Nanoparticle for synergistically promoting hepatoma ferroptosis based on mesoporous manganese dioxide, salazosulfapyridine and tumor cell biological membrane as well as preparation method and application of nanoparticle

Abstract

The invention relates to the technical field of nano-drug presentation systems, in particular to a nanoparticle for synergistically promoting hepatoma ferroptosis based on mesoporous manganese dioxide, salazosulfapyridine and a tumor cell biological membrane as well as a preparation method and application of the nanoparticle. The nanoparticle in the scheme comprises mesoporous manganese dioxide which is modified with a tumor cell membrane on the surface and loaded with salazosulfapyridine. According to the technical scheme, the nanoparticles can effectively induce hepatoma cell ferroptosis through a multi-target strategy. The salazosulfapyridine and the mesoporous manganese dioxide have a synergistic effect to enhance the ferroptosis effect; the tumor cell biological membrane has homologous targeting property, so that the accumulation capacity of the nanoparticles in liver cancer tissues is remarkably improved; the nanoparticle provided by the invention not only can be used for treatment, but also can monitor the change in the treatment process in real time. According to the technical scheme, the technical problem that a nano-drug presentation system for effectively inducing ferroptosis aiming at multiple target spots is lacked in the prior art can be solved, and the technical scheme has important significance in improving the survival rate and the life quality of liver cancer patients.

Description

Technical Field

[0001] The present invention relates to the technical field of nano drug delivery systems, and in particular to nanoparticles based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm that synergistically promote liver cancer ferroptosis, as well as a preparation method and application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. In China, the morbidity and mortality of liver cancer remain high, making it a major disease that seriously threatens public health. Although early diagnosis and surgical resection or liver transplantation can treat HCC, in the advanced stage, treatment options are limited, and the only FDA-approved drug is sorafenib. However, the efficacy of sorafenib is limited in advanced patients, and the problem of drug resistance is becoming increasingly prominent. Therefore, finding new treatment strategies and targets has become a research focus.

[0003] Ferroptosis is an iron-dependent form of programmed cell death, the core mechanism of which is the abnormal accumulation of lipid peroxides, leading to the destruction of cell membrane structure. This process is closely related to glutathione (GSH) depletion, inactivation of glutathione peroxidase 4 (GPX4), and Fenton reaction-driven reactive oxygen species (ROS) burst. In hepatocellular carcinoma (HCC), tumor cells often enhance cystine uptake to synthesize GSH by upregulating SLC7A11 (system Xc-subunit), or resist ferroptosis through abnormal iron metabolism (such as high expression of ferritin heavy chain 1), leading to treatment resistance and immunosuppressive microenvironment. Therefore, targeting ferroptosis is considered an important strategy to overcome the bottleneck of HCC treatment.

[0004] There are already some ferroptosis research strategies for HCC:

[0005] (1) Exogenous Fe supplementation 2+ / Fe 3+ Catalyze the Fenton reaction to produce reactive oxygen species (·OH). However, such materials rely on endogenous H2O2 in tumors, while the concentration of H2O2 in HCC cells is generally low, which limits the efficiency of the Fenton reaction.

[0006] (2) GSH depletion strategy: Sulfasalazine (SAS) reduces cystine uptake and GSH synthesis by inhibiting system Xc-, thereby relieving the inhibitory effect of GPX4 on lipid peroxidation. However, its single-drug efficacy is limited and may fail due to the upregulation of resistance genes such as CISD2.

[0007] (3) Manganese-based materials: manganese ions (Mn 2+) catalyzes Fenton-like reactions more efficiently than iron ions and can be controlled to release in response to the tumor microenvironment (e.g., GSH overexpression). For example, manganese-deposited iron oxide nanoparticles (FMO) release Mn in an acidic environment. 2+ and Fe 3+ , synergistically enhancing ROS generation. However, it is difficult for single metal ion delivery to simultaneously address the dual needs of GSH depletion and lipid peroxidation activation.

[0008] Although the above research has made some progress, the existing strategies mostly focus on a single target (such as system Xc- or GPX4), which fails to simultaneously destroy GSH synthesis and antioxidant defense, and is easily offset by tumor cells through metabolic reprogramming or adaptive gene expression. Nanomaterials have poor biocompatibility and targeting: Some iron / manganese-based nanoparticles have cumulative toxicity in the body and lack tumor-specific response mechanisms, resulting in technical problems such as normal tissue damage, high systemic toxicity, and poor bioavailability.

[0009] Therefore, in order to further improve the therapeutic effect of HCC and control drug toxicity at the same time, it is urgent to develop a new nanodrug delivery system that can effectively induce ferroptosis targeting multiple targets. Summary of the invention

[0010] The purpose of the present invention is to provide a nanoparticle based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm that synergistically promotes liver cancer ferroptosis, so as to solve the technical problem of the lack of a nanodrug delivery system that effectively induces ferroptosis for multiple targets in the prior art.

[0011] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0012] A nanoparticle based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm synergistically promotes liver cancer ferroptosis, comprising mesoporous manganese dioxide with a tumor cell membrane modified on the surface and loaded with sulfasalazine.

[0013] Furthermore, the tumor cell biofilm is derived from liver cancer cells.

[0014] Furthermore, the mesoporous manganese dioxide is prepared by the following method: tetraethyl orthosilicate reacts in an ethanol solution containing ammonia water to obtain silicon dioxide; after silicon dioxide is compounded with polyvinyl pyrrolidone, it is heated and condensed with formaldehyde and m-diphenol in an environment containing ammonia water to form a silicon dioxide-diphenol formaldehyde resin composite; the diphenol formaldehyde resin is etched with potassium permanganate to obtain a silicon dioxide-manganese dioxide composite; and then the mesoporous manganese dioxide is obtained by strong alkali etching;

[0015] Preferably, the mesoporous manganese dioxide is prepared by the following method: tetraethyl orthosilicate is added to a stirring ethanol solution containing aqueous ammonia to obtain silica through reaction; under ultrasonic action, the solution containing silica is dripped into a polyvinyl pyrrolidone solution to obtain a silica-polyvinyl pyrrolidone complex through reaction; under ultrasonic action, diphenol, formaldehyde and aqueous ammonia are added to obtain a silica-diphenol formaldehyde resin complex through reaction; under stirring conditions, potassium permanganate solution is dripped to obtain a silica-manganese dioxide complex through reaction; the silica-manganese dioxide complex is dispersed in a sodium hydroxide solution under ultrasonic conditions to obtain mesoporous manganese dioxide after reaction and washing.

[0016] Furthermore, the mass ratio of mesoporous manganese dioxide loaded with sulfasalazine to the tumor cell biofilm is 1:1-2; the mass ratio of mesoporous manganese dioxide modified with polyacrylamide hydrochloride to sulfasalazine is 5:2.

[0017] Furthermore, a nanoparticle based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm synergistically promotes liver cancer ferroptosis, with a particle size of 118±14.78nm and a surface potential of -41.6±0.9mV.

[0018] The technical solution also provides a method for preparing nanoparticles based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm that synergistically promote liver cancer ferroptosis, comprising the following steps performed in sequence:

[0019] S1: Preparation of mesoporous manganese dioxide;

[0020] S2: using polyacrylamide hydrochloride to modify mesoporous manganese dioxide, and then loading sulfasalazine onto the modified mesoporous manganese dioxide by electrostatic adsorption to obtain mesoporous manganese dioxide loaded with sulfasalazine;

[0021] S3: Mesoporous manganese dioxide loaded with sulfasalazine and tumor cell membrane are mixed and physically extruded through a liposome extruder for 10-15 times to obtain nanoparticles.

[0022] Further, in S1, the mesoporous manganese dioxide is prepared by the following method: tetraethyl orthosilicate is added to a stirring ethanol solution containing aqueous ammonia to obtain silica through reaction; under the action of ultrasound, the solution containing silica is dripped into a polyvinyl pyrrolidone solution to obtain a silica-polyvinyl pyrrolidone complex through reaction; under the action of ultrasound, diphenol, formaldehyde and aqueous ammonia are added to obtain a silica-diphenol formaldehyde resin complex through reaction; under stirring, potassium permanganate solution is dripped to obtain a silica-manganese dioxide complex through reaction; the silica-manganese dioxide complex is dispersed in a sodium hydroxide solution under ultrasonic conditions to obtain mesoporous manganese dioxide after reaction and washing.

[0023] Further, in S2, the mesoporous manganese dioxide loaded with sulfasalazine is obtained by the following method: dripping an aqueous dispersion of mesoporous manganese dioxide into an aqueous solution of polyacrylamide hydrochloride under ultrasonic conditions, and obtaining modified mesoporous manganese dioxide through reaction; dispersing sulfasalazine in the aqueous solution of the modified mesoporous manganese dioxide under ultrasonic conditions, and obtaining mesoporous manganese dioxide loaded with sulfasalazine through reaction.

[0024] Furthermore, in S3, mesoporous manganese dioxide loaded with sulfasalazine and tumor cell membranes were stirred and mixed in a ratio of 1-2:1, and then processed using a liposome extruder; the tumor cell membranes were derived from liver cancer cells.

[0025] The technical solution also provides an application of nanoparticles based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm that synergistically promote liver cancer ferroptosis in the preparation of drugs for treating liver cancer.

[0026] In summary, the technical principle of this technical solution is:

[0027] The present invention proposes a nanoparticle based on the synergistic effect of mesoporous manganese dioxide (H-MnO2), sulfasalazine (SAS) and tumor cell biofilm, aiming to effectively induce ferroptosis of liver cancer cells through a multi-target strategy.

[0028] Mesoporous manganese dioxide (MnO2) has the characteristics of GSH-responsive degradation. In a high GSH environment, mesoporous manganese dioxide can responsively degrade and release Mn 2+ ions. This process not only consumes GSH and weakens the antioxidant capacity of cells, but also provides a catalyst for Fenton-like reactions and promotes the generation of reactive oxygen species (ROS). 2+ It can catalyze hydrogen peroxide (H2O2) to generate hydroxyl radicals (·OH), further aggravating lipid peroxidation, leading to cell membrane damage and ferroptosis. 2+ Possessing good T1 imaging capability. In vitro, it showed MRI T1 imaging capability; in vivo, MSM nanoparticles accumulated in tumors and responded to MRI T1 imaging.

[0029] Sulfasalazine (SAS) is a system Xc-inhibitor that blocks the cystine / glutamate antiporter (SLC7A11) and reduces cysteine ​​uptake, thereby inhibiting the synthesis of glutathione (GSH). SAS combined with the GSH depletion effect of mesoporous manganese dioxide forms a "double hit", further downregulating GPX4 activity, leading to irreversible accumulation of lipid peroxidation and enhancing ferroptosis.

[0030] Tumor cell biofilms have homologous targeting properties. Nanoparticles are wrapped with tumor cell biofilms extracted from liver cancer cells, which give them homologous targeting properties, significantly improving the accumulation of nanoparticles in liver cancer tissues and reducing the risk of clearance by the immune system. Tumor cell biofilms also improve drug delivery efficiency. This targeting strategy can not only increase the effective concentration of nanoparticles at the tumor site, but also reduce toxicity to normal tissues, improving the safety and effectiveness of treatment.

[0031] The beneficial effects of this technical solution are:

[0032] (1) Efficient ferroptosis induction: Through the synergistic effect of the GSH-responsive ROS generation of mesoporous manganese dioxide and the system Xc-inhibition of SAS, the ferroptosis resistance caused by GSH upregulation is effectively overcome, and the killing power against drug-resistant liver cancer cells is improved. The nanoparticles can be specifically activated in the tumor microenvironment, achieving precise attack on tumor cells and reducing the impact on healthy tissues.

[0033] (2) Improve drug bioavailability and reduce side effects:

[0034] By encapsulating sulfasalazine in nanocarriers, its water solubility and chemical stability are improved, and the bioavailability of the drug is enhanced. Since nanoparticles have tumor targeting, the distribution of drugs in non-target areas is reduced, systemic toxicity is reduced, and patient tolerance is improved.

[0035] (3) Integration of imaging and treatment:

[0036] Generated Mn 2+ With good T1 magnetic resonance imaging capabilities, the nanoparticles can not only be used for treatment, but also monitor changes during treatment in real time, such as tumor size and location, to provide a basis for personalized treatment. Combined with imaging functions, doctors can adjust treatment plans according to the patient's specific conditions to ensure the efficiency and safety of treatment.

[0037] (4) Low dose and high efficacy:

[0038] Because nanoparticles are highly targeted and efficient, lower doses of drugs can be used to achieve the same therapeutic effect, reducing drug-related side effects. Metabolized manganese ions can be excreted from the body through the kidneys, effectively reducing the risk of toxicity to major organs.

[0039] (5) Innovation and clinical application potential:

[0040] This solution integrates metal ion catalysis, small molecule drug intervention and microenvironment responsive nanotechnology, providing a new therapy with high efficiency and low toxicity, which has clear clinical application value for the treatment of hepatocellular carcinoma. By combining multiple technical advantages, this invention not only provides new ideas for the treatment of liver cancer, but also provides a reference model for the treatment of other types of cancer, promoting the development of the field of nanomedicine.

[0041] In summary, by combining the advantages of multiple technologies and materials, the present invention designs a new type of nano drug delivery system, which not only solves the limitations of existing treatment methods, but also opens up new treatment approaches, which is of great significance for improving the survival rate and quality of life of liver cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a transmission electron microscope image of the MSM of Example 1 of the present invention.

[0043] Figure 2 This is the fitted particle size distribution image of the MSM of Example 1 of the present invention under an electron microscope.

[0044] Figure 3 1 is the potential distribution image of various nanoparticles in Example 1 of the present invention.

[0045] Figure 4 This is the iron ion XPS image of the MSM of Example 1 of the present invention.

[0046] Figure 5 This is a Fourier infrared image of the MSM of Example 1 of the present invention.

[0047] Figure 6 The hydrodynamic particle size and particle size distribution images of various nanoparticles in Example 1 of the present invention.

[0048] Figure 7 This is a Coomassie blue staining image of the gel electrophoresis of MSM of Example 1 of the present invention.

[0049] Figure 8 The hydrodynamic size change and charge change curve of MSM under physiological conditions were monitored over a period of 7 days in Example 1 of the present invention.

[0050] Fig. 9 This is an image of the experimental results of the oxygen production capacity of the MSM of Example 2 of the present invention.

[0051] Fig.10 This is an image of the Fenton-like effect evaluation experimental results of the MSM of Example 2 of the present invention.

[0052] Fig.11 This is an image showing the test results of the GSH consumption capacity of the MSM of Example 2 of the present invention.

[0053] Fig.12 This is an image of the experimental results of in vitro nuclear magnetic resonance T1 imaging of MSM according to Example 2 of the present invention.

[0054] Fig.13 This is an image of the experimental results of nuclear magnetic resonance T1 in vivo imaging of MSM in Example 2 of the present invention.

[0055] Fig.14 This is an image of the quantitative analysis results of the nuclear magnetic resonance signal intensity of the MS and MSM nanoparticles of Example 2 of the present invention.

[0056] Fig.15 This is an image of the targeting ability test results of MSM in Example 3 of the present invention (immunofluorescence confocal imaging).

[0057] Fig.16 These are fluorescent images of nanoparticles in different organs of tumor-bearing mice injected with nanoparticles according to Example 3 of the present invention.

[0058] Fig.17 These are images of fluorescent in vivo imaging results at different time points of tumor-bearing mice injected with nanoparticles according to Example 3 of the present invention.

[0059] Fig.18 This is a microscopic image of liver cancer cells treated with MSM according to Example 4 of the present invention (showing cell ferroptosis characteristics).

[0060] Fig.19 This is an image of the detection result of the cell membrane potential change after MSM treatment in Example 4 of the present invention (based on JC-1 dye).

[0061] Fig. 20 This is an image of the research results of Example 4 of the present invention on the effects of different nanoparticle treatments on the generation of ROS by cells (based on the DCFH-DA probe).

[0062] Fig.21 This is an image of the research results of the effects of different nanoparticle treatments on cellular lipid peroxidation in Example 4 of the present invention (based on the BODIPY-C11 probe).

[0063] Fig. 22 This is an image of the research results of Example 4 of the present invention showing the effects of different nanoparticle treatments on intracellular GSH levels and MDA levels.

[0064] Fig.23 This is an image of the WB experimental results of the effects of different nanoparticle treatments on the expression of ferroptosis-related proteins in cells according to Example 4 of the present invention.

[0065] Fig.24 This is an image of the in vivo biosafety and biodistribution test results of MSM in Example 5 of the present invention.

[0066] Fig.25 This is an image of the hemolysis rate detection result of MSM in Example 5 of the present invention.

[0067] Fig.26 This is an image of the in vivo therapeutic effect experimental results of MSM in Example 6 of the present invention.

[0068] Fig. 27 The images are the tunnel staining and HE staining experimental results of the tumor tissue in Example 6 of the present invention.

[0069] Fig.28 The images are the experimental results of DHE staining and C11 (C11BODIPY) staining of tumor tissue in Example 6 of the present invention.

[0070] Fig.29 This is a transmission electron microscope image of the nanoparticles of Comparative Example 3 of the present invention.

[0071] Fig.30 These are appearance images of nanoparticles obtained by different preparation methods in Comparative Example 3 of the present invention.

[0072] Fig.31 This is a statistical diagram of the particle sizes of nanoparticles obtained by different preparation methods in Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0073] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0074] Example 1: Preparation of PAH-MnO2@SAS@Membrane Nanoparticles

[0075] In order to synthesize H-MnO2 nanospheres, 98ml of anhydrous ethanol was mixed with 10ml of ultrapure water, and then 1.25ml of 25% ammonia water was added. 5ml of tetraethyl orthosilicate TEOS was added under stirring, and stirred at room temperature or 25°C (the speed of the following schemes is 300RPM unless otherwise specified) for 20h. SiO2 nanoparticles were obtained by centrifugal washing at 12000RPM for multiple times. 50mg of SiO2 nanoparticles were dropped into 500mg of 10ml PVP (polyvinyl pyrrolidone) solution under ultrasound. Stir overnight, centrifugal washing at 12000RPM for multiple times to obtain SiO2@PVP. 28ml of the obtained 50mg of SiO2@PVP was added with 20mg of meta-diphenol and 28μl of formaldehyde under ultrasound and mixed evenly, and 100μl of 2.8% ammonia water was added under stirring, and the reaction was carried out at 60°C for 2h and then at 100°C for 2h. After the reaction is cooled, the solution is centrifuged at 12000RPM and washed with water for several times to obtain SiO2@RF. Then, the m-diphenol formaldehyde resin is oxidized by the strong oxidizing property of potassium permanganate. 200μl of 0.2M KMnO4 solution is added to the 5ml (10mg / ml SiO2) solution and stirred for 4h. The excess KMnO4 is then washed with water to obtain SiO2@MnO2. Finally, the SiO2 core is etched away by NaOH. 50mg SiO2@MnO2 is dispersed in 10ml 1M NaOH solution under ultrasound and stirred overnight. After etching away the SiO2 core, the solution is washed with water for several times to remove the unreacted NaOH and obtain H-MnO2 nanoparticles (mesoporous manganese dioxide).

[0076] 1 ml of the obtained H-MnO2 (5 mg / ml, prepared with water as solvent) was dripped into 1 ml of 5 mg / ml polyacrylamide hydrochloride (PAH, adamas, CAS: 71550-12-4, molecular weight: 93.56) solution (prepared with water as solvent) under ultrasound and stirred for 4 hours. After the obtained PAH-MnO2 was washed with water by centrifugation at 12000RPM to remove excess PAH, 2 mg of sulfasalazine (SAS) was dispersed in 1 ml of PAH-MnO2 (5 mg / ml) solution (prepared with water as solvent) under ultrasound, stirred overnight (8-12 hours), and unbound SAS was washed off with anhydrous ethanol at 12000RPM to obtain PAH-MnO2@SAS (tested, using the method of this embodiment, the drug loading rate of SAS on PAH-MnO2@SAS is about 23%). The mass ratio of H-MnO2 to PAH can be 1:1-5 (preferably 1:1), and the mass ratio of PAH-MnO2 to SAS is 5:2. By adopting the overall process flow of this technical solution, the drug loading rate of SAS can reach 22-25%.

[0077] Approximately 2×10 8 After the cells were lysed by repeated freezing and thawing, the cells were placed in an ice bath to prevent overheating during the disruption process and denaturation of the membrane. The cells were disrupted by an ultrasonic cell disruptor (400W, 10min), and the supernatant was removed by centrifugation at 15000g to obtain the liver cancer cell membrane. The obtained liver cancer cell membrane was mixed with the PAH-MnO2@SAS obtained above at a mass ratio of 1-2:1 and then physically extruded 10-15 times by a lipid extruder to obtain PAH-MnO2@SAS@Membrane (MSM).

[0078] The obtained PAH-MnO2@SAS@Membrane (MSM) was tested, and the transmission electron microscope image is as follows: Figure 1 The fitted particle size distribution and potential distribution under electron microscope are shown in Figure 2 , Figure 3 The average particle size of MSM nanoparticles is 118±14.78nm, and the surface potential is -41.6±0.9mV. Figure 3 In this figure, SiO2 refers to the SiO2 nanoparticles prepared in the previous article, RF refers to SiO2@RF nanoparticles, HM refers to H-MnO2 nanoparticles, and MS refers to PAH-MnO2@SAS nanoparticles.

[0079] XPS images of MSM nanoparticles Figure 4 As shown, the Fourier infrared image is Figure 5 shown.

[0080] The hydrodynamic particle size and particle size distribution of SiO2, SiO2@RF, H-MnO2, H-MnO2@SAS and H-MnO2@SAS@M were determined by dynamic light scattering (DLS) analysis. Figure 6 As shown. The measured DLS hydrated particle size is larger than its geometric diameter, which may be related to the hydration layer formed by the groups on the surface of the nanoparticles and the solvent molecules adsorbed on the surface of the nanoparticles. This size is in the range that can significantly enhance the EPR effect (Enhanced Permeability and Retention effect), thereby improving the retention and penetration of drugs in tumor tissues.

[0081] like Figure 7 As shown, Coomassie blue staining of gel electrophoresis was performed to verify the presence of tumor cell membrane molecules in MSM.

[0082] like Figure 8As shown, the hydrodynamic size changes and charge changes of MSM under physiological conditions were monitored over a period of 7 days. In addition, the hydrodynamic size changes in different solvent systems were also observed. During the observation period, the size and potential fluctuations of MSM in dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) were not obvious, and the size fluctuations in deionized water (DI), phosphate buffered saline (PBS) and dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) were not obvious (SI), indicating that MSM has good water stability.

[0083] Example 2: Microenvironmental response and ROS generation characteristics of MSM

[0084] The liver cancer microenvironment is characterized by an acidic environment and a high GSH content due to its abnormal metabolism. Figure 4 XPS shows that MnO2 4+ In a high-valent state, it has strong oxidizing properties and can undergo redox reactions with GSH to generate low-valent Mn 2+ , the generated Mn 2+ It can further catalyze H2O2 to generate OH free radicals. The reaction process is as follows:

[0085] MnO2+GSH(glutathione)→Mn 2+ +GSSG (oxidized glutathione);

[0086] MnO2+H + →Mn 2+ +H2O;

[0087] MnO2+GSH→MnO(OH)+GSSG;

[0088] Mn2 + +H2O2→Mn(OH)2+H2O2→Mn 2+ +H2O+O2+OH.

[0089] MnO2 can catalyze the decomposition of endogenous H2O2 into O2, which is commonly used in TME for hypoxia relief. Tumor reoxygenation may inhibit angiogenesis, reduce invasiveness, and reduce the risk of metastasis. Fig. 9 The catalase mimetic activity of MSM was evaluated by monitoring the dissolved oxygen level for up to 10 minutes, indicating that the MSM in this scheme has good catalase mimetic activity. Fig.10As shown, in order to evaluate its Fenton-like effect, the ·OH yield of MSM was characterized by a colorimetric method based on methylene blue (MB). H2O2, GSH and MSM could not change the spectral signal of MB solution containing NaHCO3. "MSM+H2O2+GSH+NaHCO3" caused a sharp decrease in MB absorbance at 670nm, and the amount of ·OH produced was the largest. It can be seen that the peroxidase mimetic activity of MSM undoubtedly provides hope for CDT treatment of tumors.

[0090] like Fig.11 As shown, GSH can react with the color-developing substrate 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent) to generate yellow 2-nitro-5-thiobenzoic acid (TNB). Based on this principle, the ability of nanoparticles to consume GSH can be detected. It can be seen that the MSM in this scheme has the effect of consuming GSH.

[0091] like Fig.12 As shown, the Mn generated by the reaction 2+ It has good in vitro imaging ability, which provides hope for simultaneous monitoring of ferroptosis treatment. The experimental results show that the MSM nanoparticles of this scheme have the effect of in vitro imaging and are a potential clinically applicable imaging agent for nuclear magnetic resonance T1 imaging.

[0092] like Fig.13 In the TI in vivo imaging experiment shown, the H-MnO2@SAS@Membrane (MSM) and H-MnO2@SAS (MS) groups were injected through the tail vein, and T1 MRI was performed on the tumor area at different time intervals (0, 2, 4, 8, 12, 20h). The experimental results show that the MSM nanoparticles of this scheme have the function of TI in vivo imaging and are a potential clinically applicable imaging agent for nuclear magnetic resonance T1 imaging.

[0093] In addition, the NMR signal intensity analysis results are detailed in Fig.14 .

[0094] Example 3: In vivo and in vitro targeting of MSM

[0095] Fig.15 The results showed that biofilm-coated PAH-MnO2@SAS@Membrane (MSM) nanoparticles had superior targeting ability compared to uncoated PAH-MnO2@SAS (MS) nanoparticles. MS and MSM (50 μg mL -1 ) were co-cultured with well-growing Hepa1-6 cells in a six-well plate (2×10 cells per well). 5), co-incubated for 0.5, 1, 2, 4, and 6 hours, the cells after co-incubation were fixed with 4% paraformaldehyde, and the cell nuclei were stained with DAPI. The uptake of MS and MSM by cells was evaluated by confocal microscopy. Obviously, the phagocytosis of nanoparticles by cells showed a time-related cellular uptake behavior. Moreover, the phagocytosis of MSM by cells was better than that of MS.

[0096] In vivo fluorescence imaging was further performed using isoflurane inhalation to anesthetize Hepa1-6 tumor-bearing mice. Cy5-labeled H-MnO2@SAS and H-MnO2@SAS@Membrane nanoparticles were injected intravenously into the mice through the tail vein (50 μg mL -1 ).like Fig.17 As shown, the fluorescence images were acquired at 0, 1, 2, 4, 6, 12, and 24 hours after injection using the in vivo imaging system. 24 hours after injection, the mice were sacrificed to observe the in vitro biodistribution. Fig.16 As shown, the tumor, heart, liver, spleen, lung, and kidney were removed, and fluorescence images were acquired using an imaging system, and the mean fluorescence intensity was quantitatively analyzed.

[0097] Example 4: In vitro ferroptosis mechanism of MSM

[0098] In view of the above studies confirming that MSM has good targeting, T1 imaging and unique tumor cell microenvironment responsiveness, this example further evaluates the ferroptosis therapeutic mechanism of MSM at the cellular level. In order to observe the ferroptosis characteristics of liver cancer cells, biological electron microscopy was performed to observe the cell characteristics. Well-cultured liver cancer cells were inoculated into 10 cm culture dishes, incubated overnight with DMEM basal medium containing 100 μg / ml MSM, and then fixed with electron microscopy fixative and ultrathin sections were observed by TEM. The MSM results are shown in Figure 2. Fig.18 As shown in the results, compared with the control group, the experimental group showed obvious characteristics of ferroptosis, including mitochondrial shrinkage, reduction or disappearance of abnormal cristae, outer membrane rupture, and rupture of cell membrane integrity with visible vesicles.

[0099] To further explore the functional changes of mitochondria, we used MnO2, SAS, MS, MSM (added to DMEM culture medium) at a concentration of 100 μg / ml and the Pbs group without drug treatment to incubate liver cancer cells overnight, and then used JC-1 dye to monitor the changes in MMP (mitochondrial membrane potential). When the cell membrane potential decreases, the JC-1 dye changes from a red fluorescence aggregated state to a green fluorescence dispersed state. Confocal laser scanning microscopy results showed that compared with the control group, cells treated with MSM showed an increase in green fluorescence signals, while the higher the concentration of nanoparticles, the weaker the red fluorescence signal, indicating that the JC-1 dye changed from an aggregated state to a dispersed state ( Fig.19). The above experimental phenomenon is one of the characteristics of ferroptosis.

[0100] In order to further explore the principle, after the tumor cells engulf MnO2, it will react with GSH in the tumor cells to generate Mn 2+ . The 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect the generation of ·OH, because the non-fluorescent DCFH-DA can be converted into fluorescent DCF through ROS oxidation. Specifically, after Hepa1-6 cells were treated with DMEM (PBS was used instead of drugs), MnO2, SAS, MS, and MSM groups (drug concentration: 100μg / ml) for 6 hours, the DCFH probe was first used to co-incubate with the above groups. After incubation, the fixed cells were stained with the nuclear dye DAPI to determine that the cells were in good condition. The DCF fluorescence level was detected by CLSM. The DCF signal of the DMEM group was negligible, and MS and MSM had stronger DCF fluorescence signals than the MnO2 group and the SAS group ( Fig. 20 ). This indicates that the combination of MS and MSM enhanced the generation of ROS and the onset of ferroptosis in Hepa1-6 cells. The analysis may be because: MS and MSM simultaneously reduce the intracellular GSH level through the exogenous pathway.

[0101] In addition, excessive ROS production will lead to lipid peroxidation (LPO) and mitochondrial damage, resulting in ferroptosis. Therefore, the fat-soluble LPO fluorescent indicator BODIPY-C11 fluorescent probe (581 / 591nm) was used to evaluate the LPO produced by ·OH free radicals. Hepa1-6 cells were treated with DMEM, MnO2, SAS, MS, and MSM (drug concentration: 100μg / ml) in groups for 6 hours, and the LPO level in the cell process was detected by BODIPY-C11 fluorescent probe. The fluorescence of BODIPY-C11 was observed by confocal microscopy, and it was found that the combination of MS and MSM had a higher level of LPO fluorescence. In addition, this phenomenon was also found by FCM quantitative detection, indicating that the combination of MS and MSM produced more ·OH free radicals and produced higher levels of LPO ( Fig.21 , from left to right: control (Pbs), SAS, MnO2, MS, MSM).

[0102] The effect of nanoparticles on the intracellular GSH level was detected, and the MDA in the cells was detected by using the MDA kit. The experimental results are shown in Fig. 22 . It can be seen from the experimental data that MS and MSM simultaneously reduce the intracellular GSH level through exogenous pathways. The MS and MSM groups have higher levels of MDA, and the MDA level of MSM is higher than that of the MS group. The inventors analyzed that the reason is that MSM has an additional cell membrane, which leads to higher lipid metabolism.

[0103] Hepa1-6 cells were treated with DMEM medium containing 100 μg / ml MnO2, SAS, MS, and MSM for 24 hours, and the expression of related proteins was detected by WB. The MS and MSM groups effectively inhibited the endogenous expression of GPX4 by inhibiting the cystine / glutamate antiporter xCT / SLC7A11 on the cell membrane, leading to ferroptosis ( Fig.23 , A is the WB experimental result diagram, and B is the signal intensity statistical diagram of A).

[0104] Example 5: In vivo biosafety and biodistribution of MSM

[0105] This example studies the biosafety of MSM in 6-8 week old male C57 mice. The mice were randomly divided into 5 groups, namely the control group and the groups injected with MSM NPs at 0, 1, 3, 7, and 14 days. Each mouse received an injection of 100 μl of MSM (concentration of 100 μg mL -1 ). The mice were euthanized after 14 days. Blood samples were collected for routine blood tests and biochemical examinations, and major organs were removed for H&E staining and histological analysis. The experimental results are shown in Fig.24 , indicating that the nanoparticles in this scheme have good biosafety.

[0106] The collected blood was used to separate the red blood cells. The separated red blood cells were washed with PBS for several times and then prepared into a red blood cell suspension with physiological saline. A 2 ml EP tube was taken and the red blood cell suspension was added. It was mixed with the pre-prepared MSM physiological saline solution of different concentrations. The mixed solution was placed in a 37°C oven for 3 hours, and then centrifuged at 3000 rpm for 10 min to observe the hemolysis of the supernatant. At the same time, a negative control without nanoparticles and a positive control with only water were set up. The supernatant was taken to detect the absorbance at 577 nm. The calculation formula is: Hemolysis rate = (sample to be tested - negative absorbance) / (positive - negative) × 100%, and the experimental results are shown in Fig.25 .

[0107] Example 5: Evaluation of therapeutic effects on subcutaneous tumors in C57 mice

[0108] Based on the high tumor targeting and tumor-specific cytotoxicity of MSM, its tumor-killing effect on C57 mice carrying Hepa1-6 cell tumors in vivo was further studied. The dosing regimen was as follows: Fig.26 As shown in A. All mice (6-8 weeks) were divided into the following groups:

[0109] (1) Normal saline group; (2) MnO2 group; (3) SAS group; (4) MS group; (5) MSM group.

[0110] After the tumor was implanted subcutaneously on day -7, the tumor size was measured every other day starting from day 0 to draw a tumor volume change curve (26B). The mice were given a tail vein drug once every three days, with a dosage of 100 μl (drug concentration was 100 μg / ml). On day 14, the mice were killed, and the tumor tissue was extracted, weighed, and photographed ( Fig.26 C, 26E), and the tumor inhibition rate was calculated (26D). Compared with the saline group, the MnO2 group and the SAS group had a general tumor inhibition effect, with tumor growth inhibition (TGI) indexes of 39.69% and 42.35% on day 14, respectively, which were attributed to their single-target treatment of Fenton reaction and inhibition of GSH source, respectively. The MSM group had the most significant tumor inhibition effect, with a TGI (tumor growth inhibition) index of 73.39%, which was caused by malignant iron necrosis / apoptosis caused by oxidative stress damage induced by CDT (chemodynamic therapy) and aggravated by xCT (SLC7A11) inhibition.

[0111] The results of PCNA, TUNNEL and HE staining of tumor tissues are detailed in Fig. 27 , the MS / MSM group showed higher tumor apoptosis signals and inhibited tumor growth (scale bar 100um).

[0112] The results of DHE staining and C11 (C11BODIPY) staining of tumor tissues are detailed in Fig.28 , the MS / MSM group showed higher levels of ROS and lipid peroxides (scale 100um).

[0113] Comparative Example 1: Effect of Sulfasalazine (SAS)

[0114] This comparative example illustrates the effect of using sulfasalazine (SAS) alone to inhibit the System Xc-system for liver cancer ferroptosis treatment. Sulfasalazine (SAS) is a classic ferroptosis inducer. It blocks cysteine ​​uptake by inhibiting the System Xc-system, resulting in the blockage of intracellular glutathione (GSH) synthesis, and ultimately induces lipid peroxidation and ferroptosis. However, the use of SAS alone has the following problems:

[0115] Incomplete GSH consumption: The GSH level in hepatocellular carcinoma cells (HCC) is high, and inhibiting System Xc alone cannot completely deplete GSH. The residual GSH can still clear lipid peroxides through GPX4 (glutathione peroxidase 4), weakening the ferroptosis effect.

[0116] Drug resistance: Some liver cancer cells are resistant to SAS-induced ferroptosis due to overexpression of S1R receptors or upregulation of iron chelating factors such as LCN2.

[0117] Lack of synergistic oxidative stress: SAS cannot directly produce hydroxyl radicals (·OH) through the Fenton reaction and must rely on intracellular background iron ions, while the iron ion level in the tumor microenvironment is limited.

[0118] Different from the comparative example, the present scheme releases Mn by GSH-responsive degradation of manganese dioxide (MnO2). 2+ It triggers the Fenton reaction, directly produces a large amount of ·OH, and the degradation process of MnO2 consumes 2 molecules of GSH (one -Mn-O- bond is broken each time), which synergizes with SAS to achieve double GSH depletion (inhibition of synthesis + direct consumption) and multi-pathway ROS generation, significantly enhancing oxidative stress. 2+ The release of MnO2 can achieve real-time imaging and provide a visualization tool for efficacy evaluation. It can be seen that the technical solution combines MnO2 and SAS to achieve synergistic effects in GSH depletion, increase ROS generation, etc., and can also achieve nuclear magnetic resonance T1 imaging, achieving multiple effects.

[0119] Comparative Example 2: Manganese-doped silica nanoparticles combined with sorafenib

[0120] In the prior art, manganese-doped mesoporous silica nanoparticles (MMSNs@SO2) developed by Tang et al. consume GSH through degradation and release sorafenib to inhibit System Xc-system and induce ferroptosis (Dual GSH-exhausting sorafenib loaded manganese-silica nanodrugs for inducing the ferroptosis of hepatocellular carcinoma cells, doi.org / 10.1016 / j.ijpharm.2019.118782). However, this scheme has the following shortcomings: Single drug synergistic mechanism: Sorafenib mainly targets Raf kinase and angiogenesis, and its inhibitory effect on System Xc-system is weaker than SAS, and the Fenton activity of manganese ions is not fully utilized. Limited GSH responsiveness: The -Mn-O-bond of MMSNs only breaks when the GSH concentration is high, and the GSH level in the liver cancer microenvironment is heterogeneous, which may lead to uneven drug release. Lack of real-time monitoring: The imaging function of manganese ions is not integrated, and the distribution and efficacy of drugs in tumors cannot be dynamically evaluated.

[0121] Compared with this comparative example, this technical solution uses manganese dioxide (rather than silicon dioxide) as a carrier, and its stronger redox activity can respond to degradation in a wider range of GSH concentrations, ensuring that Mn 2+At the same time, SAS has a significantly higher inhibitory efficiency on System Xc-system than sorafenib, forming a double metabolic blow with the consumption of GSH by MnO2, and 2+ The Fenton reaction mediated by Mn amplifies lipid peroxidation. 2+ The MRI imaging function realizes visual monitoring of the treatment process and provides a basis for precise dose adjustment.

[0122] The above advantages have not been achieved in existing studies using SAS or manganese-based nanomedicines alone, highlighting the breakthrough of this solution in the treatment of liver cancer. It can be seen that the "MnO2-SAS-biofilm" ternary synergistic concept effectively makes up for the current shortcomings of liver cancer ferroptosis treatment, such as poor targeting, low ferroptosis bioavailability, and high systemic toxicity.

[0123] Comparative Example 3

[0124] This scheme uses a resin structure synthesized by a hydrothermal method as a soft template, and uses a low concentration of potassium permanganate to react to generate manganese dioxide by an oxidation-reduction method. After etching, mesoporous manganese dioxide is formed.

[0125] In the prior art, mesoporous manganese dioxide is usually prepared by the hard template method, which is a scheme of etching silicon dioxide with high concentration of potassium permanganate to generate mesoporous manganese dioxide. The hard template method is a common method for preparing mesoporous materials. It uses solid silicon dioxide particles as templates, deposits the target material (manganese dioxide) around these particles, and then removes the template to leave a mesoporous material with a specific structure and porosity. However, the hard template method has the problem of uncontrollable size and thickness.

[0126] This technical solution controls the thickness of the soft template by controlling the feed ratio of the resin raw material to the silicon dioxide, thereby controlling the thickness of the generated mesoporous manganese dioxide and the size of the generated manganese dioxide. Fig.29 As shown, from A to C, the ratios of resin raw materials (isopentylphenol and formaldehyde) to silica are 1:100, 1:50, and 1:100, respectively, and the SiO2@RF and the corresponding mesoporous manganese dioxide (H-MnO2) obtained according to the preparation method of Example 1.

[0127] Comparative Example 4

[0128] This scheme is the first attempt to load sulfasalazine (SAS, CAS: 599-79-1) on mesoporous manganese dioxide. SAS has the following limitations: poor solubility, low bioavailability and easy systemic toxicity. As a small molecule drug, SAS has a molecular structure as shown in formula (1), with a salicylic acid group and a sulfonamide group connected by a diazo bond, and has the characteristic of the sulfonamide group being difficult to dissolve in water. Through this scheme, its solubility can be effectively improved to achieve improved bioavailability.

[0129] Due to the poor water solubility of SAS, technicians in this field usually use anhydrous ethanol for drug loading. However, actual research has found that this conventional method is not ideal. After many attempts, it was finally found that only using water as a solvent to load sulfasalazine by electrostatic adsorption is the most ideal.

[0130]

[0131] Before forming this technical solution, the inventor conducted a lot of attempts on the SAS loading method, which are as follows:

[0132] Attempt 1:

[0133] According to the method of Example 1, H-MnO2 nanoparticles (mesoporous manganese dioxide) were synthesized, and then H-MnO2 and SAS were mixed and incubated according to the ratio and concentration of Example 1 (without PAH modification, 2 mg SAS was dispersed in 5 mg / ml H-MnO2 solution), and the H-MnO2 nanoparticles after adding SAS and incubation were tested to determine whether SAS was loaded on H-MnO2. The test results showed that SAS was basically not loaded on H-MnO2 nanoparticles. Therefore, directly loading SAS on H-MnO2 nanoparticles, using water as solvent, and not adding PAH poly-modification, such a process means cannot meet the requirements of this scheme, and it is necessary to explore new process schemes.

[0134] In addition, the particle size of the nanoparticles H-MnO2@SAS prepared according to this scheme is 112.3 nm, which is similar to the particle size of HMnO2 prepared in Example 1 of this scheme (the particle size of HMnO2 prepared in Example 1 is about 111.6 nm).

[0135] Attempt 2:

[0136] According to the experimental results of attempt 1, it is not feasible to use mesoporous manganese dioxide to directly load drugs, which may be caused by excessive surface charge. In this attempt, PAA (polyacrylic acid, adamas, CAS: 9003-01-4, molecular weight range 2000-3000) was used to increase the drug loading. This process flow is basically based on Example 1, except that the surface charge is reduced by PAA (polyacrylic acid) modification, and the PAH in the original Example 1 is replaced by PAA to prepare H-MnO2@SAS. The drug loading rate of the obtained H-MnO2@SAS was tested, and it was found that the drug loading rate of SAS was only about 8% (calculation method: the amount of SAS loaded into the nanoparticles / the sum of the mass of the nanoparticles and SAS × 100%), and in repeated experiments, it was found that the drug loading rate obtained in this way was unstable (the drug loading rate of the scheme in Example 1 can reach 23%). Although both PAA and PAH are polymers synthesized based on acrylic acid monomers, there are significant differences between the two in improving the drug loading effect of SAS on mesoporous manganese dioxide, and PAH has a more significant beneficial effect.

[0137] In addition, the particle size of the H-MnO2@SAS nanoparticles prepared according to this scheme is 135 nm, which is larger than the HMnO2 and MSM nanoparticles prepared in Example 1 of this scheme (the particle size of the MSM prepared in Example 1 of this scheme is about 118 nm, and the particle size of the HMnO2 prepared in Example 1 is about 111.6 nm).

[0138] Attempt three:

[0139] This attempt was made with reference to the literature (Hollow MnO2 as a tumor-microenvironment-responsive biodegradable nano-platform for combination therapy favoring antitumor immune responses). Specifically, the process flow is basically based on Example 1, with the difference that PAH is replaced by PEG (polyethylene glycol) to modify the surface of HMnO2, and the PAH in the original Example 1 is replaced by PEG in equal amounts to prepare H-MnO2@SAS. The drug loading rate of the obtained H-MnO2@SAS was tested, and it was found that the drug loading rate of SAS was only about 6%, and the conventional method was not ideal. PEG is a conventional substance for surface modification of mesoporous manganese dioxide, but when used in the specific scenario of this scheme, PEG cannot effectively increase the loading of SAS on mesoporous manganese dioxide.

[0140] In addition, the particle size of the H-MnO2@SAS nanoparticles prepared according to this scheme is 176.3 nm, which is larger than the HMnO2 and MSM nanoparticles prepared in Example 1 of this scheme (the particle size of the MSM prepared in Example 1 of this scheme is about 118 nm, and the particle size of the HMnO2 prepared in Example 1 is about 111.6 nm).

[0141] Attempt 4:

[0142] This process flow is basically based on Example 1, except that: after adding PAH for modification according to Example 1, BSA is added at a mass ratio of 1:5 (H-MnO2:BSA), and after incubation, SAS is added for drug loading according to the method of Example 1. The drug loading rate of H-MnO2@SAS prepared by this method is acceptable, but due to the addition of BSA and the easy agglomeration of BSA during the modification process, the particle size of H-MnO2@SAS is about 436nm, and a good size cannot be obtained.

[0143] Attempt Five:

[0144] In this attempt, this process adopts an original idea, skips the drug loading process, uses NHS and EDC as dehydration catalysts, and directly dehydrates and condenses SAS with H-MnO2 prepared by the reference embodiment 1. However, the dehydration reaction conditions are harsh, and a good dehydration condensation reaction cannot be carried out at room temperature and pressure, and the reaction process is uncontrollable. NHS and EDC may cause the OH and COOH of SAS to dehydrate and produce side reactions, and the drug loading effect is poor. Therefore, the preparation of nanoparticles in this scheme should not adopt the method of using NHS and EDC as dehydration catalysts. In addition, the particle size of the H-MnO2@SAS nanoparticles prepared according to this scheme is 114.3nm. Although the particle size is acceptable, the drug loading effect is not ideal. Therefore, the preparation method of this attempt is not suitable for the preparation of nanoparticles in this scheme.

[0145] Attempt six:

[0146] This process flow is basically based on Example 1, except that the solvent is replaced by anhydrous ethanol from deionized water to improve the solubility of SAS, in order to improve the drug loading effect. The prepared H-MnO2@SAS is dispersed in water after centrifugal enrichment, and it is found that the particle size of the nanoparticles increases, resulting in a relatively serious agglomeration phenomenon (the particle size of H-MnO2@SAS can reach 1466.6nm). Analysis may be because non-electrostatic adsorption destroys the surface charge of the complex and causes agglomeration. The H-MnO2@SAS prepared by the method of Example 1 using water as a solvent, after centrifugal collection and redispersion in water, the nanoparticles have good dispersibility, no agglomeration phenomenon, and after particle size measurement, no particle size increase occurs. It can be seen that using water as a solvent for the dispersion and dissolution of H-MnO2, PAH and PAH-MnO2 is very critical for the preparation of H-MnO2@SAS, which ensures the dispersibility and stability of nanoparticles in different solvent environments, and the prepared nanoparticles can be used in a variety of formulation environments, expanding the application scenarios of nanoparticles.

[0147] After sulfasalazine and PAH-MnO2 were stirred overnight, the supernatant was centrifuged and observed. If the supernatant was clearer, it meant that more sulfasalazine was successfully loaded into the nanoparticles. For details of the experimental results, see Fig.30 If the best preparation method in Example 1 is adopted, the appearance of the obtained supernatant is as follows Fig.30 As shown in A (the supernatant is taken for display). In the "Trial 1" of this comparative example, SAS is directly loaded onto the mesoporous manganese dioxide without modification of the mesoporous manganese dioxide. It is almost difficult to achieve drug loading. Therefore, the color of the supernatant is very dark, as shown in FIG. Fig.30 D. In the "Trial 2" and "Trial 3" of this comparative example, different mesoporous manganese dioxide modifications were used, but the drug loading of the nanoparticles was not ideal, and the supernatant was dark in color, such as Fig.30 B and 30E. In the "Trial 4" of this comparative example, BSA was added to modify the mesoporous manganese dioxide, and the drug loading effect was acceptable, and the supernatant was relatively clear, as shown in FIG. Fig.30 C. "Trial 5" of this comparative example used the NHS-EDC method for drug loading, but the drug loading effect was not ideal and the clarity of the supernatant was not ideal, such as Fig.30 As shown in F.

[0148] The particle size statistics of nanoparticles obtained by different preparation methods can be found in Fig.31 According to the particle size test results, the particle size of the MSM nanoparticles prepared by the method of Example 1 of this scheme is also relatively ideal, and the EPR effect can be better exerted.

[0149] It can be seen that how to effectively load SAS onto mesoporous manganese dioxide has certain technical difficulties. Referring to the conventional methods of the prior art, it is difficult to achieve effective loading of SAS on mesoporous manganese dioxide. In summary, the technical solution uses PAH to load SAS by electrostatic adsorption, which can ensure the drug loading rate of SAS and the dispersibility and particle size of nanoparticles. In addition, in the preparation process, the use of water as a reaction solvent is very critical to the successful preparation of nanoparticles.

[0150] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A nanoparticle based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm that synergistically promotes liver cancer ferroptosis, characterized in that: The invention comprises mesoporous manganese dioxide whose surface is modified with tumor cell membrane and loaded with sulfasalazine.

2. The nanoparticles based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm for synergistically promoting liver cancer ferroptosis according to claim 1, characterized in that: The tumor cell biofilm is derived from liver cancer cells.

3. The nanoparticles based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm for synergistically promoting liver cancer ferroptosis according to claim 2, characterized in that: The mesoporous manganese dioxide is prepared by the following method: tetraethyl orthosilicate reacts in an ethanol solution containing ammonia water to obtain silicon dioxide; after silicon dioxide is compounded with polyvinyl pyrrolidone, it is heated and condensed with formaldehyde and m-diphenol in an environment containing ammonia water to form a silicon dioxide-diphenol formaldehyde resin composite; the diphenol formaldehyde resin is etched with potassium permanganate to obtain a silicon dioxide-manganese dioxide composite; and then the composite is etched with a strong base to obtain the mesoporous manganese dioxide; Preferably, the mesoporous manganese dioxide is prepared by the following method: tetraethyl orthosilicate is added to a stirring ethanol solution containing aqueous ammonia to obtain silica through reaction; under ultrasonic action, the solution containing silica is dripped into a polyvinyl pyrrolidone solution to obtain a silica-polyvinyl pyrrolidone complex through reaction; under ultrasonic action, diphenol, formaldehyde and aqueous ammonia are added to obtain a silica-diphenol formaldehyde resin complex through reaction; under stirring conditions, potassium permanganate solution is dripped to obtain a silica-manganese dioxide complex through reaction; the silica-manganese dioxide complex is dispersed in a sodium hydroxide solution under ultrasonic conditions to obtain mesoporous manganese dioxide after reaction and washing.

4. The nanoparticles based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm for synergistically promoting liver cancer ferroptosis according to claim 3, characterized in that: The mass ratio of mesoporous manganese dioxide loaded with sulfasalazine to the tumor cell biofilm is 1:1-2; the mass ratio of mesoporous manganese dioxide modified with polyacrylamide hydrochloride to sulfasalazine is 5:

2.

5. The nanoparticles based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm for synergistically promoting liver cancer ferroptosis according to claim 4, characterized in that: Its particle size is 118±14.78nm and its surface potential is -41.6±0.9mV.

6. A method for preparing nanoparticles based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm for synergistically promoting liver cancer ferroptosis according to any one of claims 1 to 5, characterized in that: The method includes the following steps in sequence: S1: Preparation of mesoporous manganese dioxide; S2: using polyacrylamide hydrochloride to modify mesoporous manganese dioxide, and then loading sulfasalazine onto the modified mesoporous manganese dioxide by electrostatic adsorption to obtain mesoporous manganese dioxide loaded with sulfasalazine; S3: Mesoporous manganese dioxide loaded with sulfasalazine and tumor cell membrane are mixed and physically extruded through a liposome extruder for 10-15 times to obtain nanoparticles.

7. The method for preparing nanoparticles based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm for synergistically promoting liver cancer ferroptosis according to claim 7, characterized in that: In S1, the mesoporous manganese dioxide is prepared by the following method: tetraethyl orthosilicate is added to a stirring ethanol solution containing ammonia water, and silicon dioxide is obtained through reaction; under the action of ultrasound, the solution containing silicon dioxide is dripped into a polyvinyl pyrrolidone solution, and a silicon dioxide-polyvinyl pyrrolidone complex is obtained through reaction; under the action of ultrasound, diphenol, formaldehyde and ammonia water are added, and a silicon dioxide-diphenol formaldehyde resin complex is obtained through reaction; under stirring conditions, potassium permanganate solution is dripped, and a silicon dioxide-manganese dioxide complex is obtained through reaction; the silicon dioxide-manganese dioxide complex is dispersed in a sodium hydroxide solution under ultrasonic conditions, and mesoporous manganese dioxide is obtained after reaction and washing.

8. The method for preparing nanoparticles based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm for synergistically promoting liver cancer ferroptosis according to claim 7, characterized in that: In S2, the mesoporous manganese dioxide loaded with sulfasalazine is obtained by the following method: dripping a mesoporous manganese dioxide aqueous dispersion into a polyacrylamide hydrochloride aqueous solution under ultrasonic conditions, and obtaining modified mesoporous manganese dioxide through reaction; dispersing sulfasalazine in the aqueous solution of the modified mesoporous manganese dioxide under ultrasonic conditions, and obtaining mesoporous manganese dioxide loaded with sulfasalazine through reaction.

9. The method for preparing nanoparticles based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm for synergistically promoting liver cancer ferroptosis according to claim 8, characterized in that: In S3, mesoporous manganese dioxide loaded with sulfasalazine and tumor cell membranes were stirred and mixed in a ratio of 1-2:1 and then treated using a liposome extruder; the tumor cell membranes were derived from liver cancer cells.

10. Use of nanoparticles based on mesoporous manganese dioxide, sulfasalazine and tumor cell biofilm for synergistically promoting liver cancer ferroptosis in the preparation of drugs for treating liver cancer according to any one of claims 1 to 5.

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