Preparation method and application of composite nanoparticles loaded with AAP and manganese ions

Abstract

The invention belongs to the technical field of nanotechnology, and relates to a preparation method and application of composite nanoparticles loaded with AAP and manganese ions. Specifically, an AAP-coated HMSN-CS-Mn < 2 + > nanoparticle which takes HMSN as a carrier and is loaded with AAP and manganese ions (Mn < 2 + >) is constructed. After reaching a tumor part, the nano-particles are subjected to catalytic hydrolysis of ALP highly expressed in liver cancer cells, and AAP is released. At the tumor part, AAP is reduced to generate VitC, and tumor cells are induced to generate high-concentration H2O2, so that the tumor cells are killed. High-concentration H2O2 is catalyzed by Mn < 2 + >, highly toxic. OH is generated through Fenton-like reaction, cell apoptosis is induced, and finally the expected anti-tumor effect is achieved.

Description

Technical Field

[0001] The invention belongs to the field of nanotechnology and relates to a preparation method of composite nanoparticles loaded with AAP and manganese ions and application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the most common cancers in the world and the most common primary liver cancer. It is also the sixth most common cancer and the third most deadly cancer in the world. Common risk factors include viral hepatitis, alcoholism and non-alcoholic fatty liver disease. Liver cancer has a high incidence and strong metastasis, and patients often have a poor prognosis. Only 5%-15% of patients with early liver cancer can be treated by surgical resection, while in the middle and late stages, the main treatment is transcatheter arterial chemoembolization and chemotherapy drugs such as sorafenib. However, long-term use of chemotherapy drugs has limited effects and is prone to drug resistance and toxicity. Therefore, it is particularly urgent and important to carry out active and effective research on the prevention and treatment of liver cancer and find new methods to fight liver cancer.

[0003] In recent years, nanotechnology has made significant progress in the field of tumor treatment, especially inorganic nanocarriers with mesoporous structures have become a research hotspot. Hollow mesoporous silica nanoparticles (HMSNs) combine the advantages of nano-mesoporous materials and larger hollow cavities, connected to the outside world through mesopores. These nanoparticles can accumulate at the tumor site through the enhanced permeability and retention (EPR) effect, achieving efficient nano-drug loading and release. In addition, due to its higher drug loading capacity, specific surface area, adjustable pore size and biocompatibility, it improves the bioavailability of drugs and reduces toxicity to normal tissues.

[0004] Therefore, compared with normal cells, tumor tissues are more sensitive to the accumulation of reactive oxygen species (ROS). Vitamin C (VitC), also known as L-ascorbic acid, is one of the essential nutrients for the human body and is involved in a variety of biochemical reactions. Studies have found that VitC exerts its anti-tumor effect by producing high concentrations of hydrogen peroxide (H2O2) in tumor cells. H2O2 destroys lipids, proteins, and DNA, leading to tumor cell death. It also reacts with glutathione, consumes NADPH, disrupts glycolysis in tumor cells, and causes energy failure.

[0005] Chemodynamic therapy (CDT) involves the use of various transition metal ions to catalyze endogenous H2O2 to generate highly cytotoxic hydroxyl radicals (·OH) through Fenton or Fenton-like reactions to eliminate tumor cells. It has attracted much attention due to its high efficacy in the treatment of deep tissue hypoxia tumors and less damage to normal tissues. Tumor cells have relatively high oxidative stress levels due to high metabolic rates, mitochondrial defects and other problems. However, VitC is easily ineffective in vivo, reducing its application value in anti-tumor effects.

[0006] Therefore, it is particularly urgent and important to actively carry out effective research on liver cancer prevention and treatment and to find new methods to fight liver cancer.

[0007] Currently, there is no report on the preparation method of composite nanoparticles loaded with AAP and manganese ions and their application in the treatment of liver cancer. Summary of the invention

[0008] The purpose of the present invention is to solve the above problems and provide a method for preparing composite nanoparticles loaded with AAP and manganese ions and its application, using hollow mesoporous silica nanoparticles (HMSN) as carriers, loaded with ascorbic acid phosphate (AAP) and manganese ions (Mn 2+ ) of AAP@HMSN-CS-Mn 2+ Nanoparticles. After reaching the tumor site, the nanoparticles are catalyzed and hydrolyzed by ALP, which is highly expressed in liver cancer cells, and release AAP. At the tumor site, AAP is reduced to generate VitC, which induces tumor cells to produce high concentrations of H2O2, thereby killing tumor cells. The high concentration of H2O2 is transported by Mn 2+ Catalyzed by Fenton-like reaction, it produces highly toxic ·OH, which induces cell apoptosis and ultimately achieves the expected anti-tumor effect.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is: The preparation method of composite nanoparticles loaded with AAP and manganese ions comprises the following steps: (1) Preparation of AAP@HMSN-CS: hollow mesoporous silica nanoparticles were added to ultrapure water and ultrasonically dispersed to prepare a 5 mg / mL dispersion; ascorbic acid phosphate was added to ultrapure water to prepare a 25 mg / mL ascorbic acid phosphate solution; 10 mL of the hollow mesoporous silica nanoparticle dispersion and 10 mL of the ascorbic acid phosphate solution were added to a small brown bottle in sequence and stirred; the chitosan solution was added to the above mixed solution, stirred, centrifuged, the supernatant was discarded, and the mixture was washed with ultrapure water for 3 times. After freezing, the mixture was placed in a vacuum freeze dryer and dried to obtain the AAP@HMSN-CS powder, which was stored at 4°C for future use; (2)HMSN-CS-Mn 2+Preparation: hollow mesoporous silica nanoparticles were added to ultrapure water and ultrasonically dispersed to prepare a 5 mg / mL dispersion; an appropriate amount of MnCl2 was weighed and dissolved in ultrapure water to prepare a 5 mg / mL solution; 10 mL of the hollow mesoporous silica nanoparticle dispersion and 10 mL of the chitosan solution were added to a small brown bottle in sequence and stirred; 10 mL of the MnCl2 solution was added to the mixed solution and stirred; centrifuged, the supernatant was discarded, and the mixture was washed with ultrapure water for 3 times. After freezing, the mixture was dried in a vacuum freeze dryer to obtain HMSN-CS-Mn 2+ The powder was stored at 4℃ for future use; (3)AAP@HMSN-CS-Mn 2+ Preparation: Add AAP@HMSN-CS to ultrapure water and prepare a 1 mg / mL dispersion by ultrasonic dispersion; add MnCl2 to 1 ultrapure water to prepare a 2 mg / mL solution; add 10 mL of AAP@HMSN-CS dispersion and 10 mL of MnCl2 solution to a small brown bottle in sequence and stir; centrifuge, discard the supernatant, wash with ultrapure water 3 times, freeze, and dry in a vacuum freeze dryer to obtain AAP@HMSN-CS-Mn 2+ The powder is composite nanoparticles loaded with AAP and manganese ions and is stored at 4°C for future use.

[0010] It is further specified that in steps (1) and (2), the concentration of the chitosan solution is 0.5 mg / mL.

[0011] It is further specified that in steps (1), (2) and (3), the stirring is performed by magnetic stirring in a constant temperature magnetic stirrer at 37°C for 24 hours.

[0012] It is further specified that in steps (1), (2) and (3), the centrifugation operation is performed at 8000 rpm for 10 min.

[0013] It is further specified that in steps (1), (2) and (3), the freezing is performed in an ultra-low temperature freezer at -80°C for 1 hour.

[0014] It is further specified that in steps (1), (2) and (3), the drying time is 16-24 hours.

[0015] Another object of the present invention is to provide a composite nanoparticle material loaded with AAP and manganese ions obtained by the preparation method.

[0016] Another object of the present invention is to provide the use of the composite nanoparticle material loaded with AAP and manganese ions prepared by the preparation method in the preparation of drugs for treating liver cancer.

[0017] Due to the adoption of the above technical solution, the present invention has the following beneficial effects: The present invention Figure 1 The loads AAP and Mn are shown. 2+ Hollow mesoporous silicon nanoparticles. HMSN has good biosafety. AAP produces vitamin C in situ under the action of alkaline phosphatase highly expressed in tumor cells. The high expression of vitamin C produces more H2O2, which amplifies the activity of Mn 2+ Fenton-like reaction for tumor treatment. 2+ The nanocatalytic therapy achieved significantly enhanced anti-tumor effects in vivo while avoiding significant damage to normal tissues. The research results of this team show that AAP-based nanocatalytic therapy is a promising strategy to improve the efficacy of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the embodiment of the present invention 1AAP@HMSN-CS-Mn 2+ Schematic diagram of the preparation process and induction of tumor cell apoptosis.

[0019] Figure 2 The present invention HMSN and AAP@HMSN-CS-Mn 2+ Nanomaterial characterization diagram. Among them, (A) SEM of HMSN (B) TEM of HMSN (C) AAP@HMSN-CS-Mn 2+ SEM of (D) AAP@HMSN-CS-Mn 2+ Transmission electron microscopy (E) of AAP@HMSN-CS-Mn 2+ Energy spectrum analysis (F) FI-TR analysis of HMSN (G) Nitrogen adsorption curve of HMSN (H) AAP@HMSN-CS-Mn 2+ FI-TR analysis of (I) AAP@HMSN-CS-Mn 2+ Zata potential analysis.

[0020] Figure 3 It is the transmission electron microscopy image of HMSN after etching for 0.5h and 2h.

[0021] Figure 4 (A) is the UV absorption peak characterization diagram of AAP, and (B) AAP and AAP@HMSN-CS have the same UV absorption peak diagram at 262nm.

[0022] Figure 5 AAP@HMSN-CS-Mn 2+ The average kinetic particle size is about 306nm.

[0023] Figure 6 A is the AAP standard curve; Figure 6 B AAP@HMSN-CS encapsulation efficiency diagram.

[0024] Figure 7 Yes FG AAP@HMSN-CS-Mn 2+ Stability study diagram.

[0025] Figure 8 This is an in vitro experimental diagram of nanomaterials on HepG cells. (A) AAP@HMSN-CS-Mn 2+ Killing experiment of HepG2 cells (B) AAP@HMSN-CS-Mn 2+ Killing experiment of HUVEC cells (C) AAP@HMSN-CS-Mn 2+ Detection of ALP content in HepG2 cells (D / E / F) AAP@HMSN-CS-Mn 2+ Effects on ROS / JC-1 / live-dead cell staining of HepG2 cells (G) Expression of apoptotic proteins detected by WB in tumor cell apoptosis (H / I / J) AAP@HMSN-CS-Mn 2+ Effects on Caspase-1 / Caspase-4 / Gasdermin D in HepG2 cells.

[0026] Fig. 9 This is a graph showing the killing effects of different concentrations of HMSN on HepG2 cells and HUVEC cells at 12 hours.

[0027] Fig.10 At 12h, different concentrations of ALP had no obvious killing effect on HepG2 cells and HUVEC cells.

[0028] Fig.11 It is a statistical chart of the determination of intracellular ROS content.

[0029] Fig.12 It is a statistical chart of the determination of intracellular JC-1 content.

[0030] Fig.13 It is a statistical graph of the determination of live and dead staining content in cells.

[0031] Fig.14 This is a statistical graph of the determination of the intracellular pyroptosis markers (A) Caspase-1 (B) Caspase-4 (C) Gasdermin D content.

[0032] Fig.15 This is a graph showing the effects of nanomaterials on the in vivo treatment of HepG2 liver cancer model BALB / c mice.

[0033] Fig.16This is the effect diagram of organ index of each group of HepG2 liver cancer model mice.

[0034] Fig.17 This is a graph showing the expression of Casepase 3 in mouse tumor tissue after treatment of the HepG2 liver cancer model in each group (20×).

[0035] Fig.18 This is a graph showing the expression of Casepase 8 in mouse tumor tissue after treatment of the HepG2 liver cancer model in each group (20×).

[0036] Fig.19 This is a graph showing the expression of Bax in mouse tumor tissue after treatment of the HepG2 liver cancer model in each group (20×).

[0037] Fig. 20 This is a graph showing the expression of Bcl-2 in mouse tumor tissue after treatment of the HepG2 liver cancer model in each group (20×). Fig.21 This is the expression of Casepase 1 in mouse tumor tissue after treatment of HepG2 liver cancer model in each group (20×).

[0038] Fig. 22 This is a graph showing the expression of Casepase 4 in mouse tumor tissue after treatment of the HepG2 liver cancer model in each group (20×).

[0039] Fig.23 This is a graph showing the expression of Gasdermin DN in tumor tissue after treatment of HepG2 liver cancer model in each group (20×).

[0040] Fig.24 This is a graph showing the expression of IL-18 and IL-1β in mouse serum.

[0041] Fig.25 These are H&E staining (10×) images of the heart, liver, spleen, lung, and kidney of each group of HepG2 liver cancer model mice.

[0042] Fig.26 These are the pictures of the HepG2 liver cancer model of mice in each group; including (A) lymphocytes (B) monocytes (C) neutrophils (D) hemoglobin (E) red blood cells (F) and platelet content.

[0043] Fig. 27 This is a graph showing the levels of (A) aspartate aminotransferase (B) alanine aminotransferase (C) alkaline phosphatase (D) blood urea (E) blood uric acid (F) and blood creatinine in the serum of each group of HepG2 liver cancer model mice.

[0044] Fig.28 AAP@HMSN-CS-Mn 2+ Hemolysis rate diagram.

[0045] In the above figure, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. Figure 17-23 The scale bar is 100 μm. DETAILED DESCRIPTION

[0046] The specific implementation of the invention is further described below with reference to the accompanying drawings.

[0047] All chemicals in the present invention were used directly without further purification. 3-Aminopropyltriethoxysilane (APTS) was purchased from Aladdin Reagent (Shanghai) Co., Ltd., sodium carbonate (Na2CO3) was purchased from Aladdin Reagent (Shanghai) Co., Ltd., ammonia water (NH3·H2O) was purchased from Chongqing Chuandong Chemical (Group) Co., Ltd., tetraethyl silicate (TEOS) was purchased from Aladdin Reagent (Shanghai) Co., Ltd., ascorbyl phosphate (AAP) was purchased from Sigma, USA, manganese chloride (MnCl2) was purchased from Aladdin Reagent (Shanghai) Co., Ltd., chitosan (CS) was purchased from Aladdin Reagent (Shanghai) Co., Ltd., H&E staining kit was purchased from Beijing Solebao Technology Co., Ltd., proteinase K was purchased from Beijing Solebao Technology Co., Ltd., TUNEL cell apoptosis detection kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., Anti-Ki67 antibody was purchased from Abcam (Shanghai) Co., Ltd., ALP detection kit was purchased from Shanghai Biotech Co., Ltd., reactive oxygen species (ROS) detection kit was purchased from Wuhan Sevier Biotech Co., Ltd., SDS-PAGE gel rapid preparation kit was purchased from Shanghai Biotech Co., Ltd., transfer buffer was purchased from Beijing Solebow Technology Co., Ltd., electrophoresis buffer was purchased from Beijing Solebow Technology Co., Ltd., protease inhibitor mixture (universal, 100X) was purchased from Shanghai Biotech Co., Ltd., high-efficiency RIPA tissue / cell rapid lysis buffer was purchased from Beijing Solebow Technology Co., Ltd., protease inhibitor (PMSF) was purchased from Beijing Solebow Technology Co., Ltd., PVDF membrane was purchased from Wuhan Sevier Biotech Co., Ltd., BCA protein concentration determination kit was purchased from Beijing Solebow Technology Co., Ltd., Rabbit anti-Caspase 1 antibody was purchased from CST, USA, Rabbit anti-Caspase 3antibody was purchased from CST, USA, Rabbit anti-Caspase 4antibody was purchased from CST, USA, Rabbit anti-Caspase 8antibody was purchased from CST, USA, Rabbit anti-Bax Antibody was purchased from CST, USA; Rabbit anti-Bcl-2antibody was purchased from CST, USA; Rabbit anti-Gasdermin DN antibody was purchased from CST, USA; QuickBlock TM Western universal diluent was purchased from Shanghai Biotech Biotechnology Co., Ltd., Mouse IL-1β ELISA Kit was purchased from Hangzhou Lianke Biotechnology Co., Ltd., and Mouse IL-18 ELISA Kit was purchased from Hangzhou Lianke Biotechnology Co., Ltd.

[0048] HepG2 and HUEVC cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The healthy BALB / c mice we used, female, 6-8 weeks old, were purchased from the Animal Experiment Center of Guangxi Medical University, SPF-level laboratory. All mice were handled according to the protocol approved by the Animal Ethics Committee of Guangxi Medical University.

[0049] Example 1 The preparation method of composite nanoparticles loaded with AAP and manganese ions comprises the following steps: (1) Synthesis process of HMSN 5mL ultrapure water and 35mL anhydrous ethanol were added to the reaction vessel, and then 1.57mL ammonia water was added, and stirred in a constant temperature water bath at 30℃ for 30min. 3mL tetraethyl silicate was added to the above mixed solution, and the reaction was continued at 30℃ with magnetic stirring for 45min, and then 2.5mL TEOS and 1mL APTS were added to the above mixed solution and slowly dripped within 3min, and stirred in a constant temperature water bath at 30℃ for 80min. The silica nanoparticle solution was divided into two parts, centrifuged at 8000rpm for 10min, and the supernatant was discarded and washed 2-3 times. Each silica nanoparticle solution was evenly mixed with Na2CO3 solution (3.18g / 50mL) and added to the reaction vessel, and etched in a constant temperature water bath at 80℃ for 120min. The solution was taken out and centrifuged at 8000rpm for 10min, the supernatant was discarded, and ultrapure water was added to mix evenly by ultrasonic dispersion, and the washing was repeated 2-3 times to obtain HMSN. The prepared HMSN dispersion solution was placed in a -80°C ultra-low temperature refrigerator for 1 hour, and then placed in a vacuum freeze dryer for 16-24 hours. The obtained powder was stored at 4°C for later use.

[0050] (2) AAP@HMSN-CS synthesis process Add 50 mg of HMSN to 10 mL of ultrapure water and disperse by ultrasonic to prepare a 5 mg / mL dispersion. Add 250 mg of AAP to 10 mL of ultrapure water to prepare a 25 mg / mL AAP solution. Add the HMSN dispersion and AAP solution to the small brown bottle in turn, and stir magnetically in a constant temperature magnetic stirrer at 37°C for 24 hours. Take 10 mL of 0.5 mg / mL CS solution and add it to the above mixed solution, and stir magnetically at 37°C for 24 hours. Centrifuge at 8000 rpm for 10 minutes, discard the supernatant, wash 3 times with ultrapure water, freeze in a -80°C ultra-low temperature refrigerator for 1 hour, and then put it into a vacuum freeze dryer to dry for 16-24 hours. The obtained powder is stored at 4°C for later use.

[0051] (3)HMSN-CS-Mn 2+ Synthesis process Add 50 mg of HMSN to 10 mL of ultrapure water and prepare a 5 mg / mL dispersion by ultrasonic dispersion. Weigh an appropriate amount of manganese chloride and dissolve it in ultrapure water to prepare a 5 mg / mL solution. Add 10 mL of HMSN dispersion and 10 mL of 0.5 mg / mL CS solution to the small brown bottle in sequence, and stir magnetically in a constant temperature magnetic stirrer at 37°C for 24 hours. Take 10 mL of MnCl2 solution and add it to the above mixed solution, and stir magnetically at 37°C for 24 hours. Centrifuge at 8000 rpm for 10 minutes, discard the supernatant, wash with ultrapure water 3 times, freeze in a -80°C ultra-low temperature refrigerator for 1 hour, and then put it in a vacuum freeze dryer to dry for 16-24 hours. The obtained powder is stored at 4°C for later use.

[0052] (4)AAP@HMSN-CS-Mn 2+ Synthesis process Add 10 mg of AAP@HMSN-CS to 10 mL of ultrapure water and prepare a 1 mg / mL dispersion by ultrasonic dispersion. Add 20 mg of MnCl2 to 10 mL of ultrapure water to prepare a 2 mg / mL solution. Add 10 mL of AAP@HMSN-CS dispersion and 10 mL of MnCl2 solution to a small brown bottle in sequence and stir magnetically in a 37°C constant temperature magnetic stirrer for 24 hours. Centrifuge at 8000 rpm for 10 minutes, discard the supernatant, wash 3 times with ultrapure water, freeze in a -80°C ultra-low temperature refrigerator for 1 hour, and then dry in a vacuum freeze dryer for 16-24 hours to obtain AAP@HMSN-CS-Mn 2+ The powder is composite nanoparticles loaded with AAP and manganese ions and is stored at 4°C for future use.

[0053] The present invention is based on constructing HMSN as a carrier, loaded with AAP and manganese ions (Mn 2+ ) of AAP@HMSN-CS-Mn 2+ Nanoparticles. After reaching the tumor site, the nanoparticles are catalyzed and hydrolyzed by ALP, which is highly expressed in liver cancer cells, to release AAP. In the tumor site, AAP is reduced to generate VitC, which induces tumor cells to produce high concentrations of H2O2, thereby killing tumor cells. High concentrations of H2O2 are transported to the tumor site by Mn 2+ Catalyzed by Fenton-like reaction, it produces highly toxic ·OH, which induces cell apoptosis and ultimately achieves the expected anti-tumor effect.

[0054] The morphology of the samples was determined using a S-4800 field emission scanning electron microscope (FE-SEM). The crystal structure of the samples was detected by X-ray powder diffraction (Bruker) with Cu Kα radiation (λ=0.154nm). Transmission electron microscope (TEM) micrographs of the samples were obtained using a FEI Tecnai G2 S-Twin and a 200kV field emission gun. The UV-visible absorption spectra were measured on a U-3100 spectrophotometer (Hitachi). Fourier transform infrared (FT-IR) spectra were measured using a Vertex Perkin-Elmer 580BIR spectrophotometer (Bruker) using KBr particle technology. An inductively coupled plasma mass spectrometer (ICP-MS) was installed on an iCAP 6300 from Thermo scientific. The DLS and zeta potential of the samples in water were measured on a Malvern instrument Zetasizer Nano. The nitrogen adsorption–desorption isotherms were measured at 77 K using a Micromeritics ASAPTristar II 3020 system.

[0055] HMSN was synthesized by high temperature etching method, and SEM and TEM showed a spherical structure with a diameter of about 300nm ( Figure 2 A, B). By comparing the etching time of HMSN at different times of 0.5h and 2h, it is confirmed that with the increase of etching time, the mesoporous structure of HMSN becomes more prominent ( Figure 3 In addition, the infrared spectrum of HMSN is at 1077 cm -1 A strong absorption peak appears, which comes from the asymmetric stretching vibration of Si-O-Si. The characteristic absorption peak at 960cm-1 belongs to the bending vibration absorption characteristic peak of Si-OH on the HMSN surface. -1 The broad absorption peak at 1634 cm -1 The weaker absorption peaks at are related to the asymmetric stretching vibration of -OH and the bending vibration of water molecules in the mesopores of HMSN. Figure 2 F). Figure 2 G and Table 1, the N2 adsorption-desorption isotherms and the corresponding pore size distribution of HMSN were determined. The specific surface area of ​​HMSN is 48.376 m 2 g -1 , the pore size is 18.745nm.

[0056] Table 1 <![CDATA[Surface area (m 3 / g)]]> <![CDATA[Pore volume (cm 3 / g)]]> Pore ​​size (nm) HMSN 48.376 0.227 18.745 Subsequently, AAP nanoparticles were introduced into the mesoporous surface of HMSN through diffusion effect to obtain AAP@HMSN nanocomposite. AAP@HMSN was wrapped with chitosan to obtain AAP@HMSN-CS nanocomposite. AAP was characterized by UV-visible absorption spectroscopy and it was found that the AAP scanning spectrum showed a special absorption peak at 262nm (Figure 4A). Figure 4B tested that the UV-visible absorption spectra of AAP and AAP@HMSN-CS had the same ultraviolet absorption peak at 262nm, proving that AAP was successfully loaded. Mn was loaded in AAP@HMSN-CS nanocomposite. 2+ , construct AAP@HMSN-CS-Mn 2+ Nanocomposites, whose morphology and size have little change ( Figure 2 C, D), elemental mapping images show that all elements (Si, O, Mg, Mn, P, Cl, and N) are uniformly distributed, revealing the coexistence of different elements in the MnDIG@PEG nanocomposite, proving that AAP and Mn are successfully loaded to form AAP@HMSN-CS-Mn 2+ ( Figure 2 E). Same AAP@HMSN-CS-Mn 2+ Infrared spectroscopy also confirmed the material AAP@HMSN-CS-Mn 2+ ( Figure 2 In addition, the Zeta potential change of the obtained sample also proves that AAP@HMSN-CS-Mn 2+ Successful preparation of nanocomposites. AAP@HMSN-CS-Mn 2+ The final Zeta potential and average kinetic particle size were 50.57±2.78mV( Figure 2 I) and 306nm( Figure 5 ).

[0057] AAP@HMSN-CS-Mn 2+ The most important features of the nanocomposites are cargo delivery and TME-responsive biodegradability, which are beneficial for improving the chemical kinetics of AAP self-amplification. The released AAP, Mn 2+ The loading efficiency of AAP was 132.59±1.5μg / mg according to the standard curve of AAP. When the ratio of HMSN:AAP was 1:5, the encapsulation efficiency of AAP@HMSN-CS was the highest, which was 13.26±0.15% ( Figure 6 A, B).

[0058] Example 2: This example is to prepare AAP@HMSN-CS-Mn 2+In vitro anticancer tests of nanomaterials include the following studies: (1) AAP@HMSN-CS-Mn 2+ stability Weigh an appropriate amount of prepared AAP@HMSN-CS-Mn 2+ Ultrasonic uniform dispersion was made in an appropriate amount of ultrapure water to prepare 15 mL of 0.1 mg / mL dispersion, which was placed in a 50 mL centrifuge tube and stored at 4 °C. 2+ The dispersion was measured by laser particle size analyzer to measure its hydrodynamic particle size in water and its surface Zeta potential. All samples were measured 3 times and the results were averaged. 2+ The dispersion was stored in a 50 mL centrifuge tube at 4 °C.

[0059] (2) Cytotoxicity assay Take a certain volume of cell suspension and prepare it to a final concentration of 5×10 4 5×10 cells / mL new cell suspension, repeatedly pipetting and mixing. Inoculate into 96-well plate (do not inoculate the periphery) using 8-channel gun. 100 μL per well, 5×10 cells 3 The cells were cultured at 37°C for 24 h. The 2% FBSDMEM medium was prepared into groups: PBS, AAP@HMSN-CS-Mn 2+ 、AAP@HMSN-CS-Mn 2+ +ALP(1U),AAP@HMSN-CS-Mn 2+ +ALP (5U), concentrations were: 0, 0.25, 0.5, 1.0, 2.0, 4.0 mg / mL working solution, 100 μL / well, three replicates for each concentration, incubated in a 37°C, 5% CO2 cell culture incubator for 12 hours. 10 μL CCK-8 reagent was added to each well, incubated in a 37°C, 5% CO2 cell culture incubator for 2 hours; the OD value of each well at a wavelength of 450 nm was detected by a full-wavelength microplate reader.

[0060] (3) Determination of cellular ROS Inoculate in a 6-well plate using a pipette. 1 mL per well, 5×10 cells 5 . Incubate at 37℃ for 24h. Use a pipette to carefully aspirate the culture medium along the side wall of the 6-well plate and discard it. Rinse once with PBS and discard the PBS. 2% FBS DMEM culture medium was prepared into groups: PBS, ALP, AAP@HMSN-CS, HMSN-CS-Mn 2+ 、AAP@HMSN-CS-Mn 2+,AAP@HMSN-CS+ALP, HMSN-CS-Mn 2+ +ALP、AAP@HMSN-CS-Mn 2+ +ALP working solution, 1mL / well, placed in a 37℃, 5% CO2 cell culture incubator and cultured for 12h. Take out the ROS detection kit from the -20℃ refrigerator and equilibrate at room temperature for 30min. Dilute the positive control Rosup reagent with serum-free medium at a ratio of 1:1000 to a final concentration of 10μmol / L. Take out the six-well plate, remove the cell culture medium from the positive control well, add 1mL of diluted Rosup reagent, and incubate in the incubator for 30min. Dilute the DCFH-DA probe with serum-free medium at a ratio of 1:1000 to a final concentration of 10μmol / L. Remove the old cell culture medium from each well and add 1mL of diluted DCFH-DA probe. Incubate in a 37℃ cell culture incubator for 20min. Wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA probe that has not entered the cells. Direct observation with a laser confocal microscope or a fluorescence microscope.

[0061] (4) Determination of cell JC-1 Inoculate in a 6-well plate using a pipette. 1 mL per well, 5×10 cells 5 . Incubate at 37℃ for 24h. Use a pipette to carefully aspirate the culture medium along the side wall of the 6-well plate (be careful not to touch the bottom of the well) and discard it. Rinse once with PBS and discard PBS. 2% FBSDMEM culture medium was prepared into groups: PBS, ALP, AAP@HMSN-CS, HMSN-CS-Mn 2+ 、AAP@HMSN-CS-Mn 2+ ,AAP@HMSN-CS+ALP, HMSN-CS-Mn 2+ +ALP、AAP@HMSN-CS-Mn 2++ALP working solution, 1mL / well, placed in a 37℃, 5% CO2 cell culture incubator and cultured for 12h. Take out the mitochondrial membrane potential detection JC-1 kit from the -20℃ refrigerator and equilibrate at room temperature for 30min. Dilute the positive control CCCP reagent (10mM) with serum-free medium at a ratio of 1:1000 to a final concentration of 10μM. Take out the six-well plate, remove the cell culture medium from the positive control well, add 1mL of diluted positive control CCCP reagent (10μM), and incubate in the incubator for 30min. Dilute the JC-1 reagent at a ratio of 1mL JC-1 staining buffer per 5μL JC-1 reagent to prepare the JC-1 working solution. Remove the cell culture medium from each well, first add 1mL of JC-1 working solution, and then add 1mL of cell culture medium. Incubate in a 37℃ cell culture incubator for 20min. Aspirate the supernatant from each well and wash twice with JC-1 staining buffer. Add 2mL of cell culture medium. Directly observe with a laser confocal microscope or a fluorescence microscope.

[0062] (5) Live and dead cell staining The final concentration was 5 × 10 5 10 / mL new cell suspension, pipette repeatedly to mix. Inoculate in 6-well plate with 1mL per well, cell number 5×10 5 The cells were cultured at 37°C for 24 h. The 2% FBSDMEM medium was prepared into the following groups: PBS, ALP, AAP@HMSN-CS, HMSN-CS-Mn 2+ 、AAP@HMSN-CS-Mn 2+ ,AAP@HMSN-CS+ALP, HMSN-CS-Mn 2+ +ALP、AAP@HMSN-CS-Mn 2+ +ALP working solution, 1mL / well (concentration according to Table 4), placed in a 37℃, 5% CO2 cell culture incubator and cultured for 12h. Take out the mitochondrial membrane potential JC-1 detection kit from the -20℃ refrigerator and equilibrate at room temperature for 30min. Dilute Calcein AM and PI reagents with detection buffer at a ratio of 1:1000. Take out the six-well plate, remove the cell culture medium, and add 1mL of diluted Calcein AM and PI reagents. Incubate in a 37℃ cell culture incubator for 30min. Wash the cells 3 times with serum-free cell culture medium. Observe directly with a laser confocal microscope or a fluorescence microscope.

[0063] (6) Measurement of Cellular ALP Content HepG2 cells were seeded in a 6-well plate using a pipette. The density was 5×10 5When the cells grow to about 80%, discard the original culture medium. 2% FBSDMEM culture medium is prepared into groups: PBS, HMSN, AAP@HMSN-CS, HMSN-CS-Mn 2+ 、AAP@HMSN-CS-Mn 2+ , and the working solution were added to the culture dish and placed in a 37°C, 5% CO2 cell culture incubator for further culture for 12 hours. Discard the old culture medium of the culture dish, wash it three times with 1mL PBS, and then dry it with a 1mL pipette. Add 1mL PBS to the culture dish again, scrape the adherent cells with a cell scraper, and repeatedly and gently aspirate the cell suspension with a 1mL pipette to rinse the adherent surface and collect it in a pre-cooled 1.5mL EP tube. Pre-cool a small high-speed centrifuge in advance, centrifuge at 4°C 2000rpm for 7min, discard the supernatant, and leave only the sediment at the bottom of the tube. Prepare the lysis solution in the dark, the ratio is: high-efficiency RIPA tissue / cell rapid lysis solution: PMSF = 100:1. Add 1mL of lysis solution to each 1.5mL EP tube, sonicate the cells at an amplitude of 40%, and then place them on ice for 30min. Centrifuge at 4°C 12000rpm for 15min, and collect the supernatant in a new 1.5mL EP tube. Use a 96-well plate to set up blank control wells, standard wells, and sample wells. The amount of standard is X = 4, 8, 16, 24, 32, and 40 μL respectively. (See the table below for details) Configure blank control wells, standard wells, and sample wells Assay Buffer 50 (100-X) 0 Chromogenic substrate 50 0 50 sample 0 0 50 Standard working solution 0 X 0 ① Mix well with the help of a shaker.

[0064] ②Incubate at 37℃ for 5-10 minutes.

[0065] ③ Add 100 μL of reaction stop solution to each well to terminate the reaction.

[0066] The absorbance was measured at 405 nm using an enzyme reader.

[0067] (7) WB detection of cell apoptosis protein indicators HepG2 cells were routinely cultured in 10 cm 2 When the cells grow to about 80%, discard the original culture medium. The 2% FBSDMEM culture medium is prepared into groups: PBS, ALP, AAP@HMSN-CS, HMSN-CS-Mn 2+ 、AAP@HMSN-CS-Mn 2+ ,AAP@HMSN-CS+ALP, HMSN-CS-Mn 2+ +ALP、AAP@HMSN-CS-Mn 2++ALP working solution is added to the culture dish, and the dish is placed in a 37°C, 5% CO2 cell culture incubator for 12 hours. Observe the morphological changes of the cells. When suspended cells appear, remove the cell culture dish. Ice the sterile, enzyme-free 1.5mL EP tubes, mark the groups, and insert them on ice for later use. Discard the old culture medium of the culture dish, wash it three times with 1mL PBS, and dry it with a 1mL pipette. Add 1mL PBS to the culture dish again, scrape the adherent cells with a cell scraper, and repeatedly and gently aspirate the cell suspension with a 1mL pipette to rinse the adherent surface and collect it in a pre-cooled 1.5mL EP tube. Pre-cool a small high-speed centrifuge in advance, centrifuge at 4°C 2000rpm for 7min, discard the supernatant, and leave only the sediment at the bottom of the tube. Prepare the lysis solution in the dark, the ratio is: high-efficiency RIPA tissue / cell rapid lysis solution: PMSF = 100:1. Add 1mL of lysis buffer to each 1.5mL EP tube, sonicate the cells at 40% amplitude, and then place on ice for 30 minutes. Centrifuge at 4℃12000rpm for 15min, collect the supernatant in a new 1.5mL EP tube. Turn on the metal bath and preheat to 100℃ for use, and use a micro-spectrophotometer to preliminarily detect the protein concentration. Add protein loading buffer, the ratio is: protein sample volume: 4×protein loading buffer (containing DTT) = 4:1. Place on a vortex oscillator for 15s, centrifuge at 1000rpm for 1min to collect the tube wall liquid, place in a metal bath and boil at 100℃ for 10min, cool to room temperature and place in a -20℃ refrigerator for use.

[0068] After the gel is prepared, add the sample and add 5μL of marker on the left and right sides of the protein sample hole. Pour the outer tank liquid into the electrophoresis tank to the corresponding mark; cover the electrophoresis tank cover, pay attention to the positive and negative poles; insert the electrophoresis instrument, and also pay attention to the positive and negative poles. Adjust the voltage to 90V to start electrophoresis, about 20-30min, until the marker and sample are at the boundary between the upper and lower gel layers; change to 120V constant voltage and run for about 60-90min, until the marker runs away completely and the sample runs to the bottom of the protein gel. Then transfer the membrane at 400mA constant current for 30min to completely transfer proteins below 150kD. Take 20mL of 20×TBST, add 380mL of distilled water, and prepare 1×TBST. After the transfer is completed, recover the transfer solution, discard the gel, put all the PVDF membranes that have been transferred into a glass dish, pour an appropriate amount of 1×TBST, place it on a shaker to wash for 10min, and change the solution three times. Prepare 5% skim milk for blocking. Take a clean 50mL centrifuge tube and weigh 1g of skim milk powder, and add 20mL 1×TBST. Use a clamp to put all the PVDF membranes after washing into 5% skim milk and block them on a shaker at room temperature for 1h. Wash the membrane again after blocking. Clean the antibody incubation box with ddH2O, spray it with alcohol, and then turn it upside down on a paper towel to dry. Pour rabbit anti-Caspase-3, Caspase-8, Bax and Bcl-2 primary antibodies into the antibody incubation box at a ratio of 1:1000 and store it in a 4℃ refrigerator. After washing the membrane, shake off the TBST on the PVDF membrane, fully expose the protein surface to the primary antibody solution, and place it in a 4℃ cold storage shaker for incubation for more than 12h. After the primary antibody incubation is completed, shake off the residual primary antibody on the membrane and wash the membrane again, the same as step (4). When starting the first wash, use a pipette to recover the primary antibody into a new tube and store it in a 4℃ refrigerator. Wash the antibody incubation box and wait for it to dry. Prepare secondary antibody: Take a new 15mL centrifuge tube, add 3mL 5% skim milk and 1μL corresponding secondary antibody in a ratio of 1:3000, and mix them upside down; mark the tube cap and the tube body with the corresponding secondary antibody. Pour the secondary antibody dilution into the antibody incubation box, put the membrane into the secondary antibody incubation solution with a clamp, fully expose the protein surface to the secondary antibody incubation solution, and incubate at room temperature for 1h. The membrane washing method and secondary antibody recovery method are the same as above. After the last wash, TBST is not poured, and the membrane is soaked in it when exposed to prevent the membrane from drying out. Prepare the color development solution: According to the size and amount of the membrane, add 1mL Solution A and 1mL Solution B in a new 15mL centrifuge tube at a ratio of 1:1, mix them upside down; put it in a drawer and keep it away from light. First turn on the dehumidifier and exposure meter, then open the software, select the folder, and change the exposure time to 2 minutes; wipe the color development plate with a napkin, place the membrane on the color development plate with a clamp, use a 1mL pipette to add a small amount of color development solution back and forth, slightly tilt the color development plate to allow the color development solution to be fully and evenly distributed on the membrane, put it into the exposure meter, click "image acquire" in the software, and adjust the image color.

[0069] (8) Immunofluorescence detection of cell pyroptosis protein indicators Preparation of cell slides: HepG2 cells were routinely treated until the cells were in the logarithmic growth phase and the density reached about 80%. They were washed twice with PBS, and the cells were digested with 0.25% trypsin for 3 minutes. The digestion was terminated with complete medium, and the cells were transferred to a centrifuge tube and centrifuged at 800 rpm for 3 minutes to collect the cell precipitate. The cells were resuspended with complete medium, and 10 μL of cell suspension + 1 μL of trypan blue were mixed and the cells were counted. The calculation formula was:

[0070] According to the counting situation, the cell density was adjusted, and a certain volume of the above cell suspension was prepared to a final concentration of 2.5×10 5 Prepare a new cell suspension of 100 cells / mL and mix it by blowing repeatedly. Take a cell slide corresponding to the size of the six-well plate and pass it through the flame of an alcohol burner. After it cools thoroughly, place it in a six-well plate. Take 0.5mL of the cell suspension and slowly drip it onto the cell slide, spread it evenly, and place it in an incubator for culture. Observe it under a microscope after 3-6 hours. After the cells adhere to the wall, add 2mL of complete culture medium. Prepare 24 cell slides in this way.

[0071] Cell slide grouping and material incubation: 24 cell slides were randomly divided into 8 groups according to Table 4, with 3 slides in each group. 2% FBSDMEM medium was prepared into groups: PBS, ALP, AAP@HMSN-CS, HMSN-CS-Mn 2+ 、AAP@HMSN-CS-Mn 2+ ,AAP@HMSN-CS+ALP, HMSN-CS-Mn 2+ +ALP、AAP@HMSN-CS-Mn 2+ +ALP working solution, 1 mL / well (concentration according to Table 1), placed in a 37°C, 5% CO2 cell culture incubator and cultured for 12 h.

[0072] Immunofluorescence detection of pyroptosis markers (Gasdermin D, Caspase-1, Caspase-4): After the material is incubated for the time, observe the morphological changes of the cells. When suspended cells appear, use a pipette to remove the old culture medium in the culture well, rinse the cell slides 2-3 times with PBS, shake up and down, discard the PBS, and then fix with 4% paraformaldehyde at room temperature for 30 minutes. Use a pipette to remove the paraformaldehyde in the culture wells, add PBS to the culture wells with the slides, place on a shaker and rinse for 10 minutes × 3 times, permeabilize with 0.1% tritonX-100 at room temperature for 10 minutes (the slides must be completely covered), use a pipette to remove all the liquid in the culture wells, place on a shaker and rinse with PBS for 5 minutes × 3 times, discard the PBS, block with 5% BSA at room temperature for 30 minutes, discard the BSA, prepare the primary antibody with the primary antibody diluent at a dilution ratio of 1:100, add 100 μL of the primary antibody to each well, and incubate at 4°C overnight. On the next day, after returning to room temperature, rinse with PBS for 10 min×3 times, prepare secondary antibody with secondary antibody diluent at a dilution ratio of 1:50, add 100 μL of secondary antibody with green fluorescence, incubate at 37°C in the dark for 1 h, rinse with PBS for 10 min×3 times, discard PBS, add 50 μL of 1 μg / mL DAPI working solution, incubate at 37°C in the dark for 8 min, place on a shaker and rinse twice with PBS in the dark, add 20 μL of anti-quencher to seal the slide, and observe under a fluorescence microscope.

[0073] (9) Statistical analysis The data of this study were statistically processed by Origin 8.0 software. All experiments were repeated at least three times, and the results were presented as mean ± standard deviation (SD). The experimental data were analyzed by variance analysis (ANOVA). If there was a statistically significant difference between the two groups of data, it was marked as *P<0.05; if the difference was significant, it was marked as **P<0.01; if the difference was relatively significant, it was marked as ***P<0.001; if the difference was extremely significant, it was marked as ****P<0.0001.

[0074] (10) Results Since the biocompatibility of nanocomplexes is an important requirement in tumor therapy, the CCK8 method was used to evaluate the cytotoxicity of nanocomplexes against human umbilical vein endothelial cells (HUVEC) and human hepatoma cell (HepG2) cells. Fig. 9 As shown in the figure, even when the concentration of HMSN was 4 mg / mL, the activity of HUVEC and HepG2 cells was always high, indicating that the HMSN nanocarrier has good biocompatibility. In addition, the cytotoxicity of ALP to HUVEC and HepG2 cells was also low ( Fig.10 ).like Figure 8 As shown in A, AAP@HMSN-CS-Mn 2+As the concentration of nanoparticles increases, the cytotoxicity to HepG2 cells increases. 2+ When the nanoparticles interact with HepG2 cells expressing or adding ALP, ALP hydrolyzes AAP to generate vitC, thereby amplifying the CDT effect. Moreover, compared with 5U of ALP, 1U of ALP can effectively hydrolyze AAP to generate vitC. In addition, compared with HepG2 cells, AAP@HMSN-CS-Mn 2+ The cytotoxicity to HUVEC cells was reduced, indicating that it has certain biocompatibility ( Figure 8 B). In order to further verify the ability of AAP to be hydrolyzed, HepG2 cells were treated with nanomaterials in groups, and it was found that the nanomaterials loaded with AAP induced the most significant results in HepG2 cells consuming ALP ( Figure 8 C). Subsequently, fluorescence imaging of 2′,7′-dichlorofluorescein generated by oxidation of 2′,7′-dichlorofluorescein diacetate by ROS was used to accurately evaluate the generation of ·OH. 2+ Compared with the control group, AAP@HMSN-CS-Mn 2+ and AAP@HMSN-CS-Mn 2+ +ALP positive control group showed strong green fluorescence, among which AAP@HMSN-CS-Mn 2+ The green fluorescence of the +ALP group was the strongest, and the fluorescence was attributed to the Mn 2+ In addition, AAP@HMSN-CS+ALP, HMSN-CS-Mn 2+ +ALP observed weak green fluorescence, which is because the addition of ALP further enhanced the green fluorescence, revealing the effect of self-amplification of ROS by nanomaterials ( Figure 8 D). Fig.11 yes Figure 8 D. The decline of mitochondrial membrane potential is a landmark event in the early stage of apoptosis. JC-1 is a fluorescent probe used to detect mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 produces red fluorescence; when the mitochondrial membrane potential is low, JC-1 produces green fluorescence. Figure 8 E,AAP@HMSN-CS,HMSN-CS-Mn 2+ 、AAP@HMSN-CS-Mn 2+ The positive control group could produce green fluorescence, and the addition of ALP further enhanced the green fluorescence, confirming that the mitochondrial membrane potential JC-1 of HepG2 cells decreased, thereby inducing early apoptosis of HepG2 cells. Fig.12 yes Figure 8E. In addition, Calcein-AM / PI dual fluorescence staining was used to verify the death of HepG2 cells after treatment in each experimental group. After staining, live cells produced green fluorescence, while dead cells produced red fluorescence. Figure 8 As shown in F, compared with PBS, ALP, AAP@HMSN-CS, HMSN-CS-Mn 2+ ,AAP@HMSN-CS+ALP, HMSN-CS-Mn 2+ +ALP and other control groups, AAP@HMSN-CS-Mn 2+ and AAP@HMSN-CS-Mn 2+ +ALP positive control group showed stronger red fluorescence and weaker green fluorescence, which indicated that AAP@HMSN-CS-Mn 2+ and AAP@HMSN-CS-Mn 2+ The +ALP positive control group induced more cell death than the other groups and had significant cytotoxicity. Fig.13 yes Figure 8 F. We have analyzed the statistical diagram of AAP@HMSN-CS-Mn 2+ The effects of β-catenin on HepG2 cells were studied in depth. We detected the protein expression levels of anti-apoptotic protein Bcl-2 and apoptotic proteins Caspase-3, Caspase-8, and Bax. Figure 8 G The results showed that compared with the control group, AAP@HMSN-CS-Mn 2+ and AAP@HMSN-CS-Mn 2+ +ALP positive control group down-regulated the expression of Bcl-2 and up-regulated the expression of Caspase-3, Caspase-8 and Bax. This indicates that AAP@HMSN-CS-Mn 2+ It promoted the apoptosis of HepG2 cells, leading to cell death. 2+ To determine whether there are other anti-tumor pathways, the researchers prepared cell slides and performed immunofluorescence staining of pyroptosis markers Gasdermin D, Caspase-1, and Caspase-4 on HepG2 cells treated with different methods. Figure 8 The results showed that AAP@HMSN-CS-Mn 2+ and AAP@HMSN-CS-Mn 2+ HepG2 cells treated with +ALP positive control group showed strong green fluorescence. This confirmed the high expression of Gasdermin D, Caspase-1, and Caspase-4. This indicates that compared with the control group, AAP@HMSN-CS-Mn 2+and AAP@HMSN-CS-Mn 2+ The +ALP positive control group had a stronger effect of inducing cell pyroptosis on HepG2 cells. Fig.14 is a statistical graph of the expression of pyroptosis markers. According to the above results, the nanocomplex constructed in vitro due to AAP@HMSN-CS-Mn 2+ The self-amplified therapeutic effect showed superior anti-tumor effect.

[0075] Example 3: This example is to prepare AAP@HMSN-CS-Mn 2+ In vivo anticancer testing of nanomaterials, including the following studies: 1. Establishment of tumor model: HepG2 cells in the logarithmic growth phase were collected and the cell concentration was adjusted to 2 × 10 7 100 μL of cell suspension was injected subcutaneously into the right upper abdomen of mice. After 2-3 weeks, the tumor volume reached 60-80 cm 3 The patients were randomly divided into five groups (n = 6 in each group) and injected intratumorally (5 mg kg -1 )treat, i: PBS, ii: HMSN, iii: AAP@HMSN-CSG, iv: HMSN-CS-Mn 2+ ,v:AAP@HMSN-CS-Mn 2+ Nude mice were treated 7 times within 0-12 days, with medication administered every 2 days, and tumor size was measured and body weight was recorded. Nude mice were euthanized after 12 days, and major organs (heart, liver, spleen, lung and kidney) and tumors were collected and weighed.

[0076] Each group (n=3) of isolated tumor tissues was freeze-thawed with liquid nitrogen for 5 minutes and placed in a -80°C refrigerator for temporary storage. The remaining part was embedded in paraffin.

[0077] 2. Immunofluorescence staining to monitor apoptosis (TUNEL).

[0078] The tumor paraffin slides were defatted, autoclaved, and antigen retrieval was performed according to routine procedures. Subsequently, 3% H2O2 was sterilized, loaded with 5% bovine serum albumin (BSA), and washed 3 times with PBS for 5 minutes each. 50 μL of TUNEL reaction solution was added to the tumor tissue area. The slides were incubated at 37°C for 1 hour and washed 3 times for 5 minutes each. The sections were then placed in the tumor tissue area containing 50 μL of DAPI working solution to stain the cell nuclei for 5 minutes. After washing three times in PBS, anti-fluorescence quenchers were added to the tumor tissue area for 5 minutes and observed by laser confocal scanning microscopy (LCSM). Immunofluorescence images showed that AAP@HMSN-CS-Mn 2+ It caused the most TUNEL-positive cell apoptosis, which was manifested by the strongest green fluorescence signal.

[0079] 3. Immunofluorescence staining to detect tumor pyroptosis The separated tumor tissue was cut into blocks, freeze-thawed with liquid nitrogen for 5 minutes, and placed in a -80°C refrigerator for temporary storage.

[0080] The tissue block was placed in a freezing embedding mold, sliced ​​with an ice cutter after adjusting the section position, and stored in dry ice.

[0081] 1. Fixation of frozen sections: Take the sections out of the refrigerator and warm them up for 20-30 minutes, fix them in 4% paraformaldehyde for 30 minutes, and wash them in PBS (PH7.4) on a decolorizing shaker for 3 times, 5 minutes each time.

[0082] 2. Antigen repair: Place the tissue sections in a repair box filled with EDTA antigen repair buffer (PH6.0) in a microwave oven for antigen repair. Heat at high heat for 3 minutes, stop for 10 minutes, and switch to low heat for 3 minutes. During this process, prevent the buffer from evaporating excessively and do not dry the slides. After natural cooling, place the slides in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time. (The repair solution and repair strength are determined according to the tissue).

[0083] 3. Circle serum blocking: After the slices are slightly dried, use a histochemical pen to draw circles around the tissue (to prevent the antibody from flowing away), dry the PBS, add 5% BSA, and block for 30 minutes. (If the primary antibody is from goat, use 10% donkey serum to block, and if the primary antibody is from other sources, use 5% BSA to block).

[0084] 4. Primary antibody incubation: dilute the primary antibody to an appropriate concentration with PBS, add 100ul of primary antibody to each slide, and incubate overnight at 4°C.

[0085] 5. Secondary antibody incubation: Take the slices out of the refrigerator and warm them up for 20-30 minutes, wash them 3 times with PBST, dilute the FITC-labeled secondary antibody with 1% BSA, add 50ul of secondary antibody to each slice, and incubate in a 37°C oven for half an hour.

[0086] 6. Counterstaining with DAPI: Wash with PBST for 3 minutes*3 times, add diluted DAPI, and incubate at room temperature for 10 minutes.

[0087] 7. Covering microscopy: Add an appropriate amount of anti-fluorescence quencher to each slice and cover it with a coverslip, and observe under a fluorescence microscope, or scan it with a full-scan instrument. 12 hours after the corresponding treatment of the above groups, the mice were anesthetized and sacrificed, and the tumor tissues were collected and cut into paraffin sections. Subsequently, Gasdermin D&DAPI, Caspase-1&DAPI, and Caspase-4&DAPI immunofluorescence staining were performed, and observed under a microscope and LCSM, respectively.

[0088] 4. ALP expression in tumor tissue 1. Take out the quick-frozen tumor tissue from the -80℃ freezer, weigh it quickly and record it. According to the usage, add 10ul PMSF to every 1ml RIPA, so that the final concentration of PMSF is 1mM. Mix well and set aside (PMSF is added immediately before use). Grind the tissue into powder with liquid nitrogen. Add lysis buffer at a ratio of 150-250ul lysis buffer per 20mg tissue and lyse for 30min. Centrifuge the lysed sample at 10000-14000g for 3-5 minutes, take the supernatant, and then proceed to the subsequent protein concentration determination.

[0089] 2. Use a 96-well plate to set up blank control wells, standard wells, and sample wells. The dosage of the standard is 4, 8, 16, 24, 32, and 40 μl, respectively. Samples can usually be 3. Directly add 50 μl. If the alkaline phosphatase activity in the sample is too high, reduce the sample volume or dilute it appropriately before measuring.

[0090] 4. Use the gun tip to gently blow and mix, or use a shaker to mix.

[0091] Incubate at 37°C for 10 minutes.

[0092] 6. Add 100 μl of reaction stop solution to each well to terminate the reaction.

[0093] 7. Measure the absorbance immediately at 405 nm.

[0094] 8 Definition of alkaline phosphatase activity unit: In diethanolamine (DEA) buffer at pH 9.8, at 37°C, the amount of alkaline phosphatase required to hydrolyze para-nitrophenyl phosphate chromogenic substrate to produce 1 micromole of p-nitrophenol per minute is defined as one enzyme activity unit, also known as one DEA enzyme activity unit. In glycine buffer at pH 9.6, at 25°C, the amount of alkaline phosphatase required to hydrolyze para-nitrophenyl phosphate chromogenic substrate to produce 1 micromole of p-nitrophenol per minute is defined as one enzyme activity unit, also known as one Glyeine enzyme activity unit. One Glycine enzyme activity unit is approximately equivalent to 3 DEA enzyme activity units. This kit measures DEA enzyme activity units.

[0095] 9. According to the definition of enzyme activity, calculate the alkaline phosphatase activity in the sample.

[0096] 5. ROS staining of frozen sections of tumor tissue 1. Prepare the materials needed for the experiment: ROS staining kit (including ROS staining solution and washing buffer), sample tissue sections to be stained, microscope slides, mounting medium, distilled water, etc.

[0097] 2. Temperature control: Ensure that the laboratory temperature is suitable, usually between 20-25℃.

[0098] 3. Experimental instruments: microscope, centrifuge, pipette, oscillator, etc.

[0099] 2. Sample processing 1. Fixation of frozen sections: Take the sections out of the refrigerator and warm them up for 20-30 minutes, fix them in 4% paraformaldehyde for 30 minutes, and wash them in PBS (PH7.4) on a decolorizing shaker for 3 times, 5 minutes each time.

[0100] 2. Evenly drop the ROS staining solution on the fixed sample and keep it moist. Place the sample in a humidifying box at a suitable temperature for pretreatment. The pretreatment time depends on the sample type and experimental requirements, usually 30 minutes to 1 hour.

[0101] 3. Drop the washing buffer on the sample, gently shake the culture dish or slide to make the washing buffer evenly cover the sample surface, and remove the excess staining solution. Repeat this step 2-3 times, each washing time is 5 minutes.

[0102] 4. Seal the washed samples with sealing media to ensure that the samples will not dry out and be damaged. The sealing method can be selected according to the experimental needs, such as sealing the slide with sealing media or sealing the culture dish with sealing agent.

[0103] 5. Microscope observation Prepare the microscope: Turn on the microscope and adjust parameters such as magnification and focus to ensure a clear image is observed.

[0104] Observe the sample: Place the sealed sample on the microscope stage, adjust the focus and field of view, and observe the ROS staining signal in the sample. Different filters and excitation wavelengths can be used to enhance the staining signal and reduce background noise.

[0105] Morphological analysis: qualitative and quantitative analysis is performed based on the morphology, intensity, and distribution of the staining signal. Image processing and data extraction of staining signals can be performed using image analysis software or computer-assisted analysis methods.

[0106] Record the ROS staining signal results observed under the microscope, including the stained cell or tissue type, the morphology and intensity of the staining signal, etc. 6. Ki67 staining of tumor tissue (chromogenic method, immunofluorescence) Paraffin-fixed tumor tissue sections were dewaxed in xylene for three times for 10 minutes. Next, they were washed with 100% ethanol for three times for 5 minutes, followed by washing in 95%, 85%, and 75% ethanol for 5 minutes each. Triton-X solution was used for 10 minutes of membrane destruction, and then the sections were immersed in PBS for three times for 5 minutes. Antigens were repaired by high pressure method for 10 minutes, and then the heating was turned off and the sections were allowed to cool naturally. They were soaked in PBS on a shaker three times for 5 minutes each. After shaking out excess water, a hydrophobic circle was drawn with a histochemical pen, and 3% hydrogen peroxide solution was added and incubated at room temperature for 10 minutes to remove endogenous enzyme activity, and then soaked in PBS on a shaker three times for 5 minutes each. 5% bovine serum albumin solution was added and blocked at 37°C for 20 minutes. Each section sample was incubated with 100 μL of anti-Ki67 antibody diluted 1:250 and incubated in a humidified box at 4°C overnight. After rewarming at room temperature for 30 minutes, wash three times with PBST solution, each time for 5 minutes. Then add 100 μL of secondary antibody (fluorescent FITC / non-fluorescent label) diluted 1:500 to each section, incubate at 37°C for 30 minutes, and then wash three times with PBST. DAPI / DAB staining is performed, with a control time of 5 minutes, based on the observation of blue fluorescence or brown reaction, and examined under a microscope. After rinsing with tap water for 5 minutes, the DAB staining solution is washed away, and hematoxylin is counterstained, followed by dehydration with a gradient alcohol series (75%, 85%, 95%, 100%). After the sections are air-dried, they are sealed with neutral gum / anti-fluorescence quencher and analyzed by optical or fluorescence microscopy.

[0107] 7. WB detection of apoptosis indicators (Casepase3 / 8, Bax, Bcl-2) The tumor tissue block was dissected out, washed with PBS, and frozen in liquid nitrogen at -80°C for temporary storage. The tumor tissue was removed, placed in a homogenizer tube, and treated with an ice bath. Two homogenizer beads with a diameter of 4 mm were added to the homogenizer tube, and lysate was added, with an amount of 20 mg and 200 μL lysate (the preparation of lysate must be carried out under light-proof conditions, and is prepared according to the ratio of high-efficiency RIPA tissue / cell rapid lysis solution: PMSF = 100:1). After homogenization with a homogenizer, the homogenizer tube was removed, placed in an ice basin, and placed on a shaker for 30 minutes, followed by centrifugation at 4°C and 12000rpm for 10 minutes, and the supernatant was collected into a clean EP tube. During centrifugation, the metal bath was opened in advance to preheat to 100°C for standby use, the protein sample and the protein loading buffer were mixed in proportion, the mixed solution was placed on a vortex oscillator for 15 seconds, and then placed in a metal bath preheated to 100°C for 10 minutes, cooled to room temperature, and placed in a -20°C refrigerator for standby use. (The subsequent steps are the same as cell WB).

[0108] 8. Immunofluorescence detection of tumor apoptosis and pyroptosis protein expression in HepG2 liver cancer model mice. BALB / c tumor-bearing mice were treated twice for 12 hours, and three mice in each group were dissected after anesthesia, and the tumor tissues were taken out for embedding and sectioning.

[0109] ① Dewax the paraffin sections in xylene for 10 min × 3 times.

[0110] ② Wash three times in 100% ethanol for 5 min, and then wash in 95%, 85% and 75% ethanol for 5 min respectively.

[0111] ③Permeabilize the membrane with Triton-X permeabilization solution for 10 minutes, and wash the sample with appropriate amount of PBS for 3 times, 5 minutes each time.

[0112] ④Use high pressure method to repair antigen for 10 minutes, then turn off the heating and let the slices cool naturally. Wash the samples with appropriate amount of PBS 3 times, 5 minutes each time.

[0113] ⑤ After shaking off excess water, draw a hydrophobic circle with a histological pen, add 3% hydrogen peroxide solution and incubate at room temperature for 10 minutes to remove endogenous enzyme activity, and rinse the sample 3 times with an appropriate amount of PBS, each time for 5 minutes.

[0114] ⑥ Block with 5% BSA at room temperature for 30 minutes and discard BSA.

[0115] ⑦ Use primary antibody diluent to prepare primary antibodies (Casepase1, Casepase3, Casepase 4, Casepase 8, Bax, Bcl-2, Gasdermin D) according to the dilution ratio in the antibody manual. Add 100 μL of primary antibody to each sample and incubate at 4°C overnight.

[0116] ⑧After returning to room temperature, rinse with PBST for 5 min × 3 times.

[0117] ⑨ Prepare the secondary antibody with secondary antibody diluent at a dilution ratio of 1:50, add 100 μL of secondary antibody with green fluorescence, and incubate at 37°C in the dark for 1 hour.

[0118] ⑩Rinse with PBST for 5 min × 3 times.

[0119] The samples were counterstained with DAPI stain and incubated for 5 min.

[0120] Wash the samples 3 times with appropriate amount of PBST, 5 min each time.

[0121] The sections were treated with anti-fluorescence quencher, covered with coverslips, and observed under a fluorescence microscope.

[0122] 9. ELISA to detect the expression of inflammatory factors 12 hours after the start of the two drug treatments, 3 BALB / c tumor-bearing mice in each group were dissected after anesthesia, and blood was taken from the eyeballs. After the blood samples were agglutinated for 30 minutes, they were centrifuged at 1000×g for 10 minutes. The serum samples were collected and packaged and stored below -20°C.

[0123] Prepare the test: Bring all reagents and samples to room temperature before testing.

[0124] Preparation of standard curve: Take 230 μL of standard and dilute it by 2 times step by step.

[0125] Soak the ELISA plate: Add 300 μL of wash solution to all wells of the ELISA plate.

[0126] Add standard: add 100 μL of the configured concentration standard to the standard wells, and add 100 μL of diluent to the blank wells.

[0127] Add sample: Add sample and detection buffer to the sample wells.

[0128] Add detection antibody: Add 50 μL of detection antibody to each well.

[0129] Incubation: Seal the microplate with a sealing film and incubate at room temperature for 2 hours.

[0130] Washing: Pour out the liquid in the well and add 300 μL of washing solution for washing.

[0131] 21 Enzyme incubation: Add 100 μL of diluted enzyme-labeled streptavidin to each well.

[0132] 22 Wash again: Add 300 μL of washing solution and wash again, pat dry after each wash.

[0133] 23 Add colorimetric substrate: Add 100 μL of colorimetric substrate TMB to each well and incubate at room temperature for 5-30 minutes.

[0134] 24 Add stop solution: Add 100 μL of stop solution to each well and observe the color change.

[0135] Detection reading: Use a microplate reader to measure the OD value at the absorption wavelength.

[0136] 10. Biosafety Assessment (1) HE staining of tumor tissue and various organs After the last administration, the mice were fasted for 12 hours, anesthetized and killed by cervical dislocation. The dissected tumor tissue was placed in a 4% paraformaldehyde solution for fixation for 48 hours, and then the tumor tissue was slightly trimmed and flattened, placed in a dehydration box and marked with a pencil. The dehydration box was placed in a glass bottle, and 75% alcohol was poured until the tissue was completely immersed. After soaking overnight, it was soaked in 85% alcohol for 2 hours, 95% alcohol for 2 hours, anhydrous ethanol I for 1 hour, and anhydrous ethanol II for 1 hour, and the dehydration process of the tissue was gradually completed. Then, it was soaked in alcohol benzene for 1 hour, xylene I for 1 hour, xylene II for 30 minutes, and waxed for 3 hours to complete the transparency of the tissue. The dehydration box was taken out of the wax dipping machine and placed on the embedding machine for embedding. During the embedding process, the tumor tissue was in the center of the embedding box and no bubbles were generated. Then it was moved horizontally to the freezing table for cooling, and after the wax solidified, it was taken out and temporarily stored at room temperature.

[0137] Place the corrected wax block on a paraffin slicer and slice each slice with a thickness of 4 μm. No scratches from the blade are required on the tissue. Then spread it in 45°C warm water, pick up the slice with an adhesive slide, bake it in a 65°C baking machine for 15 minutes, and then take it out and store it at room temperature.

[0138] After the tumor paraffin sections were baked in a 65℃ baking machine for 40 minutes, they were soaked in xylene I for 10 minutes, xylene II for 10 minutes, and xylene III for 10 minutes to complete the purpose of dewaxing the sections. Then, they were soaked in anhydrous ethanol I for 10 minutes, anhydrous ethanol II for 10 minutes, anhydrous ethanol III for 10 minutes, 95% alcohol for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and distilled water for 5 minutes to complete the gradual dehydration of the sections. The tumor sections were placed in hematoxylin stain for 3-5 minutes and washed quickly with running water for 1-2 minutes. Differentiate in differentiation solution for 5 seconds and wash again with running water for 1-2 minutes. Add anti-blue solution for 30 seconds and rinse with running water for 1-2 minutes. Then, the tumor sections were placed in eosin stain for 2 minutes and rinsed with running water for 1-2 minutes. Finally, the sections were sequentially immersed in 75% alcohol for 5 min, 95% alcohol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min to complete the dehydration and transparency of the tumor sections. After drying, the sections were sealed with neutral resin, and the changes in tumor tissue morphology of each group were observed under an optical microscope.

[0139] (2) Index of various organs of mice After the mice were anesthetized and dissected, they were killed from the neck, followed by dissection to extract the heart, liver, lung, spleen and kidney. After these organs were washed with PBS solution, the surface moisture was removed using filter paper, and their weights were accurately measured and recorded.

[0140] Organ index (mg / g) = organ mass (mg) / body mass (g) (3) Material hemolysis test 1. Take 2 ml of mouse blood and add it to the anticoagulant tube. Mix well and put it into two 15 ml centrifuge tubes. Add 7 ml 1xPBS and shake back and forth gently.

[0141] 2. Centrifuge at 1500rpm for 5min, aspirate the supernatant with a pipette, and add pBS to the 8ml mark (add along the tube wall, do not pour directly on the cells) 3. Repeat the above steps 3-5 times until the supernatant becomes clear after centrifugation. Centrifuge at 1500 rpm, smin, and collect the supernatant.

[0142] 4. After washing the red blood cells, take a 5ml centrifuge tube, take out 200ml of pure red blood cells from the bottom of the 15ml tube and mark it as tube A.

[0143] 5. Take out 4 more 5ml centrifuge tubes, add 200μL pure red blood cells + 1800μL PBS to each tube, and mark them as tube B.

[0144] 6. Take 9 1.5ml centrifuge tubes, Positive: Complete hemolysis, 50 pure red blood cells + 950μL ddH2O Negative: 500μL B tube + 500μL PBS Sample: 500μL tube B + 500μL material soaked in PBS for 24h. 7. Incubate the 9 tubes in a 37°C incubator for 1 h.

[0145] 8. Take out 9 centrifuge tubes and centrifuge at 2500rpm for 5min 9. Take a 96-well plate and inject the positive lysate and sample group, negative supernatant into the well plate, inject 100 ml into each well, and inject 1 well into each centrifuge tube.

[0146] 10. Place in an ELISA reader and measure the absorbance at 540 nm.

[0147] (Generally, the empty hole absorbance is ~0.08 or 0.09. The positive absorbance is >3 and the negative absorbance is ~0.3) Hemolysis rate: (sample group - negative) / (positive - negative) x 100% < 5% qualified (4) Blood routine, liver and kidney function tests of mice Serum biochemistry tests were performed on the blood of the control group and the treatment group. After the last administration, the mice were fasted for 12 hours. After anesthesia, about 0.5 mL of blood was taken from the eyeballs, collected in anticoagulant tubes containing EDTA-K2, and stored in a refrigerator at 4°C. Within 24 hours, blood samples from each experimental group were analyzed for cell counts including lymphocytes (Lymph), monocytes (Mon), neutrophils (Gran), red blood cells (RBC), platelets (PLT), and hemoglobin (HGB) using an automatic blood cell analyzer.

[0148] After the last dose of the drug, the mice were fasted for 12 hours, and blood was collected from the eyeballs after anesthesia. After standing at room temperature for 2 hours, the blood was centrifuged at 3000 rpm for 10 minutes. After centrifugation, the upper serum was aspirated with a pipette, and the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea (UREA), blood uric acid (UA), and blood creatinine (CREA) in the serum of each group of mice were measured by an automatic biochemical analyzer to evaluate the liver and kidney function of the mice in each dosage group.

[0149] 11. Statistical analysis Quantitative data are expressed as mean ± SD. All data in this experiment were plotted by GraphPad Prism9.0.0 software. The data are expressed as mean ± standard deviation (`x±S). When *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, the differences are statistically significant, and ns is not statistically significant.

[0150] The in vivo antitumor effect was measured in a xenograft HepG2 tumor model. Xenograft HepG2 tumors were established in the right axilla of BALB / c mice to investigate the antitumor effects of AAP@HMSN-CS-Mn 2+ All tumor-bearing mice were randomly divided into five groups (n=6): (1) control group; (2) HMSN group; (3) AAP@HMSN-CS group; (4) HMSN-CS-Mn 2+ Group; (5) AAP@HMSN-CS-Mn 2+ Group. Fig.15 As shown in A, after seven intratumoral injections of different nanocomplexes, the corresponding tumor volume and weight were measured during the experimental period and then anatomically analyzed. During the entire observation period, the extracted tumor ( Fig.15 B) No significant differences were observed in body weight and major organ indices of mice treated with various nanocomposites ( Fig.15 C. Fig.16 ). Compared with the control group, AAP@HMSN-CS and HMSN-CS-Mn 2+ The group showed a certain tumor growth inhibition rate. In contrast, AAP@HMSN-CS-Mn 2+ The most significant tumor growth inhibition was observed in the group with 44 cases, indicating that self-amplified chemokine therapy can lead to effective tumor therapy ( Fig.15 D). In addition, tumor weight and tumor inhibition rate ( Fig.15 The results of E\F) further confirmed this. Fig.15 G, The expression of ALP in tumor tissue was verified and it was found that AAP@HMSN-CS-Mn 2+ The most significant results were obtained in the treatment of tumor models with ALP depletion. In addition, frozen sections of tumors were stained with DHE-labeled ROS dye, and paraffin sections of tumors were stained with H&E, terminal deoxynucleotidyl transferase-mediated dUTP tail labeling (TUNEL), and Ki67 to show the expression of AAP@HMSN-CS-Mn 2+ The anti-tumor effect of the nanocomposite was shown in Figure 2. 2+ The red fluorescence is the strongest, indicating that ROS positive cells have strong expression, which corresponds to the in vitro treatment effect. 2+ It caused severe tumor damage, as shown by obvious tissue destruction in H&E staining results, a large number of green dots in TUNEL images, and decreased Ki67 expression ( Fig.15H). Interestingly, apoptosis and pyroptosis of tumor tissues were investigated. We detected the expression levels of anti-apoptotic protein Bcl-2 and apoptotic proteins Caspase-3, Caspase-8, and Bax in tumor tissues and found that AAP@HMSN-CS-Mn 2+ The AAP@HMSN-CS-Mn 2+ The expression of pyroptosis markers, such as Gasdermin D, Caspase-1, and Caspase-4, was induced, which, together with apoptosis, provided direct evidence to explain the significant inhibition of tumor growth. Fig.15 I), and immunofluorescence experiments also verified the same results ( Figure 17-23 ). In addition, the detection of cell pyroptosis factors IL-18 and IL-1β in mouse serum also indirectly verified cell pyroptosis ( Fig.24 ). In addition, H&E images of major organs under different treatments after 14 days showed that AAP@HMSN-CS-Mn 2+ Negligible adverse effects on normal tissues ( Fig.25 In addition, the blood routine test of nude mice ( Fig.26 ) Serum biochemistry ( Fig. 27 ) and in vitro hemolysis assay ( Fig.28 ) showed no significant difference between the control group and AAP@HMSN-CS-Mn 2+ Abnormal performance between groups.

[0151] Therefore, the tumor therapeutic drug AAP@HMSN-CS-Mn 2+ It can promote apoptosis and pyroptosis of tumor cells by regulating the expression level of apoptotic proteins, thereby inhibiting tumor growth, and shows good safety and biocompatibility in animal experiments.

[0152] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method for preparing composite nanoparticles loaded with AAP and manganese ions, characterized in that: The method comprises the following steps: (1) Preparation of AAP@HMSN-CS: hollow mesoporous silica nanoparticles are added to ultrapure water, and ultrasonic dispersion is performed to prepare a 5 mg / mL dispersion; ascorbic acid phosphate is added to ultrapure water to prepare a 25 mg / mL ascorbic acid phosphate solution; 10 mL of the hollow mesoporous silica nanoparticle dispersion and 10 mL of the ascorbic acid phosphate solution are added to a small brown bottle in sequence and stirred; chitosan solution is added to the mixed solution, stirred, centrifuged, the supernatant is discarded, and the mixture is washed with ultrapure water for 3 times. After freezing, the mixture is placed in a vacuum freeze dryer and dried to obtain the AAP@HMSN-CS powder, which is stored at 4°C for future use; (2) Preparation of HMSN-CS-Mn2+: hollow mesoporous silica nanoparticles are added to ultrapure water, and ultrasonic dispersion is performed to prepare a 5 mg / mL dispersion; an appropriate amount of MnCl2 is weighed and dissolved in ultrapure water to prepare a 5 mg / mL solution; 10 mL of the hollow mesoporous silica nanoparticles is added to the mixture, and the mixture is centrifuged and the supernatant is discarded. The mixture is washed with ultrapure water for 3 times, and then frozen and dried in a vacuum freeze dryer to obtain the AAP@HMSN-CS powder. The particle dispersion and 10 mL of chitosan solution were added to a small brown bottle in sequence and stirred; 10 mL of MnCl2 solution was added to the above mixed solution and stirred; centrifuged, the supernatant was discarded, and the mixture was washed with ultrapure water for 3 times. After freezing, the mixture was placed in a vacuum freeze dryer and dried to obtain HMSN-CS-Mn2+ powder, which was stored at 4°C for later use; (3) Preparation of AAP@HMSN-CS-Mn2+: AAP@HMSN-CS was added to ultrapure water and ultrasonically dispersed to prepare a 1 mg / mL dispersion; MnCl2 was added to 1 ultrapure water to prepare a 2 mg / mL solution; 10 mL of AAP@HMSN-CS dispersion and 10 mL of MnCl2 solution were added to a small brown bottle in sequence and stirred; centrifuged, the supernatant was discarded, the mixture was washed with ultrapure water for 3 times, frozen, and placed in a vacuum freeze dryer for drying to obtain AAP@HMSN-CS-Mn2+ powder, which is composite nanoparticles loaded with AAP and manganese ions and stored at 4°C for later use.

2. The preparation method according to claim 1, characterized in that: In steps (1) and (2), the concentration of the chitosan solution is 0.5 mg / mL.

3. The preparation method according to claim 1, characterized in that: In steps (1), (2) and (3), the stirring is carried out in a constant temperature magnetic stirrer at 37° C. for 24 hours.

4. The preparation method according to claim 1, characterized in that: In steps (1), (2) and (3), the centrifugation operation is performed at 8000 rpm for 10 min.

5. The preparation method according to claim 1, characterized in that: In steps (1), (2) and (3), the freezing is performed in an ultra-low temperature freezer at -80°C for 1 hour.

6. The preparation method according to claim 1, characterized in that: In steps (1), (2) and (3), the drying time is 16-24 hours.

7. A composite nanoparticle material loaded with AAP and manganese ions obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the composite nanoparticle material loaded with AAP and manganese ions as claimed in claim 7 in the preparation of drugs for treating liver cancer.

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