Synthesis and application of a nano-drug delivery system MPL@ICC

Abstract

This invention discloses a nanoparticle-based drug delivery system, MPL@ICC, its synthesis method, and its applications. The MPL@ICC system consists of hollow, porous nano-MnO2, a hydrophilic targeting complex on its surface, and an acid-responsive drug, INH-CA, and a photosensitizer loaded internally. It possesses both targeted transport and controlled release capabilities against human hepatocellular carcinoma cells (HepG2), allowing it to accumulate within these cells and achieve photodynamic therapy (PDT) and free radical-induced immune killing within the tumor. Simultaneously, it directly kills tumor cells and transforms them from non-immunogenic to immunogenic, mediating an anti-tumor immune response and achieving immunogenic cell death (ICD). This overcomes the shortcomings of ICD-induced cancer treatment strategies, which often fail to produce strong and durable therapeutic effects, and does not induce normal cell apoptosis, thus possessing controllability, effectiveness, and safety.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology and relates to a novel targeted nanodrug delivery platform MPL@ICC and its application in cancer treatment. Specifically, it can directly kill tumor cells through PDT and free radical induction, and can also achieve ICD through Mn(III). Background Technology

[0002] Tumor immunotherapy is a cancer treatment strategy that inhibits tumor growth and recurrence by stimulating the immune system. Inducing immunogenic cell death is one of the most promising tumor immunotherapy methods because it can not only directly destroy cancer cells, but also trigger an anti-tumor immune response for long-term treatment.

[0003] Currently, there are many types of drugs for tumor immunotherapy, and their mechanisms of action vary. For example, some nanomaterials can reduce reactions with the physiological environment and prolong circulation time in the body, thus aiding in tumor treatment. Other nanomaterials can improve the tumor cell microenvironment (TME), which is beneficial for the development of intracellular drug delivery systems (ICDs).

[0004] Manganese oxide nanomaterials (MONs) have been reported (Binbin Ding, et al.) via ferroptosis and Mn 2+ The Fenton-like response mediated by MONs induces ICD. Simultaneously, as an immune adjuvant, MONs possess advantages such as strong immunogenicity, regulation of the innate immunosuppressive tumor microenvironment, and biodegradability, which can enhance the immune response induced by nanomedicines. However, Mn 2+ Fenton-like responses have low efficacy and require large amounts of reactants, which may lead to insufficient efficacy of immunotherapy.

[0005] Mn(III) is a strong oxidizing agent that can activate various organic compounds to form ·R, such as isoniazid (INH), which is currently the most widely used anti-tuberculosis drug. Cheng et al. (Yaru Cheng, et al.) utilized Mn... 2+ By leveraging the ability of isoniazid (INH) to generate highly reactive hydroxyl radicals (·OH) and the photothermal effect of WSSe nanosheets, a synergistic anticancer WSSe / MnO2-INH nanocomposite was developed. The study demonstrated the ability of INH to induce ·OH formation and the related Fenton-like reaction mechanism in the presence of manganese, as well as the excellent photothermal conversion efficiency of the WSSe / MnO2 nanocomposite. However, during the process of loading INH with a large amount of MnO2, MnO2 reacts with INH, leading to the destruction of MnO2 and affecting the therapeutic effect of the nanoparticles. Therefore, it is necessary to improve the shortcomings in the preparation process of the combined system.

[0006] Furthermore, the role of carbon-centered radicals (·R) in amplifying ICDs has been noted. For example, the combination of ·R-generating reagent azobisisobutyrazoline hydrochloride (AIPH) with photothermal therapy (PTT) (Bo Ning et al.) can enhance tumor immunotherapy by triggering ·R bursts. However, research on the role of ·R in MONs immunotherapy is currently lacking.

[0007] Photodynamic therapy (PDT), as a rapid tumor ablation therapy, is one of the best candidates for constructing curative tumor nanovaccines. However, the high GSH content and hypoxia in the tumor microenvironment (TME) limit the effectiveness of PDT. Increased hypoxia after PDT treatment not only leads to enhanced immunosuppression but also causes rapid tumor recurrence. Although tumor markers (MONs) can assist PDT-induced ICD by consuming excess GSH and catalyzing the degradation of hydrogen peroxide (H2O2) to generate oxygen (O2), the immunogenicity induced by PDT alone is insufficient.

[0008] The following are the relevant documents retrieved by the applicant:

[0009] [1] Binbin Ding, Pan Zheng, Fan Jiang, Yajie Zhao, Meifang Wang, Mengyu Chang, Ping'an Ma, and Jun Lin. MnO x Nanospikes as Nanoadjuvants and ImmunogenicCell Death Drugs with Enhanced Antitumor Immunity and Antimetastatic Effect [J]. Angewandte Chemie. International Ed., 2020, 59:16381–16384.

[0010] [2]Yaru Cheng, Fan Yang, Kai Zhang, Yiyi Zhang, Yu Cao, Conghui Liu, Huiting Lu, Haifeng Dong, and Xueji Zhang. Non-Fenton-Type Hydroxyl RadicalGeneration and Photothermal Effect by Mitochondria-Targeted WSSe / MnO2Nanocomposite Loaded with Isoniazid for Synergistic Anticancer Treatment[J].Advanced Functional Materials, 2019, 1903850:1–12.

[0011] [3]Bo Ning, Yao Liu, Boshu Ouyang, Xiaomin Su, Huishu Guo, Zhiqing Pang, Shun Shen. Low-temperature Photothermal Irradiation Triggers Alkyl RadicalsBurst for Potentiating Cancer Immunotherapy[J]. Journal of Colloid and Interface Science, 2022, 614:436–450. Summary of the Invention

[0012] In view of the advantages and shortcomings of the above-mentioned tumor immunotherapy strategies, the purpose of this invention is to develop a nano-drug delivery system MPL@ICC that combines PDT and Mn(III)-triggered anti-tumor immunotherapy. This nano-drug delivery system MPL@ICC integrates the advantages of the above-mentioned tumor immunotherapy strategies, eliminates and inhibits the shortcomings of the prior art, and is of great significance to tumor immunotherapy.

[0013] To achieve the above objectives, the present invention employs the following technical solution:

[0014] A drug delivery nanoparticle system MPL@ICC, characterized in that the prepared drug delivery nanoparticle system MPL@ICC consists of the following components:

[0015] (1) Hollow porous nano-MnO2 spheres;

[0016] (2) A cationic hydrophilic complex modified on the surface of nano-MnO2 spheres; the cationic hydrophilic complex modified on the surface of nano-MnO2 spheres uses amino-functionalized columnar [6] aromatics as the host molecule and pyridine lactose as the guest molecule, and the sugar-functionalized columnar [6] aromatic cationic hydrophilic complex is obtained through host-guest interaction; the weight ratio of the cationic hydrophilic complex to the MnO2 nanospheres is 1-1.2:0.8-1.2;

[0017] (3) The photosensitizer Ce6 loaded in the hollow microspheres and the isoniazid protected by cinnamaldehyde have loading weight ratios of 30-40% and 15-20% in the MPL@ICC nano-drug delivery system, respectively.

[0018] The synthesis method of the above-mentioned nano-drug delivery system MPL@ICC is characterized by the following steps:

[0019] Step 1: Preparation of HMnO2

[0020] TEOS was added dropwise to a mixed solution of ethanol, RO water, and ammonia water and stirred to synthesize sSiO2. The volume ratio of TEOS, ethanol, RO water, and ammonia water was 1:20-30:3-5:0.8-1.5. After washing, the mixture was vacuum dried. The sSiO2 suspension was then added dropwise to a KMnO4 aqueous solution and sonicated to synthesize KMnO4-coated sSiO2, i.e., sSiO2@MnO2. The sSiO2 suspension and KMnO4 aqueous solution were mixed in a 1:1 ratio, with a mass concentration ratio of 1:10-20. The nanoparticles were collected by centrifugation, washed with water, and then dispersed in an alkaline aqueous solution at a certain temperature. HMnO2 was collected by stirring and centrifugation. The resulting HMnO2 water was washed multiple times, dispersed in water, and stored at 2-8℃.

[0021] Step 2: Preparation of MPL

[0022] After synthesizing amino-functionalized column[6]arene AMP6 and pyridine lactose LacPy, amino-functionalized column[6]arene AMP6 was used as the host molecule and pyridine lactose LacPy was used as the guest molecule to prepare a sugar-functionalized column[6]arene cationic hydrophilic complex through host-guest interaction; the volume ratio of the hydrophilic complex solution to the HMnO2 solution was 1:0.5-1.5, and the concentration of both was 2 mg / mL. The hydrophilic complex solution and the HMnO2 solution were mixed, stirred and centrifuged to obtain MPL.

[0023] Step 3: Preparation of the MPL@ICC nanocarrier system

[0024] After mixing the photosensitizer Ce6 solution and INH-CA solution in equal volumes and at a 1:1 mass ratio, the mixture was loaded into empty MPL spheres, stirred, and the mass ratio of photosensitizer Ce6 and INH-CA to MPL in the mixed solution was controlled to be 1:1:0.5-1.0, thus obtaining the nano-drug-loaded system MPL@ICC.

[0025] According to the present invention, the hollow porous nano-MnO2 spheres are uniform nano-MnO2 spheres with a particle size of 100-300nm and an internal diameter of 80-280nm; the spheres have multiple pores with a diameter of 10-20nm connected inside and outside.

[0026] Furthermore, the hollow porous nano-MnO2 spheres have a diameter of 170 nm, an inner diameter of 150 nm, and pores inside and outside the spheres with a diameter of 15 nm.

[0027] Specifically, the weight ratio of the cationic hydrophilic complex modified on the surface of the MnO2 nanospheres to the MnO2 nanospheres is 1:1.

[0028] The photosensitizer Ce6 and cinnamaldehyde-protected isoniazid were loaded at weight ratios of 32% and 17% in the MPL@ICC nanoparticle drug delivery system, respectively.

[0029] Preferably, in the preparation of HMnO2, the volume ratio of TEOS, ethanol, RO water and ammonia solution is 1:28:4:1; the sSiO2 suspension and KMnO4 aqueous solution are mixed in a 1:1 ratio, and their mass concentration ratio is 1:15.

[0030] The process involves dispersing sSiO2@MnO2 into an alkaline aqueous solution at a certain temperature, wherein the alkaline substance refers to one or more of Na2CO3, NaHCO3, and NaOH.

[0031] In the preparation of MPL, the volume ratio of the hydrophilic complex solution to the HMnO2 solution is 1:1.

[0032] In the preparation of MPL@ICC, the photosensitizer Ce6 solution and INH-CA solution were mixed in equal volumes and at the same mass concentration (1:1), and then loaded into empty MPL spheres. The mass ratio of photosensitizer Ce6 and INH-CA to MPL in the mixed solution was controlled to be 1:1:0.9.

[0033] The applicant's experiments demonstrate that the MPL@ICC nanoparticle drug delivery system of this invention achieves targeted drug enrichment in cancer cells. The photosensitizers Ce6 and INH-CA loaded inside HMnO2 are released and, via PDT and Mn(III), trigger the immune system to kill tumors. Furthermore, ICD can be achieved through Mn(III). This enables immunotherapy for tumors.

[0034] The MPL@ICC nanoparticle drug delivery system of this invention overcomes the drawback of immunogenic cell death (ICD)-induced cancer treatment strategies, which struggle to produce potent and durable therapeutic effects. For the first time, it successfully utilizes Mn(III)-mediated ·R to generate durable ICDs for anti-tumor immunotherapy, providing a novel and inventive approach for highly efficient cancer therapeutic nanovaccines. Due to the use of biocompatible materials and reliance on photodynamic therapy and immunogenic cell death, MPL@ICC exhibits excellent biocompatibility, not inducing normal cell apoptosis, thus combining controllability, efficacy, and safety. It provides a new strategy for designing highly efficient ICD-based cancer therapeutic nanovaccines, possessing significant potential for immunotherapy of tumors. The technological innovations compared to existing technologies are:

[0035] 1. MPL@ICC nanoparticles have the ability to target liver cancer cells and can accumulate in cancer cells. The materials used have good biocompatibility and are not toxic to normal cells.

[0036] 2. The prepared MPL@ICC nanoparticles exhibit uniform particle size and good stability. In tumor cells, they can continuously consume GSH and alleviate cellular hypoxia, while also rapidly releasing INH-CA and the photosensitizer Ce6. Furthermore, the released O2 enhances the PDT-induced ICD effect. This method combines the advantages of several single ICD schemes in the comparative literature while avoiding their disadvantages, demonstrating solid theoretical justification and significant practical results.

[0037] 3. The MPL@ICC nanodrug delivery system has an ingenious structural design. The nano-MnO2 spheres are loaded with INH-CA and require continuous consumption of GSH to release INH-CA. It can also provide O2 for PDT. It is an enhanced sustained-release and controlled-release formulation that overcomes the shortcomings of immunogenic cell death (ICD)-induced cancer treatment strategies that are difficult to produce strong and lasting efficacy.

[0038] 4. After injection of the MPL@ICC nanoparticle drug delivery system, light-treated mouse hepatocellular carcinoma cells (H22) produced ICDs, which induced an immune response in mice, inhibiting and eliminating tumors in H22 cell line hepatocellular carcinoma mice. The tumor-suppressing efficiency was significant, demonstrating the role of the tumor drug delivery system. This ICD strategy and drug preparation method have broad guiding significance for the design, development, and application of anti-hepatocellular carcinoma drugs. Attached Figure Description

[0039] Figure 1 The present invention provides a nano-drug delivery system MPL@ICC and its preparation process flowchart.

[0040] Figure 2 This is a transmission electron microscope image of sSiO2.

[0041] Figure 3This is a transmission electron microscope (TEM) image of sSiO2@MnO2.

[0042] Figure 4 This is a transmission electron microscope image of HMnO2.

[0043] Figure 5 This is a transmission electron microscope image of MPL.

[0044] Figure 6 The particle size distribution of different nanoparticles is shown. The horizontal axis represents particle size, and the vertical axis represents intensity.

[0045] Figure 7 The potentials of different nanoparticles are represented on the x-axis. The x-axis represents the potentials of various particles (sSiO2, sSiO2@MnO2, HMnO2, MPL, MPL@ICC), and the y-axis represents the potentials.

[0046] Figure 8 The stability of MPL@ICC stored at 4°C for 7 days. The horizontal axis represents the number of days, and the vertical axis represents the average particle size.

[0047] Figure 9 The UV-Vis spectra of photosensitizers Ce6, INH-CA, PL, HMnO2, and MPL@ICC are shown. The horizontal axis represents wavelength, and the vertical axis represents intensity.

[0048] Figure 10 The percentage of photosensitizer Ce6 and INH-CA released by MPL@ICC over time under different conditions (pH 7.4, 5.8, and with and without 10 mM GSH). The x-axis represents time, and the y-axis represents the drug release rate.

[0049] Figure 11 The changes in O2 concentration in a 100 μM H2O2 solution after adding different concentrations of HMnO2 are shown. The horizontal axis represents time, and the vertical axis represents dissolved oxygen content.

[0050] Figure 12 ESR spectra of different solutions with DMPO as the spin trap.

[0051] Figure 13 The UV-Vis absorption spectra of methylene blue (MB) after degradation in different solutions at 37℃ are shown.

[0052] Figure 14 The consumption of GSH after adding different concentrations of MPL.

[0053] Figure 15 Confocal fluorescence microscopy images of different cells treated with MPL@ICC and confocal fluorescence microscopy images of HepG2 cells cultured for 3 h under different conditions for hypoxia detection.

[0054] Figure 16 The average fluorescence intensity of confocal fluorescence microscopy images of different cells (I: HL7702+MPL@ICC, II: HepG2+HMnO2@ICC, III: HepG2+LBA+MPL@ICC, IV: (human cervical cancer cells) Hela+MPL@ICC, V: HepG2+MPL@ICC, VI: H22+MPL@ICC).

[0055] Figure 17 Mean fluorescence intensity of HepG2 cells cultured for 3 hours under different conditions for hypoxia detection (I: normoxic, II: hypoxic, III: INH-CA, IV: Ce6, V: MPL, VI: MPL@ICC).

[0056] Figure 18 The effect of MPL treatment time on GSH content in HepG2 cells.

[0057] Figure 19 These are confocal fluorescence microscopy images of ROS generation in the experimental group under 660nm light irradiation or without irradiation.

[0058] Figure 20 The average fluorescence intensity of the confocal fluorescence microscopy image generated by ROS (I: PBS, II: MPL, III: MPL@IC, IV: MPL@ICC, V: MPL@Ce6+ light, VI: MPL@ICC+ light).

[0059] Figure 21 The relative cell viability of HL7702 and HepG2 cells after 24 hours of treatment with different doses of MPL is shown. The x-axis represents drug concentration, and the y-axis represents cell viability.

[0060] Figure 22 The relative cell viability of HepG2 cells after incubation at different drug doses is shown. The x-axis represents drug concentration, and the y-axis represents cell viability.

[0061] Figure 23 Experiments on H22 cells are shown. (a) Image shows CRT exposure on the surface of H22 cells. Scale bar: 50 μm. (b) Image shows extracellular ATP. (c) Image shows HMGB-1 release from H22 tumor cells after various treatments. (*p<0.05, **p<0.01, ***p<0.001), (n=3). (d) Image shows the program for inoculating mice with nanomedicine. (e) Image shows the survival time of mice after being re-attacked by tumor cells (*p<0.05), (n=6). (f) Image shows a photograph of fixed tumor tissue. (Left: necrotic cells, Right: ICD cells).

[0062] Figure 24The results are presented for real-time fluorescence imaging. (a) shows the real-time fluorescence image of H22 tumor-bearing mice after intravenous injection of MPL@ICC. (c) shows the in vitro fluorescence images of major organs and tumors 12 and 24 hours after intravenous injection of MPL@ICC. (b) and (d) show the relative mean fluorescence intensities of (a) and (c), respectively. (e) shows the immunofluorescence images of tumor sections 12 hours after injection of different formulations. (f) shows the relative percentage of the corresponding hypoxic area in the tumor (**p<0.01). Data are expressed as mean ± SD (n=3). Scale bar: 100 μm. (g) shows the pH values ​​of the tumors after intravenous injection of MPL and MPL@ICC.

[0063] Figure 25 The graphs show the changes in tumor volume in different treatment groups. The horizontal axis represents time, and the vertical axis represents tumor volume.

[0064] Figure 26 Tumor weight is represented by different treatment groups. The horizontal axis represents drug concentration, and the vertical axis represents tumor weight. (I: PBS, II: MPL, III: MPL@IC, IV: MPL@ICC, V: Free drug + light, VI: MPL@Ce6 + light, VII: MPL@ICC + light)

[0065] Figure 27 The graph shows the changes in mouse body weight in different treatment groups. The horizontal axis represents the various particle treatment groups, and the vertical axis represents tumor weight.

[0066] Figure 28 Photographs of tumors in mice under different treatment groups. (I: PBS, II: MPL, III: MPL@IC, IV: MPL@ICC, V: Free drug + light, VI: MPL@Ce6 + light, VII: MPL@ICC + light)

[0067] Figure 29 H&E staining of tumor sections after various treatments. (I: PBS, II: MPL, III: MPL@IC, IV: MPL@ICC, V: Free drug + light, VI: MPL@Ce6 + light)

[0068] Figure 30 TUNEL staining for tumor sections after various treatments. (I: PBS, II: MPL, III: MPL@IC, IV: MPL@ICC, V: Free drug + light, VI: MPL@Ce6 + light)

[0069] Figure 31 Figure (a) shows the percentage of CTLs (CD3+) in the tumor and flow cytometry data. + CD8 +(a) The percentage of Treg(CD25) in tumor cells; (b) The figure represents the flow cytometry plot and the percentage of Treg(CD25) in tumor cells. + FoxP3 + ) in CD4 + Percentage in (in the figure: I: PBS, II: MPL, III: MPL@IC, IV: MPL@ICC, V: Free drug + light, VI: MPL@Ce6 + light, VII: MPL@ICC + light)

[0070] Figure 32 Immunofluorescence staining of M1 macrophages and M2 macrophages was performed, with (a) showing M1 macrophages (iNOS) and (b) showing M2 macrophages (CD206). (I: PBS, II: MPL, III: MPL@IC, IV: MPL@ICC, V: Free drug + light, VI: MPL@Ce6 + light, VII: MPL@ICC + light)

[0071] Figure 33 The percentage of CTLs in the tumor. (I: PBS, II: MPL, III: MPL@IC, IV: MPL@ICC, V: Freedrug + light, VI: MPL@Ce6 + light, VII: MPL@ICC + light)

[0072] Figure 34 This represents the percentage of Tregs in the tumor. (I: PBS, II: MPL, III: MPL@IC, IV: MPL@ICC, V: Freedrug + light, VI: MPL@Ce6 + light, VII: MPL@ICC + light)

[0073] Figure 35 Percentage of M1 and M2 macrophages in the tumor. (I: PBS, II: MPL, III: MPL@IC, IV: MPL@ICC, V: Free drug + light, VI: MPL@Ce6 + light, VII: MPL@ICC + light)

[0074] Figure 36 To determine the INF-γ content in mouse tumors using RT-PCR. Data are presented as mean ± SD (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001). (I: PBS, II: MPL, III: MPL@IC, IV: MPL@ICC, V: Free drug + light, VI: MPL@Ce6 + light, VII: MPL@ICC + light)

[0075] Figure 37To determine the TNF-α content in mouse tumors using RT-PCR. Data are presented as mean ± SD (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001). (I: PBS, II: MPL, III: MPL@IC, IV: MPL@ICC, V: Free drug + light, VI: MPL@Ce6 + light, VII: MPL@ICC + light)

[0076] Figure 38 The IL-6 levels in mouse blood were determined by ELISA. Data are presented as mean ± SD (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001). (I: PBS, II: MPL, III: MPL@IC, IV: MPL@ICC, V: Free drug + light, VI: MPL@Ce6 + light, VII: MPL@ICC + light)

[0077] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Detailed Implementation

[0078] See Figure 1 This embodiment presents a nano-drug delivery system MPL@ICC, consisting of hollow porous MnO2 nanospheres and a hydrophilic targeting complex on the surface of the microspheres. It consists of three parts: abbreviated as PL), a photosensitizer loaded inside the microspheres, and isoniazid (INH-CA), an acid-responsive drug protected by cinnamaldehyde.

[0079] PL is a glycofunctionalized columnar aromatic cation hydrophilic complex prepared by host-guest interaction with AMP6 as the host molecule and LacPy as the guest molecule. The weight ratio of the cation hydrophilic complex to MnO2 nanospheres is 1-1.2:0.8-1.2. It can specifically recognize lactose of the sugar-binding protein on the surface of HepG2, so PL can endow nanoparticles with hydrophilicity and the function of targeting tumor cells.

[0080] The acid-responsive drug isoniazid (INH-CA) was loaded at weight ratios of 30-40% and 15-20% in the MPL@ICC nanoparticle drug delivery system, respectively.

[0081] The hollow porous nano-MnO2 spheres are uniform nano-MnO2 spheres with a particle size of 100-300nm and an internal diameter of 80-280nm; the spheres have multiple interconnected pores with a diameter of 10-20nm inside and outside.

[0082] Preferably, the hollow porous nano-MnO2 spheres have a diameter of 170 nm, an inner diameter of 150 nm, and pore diameters inside and outside the spheres of 15 nm.

[0083] After the MPL@ICC nanoparticle-based drug delivery system enters tumor cells, MnO2 consumes GSH and converts H2O2 into O2. This not only rapidly releases INH-CA and the photosensitizer Ce6 (hereinafter referred to as Ce6), but the released O2 also enhances the PDT-induced ICD effect. Most importantly, the consumption of GSH and H2O2 by MnO2 also raises the pH, thereby altering the tumor microenvironment (TME) to favor the presence of Mn(III). This promotes the generation of INH·R through pH-responsive release of INH-CA at the tumor site mediated by Mn(III), further enhancing the immune death of tumor cells. In other words, the MPL@ICC nanoparticle-based drug delivery system can not only directly kill tumor cells through the combined action of PDT and free radicals, but also achieve PDT- and free radical-induced immune killing within the tumor.

[0084] The synthesis method of the above-mentioned drug delivery system MPL@ICC is carried out according to the following steps:

[0085] Step 1: Preparation of HMnO2

[0086] TEOS was added dropwise to a mixed solution of ethanol, RO water, and ammonia water and stirred to synthesize sSiO2. The volume ratio of TEOS, ethanol, RO water, and ammonia water was 1:20-30:3-5:0.8-1.5. After washing, the mixture was vacuum dried. The sSiO2 suspension was then added dropwise to a KMnO4 aqueous solution and sonicated to synthesize KMnO4-coated sSiO2, i.e., sSiO2@MnO2. The sSiO2 suspension and KMnO4 aqueous solution were mixed in a 1:1 ratio, with a mass concentration ratio of 1:10-20. The nanoparticles were collected by centrifugation, washed with water, and then dispersed in an alkaline aqueous solution at a certain temperature. HMnO2 was collected by stirring and centrifugation. The resulting HMnO2 water was washed multiple times, dispersed in water, and stored at 2-8℃.

[0087] Step 2: Preparation of MPL

[0088] After synthesizing amino-functionalized column[6]arene AMP6 and pyridine lactose LacPy, amino-functionalized column[6]arene AMP6 was used as the host molecule and pyridine lactose LacPy was used as the guest molecule to prepare a sugar-functionalized column[6]arene cationic hydrophilic complex through host-guest interaction; the volume ratio of the hydrophilic complex solution to the HMnO2 solution was 1:0.5-1.5, and the concentration of both was 2 mg / mL. The hydrophilic complex solution and the HMnO2 solution were mixed, stirred and centrifuged to obtain MPL.

[0089] Step 3: Preparation of the MPL@ICC nanocarrier system

[0090] After mixing the photosensitizer Ce6 solution and INH-CA solution in equal volumes and at a 1:1 mass ratio, the mixture was loaded into empty MPL spheres, stirred, and the mass ratio of photosensitizer Ce6 and INH-CA to MPL in the mixed solution was controlled to be 1:1:0.5-1.0, thus obtaining the nano-drug-loaded system MPL@ICC.

[0091] In this embodiment, in the preparation of HMnO2, the volume ratio of TEOS, ethanol, RO water and ammonia solution is 1:28:4:1; the sSiO2 suspension and KMnO4 aqueous solution are mixed in a 1:1 ratio, and their mass concentration ratio is 1:15.

[0092] The sSiO2@MnO2 is dispersed in an alkaline aqueous solution at a certain temperature. The alkaline solution is one or more of Na2CO3, NaHCO3, and NaOH, with NaOH being preferred.

[0093] In the preparation of MPL, the volume ratio of the hydrophilic complex solution to the HMnO2 solution is 1:1.

[0094] In the preparation of MPL@ICC, the photosensitizer Ce6 solution and INH-CA solution were mixed in equal volumes and at the same mass concentration (1:1), and then loaded into empty MPL spheres. The mass ratio of photosensitizer Ce6 and INH-CA to MPL in the mixed solution was controlled to be 1:1:0.9.

[0095] The following are specific embodiments provided by the inventor.

[0096] Example 1: Preparation of MPL@ICC nanoparticles

[0097] It should be noted that experimental methods in the following specific embodiments, where specific conditions are not specified, are generally operated according to methods known in the art. For the experimental results involved in the embodiments, please refer to [link / reference needed]. Figures 2 to 38 .

[0098] Step 1: Preparation of sSiO2

[0099] 5 mL of TEOS was added dropwise to a 45°C mixed solution consisting of 140 mL of ethanol, 20 mL of water, and 5 mL of ammonia, and stirred for 3 hours. After the reaction was complete, sSiO2 was collected by centrifugation (12000 r / min, 10 min), washed three times with ethanol, and dried under vacuum.

[0100] Step 2: Preparation of sSiO2@MnO2: A suspension of sSiO2 (40 mg, 10 mL) was added dropwise to an aqueous solution of KMnO4 (300 mg, 10 mL), and the mixture was sonicated for 6 h to synthesize KMnO4-coated sSiO2 (sSiO2@MnO2). The nanoparticles were collected by centrifugation (10000 r / min, 10 min) and washed three times with water.

[0101] Step 3: Preparation of HMnO2

[0102] sSiO2@MnO2 was dispersed in a 2M Na2CO3 aqueous solution at 60℃, stirred for 12 hours, and HMnO2 was collected by centrifugation. The resulting HMnO2 water was washed several times, finally dispersed in water, and stored at 2-8℃.

[0103] Step 4: Synthesis of AMP6 (Compound 4)

[0104] The synthesis route is shown below:

[0105]

[0106] Synthesis of Compound 1: Hydroquinone (5.0 g, 45.5 mmol) and tetrabutylammonium bromide (0.84 g, 2.4 mmol) were added to ethanol (10 mL) and stirred at room temperature for 30 min. Then, 1,2-dibromoethane (80 mL, 0.94 mol) was added, and the mixture was heated under nitrogen protection and refluxed for 14 h. The reaction mixture was cooled to room temperature, and the precipitate was removed by filtration. The solvent was removed under reduced pressure, and the product was purified by column chromatography (eluent: PE:DCM = 1:1, v / v). Compound 1 was a white solid (7.510 g, 51%).

[0107] Synthesis of Compound 2: Compound 1 (500 mg, 1.54 mmol), paraformaldehyde (232 mg, 7.71 mmol), and ferric chloride (80 mg, 0.99 mmol) were added to chloroform (23 mL), and the mixture was heated to 45 °C. The mixture was cooled to room temperature, washed three times with water, and the organic phase was concentrated and subjected to column chromatography (PE / DCM / EA, 2:1:0.06). Compound 2 was finally obtained as a yellow solid (57.2 mg, 11%).

[0108] Synthesis of Compound 3: Under argon protection, sodium azide (100 mg, 1.54 mmol) was added to an anhydrous N,N-dimethylformamide (3 mL) solution of Compound 2 (220 mg, 0.09 mmol). After stirring at 100 °C for 12 hours, the mixture was cooled to room temperature and poured into water (30 mL). The precipitate was collected by filtration and washed with water to give Compound 3 as a yellow solid (156.8 mg, 92%).

[0109] Synthesis of compound 4: A suspension of compound 3 (40.65 mg, 0.026 mmol) and Pd / C (5%, 18 mg) was stirred in methanol at 50 °C under hydrogen for 48 hours. The resulting mixture was filtered, and the filtrate was concentrated under vacuum to give compound 4 as a pale brown solid (32 mg, 98%).

[0110] Step 5: Synthesis of LacPy (Compound 8):

[0111] The synthesis route is shown below:

[0112]

[0113] Synthesis of Compound 5: D-lactose monohydrate (4.82 g, 13.4 mmol) and iodine (0.247 g, 0.97 mmol) were mixed in acetic anhydride (30 mL). The mixture was stirred at room temperature for 24 hours. After the reaction was complete, the mixture was diluted with dichloromethane (100 mL) and washed with a saturated aqueous solution of Na₂CO₃. Evaporation under reduced pressure yielded Compound 5 (10.7 g, 99%) as a white powder.

[0114] Synthesis of Compound 6: Compound 5 (1.5 g, 2.21 mmol) and 2-(2-(2-chloroethoxy)ethoxy)ethanol (1.1 g, 6.63 mmol) were dissolved in dry CH2Cl2 (18.4 mL) under a nitrogen atmosphere. The mixture was cooled to 0 °C, and BF3·Et2O (1 mL, 8.29 mmol) was slowly added. The reaction was stirred overnight at room temperature. After the reaction was complete, the mixture was washed twice with saturated NaHCO3 solution, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. Column chromatography (EA / PE = 2:3–3:2, v / v) yielded Compound 6 (0.89 g, 1.134 mmol, 51%) as a white foam.

[0115] Synthesis of Compound 7: Compound 6 (207 mg, 0.263 mmol) was dissolved in 8 mL of pyridine. The solution was refluxed under nitrogen atmosphere for 12 hours, followed by concentration under reduced pressure. The crude product was purified by column chromatography (PE / EA = 1:3) to give Compound 7 as a yellow oil (125 mg, 55%).

[0116] Synthesis of Compound 8: Sodium methoxide (108 mg, 2 mmol) was added to a solution of Compound 7 (173 mg, 0.2 mmol, 10 mL MeOH). The mixture was stirred at room temperature for 4 hours, and then an ion exchange resin (Amberlite IR 120H) was added. + Neutralized to pH 7, filtered, and the solvent was removed under reduced pressure. Compound 8 was obtained as a yellow oil (103 mg, 92%).

[0117] Step 6: Synthesis of compound INH-CA

[0118] Isoniazid (274.4 mg, 2 mmol) was added to a 10 mL flask. Under anhydrous and nitrogen protection, ethanol (3 mL) and cinnamaldehyde (317.2 mg) were added. The mixture was stirred at room temperature until a milky yellow precipitate formed. The product was collected by filtration and washed with petroleum ether and water. INH-CA (260.1 mg, 52%) was obtained by vacuum drying.

[0119] Step 7: Preparation of MPL

[0120] Mix 10 mL of HMnO2 (2 mg / mL) solution with 10 mL of PL (2 mg / mL) solution. Stir for 12 hours, then centrifuge (10000 r / min, 10 min) to collect MPL.

[0121] Loading of INH-CA and Ce6: A 1 mg / mL MPL solution was mixed with Ce6 (1.1 mg / mL) and INH-CA (1.1 mg / mL) and stirred for 12 hours. Ce6 and INH-CA were co-loaded into MPL to obtain MPL@ICC for further experiments.

[0122] Example 2: Morphology, particle size and potential characterization of MPL@ICC

[0123] 1) Appearance characterization of MPL@ICC

[0124] The morphology of the nanoparticles was characterized using transmission electron microscopy (TEM, FEI Tecnai F20, acceleration voltage of 200 kV).

[0125] 2) Particle size characterization of MPL@ICC

[0126] The ultraviolet-visible spectrum was recorded at 298K using a Shimadzu 1750 UV-Vis spectrophotometer.

[0127] 3) Potential characterization of MPL@ICC

[0128] The size distribution and zeta potential of the nanoparticles were determined using a Malvem zetasizer (ZEN3600, Malvem, UK).

[0129] Example 3: Titration method for determining the MnO2 content in MPL

[0130] Add 200 mg of dried MPL, 1 g of KI, and 50 mL of water to an Erlenmeyer flask. After the KI is completely dissolved, add 10 mL of 6 M HCl and shake well until the MPL is completely dissolved. Titrate rapidly with 0.1006 M Na₂S₂O₃ solution until the solution in the Erlenmeyer flask turns pale yellow. Then add 3 mL of 0.5% starch solution and continue titrating until the blue color disappears. The reaction is as follows:

[0131] MnO2+4HCl+2KI→MnCl2+l2+2KCl+2H2O

[0132] I₂ + 2Na₂S₂O₃ → 2NaAl + Na₂S₄O₆

[0133] Example 4: Drug Release Study

[0134] To investigate the release of Ce6 and INH-CA, MPL@ICC solution (1 mg / mL) was dialyzed with PBS under different conditions (pH 7.4, 5.8, and 5.8 + 10 mg MSH) at room temperature. The release amounts of Ce6 and INH-CA at different time points were determined by UV-Vis spectroscopy.

[0135] Example 5: H2O2 degrades to produce oxygen

[0136] 200 mL of 100 μM H2O2 solution was sealed with liquid paraffin, and HMnO2 nanoparticles of different concentrations were added. The changes in dissolved oxygen concentration in the H2O2 solution were measured using an oxygen probe (JPBJ-608, Shanghai REX Instruments Factory).

[0137] Example 6: In vitro free radical generation

[0138] A PBS buffer solution containing 10 μg / mL MB, 8 mM H2O2, 8 mM INH, and 2 mM HMnO2 was incubated at 37 °C for 24 hours, and the degradation of MB was monitored by UV-Vis absorption spectroscopy. ESR studies used spin traps of 100 mM DMPO, 8 mM H2O2, 8 mM INH, and 2 mM HMnO2.

[0139] Example 7: In vitro cell experiments

[0140] The four human normal hepatocyte cell lines used in the experiment—HL7702, HepG2, H22, and Hela—were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture. Unless otherwise specified, all cells were incubated at 37°C and 5% CO2.

[0141] Example 8: Intracellular uptake of MPL@ICC or HMnO2@IC / Ce6

[0142] HepG2, HL7702, H22 and HeLa cells were cultured in 35 mm cell culture dishes (1×10⁻⁶ cells / mL). 5Cells were cultured in medium (cells / plate) for 24 hours, then incubated for another 3 hours with MPL@ICC or HMnO2@IC / Ce6 (30 μg / mL). For LBA arrest, cells were transferred to fresh medium containing 4 mM LBA and arrested for 6 hours prior to the arrest. After fixation with 4% paraformaldehyde solution (Yuanye Biotechnology Co., Ltd.), cells were stained with Hoechst 33258 (Solepro). The fluorescence of the photosensitizer Ce6 (Ex / Em: 660 / 700-750 nm) was observed using a confocal laser scanning microscope (CLSM). Cells were washed three times with PBS at each step.

[0143] Example 9: Detection of Intracellular Hypoxia

[0144] HepG2 cells were cultured in 35 mm cell culture dishes (1×10⁻⁶ cells / mL). 5 Cells were cultured in dishes (cells / plate) for 24 hours. Except for the normoxic group, all other groups were blocked with liquid paraffin. The hypoxic groups received no further treatment, but were treated with INH-CA (9 μg / mL), Ce6 (28 μM), MPL, and MPL@ICC (MPL dose 25 μg / mL), respectively. Hypoxia levels after 3 hours of cell culture were assessed using the Hypoxyprobe Plus Kit (Hypoxyprobe, Inc.) according to the manufacturer's instructions. Nuclear staining was performed using Hoechst 33258, and fluorescence was observed using the CLSM method.

[0145] Example 10: Intracellular ROS Generation

[0146] Intracellular ROS were detected according to the indicator. Simply put, H22 cells were cultured in 6-well plates (1 × 10⁻⁶). 6 Cultured in / wells, and treated with PBS, MPL, MPL@IC, MPL@Ce6, and MPL@ICC (INH-CA = 9 μg / mL, Ce6 = 28 μM, MPL = 25 μg / mL) for 4 hours, respectively, and then with or without 660 nm light (5 mW / cm²). 2 After irradiation for 30 minutes (5 min), the cells were stained with DCFH-DA (10 μM, Solarbio Science & Technology Co., Ltd.) and finally imaged with CLSM.

[0147] Example 11: For biocompatibility testing

[0148] HL7702 cells were seeded in 96-well plates (5 × 10⁶ cells per well). 3 Cells were incubated with different concentrations of MPL or MPL@ICC for 24 hours until adherence was achieved. Cell viability relative to untreated cells was measured using the MTT assay.

[0149] Example 12: Used for in vitro combined therapy

[0150] HepG2 cells were seeded in 96-well plates (5 × 10⁶ cells per well). 3 Cells were cultured in wells for 24 hours. After cell adhesion, they were incubated for 4 hours under different conditions, washed three times with PBS, and then transferred to fresh culture medium. Cells were then exposed to 660 nm light (5 mW / cm²) with or without irradiation. 2 After treatment (30 min), the cells were incubated for another 24 hours, and then MTT assay was performed to determine relative cell viability.

[0151] Example 13: CRT expression, release of ATP and HMGB-1

[0152] Immunofluorescence staining was used to detect CRT exposure on the surface of H22 tumor cells, and the changes in ICD after MPL@ICC+ light treatment were evaluated. After incubation for 3 hours with PBS, MPL, MPL@ICC, free drug, MPL@Ce6, or MPL@ICC (dosage INH-CA1 = 9 μg / mL, Ce6 = 28 μM, MPL = 25 μg / mL), the cells were treated with light at 660 nm (2 mW / cm²). 2 Cells were exposed to light for 3 minutes, then incubated for another 3 hours. After centrifugation and washing twice with PBS, cell smears were prepared. Simultaneously, culture medium was carefully collected into test tubes for HMGB-1 assay. Cells were treated with anti-CALR primary antibody (1:200 dilution, Solarbio Science & Technology Co., Ltd.) and Alexa Fluor488-labeled goat anti-rabbit secondary antibody (1:500 dilution, Beyotime). Nuclear staining was performed using Hoechst 33258, and fluorescence was observed using the CLSM method. HMGB-1 was measured using the HMGB-1 ELISA kit (Enzyme-Label Biotechnology Co., Ltd.) according to the manufacturer's instructions. ATP assay was performed using light exposure and continuous incubation on ice, with the remaining steps identical. Extracellular ATP was measured using the ATP assay kit (Beyotime) on a fluorometer (GloMax 20 / 20) according to the manufacturer's protocol.

[0153] Example 14: Animal Model

[0154] Female Balb / c mice (6-8 weeks old) were purchased from Xi'an Yifengda Biotechnology Co., Ltd., and the procedure was carried out according to the protocol approved by the National Laboratory Animal Center. H22 cells (5 × 10⁻⁶) were used. 5 The solution was suspended in 100 μL of PBS and subcutaneously injected into the back of mice. Mice carrying H22 tumors showed improvement when the tumor volume reached 100–200 mm². 3 Receive treatment promptly.

[0155] 1) In vivo imaging

[0156] The tumor volume reached 200 mm 3 Subsequently, MPL@ICC (100 μL, Ce6 78 μg / mL) was injected intravenously. Fluorescence imaging was performed using an in vivo imaging system (IVIS Lumina II, PerkinElmer) at pre-set time points (0, 2, 4, 6, 12, 24, 36, 48 hours). At the end of the experiment, tumors and major organs were obtained for fluorescence imaging (Ce6, Ex: 650 nm, Em: 700–750 nm). Semi-quantitative biodistribution analysis was performed using fluorescence methods.

[0157] 2) Detection of hypoxia in the body

[0158] H22 tumor-bearing mice were injected intravenously with PBS, free drug, and MPL@ICC 12 hours prior to treatment. Tumors were surgically removed 90 minutes after intraperitoneal injection of pipemnidazole (60 mg / kg) (Hypoxyprobe Inc.). Tumor tissue was frozen in liquid nitrogen, embedded in OCT (Sakura, Japan), and sectioned. To detect pipemnidazole, tumor sections were incubated with FITC-conjugated anti-pipermnidazole primary antibody (1:200 dilution, Hypoxyprobe Inc.). Cell nuclei were stained with DAPI (1:5000 dilution, Leagenebiotechnology). The obtained sections were observed using the CLSM method.

[0159] 3) Immunological effects of combined therapy

[0160] H22 tumor-bearing mice were randomly divided into 7 groups and intravenously injected with 100 μL PBS, MPL, Free drug + Light, MPL@IC, MPL@ICC, MPL@Ce6 + Light, and MPL@ICC + Light (MnO2 = 9 mg / kg, Ce6 = 7.8 mg / kg, INH-CA = 4.1 mg / kg). 12 hours after injection (0.4 W / cm²), the tumor response was... 2Mice were irradiated with 660 nm light for 5 min. Five days after irradiation, mice were sacrificed, and blood was collected for IL-6 measurement using ELISA (Enzyme-Label Biotechnology Co., Ltd.). Tumors were also collected for immunological evaluation. For flow cytometry, tumor tissue was cut into small pieces and placed in a glass homogenizer containing PBS solution (pH 7.4, with 1% FBS added). Single-cell suspensions were prepared by gentle pressure homogenization without the addition of digestive enzymes. Subsequently, before removing red blood cells (RBCs) using erythrocyte lysis buffer, cells were stained with fluorescently labeled antibodies according to the manufacturer's protocol. To assess regulatory T cells (Tregs), cells were stained with anti-mouse CD4-FITC, anti-mouse CD25-APC, and anti-mouse FoxP3-PE (Multisciences) antibodies. Simultaneously, cytotoxic T lymphocyte (CTL) infiltration was stained with anti-mouse CD3-APC and anti-mouse CD8-PE (Multisciences). + CD25 + FoxP3 + and CD3 + CD8 + The cells were defined as Treg and CTL, respectively.

[0161] For the detection of M1 / M2 macrophages, tumors were fixed with Bouin's fixative and prepared into paraffin sections. The sections were dewaxed with xylene, boiled with citric acid-EDTA antigen extraction buffer (Beyotime), and then labeled with antibodies. M1 macrophages were labeled with NOS2 polyclonal antibody (immunobiotechnology) and Alex 488 conjugated with rabbit anti-secondary antibody (1:200 dilution, Beyotime), and M1 macrophages were labeled with CD206 polyclonal antibody (immunobiotechnology).

[0162] The above description of the embodiments is provided to enable those skilled in the art to deeply understand and apply the present invention, and the present invention is not limited to the above embodiments. Those skilled in the art can readily make various modifications to these embodiments and apply the general principles of this description to other embodiments without expending a great deal of creative effort. Therefore, any additions and substitutions made to the technical solutions of the present invention by those skilled in the art based on the technical prompts of the present invention are all within the protection scope of the present invention.

Claims

1. A method for synthesizing a nanoparticle-based drug delivery system MPL@ICC, characterized in that, Follow these steps: Step 1: Preparation of HMnO2 TEOS was added dropwise to a mixed solution of ethanol, RO water, and ammonia water and stirred to synthesize sSiO2. The volume ratio of TEOS, ethanol, RO water, and ammonia water was 1:20-30:3-5:0.8-1.

5. After washing, the solution was vacuum dried. The sSiO2 was then made into a suspension and added dropwise to a KMnO4 aqueous solution and sonicated to synthesize MnO2-coated sSiO2. 2， That is, sSiO2@MnO2, wherein the sSiO2 suspension and KMnO4 aqueous solution are mixed in a 1:1 ratio, and their mass concentration ratio is 1:10-20; the nanoparticles are collected by centrifugation, washed with water, and then the sSiO2@MnO2 is dispersed in an alkaline aqueous solution at a certain temperature, and HMnO2 is collected by stirring and centrifugation; the obtained HMnO2 is washed multiple times, finally dispersed in water, and stored at 2-8℃; Step 2: Synthesis of AMP6 The synthetic route of AMP6 is shown below: ； The AMP6 is compound 4; Step 3: LacPy Synthesis The synthetic route for LacPy is shown below: ； The LacPy is compound 8; Step 4: Synthesis of compound INH-CA Isoniazid was added to a 10 mL flask, and ethanol and cinnamaldehyde were added under anhydrous and nitrogen protection. The mixture was stirred at room temperature until a milky yellow precipitate was formed. The precipitate was collected by filtration, washed with petroleum ether and water, and dried under vacuum to obtain INH-CA. Step 5: Preparation of MPL After synthesizing amino-functionalized columnar aromatic hydrocarbon AMP6 and pyridine lactose LacPy, amino-functionalized columnar aromatic hydrocarbon AMP6 was used as the host molecule and pyridine lactose LacPy was used as the guest molecule to prepare a sugar-functionalized columnar aromatic hydrocarbon cationic hydrophilic complex through host-guest interaction; the volume ratio of the hydrophilic complex solution to the HMnO2 solution was 1:0.5-1.5, and the concentration of both was 2 mg / mL. The hydrophilic complex solution and the HMnO2 solution were mixed, stirred and centrifuged to obtain MPL; Step 6: Preparation of the MPL@ICC nanocarrier system After mixing the photosensitizer Ce6 solution and INH-CA solution in equal volumes and at a 1:1 mass ratio, the mixture was loaded into empty MPL spheres, stirred, and the mass ratio of photosensitizer Ce6 and INH-CA to MPL in the mixed solution was controlled to be 1:1:0.5-1.0, thus obtaining the nano-drug-loaded system MPL@ICC.

2. The synthesis method according to claim 1, characterized in that, In the preparation of HMnO2, the volume ratio of TEOS, ethanol, RO water and ammonia solution is 1:28:4:1; the sSiO2 suspension and KMnO4 aqueous solution are mixed in a 1:1 ratio, and their mass concentration ratio is 1:

15. The process involves dispersing sSiO2@MnO2 into an alkaline aqueous solution at a certain temperature, wherein the alkaline substance refers to one or more of Na2CO3, NaHCO3, and NaOH.

3. The synthesis method as described in claim 1, characterized in that, In the preparation of MPL, the volume ratio of the hydrophilic complex solution to the HMnO2 solution is 1:

1. In the preparation of MPL@ICC, the photosensitizer Ce6 solution and INH-CA solution were mixed in equal volumes and at the same mass concentration (1:1), and then loaded into empty MPL spheres. The mass ratio of photosensitizer Ce6 and INH-CA to MPL in the mixed solution was controlled to be 1:1:0.

9.

4. The MPL@ICC nanoparticle drug delivery system prepared by the synthesis method according to any one of claims 1 to 3, characterized in that, The prepared drug delivery nanoparticle system MPL@ICC consists of the following components: (1) Hollow porous nano-MnO2 spheres; (2) A cationic hydrophilic complex modified on the surface of nano-MnO2 spheres; the cationic hydrophilic complex modified on the surface of nano-MnO2 spheres uses amino-functionalized columnar [6] aromatic hydrocarbons as the host molecule and pyridine lactose as the guest molecule, and the sugar-functionalized columnar [6] aromatic hydrocarbon cationic hydrophilic complex is obtained through host-guest interaction; the weight ratio of the cationic hydrophilic complex to the MnO2 nanospheres is 1-1.2:0.8-1.2; (3) The photosensitizer Ce6 loaded in the hollow microspheres and the isoniazid protected by cinnamaldehyde have loading weight ratios of 30-40% and 15-20% in the nano-drug delivery system MPL@ICC, respectively.

5. The MPL@ICC nanoparticle drug delivery system as described in claim 4, characterized in that, The hollow porous nano-MnO2 spheres are uniform nano-MnO2 spheres with a particle size of 100-300 nm and an internal diameter of 80-280 nm; the spheres have multiple interconnected pores with a diameter of 10-20 nm on the inside and outside.

6. The MPL@ICC nanoparticle drug delivery system as described in claim 5, characterized in that, The hollow porous nano-MnO2 spheres have a diameter of 170 nm, an inner diameter of 150 nm, and pores inside and outside the spheres with a diameter of 15 nm.

7. The MPL@ICC nanoparticle drug delivery system as described in claim 4, characterized in that, The cationic hydrophilic complex modified on the surface of the MnO2 nanospheres has a weight ratio of 1:1 to the MnO2 nanospheres.

8. The MPL@ICC nanoparticle drug delivery system as described in claim 4, characterized in that, The photosensitizer Ce6 and the isoniazid protected by cinnamaldehyde have loading weight ratios of 32% and 17% in the MPL@ICC nanocarrier system, respectively.

9. The application of the MPL@ICC nanocarrier system as described in claim 4 in the preparation of drugs for treating liver cancer.

10. The application as described in claim 9, characterized in that, The photosensitizers Ce6 and INH-CA loaded inside HMnO2 directly kill tumor cells through PDT and free radical induction, and achieve ICD through Mn(III).

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