Nanosystem for tumor radioimmunotherapy, preparation method and application thereof

Abstract

The present invention discloses a nanosystem for tumor radioimmunotherapy, its preparation method and application, belonging to the technical field of biomedicine. This system is engineered to modify bacterial outer membrane vesicles (OMVs) to carry magnetite nanoparticles (Fe3O4NPs) and the ATR inhibitor VE822. By using the synergistic effect of the Fenton reaction activity of Fe3O4NPs and the inhibition of DNA damage repair by the ATR inhibitor, DNA damage is amplified and the DNA damage response (DDR) is inhibited, the generation of hydroxyl radicals (·OH) under radiation is enhanced, the radiation resistance of tumor cells is reduced, the immune response is reactivated, the infiltration of effector T cells into tumors is improved, and the growth of tumors in mice is significantly inhibited, providing an effective strategy for tumor radioimmunotherapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a nanosystem for tumor radioimmunotherapy, a preparation method thereof, and an application thereof. Background Art

[0002] The nuclear DNA damage induced by radiotherapy can induce an anti-tumor immune response. The accumulation of cGAS in micronuclei can activate the intracellular inflammatory pathway, bringing a new direction for anti-tumor treatment. However, the DNA damage caused by radiotherapy will activate the DNA damage response (DDR), which blocks the occurrence of anti-tumor inflammation, making it difficult to effectively activate the immune inflammatory response and greatly limiting the radiotherapy effect.

[0003] Currently, to enhance the radiation-induced DNA damage caused by the immune inflammatory response, ATR inhibitors have been introduced to alleviate the cell cycle arrest and DNA repair problems induced by radiotherapy. Nanoparticles (such as Fe3O4 NPs) can in-situ convert hydrogen peroxide (H2O2) into highly cytotoxic ·OH due to their participation in the Fenton reaction, enhancing the radiotherapy sensitivity. However, the low uptake efficiency of these substances by cells in tumor tissues and the increased interstitial pressure affect the penetration of nanoparticles and drugs, directly affecting the treatment effect. Bacterial outer membrane vesicles (OMVs) are naturally occurring vesicle structures outside bacterial cells, with excellent ability to penetrate biological barriers and easy-to-engineer characteristics, becoming promising drug delivery candidates in tumor treatment. However, the formation mechanism of OMVs is through local budding of the bacterial outer membrane, and the vesicle size is usually between dozens and hundreds of nanometers. Nanoparticles may not be able to effectively enter or be stably encapsulated in the space, hindering their effective use as carriers for delivering nanoparticles in treatment scenarios.

[0004] In summary, it is of great significance to develop a nanosystem for tumor radioimmunotherapy to promote radioimmunotherapy through the synergistic effect of amplifying DNA damage and inhibiting DDR. Summary of the Invention

[0005] Aiming at the problems of high tumor radiation resistance, inhibited immune response, and low drug delivery efficiency existing in the current radiotherapy, the present invention proposes a nanosystem for radioimmunotherapy, a preparation method thereof, and an application thereof.

[0006] In the first aspect of the present invention, it relates to a nanosystem for radioimmunotherapy, comprising:

[0007] 1. Engineered bacterial outer membrane vesicles (OMVs): Based on Escherichia coli BL21(DE3), it is modified and surface-modified with the iRGD tumor-homing peptide. The iRGD peptide on its surface can specifically bind to integrin receptors overexpressed on tumor cells and blood vessels, achieving precise targeted delivery, increasing the drug concentration in tumor tissues, enhancing the therapeutic effect, and reducing damage to normal tissues.

[0008] 2. Magnetite nanoparticles (Fe3O4NPs): Encapsulated within OMVs, using their Fenton reaction activity, they react with overexpressed H2O2 in tumor cells under radiation conditions to generate highly cytotoxic ·OH, causing DNA damage and enhancing the radiotherapy effect.

[0009] 3. Ataxia telangiectasia and Rad3-related (ATR) inhibitor VE822: Loaded in OMVs, by inhibiting the activity of ATR kinase, it interferes with the DNA damage repair process in tumor cells, maintains the DNA break state, restores radiation-induced inflammation and apoptosis, and reduces the radiation resistance of tumor cells.

[0010] The second aspect of the present invention relates to a preparation method of a nanosystem for radioimmunotherapy, comprising the following steps:

[0011] 1. Construct engineered bacteria expressing the iRGD peptide: Artificially synthesize a double-stranded DNA fragment (ClyA-Myc-iRGD) containing the iRGD (CRGDKGPDC) sequence, and ligate it to the pGEX-4T1 plasmid through restriction enzymes. Transform the constructed plasmid into competent Escherichia coli BL21(DE3) cells, screen and purify using ampicillin, and induce expression with IPTG to obtain low-toxic engineered Escherichia coli expressing the iRGD peptide on the surface.

[0012] 2. Prepare glucose polymer-modified Fe3O4NPs: Synthesize Fe3O4 nanoparticles by the co-precipitation method, and sequentially perform amino functionalization and glucose polymer (GP) modification. Make the modified Fe3O4NPs be actively phagocytosed by engineered Escherichia coli through the specific ABC transporter pathway.

[0013] 3. Obtain OMVs containing Fe3O4NPs (iOF): Incubate the modified Fe3O4NPs with engineered Escherichia coli. After being phagocytosed, use peptidoglycan degradation to induce explosive lysis of bacteria, release iOF, and then enrich it by multiple differential centrifugations.

[0014] 4. Prepare the final nanosystem (iOFV): After performing cytotoxicity tests on the ATR inhibitor VE822, load it with iOF by ultrasonic incubation to obtain the iOFV nanosystem.

[0015] The third aspect of the present invention relates to the application of the above-mentioned nanosystem in the preparation of anti-tumor drugs for radioimmunotherapy.

[0016] The above-mentioned nanosystem can be used to prepare drugs for enhancing tumor radioimmunotherapy. By enhancing radiation-induced DNA damage, inhibiting the DNA damage response of tumor cells, promoting tumor cell apoptosis and ferroptosis, activating the immune response, and enhancing the infiltration of effector T cells into tumors, tumor growth can be inhibited.

[0017] Advantages of the present invention:

[0018] 1. Targeted delivery and efficient treatment: Engineered OMVs conjugated with iRGD peptide can precisely target tumor tissues and cells, effectively deliver Fe3O4NPs and VE822 to the tumor site, increase the local drug concentration, enhance the therapeutic effect while reducing damage to normal tissues.

[0019] 2. Synergistic radiosensitization: The synergistic effect of the Fenton reaction of Fe3O4NPs and the inhibition of DNA damage repair by VE822 amplifies the degree of DNA damage, continuously inhibits DDR, significantly reduces the radiation resistance of tumor cells, and enhances the radiotherapy effect.

[0020] 3. Activation of immune response: Sustained DNA damage triggers apoptosis and ferroptosis, while activating the cGAS-STING immune pathway, promoting dendritic cell maturation, enhancing antigen presentation and T cell activation, attracting CD 3+ 、CD 4+ and CD 8+ T cell infiltration, and enhancing the anti-tumor immune response.

[0021] 4. Good biosafety: Verified by in vivo experiments, iOFV has no adverse effects on the hematological parameters, liver and kidney functions of mice, does not induce hemolysis, and can be effectively cleared through the kidneys, showing high systemic application safety in cancer treatment. Brief description of the drawings

[0022] Figure 1 : Transmission electron microscope (TEM) images of Fe3O4 nanoparticles, engineered outer membrane vesicles (iOMV) and engineered exosome-like vesicles (iOFV). Scale bars: 200 nm, 100 nm, 500 nm.

[0023] Figure 2 : Particle size distributions of Fe3O4 nanoparticles, iOMV and iOFV.

[0024] Figure 3 : Changes in particle size and protein content of iOFV at 4°C. Data are presented as mean ± standard deviation (n = 3 independent samples).

[0025] Figure 4 : UV-visible absorption spectra of oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB) containing hydrogen peroxide (H2O2, 100 mM) and different concentrations of iOFV (pH = 6.2).

[0026] Figure 5 : Electron spin resonance (ESR) spectra of hydroxyl radicals (·OH) under different conditions (iOFV = 100 μg / mL, H2O2 = 100 mM). Data were processed using GraphPad Prism 9.3 software and are presented as mean ± standard deviation.

[0027] Figure 6 : Relative cell viability after treatment of LO2 cells and 4T1 cells with different concentrations of engineered exosome-like vesicles (iOFV). n = 3 independent samples.

[0028] Figure 7 : Relative cell survival rate after treatment of 4T1 cells with different concentrations of iOF and iOFV. n = 3 independent samples.

[0029] Figure 8 : Relative cell survival rate after treatment of 4T1 cells with different concentrations of iOF and iOFV, followed by exposure to 6 Gy radiation. n = 3 independent samples.

[0030] Figure 9 : Figure of key biomarker detection results (I).

[0031] Figure 10 : Figure of key biomarker detection results (II).

[0032] Figure 11 : Figure of key biomarker detection results (III).

[0033] Figure 12 : Figure of key biomarker detection results (IV).

[0034] Figure 13 : Internalization of fluorescein isothiocyanate (FITC)-labeled OFV and iOFV nanoparticles by 4T1 cells after incubation for 2 hours, 4 hours, and 6 hours, observed by fluorescence microscopy. Scale bar: 50 μm.

[0035] Figure 14 : Fluorescence distribution of DiR-labeled OFV and iOFV in 4T1 tumor-bearing mice at different time points after injection. n = 3 biologically independent mice per group.

[0036] Figure 15 : Distribution of residual iron in various organs of mice 48 hours after injection of iOFV. n = 3 biologically independent mice per group.

[0037] Figure 16 : Iron content in the urine of mice 48 hours after injection of iOFV. Each group had n = 3 biologically independent mice.

[0038] Figure 17 : Analysis of the blood heat map and biochemical analysis of the blood of mice at 0, 1, 3, and 5 days after injection of iOFV.

[0039] Figure 18 : Hemolysis test of iOFV in mice at 0, 1, 3, and 5 days after injection of iOFV.

[0040] Figure 19 : Measurement of the weights of the major organs of mice at 0, 1, 3, and 5 days after injection of iOFV.

[0041] Figure 20 : Radioimmunotherapy effect of iOFV in vivo (I).

[0042] Figure 21 : Radioimmunotherapy effect of iOFV in vivo (II).

[0043] Figure 22 : Radioimmunotherapy effect of iOFV in vivo (III).

[0044] Figure 23 : Radioimmunotherapy effect of iOFV in vivo (IV). Specific implementation manners

[0045] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. However, the following embodiments are only for explaining the present invention, and the protection scope of the present invention should include all the contents of the claims. Moreover, through the description of the following embodiments, those skilled in the art can fully implement all the contents of the claims of the present invention.

[0046] In the implementation of the present invention, unless otherwise specified, chemical reagents such as FeCl3·6H2O and FeSO4·4H2O are all purchased from Shanghai Chemical Reagent Co., Ltd., China. All chemicals and solvents are of analytical grade; cell culture-related reagents such as RPMI-1640 medium, DMEM medium, and fetal bovine serum (FBS) are purchased from Gibco; Escherichia coli BL21(DE3) is purchased from Anhui General Biology; the 4T1 mouse breast cancer cell line and LO2 human normal liver cell line used in the experiment are respectively cultured in RPMI-1640 and DMEM media, supplemented with 10% fetal bovine serum and 100 U / mL penicillin-streptomycin, and the cells are stored in a humidified incubator at 37°C with 21% O2 and 5% CO2. For animal experiments, 6-8-week-old female BALB / c mice are used, housed in a specific pathogen-free facility, with the temperature controlled at 22-26°C, humidity at 40-70%, and a 12-hour light / dark cycle. Tumor growth is monitored to ensure that the tumor diameter does not exceed 20 mm or the weight does not exceed 4000 mg. After the experiment, the mice are euthanized with carbon dioxide.

[0047] The present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0048] 1. Preparation and Characterization of iOFV

[0049] A preparation method of a nano-system iOFV for radioimmunotherapy includes the following steps:

[0050] (1) Construction of an engineered bacterium expressing the iRGD peptide:

[0051] Through in vitro gene synthesis technology, a 1011 bp double-stranded DNA fragment (ClyA-Myc-iRGD) containing the iRGD (CRGDKGPDC) sequence is precisely synthesized, as shown in SEQ ID NO.1 in the sequence listing. It is ligated to the pGEX-4T1 plasmid using restriction endonucleases to construct a recombinant plasmid. Subsequently, the recombinant plasmid is transformed into Escherichia coli BL21(DE3) competent cells, and high-purity transformed strains are obtained by screening and purification using ampicillin. The transformed strains are cultured. When the optical density (OD600) of the strains reaches 0.5-0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) with a final concentration of 0.2 mM is added, and the protein expression is induced by incubation overnight at 20°C. After that, the bacteria are harvested by centrifugation at 4°C and 5000×g for 30 minutes, and the expression of the Myc-tagged protein in transgenic BL21(DE3) is verified using Western blotting technology, and a low-toxic Escherichia coli expressing the iRGD peptide on the surface is successfully obtained.

[0052] (2) Preparation of glucose polymer-modified Fe3O4 NPs:

[0053] First, Fe3O4 nanoparticles were synthesized by the co - precipitation method. 0.9 g of ferric chloride hexahydrate (FeCl3·6H2O) and 0.45 g of ferrous sulfate tetrahydrate (FeCl2·4H2O) were dissolved in 150 ml of deionized water. 25% ammonium hydroxide was slowly added under continuous stirring, and the pH of the reaction system was controlled at about 10. After the reaction was completed, the Fe3O4 nanoparticles precipitated, centrifuged at 6000 rpm for 10 min, washed with deionized water until neutral, and dried in vacuum for 12 h. To introduce amino groups, 1 g of Fe3O4 nanoparticles was dispersed in 50 mL of ethanol. After ultrasonic treatment for 30 minutes, 2 mL of 3 - aminopropyltriethoxysilane (APTES) was added, and the reaction was carried out at 80 °C for 5 hours. After the reaction, it was washed with ethanol and deionized water multiple times, centrifuged to remove unreacted APTES, and then dried in vacuum for 12 hours to obtain amino - functionalized Fe3O4 nanoparticles.

[0054] Then, 1 g of amino - functionalized Fe3O4 nanoparticles was dispersed in 50 mL of deionized water. 0.5 g of glucose polymer was dissolved in 50 mL of water and slowly dropped in, and the reaction was carried out at room temperature for 6 hours. The product was centrifuged, washed with deionized water, and dried in vacuum for 12 hours to obtain glucose - polymer - modified Fe3O4 NPs (GP - Fe3O4 NPs).

[0055] (3)Obtaining OMVs containing Fe3O4 NPs (iOF):

[0056] 150 ml of engineered Escherichia coli (1.0×10 7 CFU) suspension and 8.0 mg / mL of GP - Fe3O4 NPs were incubated in a shaking incubator at 37 °C and 150 rpm for 6 hours to allow the nanoparticles to be fully phagocytosed by the bacteria. The mixture was centrifuged at 1000×g for 5 minutes, and the unbound nanoparticles were discarded. It was washed 3 times with PBS. Lysozyme (20 mg / mL) was added, and ultrasonic treatment (ice - bath for 12 hours) was used to promote bacterial lysis to release outer - membrane vesicles (OMVs). The suspension was filtered through 100 nm and 220 nm polycarbonate membranes in sequence, and then centrifuged at 12000×g to purify iRGD - OMV@Fe3O4, namely iOF.

[0057] (4)Preparing the final nano - system (iOFV):

[0058] The cytotoxicity of the ATR inhibitor VE822 was tested to determine its toxicity to tumor cells and normal cells. iOF and VE822 (320 nM) were incubated in the dark at 4 °C for 6 hours to prepare iOFV. This preparation could be stored at 4 °C for 1 week, and its stability and activity were regularly detected during this period.

[0059] (5)Material characterization:

[0060] The morphologies of Fe3O4 NPs, iOMV, and iOMV@Fe3O4 were detected using a field emission scanning electron microscope (SEM, ZEISS SIGMA) and a transmission electron microscope (TEM, JEOL JEM-1400). TEM samples were carefully deposited on carbon-coated copper grids and dried at room temperature. Energy-dispersive X-ray spectroscopy (EDS) and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) analyses were performed using Talos F200X and JEOL Arm-200F to obtain elemental composition and microstructure information. The ultraviolet-visible absorption spectra were recorded using a UV-1601 spectrophotometer, and the iron content was determined using inductively coupled plasma mass spectrometry (ICP-MS, Thermo Fisher iCAP Qc). The release of VE822 from the vesicles was monitored to evaluate the stability of VE822 encapsulated in OMVs. The Zeta potential of iOFV and its dispersibility in different polar solutions were detected, the drug loading efficiency and ultraviolet absorption characteristics were retested by ultraviolet-visible spectroscopy, and the protein concentration of iOFV stored at 4 °C for 2 weeks was detected.

[0061] 2. Performance Detection and Application

[0062] 2.1 Materials and Methods

[0063] (1) Cell Lines and Animals

[0064] The 4T1 mouse breast cancer cell line and LO2 human normal liver cell line were cultured in RPMI-1640 and DMEM media supplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin-streptomycin, respectively. The cells were cultured in a humidified incubator at 37 °C with 21% O2 and 5% CO2. According to the growth of the cells, the fresh medium was changed every 2 to 3 days, and then the cells in a stable growth and logarithmic growth phase were used for the experiment.

[0065] Female BALB / c mice aged 6-8 weeks were housed in a specific pathogen-free facility with the temperature controlled at 22-26 °C, humidity at 40-70%, and a 12-hour light / dark cycle. The tumor growth was monitored to ensure that the tumor diameter did not exceed 20 mm or the weight did not exceed 4000 mg. At the end of the experiment, the mice were euthanized using CO2.

[0066] (2) Detection of Reactive Oxygen Species Generation and ·OH Free Radicals In Vitro

[0067] 4T1 cells were inoculated and cultured under standard conditions for 12 hours, and then the medium was changed. Nanoparticles with a final concentration of 200 μg / mL (calculated as Fe3O4) were added, and the cells were incubated for another 12 hours. Then the cells were irradiated with 6 Gy and incubated for an additional 24 hours. The intracellular ROS level was evaluated by staining with DCFH-DA (a reactive oxygen species probe). The generation of ROS was observed under a confocal microscope.

[0068] 60 μL of the nanoparticle solution (800 μg / mL) was mixed with 20 μL of the superoxide dismutase (SOD) solution (1000 U / mL) and 60 μL of 200 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO). After the sample was irradiated with 6 Gy, 300 μL of acetic acid buffer (pH 3.6) and 60 μL of 10% H2O2 were added. Electron paramagnetic resonance (EPR) was used to analyze the generation of ·OH in the reaction.

[0069] (3) Cytotoxicity detection

[0070] The cytotoxicity of iOFV against LO2 and 4T1 cells was detected using the Cell Counting Kit-8 (CCK8). LO2 and 4T1 cells were respectively inoculated into 96-well plates and incubated for 12 hours under standard conditions to allow them to adhere. Different concentrations of iOFV were added, and after continued incubation for 24 hours, according to the instructions of the CCK8 kit, the absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader to evaluate cell viability. Cell viability was calculated according to the following formula:

[0071] Cell viability (%) = A sample / A negative control × 100%

[0072] where A sample is the absorbance value of LO2 and 4T1 cells after treatment with the drug, and A negative control is the absorbance value of LO2 and 4T1 cells after treatment with PBS buffer solution.

[0073] (4) Evaluation of radiotherapy sensitization effect

[0074] 4T1 cells were incubated with the same concentration of iOF and iOFV, and cell viability was detected after radiotherapy or without radiotherapy. The cell colony formation assay was used to evaluate the sensitivity of tumor cells to ionizing radiation. The 4T1 cell line was seeded at 10 3Cells were seeded at a sparse density in 6-well plates and cultured for 48 hours to form stable cell colonies. Subsequently, the medium was replaced with fresh medium containing NPs at a final concentration of 100 μg / mL (based on Fe3O4), and the cells were incubated for another 24 hours. Then, the cells were irradiated with a linear accelerator (Clinacix, Varian, USA), and after irradiation, the cells were continuously cultured, washed with PBS every two days, and fresh medium was added. After 7 days, the colonies were stained with crystal violet, counted using ImageJ, the cell survival rate was measured, and the "multi-target, single-hit" model was used to perform non-linear fitting of the survival data to calculate the quasi-threshold dose (Dq) and the sensitization enhancement ratio (SER).

[0075] (5)Detection of key markers

[0076] JC-1 staining was used to detect mitochondrial membrane potential, AnnexinV-FITC / propidium iodide (PI) staining was used to detect the proportion of apoptotic cells, C11-BODIPY staining was used to evaluate the level of lipid peroxidation, and Western blot analysis was used to detect the expression of proteins such as Bcl-2 and GPX4.

[0077] (6)Verification of cell uptake and targeting

[0078] The nanoparticles were conjugated with FITC fluorescent probes and incubated with the 4T1 cell line for 12 hours. The cells were seeded in culture plates, incubated under standard conditions for 12 hours, and then the medium was replaced. FITC-labeled nanoparticles at a final concentration of 200 μg / mL (based on Fe3O4) were added, and the cells were incubated for 0, 1, 2, and 4 hours respectively. After incubation, the medium was aspirated, and the cells were washed three times with PBS. Then the cells were incubated with FITC for 20 minutes, and the cell uptake was observed using a fluorescence microscope.

[0079] To verify the active targeting of the nano-system iOFV, 4T1 cells resistant to conventional radiotherapy were used. Fluorescein isothiocyanate (FITC)-labeled OMV was incubated with 4T1 cells, and the cell uptake was quantitatively evaluated using a fluorescence microscope, flow cytometry, and fluorescence techniques. OFV and iOFV were labeled with DiR and intravenously injected into tumor-bearing mice. In vivo imaging was performed at different time points (6, 12, 24, 48, and 72 hours), and the excised tumors and major organs were analyzed.

[0080] (7)Pharmacokinetic and biodistribution studies

[0081] Before the experiment, the mice were acclimatized in the laboratory environment for 1 week. To evaluate tumor growth, 1×10 6Four T1 cells were suspended in PBS and subcutaneously injected into the right flanks of each mouse at 37 °C. Fourteen days later, the tumor-bearing mice were randomly divided into 8 groups (n = 8) and given different treatment regimens. Specifically, 10 mg / kg of Fe3O4 was injected via the tail vein. The next day, the tumor sites were irradiated under anesthesia with a source-skin distance (SSD) of 100 cm. 6 MV X-rays were used with a dose rate of 400 cGy·min -1 , and a dose of 6 Gy was given. The maximum accumulation region of the 6 MV linear accelerator photon beam was located 1.0 cm below the skin surface. Tumor volume and body weight were monitored every 2 days; tumor volume was measured using calipers, and the calculation formula was:

[0082] V = a × b 2 / 2;

[0083] where V (mm 3 ) represents the tumor volume, and a (mm) and b (mm) represent the length and width of the tumor, respectively.

[0084] On the 8th day of treatment, 2 mice were randomly selected from each group and euthanized to evaluate immune parameters. Peripheral blood, urine, tumors, and major organs (including the heart, liver, spleen, lungs, and kidneys) were collected, dried, weighed, digested with aqua regia, and diluted with ultrapure water. The iron concentration in the samples was measured using ICP-MS.

[0085] (8) Biosafety assessment

[0086] Five-week-old healthy female ICR mice were selected and injected with iOFV via the tail vein on days 1, 3, and 5. Blood samples were collected at different time points for hematological and biochemical analysis, and the weight changes of major organs were monitored. iOFV (200 μg / ml) was incubated with mouse-derived red blood cells for 12 hours to detect whether hemolysis was induced. Real-time fluorescence quantitative PCR was used to quantitatively detect the expression levels of key genes affecting normal cell activity in various tissues.

[0087] (9) In vivo radioimmunotherapy

[0088] A subcutaneous 4T1 tumor-bearing female BALB / c mouse model was used, and the mice were randomly divided into 6 treatment groups: control group, iOF, iOFV, IR, iOF + IR, and iOFV + IR. Radiotherapy (6 Gy) was performed 12 hours after intravenous injection of the corresponding material. During the entire study, tumor growth and body weight changes were strictly monitored, and tumor volume was accurately measured using calipers. The calculation formula was:

[0089] V = a × b 2 / 2;

[0090] where V (mm 3is the tumor volume, and a (mm) and b (mm) are the tumor length and width, respectively.

[0091] After the treatment, the tumor tissues were subjected to hematoxylin and eosin (H&E) staining, TUNEL staining, detection of reactive oxygen species (ROS) levels with DCFH-DA probe, and immunofluorescence analysis of γ-H2AX expression.

[0092] (10) Immunological mechanism research

[0093] The tumor-bearing mice were divided into 7 treatment groups: control, iOF, iOFV, IR, iOF+IR, iOFV+IR, and iOMV+IR. After 14 days of treatment, the immune profiles of the tumor tissues were analyzed by flow cytometry, and the expressions of the maturation markers CD 80 and CD 86 of dendritic cells (DCs) were detected, as well as the infiltration degrees of CD 3+ , CD 4+ and CD 8+ T cells in the tumor tissues, blood, and spleen. The levels of IL-6 and TNF-α in the tumor tissues were detected by ELISA to evaluate the activation of the cGAS-STING pathway.

[0094]

[0094] (11) Statistical analysis

[0095] Unpaired t-tests were used to compare the differences between two groups, while one-way analysis of variance (one-way ANOVA) was used to compare the differences among multiple groups. The data were expressed as mean ± standard deviation (SD). A p-value < 0.05 was considered statistically significant.

[0096] 3. Result analysis

[0097] 3.1 Characterization of iOFV

[0098] The structures of Fe3O4 NPs, iOMV, and iOMV@Fe3O4 were characterized by field emission scanning electron microscopy (SEM, ZEISS SIGMA) and transmission electron microscopy (TEM, JEOL JEM-1400).

[0099] The average particle size of Fe3O4 NPs was about 5 nm, with good dispersion; iOMV and iOMV@Fe3O4 were spherical, with sizes of about 100.5 nm and 152.6 nm, respectively; the encapsulation efficiencies of Fe3O4 NPs and VE822 were 46.5% and 0.0182% (wt%), respectively, as shown in Table 1; iOFV had good stability after 12 hours in polar solutions, and the protein concentration changed little after storage at 4°C for 2 weeks, showing the characteristics of long-term storage. These results were visually demonstrated in Figure 1 , Figure 2 and Figure 3 respectively.

[0100] Table 1

[0101]

[0102] 3.2 Performance Detection

[0103] (1)Detection of the production of reactive oxygen species in vitro and ·OH free radicals

[0104] As the concentration of H2O2 increased, ROS gradually increased, showing a concentration-dependent relationship with H2O2, as Figure 4 shown. In addition, to prove that iOFV can increase ·OH through the Fenton reaction with radiotherapy-induced H2O2, an appropriate amount of SOD was added to this test system, and the ability of iOFV to generate radiotherapy-induced ·OH was tested using electron spin resonance (ESR) 13. The results are as Figure 5 shown. When H2O2 was absent, the iOFV+IR group produced more ·OH than the radiotherapy group. This is because radiotherapy induced a certain amount of H2O2, and the iOFV group could undergo the Fenton reaction to generate ·OH. Similarly, in the presence of H2O2, the radiotherapy group produced more ·OH than the non-radiotherapy group. In summary, iOFV effectively enhanced the radiation-induced Fenton reaction. The mechanism is that the H2O2 decomposed by SOD increased under radiotherapy induction, and more ·OH was generated through the Fenton reaction.

[0105] (2)Cytotoxicity detection

[0106] The results showed that the survival rate of LO2 cells co-cultured with 200 μg / mL iOFV was still higher than 80%, indicating that iOFV has good biocompatibility; while as the concentration of iOFV increased, the viability of 4T1 cells gradually decreased, showing tumor-specific toxicity. As Figure 6 shown in the relative cell viability histogram of cells treated with different concentrations of iOFV in , the influence trend of different concentrations of iOFV on the viability of the two types of cells was clearly demonstrated, intuitively reflecting the biocompatibility and tumor-specific toxicity of iOFV.

[0107] (3)Evaluation of radiotherapy sensitization effect

[0108] As Figure 7 shown, as the concentrations of iOF and iOFV increased, the viability of 4T1 cells decreased more significantly under radiotherapy; the Dq of iOFV was 0.81 Gy and the SER was 3.29, showing strong radiosensitizing ability. It can be clearly seen from Figure 8 the 4T1 cell survival rate curves and cell colony images under different treatments that the cell survival rate of the iOFV treatment group after radiotherapy was significantly lower than that of other groups, and the number of cell colonies decreased significantly, fully demonstrating the radiosensitization effect of iOFV.

[0109] (4)Detection of key markers

[0110] Combined treatment with radiotherapy and iOFV caused significant mitochondrial dysfunction. In the iOFV+IR group, the mitochondrial membrane was significantly reduced, the mitochondrial membrane potential (MMP) increased, accompanied by the release of cytochrome c and the activation of the apoptotic cascade; AnnexinV-FITC / PI staining confirmed a significant increase in the proportion of apoptotic cells in the iOFV+IR group; C11-BODIPY staining showed the highest level of lipid peroxidation in the iOFV+IR group; Western blot analysis showed that treatment with iOFV+IR significantly reduced the expression of Bcl-2 and GPX4. From Figure 9 、 Figure 10 、 Figure 11 and Figure 12 the images of mitochondrial membrane potential disruption, the analysis chart of cell apoptosis level, the images of lipid peroxidation, and the immunoblot analysis and quantitative results of the expression of Bcl-2 and GPX4 proteins, it can be seen that there are significant differences between the iOFV+IR group and other groups, strongly confirming that the combination of iOFV and radiotherapy can induce mitochondrial damage, disrupt cellular redox homeostasis, trigger lipid peroxidation, ultimately leading to tumor cell apoptosis and ferroptosis, and at the same time enhance the anti-tumor immune response by activating the cGAS-STING immune pathway.

[0111] (5)Verification of cell uptake and targeting

[0112] As Figure 13 shown by the fluorescence microscopy images of the internalization of nanoparticles by cells in Figure 14 the fluorescence signal in the cells of the iOFV-treated group was significantly stronger than that in the OFV group, intuitively demonstrating the effect of iRGD peptide in enhancing cell uptake. From

[0113] the fluorescence distribution in the tumor-bearing mice in

[0114] it can be clearly seen that the fluorescence intensity of DiR-labeled iOFV in the tumor site gradually increased within 12 hours and reached a relatively high level and continued to increase after 24 hours, further confirming that iOFV has significant tumor targeting and that the iRGD peptide can effectively guide the accumulation of the nanosystem in the tumor site. Figure 15 the column chart of organ iron distribution in Figure 16As shown, the curve of urine iron content changing with time visually demonstrates the process of its excretion through the kidneys, providing strong evidence for the safety and metabolic pathway of iOFV.

[0115] (7)Bio-safety assessment

[0116] The results showed that there were no statistically significant differences in the hematological parameters, liver function, and kidney function of the mice treated with iOFV compared with the control group. There was no obvious hemolysis induced, and there were no significant differences in the expression of key genes between the treatment group and the control group, confirming that iOFV has good bio-safety in vivo. As shown in the heatmap of mouse blood and biochemical analysis data in Figure 17 and Figure 18 , the comparison differences of various indicators between the treatment group and the control group were not obvious; the hemolysis test results showed no obvious hemolysis phenomenon, and the organ weight measurement data as shown in Figure 19 indicated that the weights of each organ were not significantly affected, all of which visually proved that iOFV has good bio-safety.

[0117] (8)In vivo radioimmunotherapy

[0118] As shown in Figure 20 , Figure 21 , Figure 22 and Figure 23 , the tumor volume of the iOFV+IR group was the smallest. H&E staining showed that the tumor cell tissue was looser, which means an increase in tumor cell necrosis; TUNEL staining indicated a significant increase in tumor cell apoptosis, and the red fluorescence in the iOFV+IR group was the strongest; the DCFH-DA probe detected that the ROS levels in the iOF+IR and iOFV+IR groups were significantly higher, confirming that Fe3O4 nanoparticles enhanced the production of ROS after radiation; immunofluorescence analysis of the expression of γ-H2AX showed more obvious DNA breaks in these two groups, indicating that iOFV significantly enhanced tumor radiosensitivity and promoted radiation-induced immune reconstruction. As shown in the tumor growth curve, the tumor volume growth of the iOFV+IR group was significantly slower than that of other groups, visually reflecting the best anti-tumor effect of this group. From the H&E-stained tumor tissue section images, it can be clearly seen that the tumor cell structure in the iOFV+IR group was loose and the nuclear morphology was abnormal, indicating a high degree of tumor cell necrosis; in the TUNEL-stained images, there were a large number of apoptotic cells marked with red fluorescence in the iOFV+IR group, and the number was significantly more than that of other groups; the ROS level images detected by the DCFH-DA probe showed that the fluorescence intensities of the iOF+IR and iOFV+IR groups were significantly higher than those of the control group, visually demonstrating the effect of Fe3O4 nanoparticles enhancing the production of ROS after radiation; in the images of immunofluorescence analysis of γ-H2AX expression, the fluorescence intensities of the iOF+IR and iOFV+IR groups were stronger, indicating more serious DNA breaks in these two groups, fully proving the role of iOFV in enhancing tumor radiosensitivity and promoting immune reconstruction.

[0119] (9) Immunomechanism research

[0120] The results showed that Fe3O4 nanoparticles promoted the production of ROS and induced ferroptosis, enhanced anti-tumor immunity, and promoted DC maturation; in the iOF+IR and iOFV+IR groups, the infiltration levels of CD 3+ , CD 4+ and CD 8+ T cells in tumor tissues, blood, and spleen were higher; the levels of IL-6 and TNF-α in these two groups were significantly increased, and the cGAS-STING pathway was significantly activated, resulting in an increase in the production of type I interferons (IFN-γ and IFN-β), further enhancing the anti-tumor immune response. From the flow cytometry results of the co-expression analysis of immune cells in tumor tissues in Figure 18 , it can be seen that the proportions of CD 80+ , CD 86+ DC cells in the iOF+IR and iOFV+IR groups were significantly higher than those in other groups, indicating that the dendritic cell maturity in these two groups was higher; the quantitative analysis chart of immune cell infiltration clearly showed that the numbers of CD 3+ , CD 4+ and CD 8+ T cells in tumor tissues, blood, and spleen in the iOF+IR and iOFV+IR groups were significantly more than those in other groups; the bar chart of the levels of IL-6 and TNF-α in tumor tissues detected by ELISA showed that the cytokine levels in the iOF+IR and iOFV+IR groups were significantly increased, intuitively reflecting the activation of the cGAS-STING pathway. These results together confirmed that iOFV combined with radiotherapy could promote immune reconstruction and enhance the anti-tumor immune response.

[0121] In summary, through a series of rigorous experiments, the present invention successfully prepared and comprehensively characterized the iOFV nanosystem. In in vitro experiments, iOFV exhibited good radiosensitizing effects and could effectively induce DNA damage and apoptosis of tumor cells; in in vivo experiments, iOFV had good biosafety, excellent tumor targeting, and significantly enhanced the effect of tumor radioimmunotherapy. The present invention provides an innovative and highly potential strategy for tumor radioimmunotherapy, which is expected to bring new breakthroughs and changes to clinical tumor treatment. In future research and applications, the preparation process and treatment plan of iOFV can be further optimized, and precise treatment can be carried out according to different tumor types and individual differences of patients to achieve more efficient and safer tumor treatment goals. At the same time, the research results of the present invention also provide important references for in-depth research in related fields and contribute to the continuous progress of tumor treatment technologies.

[0122] In terms of expanding actual application scenarios, this nanosystem can further explore the possibility of combining with other treatment means (such as chemotherapy drugs, immune checkpoint inhibitors, etc.), evaluate the efficacy of combination treatment regimens for different types of tumors, and provide more options for the comprehensive treatment of clinical tumors. In optimizing the preparation process, attempts can be made to improve the synthesis method of nanoparticles, the encapsulation technology of OMVs, and the modification strategy of iRGD peptide, so as to improve the encapsulation efficiency, stability and targeting of the nanosystem, reduce the production cost, and promote its transformation process from laboratory research to clinical application. In view of the individual differences of different tumor patients, carry out personalized treatment research, and accurately adjust the dose and treatment regimen of iOFV according to factors such as the patient's tumor type, stage, gene expression profile, etc., so as to improve the treatment effect while reducing adverse reactions and achieve precision medicine in the true sense.

[0123] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A nanosystem for tumor radioimmunotherapy, characterized in that, Comprising engineered bacterial outer membrane vesicles (OMVs), which are based on Escherichia coli BL21(DE3) and surface-modified with the iRGD tumor-homing peptide; the engineered bacterial outer membrane vesicles (OMVs) encapsulate magnetite nanoparticles (Fe3O4NPs) and the ataxia telangiectasia and Rad3-related (ATR) inhibitor VE822.

2. The nanosystem for tumor radioimmunotherapy according to claim 1, wherein The bacterial outer membrane vesicles (OMVs) are derived from the Escherichia coli BL21(DE3) strain, which has been genetically engineered to express the iRGD peptide on its surface.

3. The nanosystem for tumor radioimmunotherapy according to claim 1, characterized in that, The average particle size of the Fe3O4NPs is about 5 nm, and the size of the OMVs encapsulating the Fe3O4NPs is about 152.6 nm.

4. The nanosystem for tumor radioimmunotherapy according to claim 1, characterized in that, The encapsulation efficiency of the Fe3O4NPs is 46.5%, and the encapsulation efficiency of VE822 is 0.0182%.

5. A method for preparing the nanosystem according to any one of claims 1-4, characterized in that, Comprising the following steps: Construct a plasmid encoding the iRGD peptide and ligated to Escherichia coli cytolysin A, and transform it into Escherichia coli BL21(DE3) competent cells. After inducing protein expression, harvest the bacteria; use the co-precipitation method to synthesize Fe3O4 nanoparticles, and perform amino-functionalization and glucose polymer modification on them; incubate the modified Fe3O4 nanoparticles with engineered Escherichia coli to allow the bacteria to phagocytose the nanoparticles, and then use peptidoglycan degradation to trigger the formation of bacterial outer membrane vesicles (OMVs). Enrich the OMVs loaded with Fe3O4NPs by multiple differential centrifugations; load the ATR inhibitor VE822 into the OMVs loaded with Fe3O4NPs.

6. Use of the nanosystem according to any one of claims 1-4 in the preparation of an anti-breast tumor drug for radioimmunotherapy.

7. The application according to claim 6, characterized in that, The radioimmunotherapy includes using the nanosystem to enhance radiation-induced DNA damage, inhibit the DNA damage response of tumor cells, promote tumor cell apoptosis and ferroptosis, activate the immune response, and enhance the infiltration of effector T cells into the tumor, thereby inhibiting tumor growth.

8. The application according to claim 6, characterized in that, In the combination therapy of the nanosystem, the nanosystem combined with radiotherapy triggers mitochondrial apoptosis by activating the endoplasmic reticulum stress, TNF-α, and DNA damage-related signaling pathways; at the same time, it promotes glutathione consumption, reduces the activity of GPX4, resulting in the accumulation of lipid peroxides and triggering ferroptosis.

9. The application according to claim 6, characterized in that, In the synergistic effect of the nanosystem, the ·OH generated by the Fenton reaction of Fe3O4NPs under radiation enhances DNA damage, and the ATR inhibitor VE822 inhibits DNA damage repair. The two work together to maintain the DNA break state and continuously activate the immune response.

Images