Preparation method and application of ferrocene-loaded electroactive mesoporous silica composite nanoparticles

Abstract

The invention belongs to the technical field of nanotechnology, and relates to a preparation method and application of ferrocene-loaded electroactive mesoporous silica composite nanoparticles. Specifically, MSNs are constructed as a template, elemental platinum is subjected to in-situ reduction and loaded on the MSNs, and ferrocene is used as an electroactive group, so that the Fc / Pt-coated MSNs (FPMSN) nanoparticles with electroactivity are successfully synthesized. A HepG2 tumor-bearing nude mouse model is constructed, FPMSNs combined electrodynamic therapy antibody internal tumor effect research is carried out, and tumor growth is proved to be effectively inhibited by FPMSNs combined electrodynamic therapy through weight and tumor volume measurement of tumor-bearing nude mice in different treatment groups. The obvious effectiveness and good biological safety of the FPMSNs electrodynamic therapy provide a new way for developing a new cancer treatment method.

Description

Technical Field

[0001] The invention belongs to the field of nanotechnology and relates to a preparation method of ferrocene-loaded electroactive mesoporous silica composite nanoparticles and application thereof. Background Art

[0002] Malignant tumors (i.e. cancer), as a disease that seriously threatens human health, have brought heavy economic and psychological burdens to patients and their families. According to the data in the "World Health Statistics Report 2022" released by the World Health Organization, cancer mortality ranks among the top five non-communicable diseases in the world. According to the global cancer statistics released by the American Cancer Society in 2020, there were 19.29 million new cancer cases and 9.96 million deaths in 2020. China's cancer incidence and mortality rates ranked first in the world. Traditional clinical cancer treatments are mainly surgery, chemotherapy and radiotherapy, but the effects of these treatments are still unsatisfactory, and there are problems such as treatment limitations, easy recurrence, large adverse reactions, and narrow treatment windows. Therefore, actively developing effective new strategies for tumor prevention and treatment and finding new anti-tumor drugs are difficult problems that scientists urgently need to solve.

[0003] According to the World Health Organization, liver cancer is the fifth most common cancer in the world and the fourth leading cause of cancer-related death. Its incidence and mortality rates remain high, and the incidence rates in Asia, especially in China, are even more significant. In 2020, liver cancer caused 830,000 deaths. For patients with early liver cancer, surgical resection is one of the most effective treatments. With the development of minimally invasive surgical techniques, the safety and effectiveness of surgical treatments, including liver resection and liver transplantation, have been significantly improved. For patients with advanced or inoperable liver cancer, a comprehensive treatment plan is often used, including chemotherapy, targeted therapy, radiotherapy and other combined methods, aiming to inhibit tumor growth and metastasis to the greatest extent. Since it is difficult to achieve satisfactory therapeutic effects with current treatment methods, liver cancer treatment faces many challenges. Future research should continue to explore the pathogenesis of liver cancer in depth and strive to find effective anti-liver cancer drugs to improve patients' survival rate and quality of life.

[0004] With the advancement of technology and the deepening of medical research, various nanodynamics have emerged in the field of cancer treatment, which are known for their specificity, small side effects and significant efficacy. At present, the main nanodynamic therapies studied for tumors include photodynamic therapy, sonodynamic therapy, radiodynamic therapy, electrodynamic therapy and chemodynamic therapy. Nanodynamic therapy uses nanomaterials combined with specific forms of physical energy (such as acoustic, light, electricity, radioactivity and chemical energy) to precisely target tumor cells. One feature of these therapies is that they can convert local energy into a mechanism that triggers the production of reactive oxygen species (ROS) in tumors, leading to cell death or damage. Although these ROS-based nanodynamic therapies have shown great promise in tumor treatment, there are still many challenges in practical applications. Electrochemical reactions are widely used in the biomedical field of disease treatment because they can produce multifunctional products. For example, platinum nanoparticles injected into the body are activated under the action of square wave alternating current, which can kill tumor cells and inhibit tumor growth. Compared with traditional electrochemotherapy, electrodynamic therapy (EDT) is not only less invasive, but also has a killing effect on larger tumors. The advantage of electrodynamic therapy for tumors is that it does not rely on hydrogen peroxide in the tumor microenvironment, and can produce cytotoxic hydroxyl radicals through water electrolysis, while avoiding the side effects and complications of traditional surgery and chemoradiotherapy. Although EDT shows good tumor treatment effects, the process of inserting electrodes into the tumor during treatment will cause unnecessary tissue damage, and requires precise operation through imaging, which is difficult to practice. In addition, in order to achieve the clinical transformation of EDT, further research is needed on EDT treatment parameters, biosafety, etc., and it should be combined with other treatment strategies such as chemotherapy and immunotherapy.

[0005] Mesoporous silica nanoparticles (MSNs), as a material that has attracted much attention in the field of bio-nanomaterials, have shown broad application prospects from a medical perspective. Its unique microporous structure and large surface area make it an ideal choice for drug delivery, imaging and treatment in the medical field. In terms of medical applications, the characteristics of MSNs are particularly eye-catching. As a drug delivery system, MSNs achieve precise controlled release of drugs through controllable pore structures. This targeted release property helps to more effectively target tumor cells and reduce damage to normal tissues. In addition, MSNs can be used for imaging-guided therapy. By carrying fluorescent markers or contrast agents, doctors can more clearly observe the location and changes of tumors and improve surgical accuracy. In the treatment of liver cancer, the application of MSNs has become particularly significant. By carrying anticancer drugs, MSNs can achieve targeted delivery of drugs directly to liver cancer tissues, thereby reducing systemic toxicity, increasing the concentration of drugs in tumor tissues, and effectively enhancing the therapeutic effect. Liao et al. successfully synthesized MSNs-encapsulated alginate (Alg) microspheres using aerodynamic atomization technology. As a drug delivery carrier, MSN@Alg microspheres have excellent biocompatibility, sustained-release properties, and enhanced drug release capacity. By functionalizing them with the liver cancer cell targeting peptide K4YRGD and loading them with the anticancer drug doxorubicin (DOX), MSN@Alg-RGD showed significantly improved intracellular delivery efficiency to liver cancer cells (HepG2) through receptor-mediated endocytosis. MSNs provide strong support for the development of efficient drug delivery systems for liver cancer treatment and have important clinical application prospects.

[0006] Due to its unique DNA-damaging cytotoxicity, platinum-based chemotherapy has long been the first-line choice for clinical tumor treatment. Unfortunately, the side effects of platinum-based drugs have severely limited their clinical application. With the development of nanotechnology and chemical synthesis technology, platinum-based anticancer drugs have made great progress in cancer treatment in recent years. Platinum (Pt)-based nanoplatforms are a type of biocompatible nanoformulation that not only has photothermal antitumor properties, but also exhibits excellent radiotherapy sensitization properties. During electrodynamic therapy, platinum nanoparticles (PtNPs) undergo electrically driven catalytic reactions on their surfaces, generating reactive oxygen species under direct current (DC) or square wave alternating current (AC) electric fields. In order to further expand the potential of EDT, the Liu team designed MSNs-based nanocomposites decorated with PtNPs and loaded with the anticancer drug doxorubicin for synergistic electrodynamic chemotherapy. This silica-based nanocomposite can achieve uniform killing of large-sized tumors and achieve significant tumor destruction effects at relatively low currents. In general, platinum-based anticancer chemotherapy drugs can kill tumor cells by damaging DNA through chemotherapy. At the same time, Pt-based nanoplatforms also have good electrocatalytic activity and can mediate new electrodynamic therapy. Pt-based compounds also have the potential to be used as photosensitizers for photodynamic therapy of malignant tumors. Platinum-based nanoplatforms can also regulate the immunosuppressive environment and combine with immune checkpoint inhibitors to synergistically ablate tumor cells. Platinum-based nanoplatforms have broad research prospects in the field of cancer treatment due to their high selectivity, low damage, minimally invasive treatment mode, imaging monitoring, synergistic drug delivery and other integrated diagnosis and treatment modes.

[0007] Ferrocene (Fc) is an iron complex containing two cyclopentadiene groups, in which the central iron atom is combined with two cyclopentadiene rings in a sandwich configuration. Fc can quickly transfer electrons on the electrode surface, providing good conductivity for the sensor, so ferrocene is often used as an electron transfer medium in electrochemical biosensors. As a metal-organic complex with an electron donor-acceptor conjugated structure, Fc shows broad application prospects in drug delivery and tumor treatment due to its reversible redox properties. For example, Xu Jie et al. reported an amphiphilic polyferrocene (BPnbs-Fc) fragment that can quickly consume intracellular glutathione (GSH) and cascade release aminoferrocene (AFc). The synergistic effect of GSH depletion and hydroxyl radical (OH) burst effectively inhibits tumor growth. AFc itself acts as a catalyst, and the OH generated by the reaction with H2O2 can react with Fe 3+ / Fe 2+ , followed by Fe 2+ Reacts with H2O2 to generate OH and Fe 3+, then Fe 3+ Reacts with GSH to generate GSSH and Fe 2+ The whole process realizes the recycling of iron ions. In addition, FcPBn-MI is designed by using phenyl borate and ferrocene as the repeating main unit. x -ICG 1-x Nanoparticles demonstrate the advantages of Fc-based nanoparticles in tumor-targeted delivery. x -ICG 1-x After intravenous injection, the nanoparticles can effectively covalently bind to serum albumin to form an albumin-rich protein corona, significantly improving the efficiency of tumor-targeted delivery. After being taken up by tumor cells, endogenous H2O2 triggers the phenylboronic acid ester in the polyferrocene skeleton, and ferrocene is released and oxidized. OH and hydroxide ions are generated in the ferrocene aggregates, thereby inhibiting tumor cell growth. In summary, the application of Fc and its derivatives in oncology research demonstrates its unique value in tumor treatment, drug delivery system design, and tumor microenvironment-specific response.

[0008] Currently, there is no report on the preparation method of ferrocene-loaded electroactive mesoporous silica composite nanoparticles and their application in the treatment of liver cancer. Summary of the invention

[0009] The purpose of the present invention is to provide a method for preparing electroactive mesoporous silica composite nanoparticles loaded with ferrocene and its application, using aminated MSNs as templates, reducing H2PtCl6 to platinum and uniformly loading it on MSNs, and then combining the amino groups on MSNs with the carboxyl groups on ferrocene carboxylic acid to load Fc on the surface of platinum-loaded mesoporous silica (Pt@MSNs) to synthesize electroactive nanoparticles FPMSNs (Fc / Pt@MSNs). Combined with electrodynamic therapy, water molecules on the surface of FPMSN nanoparticles react under the drive of square wave alternating current electric field to produce cytotoxic OH, and at the same time catalyze intracellular hydrogen peroxide to generate ROS through Fenton reaction, because the presence of ferrocene causes the rapid conversion of iron ions between +2 and +3, which increases the speed of electron transfer, expands the current in tumor cells, increases the generation of ROS and OH, and promotes apoptosis and pyroptosis of tumor cells. By constructing a HepG2 tumor-bearing nude mouse model and studying the in vivo anti-tumor activity of FPMSNs combined with electrodynamic therapy, we found that the significant effectiveness and good biosafety of FPMSNs electrodynamic therapy provide a new approach for the development of new cancer treatments.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is: The method for preparing electroactive mesoporous silica composite nanoparticles loaded with ferrocene comprises the following steps: (1) Preparation of modified MSNs: 1) Anhydrous ethanol, deionized water and aqueous ammonia are mixed and stirred in a constant temperature magnetic stirring water bath to obtain system I; 2) adding tetraethyl silicate to system I and reacting in a constant temperature water bath to obtain system II; 3) 2.5 mL of tetraethyl silicate and 1.5 mL of 3-aminopropyltriethoxysilane were mixed and slowly added dropwise to system II, and then reacted in a constant temperature magnetic stirring water bath; 3) repeatedly washing with anhydrous ethanol for 3 times to obtain mesoporous silica; 4) The mesoporous silica obtained above was mixed with 50 mL of 0.6 M Na2CO3 solution and stirred in a water bath at 80° C. for 2 h, then washed with anhydrous ethanol for 3 times and freeze-dried for 24 h to obtain surface amino-modified MSNs powder for later use; (2) Preparation of Pt@MSNs: Take H2PtCl6 and quickly add it to anhydrous ethanol to obtain system III, then add the surface amino-modified MSNs powder obtained in step (1) to system III, stir and place in a flat-bottom flask, stir in a constant temperature magnetic stirring water bath at 80°C, condense and reflux, and stop heating after observing that the reaction system turns black; then repeatedly use anhydrous ethanol to wash 3 times, freeze-dry for 24 hours, and obtain amino-modified platinum-loaded MSNs powder, namely Pt@MSNs, for later use; (3) Preparation of Fc / Pt@MSNs: S1, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide to anhydrous ethanol to obtain a mixed solution I for standby use; S2, measure a certain amount of H2O2 and set aside; S3, respectively measuring a certain amount of p-hydroxybenzoic acid, horseradish peroxidase, ferrocenecarboxylic acid and tyramine ethanol solution, mixing them in a flat-bottom flask to obtain a mixed solution II for later use; S4, mixing the mixed solution I of S1, the H2O2 of S2, and the mixed solution II of S3 to obtain a mixed solution III; S5. Weigh the Pt@MSNs powder obtained in step (2) and add it to the mixed solution III, and place it in a constant temperature magnetic stirring water bath for stirring; then wash it alternately with anhydrous ethanol and deionized water for a total of 3 times, and freeze-dry it for 24 hours to obtain electroactive mesoporous silica composite nanoparticles loaded with ferrocene, namely Fc / Pt@MSNs, which are set aside.

[0011] Further, in step (1), the amount of anhydrous ethanol, deionized water and ammonia water is 10 mL; the reaction condition of system I is stirring in a constant temperature magnetic stirring water bath at 30°C for 20 min; the reaction condition of system II is adding 2.5 mL of tetraethyl silicate and reacting in a constant temperature water bath at 30°C for 45 min.

[0012] Further, in step 3), the reaction condition is to react in a constant temperature magnetic stirring water bath at 30° C. for 80 min.

[0013] Further, in step (2), the amount of H2PtCl6 used in the system III is 0.275 mg / mL, 180 μL, and the amount of anhydrous ethanol used is 50 mL.

[0014] Further, in step (3), the amounts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and anhydrous ethanol are 4 mL, 1 mL, and 15 mL, respectively; the concentration and amount of H2O2 are 5 mmol and 10 mL, respectively.

[0015] Further, in step (3), the dosage of the ethanol solution of p-hydroxybenzoic acid, horseradish peroxidase, ferrocenecarboxylic acid and tyramine is 10 mL at 1 mg / mL each.

[0016] Further, in step S5, the amount of the Pt@MSNs powder used is 30 mg; and the stirring condition in the constant temperature magnetic stirring water bath is stirring at 37° C. for 24 h.

[0017] Another object of the present invention is to provide an electroactive mesoporous silica composite nanoparticle material loaded with ferrocene obtained by the preparation method.

[0018] Another object of the present invention is to provide the use of the ferrocene-loaded electroactive mesoporous silica composite nanoparticle material prepared by the preparation method in the preparation of drugs for treating liver cancer.

[0019] Due to the adoption of the above technical solution, the present invention has the following beneficial effects: The present invention is a method for preparing electroactive mesoporous silica composite nanoparticles loaded with ferrocene and its application. With aminated MSNs as templates, H2PtCl6 is reduced to platinum and uniformly loaded on MSNs. The amino groups on MSNs are then combined with the carboxyl groups on ferrocene carboxylic acid, and Fc is loaded on the surface of platinum-loaded mesoporous silica (Pt@MSNs) to synthesize electroactive nanoparticles FPMSNs (Fc / Pt@MSNs). Combined with electrodynamic therapy, water molecules on the surface of FPMSN nanoparticles react under the drive of square wave alternating current electric field to produce cytotoxic OH, and at the same time catalyze intracellular hydrogen peroxide to generate ROS through Fenton reaction. Because of the presence of ferrocene, the rapid conversion of iron ions between +2 and +3 increases the speed of electron transfer, expands the current in tumor cells, increases the generation of ROS and OH, thereby promoting apoptosis and pyroptosis of tumor cells, and finally achieves the expected anti-tumor effect. By constructing a HepG2 tumor-bearing nude mouse model, the anti-tumor effect of FPMSNs combined with electrodynamic therapy was studied in vivo. The weight and tumor volume measurements of tumor-bearing nude mice in different treatment groups proved that FPMSNs combined with electrodynamic therapy effectively inhibited tumor growth. The applicant successfully demonstrated the anti-tumor mechanism of electroactive nanoparticles FPMSNs combined with electrodynamic therapy in nude mice, providing substantial evidence for the application of nanomaterials combined with electrodynamic therapy in cancer treatment. The significant effectiveness and good biosafety of FPMSNs electrodynamic therapy provide a new way to develop new cancer treatment methods. . BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the anti-tumor principle of the electroactive nanomaterial FPMSNs combined with electrodynamic therapy of the present invention.

[0021] Figure 2 It is a characterization result diagram of the FPMSN nanoparticles of the present invention.

[0022] Figure 3 A. Biocompatibility of FPMSN nanoparticles on HepG2 cells; B. Biocompatibility of FPMSN nanoparticles on HUVEC cells; C. Effect of direct current on HepG2 cell viability; D. Effect of FPMSN nanoparticles on HepG2 cell viability under direct current; E. Changes in pH value of PBS solution near the two electrodes after applying direct current for 10 minutes; F. Effect of square wave alternating current electric field on HepG2 cell viability; G. Effect of square wave alternating current electric field on HepG2 cell viability; H. Graph showing the effect of FPMSN nanoparticles on HepG2 cell viability under square wave alternating current electric field.

[0023] Figure 4A. The DCFH-DA fluorescence intensity produced by HepG2 cells under different concentrations of FPMSN nanoparticles; B. The DCFH-DA fluorescence intensity produced by HepG2 cells under square wave AC electric fields of different voltages; C. The DCFH-DA fluorescence intensity produced by HepG2 cells under square wave AC electric fields of different currents; D. The DCFH-DA fluorescence intensity graph produced by HepG2 cells under square wave AC or DC electric fields of different current durations.

[0024] Figure 5 A. The relative survival rate of HepG2 cells subjected to electrodynamic therapy under square wave AC electric fields of different currents; B. The relative survival rate of HepG2 cells subjected to electrodynamic therapy under square wave AC electric fields of different voltages; C. The relative survival rate of HepG2 cells subjected to electrodynamic therapy under square wave AC electric fields with direct current and current durations of 1, 10, 30, and 60 s, respectively; D. A graph of the relative survival rate of HepG2 cells subjected to electrodynamic therapy under square wave AC electric fields of different durations.

[0025] Figure 6 This is a study diagram of FPMSN nanoparticles causing apoptosis of liver cancer cells.

[0026] Figure 7 These are confocal microscopy images of HepG2 cells stained with pyroptosis antibodies after different treatments. A. Gasdermin D; B. Caspase-1; C. Caspase-4.

[0027] Figure 8 The in vivo anti-tumor effect of FPMSNs nanoparticles combined with EDT. A. Tumor images of nude mice in each group after treatment; B. Schematic diagram of the treatment cycle of nude mice; C. Weight changes of nude mice in each group; D. Tumor volume changes of nude mice in each group; E. H&E staining of tumor tissues of nude mice in different nanomaterial treatment groups (scale bar is 100μm); F. Ki67 staining results of tumor sections of nude mice in different nanomaterial treatment groups (scale bar is 100μm).

[0028] Fig. 9 These are HE staining images of the heart, liver, spleen, lung and kidney of nude mice in different nanomaterial treatment groups (scale bar is 100 μm).

[0029] Fig.10 A. TUNEL staining results of nude mouse tumor sections in different nanomaterial treatment groups (scale bar is 50 μm); B. ROS staining results of nude mouse tumor sections in different nanomaterial treatment groups (scale bar is 50 μm).

[0030] Fig.11A. WB blotting of different treatment groups; B. Expression analysis of Caspase-1, Caspase-4 and Gasdermin DN in different treatment groups; ELISA results of pyroptotic cytokines in serum in different nanomaterial treatment groups; C. IL-1β content and standard curve; D. IL-18 content and IL-18 standard curve.

[0031] Fig.12 This is the immunofluorescence statistical analysis of A. Caspase-1, B. Caspase-4 and C. Gasdermin D in different nanomaterial treatment groups.

[0032] Fig.13 This is a schematic diagram of the contact angle measurement of FPMSNs. A. PBS solution; B. PBS solution of FPMSNs; C. Hemolysis of FPMSNs at different concentrations; D. Hemolysis percentage of FPMSNs at different concentrations; E. Schematic diagram of the distribution of nanomaterials in various organs of nude mice after FPMSNs combined with electrodynamic therapy; F. Schematic diagram of the survival time of nude mice in different nanomaterial treatment groups.

[0033] Fig.14 A. Schematic diagram of the effects of routine blood tests on nude mice in different nanomaterial treatment groups: white blood cell count (WBC), lymphocyte count (LYM), monocyte count (MONO), neutrophil count (NEU), hemoglobin content (HCB) and red blood cell count (RBC); B. Schematic diagram of the effects of renal function on nude mice in different nanomaterial treatment groups: creatinine (CREA), urea nitrogen (UREA), uric acid (UA); C. Schematic diagram of the effects of liver function on nude mice in different nanomaterial treatment groups: albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DBIL), total bile acid (TBA), total bilirubin (TBIL), γ-glutamyl transferase (γ-GT).

[0034] In the above figure, ns P>0.05, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001. DETAILED DESCRIPTION

[0035] The specific implementation of the invention is further described below with reference to the accompanying drawings.

[0036] Chemicals and reagents: ultrapure water, anhydrous ethanol, ammonia (NH3·H2O), tetraethyl silicate, sodium carbonate, 3-aminopropyltriethoxysilane, chloroplatinic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide, ferrocenecarboxylic acid. Instruments: 1.5mL, 15mL, 50mL centrifuge tubes, 1mL syringes, 50mL flat-bottom flasks, stirring rods, 5mL pasteurized pipettes, digital constant temperature magnetic stirring water bath, ultrapure water machine, constant temperature magnetic stirrer, small and medium-sized desktop centrifuges, ultrahigh-speed centrifuges, vortex oscillators, scanning electron microscopes, laser particle size analyzers, ultraviolet spectrophotometers, Fourier transform infrared spectrometers, biological safety cabinets, tray balances, electronic balances, vertical electric heating constant temperature blast drying ovens, ultra-low temperature freezers, vacuum freeze dryers, ultrasonic cleaners. The above reagents and instruments can all be purchased on the market.

[0037] HepG2 and HUEVC cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The healthy BALB / c mice used, female, 6-8 weeks old, were purchased from the Animal Experiment Center of Guangxi Medical University, SPF-level laboratory. All mice were treated according to the protocol approved by the Institutional Animal Care and Use Committee of Guangxi Medical University (approval number:).

[0038] Example 1 The method for preparing electroactive mesoporous silica composite nanoparticles loaded with ferrocene comprises the following steps: (1) Preparation of modified MSNs: 1) Mix 10 mL each of anhydrous ethanol, deionized water and ammonia water, and stir in a constant temperature magnetic stirring water bath at 30° C. for 20 min to obtain system I; 2) Add 2.5 mL of tetraethyl silicate to system I and react in a constant temperature water bath at 30° C. for 45 min to obtain system II; 3) Mix 2.5 mL of tetraethyl silicate and 1.5 mL of 3-aminopropyltriethoxysilane, slowly add the mixture into system II using a Pasteur pipette, and then react in a constant temperature magnetic stirring water bath at 30° C. for 80 min; 3) repeatedly washing with anhydrous ethanol for 3 times to obtain mesoporous silica; 4) The mesoporous silica obtained above was mixed with 50 mL of 0.6 M Na2CO3 solution and stirred in a water bath at 80° C. for 2 h, then washed with anhydrous ethanol for 3 times and freeze-dried for 24 h to obtain surface amino-modified MSNs powder for later use; (2) Preparation of Pt@MSNs: 180 μL of 0.275 mg / mL H2PtCl6 was quickly added to 50 mL of anhydrous ethanol to obtain system III, and then the surface amino-modified MSNs powder obtained in step (1) was added to system III, stirred and placed in a flat-bottomed flask, stirred in a constant temperature magnetic stirring water bath at 80°C, condensed and refluxed, and the heating was stopped after the reaction system turned black; then, the system was repeatedly washed with anhydrous ethanol for 3 times, freeze-dried for 24 h, and amino-modified MSNs powder loaded with platinum element, namely Pt@MSNs, was obtained for later use; (3) Preparation of Fc / Pt@MSNs: S1, adding 4 mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1 mL of N-hydroxysuccinimide to 10 mL of anhydrous ethanol to obtain a mixed solution I for standby use; S2, measure 10mL of 5mmol H2O2 and set aside; S3, measuring 10 mL of 1 mg / mL ethanol solution of p-hydroxybenzoic acid, horseradish peroxidase, ferrocenecarboxylic acid and tyramine, and mixing them in a 100 mL flat-bottom flask to obtain a mixed solution II for later use; S4, mixing the mixed solution I of S1, the H2O2 of S2, and the mixed solution II of S3 to obtain a mixed solution III; S5. Weigh 30 mg of Pt@MSNs powder obtained in step (2) and add it to mixed solution III. Place it in a 37°C constant temperature magnetic stirring water bath and stir for 24 h. Then wash it alternately with anhydrous ethanol and deionized water for a total of 3 times. Freeze-dry it for 24 h to obtain electroactive mesoporous silica composite nanoparticles loaded with ferrocene, namely Fc / Pt@MSNs, which are set aside.

[0039] Example 2: This example conducts an in vitro anticancer test on the composite nanomaterial prepared in the example of the present application, including the following studies: The 4-week-old male BALB / c nude mice used in this experiment were purchased from the Animal Experiment Center of Guangxi Medical University. 48 nude mice were purchased and each nude mouse was inoculated with 1×10 6 After 14 days of tumor inoculation, the cells were randomly divided into eight groups: Control, Pt@MSNs, Fc@MSNs, Fc / Pt@MSNs, E, Pt@MSNs+E, Fc@MSNs+E, and Fc / Pt@MSNs+E, with six cells in each group.

[0040] (1) Characterization The above steps are adopted The MSNs nanoparticles with controllable particle size were prepared by this method. The method is a preparation method of mesoporous silica nanoparticles based on the sol-gel method. The silanol molecules are polymerized by hydrolysis and polycondensation under alkaline conditions to form spherical silica particles with orderly structure. The synthesis principle and interface nucleation growth mechanism are reasonably analyzed by studying the influence of experimental parameters. The changes in product morphology. Subsequent experiments all have good shape and uniform particle size. Then chloroplatinic acid is reduced to elemental platinum loaded on the surface of mesoporous silica nanoparticles by anhydrous ethanol. The carboxyl group on ferrocene is activated by EDC, and NHS forms a stable active ester intermediate to connect ferrocene to the surface of mesoporous silica nanoparticles. Subsequently, the FPMSN nanoparticles were analyzed by UV-visible spectrophotometer, X-ray energy dispersive spectroscopy, X-ray diffraction analysis, Zeta potential analysis and inductively coupled plasma spectroscopy to analyze the main components, particle size distribution, elemental composition, specific surface area, pore volume and pore size of the FPMSN nanoparticles. The results show that the successfully synthesized Fc / Pt@MSNs have an average particle size of about 200 nm, good dispersibility and size uniformity, regular pore structure, and O, Si, Fe and Pt elements are evenly distributed on FPMSN nanoparticles (Fc / Pt@MSNs) (see Figure 2 ).

[0041] (2) Cytotoxicity assay We used a double salt bridge system. In a 96-well plate, 5 × 10 4 / well density plating, add the same volume of pure water or Fc / Pt@MSNs nanoparticle aqueous solution for incubation for 2h, and add the same volume of PBS solution to two adjacent wells. After the reaction system is established, insert the platinum electrode and the double salt bridge between the two PBS wells, and input a DC or square wave AC signal of different durations. Use a multimeter to monitor the current and keep it at 3mA, and keep the power on for 10min. After 24h, the cell viability is measured using the standard CCK8 colorimetric method.

[0042] When the concentration increased from 1 μg / mL to 10 mg / mL, there was no significant effect on the viability of HepG2 cells and HUVEC cells. Even at a concentration of up to 10 mg / mL and a cell viability of more than 80%, Fc / Pt@MSNs had no obvious cytotoxic effect on these two cells, and had good biocompatibility, and could continue to be used in subsequent in vitro experiments. Secondly, applying 5 and 10 minutes of direct current had a significant inhibitory effect on the viability of HepG2 cells. When DC was applied for 5 minutes, the cell viability was less than 40%; when DC was applied for 10 minutes, the cell survival rate was less than 30%, indicating that DC had a significant killing effect on HepG2 cells. In addition, when Fc / Pt@MSNs was combined with direct current, the concentration increased from 1 μg / mL to 10 mg / mL, and the cell activity decreased significantly and was greatly inhibited. Under the action of direct current, the effects of different concentrations of Fc / Pt@MSNs on the viability of HepG2 cells were very significant, and the cell survival rate was less than 10%. This is because under the action of direct current, the electrolysis of water causes a sharp change in the pH value of the environment around the electrode, which is the main cause of tumor cell death. In addition, the experimental results show that after 10 minutes of power-on in the double salt bridge device, the square wave alternating current has no significant effect on the viability of HepG2 cells and HUVEC cells, and the cell survival rates of both cells are higher than 80%, indicating that square wave alternating current has no obvious cytotoxic effect on these two cells and can continue to be used in subsequent in vitro experiments. Finally, the cytotoxic effect of HepG2 cells under different concentrations of Fc / Pt@MSNs was evaluated, and it was found that the cell viability of HepG2 cells showed a significant downward trend as the concentration increased from 1μg / mL to 10mg / mL. The cell viability was less than 50% at a concentration of 0.1mg / mL, and the cell viability was less than 40% at a concentration of 1mg / mL. This shows that FPMSN nanoparticles combined with electrodynamic therapy can achieve a good tumor cell killing effect (see Figure 3 ).

[0043] (3) Effect of square wave AC electric field on electrodynamic behavior In order to quantify the level of intracellular reactive oxygen species, we used DCFH-DA as a fluorescent probe. HepG2 cells were first cultured at 5×10 5 / well concentration in a 6-well plate overnight. Before electrodynamic treatment, Fc / Pt@MSNs nanoparticles or the same volume of ultrapure water were added to the culture dish, followed by a 20μM DMSO solution of DCFH-DA. After electrodynamic treatment at different concentrations of Fc / Pt@MSNs, different voltages, different currents, and different current durations, the cells were incubated for 1h and digested, and finally the fluorescence intensity of DCFH-DA was quantitatively detected using a fluorescence spectrophotometer.

[0044] Driven by a DC electric field, FPMSN nanoparticles have a strong killing effect on tumor cells, but the application of DC in the field of tumor treatment needs to solve this problem. For example, directly inserting electricity into the body will generate H + , H2, etc., causing damage to tissues. In order to reduce side effects, we tried to use square wave alternating current instead of an applied electric field. The FPMSN nanoparticles were verified to catalyze water decomposition under square wave alternating current. By controlling the positive and negative duration of the current and recording the waveform, square wave output waveforms of different durations were obtained. The intracellular reactive oxygen species fluorescent probe DCFH-DA was used to quantitatively detect the production of intracellular reactive oxygen species. The production of intracellular ROS under square wave alternating electric fields of different concentrations, voltages, and currents was studied. Figure 4 The results showed that the use of 0.4 mg / mL Fc / Pt@MSNs and 20 V square wave AC was suitable for subsequent experiments. Compared with direct current, the fluorescence intensity of DCFH-DA was better under 60 s square wave AC exchange, which may be related to the change in the direction of the electric field and the ion migration process. Therefore, the 60 s square wave AC electric field was selected as the condition for subsequent experiments.

[0045] (4) Anti-tumor activity test of Fc / Pt@MSNs at the cellular level To evaluate its antitumor ability in vitro, 5 × 10 4 / well density plating, add the same volume of pure water or Fc / Pt@MSNs nanoparticle aqueous solution for incubation for 2h, and add the same volume of PBS solution to two adjacent wells. After the reaction system is established, insert the platinum electrode and the double salt bridge between the two PBS wells, and input direct current with different voltages and currents or square wave alternating current with different durations and frequencies. After 24h, the cell survival rate is measured using the standard CCK8 colorimetric method.

[0046] Cell experiments were conducted using the HepG2 cell line as a model to verify the killing ability of Fc / Pt@MSNs under square wave AC electric fields at different currents and voltages. First, HepG2 cells and Fc / Pt@MSNs were incubated together in an intermediate reaction vessel for 2 hours, and then platinum wire electrodes were inserted through a double salt bridge connection to apply square wave AC with different currents, and the voltage was maintained at 20V for 10 minutes. The culture was then continued for 24 hours, and the cell viability was tested using the CCK8 method. Figure 5The results showed that the cell death rate in each group increased after the addition of Fc / Pt@MSNs, and the killing effect of Fc / Pt@MSNs on cells was independent of the current size. Therefore, the square wave AC electric field current used in subsequent experiments was 3mA. Secondly, the killing ability of FPMSN nanoparticles under different voltages under square wave AC electric field was verified; the experimental process was similar to the above, except that square wave AC with different voltages was applied and the current was kept at 3mA for 10 minutes. The results showed that after the addition of Fc / Pt@MSNs, as the voltage increased from 3V to 20V, the cell survival rate decreased significantly, and the effect difference between concentrations was significant. Therefore, a square wave AC electric field with a voltage of 20V was selected as a parameter in subsequent experiments. In order to verify the cell killing effect of Fc / Pt@MSNs at different durations under square wave AC electric field, we used a double salt bridge device for cell level experiments. The experimental results showed that under the conditions of voltage of 20V and current of 3mA, square wave AC with positive and negative switching after 60 seconds had the best cell killing effect. Therefore, a square wave AC electric field with a voltage of 20 V and a duration of 10 minutes was selected for subsequent experiments. In summary, the experimental results show that FPMSN nanoparticles exhibit the best cell killing effect when subjected to a 60-second positive-negative alternating square wave AC electric field and a voltage of 20 V for 10 minutes. This parameter will be used in subsequent cell-level experiments.

[0047] (5) Study on apoptosis of liver cancer cells induced by FPMSN nanoparticles 5 × 10 5 / well concentration was added to HepG2 cells and incubated overnight. Subsequently, 0.4 mg / mL of FPMSN nanoparticles or the same volume of complete culture medium was added to two different culture dishes. After culturing for 2 hours, a culture dish with Fc / Pt@MSNs nanoparticles and a culture dish with culture medium were placed on a double salt bridge, and a 60s square wave alternating current was passed through a platinum wire electrode to maintain the system current at 3mA for 10 minutes. After replacing the new culture medium, the culture was continued for 12 hours. Subsequently, Calcein-AM and PI were used to fluorescently stain the cells for 15 minutes. Finally, the cells were observed using a laser confocal microscope.

[0048] Fluorescent staining of live and dead cells is a commonly used apoptosis assay that distinguishes the life and death status of cells by detecting the distribution of fluorescent dyes in cells. Common fluorescent staining reagents for live and dead cells include calcein-AM and PI dyes. Among them, calcein-AM is a green fluorescent dye that can be used to evaluate indicators such as cell viability and cytotoxicity. It is hydrolyzed into calcein by intracellular esterases, releasing a strong green fluorescence signal. In living cells, calcein-AM is stable in the cytoplasm and cannot enter the cell nucleus for staining due to the integrity of the cell membrane. In dead cells with damaged membranes, calcein-AM can penetrate into dead cells and bind to DNA in the cell nucleus for staining. In addition, PI dye can mark the location of dead cells by penetrating the cell membrane and entering the nucleus of dead cells to bind to DNA. After PI binds to DNA in the cell nucleus, it presents a red fluorescence signal under a fluorescence microscope, which can be used to distinguish the survival and death status of cells. Such as Figure 6 As shown in the figure, from the pictures taken by confocal fluorescence microscope, it can be seen that the cells in the control group, Fc@MSNs group, Pt@MSNs group, and EchT group grew well, and strong green fluorescence and only weak red fluorescence could be seen in the field of view, which means that the cells in these four groups are alive, and it also means that Fc@MSNs, Pt@MSNs, and EchT have basically no toxicity to cells. However, in the Fc / Pt@MSNs group and the Fc@MSNs+E group, there are fewer dead cells visible in the fluorescence field of view. The Fc / Pt@MSNs group may be that the platinum-catalyzed ferrocene enhances its Fenton reaction to generate reactive oxygen species ROS to kill tumor cells, while the Fc@MSNs+E group may be that the Fenton reaction of ferrocene increased under the action of the electric field to produce more reactive oxygen species ROS to kill tumor cells. The Fc / Pt@MSNs+E group has the most dead cells in the fluorescence field of view, which may be due to the enhanced ferritin reaction under the action of the electric field to produce more reactive oxygen species ROS to kill tumor cells.

[0049] (6) FPMSN nanoparticles induce pyroptosis of liver cancer cells HepG2 cells in the logarithmic growth phase were cultured at 5×10 5 / well was added to a 6-well plate to make a cell slide and incubated overnight. 0.4 mg / mL Fc / Pt@MSNs nanoparticles or the same volume of ultrapure water were added to the culture dish. The electrodynamic treatment method was the same as above. After incubation for 1 hour, serum-free diluted Gasdermin D, Caspase-1, and Caspase-4 antibody solutions were added respectively, incubated at 37°C for 30 minutes, washed 3 times with PBS, and then added with DAPI working solution for incubation at room temperature for 15 minutes, washed 3 times with PBS, and observed under a laser confocal microscope.

[0050] Necroptosis is a newly discovered atypical form of programmed cell death and a special form of programmed necrosis. The characteristic of pyroptosis is that it is triggered by the activation of other signaling pathways when the apoptotic signaling pathway is blocked or inactivated, and exhibits characteristics similar to apoptosis. In pyroptosis, cells will undergo size deformation, capillary bursting, and overflow of cell contents. In pyroptosis, Caspase-1 and Caspase-4 are mainly involved in the interaction with Gasdermin D, thereby participating in the formation and regulation of cell membrane channels. When mLKL is phosphorylated and forms aggregates, these aggregates will bind to Gasdermin D in the cell membrane and cause it to promote the formation of cell membrane channels, ultimately causing the cell to overflow its contents and undergo cell death. At the same time, Caspase-1 and Caspase-4 can also directly or indirectly affect the phosphorylation of mLKL and its binding to Gasdermin D, thereby participating in the regulation of pyroptosis. Such as Figure 7 As shown in the figure, the results of the Fc@MSNs group, Pt@MSNs group, Fc / Pt@MSNs group, EchT group, and Fc@MSNs+E group were the same as those of the blank control group. No green fluorescence or only weak green fluorescence was observed in the cells, while the Pt@MSNs+E group and Fc / Pt@MSNs group had obvious green fluorescence. However, the intracellular green fluorescence of the Fc / Pt@MSNs+E group was significantly stronger than that of the Pt@MSNs+E group. The above results prove that electrodynamic therapy activates Caspase-1 and Caspase-4, cuts pro-IL-1β and pro-IL-18 into mature forms, and cuts Gasdermin D (encoded by GSDMD) to induce cell pyroptosis.

[0051] Example 3: This example is an in vivo anticancer test on the composite nanomaterial prepared in the examples of this application, including the following studies: 1. Establishment of tumor model: The 4-week-old male BALB / c nude mice used in this experiment were purchased from the Animal Experiment Center of Guangxi Medical University. 48 nude mice were purchased and each nude mouse was inoculated with 1×10 6 After 14 days of tumor inoculation, the cells were randomly divided into eight groups: Control, Pt@MSNs, Fc@MSNs, Fc / Pt@MSNs, E, Pt@MSNs+E, Fc@MSNs+E, and Fc / Pt@MSNs+E, with six cells in each group.

[0052] 2. Analysis of body weight and volume of HepG2 tumor-bearing nude mice The anti-tumor therapeutic effect of electroactive FPMSNs combined with electrodynamic therapy was studied in vivo. Tumor-bearing nude mice were randomly divided into 8 groups (n=6): Control, Pt@MSNs, Fc@MSNs, Fc / Pt@MSNs, E, Pt@MSNs+E, Fc@MSNs+E, and Fc / Pt@MSNs+E. 100 μL of nanoparticles in each group was injected intratumorally, and 10 mA, 20 V square wave alternating current was applied for 10 min. The body weight and tumor size of the nude mice were recorded every other day during the entire treatment cycle. Figure 8 C and 8D. The body weight of nude mice is an important indicator for evaluating drug systemic toxicity and the health status of nude mice. The results showed that although the tumor volume of nude mice in the final treatment group was reduced, there was no significant difference in their body weight changes compared with the control group, indicating that the treatment did not cause serious systemic toxicity. Figure 7 A is the tumor image of each treatment group after the treatment. The experimental results show that the tumor size of the four groups of Pt@MSNs, Fc@MSNs, Fc / Pt@MSNs and E without electric field is not much different from that of the Control group. After the electric field is applied, the tumor of the Fc / Pt@MSNs+E group is the smallest among the three groups of Pt@MSNs+E, Fc@MSNs+E and Fc / Pt@MSNs+E, which verifies that Fc / Pt@MSNs combined with electrodynamic therapy has significant anti-tumor activity.

[0053] 3. H&E staining analysis After the treatment, the nude mice in each group were euthanized, and then the nude mice were dissected, and the hearts, livers, spleens, lungs, kidneys and tumors were taken and fixed in 4% paraformaldehyde. The tissues fixed for 24 hours were sequentially immersed in different concentrations of ethanol (70%, 85%, 95%, 100%) for dehydration, and then immersed in xylene overnight to make them transparent. The transparent samples were immersed in paraffin for 8 hours and cut longitudinally on a paraffin slicer. The slices were placed in hot water to remove wrinkles, attached to a slide, and dried. The slices after the above treatment were immersed in xylene twice (10 minutes each), and then immersed in different concentrations of ethanol (the opposite order of dehydration) in sequence, and the slices were stained in hematoxylin for 5 minutes, washed with water and PBS to turn blue, and then washed repeatedly and stained in eosin for 1-3 minutes. After blood staining, the sections were immersed in 95% and 100% ethanol for 10 minutes (changed every 5 minutes), then immersed in xylene, and sealed with neutral gum after drying slightly to avoid bubbles, and observed under a microscope.

[0054] HE staining was used to evaluate the effect of Fc / Pt@MSNs combined with electrodynamic therapy on tumor tissue structure. Figure 8As shown in E, the tumor tissue of the Control group showed typical malignant characteristics, including obvious cell atypia, enlarged nuclei, and increased nuclear division images. In contrast, in the tumor tissue of Fc / Pt@MSNs+E, these malignant characteristics were significantly reduced, the cells were arranged more regularly, and the nuclear division images were less, which verified that FPMSNs effectively inhibited the proliferation of tumor cells. In order to evaluate the potential systemic toxicity of the treatment, HE staining was performed on the main organs (heart, liver, spleen, lung, and kidney) of nude mice in each treatment group. Fig. 9 Organ tissue sections of nude mice in the control and treatment groups were displayed. Nude mice in the control group showed normal tissue structure, and no obvious pathological changes were observed. In the other treatment groups, no obvious changes in tissue structure were observed, which comprehensively demonstrated that Fc / Pt@MSNs and electrodynamic therapy were not only effective, but also showed high tissue specificity, that is, the therapy had little effect on normal tissues while inhibiting tumor growth.

[0055] , Ki67 staining analysis Place the tumor tissue sections in a repair box, fill with antigen repair buffer, place in a pressure cooker for antigen repair, heat on medium heat for 8 minutes, turn off the heat and keep warm for 8 minutes, and then heat on medium-low heat for 7 minutes. After the slides cool naturally, use PBS for decolorization and shake and wash on a shaker 3 times, 5 minutes each time. Next, use endogenous peroxidase blocking solution to stand for 10 minutes at room temperature and away from light to remove the interference of peroxidase. Subsequently, use 5% BSA to block at room temperature for 20 minutes. Prepare an appropriate concentration of primary antibody and drop it on the sections, then place the sections in a wet box and incubate at 4°C for 12 hours. Rewarm at room temperature for 30 minutes, wash 3 times with PBST for 5 minutes each time, add the diluted secondary antibody and incubate at room temperature for 1 hour. After washing with PBST, add DAB colorimetric solution to the tissue area, observe the brown-yellow positive cells under a microscope, and stop the reaction immediately after the color development reaches the required level. After nuclei were stained with hematoxylin, the sections were dehydrated in 75%, 85%, 95% and 100% ethanol in turn, each time for 5 min, and then transparentized with xylene twice for 5 min each time. After drying, the sections were sealed with neutral gum and observed under a microscope.

[0056] Ki67 is an important marker of cell proliferation and is widely used to evaluate proliferation activity in tissue samples by immunohistochemical staining. We analyzed the proliferation activity of tumor cells in nude mice in different treatment groups by Ki67 immunohistochemistry. Ki67-positive cells appeared as brown nuclear markers under the microscope, which reflects the proliferation status of cells. Figure 8As shown in F, compared with the Control group, the brown nuclear labeling in the Fc@MSNs+E and Pt@MSNs+E groups was significantly reduced. In addition, the Fc / Pt@MSNs+E group had the least positive expression of ki67. The results showed that Fc / Pt@MSNs combined with electrodynamic therapy effectively inhibited the proliferation of tumor cells.

[0057] , TUNEL staining analysis After the proteinase K stock solution was diluted with PBS, it was added to the dewaxed and rehydrated tissue sections of the tumor tissue sections, and incubated in a 37°C incubator for 20 minutes, followed by three washes. Next, 0.1% Triton working solution was used for membrane permeabilization, incubated at room temperature for 20 minutes and then washed. Buffer was added until the tissue was completely covered and equilibrated at room temperature for 10 minutes. The TdT enzyme, dUTP and buffer in the TUNEL kit were prepared into a working solution at a ratio of 1:5:50, added until the tissue was completely covered, and then the sections were placed flat in a wet box with a small amount of water and incubated in a 4°C refrigerator overnight with shaking. DAPI was then added for nuclear counterstaining, incubated at room temperature for 10 minutes under light-proof conditions, and then washed three times and PBS was dried. After drying, anti-fluorescence quenching agent was added to seal the sections and photographed using a laser confocal microscope.

[0058] To evaluate the apoptosis of tumor cells induced by Fc / Pt@MSNs combined with electrodynamic therapy, we performed TUNEL staining on the tumor tissues of nude mice in each treatment group. Fig.10 As shown in A, TUNEL staining of the Control, Pt@MSNs, Fc@MSNs, and Fc / Pt@MSNs groups showed almost no fluorescent signal, and the fluorescent signal of group E was very weak, suggesting that the apoptosis rate of tumor cells was extremely low. In contrast, a significant increase in TUNEL positive signals was observed in the tumor tissues of each treatment group of nanomaterials combined with electrodynamic therapy, indicating a high level of cell apoptosis. Among them, the fluorescence signal of the Fc / Pt@MSNs+E group was the strongest, indicating that the tumor cells in this treatment group had the most apoptosis. These results show that Fc / Pt@MSNs combined with electrodynamic therapy significantly enhanced the apoptosis of tumor cells.

[0059] , ROS generation analysis Tumor sections were rewarmed at room temperature to slowly restore the temperature of the tissue, and then the surface moisture was gently wiped off. Then, a leak-proof histochemical marker pen was used to draw a closed boundary around the tissue sample. Next, an autofluorescence quencher was added and incubated for 5 minutes to reduce the autofluorescence background of the tissue, and then rinsed with running water for 10 minutes to remove excess quencher. ROS-specific dye was added to the circled area and incubated at 37°C in a light-proof incubator for 30 minutes to achieve specific labeling of ROS. Subsequently, nuclear staining was performed using DAPI solution to enhance the fluorescence signal of the cell nucleus, incubated for 10 minutes, and room temperature and light-proof conditions were adopted. After that, the slides were transferred to PBS solution and gently shaken on a decolorization shaker, and washed three times for 5 minutes each time to remove unbound dye. After the slides were dried, they were sealed with anti-fluorescence quenching sealing agent and imaged using a laser confocal microscope.

[0060] To verify the level of ROS produced by FPMSNs combined with electrodynamic therapy, we took pictures using a laser confocal microscope and obtained the localization and quantitative analysis of ROS produced in the tumor tissues of nude mice in each treatment group. Fig.10 As shown in B, there is only weak red fluorescence in the tumor tissues of the Control group, Pt@MSNs group, Fc@MSNs group, Fc / Pt@MSNs group and E group, indicating that the generation of ROS is low. From the fluorescence quantitative statistical graph, it can be seen that the intensity of red fluorescence in the Fc / Pt@MSNs+E group is about 1.5 times that of the Fc@MSNs+E and Pt@MSNs+E groups. The experimental results show that mesoporous silica loaded with ferrocene and platinum nanoparticles can generate a large amount of ROS under square wave alternating current, and the effect of inhibiting tumor growth is obvious.

[0061] 、 Western blot detection of pyroptosis proteins Weigh the frozen tumor tissue, add liquid nitrogen and grind the tumor tissue into powder in a mortar, and place the ground tumor tissue in a 50mL centrifuge tube. Prepare a mixed solution by adding 20μL of protease and phosphatase inhibitors per milliliter of RIPA lysis buffer, then add 150μL of the prepared mixed solution for every 20mg of tumor tissue, and place the centrifuge tube on an ice basin for lysis for 30min. Precool the centrifuge to 4℃, 10000g, and centrifuge for 10min before taking the supernatant. Determine the protein content in the supernatant by BCA protein quantification method, and add 4× instant protein loading buffer according to the volume. Dispense into EP tubes, denature in a boiling water bath for 10min, cool to room temperature, and store in a -20℃ refrigerator. Prepare a 10% gel using a one-step PAGE gel rapid preparation kit. After the upper and lower gels are completely solidified, add the marker and the melted sample, adjust the voltage to 110V, and perform electrophoresis for 80min. After electrophoresis, the separated proteins were transferred from the gel to a polyvinylidene fluoride (PVDF) membrane with a transfer current of 100 mA for 55 minutes. After the transfer, use a quick blocking solution to block for 10 minutes to block sites on the membrane that are not occupied by proteins and reduce nonspecific binding. After blocking, wash with TBST three times for 5 minutes each. Use antibody diluent to prepare β-actin, Caspase-1, Caspase-4, Gasdermin D-FL and Gasdermin DN antibodies respectively, place the blocked PVDF membrane in the prepared antibody diluent, and incubate on a shaker at 4°C overnight. Wash off the primary antibody with TBST, place the PVDF membrane in a fluorescent secondary antibody, incubate at 4°C for 2 hours, and finally wash off the excess secondary antibody with TBST and place Imaging using the CLX dual-color infrared laser imager.

[0062] To explore the anti-tumor mechanism of FPMSNs combined with electrodynamic therapy, we lysed the tumor tissues of the Control group, FPMSNs group, E group and FPMSNs+E group to extract proteins. The extracted protein mixture was then separated by gel electrophoresis. Under the action of the electric field, proteins are separated from large to small according to molecular weight. The separated proteins are transferred from the gel to a polyvinylidene fluoride (PVDF) membrane, a step that ensures that the proteins can be detected by specific antibodies. To prevent nonspecific binding, skim milk powder is used for blocking. Then, primary antibodies Caspase-1, Caspase-4, Gasdermin D-FL and Gasdermin DN are added to bind to the target protein, and a fluorescent secondary antibody matching the primary antibody is selected to combine, and the protein blot is detected by a scanner.

[0063] like Fig.11As shown in A and B, the expression levels of Caspase-1 in the Control group, FPMSNs group and E group were similar, and the expression level of Caspase-1 in the FPMSNs+E group was the highest; the expression levels of Caspase-4 in the Control group, FPMSNs group and E group were similar, and the expression level of Caspase-4 in the FPMSNs+E group was the highest; the expression levels of Gasdermin D in the Control group and FPMSNs group were similar, and the expression level of Gasdermin D in the FPMSNs+E group was higher than that in the E group. The experimental results show that FPMSNs combined with electrodynamic therapy can effectively induce pyroptosis of tumor cells and inhibit tumor growth.

[0064] ELISA detection of serum pyroptosis factor In order to verify the effect of electrodynamic therapy on tumor cell pyroptosis factors, we used ELISA to measure IL-1β and IL-18. Take out the frozen serum and thaw it, prepare the standard solution with ultrapure water and let it stand for 10 minutes. Add the sample and standard to the wells of the ELISA plate, add 100μL to each well. Cover the ELISA plate and incubate at 37℃ for 90min. Shake off the liquid in the well, add 100μL of biochemical antibody working solution to each well, and incubate at 37℃ for 60min. Shake off the liquid in the well, pat dry on absorbent paper, and wash 3 times with washing solution. Then add 100μL of enzyme conjugate working solution to each well, incubate at 37℃ for 30min, and wash the plate 5 times. Add substrate solution to each well, incubate at 37℃ in the dark for about 15min, add 50μL of stop solution to each well to stop the reaction, and then immediately measure the OD value of each well with an ELISA reader.

[0065] Caspase-1 precursor can be converted into active Caspase-1 by self-cleavage. The activated form of Caspase-1 has efficient protease activity and can cleave a variety of substrate proteins. Activated Caspase-1 directly acts on pro-IL-1β and pro-IL-18, releasing mature, biologically active cytokines by cleaving their precursor forms. After cleavage, IL-1β and IL-18 become active and are released to the outside of the cell through cell membrane pores. Fig.11As shown in Figures C and 11D, we detected the inflammatory factors IL-1β and IL-18 in the serum of nude mice in different treatment groups by ELISA. The trends of IL-1β and IL-18 in different treatment groups were the same. The Fc@MSNs group, Pt@MSNs group, and Fc / Pt@MSNs group were slightly higher than the blank control group, and the E group, Fc@MSNs+E group, Pt@MSNs+E group, and Fc / Pt@MSNs+E group were significantly higher than the blank control group. Among them, the IL-1β and IL-18 content in the Fc / Pt@MSNs+E group was the highest, nearly twice that of the blank control group. The above results show that FPMSNs combined with electrodynamic therapy can promote the activation of Caspase-1, cleave pro-IL-1β and pro-IL-18 into mature forms, and release inflammatory factors IL-1β and IL-18 through the pores on the cell membrane, thereby inducing cell pyroptosis.

[0066] , Immunofluorescence detection of pyroptosis proteins Paraffin sections of tumor tissue were dewaxed and hydrated; frozen sections were restored to room temperature and fixed with paraformaldehyde for 30 minutes. Sufficient antigen retrieval buffer was added and antigen retrieval was performed in a pressure cooker. The pressure cooker was cooled to room temperature, and the slides were taken out and washed three times with PBS for 5 minutes each time. After drying the slides, the tissues were circled with a brush, and 100 μL of endogenous peroxidase blocking solution was added to each tissue, and the tissues were left to stand at room temperature for 10 minutes and then dried. Diluted Caspase-1, Caspase-4, and Gasdermin D antibody solutions were added respectively, incubated at 4°C overnight, and washed 3 times with PBST for 5 minutes each time. The prepared secondary antibody with FITC was added, incubated at 37°C for 30 minutes, and then washed 3 times with PBST for 5 minutes each time. Then, a DAPI anti-fluorescence quenching sealing agent was added and imaging was performed using a laser confocal microscope.

[0067] To further verify the anti-tumor pyroptosis mechanism of FPMSNs combined with electrodynamic therapy, we used immunofluorescence to detect the expression of Caspase-1, Caspase-4 and Gasdermin D in tumor sections of nude mice in different treatment groups. Fig.12As shown in the figure, in each experimental group, no or only weak green fluorescence was observed in the cells of the Fc@MSNs group, Pt@MSNs group, Fc / Pt@MSNs group, and E group, indicating that these treatments were similar to the results of the blank control group. However, in the Pt@MSNs+E group, the Fc@MSNs+E group, and the Fc / Pt@MSNs group, obvious green fluorescence appeared in the cells, and in the Fc / Pt@MSNs+E group, the intensity of green fluorescence exceeded that of the other groups. These results indicate that FPMSNs combined with electrodynamic therapy can effectively activate Caspase-1 and Caspase-4, and cut Gasdermin D, thereby inducing cell pyroptosis. These results are consistent with the results of the WB experiment, proving that FPMSNs combined with electrodynamic therapy can effectively induce pyroptosis in tumor cells. pro-Caspase-1 is converted into active Caspase-1 by self-cleavage. This activated form of Caspase-1 has efficient protease activity and can cut a variety of substrate proteins.

[0068] , Biosafety Analysis Contact angle detection: Prepare PBS solution and PBS solution of FPMSNs with a concentration of 0.4 mg / mL, and use a 100 μL injection needle to draw PBS and FPMSNs solution. Use a level to adjust the level of the stage of the contact angle analyzer, place a glass slide as a solid contact surface on the stage, adjust the camera focus and light brightness, set the automatic dripping liquid on the glass slide, and then measure its contact angle using the ring method.

[0069] Hemolysis test: After nude mice were anesthetized, 5 mL of eyeball blood was collected in an anticoagulant tube and mixed by inversion. The whole blood was added to PBS and mixed by inversion. Centrifuged at 4°C, 1500 rpm for 5 min, and the supernatant was removed with a Pasteur pipette. Repeat the above steps until the supernatant was clear. 200 μL of red blood cells at the bottom of the centrifuge tube were removed and resuspended in 1.8 mL of PBS. PBS solutions of FPMSNs were prepared with concentrations of 0.1 mg / mL, 0.4 mg / mL, 1 mg / mL, and 2 mg / mL, respectively. Pure water was used as a positive control and PBS as a negative control. The diluted whole blood was mixed with FPMSNs samples of various concentrations, positive controls, and negative controls, and the volume of each group was kept consistent. Incubate at 37°C for 1 h and centrifuge at 4°C, 2500 rpm for 5 min. The absorbance of the supernatant was measured at 540 nm using a spectrophotometer.

[0070] Distribution of Pt in vivo and plotting of survival curves: 9 BALB / c nude mice were purchased and each nude mouse was inoculated with 1×10 6HepG2 cells. After injection, the nude mice were managed and monitored for health on a daily basis, and their diet, activities, etc. were recorded to ensure the success of tumor implantation and monitor possible abnormal reactions. After 14 days of tumor implantation, 100 μL (0.4 mg / mL FPMSNs in PBS solution) was administered intratumorally to each nude mouse. Three nude mice were dissected at 0h, 24h, and 72h, and their hearts, livers, spleens, lungs, kidneys, brains, and tumors were taken. The removed tissues were immediately weighed and clearly marked, and then stored in a -80°C refrigerator until all samples were collected. ICP-MS (inductively coupled plasma mass spectrometry) was used to measure the Pt content in each tissue. Each nude mouse was inoculated with 1×10 6 HepG2 cells. After injection, the nude mice were managed and monitored for health on a daily basis, and their diet, activities, etc. were recorded to ensure the success of tumor implantation and monitor possible abnormal reactions. Treatment began on the fourteenth day after tumor implantation. Before treatment, each nude mouse was injected intramuscularly with 10% chloral hydrate at a dose of 350 mg / kg. After the nude mice were completely anesthetized, 100 μL of each group of drugs were injected intratumorally according to the group. Then the platinum electrode was inserted into the tumor, and a square wave alternating current of 10 mA and 20 V was applied for 10 minutes. The treated nude mice were placed in an incubator at 37°C for rewarming, and returned to the cage after they woke up. The survival status of the nude mice was recorded every day until the 50th day.

[0071] Routine blood analysis and liver and kidney function analysis: The whole blood of nude mice in each treatment group was placed in an EDTA anticoagulant tube. The optimal concentration ratio of EDTA to blood is 1.5 mg / mL. The sample volume should not be too much or too little. After sampling, gently invert the blood several times to fully mix the blood with the anticoagulant and store it at 4°C. The number of white blood cells (Leukocyte count, WBC), lymphocyte count (Lymphocyte count, LYM), monocyte count (Monocyte count, MONO), neutrophil number (Neutrophil number, NEU), hemoglobin content (Hemoglobin content, HGB) and red blood cell count (Erythrocyte count, RBC) of each sample were measured using a fully automatic blood cell analyzer. The serum of nude mice in each treatment group was taken out of the -80°C refrigerator and melted in running water. The liver function and kidney function of each sample were measured using a fully automatic biochemical analyzer. Liver function indexes: Albumin (ALB), alkaline phosphatase (ALP), glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), direct bilirubin (DBIL), total bile acid (TBA), total bilirubin (TBIL), gamma-glutamyltransferase (γ-GT). Kidney function indexes: urea nitrogen (UREA), creatinine (CREA), uric acid (UA).

[0072] like Fig.13 As shown in A and B, we used a glass slide as the solid phase and a PBS solution as the liquid phase to measure the contact angle of the solid-liquid interface. The contact angle between the PBS solution and the glass slide is 33.215°, and the contact angle between the PBS solution of FPMSNs and the glass slide is 34.639°. There is no significant change in the contact angle between the two groups. Since PBS is an isotonic solution of the human body, FPMSNs will not change the contact angle of the solution. The hydrophilicity of the solution is close to that of the PBS solution, so FPMSNs have good biosafety.

[49] .

[0073] like Fig.13As shown in C and D, we set up the concentrations of FPMSNs in the positive group, negative group, and experimental group to be 0.1 mg / mL, 0.4 mg / mL, 1 mg / mL, and 2 mg / mL, respectively. The results showed that the positive group was completely hemolyzed, and the results of the nanomaterial groups with different concentrations were similar to those of the negative group. After measuring the absorbance value of each group by an ELISA instrument, the hemolysis rate of each group was calculated. The hemolysis rate of the experimental 0.1 mg / mL FPMSNs was 0.37%, the hemolysis rate of 0.4 mg / mL FPMSNs was 0.24%, the hemolysis rate of 1 mg / mL FPMSNs was 0.21%, and the hemolysis rate of 2 mg / mL FPMSNs was 0.35%. According to ISO standards, a hemolysis rate of less than 5% is generally considered biocompatible. The hemolysis rates of all our experimental groups were less than 0.5%, indicating that FPMSNs have good biocompatibility.

[0074] To investigate the distribution of FPMSNs in vivo, we injected 0.4 mg / mL FPMSNs into nude mice and used ICP-MS to measure the concentration of Pt in the heart, liver, spleen, lung, kidney, tumor, and brain tissues of nude mice in three groups at different time periods. Fig.13 As shown in E, the concentrations of Pt in the heart, liver, spleen, lung, kidney, and brain remained basically unchanged at 0h, 24h, and 72h, while the concentration of Pt in the tumor tissue gradually increased. The results showed that FPMSNs nanomaterials were mainly enriched in the tumor site and had little effect on the normal tissues of nude mice. The survival curve is a key experiment to evaluate the long-term effects and potential toxicity of treatment. By recording the proportion of surviving animals over a certain period of time, it provides important data for the safety and effectiveness of treatment. These curves can not only show the effects of different treatment regimens on survival rate, but can also be used to compare the efficacy of the treatment group with the control group or other treatment groups. Fig.13 As shown in F, all the nude mice in the Control group died on the 32nd day, and the survival rates of the nude mice in the Fc@MSNs+E group, the Pt@MSNs+E group, and the Fc / Pt@MSNs+E group were 40% and 20% respectively on the 50th day. The survival rates of the nude mice in the Fc / Pt@MSNs+E group were 80%, and all the nude mice in the other groups died. The results show that electroactive nanoparticles FPMSNs combined with electrodynamic therapy can effectively prolong the survival period of nude mice and have good biosafety.

[0075] like Fig.14 As shown in A, the number of white blood cells, lymphocytes, monocytes and neutrophils in the Fc / Pt@MSNs+E group was lower than that in the Control group, while the hemoglobin content and red blood cell number did not change significantly in different treatment groups. The blood routine tests of all treatment groups were within the normal range and there was no significant difference. The results showed that FPMSNs combined with electrodynamic therapy did not affect the blood routine indicators of nude mice and had good biosafety.

[0076] In order to verify the biosafety of FPMSNs combined with electrodynamic therapy, we tested the liver and kidney functions of nude mice in different treatment groups. Liver functions include albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DBIL), total bile acid (TBA), total bilirubin (TBIL), and γ-glutamyl transferase (γ-GT); kidney functions include creatinine (CREA), urea nitrogen (UREA), and uric acid (UA). Fig.14 As shown in B, there was no significant difference in renal function between the Control group and the different treatment groups, and all were within the normal range. Fig.14 As shown in C, there was no significant difference in liver function between the control group and the different treatment groups, and all were within the normal range. The results showed that FPMSNs combined with electrodynamic therapy did not cause damage to the liver and kidney function of nude mice.

[0077] 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 electroactive mesoporous silica composite nanoparticles loaded with ferrocene, characterized in that: The method comprises the following steps: (1) preparing modified MSNs: 1) mixing anhydrous ethanol, deionized water and ammonia water, and stirring in a constant temperature magnetic stirring water bath to obtain system I; 2) adding tetraethyl silicate to system I, and reacting in a constant temperature water bath to obtain system II; 3) mixing 2.5 mL of tetraethyl silicate and 1.5 mL of 3-aminopropyltriethoxysilane, and then slowly dropping the mixture into system II, and then reacting in a constant temperature magnetic stirring water bath; 3) Repeatedly wash with anhydrous ethanol for 3 times to obtain mesoporous silica; 4) Stir the obtained mesoporous silica with 50 mL of 0.6 M Na2CO3 solution, and stir in a water bath at 80°C for 2 h, then wash with anhydrous ethanol for 3 times, and freeze-dry for 24 h to obtain MSNs powder with surface amino modified, which is set aside; (2) Prepare Pt@MSNs: Take H2PtCl6 and quickly add it to anhydrous ethanol to obtain system III, then add the MSNs powder with surface amino modified obtained in step (1) to system III, stir and place it in a flat-bottom flask, stir in a water bath with magnetic stirring at 80°C, condense and reflux, and stop heating after observing that the reaction system appears black; then repeatedly wash with anhydrous ethanol for 3 times, freeze-dry for 24 h, and obtain MSNs powder with amino modified platinum element, which is Pt@MSNs, which is set aside; (3) Prepare Fc / Pt@MSNs: S1. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide to anhydrous ethanol to obtain a mixed solution I for use; S2. Measure a certain amount of H2O2 for use; S3. Measure a certain amount of ethanol solution of p-hydroxybenzoic acid, horseradish peroxidase, ferrocenecarboxylic acid and tyramine respectively, mix them in a flat-bottom flask to obtain a mixed solution II for use; S4. Mix the mixed solution I of S1 and the H2O2.S3 mixed solution II of S2 to obtain a mixed solution III; S5. Weigh the Pt@MSNs powder obtained in step (2) and add it to the mixed solution III, place it in a constant temperature magnetic stirring water bath and stir; then use anhydrous ethanol and deionized water to wash alternately for a total of 3 times, freeze-dry for 24 hours, and obtain ferrocene-loaded electroactive mesoporous silica composite nanoparticles, namely Fc / Pt@MSNs, for use.

2. The preparation method according to claim 1, characterized in that: In step (1), the amount of anhydrous ethanol, deionized water and ammonia water is 10 mL; the reaction condition of system I is stirring in a constant temperature magnetic stirring water bath at 30°C for 20 minutes; the reaction condition of system II is adding 2.5 mL of tetraethyl silicate and reacting in a constant temperature water bath at 30°C for 45 minutes.

3. The preparation method according to claim 1, characterized in that: In step 3), the reaction condition is to react in a constant temperature magnetic stirring water bath at 30° C. for 80 min.

4. The preparation method according to claim 1, characterized in that: In step (2), the amount of H2PtCl6 used in the system III is 0.275 mg / mL, 180 μL, and the amount of anhydrous ethanol used is 50 mL.

5. The preparation method according to claim 1, characterized in that: In step (3), the amounts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and anhydrous ethanol are 4 mL, 1 mL and 15 mL respectively.

6. The preparation method according to claim 1, characterized in that: In step (3), the concentration and amount of H2O2 are 5 mmol and 10 mL respectively.

7. The preparation method according to claim 1, characterized in that: In step (3), the dosage of the ethanol solution of p-hydroxybenzoic acid, horseradish peroxidase, ferrocenecarboxylic acid and tyramine is 10 mL each at 1 mg / mL.

8. The preparation method according to claim 1, characterized in that: In step S5, the amount of the Pt@MSNs powder used is 30 mg; the stirring condition in the constant temperature magnetic stirring water bath is stirring at 37° C. for 24 hours.

9. The electroactive mesoporous silica composite nanoparticle material loaded with ferrocene obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the ferrocene-loaded electroactive mesoporous silica composite nanoparticle material according to claim 9 in the preparation of drugs for treating liver cancer.

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