055] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following embodiments.

[0056] Example 1

[0057] Preparation of diselenide bond-doped mesoporous silica SeMSNs:

[0058] First, 5 mL of deionized water was added to a round-bottom flask, and then 75 mg of CTAT and 16.74 μL of TEAH3 were dissolved in it. The mixture was placed in an oil bath and stirred thoroughly until the temperature rose to 80°C. At the same time, 533 μL of TEOs and 100 μL of BTESePD were added dropwise, and the mixture was stirred continuously at 80°C for 4 h. Finally, the synthesized product was collected by centrifugation, washed three times with ethanol, and then dried in a vacuum. To remove the template agent CTAT, the dried product was dissolved in a mixed solvent (the mixed solvent consisted of ethanol and hydrochloric acid in a ratio of 24 mL:3 mL) at a ratio of 100 mg:27 mL and stirred and refluxed at 70°C for 24 h. Subsequently, the precipitate after centrifugation was washed with water several times and dried in a vacuum to obtain SeMSNs.

[0059] Preparation of nano drug delivery system SMON-P@CuT / LipD:

[0060] SMON loaded with Pem: First, amino groups were implanted on the surface of SeMSN to impart a positive charge, allowing the negatively charged Pem to be loaded via electrostatic adsorption. The specific steps are as follows: 250 mg of SeMSN was stirred and refluxed with 40 mL of toluene and 1 mL of APTES at 110°C for 24 hours. The product was centrifuged and washed three times with ethanol, then vacuum-dried to obtain SMON. For drug loading, SMON and Pem were dissolved in 5 mL of methanol at a mass ratio of 3:2. The mixture was then magnetically stirred at 30°C for 24 hours, with a 12-hour volatilization stirring time. The product was collected by centrifugation to obtain Pem-loaded amino-diselenide-doped mesoporous silica (SMON-P). The Pem content in the supernatant was measured by UV-Vis spectroscopy at 226 nm. The Pem loading rate was calculated to be 10.31% using a standard curve.

[0061] Synthesis and characterization of ammoniated diselenide bond-doped mesoporous silica with a copper-based shell and loaded with Pem (SMON-P@CuT): To synthesize SMON-P@CuT, 2 mL of 10 mg / mL SMON-P as prepared was added to 10 mL of ethanol containing 0.8 mg / mL 3,3'-dithiobis(propionylhydrazide), followed by the addition of 0.24 mL of 32.56 mg / mL Cu 2+ The aqueous solution was stirred at 37 ° C for 19 h. Finally, the product was centrifuged, washed three times with deionized water, vacuum dried and collected to obtain SMON-P@CuT. The Cu 2+ The loading amount is 8.25%.

[0062] Synthesis and Characterization of SMON-P@CuT / LipD: 4 mg of phospholipids, 3 mg of cholesterol, 8 mg of DOPE, 5 mg of polyethylene glycol (DSPE-mPEG2000), and 3 mg of DC were dissolved in ethanol and mixed thoroughly. The mixture was then rotary evaporated under vacuum to form a thin film. The lipid film was hydrated by adding 5 mL (1 mg / mL) of SMON-P@CuT nanoparticles dispersed in water to form a nanoparticle coating. The mixture was centrifuged at 8000 rpm and collected to obtain SMON-P@CuT / LipD. The DC content in the supernatant was measured by UV-Vis spectroscopy at 283 nm. Using a standard curve, the DC loading rate was calculated to be 6.32%.

[0063] Testing and Characterization:

[0064] 1) Morphology and structure of amination-diselenide bond-doped mesoporous silica nanocarrier and SMON-P@CuT / LipD:

[0065] The microscopic morphology of the amino diselenide bond-doped mesoporous silica nanocarrier is shown in Figure 2. Figure 1 As shown in the TEM image, the nanoparticles are spherical in shape, with uniform particles and a particle size of about 80 nm. However, after being coated with a copper shell, the surface of the nanoparticles becomes rough and the particle size is about 95 nm.

[0066] The micromorphology and elemental mapping of SMON-P@CuT / LipD are shown in Figure 2. Figure 1 As shown in the TEM image, the morphology of the nanodrug delivery system is spherical, with an obvious smooth membrane covering the outer layer and a particle size of about 110 nm. The mapping diagram shows that Si, O, Se, Cu, S, N, P and Cl elements are present in the nanodrug delivery system, among which the elements Si, O and Se representing SeMSN are evenly distributed in the middle part of the nanoparticles; the elements Cu and S representing CuT are distributed in a circle around the nanoparticles; and the elements P and Cl representing the liposome membrane and the drug DC it carries are distributed in the outermost circle of the nanoparticles.

[0067] Mesoporous structure analysis of amino diselenide bond-doped mesoporous silica nanocarriers Figure 2 As shown in Figure 2, the nitrogen adsorption and desorption curve of SMON shows a typical IV type isotherm and has an H2 type hysteresis loop, indicating that it has a clear mesoporous structure. In addition, the pore size distribution shows that the pore size of SMON is relatively uniform, which is 3.41 nm. The specific surface area of ​​SMON is calculated to be 438.71 m 2 / g, pore volume 0.27cm 3 / g, suitable for encapsulation of drug molecules.

[0068] 2) Glutathione consumption ability of SMON-P@CuT / LipD:

[0069] The glutathione consumption abilities of SMON-P, SMON-P@CuT, and SMON-P@CuT / LipD are shown in Figure 4. Figure 3 As shown, the diselenide bonds in SMON-P led to a cumulative glutathione consumption of 0.56 mmol / g within 72 hours. SMON-P@CuT, coated with a copper-based shell with an SS structure, rapidly and significantly consumed glutathione within 12 hours, reaching 4.6 mmol / g. This consumption gradually stabilized after 12 hours, reaching 6.36 mmol / g after 72 hours. SMON-P@CuT / LipD, due to its outermost liposome membrane, consumed only 0.99 mmol / g of glutathione within the first 8 hours. However, this consumption trended rapidly after 12 hours, reaching 6.08 mmol / g by 72 hours. This indicates that glutathione can fully react with SS and Se-Se over time, laying the foundation for a series of subsequent therapeutic effects.

[0070] 3) Drug loading and release of SMON-P@CuT / LipD:

[0071] The infrared spectra of the relevant materials during the synthesis of SMON-P@CuT / LipD are shown in Figure 2. Figure 4 As shown in the figure, after loading Pem, the FTIR spectrum of SMON-P is at 950cm -1 The characteristic peak of Pem appeared at 1532 cm, which was due to the stretching vibration of the CH bond on the benzene ring, confirming the successful loading of Pem. After adsorbing the copper-based shell CuT, the FTIR spectrum of SMON-P@CuT was at 1532 cm -1 The NH characteristic peak of TPH appeared at 716 cm, confirming the successful adsorption of CuT. After coating the outermost liposome membrane loaded with DC, the FTIR spectrum of SMON-P@CuT / LipD was -1 The DC-specific C-Cl peak appeared at , confirming the successful loading of DC.

[0072] The drug release curve of SMON-P@CuT / LipD is shown in Figure 2. Figure 5 、 Figure 6 As shown in the figure, the DC release from SMON-P@CuT / LipD is pH-dependent, and the release rate increases with decreasing pH value. This is because the DOPE contained in the coated liposome membrane can be cleaved in response to acidic conditions. 2+ The release of CuT and Pem not only depends on acidic pH, but is also significantly affected by high levels of GSH. This is because the SS structure in TPH reacts with GSH to cause the instability of the CuT structure, which in turn triggers the release of CuT. 2+ The release of Pem was related to the degradation of the nanocarriers caused by the reaction of diselenide bonds and GSH.

[0073] 4) ROS production ability of SMON-P@CuT / LipD:

[0074] The UV absorption peaks of SMON-P@CuT / LipD after co-incubation with 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 for different time periods are shown in the figure. Figure 7 As shown. The colorless TMB can be oxidized by ROS to blue-green, and its corresponding maximum UV absorption peak appears at 650nm. This feature can be used to evaluate the production of OH. After 1 hour of incubation, the mixed solution turned light blue, and the solution was detected by UV spectrophotometer to have a clear UV absorption peak at 650nm. As the incubation time increases, the absorbance gradually increases. In addition, pH value will also affect Cu 2+The degree of Fenton-like reaction occurs, and low pH promotes the forward direction of the Fenton-like reaction. The above shows that SMON-P@CuT / LipD can continuously decompose H2O2 through the Fenton-like reaction in the acidic microenvironment of the tumor, producing toxic ·OH, which disrupts the redox balance in the tumor.

[0075] 5) In vitro cytotoxicity of SMON-P@CuT / LipD:

[0076] Cell survival of normal MOVAS cells treated with different concentrations of SMON-P@CuT / LipD Figure 8 As shown in the results, even when normal cells were cultured at a high dose of 100 μg / mL for 48 h, more than 85% of the cells survived, indicating that SMON-P@CuT / LipD has good biocompatibility.

[0077] Cell survival of mouse breast cancer cells 4T1 treated with different concentrations of SMON-P@CuT / LipD Figure 9 As shown in the figure, SMON-P@CuT / LipD exhibited highly potent and dose-dependent cytotoxicity against 4T1 cells after 48 hours of incubation, based on cell viability. The IC50 value of SMON-P@CuT / LipD was 25.67 μg / mL, significantly lower than that of SMON (376.6 μg / mL), indicating that SMON-P@CuT / LipD possesses a potent antitumor effect.

[0078] 6) In vivo efficacy of SMON-P@CuT / LipD:

[0079] The curves of tumor volume and weight changes over time in mice in different experimental groups are as follows Figure 10 As shown, with the normal saline group as the control group, eight experimental groups were set up, including free DC, blank carrier SMON and chemotherapy drug Pem alone, drug-loaded Pem group (SMON-P), unloaded Pem coated with copper shell group (SMON@CuT), drug-loaded Pem coated with copper shell group (SMON-P@CuT), drug-loaded Pem coated with copper shell group and liposome membrane group (SMON-P@CuT / Lip), and nano drug delivery system (SMON-P@CuT / LipD). On the 11th day after treatment, the tumor volume of the normal saline group was close to 2000mm 3 , which is about 10 times larger than the initial volume. Compared with the saline group, free DC did not show obvious tumor inhibitory effect, which may be due to its poor water solubility. SMON showed a certain tumor inhibitory effect, which was due to the fact that the diselenide bond could destroy the redox balance in the tumor by reacting with GSH. The anti-cancer effect of SMON-P@CuT was significantly enhanced, indicating that Cu 2+Copper-mediated ferroptosis and excessive GSH clearance, which leads to reduced GPX4 activity and ferroptosis, play a significant role. After SMON-P@CuT / Lip treatment, tumor volume and weight further decreased, indicating that the liposome coating enhanced the blood circulation of the nanodelivery system in mice. The tumor inhibition rate of SMON-P@CuT / LipD reached 88% on day 11, indicating that the presence of DCs can further enhance the anti-tumor effect.

[0080] Example 2

[0081] Preparation of diselenide bond-doped mesoporous silica SeMSNs:

[0082] First, 5 mL of deionized water was added to a round-bottom flask, and then 75 mg of CTAT and 16.74 μL of TEAH3 were dissolved therein. The mixture was placed in an oil bath and stirred thoroughly until the temperature rose to 85°C. At the same time, 400 μL of TEOs and 200 μL of BTESePD were added dropwise, and the mixture was stirred continuously at 85°C for 5 h. Finally, the synthesized product was collected by centrifugation, washed three times with ethanol, and then dried in a vacuum. To remove the template agent CTAT, the dried product was dissolved in a mixed solvent (the mixed solvent consisted of ethanol and hydrochloric acid in a ratio of 24 mL:3 mL) at a ratio of 100 mg:27 mL and stirred and refluxed at 70°C for 48 h. Subsequently, the precipitate after centrifugation was washed with water several times and dried in a vacuum to obtain SeMSNs.

[0083] Preparation of SMON-P@CuT / LipD nanodrug delivery system:

[0084] SeMSN loaded with Pem: First, amino groups were implanted on the surface of SeMSN to make its surface positively charged, and negatively charged Pem was loaded by electrostatic adsorption. The specific steps are as follows: 250 mg of SeMSN was stirred and refluxed with 40 mL of toluene and 1 mL of APTES at 110 ° C for 24 hours. The product was centrifuged and washed three times with ethanol, and then vacuum dried to obtain SMON. For drug loading, 30 mg of SMON and 20 mg of Pem were dissolved in 5 mL of methanol, and the mixture was magnetically stirred at 30 ° C for 23 hours, with a volatilization stirring time of 12 hours, and then the product Pem-loaded SMON (SMON-P) was collected by centrifugation. The Pem content in the supernatant was measured by UV-Vis at 226 nm, and the Pem loading rate was calculated to be 12.7% after being incorporated into the standard curve.

[0085] Synthesis and characterization of SMON-P@CuT: To synthesize SMON-P@CuT, the prepared SMON-P (2 mL, 10 mg / mL) was added to 10 mL of ethanol containing 3,3'-dithiobis(propionylhydrazide) (0.9 mg / mL), and then 0.25 mL of 32.5 mg / mL of Cu was added. 2+ The aqueous solution was stirred at 36 ° C for 19 h. Finally, the product was centrifuged, washed three times with deionized water, vacuum dried and collected to obtain SMON-P@CuT. The Cu 2+ The loading amount is 8.53%.

[0086] Synthesis and Characterization of SMON-P@CuT / LipD: 5 mg of phospholipids, 4 mg of cholesterol, 8 mg of DOPE, 6 mg of polyethylene glycol (DSPE-mPEG2000), and 3 mg of DC were dissolved in ethanol and mixed thoroughly. The mixture was then rotary evaporated under vacuum to form a thin film. 5 mL of SMON-P@CuT nanoparticles (1.2 mg / mL) dispersed in water was added to hydrate the lipid film, forming a nanoparticle coating. The mixture was then centrifuged at 8000 rpm and collected to yield SMON-P@CuT / LipD. The DC content in the supernatant was measured by UV-Vis spectroscopy at 283 nm. Using a standard curve, the DC loading rate was calculated to be 5.12%.

[0087] The various technical features in the above-described embodiments can be arbitrarily combined. To make the description concise, not all possible combinations of the various technical features in the above-described 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.

[0088] The above-described embodiments merely represent a number of embodiments of the present invention. While the 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, and these variations and improvements 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 multifunctional anti-tumor nanodrug delivery system based on copper death, characterized in that: The nano drug delivery system comprises: an amino diselenide bond-doped mesoporous silica loaded with a chemotherapy drug, a copper-based shell coated on the surface of the amino diselenide bond-doped mesoporous silica loaded with a chemotherapy drug, and a liposome membrane loaded with a small molecule inhibitor coated on the surface of the copper-based shell; the copper-based shell is formed by self-assembly of copper ions and a ligand 3,3'-dithiobis(propionylhydrazine); The mass ratio of the amino diselenide bond-doped mesoporous silica to the chemotherapy drug is (28-32): (19-21); The amine diselenide bond doped mesoporous silica loaded with chemotherapy drugs and Cu 2+ , the mass ratio of 3,3'-dithiobis(propionylhydrazide) is (18~22):(7~9):(7~9); The mass ratio of amination diselenide bond-doped mesoporous silica coated with a copper-based shell and loaded with chemotherapy drugs to small molecule inhibitors is (4~6):(2~4); The liposome membrane is made of four raw materials: phospholipid, cholesterol, dioleoylphosphatidylethanolamine, and polyethylene glycol; the mass ratio of the phospholipid, cholesterol, dioleoylphosphatidylethanolamine, and polyethylene glycol is (3-5): (2-4): (7-9): (4-6); The chemotherapy drug includes pemetrexed; the small molecule inhibitor includes diclofenac.

2. The nano drug delivery system according to claim 1, characterized in that Based on the total mass of the amino diselenide bond-doped mesoporous silica, the selenium doping amount is 15-25 wt%; the average particle size of the amino diselenide bond-doped mesoporous silica is 80-90 nm, and the specific surface area is 400-450 m 2 / g, pore volume of 0.2~0.4 cm 3 / g, and the pore size is 2~4 nm.

3. The nano drug delivery system according to claim 1, characterized in that The average molecular weight of the polyethylene glycol is 1900-2100.

4. A method for preparing the nano drug delivery system according to any one of claims 1 to 3, comprising the following steps: 1) Adding the template agent hexadecyltrimethylammonium p-toluenesulfonate and triethanolamine to deionized water, followed by heating and stirring, then dropwise adding ethyl orthosilicate and bis[3-(triethoxysilyl)propyl] diselenide, and continuing to stir and react. After the reaction is completed, centrifugation is performed to obtain a precipitate; the precipitate is then dissolved in solvent 1 and stirred under reflux to remove the template agent, thereby obtaining diselenide bond-doped mesoporous silica; 2) performing an amination treatment on the diselenide bond-doped mesoporous silica obtained in step 1) to obtain amination-diselenide bond-doped mesoporous silica; 3) dispersing the amino diselenide bond-doped mesoporous silica obtained in step 2) in solvent 2, then adding the chemotherapy drug solution, stirring at room temperature for a set time, opening the lid and stirring to evaporate half of the solvent, and then centrifuging to collect the product to obtain the amino diselenide bond-doped mesoporous silica loaded with the chemotherapy drug; 4) The amine diselenide bond-doped mesoporous silica loaded with chemotherapy drugs obtained in step 3) is dispersed in solvent 3, and then Cu is added. 2+ An aqueous solution and a 3,3'-dithiobis(propionylhydrazide) solution are stirred for reaction. After the reaction is completed, the product is collected by centrifugation to obtain an amino diselenide bond-doped mesoporous silica coated with a copper-based shell and loaded with chemotherapy drugs; 5) mixing phospholipids, cholesterol, dioleoylphosphatidylethanolamine, polyethylene glycol, and a small molecule inhibitor solution, rotary evaporating under vacuum conditions to form a thin film, adding amino diselenide bond-doped mesoporous silica loaded with a chemotherapeutic drug and coated with a copper-based shell dispersed in water to hydrate the lipid film to form a nanoparticle coating layer, and then collecting by centrifugation to obtain the nano drug delivery system.

5. The preparation method according to claim 4, characterized in that In step 1), the ratio of hexadecyltrimethylammonium p-toluenesulfonate, triethanolamine, and deionized water is (70-80) mg: (14-18) μL: (4-6) mL; the volume ratio of ethyl orthosilicate and bis[3-(triethoxysilyl)propyl] diselenide is (400-600): (100-200); the reaction temperature is 75-85°C, and the reaction time is 3-5 h; the temperature for removing the template and stirring under reflux is 65-75°C, and the stirring and reflux time is 24-48 h; the solvent 1 is a mixture of ethanol and hydrochloric acid.

6. The preparation method according to claim 4, characterized in that In step 2), the diselenide bond-doped mesoporous silica is subjected to an amination treatment, specifically comprising the following steps: stirring and refluxing a mixed solution of the diselenide bond-doped mesoporous silica, toluene, and (3-aminopropyl)triethoxysilane. After the reflux, the product is collected by centrifugation, washed, and vacuum-dried to obtain the amination-diselenide bond-doped mesoporous silica. The ratio of the diselenide bond-doped mesoporous silica, toluene, and (3-aminopropyl)triethoxysilane is (200-300) mg: (32-48) mL: (0.8-1.2) mL; The stirring and reflux temperature is 105-115° C., the stirring and reflux time is 23-25 ​​h, and the vacuum drying time is 8-12 h.

7. The preparation method according to claim 4, characterized in that In step 3), the mass ratio of amino diselenide bond-doped mesoporous silica and the chemotherapy drug is (28~32):(19~21); the concentration of amino diselenide bond-doped mesoporous silica dispersed in solvent 2 is 5~7 mg / mL, and the concentration of the chemotherapy drug solution is 3~5 mg / mL; the total stirring time is 22~26 h, of which the volatilization stirring time is 11~13 h; the solvent 2 includes methanol; and the chemotherapy drug includes pemetrexed.

8. The preparation method according to claim 4, characterized in that In step 4), the solvent 3 includes ethanol; the amino diselenide bond-doped mesoporous silica loaded with chemotherapy drugs, Cu 2+ The mass ratio of 3,3'-dithiobis(propionylhydrazide) is (18~22):(7~9):(7~9); the concentration of the amino diselenide bond-doped mesoporous silica loaded with chemotherapy drugs dispersed in solvent 3 is 9~11 mg / mL, Cu 2+ The concentration of the aqueous solution is 32-33 mg / mL, and the concentration of the 3,3'-dithiobis(propionylhydrazide) solution is 0.5-1 mg / mL; the stirring time is 18-20 h, and the stirring temperature is 30-37 °C.

9. The preparation method according to claim 4, characterized in that In step 5), the mass ratio of phospholipid, cholesterol, dioleoylphosphatidylethanolamine, polyethylene glycol and small molecule inhibitor is (3-5): (2-4): (7-9): (4-6): (2-4); the concentration of the phospholipid solution is 9-11 mg / mL, the concentration of the cholesterol solution is 1-3 mg / mL, the concentration of the dioleoylphosphatidylethanolamine solution is 3-5 mg / mL, the concentration of the polyethylene glycol solution is 2-3 mg / mL, and the concentration of the small molecule inhibitor solution is 2-4 mg / mL; the rotary evaporation time is 10-20 min; the concentration of the amino-diselenide bond-doped mesoporous silica loaded with a copper-based shell and loaded with chemotherapy drugs dispersed in water is 0.8-1.2 mg / mL; the hydration time is 25-35 min, and the hydration temperature is 36-38°C; the small molecule inhibitor includes diclofenac.

10. Use of the nano drug delivery system according to any one of claims 1 to 3 or the nano drug delivery system prepared by the preparation method according to any one of claims 4 to 9 in preparing tumor-targeted therapeutic drugs; the tumor is breast cancer.

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