Drug-loaded nanovaccine, preparation method and application thereof

Abstract

The present application relates to the field of nanobiomaterials, in particular to a drug-loaded nanovaccine and a preparation method and application thereof, so as to improve tumor treatment effect and biological safety. The drug-loaded nanovaccine comprises MIL-100(Fe) and a manganese-doped mesoporous silica carrier, the MIL-100(Fe) is coated on the outer surface of the manganese-doped mesoporous silica carrier, and the manganese-doped mesoporous silica carrier is loaded with sorafenib. The MF@SOR nanovaccine of the present application has very high biological safety. Meanwhile, the carrier of the present application is wrapped with a layer of MIL-100(Fe), the coating is very uniform and stable, which can further improve the bioavailability of sorafenib, maximize the use of drugs and reduce the toxic and side effects of drugs. In addition, the MF@SOR nanovaccine can also induce the body to produce immune memory, inhibit the recurrence and metastasis of tumors, the recurrence and metastasis of tumors are significantly reduced, and the treatment effect of tumors is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of nanobiomaterials, specifically to a drug-loaded nanovaccine, its preparation method, and its application. Background Technology

[0002] The rapid development of the 20th century has greatly improved human living standards. However, the global environment is also deteriorating, with pollution increasing year by year, leading to a rise in cancer incidence. Cancer has become one of the leading causes of death worldwide. Currently, the main treatment methods include radiotherapy, chemotherapy, interventional therapy, and surgical resection. However, due to the complexity, heterogeneity, recurrence rate, and metastasis of tumors, current clinical treatment options are very limited, and cancer cannot be fundamentally cured. Therefore, developing new and effective treatment methods remains a direction that requires continued exploration.

[0003] In recent years, studies have shown that metal immunotherapy can simultaneously activate multiple anti-tumor immune-related signaling pathways, and its potential in tumor immune regulation has been recognized. Current metal immunotherapy mainly relies on increasing the intratumoral metal ion content to initiate anti-tumor immunity. However, excessive free metal ions entering the circulatory system may cause a series of side effects. In addition, Gasdermin family proteins are widely expressed in normal tissues, and non-specific activation of the pyroptosis pathway in these tissues may initiate harmful inflammatory responses, causing irreversible tissue damage. How to effectively deliver metal ions to the tumor region and release them specifically, reducing their damage to normal tissues and enhancing their tumor-killing effect, is an urgent problem to be solved.

[0004] The development of nanomedicine has brought new ideas to the application of metal immunotherapy. Current technologies load therapeutic drugs onto carriers, enabling them to release the drugs at the tumor site. However, most carriers, such as Prussian blue, iron oxide nanoparticles, and template-prepared SiO2, are highly toxic, causing toxic reactions or side effects in humans and exhibiting low biosafety. Furthermore, they suffer from low targeting and small drug-carrying space, resulting in suboptimal therapeutic effects. In addition, to improve the stability of drug delivery, current technologies coat the carrier with a layer to prevent drug leakage. However, current coating methods still suffer from instability and unevenness. In conclusion, further exploration and development of new nanomedicine delivery systems are needed to improve drug biosafety and carrier drug loading capacity, while simultaneously addressing the problem of inadequate coating and enhancing the therapeutic efficacy against tumors. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a drug-loaded nanovaccine to improve the efficacy and biosafety of tumor treatment.

[0006] The basic solution provided by this invention is: a drug-loaded nanovaccine, comprising MIL-100(Fe) and a manganese-doped mesoporous silica support, wherein MIL-100(Fe) coats the outer surface of the manganese-doped mesoporous silica support, and sorafenib is loaded within the manganese-doped mesoporous silica support. (Metal-organic frameworks (MOFs) are a type of nanomaterial with extremely wide applications. There are many types of MOFs, and MIL-100(Fe) is one of them. MIL-100(Fe) is a technical term in this field, referring to one type of metal-organic framework. MIL-100(Fe) is a single, integral unit.)

[0007] The working principle and advantages of this invention are as follows:

[0008] In this document, MF@SOR stands for MSN, which is an abbreviation for manganese-doped mesoporous silica in this application; F stands for MIL-100 (Fe); and SOR is an abbreviation for sorafenib.

[0009] After being phagocytosed by tumor cells, the MF@SOR nanovaccine responds to a low-pH, high-GSH environment, releasing iron ions, manganese ions, and sorafenib. Iron ions and sorafenib activate pyroptosis in both classical and non-classical pathways, respectively, causing severe pyroptosis in tumor cells and damaging their DNA. Simultaneously, a large number of inflammatory factors and damage-related molecular patterns are released. The damaged DNA and manganese ions jointly activate the cGAS-STING pathway, releasing type I interferon. Together with inflammatory factors and damage-related molecular patterns, this promotes dendritic cell maturation, increases the number of CD8+ T cells, and decreases the number of Treg cells, thus reshaping the immune microenvironment in the tumor region.

[0010] The MF@SOR proposed in this application is the result of extensive experimentation and analysis by the inventors. The inventors' initial idea was to construct a new metal-organic framework by coordinating iron and manganese with trimesic acid, based on MIL-100 (Fe). The inventors synthesized the new substance using a hydrothermal method in a high-temperature reaction chamber. However, upon centrifugation and resuspending, the synthesized substance completely dissolved. The inventors believe this was due to the high instability of the new substance formed by the simultaneous coordination of iron and manganese. In existing technologies, MIL-100 (Fe) is often chosen as a support. If the simultaneous coordination of iron and manganese fails in experiments, MIL-100 (Fe) is generally not used again; the conventional approach is to use a different substance and re-coordinate it with manganese. However, the inventors of this application were eager to load iron and manganese into the same carrier to achieve the subsequent synergistic anti-tumor function of iron-manganese bimetallic compounds. Therefore, the inventors adopted a new approach, loading iron and manganese onto the carrier stepwise. Using SiO2 as the carrier, they first created pores and successfully doped manganese into the carrier, then loaded sorafenib, and finally coated the manganese-doped mesoporous SiO2 surface with MIL-100 (Fe). This allowed iron ions to successfully coordinate with the carboxyl groups on the manganese-doped mesoporous SiO2 surface. The inventors broke with the conventional practice of using MIL-100 (Fe) as the carrier, instead coating the manganese-doped mesoporous SiO2 surface with a thin film of MIL-100 (Fe), ultimately successfully incorporating iron, manganese, and sorafenib into MF@SOR.

[0011] Furthermore, the carrier of this invention is coated with a layer of MIL-100 (Fe). MIL-100 (Fe) not only releases iron ions but also prevents sorafenib leakage through mesopores. The coating of MIL-100 (Fe) is highly uniform and stable, and it does not cleave during drug delivery, preventing premature release of the drug before reaching the lesion and further improving the bioavailability of sorafenib. Simultaneously, the moderate thickness of MIL-100 (Fe) prevents the MF@SOR nanoparticles from becoming excessively large. The overall size of the MF@SOR nanoparticles is highly favorable for delivery, exhibiting a strong enhanced permeability and retention effect (EPR). By improving passive targeting, they reach and accumulate in the tumor region through the incomplete tumor vascular wall, maximizing drug utilization.

[0012] Compared to sorafenib's IC50 value, the MF@SOR nanovaccine has a significantly lower IC50 value. The MF@SOR of this application can exert a stronger anti-tumor effect with a lower concentration of SOR, reducing the dosage of sorafenib and further mitigating potential side effects. Furthermore, the MF@SOR nanovaccine can induce immune memory in the body, inhibiting tumor recurrence and metastasis, significantly reducing tumor recurrence and metastasis, and significantly improving the therapeutic effect of tumor treatment.

[0013] Furthermore, existing technologies typically employ template methods to prepare mesoporous SiO2. Template agents are highly toxic and difficult to completely remove, potentially causing toxic reactions or side effects in humans, which is detrimental to human health. In contrast, the manganese-doped mesoporous silica support prepared in this application utilizes a pore-drilling method, avoiding the use of template agents, resulting in very low toxicity and extremely high biosafety. Simultaneously, current SiO2 supports have limited drug-carrying space, while the manganese-doped mesoporous silica support prepared in this application has a large mesoporous drug-carrying space, significantly increasing the drug loading capacity. To load the same amount of drug, this application uses fewer supports, improving bioavailability and reducing costs.

[0014] In addition, the Mn released after the nano vaccine response 2+ With T1 imaging capabilities, the nanovaccine of this application can perform responsive imaging in the tumor region, effectively improving the accuracy of tumor detection and localization. Furthermore, the nanovaccine can remain in the tumor region for a relatively long time, possessing the potential to continuously stimulate the body to produce anti-tumor immunity within the tumor area.

[0015] This invention also provides a method for preparing a drug-loaded nanovaccine, including the preparation of non-porous silica and the preparation of MF@SOR, wherein the preparation of MF@SOR includes the following steps:

[0016] Step 1: MSN Setup

[0017] (1) Dissolve MnCl2·4H2O and NH4Cl, then add SiO2 nanoparticle aqueous solution and NH3·H2O to the mixture, and then transfer the mixture to the reaction vessel for reaction;

[0018] (2) The product obtained after the reaction was washed and centrifuged to obtain MSN nanoparticles;

[0019] Step 2: Preparation of MSN@SOR

[0020] (1) MSN nanoparticles were dispersed in a mixed solution of ethanol and 3-aminopropyltriethoxysilane, stirred, washed and collected by centrifugation to obtain MSN-NH2 nanoparticles;

[0021] (2) Disperse MSN-NH2 nanoparticles in a mixed solution of ethanol, then completely dissolve succinic anhydride in DMF and add it to the above ethanol solution, and stir; then centrifuge the mixture, collect the precipitate and wash it to obtain MSN-COOH nanoparticles;

[0022] (3) Disperse MSN-COOH nanoparticles in ethanol containing sorafenib, stir, centrifuge the mixture, collect the precipitate and wash it to obtain MSN@SOR nanoparticles;

[0023] Step 3: Preparation of MF@SOR

[0024] (1) Dissolve the prepared MSN@SOR nanoparticles in water, then stir with FeCl3 ethanol solution, then add H3BTC ethanol solution and stir again;

[0025] (2) The product MF@SOR can be obtained by centrifugation and washing.

[0026] Furthermore, in step two (1), the volume ratio of ethanol to 3-aminopropyltriethoxysilane is 50:1. Excessive volume of 3-aminopropyltriethoxysilane leads to poor dispersion of the nanoparticles; insufficient volume results in a small amount of surface-modifying groups, making it difficult for MIL-100(Fe) to be applied. Therefore, when the volume ratio of ethanol to 3-aminopropyltriethoxysilane is 50:1, the nanoparticles are well-dispersed, and MIL-100(Fe) is also more easily applied.

[0027] Furthermore, the stirring time in step two (2) is 8-14 h. In the process of modifying the carboxyl group onto the MSN surface in step two (2), the inventors discovered through long-term practice and analysis that shortening the stirring time and achieving the same modification effect without N2 protection can save production costs and shorten the drug production time.

[0028] Furthermore, the stirring time in step two (3) is 6-12 h; the stirring speed is 200-250 rpm. If the stirring speed is lower than 200 rpm, the stirring will be uneven and the stirring effect will be poor; if the speed is higher than 250 rpm, the nanoparticles will be easily broken. Therefore, the stirring speed is 200-250 rpm, which can ensure uniform stirring and intact nanoparticle morphology.

[0029] Furthermore, in step three (1), the stirring speed for the first stirring is 1000-1500 rpm. A stirring speed of 1000-1500 rpm can make the coating of MIL-100 (Fe) more uniform. When the stirring speed is lower than 1000 rpm, the iron ion modification of the innermost layer is uneven, which leads to uneven subsequent MIL-100 (Fe) modification; when the stirring speed is higher than 1500 rpm, the iron ions cannot coordinate with the carboxyl groups on the MSN surface, and it is difficult for MIL-100 (Fe) to be coated on the surface.

[0030] Furthermore, the second stirring time in step three (1) is 1.5-2 hours. In this scheme, if the stirring time is less than 1.5 hours, the coating of MIL-100 (Fe) is unstable; if the stirring time exceeds 2 hours, the coating of MIL-100 (Fe) is too thick, resulting in an excessively large size of the MF@SOR nanovaccine, which limits its passive targeting function and is not conducive to delivering the drug to the tumor. When the stirring time is 1.5-2 hours, the coating of MIL-100 (Fe) is stable and can significantly improve the passive targeting function and enhance drug utilization.

[0031] Furthermore, the concentration of the FeCl3 ethanol solution in step three (1) is 7.5-8.5 mg / mL. −1 The concentration of the H3BTC ethanol solution is 8-12 mg / mL. −1 Too low a concentration of FeCl3 and H3BTC will result in insufficient iron content; too high a concentration of FeCl3 and H3BTC will cause nanoparticles to easily aggregate. Therefore, the optimal concentration of FeCl3 in ethanol solution is 7.5-8.5 mg / mL. −1 The concentration of H3BTC ethanol solution is 8-12 mg / mL. −1 It has a high iron content and good nanoparticle dispersibility.

[0032] This invention also provides an application of a drug-loaded MF@SOR nanovaccine in the preparation of drugs for treating tumors.

[0033] Furthermore, the tumor is liver cancer. Attached Figure Description

[0034] Figure 1The image shows the characterization properties of the nanoparticles. (A) is a scanning electron microscope (SEM) image of SiO2, (B) is a scanning electron microscope (SEM) image of MSN, (C) is a scanning electron microscope (SEM) image of MF@SOR, (D) is a scanning electron microscope (SEM) image of MF@SOR (scale bar: 200 nm), (E) is a TEM image of MF@SOR under pH=5 and GSH conditions (scale bar: 100 nm), (F) is a high-angle annular dark-field image and elemental spectrum of MF@SOR (scale bar: 50 nm), (G) shows the particle size of different nanoparticles and (H) zeta potential (n = 3), (I) is the X-ray diffraction pattern of MSN, (J) is the X-ray photoelectron spectrum of Mn in MF@SOR, (K) is the nitrogen adsorption curve of MSN and (L) is the corresponding pore size distribution, (M) is the Fourier transform infrared spectrum of SOR, MSN, and MF@SOR, and (N) is the TEM image of MF@SOR in PBS at pH= The cumulative release of SOR under the conditions of pH 5.0 (containing GSH), pH = 6.5 (containing GSH), pH = 7.4 (containing GSH) and pH = 7.4 (n = 3), (O) represents the iron and manganese content in MF@SOR measured by ICP-OES.

[0035] Figure 2 (A) represents the stability of the nano-vaccine in different solutions. Figure 2 (B) is the X-ray photoelectron spectrum of Fe in MF@SOR. Figure 2 (C) shows the in vitro MRI imaging results. Figure 2 (D) represents the r1 value after MRI scans under different conditions.

[0036] Figure 3 The mechanism of pyroptosis and STING pathway activation is shown in (A) immunofluorescence imaging of cleaved caspase-1 and cleaved caspase-3 expression in different tumor samples (scale bar: 50 μm), (B) immunofluorescence imaging of CRT and HMGB1 expression in hepa1-6 tumors after different treatments (scale bar: 50 μm), (C) immunofluorescence imaging of p-STING and (D) p-TBK1 expression in hepa1-6 tumors after different treatments (scale bar: 25 μm), semi-quantitative fluorescence intensity analysis of CRT (E), HMGB1 (F), p-STING (G) and p-TBK1 (H) staining (n = 3), and the levels of (I) IL-18, (J) IL-1β, (K) IFN-β and (L) CXCL10 in mouse serum after different treatments (n = 3).

[0037] Figure 4The images show the endocytosis of MF@DiI, the cytotoxicity of different nanoparticles, and the in vitro antitumor effects of different nanoparticles. (A) shows the cell uptake of MF@DiI and hepa1-6 cells after incubation for different times (scale bar: 50 μm). (B) shows the intracellular uptake of MF@DiI and hepa1-6 cells after different incubation times by flow cytometry. The cell viability (n = 3) of different concentrations of MSN, MF, MF@SOR and (C) hepa1-6 cells and (D) HUVECs after 12 hours of culture. (E) shows the fluorescence image of cells stained with a live / dead cell kit. (F) shows the corresponding fluorescence percentage (scale bar: 100 μm).

[0038] Figure 5 The images show the in vivo antitumor effects and magnetic resonance imaging. (A) is a schematic diagram of the experimental design of the primary tumor model (created using BioRender.com). (B) is a curve showing the relative tumor volume of different treatment groups over time (n = 5). (C) is a curve showing the weight of mice in different treatment groups over time (n = 5). (D) is a curve showing the survival rate of mice in different treatment groups (n = 5). (E) is an immunofluorescence image of TUNEL, PCNA, and H&E in each group (scale bar: 50 μm). (F) is a semi-quantitative fluorescence intensity analysis of PCNA and (G)TUNEL staining (n = 3). (H) is an H&E staining image of the major organs of mice in each group after different treatments (scale bar: 100 μm). (I) Photographs and (J) weights of tumor tissues after treatment in different groups (n = 5). (K) is a T1 MRI of tumor-bearing mice at different time points. (L) is a change in signal intensity of the tumor region of tumor-bearing mice at different time points (n = 3).

[0039] Figure 6 To investigate DNA damage and in vitro DC cell maturation, (A) shows immunofluorescence images of γ-H2AX staining after different treatments (scale bar = 25 μm), (B) shows the corresponding quantitative analysis of γ-H2AX expression (n = 3), (C) shows the co-culture system protocol, (D) shows representative bright-field microscopy images of JAWS II cells after treatment (scale bar = 100 μm), (E) shows representative flow cytometry results of mature DCs (CD11c+CD80+ CD86+) after different treatments, (F) shows the corresponding quantitative analysis of flow cytometry results (n = 3), and (G) shows the levels of (H) IFN-β and (C) CXCL10 measured in the co-culture system after different treatments (n = 3).

[0040] Figure 7For the immunomodulatory analysis of the primary tumor model, (A) the content of mature DC cells in the spleen and (B) tumor-associated lymphoid tissue, (F) the quantitative analysis of the content of mature DC cells in the spleen and (G) tumor-associated lymphoid tissue (n=3), (C) the flow cytometry analysis of CD3+CD8+ T cells in the spleen, (D) tumor-associated lymphoid tissue, and (E) tumor after different treatments, and the corresponding quantitative analysis in (H, I, and J) (n=3), (K) the immunofluorescence image of CD8+ T cells in hepa1-6 tumors after different treatments (scale bar: 50μm), and (L) the immunohistochemical image of Foxp3+ expression in each group of tumor samples (scale bar: 50μm).

[0041] Figure 8 To establish relapse and metastasis models and monitor long-term immune effects, (A) is a schematic diagram of the animal experimental design for the relapse tumor model (drawn using BioRender.com), (B) shows the weight changes of mice with relapsed tumors (n = 3), (C) shows the growth curves of relapsed tumor volume in each group (n = 3), (D) shows the weight of relapsed tumors in each group (n = 3), (E) shows flow cytometry images of CD3+CD8+ T cells in each group of relapsed tumors and (F) shows the corresponding quantitative analysis, (G) is a schematic diagram of the animal experimental design for the lung metastasis model (created using BioRender.com), and (H) shows representative photographs of mouse lung tissue and H&E staining images of mouse lung tissue. Detailed Implementation

[0042] The following detailed explanation illustrates the specific implementation methods:

[0043] Example 1: MF@SOR

[0044] A drug-loaded nanovaccine MF@SOR comprises MIL-100 (Fe) and a manganese-doped mesoporous silica carrier, wherein the MIL-100 (Fe) is coated on the outer surface of the manganese-doped mesoporous silica carrier, and sorafenib is loaded within the manganese-doped mesoporous silica carrier.

[0045] The preparation method of MF@SOR includes the following steps:

[0046] Step 1: Preparation of non-porous silica

[0047] Mix 98 mL of ethanol and 10 mL of water, add 1.25 mL of ammonia (28% by mass) and 5 mL of TEOS to the mixture; stir at 300 rpm for 20 h at room temperature; then collect the SiO2 nanoparticles, centrifuge at 13000 rpm for 10 min, and wash three times with ethanol and water.

[0048] Step 2: MSN Preparation

[0049] (1) Dissolve 70-90 mg MnCl2·4H2O (80 mg MnCl2 in this example) and 400 mg NH4Cl in 77.4 mL of deionized water, and then add 1-4 mL of the above-mentioned SiO2 nanoparticle aqueous solution (SiO2 nanoparticle aqueous solution concentration is 80 mg / mL) to the mixture. –1 In this embodiment, the amount of SiO2 nanoparticle aqueous solution used is 1 mL) and 1.5-3.5 mL of NH3·H2O (mass fraction 28%, in this embodiment, the amount of NH3·H2O used is 1.6 mL). The mixture is then transferred to a 150 mL high-temperature reactor and heated at 180°C for 10 hours.

[0050] (2) After washing with water three times, MSN nanoparticles were obtained by centrifugation at 13,000 rpm for 5 minutes.

[0051] Step 3: Preparation of MSN@SOR

[0052] (1) 40 mg of MSN nanoparticles were dispersed in a mixture of ethanol and 3-aminopropyltriethoxysilane (the volume ratio of ethanol to 3-aminopropyltriethoxysilane was 50:1, and in this example, 20 mL of ethanol and 400 μL of 3-aminopropyltriethoxysilane were dispersed), and then stirred at 600 rpm for 12 h. After washing and centrifugation, MSN-NH2 nanoparticles were obtained.

[0053] (2) 40 mg MSN-NH2 nanoparticles were dispersed in a mixed solution of 15 mL ethanol, and then 140 mg succinic anhydride was completely dissolved in DMF and added to the above ethanol solution. After stirring at 20 °C for 8-14 h (the stirring time here is 12 h in this example), the MSN-COOH nanoparticles were collected by centrifugation at 13000 rpm for 5 minutes, and then washed several times with ethanol.

[0054] (3) Disperse 8 mg of MSN-COOH nanoparticles in 5 mL of ethanol containing 3.5 mg of SOR, and gently stir at 200-250 rpm (250 rpm in this example) for 6-12 h (12 h in this example). Centrifuge the mixture (13000, 5 min), collect the precipitate and wash it three times.

[0055] Step 4: Preparation of MF@SOR

[0056] (1) Prepare an aqueous solution of MSN@SOR nanoparticles (20 ml, 1 mg / mL) −1 (and concentrations of 7.5-8.5 mg / mL) −1 The FeCl3 ethanol solution (volume of FeCl3 ethanol solution is 10 mL, concentration of FeCl3 ethanol solution in this example is 8.1 mg / mL) −1 At room temperature, stir vigorously at 1000-1500 rpm (1000 rpm in this example) for 10-20 minutes (15 minutes in this example); then add 8-12 mg / mL at 40°C. −1 The H3BTC ethanol solution (volume of H3BTC ethanol solution is 10 mL, concentration of H3BTC ethanol solution in this example is 10.5 mg / mL) −1 Stir for 1.5-2 hours (in this embodiment, the stirring time is 2 hours).

[0057] (2) Then, the product is separated by centrifugation and washed repeatedly with ethanol and water to obtain the product MF@SOR, which is then stored at 4°C.

[0058] Comparative Example 1: MSN (MSN is an abbreviation for manganese-doped mesoporous silica)

[0059] The difference between the preparation method of MSN in Comparative Example 1 and Example 1 is that all steps three and four in Example 1 are omitted.

[0060] Comparative Example 2: MF (In this application, MF stands for MSN and F stands for MIL-100 (Fe))

[0061] The difference between the preparation method of MF in Comparative Example 2 and Example 1 is that step 3 (3) of Example 1 is omitted, and MSN@SOR in step 4 is replaced with MSN-COOH.

[0062] Comparative Example 3: SOR

[0063] SOR drug, purchased from GLPBIO.

[0064] Comparative Example 4: MSN@SOR (MSN is an abbreviation for manganese-doped mesoporous silica, and SOR is an abbreviation for sorafenib)

[0065] The preparation method of MSN@SOR in Comparative Example 4 differs from that in Example 1 in that all steps in step four of Example 1 are omitted.

[0066] I. Experimental Apparatus and Reagents

[0067] (1) Experimental apparatus

[0068] Transmission electron microscopy (TEM, Talos F200X, USA), scanning electron microscopy (SEM, HITACHI S4800, Japan), laser particle size analyzer (Zetasizer Nano ZS90, Malvern Instruments Ltd., Worcs, UK), X-ray diffractometer (BRUKERD8 ADVANCE, Germany), fully automated surface area and porosity analyzer BET (QuantachromeAutosorb IQ3 apparatus, USA), Fourier-transform infrared spectroscopy (FTIR, iS10 FT-IR spectrometer, USA), high-performance liquid chromatography (HPLC, Shimadzu LC-2010AHt, Japan), X-ray photoelectron spectroscopy (Thermo Scientific K-Alpha, USA), inductively coupled plasma optical emission spectrometry (ICP-OES, Thermo ICP-OES7200 (USA).

[0069] (2) Experimental reagents

[0070] Tetraethyl orthosilicate (TEOS) (T819505), Trimesic acid (H3BTC) (T819407), Iron(III) chloride (FeCl3) (I811935), Succinic anhydride (S817605), 3-Aminopropyltriethoxysilane (A800523), Ammonium chloride (NH4Cl) (A801305), N,N-dimethylformamide (DMF) (N807505), and ammonia hydroxide solution (NH3·H2O, 28%) were purchased from Macklin (Shanghai, China); Manganese chloride tetrahydrate (MnCl2·4H2O) (R000534) was purchased from Rhawn (Shanghai, China). (China); Fetal bovine serum (FBS) purchased from Cell-Box (HK) Biological Products Trading Co.The following cell culture media were purchased from Booster (Wuhan, China): Dulbecco's modified Eagle's medium (DMEM) (PYG0074), 5% blocking solution (5% BSA-confining-liquid) (BSA) (AR0004), and phosphate-buffered saline (PBS) (PYG0021); Streptomycin-penicillin combination (C0222), Trypsin-EDTA solution (C0201), DAPI staining agent (C1005) (for staining cell nuclei), DNA Damage Assay Kit by γ-H2AX Immunofluorescence, Triton X-100 (P0096), and DiI (C1036) were purchased from Beyotime Biotechnology (Jiangsu, China); Enzyme-linked immunosorbent assays (ELISAs), including... IL-6 (MM-0163M1), IL-12 (MM-0105M1), IFN-β (MM-0124M1), TNF-α (MM-0132M2), IL-18 (MM-0169M1), and IL-1β (MM-0040M1) were purchased from MEIMIAN (Jiangsu, China); the Calcein-AM / PI Double Staling Kit (C542) was purchased from Dojindo Laboratories (Kumamoto, Japan); sorafenib (GC17369) and the Cell Counting Kit-8 (CCK8) (GK10001) were purchased from GLPBIO (Montclair, CA, USA); PE anti-mouse CD3 (100206), APC anti-mouse CD8a (100712), and FITC anti-mouse CD44 were also purchased. (103006), PerCP / Cyanine5.5 anti-mouse CD62L (104432), FITC anti-mouse CD11c (117306), PE anti-mouse CD80 (104708), and APC anti-mouse CD86 were purchased from Biolegend (San Diego, CA, USA); HMGB1 polyclonal antibody (10829-1-AP), calreticulin polyclonal antibody (10292-1-AP), CoraLited488-conjugated Goat Anti-Rabbit IgG (H+L) (S00013-2) and CoraLited594-conjugated Goat Anti-Rabbit IgG (H+L) (S00013-4) were purchased from Proteintech (Wuhan, China).

[0071] II. Experiments demonstrating the properties of nanoparticles

[0072] (I) Characterization of nanoparticles

[0073] (1) Morphological characterization and elemental spectrum analysis of MF@SOR

[0074] Experiment 1

[0075] The morphology of MF@SOR in Example 1 was observed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). SiO2 nanoparticles were used as a precursor, such as... Figure 1 As shown in Figure A, the TEM image reveals that the synthesized SiO2 is spherical and uniformly dispersed. MSN was prepared by hydrothermal drilling of SiO2 and doping with manganese ions. Figure 1 As shown in Figure B, the mesoporous structure can be clearly observed through TEM images. MSN@SOR was coated with MIL-100(Fe) and then stirred to obtain MF@SOR, as shown in Figure B. Figure 1 As shown in C and 1D, TEM and SEM images show that the MF@SOR of Example 1 is spherical, with a regular shape and uniform size. Subsequently, the MF@SOR of Example 1 was exposed to a low-pH environment containing GSH, as... Figure 1 As shown in Figure E, the TEM image reveals that the morphology of the MF@SOR from Example 1 has changed; it has lost its original shape and fragmented. Figure 1 As shown in F, the high-angle annular dark field and elemental spectrum show a uniform distribution of iron, manganese, silicon, and oxygen.

[0076] (2) Particle size and potential analysis of MF@SOR

[0077] Experiment 2

[0078] The particle size and zeta potential of MF@SOR in Example 1 were determined using a laser particle size analyzer. Figure 1 As shown in G and 1H, the particle size distribution of MSN is approximately 131.37 ± 0.58 nm, and the zeta potential is approximately -18.63 ± 1.06 mV. After the addition of sorafenib, the particle size distribution of MSN@SOR increases to approximately 135.93 ± 1.35 nm, and the zeta potential changes to -19.57 ± 0.71 mV. The results indicate that loading with SOR slightly alters the diameter and zeta potential of MSN nanoparticles. After coating with MIL-100(Fe), the pores of MSN are covered, the particle size changes significantly, increasing to approximately 153.87 ± 1.8 nm, and the zeta potential reverses to 10.31 ± 1.53 mV, indicating successful coating with MIL-100(Fe).

[0079] (3) Stability of MF@SOR nanovaccine

[0080] Experiment 3

[0081] The particle size stability of MF@SOR from Example 1 was tested using a laser particle size analyzer after different number of days. Figure 2 As shown in Figure A, after the MF@SOR nanovaccine of Example 1 was suspended in double-distilled water, physiological saline, DMEM, and FBS for 1, 3, 5, 7, 9, 11, and 13 days, no significant size change was observed, indicating that the MF@SOR of Example 1 has good colloidal stability.

[0082] (4) X-ray diffraction pattern analysis of MF@SOR nanovaccine

[0083] Experiment 4

[0084] The material composition and structure of MF@SOR in Example 1 were determined using X-ray diffraction. Figure 1 As shown in Figure I, the X-ray diffraction pattern of MF@SOR reveals the presence of manganese oxide (MnO4) within the silicate framework. 2+ Mn6 3+ SiO 12 (JCPDS No.: 89-5661) and other manganese silicate phases (Mn2SiO4, JCPDS No.: 02-1327 and Mn5Si3O) 12(JCPDS No.: 37-0221), confirming the presence of manganese covalent bonds in the silica framework. The characteristic peak near 2θ = 11° is consistent with the MIL-100 (Fe) standard mode, indicating that the MIL-100 (Fe) shell coating was successful.

[0085] (5) Drug loading potential of MSN nanoparticles

[0086] Experiment 5

[0087] like Figure 1 As shown in K, the nitrogen adsorption-desorption curves further demonstrate that MSN nanoparticles exhibit a type IV isotherm with an H3-type hysteresis loop at P / P0 = 0.4–1.0, indicating that MSN nanoparticles possess a mesoporous structure. Furthermore, as shown in K... Figure 1 As shown in Figure L, the average pore size of the MSN nanoparticles is 5.6284 nm, and the pore volume is 0.3213 cm³g. -1 This indicates that MSN nanoparticles have the potential to load SOR.

[0088] (6) Fourier transform infrared spectroscopy analysis of MF@SOR

[0089] Experiment 6

[0090] Fourier transform infrared spectroscopy was used to confirm the successful synthesis of the MF@SOR nanovaccine in Example 1. As shown in Figure 1M, overall, the infrared spectrum of MF@SOR in Example 1 retains the typical characteristics of MSN, with a wavelength of 1017 cm⁻¹. -1 The absorption peak at 2959 cm⁻¹ is a typical Si-O asymmetric stretching vibration mode of mesoporous silica, indicating that the main structure of the synthesized material is silica. -1 Several absorption peaks appeared nearby, mainly originating from the CH structure of SOR. (1561 cm⁻¹) -1 The nearby absorption peaks are related to the NH and C=C structures in the SOR structure, while the 1442 cm⁻¹ peak is... -1 The nearby absorption peaks are also related to methyl stretching vibrations. The appearance of these characteristic peaks indicates that SOR has been successfully loaded onto the mesoporous silica surface. Furthermore, the 465 cm⁻¹ peak in the sample... -1 A sharp absorption peak was observed nearby, mainly due to the Fe-O bending vibration mode, confirming the successful modification of MIL-100 (Fe).

[0091] (7) X-ray photoelectron spectroscopy analysis of MF@SOR

[0092] Experiment 7

[0093] X-ray photoelectron spectroscopy was used to analyze the valence states of manganese and iron in the MF@SOR nanovaccine of Example 1. Figure 1 As shown in J and 2B, 43 and 643.29 eV represent Mn, respectively. 2+ and Mn 3+ Similarly, the iron 2p3 / 2 spectrum was decomposed into two characteristic bands at 710.19 and 711.84 eV, representing Fe, respectively. 2+ and Fe 3+ These results indicate that manganese and iron ions are present in the MF@SOR nanovaccine of Example 1.

[0094] (8) Analysis of manganese and iron content in MF@SOR nano-vaccines

[0095] Experiment 8

[0096] The manganese and iron content in the MF@SOR nanovaccine of Example 1 was evaluated using inductively coupled plasma-optical emission spectrometry (ICP-OES). Figure 1 As shown in O, the values ​​are 0.1578 ± 0.0009 mg mg. -1 and 0.073 ± 0.0004 mg mg -1 .

[0097] (ii) Drug release capacity of MF@SOR

[0098] Experiment 9

[0099] The magnetic resonance imaging (MRI) performance of the MF@SOR nanovaccine from Example 1 was validated in vitro under different simulated tumor microenvironment (TME) conditions. Figure 2 As shown in Figure C, in environments with low pH or high GSH, the contrast signal of T1 MRI increases with increasing concentration, and this trend is particularly pronounced when both low pH and high GSH are present simultaneously. However, in a physiological environment (pH 7.4), the increasing trend of T1 MRI contrast signal is relatively weak. Furthermore, as... Figure 2 As shown in Figure D, there is a clear linear relationship between the 1 / T1 value and the manganese concentration. Compared with the pH 7.4 group, the r1 values ​​of all other groups increased significantly, with the r1 value of the pH 5.0 + 10 mM GSH group reaching as high as 54.677 mM. -1 S -1 This is because MIL-100(Fe) degrades under acidic conditions, and the internal Mn... 2+The dissociation from MSN nanoparticles under the reducing environment of GSH indicates that the MF@SOR nanovaccine of Example 1 can be used as a TME-stimulated T1 MRI contrast agent.

[0100] Experiment 10

[0101] According to the high-performance liquid chromatography (HPLC) results, the encapsulation efficiency and drug loading of SOR were 35% and 10.7%, respectively. Subsequently, the ability of the MF@SOR nanovaccine from Example 1 to release SOR was verified. As shown in Figure 1N, after incubation for 48 hours in phosphate-buffered saline (PBS) at pH 5.0 containing 10 mM GSH, approximately 65% ​​of the SOR was released from the MF@SOR nanovaccine from Example 1. In PBS at pH 6.5 containing 10 mM GSH, approximately 40% of the SOR was released. Under the conditions of pH 7.4 and 10 mM GSH, approximately 20% of the total SOR was released, while under the same incubation time at pH 7.4 and 10 mM GSH, less than 10% of the total SOR was released. Clearly, in the presence of GSH, the amount of SOR released by the MF@SOR nanovaccine in Example 1 gradually increased as the ambient pH decreased. This indicates that the amount of SOR released is higher under acidic and reducing conditions compared to neutral conditions. This result demonstrates that the nanovaccine is highly stable in circulation, releasing almost no drug; conversely, it releases a large amount of drug in the tumor region, thus enhancing efficacy and reducing side effects.

[0102] III. Mechanisms of Pyroptosis and STING Pathway Activation

[0103] Experiment 11

[0104] Chemotherapy induces pyroptosis via caspase-3 (a non-classical pathway), while regulation of intracellular iron-induced pyroptosis is achieved via caspase-1 (a classical pathway). Cleaved caspase-3 cleaves GSDME, leading to the release of GSDME-N, while cleaved caspase-1 cleaves GSDMD, leading to the release of GSDMD-N. To investigate the mechanism of pyroptosis induced by nanovaccines, this invention performed immunohistochemistry (IHC) to detect cleaved caspase-1 and cleaved caspase-3 in tumor tissues after different treatments. Figure 3As shown in Figure A, the expression of cleaved caspase-1 was significantly increased in the MIL-100(Fe) treatment group (MF@SOR of Example 1), while the expression of cleaved caspase-3 was significantly increased in all SOR treatment groups (SOR of Comparative Example 3, MSN@SOR of Comparative Example 4, and MF@SOR of Example 1). These results indicate that MF@SOR of Example 1 can induce tumor cell thermophagy through both classical and non-classical pathways. GSDME-N and GSDMD-N translocate to the cell membrane and form membrane pores, thereby driving cell swelling, membrane rupture, and DAMP release, which in turn induces a pyroptosis-induced immune response. Figure 3 As shown in B, 3E, and 3F, calreticulin (CRT) expression gradually increases, while the expression of high mobility group box 1 (HMGB1) in the nucleus gradually decreases. Figure 3 As shown in I and 3J, the increase in serum IL-18 and IL-1β concentrations also indicates that pyroptosis has occurred.

[0105] Activation of the cGAS-STING pathway depends on phosphorylation of STING and TBK1. Therefore, p-STING was used to test tumor tissues after different treatments. Figure 3 C and 3G) and p-TBK1 ( Figure 3 Immunofluorescence staining (D and 3H) was performed. The results showed that mouse tumor tissue sections treated with Comparative Example IV MSN@SOR and Example 1 MF@SOR showed increased expression of p-STING and p-TBK1, indicating increased expression of cGAS-STING-related proteins in tumor tissues. Furthermore, as... Figure 3 As shown in K and 3L, the concentrations of IFN-β and CXCL10 in serum were detected using an enzyme-linked immunosorbent assay kit, and the changes in their concentrations also indicated that the cGAS-STING pathway was activated.

[0106] IV. Experiments demonstrating the effectiveness of nanoparticles

[0107] (I) Cytotoxicity and in vitro antitumor effects

[0108] Experiment 12

[0109] In this application, the MF@SOR nanovaccine in Example 1 was designed to kill cancer cells. Therefore, confocal laser scanning microscopy (CLSM) and flow cytometry were used to preliminarily verify the absorption of MF@DiI nanoparticles (MF@DiI refers to DiI fluorescence loaded onto MF nanoparticles) by hepa1-6 cells. Figure 4 As shown in Figure A, the MF@DiI fluorescence signal around the nuclei of DAPI-stained cells increased over time at 0.5 hours, 1 hour, 2 hours, 3 hours, and 4 hours. Figure 4 As shown in Figure B, flow cytometry results showed that after 4 h of treatment, the intracellular uptake rate of MF@DiI nanoparticles by hepa1-6 cells was approximately 73%, which was consistent with the CLSM images, quantitatively confirming that hepa1-6 cells have a strong phagocytic capacity for MF@DiI nanoparticles.

[0110] Experiment 13

[0111] Hepa1-6 cells were exposed to different concentrations of free Comparative Example 3 SOR, Comparative Example 1 MSN, Comparative Example 2 MF, Comparative Example 4 MSN@SOR, or Example 1 MF@SOR. Human umbilical vein endothelial cells (HUVECs) were exposed to different concentrations of Comparative Example 1 MSN, Comparative Example 2 MF, Comparative Example 4 MSN@SOR, or Example 1 MF@SOR. Figure 4 As shown in Figure C, the survival rate of hepa1-6 decreased with increasing concentrations of MSN (Comparative Example 1) or MF (Comparative Example 2) nanoparticles. This is because the presence of acidity and high GSH in tumor cells favors nanoparticle degradation and promotes Fe2+ degradation. 3+ and Mn 2+ The release of these molecules activates the classical pyroptosis pathway in hepa1-6 cells. Conversely, when MSN (Comparative Example 1) or MF (Comparative Example 2) are internalized by HUVECs, they are slowly degraded and cleared within the acidic environment of lysosomes, thus exhibiting weaker cytotoxicity towards HUVECs. Figure 4 As shown in C and 4D, after loading SOR, the survival rates of hepa1-6 and HUVEC were lower than those of MSN and MF at the same concentration. This is because SOR can activate the non-classical pyroptosis pathway and has chemotherapeutic effects. The IC50 value of MSN@SOR against hepa1-6 in Comparative Example 4 was 85.13 μg / mL (corresponding to an SOR concentration of 13.04 μg / mL). -1 The IC50 value of MF@SOR in Example 1 against hepa1-6 was 42.72 μg / mL (corresponding to a SOR concentration of 6.54 μg / mL). -1The concentrations of SOR were all lower than the IC50 value of free SOR (26.04 μg / mL). In other words, when used with MSN (Comparative Example 1) or MF (Comparative Example 2), lower concentrations of SOR could exert a stronger antitumor effect, which may be due to the carrier increasing the influx of SOR into hepa1-6 cells. These findings are significant for the development of antitumor drugs because reducing the amount of SOR used helps to mitigate potential side effects. When the concentration of the MF@SOR nanovaccine in Example 1 was increased to 200 μg / mL... -1 At that time (the corresponding SOR concentration was 30.63 μg / mL) -1 The survival rate of Hepa1-6 cells decreased to about 10%, indicating that it has a highly efficient killing effect. Therefore, in subsequent experiments, 200 μg / mL was used. -1 MSN, MF, MSN@SOR, and MF@SOR nanovaccines were evaluated for their antitumor mechanisms of action in vitro and activated the immune system in an in vitro co-culture system.

[0112] Experiment 14

[0113] In addition, calcein acetoxymethyl ester (calcein-AM) was used to stain live cells, propidium iodide (PI) was used to stain dead cells, and finally the distribution of live and dead cells in the hepa1-6 cells was observed using an inverted fluorescence microscope. Figure 4 As shown in E and 4F, the results indicate that the control group had the most live cells and almost no dead cells, while the MF@SOR in Example 1 had the fewest live cells and a large number of dead cells. (In this application, the control groups in Experiments 15, 16, and 17 refer to the groups treated with physiological saline, while the control groups in the other experiments refer to the groups treated with DMEM basal medium.)

[0114] (II) In vivo antitumor effects of MF@SOR

[0115] Experiment 15

[0116] Figure 5 A illustrates the treatment regimen used in C57BL / 6J mice to evaluate the effects of nanoparticles on tumor-bearing mice. Throughout the treatment period, mouse body weight and tumor volume were measured every two days. Figure 5 As shown in Figure C, no weight abnormalities occurred in any of the treatment groups. Figure 5 As shown in B, 5I, and 5J, compared with the control group treated with saline, tumor growth was inhibited in the intervention group treated with Comparative Example 1 MSN. Tumor growth was also significantly inhibited in the mouse groups treated with Comparative Example 3 SOR, Comparative Example 4 MSN@SOR, or Example 1 MF@SOR, with Example 1 MF@SOR showing the best effect in inhibiting tumor growth.

[0117] Experiment 16

[0118] PCNA, TUNEL, and H&E staining assays were performed to evaluate the antitumor efficacy. Figure 5 As shown in Figures E, 5F, and 5G, PCNA images revealed that the MF@SOR in Example 1 exhibited the weakest proliferation-related FL signal, the MSN@SOR in Comparative Example 4 showed a weak proliferation-related FL signal, the SOR treatment group in Comparative Example 3 showed an increase in proliferation-related FL signal, while the control group and the MSN treatment group in Comparative Example 1 showed a significant increase in fluorescence (FL) signal. In contrast, TUNEL images showed that among the two groups receiving either MSN@SOR in Comparative Example 4 or MF@SOR in Example 1, the MF@SOR in Example 1 showed the strongest apoptotic FL signal, the MSN@SOR in Comparative Example 4 showed a relatively strong apoptotic FL signal, the SOR in Comparative Example 3 showed a weak FL signal, and the apoptotic FL signal in the control group and the MSN treatment group in Comparative Example 1 was almost negligible. H&E staining showed that excessive apoptosis and necrosis were observed in MSN@SOR of Comparative Example 4 and MF@SOR of Example 1, while the incidence of necrosis and apoptosis gradually decreased in SOR of Comparative Example 3 and MSN of Comparative Example 1. Almost no necrosis and apoptosis were observed in the control group. The results were consistent with the results of PCNA and TUNEL staining.

[0119] Simultaneously, the survival time of the mice was recorded to verify that MF@SOR in Example 1 could prolong the survival time of the mice. Figure 5 As shown in Figure D, the survival time of mice in Comparative Example 1 (MSN), Comparative Example 3 (SOR), Comparative Example 4 (MSN@SOR), and Example 1 (MF@SOR) was significantly prolonged, with the longest survival time observed in mice in Example 1 (MF@SOR). Specifically, all mice in the control group died after 31 days, all mice in the MSN group of Comparative Example 1 died after 33 days, all mice in the SOR group of Comparative Example 3 died after 43 days, 4 mice in the MSN@SOR group of Comparative Example 4 died after 53 days, and one mouse survived until the end of the 60-day observation period. In the MF@SOR group of Example 1, only one mouse died at 56 days, and the remaining four mice survived until the end of the 60-day observation period. This is attributed to the additional effect of the MF vector activating the systemic anti-tumor immune response. Furthermore, as... Figure 5 As shown in Figure H, after 14 days of various treatments, no obvious pathological damage or inflammatory lesions were found in the H&E-stained anatomical images of the major organs (heart, liver, spleen, lungs, and kidneys) of mice, indicating that the side effects during tumor treatment were negligible. In summary, these results demonstrate that the MF@SOR nanovaccine of Example 1 of this application has a strong anti-tumor effect and can also significantly prolong the lifespan of mice.

[0120] (III) Imaging performance of MF@SOR

[0121] Experiment 17

[0122] This invention verified the T1 MRI characteristics of MF@SOR from Example 1 in a subcutaneous tumor model. Figure 5 As shown in K and 5L, the T1 MRI contrast signal reached its maximum at 2 h, then gradually decreased, and observation ceased at 20 h, indicating that the nanoparticles rapidly accumulated at the tumor site through the EPR effect. These results demonstrate that the MF@SOR nanovaccine of Example 1 possesses excellent T1 MRI characteristics. Imaging images also revealed that the T1 MRI contrast signal in the tumor region was significantly higher than that in the surrounding tissue after injection. These results indicate that the MF@SOR nanovaccine of Example 1 exhibits responsive imaging capabilities, improving the accuracy of tumor detection and localization, and providing a novel method for tumor-specific diagnosis.

[0123] (iv) Immunotherapy with MF@SOR

[0124] Experiment 18

[0125] This invention utilizes γ-H2AX to detect DNA damage occurring during pyroptosis, and observes the positivity rate of γ-H2AX using immunofluorescence. Figure 6 As shown in Figures A and 6B, compared to the control group with the lowest fluorescence, the MF@SOR treatment group in Example 1 showed significantly more γ-H2AX positive foci, with a trend similar to that of CRT fluorescence, indirectly indicating that DNA is damaged through pyroptosis. Damaged DNA escaping from the nucleus to the cytoplasm can be recognized by cGAS and activate the STING pathway. Therefore, the corresponding pro-inflammatory cytokines IFN-β and CXCL10 in the supernatant of cancer cells treated with DMEM, Comparative Example 1 MSN, Comparative Example 2 MF, Comparative Example 3 SOR, Comparative Example 4 MSN@SOR, and Example 1 MF@SOR were detected using an ELISA kit. Figure 6 As shown in G and 6H, the concentrations of IFN-β and CXCL10 in Comparative Example 2 MF, Comparative Example 4 MSN@SOR, and Example 1 MF@SOR were significantly increased, indicating the activation of the cGAS-STING pathway.

[0126] Experiment 19

[0127] Dendritic cells (DCs), as the most important antigen-presenting cells (APCs), play a crucial role in both innate and adaptive immunity. This application demonstrates that pyroptosis can trigger the release of CRT, HMGB1, and several pro-inflammatory cytokines. Once immature DCs are stimulated by inflammatory substances or tumor-associated antigens released by dying tumor cells, they migrate to nearby draining lymph nodes and mature. Mature DCs exhibit changes in receptors and molecules on their surface, enabling them to better interact with other immune cells and present antigens. Mature DCs present antigen fragments to T cells via antigen-presenting molecules on their surface, thereby activating the T cell immune response. DC maturation is typically accompanied by the upregulation of co-stimulatory molecules (CD80 and CD86) and the secretion of pro-inflammatory cytokines. To investigate the effects of pyroptosis on in vitro DC maturation, such as... Figure 6 As shown in Figure C, in this application, hepa1-6 cells treated with various nanoparticles were seeded in the upper cavity of a Transwell system, and JAWS II (immature DCs) were seeded in the lower cavity and co-cultured for 24 hours. The morphology of the lower cavity cells was then observed under a microscope (control group and MF@SOR treatment group in Example 1). These cells were then collected for flow cytometry (FCM) analysis to assess the expression of CD80 and CD86. Simultaneously, the supernatant was collected, and the release of pro-inflammatory cytokines, such as IL-6 and TNF-α, which are also indicators of DC activation, was detected using appropriate ELISA kits.

[0128] like Figure 6 As shown in Figure D, compared to the control group where cells were small and uniform in size, the cells in the MF@SOR treatment group were larger and showed angular features, which visually reflects the maturation of DC cells. Figure 6 As shown in E and 6F, compared with the control group (43.64% ± 0.39%), the percentage of mature DCs in Comparative Example 1 MSN (48.5% ± 0.51%), Comparative Example 3 SOR (53.54% ± 1.04%), Comparative Example 4 MSN@SOR (62.77% ± 0.63%), and Example 1 MF@SOR (69.07% ± 1.11%) increased sequentially. Furthermore, the percentage of mature DCs in Example 1 MF@SOR was higher than that in the other comparative examples. This is because the activation of the pyroptosis and cGAS-STING pathway led to the release of pro-inflammatory substances.

[0129] Experiment 20

[0130] After determining that the nanoparticles of this application can promote the maturation of dendritic cells (JAWS II), blood, tumors, spleens, and tumor draining lymph nodes (TDLNs) tissues of mice treated with different nanoparticles were collected to evaluate relevant immune markers and thus study their effects on immune system activation.

[0131] As previously mentioned, immature dendritic cells (DCs) migrate to immune organs (such as nearby draining lymph nodes and the spleen) upon stimulation, where they mature and expand into activated T cell clones. Therefore, flow cytometry was used to analyze the maturation status of DCs in tumors, TDLNs, and the spleen of mice treated with different drugs. Figure 7 As shown in A and 7F, the results indicated that the proportion of mature DC cells in the spleen of mice in the MF@SOR treatment group of Example 1 was approximately (24.17% ± 1.88%), more than twice that of the control group (8.9% ± 1.08%). Furthermore, as... Figure 7 As shown in B and 7G, the proportions of mature DC cells (CD80+CD86+CD11c+) in mouse lymph nodes treated with different nanoparticles were as follows: Comparative Example 1 MSN (18.8% ± 0.28%), Comparative Example 3 SOR (22.47% ± 0.46%), Comparative Example 4 MSN@SOR (26.23% ± 1.24%), and Example 1 MF@SOR (33.33% ± 1.1%). Furthermore, the percentage of mature DCs in Example 1 MF@SOR was significantly higher than in the other comparative examples. These results clearly demonstrate that the MF@SOR nanovaccine in Example 1 can promote DC cell maturation.

[0132] Experiment 21

[0133] Since mature dendritic cells (DCs) can stimulate an effective adaptive immune response by activating T lymphocytes, this invention investigated the relative proportions of CD3+CD8+ T cells in mouse tumor tissues, TDLNs, and spleen after different treatments. Compared with the control group, the spleen of MF@SOR mice in Example 1 ( Figure 7 C and 7 H), TDLNs ( Figure 7 D and 7 I) and tumors ( Figure 7 The frequency of CD3+CD8+ T cells was increased most in E and 7J, followed by comparative examples of MSN@SOR, SOR, and MSN. These results indicate that MSN, SOR, MSN@SOR, or MF@SOR can induce systemic immune responses to varying degrees. Meanwhile, as Figure 7As shown in K, immunofluorescence results indicated that the content of CD8+ T cells in the tumor was consistent with that in FCM. Furthermore, IHC analysis was used to analyze immunosuppressive Tregs within the tumor, such as... Figure 7 As shown in Figure L, the results indicate that MF@SOR in Example 1 significantly reduced the number of Foxp3+ cells, indicating that immunosuppression was alleviated.

[0134] In summary, compared with several other comparative examples, the results show that MF@SOR in Example 1 has the strongest ability to stimulate and alleviate immunosuppression, further elucidating the synergistic immune-enhancing effect of pyroptosis and the cGAS-STING pathway. This powerful immune response can play a crucial role in prolonging the survival rate of mice.

[0135] (v) Long-term immune effects of MF@SOR on tumor recurrence and metastasis

[0136] Experiment 22

[0137] like Figure 8 As shown in A and 8G, following the observation of significant immunostimulatory effects and marked inhibition of tumor growth in primary tumors, this application investigated long-term immune memory in a tumor recurrence and metastasis model. Figure 8 As shown in Figure C, the mice showed no significant abnormalities in body weight, and compared to several control groups, the mice treated with MF@SOR in Example 1 exhibited significantly reduced tumor recurrence and growth; simultaneously, as Figure 8 As shown in H, the number of lung metastases treated with MF@SOR in Example 1 was also significantly reduced, indicating that the nanovaccine of the present invention generated immune memory, thereby providing long-term protection against tumor recurrence and metastasis.

[0138] This application also used FCM to quantify the proportion of CD3+CD8+ T cells in the re-invading tumor; these cells are the main effector immune cells that kill cancer cells. Figure 8 As shown in E and 8F, the FCM results indicated that CD3+CD8+ T cells were most significantly increased in mice previously treated with MF@SOR (Example 1) (38.97% ± 2%), followed by mice treated with MSN@SOR (Comparative Example 4) (28.43% ± 1.72%), SOR (Comparative Example 3) (23.37% ± 1.09%), MSN (Comparative Example 1) (16.03% ± 1.08%), and the control group (8.03% ± 0.81%), consistent with the results from the aforementioned primary tumor model. These results demonstrate that MF@SOR (Example 1) can generate durable immune memory and elicit a robust immune response against tumor recurrence and metastasis.

[0139] In summary, MF@SOR in Example 1 activated two pyroptosis pathways (classical and non-classical) and the cGAS-STING pathway, effectively triggering a systemic anti-tumor immune response, demonstrating long-term immune memory protection, alleviating immunosuppression in the tumor region, and playing a role in reshaping the tumor's immune microenvironment.

Claims

1. A drug-loaded nanovaccine, characterized in that: The invention comprises MIL-100 (Fe) and a manganese-doped mesoporous silica support, wherein the MIL-100 (Fe) is coated on the outer surface of the manganese-doped mesoporous silica support and the manganese-doped mesoporous silica support is loaded with sorafenib. The preparation method of the drug-loaded nanovaccine, namely the preparation of MF@SOR, includes the following steps, where M refers to MSN, which is the abbreviation for manganese-doped mesoporous silica in this application; F refers to MIL-100 (Fe); and SOR is the abbreviation for sorafenib. Step 1: Preparation of non-porous silica; Step 2: MSN Preparation (1) Dissolve MnCl2·4H2O and NH4Cl, then add SiO2 nanoparticle aqueous solution and NH3·H2O to the mixture, and then transfer the mixture to the reaction vessel for reaction; (2) The product obtained after the reaction was washed and centrifuged to obtain MSN nanoparticles; Step 3: Preparation of MSN@SOR (1) MSN nanoparticles were dispersed in a mixed solution of ethanol and 3-aminopropyltriethoxysilane, stirred, washed and centrifuged to obtain MSN-NH2 nanoparticles. (2) Disperse MSN-NH2 nanoparticles in a mixed solution of ethanol, then completely dissolve succinic anhydride in DMF and add it to the above ethanol solution, and stir; then centrifuge the mixture, collect the precipitate and wash it to obtain MSN-COOH nanoparticles; (3) Disperse MSN-COOH nanoparticles in ethanol containing sorafenib, stir, centrifuge the mixture, collect the precipitate and wash it to obtain MSN@SOR nanoparticles; Step 4: Preparation of MF@SOR (1) Dissolve the prepared MSN@SOR nanoparticles in water, then stir with FeCl3 ethanol solution, then add H3BTC ethanol solution and stir again; (2) The product MF@SOR can be obtained by centrifugation and washing.

2. The drug-loaded nanovaccine as described in claim 1, characterized in that: The volume ratio of ethanol and 3-aminopropyltriethoxysilane in step two (1) is 50:

1.

3. The drug-loaded nanovaccine as described in claim 2, characterized in that: The stirring time in step 2 (2) is 8-14 h.

4. The drug-loaded nanovaccine as described in claim 3, characterized in that: The stirring time in step 2 (3) is 6-12 h; the stirring speed is 200-250 rpm.

5. The drug-loaded nanovaccine as described in claim 4, characterized in that: In step 3 (1), the stirring speed for the first stirring is 1000-1500 rpm.

6. The drug-loaded nanovaccine as described in claim 5, characterized in that: The second stirring time in step 3 (1) is 1.5-2 hours.

7. The drug-loaded nanovaccine as described in claim 6, characterized in that: The concentration of the FeCl3 ethanol solution mentioned in step 3 (1) is 7.5-8.5 mg / mL. −1 The concentration of the H3BTC ethanol solution is 8-12 mg / mL. −1 .

8. The use of the drug-loaded nanovaccine according to any one of claims 1-7 in the preparation of a drug for treating liver cancer.

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