Liposome composite material for enhancing breast cancer radioimmunotherapy as well as preparation method and application of liposome composite material

Abstract

The invention discloses a liposome composite material for enhancing breast cancer radioimmunotherapy as well as a preparation method and application of the liposome composite material. The liposome composite material is prepared by encapsulating RudZ and MnB in a lipid phase and a water phase through folic acid (FA) modified liposome (Lipo), wherein MnB refers to MnO2 nanoparticles, RudZ refers to cis-[Ru (DIP) 2dppz] < 2 + > Cl2, and the structural formula of the RudZ is as follows: # imgabs0 #. The RuII-MnIV-based liposome composite material has the advantages that under the combined irradiation of 4Gy X-rays, the RudZ is Cis-[Ru (DIP) 2dppz] < 2 + > Cl2; according to the present invention, active breast cancer targeting, fluorescence imaging tracing, active oxygen generation promotion, tumor microenvironment oxygen deficit relieving, mitochondrial DNA release caused by mitochondrial injury, and cell cycle G2 / M period retardation can be achieved, and the cGAS-STING immune signal channel can be double activated in cooperation with Mn < 2 + > release, such that the liposome composite material has the breast cancer radioimmunotherapy effect enhancing function.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a liposome composite material for enhancing radioimmunotherapy of breast cancer, and a preparation method and application thereof. Background Art

[0002] Radiotherapy is one of the most commonly used and effective means of treating malignant tumors in clinical practice. It is the preferred method for treating breast cancer before and after breast cancer surgery and in the middle and late stages. However, there are still two major bottlenecks that greatly limit the effectiveness of radiotherapy. First, water molecules in cancer tissues rarely produce hydroxyl radicals through ionizing radiation. This is because the hydrogen-oxygen bond energy of water molecules is high. Therefore, at lower radiation doses, water molecules rarely produce free radicals through radiation. While high doses of X-rays improve the effect of radiotherapy, they also have greater toxic side effects and immunosuppressive effects on nearby normal tissues during treatment. Second, the hypoxic tumor microenvironment greatly reduces the efficacy of radiotherapy on tumor tissues, because the hypoxic state is conducive to the repair of DNA damaged by radiotherapy, thus producing radiotherapy resistance. Secondly, for some tumors that have already metastasized, radiotherapy is often helpless. Therefore, in order to improve the effectiveness of radiotherapy, people have proposed strategies such as using high atomic number metals, increasing the production of reactive oxygen during radiotherapy, consuming glutathione in tumor cells, improving the hypoxic microenvironment of tumors, and cell cycle arrest to enhance radiotherapy sensitization. However, the lack of effective delivery specifically for radiosensitizers has resulted in the inability of radiosensitizers to target tumor sites, thus limiting their clinical application. Therefore, in order to solve the problems of radiotherapy resistance and targeting and improve the efficacy of radiotherapy, designing a targeted delivery carrier for radiosensitizers is an urgent and important clinical need.

[0003] In recent years, with the continuous development of tumor immunology, people's understanding of tumors and the immune system has gradually deepened, and immunotherapy has become a hot topic in current research. At present, it is generally believed that radiotherapy can stimulate systemic immune responses and has distal effects, which provides a strong theoretical basis for radioimmunity. Considering that the cGAS-STING signaling pathway plays an important role in various pathological and physiological processes such as autoimmunity and tumors, activating the cGAS-STING innate immune signaling pathway is an effective radioimmunotherapy strategy. Radiotherapy can lead to the release of proinflammatory cytokines through the cGAS-STING immune signaling pathway, and the anti-tumor effect of mitochondria on immunity is also crucial. Recent studies have shown that mitochondria are also closely related to the occurrence and development of immune responses. Mitochondrial DNA can cause the activation of the cGAS-STING immune signaling pathway and induce anti-tumor immunity. Therefore, it is urgent to design a radiosensitizer that can be targeted to tumor mitochondria and effectively activate the cGAS-STING immune signaling pathway to enhance breast cancer radioimmunotherapy. Summary of the invention

[0004] The purpose of the present invention is to provide a Ru-based method for enhancing radioimmunotherapy of breast cancer. II -Mn IV The nanoliposome composite material is as follows:

[0005] A liposome composite material for enhancing radioimmunotherapy of breast cancer, wherein the liposome composite material is prepared by encapsulating RudZ and MnB in a lipid phase and an aqueous phase respectively through liposomes modified with folic acid; MnB is MnO2 nanoparticles; RudZ is cis-[Ru(DIP)2dppz] 2+ Cl2, the structural formula is as follows:

[0006]

[0007] The present invention also provides a method for preparing the above-mentioned liposome composite material for enhancing radioimmunotherapy of breast cancer, comprising the following steps:

[0008] DPPC, DSPE-PEG 3400 -COOH, CHO and DSPE-PEG 3400 -FA were dissolved in chloroform / methanol mixed solvent, and ultrasonicated to fully dissolve, then chloroform / methanol mixed solvent was added to the eggplant-shaped bottle, and then DPPC and DSPE-PEG with a molar ratio of 60:10:10:(5-10) were added. 3400 -COOH, CHO, DSPE-PEG 3400 -FA forms folic acid-modified liposomes, and then RudZ is added for ultrasonic treatment, and then the residual solvent is removed by rotary evaporation, and the obtained lipid membrane is hydrated with MnB to prepare a uniform liposome composite material.

[0009] Preferably, the drug-lipid ratio of RudZ and folic acid modified liposomes is 1:(10-20).

[0010] Preferably, the concentration of RudZ is 2-4 μM, and the [Mn] concentration of MnB is 10-20 μg / mL.

[0011] Preferably, the preparation method of RudZ is as follows:

[0012] (1) Synthesis of dppz ligand:

[0013] Use anhydrous ethanol to prepare an o-phenylenediamine solution and a 1,10-phenanthroline-5,6-dione solution with a molar ratio of 13.868:(7.135-10.125), slowly add the o-phenylenediamine solution to the 1,10-phenanthroline-5,6-dione solution while stirring, heat to boiling in a warm water bath, boil for 5 minutes, cool in an ice water bath, crystallize, filter, and recrystallize to obtain a brown needle-shaped dppz ligand;

[0014] (2) Synthesis of cis-Ru(DIP)2Cl2:

[0015] Dissolve 4,7-diphenyl-1,10-phenanthroline and ruthenium chloride hydrate in a molar ratio of 3.06:(2.13-3.26) in DMF under N2 protection and mix, heat to 151°C-181°C and condense under reflux for 8 hours, and terminate the reaction when the solution turns purple. After cooling, add acetone and stir, store at -20°C overnight, filter, wash and vacuum dry the precipitate to obtain cis-Ru(DIP)2Cl2;

[0016] (3) Synthesis of cis-[Ru(DIP)2dppz] 2+ Cl2:

[0017] The cis-Ru(DIP)2Cl2 and dppz ligand with a molar ratio of 0.375:(0.718-1.536) were dissolved in DMF and mixed under N2 protection, heated to 151°C-181°C and condensed under reflux for 8-10h. The reaction was terminated when the solution turned reddish brown. After cooling, DMF was removed by vacuum at 75°C and 100rpm. The reddish brown metal complex cis-[Ru(DIP)2dppz] was obtained by elution purification and vacuum drying. 2+ Cl2.

[0018] Preferably, the preparation method of MnB is as follows:

[0019] Add BSA and MnCl2·4H2O in a mass ratio of (1-5):1 into a hydrothermal tube, stir for 10 min in an oil bath at 37°C-40°C, adjust the pH to 9.0-11.0, continue stirring and reacting for 2-4 h, and then dialyze for 24 h to purify to obtain MnB.

[0020] The present invention also provides the use of a liposome composite material for enhancing radioimmunotherapy of breast cancer prepared by the above preparation method in the preparation of a drug for increasing the generation of active oxygen in the process of tumor radiotherapy.

[0021] The present invention also provides the use of a liposome composite material for enhancing radioimmunotherapy of breast cancer prepared by the above preparation method in the preparation of a drug for improving hypoxia in the tumor microenvironment.

[0022] The present invention also provides a liposome composite material for enhancing breast cancer radioimmunotherapy prepared by the above preparation method, and its use in preparing a drug that targets breast cancer mitochondria, cooperates with high-energy ionizing radiation to cause mitochondrial DNA oxidative damage, and activates the cGAS-STING innate immune signaling pathway.

[0023] Preferably, the liposome composite material induces a strong anti-tumor immune response while being used for radiosensitization.

[0024] Compared with the prior art, the advantages of the present invention are:

[0025] (1) The present invention is based on Ru II -Mn IV The liposome composite material, under combined 4Gy X-ray irradiation, can achieve active targeting of breast cancer, fluorescence imaging tracing, promote the generation of reactive oxygen species, alleviate hypoxia in the tumor microenvironment, damage mitochondria to cause mitochondrial DNA release, arrest the cell cycle G2 / M phase, and dual activate the cGAS-STING immune signaling pathway. These characteristics enable the liposome composite material to enhance the radioimmunotherapy of breast cancer.

[0026] (2) The present invention uses an ultrasonic thin film dispersion method and liposomes (Lipo) as a loading and assembly medium to encapsulate RudZ and MnB in a lipid phase and an aqueous phase, respectively, and constructs a liposome composite material RMLF by modifying folic acid (FA) with a function of targeting the folate receptor highly expressed on the surface of breast cancer. This liposome has good biocompatibility in vivo and in vitro, can be quickly taken up by 4T1 cells, and can promote the generation of reactive oxygen species during radiotherapy in conjunction with 4Gy X-rays. It can also catalyze endogenous hydrogen peroxide in the tumor to generate oxygen, thereby improving the hypoxia of the tumor microenvironment. In addition, RMLF combined with X-rays can cause an increase in γ-H2AX foci and downregulate HIF-1α expression, block the cell cycle of tumor cells in the G2 / M phase sensitive to radiotherapy, show the highest level of apoptosis rate, and significantly inhibit cell proliferation. The synergistic effect of RudZ and MnB effectively inhibits cell division. The synergistic sensitization of RudZ and MnB and the active targeting effect of modified folic acid ultimately enhance the therapeutic effect of radiotherapy and play a role in radiosensitization.

[0027] (3) Subcellular localization showed that RMLF targeting mitochondria caused mitochondrial damage and promoted the escape of mitochondrial DNA into the cytoplasm. The mitochondrial DNA in the cytoplasm could serve as a damage-associated molecular pattern, activate the cGAS-STING immune signaling pathway, and induce anti-tumor immune response. In addition, the escaped mitochondrial DNA could improve the immunosuppressive tumor microenvironment and promote the polarization of tumor-associated macrophages from M2 to M1.

[0028] (4) By injection into the tail vein of mice, RMLF showed effective tumor targeting, good biocompatibility, and up-regulated γ-H2AX and down-regulated HIF-1α in the tumor, indicating that RMLF can effectively alleviate the hypoxia of the tumor microenvironment and enhance the radiation damage of tumor cell DNA, thereby playing its role in radiosensitization. RMLF combined with radiotherapy can induce the maturation of dendritic cells and polarization of tumor-associated macrophages, induce cell apoptosis, and produce Mn 2+ and mitochondrial DNA can dually activate the cGAS-STING immune signaling pathway and significantly increase CD8 + T cells and CD4 + The proportion of T cells and the long-term effect of induced anti-tumor immunity can effectively inhibit the growth, recurrence and metastasis of primary tumors. This shows that RMLF can significantly improve the effect of breast cancer radiotherapy and also has the effect of long-term immune memory. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The synthesis process of RMLF and its induced mitochondrial DNA damage and Mn 2+ Schematic diagram of radioimmunotherapy for breast cancer achieved by release.

[0030] Figure 2 Synthetic route of RudZ.

[0031] Figure 3 Characterization of RMLF and MnB; wherein, (a) X-ray diffraction (XRD) patterns of MnB and RMLF nanoparticles; (b, c) X-ray photoelectron spectra of MnB nanoparticles; (d, e, f) X-ray photoelectron spectra of RMLF.

[0032] Figure 4 Characterization of RMLF; (a) encapsulation efficiency and drug loading of RL, RML and RMLF (the inset is the standard curve of RudZ, EE: Encapsulation efficiency is the encapsulation efficiency, LC: loading of drug is the drug loading); (b) particle size of RMLF at different time points in PBS, RPMI 1640 and RPMI 1640+10% fetal bovine serum (FBS); (c) measurement of the release of RudZ in RMLF at 37°C using PBS buffer solution (pH=5.5) containing 2% (w / v) SDS.

[0033] Figure 5Characterization of RMLF; (a, b) are transmission electron microscopy (TEM) images of MnB and RL, respectively; (c, d) are transmission electron microscopy (TEM) images of RMLF at different sizes, respectively; (e, f, g) are particle size distribution, polydispersity coefficient analysis and Zeta potential distribution of Lipo, MnB, RL, RML and RMLF, respectively; (h) energy dispersive spectrum (EDX) of RMLF; (i) UV-visible absorption spectra of different components.

[0034] Figure 6 Typical oxygen generation kinetic curves of RL, RML, RMLF and MnB nanoparticles in the presence of H2O2.

[0035] Figure 7 In vitro cytotoxicity, cellular uptake, in vitro targeting, reactive oxygen species generation, and relief of hypoxia in the tumor microenvironment; Fig. 7A Viability of 4T1 cells treated with (a) RudZ (b) MnB (c) RL, RML and RMLF (d) CDDP (e) H2O2 at different concentrations. Figure 7B (a) Quantitative analysis of fluorescence intensity at different uptake times was performed using ImageJ; Figure 7B (b) The in vitro uptake of RudZ and different liposome samples by 4T1 cells was analyzed by inverted fluorescence microscopy, and their fluorescence intensity was quantitatively analyzed using ImageJ. Figure 7C (a) The ROS production of 4T1 cells co-cultured with RudZ, MnB and RMLF or the control group was detected. The fluorescence intensity of DCF was correlated with the ROS production. The concentration of RudZ was 2 μM, [Mn] = 10 μg / mL, and the scale bar represents 5 μm; (b) Image J was used to quantitatively analyze the DCF fluorescence intensity of more than 100 cells in each group.

[0036] FIG8 shows the radiosensitizing activity of RMLF in vitro; Fig. 8A (a) Cell cycle analysis of 4T1 cells after different treatments was performed by flow cytometry; (b) Flow cytometric analysis of 4T1 cells after different treatments using Annexin V / PI double staining. Figure 8B (a) Optical photographs of the survival rate of clone formation under different treatment groups; (b) Quantitative analysis of the survival rate of clone formation. Figure 8C (a) Cell viability of 4T1 cells treated with RudZ, MnB, RML, RMLF and CDDP, with a radiation dose of 4 Gy, a RudZ / CDDP concentration of 2 μM, [Mn] = 10 μg / mL, and RMLF (RudZ: 2 μM, [Mn] = 10 μg / mL); (b) Cumulative contribution of each RMLF component to the efficacy of radiotherapy.

[0037] Figure 9 RMLF targets mitochondria, damages mitochondria, causes mitochondrial DNA escape, and activates the cGAS-STING immune signaling pathway, thereby generating an anti-tumor immune response; Fig.9A Co-localization images of RudZ, RL and RMLF with MitoTracker Green-stained mitochondria, the scale bar represents 10 μm. Fig. 9B Flow cytometry was used to detect mitochondrial membrane potential of 4T1 cells after different treatments. Fig. 9C RT-PCR analysis was used to examine the damage levels of various regions of mitochondrial DNA (D-loop, ND6, ATP6, and CO I) in 4T1 cells after treatment with different working groups. Fig.9D (a) Using mtTFA rabbit monoclonal antibody to distinguish mtDNA (TFAM-mtTFAGreen) and Hoechst-stained cell nuclei, 4T1 cells were treated with RMLF and then treated with 4Gy X-rays to detect the escape of mitochondrial DNA into the cytoplasm. The scale bar represents 10 μm. (b) qRT-PCR analysis of the escape level of mitochondrial DNA in the supernatant of 4T1 cells after treatment with different working groups. Fig.9E qRT-PCR analysis was used to analyze the relative expression levels of cGAS, STING, NFκB, and IRF3 mRNA in 4T1 cells after different treatments.

[0038] Fig.10 Biocompatibility analysis of RMLF in mice; hemolysis test results of mouse red blood cells in the presence of different concentrations of RMLF samples.

[0039] Fig.11 Fluorescence imaging and in vivo biodistribution of RML and RMLF in mouse organs; (a) ex vivo tissue distribution and tumor aggregation images; (b, c) fluorescence signals of the heart, liver, spleen, lung, kidney and tumor extracted from tumor-bearing mice after treatment with RML or RMLF for different time periods (2, 6, 12 and 24 h).

[0040] Figure 12 shows the in vivo radioimmunotherapy activity of RMLF combined with X-ray; Fig. 12A (a) Relative tumor volume change curve during the treatment of different working groups; (b) Body weight of mice during the treatment of different working groups; (c) Tumor photos at the end of treatment of different working groups; (d) Tumor weight at the end of treatment of different working groups (n=5). Fig. 12B Representative γ-H2AX and HIF-1α immunofluorescence images of tumor tissues at the end of treatment, scale bar represents 50 μm. Fig. 12C(a) The proportion of cells expressing CD80 and CD86 in tumor-draining lymph nodes at the end of treatment with different working groups was quantified by flow cytometry; (b, c) The proportion of cells expressing CD86 and CD206 in tumor tissues at the end of treatment with different working groups was quantified by flow cytometry. Fig.12D The levels of cGAMP and TNF-α, IFN-β and IL-6 cytokines in tumors of different groups of mice were analyzed by ELISA (n=3). Fig.12E Histological analysis of tumors using immunohistochemical staining with TUNEL, CD4, and CD8, scale bar represents 50 μm. Fig.12F HE-stained sections of different tissues (heart, liver, spleen, lung, kidney, and tumor) of mice at the end of treatment. The scale bar represents 40 μm. Figure 12G Representative immunohistochemistry (IHC) images of CD86 and CD206 macrophages in tumor tissues after treatment completion, scale bar represents 40 μm. Fig.12H (a, b, c, d, e, f, g) WBC, RBC, PLT, ALT, AST, BUN, and CREA of mice were detected at the end of treatment.

[0041] Figure 13 Long-term effects of anti-tumor immune response induced by RMLF combined with radiotherapy; Fig.13A (a) Relative tumor volume change curves of different treatment groups during the treatment period; (b) Body weight of mice in different treatment groups during the treatment period; (c) Tumor photos of different treatment groups at the end of treatment; (d) Tumor weight of different treatment groups at the end of treatment (n=5). Fig. 13B Tumor tissues were histologically analyzed using TUNEL, CD4, CD8 immunofluorescence staining and HE staining. Fig. 13C (a, b) Detection of CD8 in spleen of 4T1 tumor-bearing mice + T cells and CD4 + T cell effector memory cells (CD45 + CD3 + CD4 + CD44 + CD62 - and CD45 + CD3 + CD8 + CD44 + CD62 - ) and central memory T cells (CD45 + CD3 + CD4 + CD44 + CD62 + and CD45 + CD3 + CD8 +CD44 + CD62 + ) expression.

[0042] Fig.14 The H NMR spectrum of RudZ ( 1 H-NMR) chart. DETAILED DESCRIPTION

[0043] The disclosure below provides many different embodiments or examples to implement different structures of the present invention. In order to simplify the disclosure of the present invention, the parts and settings of specific examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention. In addition, the present invention can repeat reference numbers and / or reference letters in different examples, and this repetition is for the purpose of simplification and clarity, which itself does not indicate the relationship between the various embodiments and / or settings discussed.

[0044] The present invention is further described with reference to the accompanying drawings and implementation examples.

[0045] 1. Based on Ru II -Mn IV Synthesis and characterization of liposomal composites RMLF

[0046] 1.1cis-[Ru(DIP)2dppz] 2+ Synthesis of Cl2(RudZ)

[0047] A metal ruthenium polypyridine complex has a molecular structure shown in the following formula (I):

[0048]

[0049] The synthetic route of the above-mentioned metal ruthenium polypyridine complex (RudZ) is as follows: Figure 2 , the preparation method comprises the following steps:

[0050] (1) Synthesis of dppz ligand:

[0051] First, weigh 1.4997 g (13.868 mM) of o-phenylenediamine and 1.4997 g (7.135 mM) of 1,10-phenanthroline-5,6-dione respectively into a beaker, add about 30 mL of anhydrous ethanol and heat slightly to completely dissolve it, slowly stir the o-phenylenediamine solution into the 1,10-phenanthroline-5,6-dione solution, and heat to boiling in a warm water bath for about 5 minutes; cool and crystallize in an ice water bath to obtain a reddish-brown solid, filter and recrystallize to obtain a brown-yellow needle-shaped product, namely the dppz ligand.

[0052] (2)cis-Ru(DIP)2Cl2 and cis-[Ru(DIP)2dppz] 2+ Synthesis of Cl2:

[0053] First, 1.0197g (3.06mmol) of 4,7-diphenyl-1,10-phenanthroline (DIP) and 0.4435g (2.13mmol) of ruthenium chloride hydrate (RuCl3·nH2O) were dissolved in 50mL of DMF under the protection of N2, heated to 151°C, and condensed and refluxed for 8h. After the solution turned purple, the reaction was terminated and cooled to room temperature, and 100mL of acetone was added to the above mixture. The mixture was stored in a -20°C refrigerator overnight. The precipitate was then filtered and washed with ultrapure water and ether. Finally, cis-Ru(DIP)2Cl2 was obtained by vacuum drying.

[0054] Then Ru(DIP)2Cl2 (315.7 mg, 0.375 mmol) and dppz ligand (201.2 mg, 0.708 mmol) were dissolved in 50 mL DMF under the protection of N2, heated and condensed under reflux for 8 h, and the solution changed from purple to reddish brown. After the solution was cooled to room temperature, a vacuum oil pump was used to remove DMF at 75 ° C and 100 rpm to obtain a reddish brown crude product. The crude product was purified by silica gel column chromatography using dichloromethane and methanol (CH2Cl2: CH3OH = 10: 1 v / v) as eluents. The reddish brown metal complex cis-[Ru(DIP)2dppz] was obtained by vacuum drying. 2+ Cl2(RudZ).

[0055] Yield of RudZ: 40.5%. 1 H NMR (400MHz, DMSO) δ = 9.69 (d, 2H), 8.56 (d,

[0056] 2H),8.42(d,2H),8.38(m,4H),8.29(m,4H),8.04(d,2H),7.85(s,2H),7.71(m,4H),7.66ppm(m,20H), such as Fig.14 shown.

[0057] 1.2 Synthesis and characterization of MnB nanoparticles

[0058] Add 9mL BSA solution (2mg / mL) and 3.6mL MnCl2·4H2O solution (1mg / mL) to the hydrothermal tube, stir for 10min in an oil bath at 37℃-40℃, adjust the pH to 9.0-11.0, continue stirring and reacting for 2-4h, and then dialyze for 24h for purification to obtain MnB solution, that is, MnO2 nanoparticles made by mineralization with BSA as a biological template. The obtained MnB solution was placed in a freeze dryer for freeze drying and preservation, and the X-ray diffraction spectrum of the freeze-dried sample was obtained by X-ray diffractometer to analyze its structure. Its element content and valence state were analyzed by X-ray photoelectron spectrometer, such as Figure 3 As shown in b and c.

[0059] 1.3 Synthesis and structural characterization of RMLF liposome composites

[0060] (1) RMLF was prepared by thin film ultrasonic dispersion method, specifically by the following steps:

[0061] DPPC, DSPE-PEG 3400 -COOH, CHO (cholesterol) and DSPE-PEG 3400 -FA was dissolved in a mixed solvent of chloroform and methanol (v:v=3:1) and ultrasonically treated for 3 min to fully dissolve the phospholipids. Then, 5 mL of chloroform / methanol mixed solvent was added to a 50 mL eggplant-shaped bottle, and then different volumes of DPPC, DSPE-PEG 3400 -COOH, CHO, DSPE-PEG 3400 -FA (molar ratio 60:10:10:5) and RudZ (2 mg / mL, RuDZ to all phospholipids drug lipid ratio (molar ratio) is 20:1) were ultrasonically treated for 5 min. Then, a rotary evaporator was used to rotate at 45 ° C, 85 rpm for 30 min under reduced pressure to remove the residual mixed solvent. The spin-dried lipid film was hydrated with 5 mL of MnB nanoparticles (1.6 mg / mL) at 45 ° C, 200 rpm under normal pressure, and then a uniform liposome RMLF was prepared by ultrasonic extrusion and other methods at room temperature. The liposomes were then dialyzed with ultrapure water (MW = 3500 Da) to remove free drugs for 24 hours. The purified nanoliposomes were prepared for subsequent use.

[0062] (2) The hydrated particle size, Zeta potential and morphology of RMLF liposomes were investigated by dynamic light scattering and transmission electron microscopy, respectively. The UV-visible spectrum of RMLF was measured by UV spectrophotometer. The X-ray diffraction pattern of the freeze-dried material was obtained by X-ray diffractometer. The elemental composition of RMLF was analyzed by X-ray photoelectron spectrometer and energy dispersive spectrometer. Fluorescence images of RudZ, Lipo and other composite materials were taken by inverted fluorescence microscope. In order to investigate its stability, RMLF was dispersed in different physiological media (PBS, RPMI 1640, RPMI 1640+10% FBS) to investigate the changes in its hydrated particle size within one week.

[0063] (3) in vitro drug release;

[0064] The in vitro drug release pattern of RMLF was studied by dialysis. 1 mL of RMLF sample was placed in a dialysis bag (MW = 3500Da) and the two ends were tied with surgical cotton thread. Then PBS (pH = 5.5) containing 2% (w / v) SDS was used as the release medium to simulate the in vivo environment at 37°C and 110rpm. At the specified time interval, 1 mL of sample was collected for detection (λ = 458nm) and replaced with an equal volume of fresh PBS. The drug release rate at different time intervals was calculated using a UV spectrophotometer and the standard curve of RudZ. The drug release rate was calculated according to the following formula:

[0065]

[0066] Where M is the total amount of drug; C n , C i represents the concentration of RudZ at the nth and ith sampling times; V0, V i Represents the initial volume of the release medium and the volume of the i-th sampling.

[0067] (4) extracorporeal oxygen production level;

[0068] Add 2 mL of sample to a 10 mL EP tube, and then add an appropriate volume of 30% H2O2 solution to make the final H2O2 concentration 1 mM. Use a portable dissolved oxygen meter to record the oxygen concentration every 1 min until the oxygen concentration is balanced.

[0069] 2. Performance testing and application

[0070] 2.1 Materials and methods

[0071] (1) Cell lines and animals

[0072] 4T1 (mouse breast cancer tumor) cell line was cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 1% 10U / mL double antibiotics (penicillin and streptomycin), and in an atmosphere of 5% CO2, 37°C, and 95% relative humidity. According to the growth of the cells, the medium was replaced with new medium every 2 to 3 days, and cells with stable growth and in the logarithmic growth phase were used for the experiment.

[0073] Balb / c mice were raised to 4 to 6 weeks old according to the regulations approved by the Experimental Animal Center of Southern Medical University. 2×10 6 4T1 cells (resuspended in 100 μL PBS buffer solution) were used to establish a breast cancer (4T1) tumor model.

[0074] (2) In vitro cytotoxicity

[0075] 4T1 cells were inoculated in a 96-well cell culture plate, 100 μL (5000 cells per 100 μL) was added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere. Samples of different concentrations (RudZ, MnB, RL (liposome material loaded with RudZ alone), RML (liposome material loaded with RudZ and MnB but not modified with folic acid), RMLF, CDPP, H2O2) were added to the 96-well plate in sequence, and each sample was paralleled to 3 replicates. The control wells were replaced with an equal amount of PBS buffer solution, and continued to be incubated in a 37°C, 5% CO2 cell culture incubator for 24 hours. The culture medium in the 96-well plate was removed, 100 μL MTT solution was added to each well, and the culture was continued for 4 hours. The MTT solution in the well was aspirated, and 100 μL DMSO solution was added to each well to dissolve the formazan crystals formed, and the absorbance value of each well at 490 nm was detected using an ELISA reader. Cell viability was calculated according to the following formula:

[0076] Cell viability (%) = A sample / A negative control × 100%

[0077] Among them, sample A is the absorbance value of 4T1 cells after drug treatment, and negative control A is the absorbance value of 4T1 cells after PBS buffer solution treatment.

[0078] (3) Cellular uptake and targeting verification

[0079] 4T1 cells were cultured at 3 × 10 5The cells were inoculated in a 6-well plate at a density of 100 μM / well and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours to allow the cells to adhere to the wall. Then RMLF (RudZ concentration was 2 μM, [Mn] concentration was 10 μg / mL, and this concentration was used in subsequent experiments) was added to each well, and an equal amount of PBS buffer solution was added as a negative control. The supernatant was discarded at different time points (1, 3, 6, 9, 12 hours), washed 3 times with PBS buffer solution, and then 1 mL of Hoechst 33342 (10 μM) was added to stain the cell nucleus, and the reaction was protected from light for 15 minutes. 1 mL of 4% paraformaldehyde was added to fix for 10 minutes. Finally, the sample was placed under an inverted fluorescence microscope to observe the fluorescence intensity of the cells. Image J was used to statistically and quantitatively analyze the differences in different components of RudZ fluorescence intensity in more than 100 cells.

[0080] In order to verify the active targeting effect of liposome RMLF modified with folic acid, one group of cells was incubated with free folic acid (free FA, 1mM) in a cell culture incubator for 30min before adding the sample. Then, free RudZ and the corresponding composite nanomaterial containing RudZ were added to the relative wells, and the negative control group was replaced with an equal amount of PBS buffer solution. The drug and 4T1 cells were incubated in the cell culture incubator for another 6h, and the supernatant was discarded and washed 3 times with PBS buffer solution. Finally, the sample was placed under an inverted fluorescence microscope to observe the fluorescence intensity of the cells. Image J was used to perform statistical quantitative analysis of the fluorescence intensity of different treatment groups.

[0081] (4) Production of reactive oxygen species in vitro

[0082] 4T1 cells were cultured at 3 × 10 5Cells were inoculated at a density of 100 μM / well in a 6-well plate and incubated for 24 h in an anaerobic bag at 37 °C with 1% O2, 5% CO2 and N2 balance to allow the cells to adhere to the wall. RudZ (RudZ concentration is 2 μM), MnB nanoparticles ([Mn] 10 μg / mL), RMLF (concentration as above) and 100 μM H2O2 were added to each well and incubated with the cells for 12 h. The cells were washed 3 times with PBS buffer solution to remove the unphagocytosed materials. The negative control group was replaced with an equal amount of PBS buffer solution (the experimental conditions at the subsequent cell level were basically the same as above). After washing, 2 mL of DCFH-DA reactive oxygen fluorescent probe (10 μM) was added to the 6-well plate, which was sealed with sealing film and wrapped with tin foil, and subjected to 0 Gy or 4 Gy X-ray treatment. Finally, the medium containing DCFH-DA dye was aspirated, the cells were washed 3 times with PBS buffer solution, and redispersed in PBS buffer solution. The samples were placed under an inverted fluorescence microscope to observe the fluorescence intensity of the cells, and Image J was used to perform statistical quantitative analysis on the DCF fluorescence intensity in more than 100 cells to determine the differences in ROS generation by different components.

[0083] (5) Evaluation of radiosensitization effect

[0084] 4T1 cells were cultured at 1×10 5 Cells were inoculated at a density of 1000 / well in a 12-well plate. Different materials were added to the cells for 12 hours under the same conditions as above. After receiving 0Gy or 4Gy X-ray treatment, they were incubated in a cell culture incubator overnight. The supernatant was then discarded and washed three times with PBS buffer solution. The cells were collected by centrifugation (1000rpm, 3min) using EDTA-free trypsin digestion. 1mL of PBS buffer was added to resuspend the cells, and the cells were added to a centrifuge tube and centrifuged again, and the cell pellet was retained. 1mL of pre-cooled 75% ethanol was added to the cell pellet, blown evenly to a single cell suspension, and fixed in a 4°C refrigerator overnight. Centrifugation (1000rpm, 3min) was used to collect the cells, 500μL of staining buffer and 10μL of iodinated pyridinium (PI) staining solution (20×) were added to each tube, and 10μL of RNaseA (50×) was added. Gently blown to a single cell suspension, and stained at room temperature in the dark for 15-20min. Red fluorescence was detected by flow cytometry at 488 nm, and Flow jo flow analysis software was used to analyze the DNA content and cell cycle of the cells, and Annexin V-FITC and PI staining were used to detect cell apoptosis.

[0085] 4T1 cells were inoculated in 6-well plates at 2000 cells / well, shaken, and different materials were added to the cells for 12 hours under the same conditions as above, and then treated with 0Gy or 4Gy X-rays, and continued to be incubated in a cell culture incubator for 1-2 hours. The supernatant was then discarded, washed 3 times with PBS buffer solution, and continued to be cultured for 7-14 days, with the solution changed every 3 days. Finally, the culture medium was aspirated, washed 3 times with PBS buffer solution, 1mL of 4% paraformaldehyde was added to each well to fix the cells for 15 minutes, the paraformaldehyde was discarded, and the cells were stained with Wright-Giemsa staining solution for 5 minutes, and then washed 3 times with water immersion until the attached dye was washed away, dried for 6 minutes, photographed and counted, and the clone formation rate and survival fraction (survival fraction, SF) were calculated.

[0086] Clone formation rate (%) = number of clones formed in the blank control group / number of cells inoculated in the blank control group × 100%

[0087] Survival fraction (SF) = number of clones formed in a certain dose treatment experimental group / number of inoculated cells × clone formation rate

[0088] (6) Mitochondrial localization, mitochondrial damage, mitochondrial membrane potential changes, mitochondrial DNA damage and escape

[0089] MitoTracker Green mitochondrial green fluorescent probe was used to analyze the co-localization of drugs in 4T1 cells. 4 The cells were inoculated at a density of 100 / well in a confocal dish and incubated for 48 hours at 37°C in an anaerobic bag balanced with 1% O2, 5% CO2 and N2 to allow the cells to adhere to the wall. After adding RudZ (RudZ concentration is 2μM), MnB nanoparticles ([Mn] 10μg / mL), and RMLF (concentration as above) and incubating with the cells for 6 hours, the waste liquid was discarded and the cells were washed 3 times with PBS buffer solution (unless otherwise specified, the experimental conditions at the subsequent cell level were basically the same as above). Then, 1mL Hoechst 33342 (10μM) was used to stain the nucleus, and MitoTracker Green FM (100nM) was used to stain the mitochondria. The two were co-stained for 30 minutes, the dye was discarded, the cells were washed 3 times with PBS buffer solution, and redispersed in PBS buffer solution, and observed on an inverted laser confocal microscope.

[0090] 4T1 cells were cultured at 3 × 10 5The cells were inoculated at a density of 1000 μg / well in a 6-well plate. After co-incubation with different materials and cells according to the above experimental conditions, they were treated with 4Gy X-rays and continued to be incubated in a cell culture incubator overnight. Add an appropriate amount of EDTA-free trypsin to digest the cells for 4 minutes, add 500 μL of full culture to terminate the digestion, transfer to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 3 minutes, discard the supernatant to collect the cells. Then wash twice with PBS buffer solution and discard the supernatant. Add 500 μL of mitochondrial buffer to suspend the cells, add 500 μL of JC-1 staining working solution to stain at 37°C in the dark for 20 minutes, collect the cells by centrifugation and wash twice with pre-cooled JC-1Buffer (1×). Finally, the stained cells were transferred to a flow tube for detection using a flow cytometer, and then analyzed using Flow jo flow analysis software.

[0091] qRT-PCR was used to quantitatively detect the damage and escape of mtDNA after different treatments. 4T1 cells were cultured at 3×10 5 The cells were inoculated at a density of 1000 cells / well in a 6-well plate. After co-incubation with different materials and cells according to the above experimental conditions, they were treated with 4Gy X-rays and continued to be incubated overnight in a cell culture incubator. The cells were collected by trypsin digestion, and DNA was extracted according to the operating procedures of the genomic DNA extraction and purification kit. The obtained DNA was measured for concentration and subjected to subsequent qRT-PCR experiments or stored in a -80°C refrigerator for later use.

[0092] (7) RMLF hemolysis test

[0093] Blood was collected from the mouse orbits and collected in anticoagulant tubes containing 1 mg / mL sodium heparin solution. The whole blood of the mice was then centrifuged at 2000g and 4°C for 10 min, the supernatant was discarded, the serum and red blood cells were separated, and the PBS buffer solution was added and centrifuged and washed 3 times until the supernatant became colorless. Finally, the lower red blood cell pellet was resuspended and dispersed in 1× PBS buffer solution. The RMLF liposome sample was diluted to different concentrations with PBS buffer solution, and then 900μL RMLF sample and 100μL red blood cell suspension were mixed and shaken in a constant temperature air bath shaker at 37°C and 110rpm for 2h. PBS buffer solution was used as the negative control group, and deionized water was used as the positive control group. Then, the mixed solution of different components was centrifuged at 6000g and 4°C for 10min, and the absorbance of the supernatant at 540nm was measured by an enzyme marker. The hemolysis rate was calculated by the following formula:

[0094] Hemolysis rate (%) = (A 540样品 -A 540阴性对照 ) / (A 540阳性对照 -A 540阴性对照 )×100%

[0095] (8) Fluorescence imaging and in vivo biodistribution

[0096] When the tumor volume of mice reached 150 mm 3 At 14:1, 200 μL of RMLF solution (2 mg / kg [RudZ], 16 mg / kg MnB, n=3) was injected into the tail vein of each mouse. The IVIS Lumina III imaging system was used to perform in vitro fluorescence imaging of 4T1 tumor-bearing mice injected with different radiosensitizers at different time points, and the biodistribution of RMLF in mice was observed. The fluorescence excitation wavelength was 500 nm and the emission wavelength was 620 nm.

[0097] (9) In vivo radioimmunotherapy

[0098] For tumor treatment, 4T1 tumor-bearing mice were randomly divided into 7 groups (n = 5 in each group): (1) PBS group, (2) RMLF group, (3) X-ray group, (4) RudZ + X-ray group, (5) MnB + X-ray group, (6) RMLF + X-ray group, and (7) CDDP + X-ray group. When the tumor volume reached 80 mm 3 At 14:1, all mice were injected with various radiosensitizers (2 mg / kg [Rudz], 16 mg / kg MnB) through the tail vein, and then exposed to 4 Gy X-ray irradiation 12 hours later. The treatment cycle was once every 3 days. Then, the length and width of each tumor were measured with a vernier caliper every 2 days, and the weight of the mice was weighed with an electronic analytical balance for a total of 12 days. The relevant indicators were calculated according to the following formula: Tumor volume (V) = ab 2 / 2, where a is the maximum value and b is the minimum value. Relative tumor volume (RTV) = V / V0, where V0 is the initial tumor volume. In order to evaluate the therapeutic effect of each group, the mice were killed on the 12th day after tumor treatment, the tumors were removed and photographed. The excised tumors and major organs were cut into thin slices, and then HE staining, TUNEL analysis, CD4 / CD8 immunofluorescence sections, and CD86 / CD206 immunohistochemistry analysis were performed. In addition, in order to evaluate the biocompatibility of the prepared radiosensitizer, the mouse orbital blood was collected after the treatment, and blood routine and liver and kidney function indexes were analyzed.

[0099] To examine the DNA damage and hypoxia status in the tumor, 24 h after tail vein injection of various radiosensitizers (2 mg / kg [RudZ], 16 mg / kg MnB), the mice were killed and the tumor tissues were collected. The sections were then prepared for γ-H2AX and HIF-1α immunofluorescence staining and CD86 / CD206 immunohistochemical section analysis.

[0100] (10) Flow cytometry analysis of DC cell maturation and TAMs polarization, and ELISA analysis of related cytokine expression

[0101] In order to evaluate the maturation of dendritic cells (DCs) and the phenotypic polarization of macrophages (TAMs) after RMLF combined with radiotherapy, flow cytometry antibody staining analysis was performed. The operation method is as follows: After the treatment, collect tumor tissue or tumor-draining lymph nodes. Wash away the blood stains with PBS buffer solution, cut the tissue with ophthalmic scissors, place the tissue on a nylon mesh, grind it with a syringe, and rinse it with PBS buffer solution to obtain a single cell suspension. Carefully transfer it to a 15mL centrifuge tube and centrifuge it, and discard the supernatant. Add 3mL of red blood cell lysis buffer, pipet evenly, let it stand for 3min, centrifuge it, and discard the supernatant (tumor lymph nodes do not need the step of lysing red blood cells). Add an appropriate amount of 1% BSA solution to resuspend the cells (approximately 2 to 4 million cells / 100μL). Take 100μL of cell suspension in a 1.5mL centrifuge tube, add 5μL of various flow cytometry antibodies in turn, and stain for 30min at 4℃. Finally, the cells were washed twice with 1% BSA solution, dispersed in 400 μL 1% BSA solution, transferred to a flow tube, and detected using a flow cytometer. After treatment, the tumor tissue was added with an appropriate amount of PBS buffer solution and mashed. The supernatant was collected by centrifugation at 3000 rpm for 10 min. The expression of cGAMP, TNF-α, IFN-β, and IL-6 was analyzed by double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA).

[0102] (11) Long-term immune memory experiment

[0103] In order to evaluate the long-term effect of RMLF combined with radiotherapy, the antigen-specific immune memory effect after treatment was further investigated. First, 4T1 cells were inoculated subcutaneously in the right leg of female BALB / c mice to establish a 4T1 tumor-bearing mouse model (primary tumor). According to the above experimental method, the mice were divided into 7 groups, and RMLF was injected into the tail vein on the 1st, 3rd and 5th days, and 4Gy X-ray treatment was given 12h later. On the 7th day, the primary tumor was removed by surgery. After 12 days of treatment, 4T1 cells were inoculated into the contralateral side of the mouse (distal tumor). Then, a long-term monitoring period began, and the tumor volume and mouse weight were measured every 2 days. The mice were killed on the 24th day, and the tumors were taken for photographing and weighing. In addition, secondary tumor tissues were taken, fixed with 4% paraformaldehyde, and HE staining, TUNEL analysis, and CD4 / CD8 immunofluorescence analysis were performed. After the treatment, the spleen of the mice was obtained and prepared into a single cell suspension, which was then subjected to flow cytometry antibody staining analysis. The flow cytometry antibodies used were EV450 Anti-Mouse CD4 Antibody, PerCP / Cyanine5.5 Anti-Mouse CD8a Antibody, APC Anti-Human / Mouse CD44 Antibody, and PE Anti-Mouse CD62L Antibody.

[0104] (12) Statistical analysis

[0105] The statistical significance of the data was evaluated by univariate analysis. The results were expressed as mean ± standard deviation (SD) (n ≥ 3). The difference was considered statistically significant when P < 0.05 and extremely significant when P < 0.01.

[0106] 3. Results Analysis

[0107] 3.1 Characterization of RudZ

[0108] By hydrogen nuclear magnetic resonance spectroscopy ( 1 H-NMR) was used to characterize the structure of the synthesized compound RudZ. Fig.14 ), and RudZ was scanned at full wavelength using an ultraviolet spectrophotometer.

[0109] 3.2 Characterization of MnB and RMLF liposomes

[0110] The structures of the freeze-dried MnB nanoparticles and RMLF were analyzed by XRD. The XRD spectrum of the MnB nanoparticles has an obvious peak at 2θ = 19.08° ( Figure 3 a), which is consistent with the characteristic peak of MnO2 ( Figure 3a-Simulated). The RMLF sample also has a sharp peak at 19.08°, which indicates the presence of MnB nanoparticles in RMLF. Subsequently, the X-ray photoelectron spectroscopy (XPS) analysis of MnB revealed that the two characteristic peaks of Mn in MnB at 641.48 eV and 653.98 eV are respectively attributed to Mn(IV)2p 3 / 2 and Mn(IV)2p 1 / 2 , confirming the formation of MnO2 ( Figure 3 b,c). In addition, two characteristic peaks of Ru and Mn were also detected in RMLF, which also confirmed the existence of RudZ and MnO2 ( Figure 3 d, e). XPS narrow spectrum scanning revealed that the Ru 3d signal was detected at 284.48 eV ( Figure 3 f). These characteristic peaks have a good correspondence with the elements C, O, N, Mn, and Ru, further verifying the successful construction of RMLF.

[0111] The encapsulation efficiency and drug loading of liposomes in different composite materials were measured by UV spectrophotometer. The results showed that the liposome encapsulation efficiency was similar, about 75%. The drug loading was also about 3% ( Figure 4 a). RMLF was dispersed in different physiological media (PBS, RPMI 1640, RPMI 1640 + 10% FBS) to investigate the changes in hydrated particle size within one week ( Figure 4 b). The results showed that under such physiological conditions, the hydrated size of RMLF remained within 200 nm, effectively avoiding the occurrence of aggregation and maintaining its inherent high permeability and retention effect. In order to simulate the physiological environment in vivo, phosphate buffer (pH 5.5) containing 2% (w / v) SDS was selected as the release medium for in vitro drug release, and the dialysis method was used to evaluate the release of RudZ from RMLF ( Figure 4 c). RMLF released only a small amount (28%) of RudZ in the initial 12 h, and the liposomes in the dialysis bag still had an obvious Tyndall effect, which indicated that the polyethylene glycol bilayer protected RMLF from biomolecular interactions and improved the stability of RMLF.

[0112] The morphologies of MnB nanoparticles, RL, and RMLF were observed by TEM. Figure 5 a, b, c, d). TEM images of RMLF at different sizes ( Figure 5 c, d), it can be seen that the MnB nanoparticles are encapsulated in the liposome membrane. These results all prove the successful construction of the composite radiosensitizer RMLF. At the same time, the elements in RMLF were characterized by energy dispersive spectrometer, and the elemental analysis results further confirmed the presence of Ru and Mn in RMLF ( Figure 5h). DLS was used to measure the nanosize, surface charge and polydispersity index (PDI) of MnB nanoparticles and different composites. Figure 5 e, f, g). As shown in the figure, the average hydrated particle size and Zeta potential of MnB nanoparticles are 40.81nm and -14.23mV, respectively. The average hydrated particle sizes of Lipo, RL, RML, and RMLF are 121.1nm, 98.07nm, 112.4nm, and 137.7nm, respectively; the Zeta potentials are -25.8mV, -12.56mV, -31.03mV, and -36.70mV, respectively. The UV-visible spectra of RL, RML, and RMLF show that RudZ and MnB nanoparticles are successfully encapsulated in the liposome membrane ( Figure 5 i).

[0113] 3.3 Performance Testing

[0114] (1) Extracorporeal oxygen production level

[0115] In order to determine whether the constructed composite material RMLF can catalyze hydrogen peroxide to produce oxygen, the tumor environment in vivo was simulated and the catalytic oxygen production capacity of MnB nanoparticles, RL, RML, and RMLF was studied ( Figure 6 ). Compared with RL without MnB loading, RML and RMLF loaded with MnB have better oxygen production capacity; in addition, compared with MnB nanoparticles, the oxygen release rate is slightly slower after being encapsulated by liposomes, which indicates that liposome encapsulation can play a role in slowly releasing oxygen. The generated oxygen can regulate the hypoxic microenvironment of the tumor, thereby fixing the DNA damage caused by radiotherapy and playing a role in radiotherapy sensitization.

[0116] (2) In vitro cytotoxicity

[0117] Before any in vitro and in vivo experiments were performed, the cytotoxicity of RudZ and MnB nanoparticles was first evaluated. RudZ alone was not very toxic to 4T1 cells, and when the RudZ concentration was 8 μM, the cell viability was still close to 80% ( Fig. 7A -a), the toxicity is significantly lower than that of cisplatin (CDDP) at the same concentration ( Fig. 7A -d). In addition, even high concentrations of MnB nanoparticles ([Mn] 20 μg / mL) did not cause significant effects on 4T1 cells, showing good biocompatibility ( Fig. 7A -b). Then, the cytotoxicity of RL, RML, and RMLF on 4T1 cells was investigated in turn. When the concentration of RudZ in RMLF reached 4 μM and [Mn] reached 20 μg / mL, it did not cause obvious toxicity to 4T1 cells ( Fig. 7A-c). In addition, in order to better simulate the tumor microenvironment, a small amount of H2O2 was added in some cell experiments. Therefore, the cytotoxicity of different concentrations of H2O2 was also evaluated ( Fig. 7A -e). The results showed that when the concentration of H2O2 was 100 μM, it had little effect on 4T1 cells. Therefore, the concentration of H2O2 added in subsequent in vitro cell experiments was selected to be 100 μM.

[0118] (3) Cellular uptake and targeting verification

[0119] The red fluorescence of RudZ was observed by inverted fluorescence microscopy to evaluate the cellular uptake of different composite materials. After RMLF and 4T1 cells were incubated for 6 hours, the cell uptake was almost saturated, which means that at the 6-hour time point, the drug can enter the cells well. The fluorescence intensity quantitative experiment further verified this experimental result. Figure 7B -a). In order to further verify the effect of liposome encapsulation and subsequent folic acid functionalization on the endocytosis of RudZ, the cellular uptake of RudZ, RL, RML, RMLF and RMLF+free FA was investigated after incubation with 4T1 cells for 6 h, and the fluorescence intensity was quantitatively analyzed ( Figure 7B -b). As shown in the figure, after encapsulating liposomes, the uptake of RudZ increased significantly. When liposomes were further modified with folic acid (FA), the uptake of liposomes was further increased due to its active targeting effect. In addition, free folic acid was used to block the folate receptors on the surface of tumor cells, and it was found that after treatment under the same uptake time conditions, its uptake was significantly reduced, which further illustrates that the modification of folic acid plays an irreplaceable role in active targeting.

[0120] (4) Promote the generation of reactive oxygen species

[0121] The reactive oxygen species fluorescent probe DCFH-DA was used to detect the changes in intracellular ROS content. Figure 7C -a,b), DCFH-DA itself does not have fluorescent properties, but after entering the cell, it is cut by intracellular esterase and cannot be transported out of the cell again. Under the oxidation of intracellular ROS, DCF that can emit green fluorescence is formed. Therefore, the fluorescence intensity of DCF is positively correlated with the intracellular ROS content. At the same concentration and the same culture conditions, compared with the negative control group and the single X-ray radiotherapy group (4Gy), the ROS generated by RudZ at a concentration of 2μM combined with X-rays was significantly increased. In contrast, MnB nanoparticles combined with X-rays hardly promoted the production of ROS. The DCF fluorescence intensity produced by RMLF combined with X-ray treatment was the strongest among all groups, which may be because the encapsulation of liposomes and the modification of folic acid greatly increased the tumor's uptake of drugs, thereby further increasing the yield of ROS. Quantitative analysis of fluorescence intensity also further verified this experimental result.

[0122] (5) Cell cycle and apoptosis analysis

[0123] In order to determine whether the DNA damage caused by RMLF would cause cell cycle arrest in 4T1 cells, flow cytometry was used to detect the changes in the cell cycle after different treatment groups. Fig. 8A -a shows that compared with the negative control group, the drug-only group and the X-ray group, the proportion of G2 / M phase increased after RudZ and MnB nanoparticles combined with 4Gy X-ray treatment. When 4T1 cells were treated with RMLF combined with X-rays, the proportion of G2 / M phase increased significantly, and the proportion of S phase decreased significantly. Tumor cells in G2 / M phase were more sensitive to radiotherapy, and RMLF combined with X-rays could play a role in radiosensitization. At the same time, cell apoptosis experiments showed ( Fig. 8A -b), compared with the negative control group, 4T1 cells did not show obvious apoptosis in the RMLF treatment group and the X-ray treatment group alone. However, the highest apoptosis rate was observed after RMLF combined with X-ray treatment.

[0124] (6) Cloning and cytotoxicity after radiotherapy

[0125] The ability of RMLF to enhance radiotherapy sensitivity at the cellular level was verified by cell cloning. After 4T1 cells were incubated with different groups of drugs, they were irradiated with 4Gy X-rays. After 7-10 days of culture, the proliferation of cells after different treatments was observed. Compared with cells not incubated with materials, the proliferation of 4T1 cells incubated with different radiosensitizers was inhibited to a certain extent after 4Gy X-ray irradiation ( Figure 8B -a). It is worth noting that the RMLF+X-ray group showed the least number of cell clones, cell proliferation was significantly inhibited, and its survival score (SF) was the lowest, just less than 20% ( Figure 8B -b). This may be due to the synergistic effect of RudZ and MnB nanoparticles, which effectively inhibits cell division.

[0126] The toxicity of RMLF combined with 4Gy X-ray treatment on 4T1 cells was investigated and compared with the effect of CDDP, a commonly used radiosensitizer in clinical practice. Figure 8C -a shows that after 4Gy X-ray irradiation, the cell viability of 4T1 cells co-incubated with RMLF was the lowest, only less than 40%, which was significantly better than the sensitization effect of CDDP. This may be due to the synergistic sensitization of RudZ and MnB nanoparticles and the active targeting effect of modified folic acid, which ultimately enhanced the therapeutic effect of radiotherapy. Subsequently, the different components in RMLF were analyzed, and the results are shown in Figure 8C-b, it can be found that the contribution rates of RudZ and MnB nanoparticles account for 39.02% and 40.59%, respectively, while the effects of 4Gy X-rays alone and folic acid are relatively small.

[0127] (7) Mitochondrial localization, mitochondrial damage, mitochondrial membrane potential changes, mitochondrial DNA damage and escape

[0128] like Fig.9A As shown in the figure, the subcellular organelle localization experiment shows that RudZ can be localized in mitochondria, but its colocalization coefficient is low, only 0.338, which may be caused by the low amount of drug entering the cell. Interestingly, after being encapsulated by liposomes, the drug uptake increased, and the colocalization coefficient of RL increased to 0.474. When folic acid was further modified on the liposomes, it can be clearly seen that due to the active targeting effect of RMLF, its colocalization coefficient was the highest, 0.727. The constructed liposome composite material RMLF (red fluorescence) colocalized well with MitoTracker Green (green fluorescence), and the red and green overlapped to a large extent. The overlapping part of the cell showed yellow fluorescence as a whole. Therefore, it can be inferred that after the cell takes up RMLF, a large part of the drug entering the cell acts on the mitochondria, thereby generating more reactive oxygen species during radiotherapy.

[0129] The JC-1 probe was used to detect changes in Δψm after administration of radiosensitizers. JC-1 dye aggregates in the matrix of normal mitochondria and emits red fluorescence. In contrast, in unhealthy or damaged mitochondria, JC-1 monomers are localized in the mitochondrial matrix at low concentrations, thereby producing green fluorescence. Fig. 9B As shown, RMLF administration alone and radiotherapy alone had no significant effect on the JC-1 signal. However, when 4T1 cells were treated with RudZ and 4Gy X-rays, the membrane potential depolarized due to the ROS produced, and the cells showed a weak red signal. After exposure to 4Gy X-rays combined with RMLF treatment, the red light displayed by 4T1 cells was significantly weakened and the green light was enhanced, showing the greatest mitochondrial membrane potential damage. The damage to representative regions of mitochondrial DNA was quantitatively detected by qRT-PCR, further verifying the damaging effect of RMLF combined with X-rays on mitochondrial DNA ( Fig. 9C ). In summary, RMLF combined with X-rays can induce mitochondrial oxidative stress, thereby damaging mitochondrial DNA.

[0130] Immunofluorescence staining analysis was performed using mtTFA antibody and goat anti-mouse IgG secondary antibody to investigate whether mitochondrial DNA would escape to the cytoplasm after RMLF combined with X-ray treatment. Fig.9D-a, the green fluorescence of mitochondrial DNA in the control group was evenly distributed and tightly accumulated around the nucleus. The green fluorescence of mitochondrial DNA in the RMLF+X-ray group was dispersed, and most of it escaped to the cytoplasm, which indicated that the constructed liposome composite material RMLF could promote the escape of mitochondrial DNA to the cytoplasm. Subsequently, qRT-PCR was further used to quantitatively detect the escape of mitochondrial DNA in the supernatant of 4T1 cells after different treatments, further verifying the effect of RMLF combined with X-ray treatment on inducing mitochondrial DNA escape ( Fig.9D -b). In summary, RMLF combined with X-rays can induce mitochondrial oxidative stress and damage mitochondrial DNA, causing mitochondrial DNA to escape into the cytoplasm and even into the cell supernatant.

[0131] (8) qRT-PCR verification of activation of cGAS-STING innate immune signaling pathway

[0132] qRT-PCR was used to verify whether RMLF combined with X-rays can activate the cGAS-STING immune signaling pathway, thereby generating an anti-tumor immune response. Fig.9E As shown in the figure, after treatment with medium-dose radiotherapy alone, the activation effect on cGAS-STING is basically negligible, which also confirms the phenomenon that the efficiency of radiotherapy alone in stimulating immunity is indeed very low in clinical practice. After treatment with RMLF alone, the activation effect on cGAS-STING is weak, which may be due to the released Mn 2+ It directly activates the cGAS-STING immune signaling pathway. Similarly, the MnB+X-ray group further activated the cGAS-STING pathway. It is worth noting that the RudZ+X-ray group and the RMLF+X-ray group had a stronger activation effect on cGAS-STING, among which the RMLF+X-ray group had the strongest activation effect on the pathway. This shows that the constructed liposome composite material RMLF can escape mitochondrial DNA and Mn 2+ The release of TNF-α effectively activates the cGAS-STING immune signaling pathway, thereby inducing anti-tumor immune response.

[0133] (9) RMLF hemolysis test

[0134] The blood biocompatibility of the constructed liposome composite material RMLF was evaluated, and the experimental results are as follows Fig.10 As shown. PBS buffer solution was used as the negative control group and deionized water was used as the positive control group. From the photos of mouse red blood cell hemolysis, it can be observed that almost no obvious hemolysis occurred. After treatment with different concentrations of RMLF, the highest hemolysis rate was less than 3%. Therefore, it can be inferred that RMLF has low toxicity to red blood cells and has good biocompatibility, which lays the foundation for subsequent animal experiments.

[0135] (10) Fluorescence imaging and in vivo biodistribution

[0136] By establishing a female 4T1 tumor-bearing mouse model, fluorescence imaging and in vivo biodistribution experiments were performed to evaluate the in vivo biodistribution of RMLF at different times. After RMLF was injected into the tail vein of 4T1 tumor-bearing mice, the mice were killed at different time points (2h, 6h, 12h, and 24h) for in vitro small animal fluorescence imaging and in vivo biodistribution analysis. Fig.11 As shown in the figure, as time goes by, the red fluorescence signal of RudZ increases continuously at the tumor site, and 12 hours after drug injection, the accumulation of RMLF at the tumor site reaches a peak. Therefore, 12 hours after drug administration was selected as the optimal treatment time point for radiotherapy. It is worth noting that compared with RMLF, the accumulation of RML at the 12h time point is significantly reduced, and the 24h fluorescence intensity is also significantly weaker than that of RMLF. This shows that RMLF can be enriched in the tumor more quickly and efficiently through the EPR effect and folate-mediated active targeting.

[0137] (11) In vivo radioimmunotherapy

[0138] Radioimmunotherapy was further studied on 4T1 tumor-bearing mice. The mice were randomly divided into 7 groups and given different treatments 12 hours after drug injection. The tumor volume was recorded regularly with a vernier caliper. Fig. 12A -a, the tumor size of the PBS group and the RMLF group increased rapidly over time, and there was almost no significant difference in the tumor size between the two groups, indicating that RMLF is a radiotherapy sensitizer with good biocompatibility and will not cause obvious damage to mice. The tumor growth rate of the X-ray group was slower than that of the PBS and RMLF groups, but it still had a faster growth rate, indicating that single radiotherapy can only inhibit tumor growth to a certain extent, and single radiotherapy is not enough to destroy the growth of tumor cells without the administration of therapeutic drugs. The RudZ+X-ray group and the MnB+X-ray group had a more obvious tumor growth inhibition effect, which was better than the CDDP+X-ray group. However, among all the groups, the RMLF+X-ray group had the most obvious effect in inhibiting tumor growth, which may be because folic acid enhanced the active targeting effect of RMLF, allowing more nanoparticles to be effectively enriched in the tumor site. After 12 days of treatment, all mice were dissected and the tumors were weighed, as shown in the figure. Fig. 12A -c and Fig. 12A -d, compared with the tumor volume of the control group, the tumors in the treatment group were significantly reduced, among which the RMLF+X-ray group had the smallest volume and the lightest tumor weight, showing the best anti-tumor effect, which indicates that RMLF combined with radiotherapy has a better anti-tumor effect than single radiotherapy. It is worth noting that during all the treatment processes, such as Fig. 12A As shown in Figure 5-b, compared with the PBS group, there was no significant change in the body weight of mice in all treatment groups, indicating that RMLF had no obvious toxicity and would not cause other harm to mice.

[0139] In order to evaluate the effect of RMLF+X-ray in causing DNA damage and alleviating hypoxia in the tumor site in 4T1 tumor-bearing mice, the mice were randomly divided into 7 groups, and then RMLF was injected into the 4T1 tumor-bearing mice through the tail vein. The tumors in the PBS group grew well, with almost no γ-H2AX foci and the strongest HIF-1α fluorescence expression. After 24 hours, the mice were killed and the tumor tissues were collected to prepare sections for γ-H2AX and HIF-1α immunofluorescence staining analysis. Fig. 12B As shown, among all treatment groups, the RMLF+X-ray group showed significantly higher levels of γ-H2AX foci (red) and lower HIF-1α fluorescence (green). This indicates that the DNA of tumor tissue cells is severely damaged, which suggests that RMLF can effectively alleviate the hypoxia of the tumor microenvironment and cause DNA damage in tumor cells, thereby playing its role in radiosensitization.

[0140] (12) Flow cytometry analysis of DC cell maturation and TAMs polarization, ELISA analysis of related cytokine expression, TUNEL apoptosis, CD4 / CD8 immunofluorescence, HE staining, and blood routine and liver and kidney function analysis

[0141] Flow cytometry was used to analyze the maturation of dendritic cells (DCs) and the polarization of tumor-associated macrophages (TAMs). After the treatment, single cell suspensions were prepared from tumor-draining lymph nodes and tumor tissues to analyze the maturation of DCs and the polarization of TAMs after different treatments. Fig. 12C As shown in the figure, compared with the PBS, RMLF, X-ray, and CDDP+X-ray groups, the RMLF+X-ray group had the highest expression levels of CD80 and CD86, which are markers of DC maturation. At the same time, the proportion of M1 macrophages in the RMLF+X-ray group increased significantly, while the proportion of M2 macrophages decreased significantly. This suggests that RMLF combined with X-ray therapy can reverse the immunosuppressive tumor microenvironment by converting M2 phenotype macrophages into M1 phenotype macrophages.

[0142] Enzyme-linked immunosorbent assay showed that RMLF combined with X-rays could increase the levels of cGAMP, TNF-α, IFN-β, and IL-6 ( Fig.12D). However, in the CDDP+X-ray group, compared with X-ray, cisplatin had a smaller effect on cGAMP levels and had no significant effect on the measured cytokines. These results indicate that RMLF combined with X-ray treatment can induce a strong anti-tumor immune response in 4T1 tumor-bearing mice by dual activation of the cGAS-STING innate immune signaling pathway.

[0143] like Fig.12E As shown in the TUNEL analysis of all groups after treatment, the RMLF+X-ray group showed the highest level of cell apoptosis, which indicates that RMLF has an excellent radiosensitization effect and can effectively inhibit the growth of primary tumors. In the immunofluorescence sections of all groups after treatment, the fluorescence intensity of CD8 and CD4 in the RMLF+X-ray group was the strongest, which indicates that CD8 + T cells and CD4 + The proportion of T cells was significantly higher than that of the PBS group. In addition, the expression of CD86 in immunohistochemical sections of the RMLF+X-ray group was significantly upregulated, and the expression of CD206 was significantly downregulated, which was also consistent with the flow cytometry results ( Figure 12G ), which indicates that the RMLF+X-ray group can induce TAMs polarization in vivo, achieve the regulation of tumor immunosuppressive microenvironment, enhance the efficacy of radiotherapy, and better promote the immune effect.

[0144] After the treatment, HE staining analysis was performed on the resected tumor tissues to analyze the therapeutic effects of different treatment methods on the tumor. At the same time, to further confirm the in vivo biological safety of the constructed RMLF, HE staining analysis was performed on the main organs (heart, liver, spleen, lung, and kidney) of the treated mice. Fig.12F As shown in the figure, compared with the PBS group, the main organs of mice receiving different treatments had no obvious pathological changes, indicating that RMLF has good biocompatibility and will not cause significant damage to mice. In addition, among all the HE sections of the tumors, the RMLF+X-ray group had the most serious damage, which further illustrates its excellent therapeutic effect.

[0145] In order to further investigate the biological safety of RMLF during the treatment process, and to ensure that the drug does not cause toxic side effects to its tissues and systems, blood routine and liver and kidney function analysis were performed. ALT and AST are the main indicators for detecting liver function; BUN and CREA are the main indicators for detecting kidney function. WBC, RBC, and PLT are the main indicators for detecting blood routine. Fig.12H As shown in the figure, compared with the PBS group, there were no significant changes in the blood routine and liver and kidney function indicators in the RMLF+X-ray group, which shows that RMLF has good biocompatibility.

[0146] (13) Long-term immune memory experiment

[0147] In order to evaluate the long-term effect of anti-tumor immune response induced by RMLF combined with radiotherapy, a recurrence and metastasis model was established using 4T1 tumor-bearing mice to evaluate the long-term immune effect of the RMLF+X-ray group. Fig.13A As shown in Figure 2, the tumor size of the PBS group increased rapidly over time, while the growth rate of the tumor size of the RMLF group was slightly slower than that of the PBS group, which may be due to the fact that Mn 2+ Directly activate the cGAS-STING innate immune signaling pathway, thereby inducing a certain anti-tumor immune effect. Secondly, the tumor growth rate of the RudZ+X-ray group was significantly slower than that of the X-ray group, and was better than that of the CDDP+X-ray group. It is worth noting that the RMLF+X-ray group had the best long-term immune memory effect. After the second inoculation of tumor cells, after long-term observation, its tumor volume was the smallest, tumor weight was the lightest, and anti-tumor immune effect was the strongest ( Fig.13A -c and Fig.13A -d). This is because the RudZ and MnB nanoparticles in the RMLF+X-ray group played a dual role in activating the cGAS-STING signaling pathway, while catalyzing the oxygen produced by endogenous H2O2 to reshape the immunosuppressive TME, thereby showing a strong anti-tumor immune effect. The results of TUNEL analysis, CD4 / CD8 immunofluorescence staining sections, and tumor tissue HE staining also further verified the above conclusions ( Fig. 13B In addition, there was no significant change in the body weight of the mice during the entire long-term immune memory treatment experiment ( Fig.13A -b), indicating that the treatment measures taken are very safe.

[0148] The content of effector memory T cells (Tem) and central memory T cells (Tcm) is a key indicator for evaluating long-term immune memory effects. The expression of relevant T cells in the spleen of mice after treatment was detected by flow cytometry. Fig. 13C As shown in the results, compared with other groups, the content of effector memory T cells (Tem) and central memory T cells (Tcm) in the RMLF+X-ray group was significantly increased, indicating that a strong immune memory effect was produced after treatment, which can provide effective protection for inhibiting tumor recurrence and metastasis. In summary, RMLF is not only an effective radiosensitizer, but also can effectively activate anti-tumor immune response and produce long-term memory effect.

[0149] In summary, the present invention provides a Ru-based II -Mn IVThe liposome composite material has good biocompatibility and tumor targeting, and has good radioimmunotherapy effect under X-ray irradiation. At the cellular level, it was proved that RMLF has the ability to actively target mitochondria, cause mitochondrial oxidative stress, lead to mitochondrial DNA damage and release, induce macrophage M1 polarization, cell cycle G2 / M arrest, and lead to cell apoptosis, which has an excellent radiosensitization effect. At the same time, the released mtDNA and Mn 2+ It can effectively and synergistically activate the cGAS-STING innate immune signaling pathway and activate the body's immune response. At the animal level, RMLF has the ability to actively target tumors, has a good long-term immune memory effect in the body, and can effectively inhibit tumor recurrence and metastasis.

[0150] Those skilled in the art will readily appreciate other embodiments of the invention after considering the specification and practicing the invention herein. The invention is intended to cover any variations, uses or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art of the invention. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the claims.

[0151] It should be understood that the present invention is not limited to the exact construction that has been described above and shown in the drawings and that various modifications and changes may be made without departing from the scope thereof. The scope of the present invention is limited only by the appended claims.

Claims

1. A liposome composite material for enhancing radioimmunotherapy of breast cancer, characterized in that: The liposome composite material is prepared by encapsulating RudZ and MnB in a lipid phase and an aqueous phase respectively through folic acid-modified liposomes; the MnB is MnO2 nanoparticles; the RudZ is cis-[Ru(DIP)2dppz] 2+ Cl2, the structural formula is as follows:

2. The method for preparing the liposome composite material for enhancing radioimmunotherapy of breast cancer according to claim 1, characterized in that: The following steps are involved: DPPC, DSPE-PEG 3400 -COOH, CHO and DSPE-PEG 3400 -FA were dissolved in chloroform / methanol mixed solvent, and ultrasonicated to fully dissolve, then the chloroform / methanol mixed solvent was added to the eggplant-shaped bottle, and then the DPPC and the DSPE-PEG in a molar ratio of 60:10:10:(5-10) were added. 3400 -COOH, the CHO, the DSPE-PEG 3400 -FA is used to form the folic acid-modified liposomes, and then the RudZ is added for ultrasonic treatment, and then the residual solvent is removed by rotary evaporation, and the obtained lipid membrane is hydrated with the MnB to prepare a uniform liposome composite material.

3. The method for preparing the liposome composite material for enhancing radioimmunotherapy of breast cancer according to claim 2, characterized in that: The drug-lipid ratio of the RudZ and folic acid modified liposomes is 1:(10-20).

4. The method for preparing the liposome composite material for enhancing radioimmunotherapy of breast cancer according to claim 2, characterized in that: The concentration of RudZ is 2-4 μM, and the [Mn] concentration of MnB is 10-20 μg / mL.

5. The method for preparing the liposome composite material for enhancing radioimmunotherapy of breast cancer according to claim 2, characterized in that: The preparation method of RudZ is as follows: (1) Synthesis of dppz ligand: Use anhydrous ethanol to prepare an o-phenylenediamine solution and a 1,10-phenanthroline-5,6-dione solution with a molar ratio of 13.868:(7.135-10.125), slowly add the o-phenylenediamine solution to the 1,10-phenanthroline-5,6-dione solution while stirring, heat to boiling in a warm water bath, boil for 5 minutes, cool in an ice water bath for crystallization, filter and recrystallize to obtain a brown needle-shaped dppz ligand; (2) Synthesis of cis-Ru(DIP)2Cl2: Dissolve 4,7-diphenyl-1,10-phenanthroline and ruthenium chloride hydrate in a molar ratio of 3.06:(2.13-3.26) in DMF under N2 protection and mix, heat to 151°C-181°C and condense under reflux for 8 hours, and terminate the reaction when the solution turns purple. After cooling, add acetone and stir, store at -20°C overnight, filter, wash and vacuum dry the precipitate to obtain cis-Ru(DIP)2Cl2; (3) Synthesis of cis-[Ru(DIP)2dppz] 2+ Cl2: The cis-Ru(DIP)2Cl2 and the dppz ligand in a molar ratio of 0.375:(0.718-1.536) are dissolved in DMF under N2 protection and mixed, heated to 151°C-181°C and condensed under reflux for 8-10h, and the reaction is terminated when the solution turns reddish brown. After cooling, DMF is removed by vacuum at 75°C and 100rpm, and the reddish brown metal complex cis-[Ru(DIP)2dppz] is obtained by elution purification and vacuum drying. 2+ Cl2.

6. The method for preparing the liposome composite material for enhancing radioimmunotherapy of breast cancer according to claim 2, characterized in that: The preparation method of the MnB is as follows: BSA and MnCl2·4H2O in a mass ratio of (1-5):1 were added to a hydrothermal tube, and the mixture was stirred for 10 minutes in an oil bath at 37°C-40°C, and the pH was adjusted to 9.0-11.

0. The mixture was stirred and reacted for 2-4 hours, and then dialyzed for 24 hours for purification to obtain the MnB.

7. Use of a liposome composite material for enhancing radioimmunotherapy of breast cancer prepared by the preparation method of claim 2 in the preparation of a drug for increasing the generation of active oxygen during tumor radiotherapy.

8. Use of a liposome composite material for enhancing radioimmunotherapy of breast cancer prepared by the preparation method of claim 2 in preparing a drug for improving hypoxia in the tumor microenvironment.

9. Use of a liposome composite material for enhancing radioimmunotherapy of breast cancer prepared by the preparation method of claim 2 in the preparation of a drug that targets breast cancer mitochondria, cooperates with high-energy ionizing radiation to cause mitochondrial DNA oxidative damage, and activates the cGAS-STING innate immune signaling pathway.

10. The use according to any one of claims 7 to 9, characterized in that: The liposome composite material can induce a strong anti-tumor immune response while being used for radiotherapy sensitization.

Images