Multifunctional anti-tumor nano-drug delivery system based on copper death as well as preparation method and application of multifunctional anti-tumor nano-drug delivery system

Abstract

The invention discloses a multifunctional anti-tumor nano-drug delivery system based on copper death as well as a preparation method and application of the multifunctional anti-tumor nano-drug delivery system. The nano-drug delivery system comprises chemotherapy drug-loaded aminated diselenide bond doped mesoporous silica, a copper-based shell covering the surface of the chemotherapy drug-loaded aminated diselenide bond doped mesoporous silica, and a liposome membrane covering the surface of the copper-based shell and loaded with a small-molecule inhibitor, the copper-based shell is formed by self-assembly of copper ions and a ligand 3, 3 '-dithiobis (propionyl hydrazide). In the nano-drug delivery system, diselenide bond doped mesoporous silica has GSH responsive matrix degradation behavior, shows good biological safety, and can realize on-demand release of chemotherapeutic drugs; the nano-drug delivery system can realize acid and GSH dual-response degradation and gradually release a small-molecule inhibitor, Cu < 2 + > and a chemotherapeutic drug, has good Fenton reaction activity, can effectively block tumor cell energy supply and massive GSH consumption, and provides conditions for ferroptosis and copper hyperplasia. In-vivo and in-vitro experiments show that the combination of ferroptosis and copper death and synergistic chemotherapy can show a remarkable anti-tumor curative effect. The preparation method of the nano-drug delivery system is simple and easy to operate.

Description

Technical Field

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

[0002] Cancer has a high incidence and mortality rate, and is a major threat to human health today. However, traditional cancer treatment methods are often limited. In order to protect human life and health, more effective and safer cancer treatment strategies need to be explored. In recent years, mesoporous silica nanoparticles (MSNs) have received extensive attention in drug delivery due to their adjustable pore size, morphology, and surface functionalization. However, the slow biodegradability of their pure inorganic framework limits their application under the condition of preferred matrix degradation controlled release mechanism. Current studies have shown that integrating functional groups into the mesoporous silica framework at the molecular level can solve this shortcoming. Among them, the bond energy of the diselenide bond is relatively low, and it is easily oxidized to form selenious acid or reduced to form selenol, showing good redox reaction biodegradability.

[0003] Pemetrexed (Pem) is an antifolate preparation with a pyrrolopyrimidine group as its core structure. It can inhibit tumor replication, growth and proliferation by destroying the normal metabolic process of folate dependence in tumor cells. It is a first-line chemotherapy drug for many cancers. However, Pem can only be internalized by cells through folate carriers and is difficult to accumulate in tumor sites, so the therapeutic effect is often unsatisfactory. In addition, a single chemotherapy strategy is also difficult to effectively eliminate tumors, and the combined use of multiple treatment methods is conducive to overcoming the defects of a single treatment strategy and achieving a more comprehensive and thorough treatment effect.

[0004] Ferroptosis is a non-apoptotic cell death mode with unique advantages over apoptosis. It mainly relies on lipid peroxidation caused by intracellular iron overload and imbalance of the antioxidant system to kill tumors. However, the expression level of antioxidants in the redox homeostasis of tumor cells is significantly upregulated compared with that of normal cells, which severely limits the generation of reactive oxygen species (ROS). Among them, glutathione (GSH) can not only directly scavenge ROS, but also serve 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, thereby effectively inhibiting the occurrence of ferroptosis. Therefore, in order to induce ferroptosis with a strong 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 tumor treatment strategy. Excess 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. This 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 carriers are a type of ion carrier that can bind to Cu 2+ Ligands that transport copper into cells are mostly small molecules with a short half-life in the blood, making it difficult to deliver enough copper to cancer cells. To overcome the above obstacles, copper ion carriers and nanoplatforms have been combined for 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 copper in the cell is maintained at a very low level. Therefore, how to prepare a nanodelivery 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 provide a multifunctional anti-tumor nano drug delivery system based on copper death and its preparation method and application in view of the deficiencies in the prior art. The nano drug delivery system uses amino diselenide bond-doped mesoporous silica (SMON) as a carrier, loaded with chemotherapeutic drugs, and a copper-based shell (CuT) formed by electrostatic adsorption of copper ions and self-assembly of the ligand 3,3'-dithiobis(propionylhydrazine) (TPH). 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, which consume a large amount of GSH and reduce the activity of GPX4, leading to an imbalance of the tumor antioxidant system (ferroptosis), Cu 2+ Reduced to Cu + The three aspects of synergistic tumor treatment are destruction of mitochondrial tricarboxylic acid cycle (copper death) and inhibition of tumor replication, growth and proliferation by chemotherapeutic drugs (chemotherapy).

[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 death provided by the present invention comprises: aminated diselenide bond-doped mesoporous silica loaded with chemotherapeutic drugs, a copper-based shell coated on the surface of the aminated diselenide bond-doped mesoporous silica loaded with chemotherapeutic 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(propionylhydrazide).

[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 amination diselenide bond doped mesoporous silica to the chemotherapeutic drug is (28-32): (19-21); the amination diselenide bond doped mesoporous silica loaded with the chemotherapeutic drug and Cu 2+ , 3,3'-dithiobis(propionylhydrazide) in a mass ratio of (18-22):(7-9):(7-9); and the mass ratio of amidated diselenide bond-doped mesoporous silica coated with a copper-based shell and loaded with chemotherapeutic drugs to small molecule inhibitors 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 comprises 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 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) Add template agents hexadecyltrimethylammonium p-toluenesulfonate (CTAT) and triethanolamine (TEAH3) to deionized water, then heat and stir, then dropwise add tetraethyl orthosilicate (TEOs) and bis[3-(triethoxysilyl)propyl] diselenide (BTESePD), continue stirring to react, centrifuge after the reaction is completed to obtain a precipitate; then dissolve the precipitate in solvent 1 and stir and reflux to remove the template agent to obtain 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 aminated diselenide bond-doped mesoporous silica obtained in step 2) in solvent 2, then adding the chemotherapeutic drug solution, stirring at room temperature for a set time, opening the lid and stirring to volatilize half of the solvent, and then centrifuging and collecting the product to obtain aminated diselenide bond-doped mesoporous silica loaded with chemotherapeutic drugs;

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

[0022] 5) mixing phospholipids, cholesterol, dioleoylphosphatidylethanolamine, polyethylene glycol and small molecule inhibitor solution evenly, rotary evaporating under vacuum conditions to form a thin film, adding amino diselenide bond-doped mesoporous silica loaded with chemotherapeutic drugs and coated with a copper-based shell on the surface 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.

[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; 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; 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 step 2), the temperature of the amination treatment with stirring and reflux is 110° C., the time of stirring and reflux is 24 h, and the time of 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 chemotherapeutic 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 chemotherapeutic 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 the chemotherapeutic drug is 3:2; the concentration of amino diselenide bond-doped mesoporous silica dispersed in solvent 2 is 6 mg / mL, and the concentration of the chemotherapeutic 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 amine diselenide bond-doped mesoporous silica, Cu 2+ , 3,3'-dithiobis(propionylhydrazide) in a mass ratio of (18-22):(7-9):(7-9); the concentration of the amine 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 hours, and the stirring temperature is 30-37°C.

[0038] Further preferably, in step 4), the amine diselenide bond-doped mesoporous silica, Cu 2+ , 3,3'-dithiobis(propionylhydrazide) in a mass ratio of 20:7.8:8; the concentration of the amine diselenide bond-doped mesoporous silica loaded with chemotherapeutic 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 bond-doped mesoporous silica loaded with chemotherapeutic 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 bond-doped mesoporous silica loaded with chemotherapeutic 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 phospholipids, cholesterol, dioleoylphosphatidylethanolamine, polyethylene glycol and small molecule inhibitors 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 bond-doped mesoporous silica loaded with chemotherapeutic 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 comprises diclofenac.

[0042] The present invention also provides an application of the multifunctional anti-tumor nano drug delivery system based on copper death 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 cleaved by the reaction of SS in TPH with GSH, consuming a large amount of GSH and releasing Cu 2+ , in order to achieve 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 toxic stress and causing copper death in cells. In addition, SMON can also release the chemotherapeutic 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 at multiple levels, multiple directions and multiple angles through iron death, 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 (A) of the amino diselenide bond-doped mesoporous silica nanocarrier prepared in Example 1, TEM image after copper shell coating (B), and TEM and mapping images (C) of the SMON-P@CuT / LipD nanodrug delivery system;

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

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

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

[0049] Figure 5 The DC release of the SMON-P@CuT / LipD nano drug 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 The UV absorption graphs of the SMON-P@CuT / LipD nano drug delivery system prepared in Example 1 after co-incubation with TMB and H2O2 at different time and different pH;

[0052] Figure 8 This is a cell survival bar graph 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] Fig. 9 This is a cell survival bar graph of mouse breast cancer cells (4T1) treated with different concentrations of SMON and SMON-P@CuT / LipD nanodrug delivery systems prepared in Example 1 for 48 hours;

[0054] Fig.10 The figure is a graph showing the 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 in conjunction with 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 therein, placed in an oil bath and stirred thoroughly until the temperature rose to 80 ° C, and 533 μL of TEOs and 100 μL of BTESePD were added dropwise at the same time, 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. In order to remove the template 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 inserted into 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 h. The product was centrifuged and washed three times with ethanol, and 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, and then the mixture was magnetically stirred at 30 ° C for 24 h, where the volatilization stirring time was 12 h, and the product was collected by centrifugation to obtain amino diselenide bond-doped mesoporous silica loaded with Pem (SMON-P). The content of Pem in the supernatant was measured by UV-Vis at 226 nm, and the Pem loading rate was calculated to be 10.31% after being brought into the standard curve.

[0061] Synthesis and characterization of aminated diselenide bond-doped mesoporous silica with 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 evenly, and then rotary evaporated under vacuum to form a film. 5 mL (1 mg / mL) of SMON-P@CuT nanoparticles dispersed in water were added to hydrate the lipid film to form a nanoparticle coating layer, which was collected after centrifugation at 8000 rpm to obtain SMON-P@CuT / LipD. The content of DC in the supernatant was measured by UV-Vis at 283 nm, and the DC loading rate was calculated to be 6.32% after being brought into the standard curve.

[0063] Testing and Characterization:

[0064] 1) The 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 microstructure and elemental mapping of SMON-P@CuT / LipD are shown in Figure 2. Figure 1 As shown in the figure, from the TEM image, we can see that the morphology of the nano drug delivery system is spherical, with an obvious smooth film covering the outer layer and a particle size of about 110nm; from the mapping image, we can see that there are Si, O, Se, Cu, S, N, P and Cl elements in the nano drug 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 the figure, the nitrogen adsorption-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 3.41nm. The specific surface area of ​​SMON is calculated to be 438.71m 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 2 Figure 3 As shown in the figure, the diselenide bond in SMON-P can consume 0.56mmol / g of glutathione in 72h, while SMON-P@CuT coated with a copper-based shell with an SS structure can quickly and massively consume glutathione within 12h, with the consumption reaching 4.6mmol / g. After 12h, it gradually becomes stable, and the consumption after 72h is 6.36mmol / g. Due to the coating of the outermost liposome membrane, the glutathione consumption of SMON-P@CuT / LipD was only 0.99mmol / g in the first 8h, but it showed a trend of rapid consumption at 12h, and by 72h, its glutathione consumption had reached 6.08mmol / g. It can be seen that glutathione can fully react with SS and Se-Se over time, laying the foundation for the subsequent series of therapeutic effects.

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

[0071] Infrared spectra of relevant materials during the synthesis of SMON-P@CuT / LipD 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 1532cm, 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 graph of SMON-P@CuT was -1 The NH characteristic peak of TPH appeared at 716 cm -1 The unique C-Cl peak of DC appeared at , confirming the successful loading of DC.

[0072] The drug release curve of SMON-P@CuT / LipD is shown in Figure 5 , Figure 6 As shown in the figure, it can be seen that the DC release in SMON-P@CuT / LipD is pH-dependent, and the release rate increases with the decrease of 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 Cu. 2+ The release of Pem is related to the degradation of 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. The UV absorption peaks at different pH values ​​are shown in Figure 2. Figure 7 As shown. The colorless TMB can be oxidized to blue-green by ROS, and its corresponding maximum UV absorption peak appears at 650nm. This feature can be used to evaluate the generation of ·OH. After 1 hour of incubation, the mixed solution turned light blue, and the solution was detected by UV spectrophotometer to have an obvious 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, low pH value will promote the Fenton-like reaction to proceed in the positive direction. The above shows that SMON-P@CuT / LipD can continuously decompose H2O2 through Fenton-like reaction in the acidic microenvironment of tumor, produce toxic ·OH, and destroy the redox balance in 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, 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 cell 4T1 treated with different concentrations of SMON-P@CuT / LipD Fig. 9 As shown in the figure, according to the cell survival rate, SMON-P@CuT / LipD showed efficient and dose-dependent cytotoxicity to 4T1 cells after incubation of tumor cells for 48 hours. The IC50 value of SMON-P@CuT / LipD was 25.67μg / mL, which was much lower than SMON (376.6μg / mL), indicating that SMON-P@CuT / LipD had a strong anti-tumor 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 shown in Figure 2. Fig.10 As shown in the figure, the saline group was used as the control group, and 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 and coated with copper shell group (SMON-P@CuT), drug-loaded Pem and 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 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 inhibition, which may be due to its poor water solubility. SMON showed a certain tumor inhibition effect, which was due to the fact that the diselenide bond could destroy the redox balance in the tumor by reacting with GSH. The anticancer effect of SMON-P@CuT was significantly enhanced, indicating that Cu 2+Copper death mediated by GSH and excessive clearance of GPX4, which leads to ferroptosis, played an important role. After SMON-P@CuT / Lip treatment, the volume and weight of the tumor were further reduced, indicating that the coating of the liposome membrane enhanced the blood circulation ability of the nanodelivery system in mice. The tumor inhibition rate of SMON-P@CuT / LipD reached 88% on the 11th day, indicating that the presence of DC can more effectively enhance the anti-tumor effect on the above basis.

[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, placed in an oil bath and stirred thoroughly until the temperature rose to 85 ° C, and 400 μL of TEOs and 200 μL of BTESePD were added dropwise at the same time, 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. In order to remove the template 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 inserted into 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 h. 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 then the mixture was magnetically stirred at 30 ° C for 23 h, with a volatilization stirring time of 12 h, and then the product Pem-loaded SMON (SMON-P) was collected by centrifugation. The content of Pem in the supernatant was measured by UV-Vis at 226 nm, and the Pem loading rate was calculated to be 12.7% after being brought 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 Cu 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 evenly, and then rotary evaporated under vacuum to form a film. 5 mL (1.2 mg / mL) of SMON-P@CuT nanoparticles dispersed in water were added to hydrate the lipid film to form a nanoparticle coating layer, which was collected after centrifugation at 8000 rpm to obtain SMON-P@CuT / LipD. The content of DC in the supernatant was measured by UV-Vis at 283 nm, and the DC loading rate was calculated to be 5.12% after being brought into the standard curve.

[0087] The technical features in the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the 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-mentioned embodiments only express the concentrated implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A multifunctional anti-tumor nanodrug delivery system based on copper death, characterized in that: The nano drug delivery system comprises: an amined diselenide bond-doped mesoporous silica loaded with a chemotherapeutic drug, a copper-based shell coated on the surface of the amined diselenide bond-doped mesoporous silica loaded with a chemotherapeutic 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(propionylhydrazide); The mass ratio of the amino diselenide bond-doped mesoporous silica to the chemotherapeutic drug is (28-32): (19-21); The amine diselenide bond doped mesoporous silica loaded with chemotherapeutic drugs and Cu 2+ , 3,3'-dithiobis(propionylhydrazide) in a mass ratio of (18-22):(7-9):(7-9); The mass ratio of the aminated diselenide bond-doped mesoporous silica coated with a copper-based shell and loaded with chemotherapeutic drugs to the small molecule inhibitor 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).

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 doping amount of selenium is 15-25wt%; the average particle size of the amino diselenide bond-doped mesoporous silica is 80-90nm, and the specific surface area is 400-450m 2 / g, pore volume is 0.2~0.4cm 3 / g, pore size is 2~4nm.

3. The nano drug delivery system according to claim 1, characterized in that The chemotherapy drug includes pemetrexed; the small molecule inhibitor includes diclofenac; and 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 template agents hexadecyltrimethylammonium p-toluenesulfonate and triethanolamine to deionized water, then heating and stirring, then dropping ethyl orthosilicate and bis[3-(triethoxysilyl)propyl] diselenide, continuing to stir and react, centrifuging after the reaction is completed to obtain a precipitate; then dissolving the precipitate in solvent 1 and stirring and refluxing 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 an amination diselenide bond-doped mesoporous silica; 3) dispersing the aminated diselenide bond-doped mesoporous silica obtained in step 2) in solvent 2, then adding the chemotherapeutic drug solution, stirring at room temperature for a set time, opening the lid and stirring to volatilize half of the solvent, and then centrifuging and collecting the product to obtain aminated diselenide bond-doped mesoporous silica loaded with chemotherapeutic drugs; 4) dispersing the chemotherapeutic drug-loaded aminated diselenide bond-doped mesoporous silica obtained in step 3) in solvent 3, and then adding Cu 2+ aqueous solution and 3,3'-dithiobis(propionylhydrazide) solution, stirring for reaction, and after the reaction is completed, collecting the product by centrifugation to obtain an amino diselenide bond-doped mesoporous silica loaded with a copper-based shell and loaded with chemotherapy drugs; 5) mixing phospholipids, cholesterol, dioleoylphosphatidylethanolamine, polyethylene glycol and small molecule inhibitor solution evenly, rotary evaporating under vacuum conditions to form a thin film, adding amino diselenide bond-doped mesoporous silica loaded with chemotherapeutic drugs and coated with a copper-based shell on the surface 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 and 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, and the specific operation is: a mixed solution of the diselenide bond-doped mesoporous silica, toluene and (3-aminopropyl)triethoxysilane is stirred and refluxed, and after the reflux is completed, 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 ​​hours, and the vacuum drying time is 8-12 hours.

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 chemotherapeutic 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 chemotherapeutic 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 chemotherapeutic drug includes pemetrexed.

8. The preparation method according to claim 4, characterized in that: In step 4), the solvent 3 comprises ethanol; the amine diselenide bond-doped mesoporous silica loaded with chemotherapeutic drugs, Cu 2+ , 3,3'-dithiobis(propionylhydrazide) in a mass ratio of (18-22):(7-9):(7-9); the concentration of the amine 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 hours, 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 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; the rotary evaporation time is 10-20 min; the concentration of amino diselenide bond-doped mesoporous silica loaded with chemotherapeutic drugs coated with a copper-based shell 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 as claimed in any one of claims 1 to 3 or the nano drug delivery system prepared by the preparation method as claimed in any one of claims 4 to 9 in the preparation of tumor targeted therapeutic drugs; the tumor is breast cancer.

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