Nano system for tumor radioimmunotherapy and preparation method and application thereof

Abstract

The invention discloses a nano system for tumor radioimmunotherapy and a preparation method and application thereof, and belongs to the technical field of biological medicines.The system modifies bacteria outer membrane vesicles (OMVs) through engineering to enable the bacteria outer membrane vesicles to carry magnetite nanoparticles (Fe3O4NPs) and an ATR inhibitor VE822, the Fenton reaction activity of the Fe3O4NPs and the synergistic effect of the ATR inhibitor for inhibiting DNA damage repair are utilized, and the tumor radioimmunotherapy effect is improved. DNA damage is amplified, DNA damage reaction (DDR) is inhibited, generation of hydroxyl radicals (. OH) under radiation is enhanced, radiation resistance of tumor cells is reduced, immune reaction is reactivated, infiltration of effector T cells to tumors is improved, growth of mice tumors is remarkably inhibited, and an effective strategy is provided for tumor radioimmunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a nanometer system for tumor radioimmunotherapy and a preparation method and application thereof. Background Art

[0002] Nuclear DNA damage caused by radiotherapy can induce anti-tumor immune responses, and cGAS accumulation in micronuclei can activate intracellular inflammatory pathways, bringing new directions for anti-tumor treatment. However, DNA damage caused by radiotherapy can activate the DNA damage response (DDR), which blocks the occurrence of anti-tumor inflammation, making it difficult to effectively activate the immune inflammatory response, greatly limiting the effect of radiotherapy.

[0003] At present, in order to enhance radiation-induced DNA damage caused by immune inflammatory response, ATR inhibitors have been introduced to alleviate radiotherapy-induced cell cycle arrest and DNA repair problems. Nanoparticles (such as Fe3O4NPs) can convert hydrogen peroxide (H2O2) into highly cytotoxic OH in situ due to their participation in Fenton reaction, thereby enhancing radiotherapy sensitivity. However, the inefficient uptake of these substances by cells in tumor tissue and the increased interstitial pressure affect the penetration of nanoparticles and drugs, which directly affects the therapeutic effect. Bacterial outer membrane vesicles (OMVs) are naturally occurring vesicle structures outside bacterial cells. They have excellent ability to penetrate biological barriers and are easy to engineer, making them promising drug delivery candidates in tumor treatment. However, the formation mechanism of OMVs is through local budding of the bacterial outer membrane. The vesicle size is usually between tens and hundreds of nanometers. Nanoparticles may not be able to effectively enter or stably encapsulate in space, which hinders their effective use as carriers to deliver nanoparticles in treatment scenarios.

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

[0005] Aiming at the problems existing in the existing radiotherapy, such as high tumor radiation resistance, suppressed immune response and low drug delivery efficiency, the present invention proposes a nanosystem for radioimmunotherapy and its preparation method and application.

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

[0007] 1. Engineered bacterial outer membrane vesicles (OMVs): Based on E. coli BL21 (DE3), the iRGD tumor homing peptide is modified on the surface. The iRGD peptide on its surface can specifically bind to the integrin receptors overexpressed on tumor cells and blood vessels, achieve precise targeted delivery, increase the drug concentration in tumor tissue, enhance the therapeutic effect, and reduce damage to normal tissues.

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

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

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

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

[0012] 2. Preparation of Fe3O4NPs modified with glucose polymer: Fe3O4 nanoparticles were synthesized by coprecipitation method, and amino functionalization and glucose polymer (GP) modification were performed in sequence. The modified Fe3O4NPs can be actively engulfed by engineered Escherichia coli through the specific ABC transporter pathway.

[0013] 3. Obtain OMVs (iOF) containing Fe3O4NPs: The modified Fe3O4NPs were incubated with engineered Escherichia coli. After they were engulfed, peptidoglycan degradation was used to induce explosive lysis of bacteria to release iOF, which was then enriched by multiple differential centrifugation.

[0014] 4. Preparation of the final nanosystem (iOFV): After the cytotoxicity test of the ATR inhibitor VE822, it was loaded with iOF by ultrasonic incubation to obtain the iOFV nanosystem.

[0015] The third aspect of the present invention relates to the use 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, which can inhibit tumor growth by enhancing radiation-induced DNA damage, inhibiting the DNA damage response of tumor cells, promoting tumor cell apoptosis and ferroptosis, activating immune response, and enhancing the infiltration of effector T cells into tumors.

[0017] Beneficial effects of the present invention:

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

[0019] 2. Synergistic sensitization to radiotherapy: The Fenton reaction of Fe3O4NPs and the inhibition of DNA damage repair by VE822 synergize to amplify the degree of DNA damage, continuously inhibit DDR, significantly reduce the radiation resistance of tumor cells, and enhance the effect of radiotherapy.

[0020] 3. Activate immune response: Continuous DNA damage triggers cell apoptosis and ferroptosis, while activating the cGAS-STING immune pathway, promoting dendritic cell maturation, enhancing antigen presentation and T cell activation, and attracting CD4 T cells. 3+ , CD 4+ and CD 8+ T cell infiltration and enhanced anti-tumor immune response.

[0021] 4. Good biosafety: In vivo experiments have shown that iOFV has no adverse effects on the hematological parameters and liver and kidney functions of mice, does not induce hemolysis, and can be effectively cleared by the kidneys. It has a high systemic application safety in cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0023] Figure 2 : Particle size distribution 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 ± SD (n = 3 independent samples).

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

[0026] Figure 5 : Electron spin resonance (ESR) spectra of hydroxyl radical (·OH) under different conditions (iOFV=100μg / mL, H2O2=100mM). The data were processed using GraphPadPrism9.3 software and expressed as mean±SD.

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

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

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

[0030] Fig. 9 :Key marker detection results diagram (a).

[0031] Fig.10 :Key marker detection results diagram (II).

[0032] Fig.11 :Key marker detection results diagram (III).

[0033] Fig.12 :Key marker detection results diagram (IV).

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

[0035] Fig.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 in each group.

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

[0037] Fig.16 : Iron content in mouse urine 48 hours after iOFV injection. n = 3 biologically independent mice per group.

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

[0039] Fig.18 : Hemolytic assay of iOFV in mice at 0, 1, 3, and 5 days after iOFV injection.

[0040] Fig.19 : Weight measurement of major organs of mice at 0, 1, 3, and 5 days after iOFV injection.

[0041] Fig. 20 :Radioimmunotherapeutic effect of iOFV in vivo (I).

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

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

[0044] Fig.23 :Radioimmunotherapy effect of iOFV in vivo (IV). DETAILED DESCRIPTION

[0045] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. However, the following embodiments are limited to explaining the present invention, and the protection scope of the present invention should include the entire contents of the claims, and through the description of the following embodiments, those skilled in the art can fully implement the entire contents of the claims of the present invention.

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

[0047] The present invention is further described with reference to the accompanying drawings and embodiments.

[0048] 1. Preparation and characterization of iOFV

[0049] A method for preparing a nanosystem iOFV for radioimmunotherapy comprises the following steps:

[0050] (1) Construction of engineered bacteria expressing iRGD peptide:

[0051] Through in vitro gene synthesis technology, a 1011bp double-stranded DNA fragment (ClyA-Myc-iRGD) containing the iRGD (CRGDKGPDC) sequence was precisely synthesized, as shown in SEQ ID NO.1 in the sequence table, and it was connected to the pGEX-4T1 plasmid using restriction endonucleases to construct a recombinant plasmid. Subsequently, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, and ampicillin was used for screening and purification to obtain a high-purity transformed strain. The transformed strain was cultured, and when the optical density (OD600) of the strain reached 0.5-0.6, isopropyl β-D-1-thiogalactoside (IPTG) was added at a final concentration of 0.2mM, and incubated at 20°C overnight to induce protein expression. Afterwards, the bacteria were harvested by centrifugation at 4°C and 5000×g for 30 minutes, and the expression of Myc-tagged protein in transgenic BL21 (DE3) was verified by Western blotting technology, and low-toxic Escherichia coli expressing iRGD peptide on the surface was successfully obtained.

[0052] (2) Preparation of Fe3O4NPs modified with glucose polymer:

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

[0054] Then 1g of amino-functionalized Fe3O4 nanoparticles was dispersed in 50mL of deionized water, and 0.5g of glucose polymer was dissolved in 50mL of water and slowly added dropwise, and reacted at room temperature for 6 hours. The product was centrifuged, washed with deionized water, and vacuum dried for 12 hours to obtain glucose polymer-modified Fe3O4NPs (GP-Fe3O4NPs).

[0055] (3) Obtaining OMVs containing Fe3O4NPs (iOF):

[0056] 150 ml of engineered Escherichia coli (1.0 × 10 7 The suspension of 1000 CFU (1000 μg / mL) and 8.0 mg / mL GP-Fe3O4NPs were incubated in a shaking incubator at 37°C and 150 rpm for 6 hours to allow the nanoparticles to be fully engulfed by bacteria. The mixture was centrifuged at 1000 × g for 5 minutes, the unbound nanoparticles were discarded, and the mixture was washed 3 times with PBS. Lysozyme (20 mg / mL) was added and ultrasonic treatment (ice bath for 12 hours) was performed to promote bacterial lysis and release of outer membrane vesicles (OMVs). The suspension was filtered through 100 nm and 220 nm polycarbonate membranes in turn, and then centrifuged at 12000 × g to purify iRGD-OMV@Fe3O4 to obtain iOF.

[0057] (4) Preparation of the final nanosystem (iOFV):

[0058] The ATR inhibitor VE822 was tested for cytotoxicity to tumor cells and normal cells. iOF was incubated with VE822 (320 nM) at 4°C in the dark for 6 hours to prepare iOFV. The preparation can be stored at 4°C for 1 week, during which the stability and activity are regularly tested.

[0059] (5) Material characterization:

[0060] The morphology of Fe3O4NPs, iOMVs and iOMV@Fe3O4 was examined by field emission scanning electron microscopy (SEM, ZEISSSIGMA) and transmission electron microscopy (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) were performed using Talos F200X and JEOLArm-200F to obtain elemental composition and microstructural information. UV-visible absorption spectra were recorded using a UV-1601 spectrophotometer, and iron content was determined by inductively coupled plasma mass spectrometry (ICP-MS, ThermoFisheriCAPQc). 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 solutions of different polarities were detected, the drug loading efficiency and UV absorption properties were retested by UV-visible spectroscopy, and the protein concentration of iOFV stored at 4°C for 2 weeks was detected.

[0061] 2. Performance testing and application

[0062] 2.1 Materials and methods

[0063] (1) Cell lines and animals

[0064] 4T1 mouse breast cancer cell line and LO2 human normal liver cell line were cultured in RPMI-1640 and DMEM medium supplemented with 10% fetal bovine serum (FBS) and 100U / mL penicillin-streptomycin, respectively. The cells were cultured in a humidified incubator at 37°C with 21% O2 and 5% CO2. Depending on the growth of the cells, the culture medium was replaced every 2 to 3 days, and cells with stable growth and in the 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 a temperature of 22-26°C, humidity of 40-70%, and a light / dark cycle of 12 hours. 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, mice were euthanized using CO2.

[0066] (2) In vitro detection of reactive oxygen species and OH free radicals

[0067] 4T1 cells were seeded and cultured under standard conditions for 12 hours, and then the medium was changed. Nanoparticles were added at a final concentration of 200 μg / mL (in terms of Fe3O4), and the cells were incubated for another 12 hours. The cells were then irradiated with 6 Gy and incubated for an additional 24 hours. Intracellular ROS levels were assessed using DCFH-DA (a reactive oxygen species probe) staining. ROS generation was observed under a confocal microscope.

[0068] 60 μL of nanoparticle solution (800 μg / mL) was mixed with 20 μL of 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 acetate buffer (pH 3.6) and 60 μL of 10% H2O2 were added. The generation of ·OH in the reaction was analyzed using electron paramagnetic resonance (EPR).

[0069] (3) Cytotoxicity assay

[0070] The cell counting kit 8 (CCK8) was used to detect the cytotoxicity of iOFV to LO2 and 4T1 cells. LO2 and 4T1 cells were seeded in 96-well plates and incubated for 12 hours under standard conditions to allow them to adhere. Different concentrations of iOFV were added and incubated for 24 hours. According to the instructions of the CCK8 kit, the absorbance at 450nm was measured with a microplate reader to evaluate cell viability. Cell viability was calculated according to the following formula:

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

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

[0073] (4) Evaluation of radiosensitization effect

[0074] 4T1 cells were incubated with the same concentrations of iOF and iOFV, and cell viability was measured after or without radiotherapy. The sensitivity of tumor cells to ionizing radiation was evaluated by cell colony formation assay. 4T1 cells were plated at 10 3The cells were seeded in 6-well plates at a sparse density and cultured for 48 hours to allow the cells to form a stable cell colony. After that, the culture medium was replaced with fresh medium containing NPs at a final concentration of 100 μg / mL (based on Fe3O4) and incubated for another 24 hours. The cell accelerator (Clinacix, Varian, USA) was then irradiated with linear radiation. After irradiation, the cells continued to be 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, and the cell survival rate was determined. The survival data were nonlinearly fitted using the "multi-target-single hit" model to calculate the quasi-threshold dose (Dq) and sensitization enhancement ratio (SER).

[0075] (5) Key marker detection

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

[0077] (6) Cellular uptake and targeting verification

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

[0079] To verify the active targeting effect of the nanosystem iOFV, 4T1 cells resistant to conventional radiotherapy were used, and fluorescein isothiocyanate (FITC)-labeled OMVs were incubated with 4T1 cells, and cellular uptake was assessed quantitatively using fluorescence microscopy, flow cytometry, and fluorescence techniques. OFV and iOFV were labeled with DiR and intravenously injected into breast tumor-bearing mice, and 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) Pharmacokinetics and biodistribution studies

[0081] Before the experiment, mice were acclimated to the laboratory environment for 1 week. To evaluate tumor growth, 1×10 64T1 cells were suspended in PBS and injected subcutaneously into the right flank of each mouse at 37°C. After 14 days, 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 through the tail vein. The next day, the tumor site was irradiated under anesthesia of the mice, with a source skin distance (SSD) of 100 cm. 6 MVX-rays were used with a dose rate of 400 cGy min -1 , giving a dose of 6Gy. The maximum accumulation area of ​​the 6MV linear accelerator photon beam is located 1.0cm below the skin surface. Tumor volume and body weight are monitored every 2 days; tumor volume is measured with a caliper and calculated using the formula:

[0082] V=a×b 2 / 2;

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

[0084] On the eighth day of treatment, two mice from each group were randomly selected for euthanasia to evaluate immune parameters. Peripheral blood, urine, tumors, and major organs (including heart, liver, spleen, lungs, and kidneys) were collected, dried, weighed, digested with aqua regia, and diluted with ultrapure water. Iron concentrations in the samples were determined using ICP-MS.

[0085] (8) Biosafety assessment

[0086] Healthy female ICR mice aged 5 weeks were selected and iOFV was injected through 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 that affect 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, iOF, iOFV, IR, iOF+IR, and iOFV+IR. Radiotherapy (6Gy) was performed 12 hours after intravenous injection of the corresponding material. Throughout the study, tumor growth and body weight changes were strictly monitored, and tumor volume was accurately measured with a caliper, and the calculation formula was:

[0089] V=a×b 2 / 2;

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

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

[0092] (10) Research on immune mechanisms

[0093] The tumor-bearing mice were divided into seven treatment groups: control, iOF, iOFV, IR, iOF+IR, iOFV+IR, and iOMV+IR. After 14 days of treatment, the immune profile of tumor tissues was analyzed by flow cytometry, and the maturation markers of dendritic cells (DCs) were detected. 80 , CD 86 The expression of CD4+ T cells in tumor tissue, blood and spleen 3+ , CD 4+ and CD 8+ The infiltration degree of T cells. The levels of IL-6 and TNF-α in tumor tissues were detected by ELISA to evaluate the activation of the cGAS-STING pathway.

[0094] (11) Statistical analysis

[0095] Unpaired t-test was used to compare the differences between two groups, while one-way ANOVA was used to compare the differences between multiple groups. Data are presented as mean ± standard deviation (SD). A p value < 0.05 was considered statistically significant.

[0096] 3. Results Analysis

[0097] 3.1 Characterization of iOFV

[0098] The structures of Fe3O4NPs, iOMV and iOMV@Fe3O4 were characterized by field emission scanning electron microscopy (SEM, ZEISSSIGMA) and transmission electron microscopy (TEM, JEOLJEM-1400).

[0099] The average particle size of Fe3O4NPs is about 5nm, with good dispersibility; iOMV and iOMV@Fe3O4 are spherical, with sizes of about 100.5nm and 152.6nm, respectively; the encapsulation efficiencies of Fe3O4NPs and VE822 are 46.5% and 0.0182% (wt%), respectively, as shown in Table 1; iOFV has good stability in polar solution after 12 hours, and the protein concentration changes little after being stored at 4℃ for 2 weeks, which shows that it has long-term storage characteristics. These results are all in Figure 1 , Figure 2 and Figure 3 is displayed intuitively.

[0100] Table 1

[0101] 3.2 Performance Testing

[0102] (1) In vitro detection of reactive oxygen species and OH free radicals

[0103] As the H2O2 concentration increases, ROS gradually increases, showing a H2O2 concentration-dependent relationship, such as Figure 4 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 the test system, and the ability of iOFV to produce radiotherapy-induced ·OH was tested using electron spin resonance (ESR) 13. The results are shown in Figure 5 As shown. In the absence of H2O2, 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 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 under the induction of radiotherapy increases, and more ·OH is generated through the Fenton reaction.

[0104] (2) Cytotoxicity assay

[0105] The results showed that the survival rate of LO2 cells co-cultured with 200 μg / mL LiOFV was still higher than 80%, indicating that iOFV has good biocompatibility; however, as the concentration of iOFV increased, the viability of 4T1 cells gradually decreased, showing tumor-specific toxicity. Figure 6 The bar graph of relative cell activity after cells were treated with different concentrations of iOFV clearly shows the trend of the impact of different concentrations of iOFV on the viability of the two cells, and intuitively reflects the biocompatibility and tumor-specific toxicity of iOFV.

[0106] (3) Evaluation of radiosensitization effect

[0107] like Figure 7 As shown in the figure, with the increase of iOF and iOFV concentrations, the viability of 4T1 cells decreased more significantly under radiotherapy; the Dq of iOFV was 0.81Gy and the SER was 3.29, showing strong radiosensitization ability. Figure 8 It can be clearly seen from the 4T1 cell survival rate curves and cell colony images under different treatments that the cell survival rate of the iOFV-treated group after radiotherapy was significantly lower than that of other groups, and the number of cell colonies was significantly reduced, which fully demonstrated the radiosensitization effect of iOFV.

[0108] (4) Key marker detection

[0109] Radiotherapy plus iOFV combined treatment caused obvious mitochondrial dysfunction. The mitochondrial membrane in the iOFV+IR group was significantly reduced, the cell potential (MMP) was increased, accompanied by the release of cytochrome c and the activation of the apoptotic cascade; AnnexinV-FITC / PI staining confirmed that the proportion of apoptotic cells in the iOFV+IR group increased significantly; C11-BODIPY staining showed that the iOFV+IR group had the highest level of lipid peroxidation; Western blot analysis showed that iOFV+IR treatment significantly reduced the expression of Bcl-2 and GPX4. Fig. 9 , Fig.10 , Fig.11 and Fig.12 The images of mitochondrial membrane potential destruction, cell apoptosis level analysis, lipid peroxidation images, and immunoblot analysis and quantitative results of Bcl-2 and GPX4 protein expression showed that the iOFV+IR group was significantly different from the other groups, which strongly confirmed that iOFV combined with radiotherapy can induce mitochondrial damage, destroy cellular redox homeostasis, trigger lipid peroxidation, and ultimately lead to tumor cell apoptosis and ferroptosis, while enhancing the anti-tumor immune response by activating the cGAS-STING immune pathway.

[0110] (5) Cellular uptake and targeting verification

[0111] like Fig.13 As shown in the fluorescence microscopy images of the internalization of nanoparticles by cells, the fluorescence signal in the iOFV-treated group was significantly stronger than that in the OFV group, which intuitively demonstrated the effect of iRGD peptide in enhancing cellular uptake. Fig.14 It can be clearly seen from the fluorescence distribution in tumor-bearing mice that the fluorescence intensity of DiR-labeled iOFV at the tumor site gradually increased within 12 hours, reached a higher level after 24 hours and continued to increase, further confirming that iOFV has significant tumor targeting and that the iRGD peptide can effectively guide the nanosystem to accumulate at the tumor site.

[0112] (6) Pharmacokinetics and biodistribution studies

[0113] The results showed that the half-life of iOFV was about 3.8 hours, and it preferentially accumulated in the liver, kidneys, and tumor tissues. It was effectively cleared by the kidneys and excreted through urine to prevent long-term accumulation. It has good safety and systemic application potential. Fig.15 The histogram of organ iron distribution clearly shows the high accumulation of iOFV in liver, kidney and tumor tissues; Fig.16 As shown in the figure, the curve of urine iron content changing over time intuitively shows its excretion process through the kidneys, providing strong evidence for the safety and metabolic pathway of iOFV.

[0114] (7) Biosafety assessment

[0115] The results showed that the hematological parameters, liver function and renal function of mice treated with iOFV were not statistically different from those of the control group, no obvious hemolysis was induced, and there was no significant difference in the expression of key genes between the treatment group and the control group, confirming that iOFV has good biosafety in vivo. Fig.17 and Fig.18 The blood heat map and biochemical analysis data of mice shown in the figure show that there is no significant difference in various indicators between the treatment group and the control group; the hemolysis test results show no obvious hemolysis phenomenon, and the organ weight measurement data are shown in the figure. Fig.19 As shown, the weight of each organ was not significantly affected, which intuitively proves that iOFV has good biosafety.

[0116] (8) In vivo radioimmunotherapy

[0117] like Fig. 20 , Fig.21 , Fig. 22 and Fig.23 As shown, the iOFV+IR group had the smallest tumor volume, and H&E staining showed that the tumor cell organization was looser, which meant that tumor cell necrosis increased; TUNEL staining showed that tumor cell apoptosis increased significantly, among which the iOFV+IR group had the strongest red fluorescence; DCFH-DA probe detected significantly higher ROS levels in the iOF+IR and iOFV+IR groups, confirming that Fe3O4 nanoparticles enhanced the production of ROS after radiation; Immunofluorescence analysis of γ-H2AX expression showed that DNA breaks in these two groups were more obvious, indicating that iOFV significantly enhanced tumor radiosensitivity and promoted radiation-induced immune reconstruction. As shown in the tumor growth curve, the tumor volume growth in the iOFV+IR group was significantly slower than that in other groups, which intuitively reflected that this group had the best anti-tumor effect. 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 cell nucleus morphology was abnormal, indicating a high degree of tumor cell necrosis; in the TUNEL staining images, the iOFV+IR group showed a large number of red fluorescently labeled apoptotic cells, which were significantly more than those in other groups; the ROS level images detected by the DCFH-DA probe showed that the fluorescence intensity of the iOF+IR and iOFV+IR groups was significantly higher than that of the control group, intuitively demonstrating the effect of ROS production after Fe3O4 nanoparticle-enhanced radiation; in the immunofluorescence analysis of γ-H2AX expression, the fluorescence intensity of the iOF+IR and iOFV+IR groups was stronger, indicating that the DNA breakage in these two groups was more serious, which fully demonstrated the role of iOFV in enhancing tumor radiosensitivity and promoting immune reconstruction.

[0118] (9) Research on immune mechanisms

[0119] The results showed that Fe3O4 nanoparticles promoted the production of ROS and induced ferroptosis, enhanced antitumor immunity, and promoted DC maturation; in the iOF+IR and iOFV+IR groups, CD4+ / -2 in tumor tissue, blood, and spleen increased significantly. 3+ , CD 4+ and CD 8+ The infiltration of T cells was higher; the levels of IL-6 and TNF-α were significantly increased in these two groups, and the cGAS-STING pathway was significantly activated, leading to increased production of type I interferons (IFN-γ and IFN-β), further enhancing the anti-tumor immune response. Fig.18 The flow cytometry results of immune cell co-expression analysis in tumor tissue showed that CD 80+ , CD 86+ The proportion of DC cells in the iOF+IR and iOFV+IR groups was significantly higher than that in the other groups, indicating that the DCs in these two groups were more mature. The quantitative analysis chart of immune cell infiltration clearly showed that the CD4+CD26+ cells in the tumor tissue, blood and spleen in the iOF+IR and iOFV+IR groups 3+ , CD 4+ and CD 8+ The number of T cells was significantly higher than that in other groups. The bar graph of IL-6 and TNF-α levels in tumor tissues detected by ELISA showed that the cytokine levels in the iOF+IR and iOFV+IR groups were significantly increased, which intuitively reflected the activation of the cGAS-STING pathway. These results jointly confirmed that iOFV combined with radiotherapy can promote immune reconstitution and enhance anti-tumor immune response.

[0120] In summary, the present invention successfully prepared and fully characterized the iOFV nanosystem through a series of rigorous experiments. In in vitro experiments, iOFV showed good radiation sensitization 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 regimen of iOFV can be further optimized, and precise treatment can be performed 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 and references for in-depth research in related fields, which will help promote the continuous advancement of tumor treatment technology.

[0121] In terms of the expansion of practical application scenarios, this nanosystem can further explore the possibility of combined use with other treatment methods (such as chemotherapy drugs, immune checkpoint inhibitors, etc.), evaluate the efficacy of combined treatment plans on different types of tumors, and provide more options for comprehensive clinical tumor treatment. In terms of preparation process optimization, attempts can be made to improve the synthesis method of nanoparticles, the encapsulation technology of OMVs, and the modification strategy of iRGD peptides to improve the encapsulation efficiency, stability and targeting of the nanosystem, reduce production costs, and promote its transformation from laboratory research to clinical application. In view of the individual differences of different tumor patients, personalized treatment research is carried out, and the dosage and treatment plan of iOFV are accurately adjusted according to factors such as the patient's tumor type, stage, and gene expression spectrum, so as to improve the treatment effect while reducing adverse reactions and achieve true precision medicine.

[0122] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent 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 the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

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

2. The nanosystem for tumor radioimmunotherapy according to claim 1, characterized in that: The bacterial outer membrane vesicles (OMVs) are derived from the Escherichia coli BL21 (DE3) strain, which is 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 5nm, and the size of the OMVs (iOMV@Fe3O4) encapsulating the Fe3O4NPs is about 152.6nm.

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 to 4, characterized in that: The following steps are involved: A plasmid encoding the iRGD peptide and linked to Escherichia coli lysin A was constructed and transformed into Escherichia coli BL21 (DE3) competent cells. The bacteria were harvested after inducing protein expression. Fe3O4 nanoparticles were synthesized by coprecipitation and amino-functionalized and modified with glucose polymers. The modified Fe3O4 nanoparticles were incubated with engineered Escherichia coli to allow the bacteria to engulf the nanoparticles. The formation of outer membrane vesicles (OMVs) was triggered by peptidoglycan degradation, and the OMVs loaded with Fe3O4NPs were enriched by multiple differential centrifugation. The ATR inhibitor VE822 was loaded into the OMVs loaded with Fe3O4NPs.

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

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

8. The use according to claim 6, characterized in that: In the combined treatment of the nanosystem, iOFV combined with radiotherapy induces mitochondrial apoptosis by activating related signaling pathways such as endoplasmic reticulum stress, TNF-α, and DNA damage; at the same time, it promotes glutathione consumption, reduces GPX4 activity, leads to lipid peroxide accumulation, and induces ferroptosis.

9. The use according to claim 6, characterized in that: In the synergistic effect of the nanosystem, Fe3O4NPs generate ·OH through Fenton reaction under radiation to enhance 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.

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