Medical reagent based on cell membrane vesicle and preparation method and application thereof

Abstract

The application discloses a medical reagent based on cell membrane vesicles and a preparation method and application thereof, and relates to the technical field of biological medicine. The medical reagent comprises a nano-carrier modified with a metal-phenol network; the metal is a high-atomic-number metal. The medical reagent has good tumor targeting property, and has multiple functions such as relieving the hypoxic state of a tumor microenvironment, radiosensitization and immune activation, and can be used for tumor radioimmunotherapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a medical reagent based on cell membrane vesicles, its preparation method, and its application. Background Technology

[0002] High atomic number metal-based nanomaterials can produce a series of physical effects (such as photoelectric effect, Compton scattering, Rayleigh scattering, etc.) under X-ray irradiation, and are often used in the preparation of medical reagents. Among them, the NBTXR3 radiosensitizer based on hafnium oxide (HfO2) nanoparticles has been extensively studied in clinical practice, but its intratumoral injection method limits the types of cancer it can be used for and its treatment efficiency. Therefore, it is very important to provide a new medical reagent. Summary of the Invention

[0003] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a medical reagent that can effectively improve the therapeutic effect of radiotherapy.

[0004] The present invention also provides a method for preparing the above-mentioned medical reagent.

[0005] The present invention also provides a medicine comprising the above-described medical reagent.

[0006] The present invention also provides the application of the above-mentioned medical reagents or drugs in combination with radiation irradiation.

[0007] The present invention also provides applications of the above-mentioned medical reagents or drugs.

[0008] According to a first aspect of the present invention, the medical reagent comprises a nanocarrier modified with a metal-phenol network;

[0009] The metal is a high atomic number metal.

[0010] The medical reagents according to embodiments of the present invention have at least the following beneficial effects:

[0011] The medical reagents described in this embodiment have excellent tumor targeting properties, can alleviate the hypoxic state of the tumor microenvironment, promote the maturation of dendritic cells (DCs), and the infiltration and activation of cytotoxic T cells. They can be used as radiosensitizers and immune adjuvants to enhance radioimmunotherapy, effectively inhibit tumor growth, and are used for radioimmunotherapy of tumors.

[0012] According to some embodiments of the present invention, the atomic number of the high atomic number metal is not less than 60.

[0013] According to some embodiments of the present invention, the atomic number of the high atomic number metal is 60-85. For example, it can be 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 or 85.

[0014] According to some embodiments of the present invention, the high atomic number metal includes at least one selected from neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, thallium, lead, bismuth, and uranium. Isotopes of the high atomic number metal are also within the scope of protection of the present invention.

[0015] According to some embodiments of the present invention, the high atomic number metal includes at least one selected from gadolinium, hafnium, tungsten, gold, bismuth, palladium, platinum, tantalum, gallium, tellurium, and zirconium.

[0016] According to some embodiments of the present invention, the phenol includes polyphenols.

[0017] According to some embodiments of the present invention, the polyphenols include compounds with molecular chemical structures conforming to the definition of White-Bate-Smith-Swain-Haslam (WBSSH).

[0018] According to some embodiments of the present invention, the polyphenols include tannic acid, catechins, dopamine and its condensation forms, tannin molecules, epicatechin, galloglycolic acid catechin, epigallocatechin, anthocyanins, anthraquinones, chalcones, lignans, naphthoquinones, neolignans, proanthocyanidins, pigment tannic acid, xanthones, condensates of polyphenols synthesized by plant biosynthesis or artificial fermentation, theaflavins, polymers of anthocyanins, highly condensed tannins, and artificially synthesized molecules containing polyphenol groups, or combinations thereof.

[0019] According to some embodiments of the present invention, the catalase activity of the nanocarrier is not less than 15 U / mg protein. For example, it can be 15 U / mg, 16 U / mg, 17 U / mg, 18 U / mg, 19 U / mg, 20 U / mg, 21 U / mg, 22 U / mg, 23 U / mg, 24 U / mg, 25 U / mg, 26 U / mg, 27 U / mg, 28 U / mg, 29 U / mg, or 30 U / mg.

[0020] According to some embodiments of the present invention, the nanocarrier includes at least one of vesicles and synthetic nanoparticles.

[0021] According to some embodiments of the present invention, the vesicles are isolated from biological cells.

[0022] According to some embodiments of the present invention, the biological cells include at least one of prokaryotic cells and eukaryotic cells.

[0023] According to some embodiments of the present invention, the prokaryotic cells include at least one of cyanobacteria and bacteria. The bacteria include Gram-negative bacteria. The bacteria include, but are not limited to, Escherichia coli, Salmonella, or Lactobacillus.

[0024] According to some embodiments of the present invention, the eukaryotic cells include at least one of yeast, insect cells, mammalian cells, and plant cells. The yeast includes, but is not limited to, *Saccharomyces cerevisiae* or *Pichia pastoris*. The insect cells include, but are not limited to, Sf9 cells or SF21 cells. The mammalian cells include, but are not limited to, HEK293 cells, CHO cells, or Vero cells. The plant cells are derived from, but are not limited to, lemon, tobacco, *Arabidopsis thaliana*, or rice.

[0025] According to some embodiments of the present invention, the average diameter of the nanocarrier is 100 nm to 150 nm. The average diameter is obtained by nanoparticle tracking and analysis (NTA). For example, it can be 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.

[0026] According to some embodiments of the present invention, the hydrated particle size of the nanocarrier is 50nm-200nm. For example, it can be 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, 150nm, 155nm, 160nm, 165nm, 170nm, 175nm, 180nm, 185nm, 190nm, 195nm, or 200nm.

[0027] According to some embodiments of the present invention, the hydrated particle size of the nanocarrier is 150nm-180nm.

[0028] According to some embodiments of the present invention, those skilled in the art can select methods known in the art to separate the vesicles. These methods include, but are not limited to, differential centrifugation, density gradient centrifugation, ultrafiltration, size exclusion chromatography, immunoaffinity chromatography, microfluidics, or acoustic separation.

[0029] According to some embodiments of the present invention, each 100 μg nanocarrier in the medical reagent contains 1 μg-25 μg of high atomic number metal. For example, each 100 μg of nanocarrier in the medical reagent may contain 1 μg, 1.5 μg, 2 μg, 2.5 μg, 3 μg, 3.5 μg, 4 μg, 4.5 μg, 5 μg, 5.5 μg, 6 μg, 6.5 μg, 7 μg, 7.5 μg, 8 μg, 8.5 μg, 9 μg, 9.5 μg, 10 μg, 10.5 μg, 11 μg, 11.5 μg, 12 μg, 12.5 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22 μg, 23 μg, 24 μg, or 25 μg of high atomic number metal.

[0030] According to some embodiments of the present invention, each 100 μg nanocarrier in the medical reagent contains 5 μg-25 μg of high atomic number metal.

[0031] According to some embodiments of the present invention, the hydrated particle size of the nanocarrier modified with the metal-phenol network is 150nm-300nm. For example, it can be 150nm, 155nm, 160nm, 165nm, 170nm, 175nm, 180nm, 185nm, 190nm, 195nm, 200nm, 205nm, 210nm, 215nm, 220nm, 225nm, 230nm, 235nm, 240nm, 245nm, 250nm, 255nm, 260nm, 265nm, 270nm, 275nm, 280nm, 285nm, 290nm, 295nm, or 300nm.

[0032] According to some embodiments of the present invention, the zeta potential of the nanocarrier modified with the metal-phenol network is -40 mV to -30 mV. For example, it can be -40 mV, -39 mV, -38 mV, -37 mV, -36 mV, -35 mV, -34 mV, -32 mV, -31 mV, or -30 mV.

[0033] According to some embodiments of the present invention, the average diameter of the nanocarrier modified with the metal-phenol network is 150 nm to 300 nm. The average diameter is obtained by NTA analysis. For example, it can be 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, or 250 nm.

[0034] According to some embodiments of the present invention, the medical reagent includes at least one of a sensitizer, an adjuvant, and a cytotoxic substance.

[0035] According to some embodiments of the present invention, the sensitizer includes a radiotherapy sensitizer.

[0036] According to some embodiments of the present invention, the adjuvant includes an immune adjuvant.

[0037] The method for preparing the medical reagent according to the first aspect embodiment of the second aspect of the present invention includes the following steps:

[0038] A mixture of the nanocarrier, phenol, and a high atomic number metal ion source was prepared and reacted to obtain the medical reagent.

[0039] The preparation method according to embodiments of the present invention has at least the following beneficial effects:

[0040] The preparation method of the embodiment is simple and fast.

[0041] According to some embodiments of the present invention, the preparation method includes:

[0042] A first mixture comprising the nanocarrier and phenol is prepared; a second mixture comprising a high atomic number metal ion source and the first mixture is prepared, and the mixture is reacted to obtain the medical reagent.

[0043] According to some embodiments of the present invention, the mass ratio of the nanocarrier to phenol is 1:(2-4). For example, it can be 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, or 1:4.

[0044] According to some embodiments of the present invention, the mass ratio of the phenol to the high atomic number metal ion source is (2-4):1. For example, it can be 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, or 4:1.

[0045] According to some embodiments of the present invention, the reaction temperature is 1℃-10℃. For example, it can be 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃ or 10℃.

[0046] According to some embodiments of the present invention, the reaction time is 3 min to 20 min. For example, it can be 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, or 20 min.

[0047] According to some embodiments of the present invention, the ultrasonic power of the reaction is 50W-90W. For example, it can be 50W, 52W, 54W, 56W, 58W, 60W, 62W, 64W, 66W, 68W, 70W, 72W, 74W, 76W, 78W, 80W, 82W, 84W, 86W, 88W, or 90W.

[0048] According to some embodiments of the present invention, the phenol includes polyphenols.

[0049] According to some embodiments of the present invention, the polyphenols include compounds with molecular chemical structures conforming to the definition of White-Bate-Smith-Swain-Haslam (WBSSH).

[0050] According to some embodiments of the present invention, the polyphenols include tannic acid, catechins, dopamine and its condensation forms, tannin molecules, epicatechin, galloglycolic acid catechin, epigallocatechin, anthocyanins, anthraquinones, chalcones, lignans, naphthoquinones, neolignans, proanthocyanidins, pigment tannic acid, xanthones, condensates of polyphenols synthesized by plant biosynthesis or artificial fermentation, theaflavins, polymers of anthocyanins, highly condensed tannins, and artificially synthesized molecules containing polyphenol groups, or combinations thereof.

[0051] According to some embodiments of the present invention, the high atomic number metal ion source includes a compound containing the high atomic number metal.

[0052] According to some embodiments of the present invention, compounds containing the high atomic number metal are soluble in water.

[0053] According to some embodiments of the present invention, compounds containing the high atomic number metal include at least one of halides (including but not limited to chlorides, bromides or iodides) and sulfates.

[0054] According to some embodiments of the present invention, when the nanocarrier is a vesicle derived from biological cells, the method for preparing the nanocarrier includes the following steps:

[0055] The nanocarrier is obtained by contacting biological cells with a catalase inducer and reacting them.

[0056] According to some embodiments of the present invention, the biological cells include at least one of prokaryotic cells and eukaryotic cells.

[0057] According to some embodiments of the present invention, the prokaryotic cells include at least one of cyanobacteria and bacteria. The bacteria include, but are not limited to, Escherichia coli, Salmonella, or Lactobacillus.

[0058] According to some embodiments of the present invention, the eukaryotic cells include at least one of yeast, insect cells, mammalian cells, and plant cells. The yeast includes, but is not limited to, *Saccharomyces cerevisiae* or *Pichia pastoris*. The insect cells include, but are not limited to, Sf9 cells or SF21 cells. The mammalian cells include, but are not limited to, HEK293 cells, CHO cells, or Vero cells. The plant cells are derived from, but are not limited to, lemon, tobacco, *Arabidopsis thaliana*, or rice.

[0059] According to some embodiments of the present invention, the biological cells are *Escherichia coli*; the OD of the biological cells in the reaction system 600 The range is 0.5-2. For example, it can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.

[0060] According to some embodiments of the present invention, the catalase inducer includes an oxidant. Those skilled in the art may also select other substances that can affect the catalase synthesis pathway to act on biological cells to increase the catalase level in the nanocarrier.

[0061] According to some embodiments of the present invention, the oxidant includes, but is not limited to, hydrogen peroxide (H2O2) or molecular oxygen.

[0062] According to some embodiments of the present invention, the effective concentration of hydrogen peroxide is not less than 5 μM. For example, it can be 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, or 200 μM.

[0063] According to some embodiments of the present invention, the effective concentration of the hydrogen peroxide is 5 μM-200 μM.

[0064] According to some embodiments of the present invention, when the nanocarrier is a vesicle derived from biological cells, the method for preparing the nanocarrier includes: the contact time being 12 hours or more.

[0065] According to some embodiments of the present invention, the contact time is 12h-48h. For example, it can be 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h, or 48h.

[0066] According to some embodiments of the present invention, the preparation method further includes post-reaction processing. The post-reaction processing includes separating the biological cells to obtain a liquid phase and separating at least one of the nanocarriers from the liquid phase. The method for separating the nanocarriers from the liquid phase includes centrifugation.

[0067] According to some embodiments of the present invention, the centrifugation temperature is 1℃-10℃. For example, it can be 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃ or 10℃.

[0068] According to some embodiments of the present invention, the centrifugation speed is 4000×g-6000×g. For example, it can be 4000×g, 4500×g, 5000×g, 5500×g or 6000×g.

[0069] According to some embodiments of the present invention, the centrifugation time is 10 min to 30 min. For example, it can be 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, or 30 min.

[0070] A medicament according to a third aspect of the present invention comprises the medical reagent described in the first aspect embodiment and pharmaceutically acceptable excipients or auxiliary ingredients. Since the medicament employs all the technical solutions of the medical reagent described above, it possesses at least all the beneficial effects brought about by the technical solutions of the above embodiments.

[0071] According to some embodiments of the present invention, the auxiliary component includes at least one of other tumor radiosensitizers and immune checkpoint inhibitors. The auxiliary component is preferably chosen so as not to affect the efficacy of the medical reagent described in the first aspect embodiment.

[0072] According to some embodiments of the present invention, the immune checkpoint inhibitor includes at least one of PD-1 antibody, PD-L1 antibody, CTLA4 antibody, LAG3 antibody, TIM3 antibody, and TIGIT antibody.

[0073] According to some embodiments of the present invention, the dosage form of the drug includes at least one of tablets, injections, capsules, granules, and powders.

[0074] According to some embodiments of the present invention, the drug is administered by injection or oral administration.

[0075] The application of the medical reagent described in the first aspect embodiment of the fourth aspect of the present invention or the drug combined with radiation irradiation described in the third aspect embodiment of the present invention in any one of A1) to A4):

[0076] A1) Preparation of cell-killing products;

[0077] A2) Prepare products that induce apoptosis;

[0078] A3) Prepare products that inhibit cell proliferation;

[0079] A4) Prepare products that induce cellular DNA damage.

[0080] According to some embodiments of the present invention, the cells in A1) to A4) include abnormal cells.

[0081] According to some embodiments of the present invention, the abnormal cells include at least one of tumor cells and inflammatory cells.

[0082] According to some embodiments of the present invention, the tumor cells include at least one of breast cancer cells, thyroid cancer cells, pituitary tumor cells, gastric cancer cells, melanoma cells, non-small cell lung cancer cells, cervical cancer cells, liver cancer cells, nasopharyngeal cancer cells, and glioma cells.

[0083] According to some embodiments of the present invention, the radiation includes at least one of alpha rays, beta rays, gamma rays, X-rays, neutrons, electron beams, proton beams, and particle beams.

[0084] According to some embodiments of the present invention, the single radiation dose is 1 Gy-8 Gy. For example, it can be 1 Gy, 1.5 Gy, 2 Gy, 2.5 Gy, 3 Gy, 3.5 Gy, 4 Gy, 4.5 Gy, 5 Gy, 5.5 Gy, 6 Gy, 6.5 Gy, 7 Gy, 7.5 Gy, or 8 Gy.

[0085] According to some embodiments of the present invention, the medical reagent or the drug is used 8-24 hours before radiation irradiation. For example, it can be 8h, 8.5h, 9h, 9.5h, 10h, 10.5h, 11h, 11.5h, 12h, 12.5h, 13h, 13.5h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, or 24h.

[0086] According to some embodiments of the present invention, the product includes at least one of a drug, a reagent, and a reagent kit.

[0087] The use of the medical reagent described in the first aspect embodiment of the fifth aspect of the present invention or the drug described in the third aspect embodiment of the present invention in any one of B1) to B2):

[0088] B1) Prepare drugs to improve the radiosensitivity of tumors;

[0089] B2) Prepare drugs to improve the effect of tumor radiotherapy.

[0090] According to some embodiments of the present invention, the improvement in tumor radiotherapy efficacy in B2) includes at least one of C1) to C3):

[0091] C1) inhibits tumor growth;

[0092] C2) Increase the proportion of mature dendritic cells (DCs) and CD3 in the tumor microenvironment. + CD45 + CD8 + T cell percentage, GranB + CD8 + At least one of the following: the proportion of T cells and the proportion of memory T cells;

[0093] C3) Increases at least one of the following levels in tumors: TNF-α, IFN-γ, and IL-6.

[0094] According to some embodiments of the present invention, the tumor includes a solid tumor.

[0095] According to some embodiments of the present invention, the solid tumor includes at least one of breast cancer, thyroid cancer, pituitary adenoma, gastric cancer, melanoma, non-small cell lung cancer, cervical cancer, liver cancer, nasopharyngeal carcinoma, and glioma.

[0096] According to some embodiments of the present invention, the radiation involved in the radiotherapy includes at least one of alpha rays, beta rays, gamma rays, X-rays, neutrons, electron beams, proton beams, and particle beams.

[0097] According to some embodiments of the present invention, the radiotherapy is selected from external beam radiation therapy.

[0098] According to some embodiments of the present invention, the radiotherapy method is selected from conventional fractionated radiotherapy.

[0099] According to some embodiments of the present invention, the single irradiation dose of the radiotherapy is 1 Gy-8 Gy. For example, it can be 1 Gy, 1.5 Gy, 2 Gy, 2.5 Gy, 3 Gy, 3.5 Gy, 4 Gy, 4.5 Gy, 5 Gy, 5.5 Gy, 6 Gy, 6.5 Gy, 7 Gy, 7.5 Gy or 8 Gy.

[0100] According to some embodiments of the present invention, the medical reagent or the drug is used within 72 hours before radiotherapy. For example, it can be used at 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, or 72 hours.

[0101] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0102] Figure 1 This is a schematic diagram illustrating the application of the metal-phenol outer membrane vesicles (Hf-OMVs) of the present invention in tumor radioimmunotherapy; (A) Schematic diagram of the preparation process of Hf-OMVs; (B) Schematic diagram of the principle of Hf-OMVs in tumor radioimmunotherapy;

[0103] Figure 2 The following are the preparation and characterization results of Hf-OMVs: (A) Schematic diagram of OMVs secreted by Escherichia coli; (B) TEM image of OMVs after uranyl acetate staining (the inset in the lower left corner represents the magnified TEM image, scale bar, 50 nm); (C) NTA characterization results of OMV particle size distribution; (D) UV-Vis absorption spectra of TA, OMVs, and OMVs@TA (dashed line indicates the absorption peak of TA at 280 nm); (E) Inductively coupled plasma optical emission spectrometry (ICP-OES) analysis results of medical reagents prepared with different concentrations of HfCl4; (F) Fourier transform infrared (FT-IR) spectra of TA, OMVs, and Hf-OMVs (dashed lines indicate the 2925 cm⁻¹). -1 and 1710cm -1 (G) X-ray photoelectron spectroscopy (XPS) images of OMVs and Hf-OMVs, (H) High-resolution XPS image of Hf 4f in Hf-OMVs, (I) SDS-PAGE analysis results of OMVs and Hf-OMVs, (J) TEM image of Hf-OMVs after uranyl acetate staining (the inset in the lower left corner represents the magnified TEM image, scale bar, 50 nm), (K) DLS characterization results of particle size distribution of OMVs and Hf-OMVs, (L) Hydrated particle size characterization results of OMVs and Hf-OMVs, (M) Zeta potential characterization results of OMVs and Hf-OMVs;

[0104] Figure 3 The UV-Vis absorption spectra of TA and Hf-OMVs are shown (the dashed lines represent the absorption peaks at 280 nm and 307 nm, respectively).

[0105] Figure 4 XPS spectra of OMVs;

[0106] Figure 5 High-angle annular dark field (HAADF) images of Hf-OMVs and corresponding energy-dispersive spectroscopy (EDS) elemental distribution maps;

[0107] Figure 6 The results of relative catalase activity analysis of OMVs produced by Escherichia coli with and without H2O2 stress;

[0108] Figure 7 The results of the performance test of different OMVs catalyzing oxygen production from H2O2 are as follows: (A) Oxygen production curves of OMVs extracted under H2O2 stress with and without H2O2 stress; (B) Statistical graph of O2 concentration after 5 min of reaction in Figure A; (C) O2 production curves of OMVs catalyzing different concentrations of H2O2; (D) Statistical graph of O2 concentration after 5 min of reaction in Figure C; (E) O2 production curves of OMVs catalyzing different concentrations of H2O2; (F) Statistical graph of O2 concentration after 5 min of reaction in Figure E.

[0109] Figure 8 The results show the performance of different OMVs and Hf-OMVs in relieving tumor cell hypoxia in vitro; (A) Fluorescence pattern of HIF-1α in 4T1 cells after 8 h of treatment (scale bar: 20 μm), (B) Statistical analysis results of the relative fluorescence intensity of pattern A, (C) Fluorescence pattern of HIF-1α in 4T1 cells after 24 h of treatment (scale bar: 10 μm), (D) Statistical analysis results of the relative fluorescence intensity of pattern C;

[0110] Figure 9 The results of the in vivo performance test of OMVs prepared in Example 1 and Hf-OMVs prepared in Example 1 in relieving tumor hypoxia; (A) Immunofluorescence map of HIF-1α in tumor tissue after different treatments (scale bar: 100 μm), (B) Statistical analysis results of the relative fluorescence intensity of map A.

[0111] Figure 10 The data show the cellular uptake of Hf-OMV by 4T1 cells; (A) Flow cytometry results, (B) ICP-OES analysis results, and (C) Confocal fluorescence microscopy analysis results (scale bar: 10 μm).

[0112] Figure 11 The results of the cytotoxicity test of Hf-OMV prepared in Example 1 on 4T1 cells;

[0113] Figure 12The results of the in vitro radiotherapy (RT) sensitization effect test of Hf-OMVs prepared in Example 1 are shown; (A) Schematic diagram of the radiotherapy sensitization experiment, (B) IC50 of Hf-OMVs on 4T1 cells after combined radiotherapy. 50 (C) Relative viability of 4T1 cells after radiotherapy with different concentrations of OMVs, Hf-TA and Hf-OMVs; (D) Flow cytometry representation of apoptosis after different treatments; (E) Statistical results of apoptosis rate in Figure D; (F) Representative photographs of cell colony formation experiment and statistical analysis results of relative clonal area; (G) Fluorescence map of γ-H2AX in 4T1 cells after different treatments; (H) Statistical results of relative fluorescence intensity in Figure G.

[0114] Figure 13 The hemolytic rate of erythrocytes at different concentrations of Hf-OMVs;

[0115] Figure 14 Blood biochemistry and complete blood count results of mice after intravenous injection of Hf-OMVs; (A) Alanine aminotransferase (ALT), (B) Aspartate aminotransferase (AST), (C) Alkaline phosphatase (ALP), (D) Uric acid (UA), (E) White blood cells (WBC), (F) Red blood cells (RBC), (G) Platelets (PLT), (H) Hemoglobin (HGB), (I) Hematocrit (HCT), (J) Mean corpuscular volume (MCV), (L) Mean corpuscular hemoglobin concentration (MCHC), (M) Mean corpuscular hemoglobin (MCH) (N) Hematoxylin-eosin (H&E) stained sections of heart, liver, spleen, lung and kidney (scale bar: 100 μm);

[0116] Figure 15 The results of in vivo tumor targeting analysis of OMVs; (A) IVIS fluorescence imaging and quantitative analysis of tumor sites at different time points; (B) IVIS fluorescence imaging and quantitative analysis of tumors and major organs.

[0117] Figure 16 Results of in vivo tumor targeting analysis of Hf-OMVs; (A) Schematic diagram of experimental procedure; (B) Hf in tumors 4+ Content analysis results;

[0118] Figure 17 The following are the in vivo anti-tumor effects of mice after different treatments: (A) Flowchart of the anti-tumor experiment, (B) Tumor growth curves of each mouse under different treatments, (C) Average tumor growth curves of each group, (D) Photographs of tumor tissues of mice in each group after the experiment, (E) Statistical results of tumor weight of mice in each group after the experiment, (F) Weight change curves of mice in each group, (G) TUNEL, Ki67 and H&E stained sections of tumor tissues of mice after different treatments (scale bar: 50 μm).

[0119] Figure 18 The results show the statistical results of tumor growth inhibition rate in each group of mice after the experiment.

[0120] Figure 19 (A) Results of antitumor immune response in mice after different treatments; (A) Dendritic cell maturation (CD80) in tumor tissues of each group. + CD86 + (A) Flow cytometry representation of tumor tissues in each group; (B) CD8+ of tumor tissues in each group. + GranB in T cells + (C) Flow cytometry representation of T cells; (D) Statistical analysis of mature dendritic cells in each group of tumor tissues; (E) CD3+ in each group of tumor tissues. + CD45 + CD8 in cells + Statistical analysis of T cells (E) CD8 in tumor tissues of each group + GranB in cells + Statistical analysis of T cells, (F) level of tumor necrosis factor-α (TNF-α) in each group of tumor tissues, (G) level of interferon-γ (IFN-γ) in each group of tumor tissues, (H) level of interleukin-6 (IL-6) in each group of tumor tissues;

[0121] Figure 20 The results of the detection of the remote effect induced by Hf-OMVs combined with radiotherapy are as follows: (A) Schematic diagram of the experimental procedure for assessing the remote effect; (B) Curve showing the change in the average tumor size of the primary tumor; (C) Curve showing the change in the average tumor size of the distal tumor; (D) Statistical analysis results of the distal tumor weight; (E) CD8+ in the distal tumor tissue. + Representative flow cytometry image of T cells, (F) memory T cells (CD44) in distal tumor tissue. + CD62L - Representative flow cytometry image of (G) mature DCs (CD80) in distal tumor tissue. + CD86 + (H) Quantitative analysis results of cells, (I) Quantitative analysis results of CD8+ T cells in distal tumor tissue, (II) Quantitative analysis results of memory T cells in distal tumor tissue.

[0122] Figure 21 The results are based on a statistical analysis of the weight of the primary tumor.

[0123] Figure 22 The curves showing the changes in body weight of mice in different groups;

[0124] Figure 23 Individual growth curves of distal tumors in different groups of mice;

[0125] Figure 24 Photos of primary and distal tumors in different groups of mice;

[0126] Figure 25 The results show the statistical results of tumor inhibition rates in different groups of mice. Detailed Implementation

[0127] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0128] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0129] In the description of this invention, the use of terms such as "first," "second," etc., is for the purpose of distinguishing technical features only and should not be construed as indicating or implying relative importance, or implicitly indicating the number of technical features indicated, or implicitly indicating the order of the technical features indicated.

[0130] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0131] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0132] Unless otherwise specified, "room temperature" in this invention means (25±5)℃.

[0133] In this invention, unless otherwise specified, "particle size" in NTA analysis and dynamic light scattering analysis refers to "diameter".

[0134] In this article, "outer membrane vesicles" (OMVs) are spherical, lipid bilayered vesicles (20-250 nm in diameter) secreted by Gram-negative bacteria, containing bioactive substances such as proteins, nucleic acids, and metabolites. As intercellular communication carriers, they participate in immune regulation, substance transport, and disease development, and show potential in the biomedical field (such as drug delivery and vaccine development). OMVs are mainly released by Gram-negative bacteria through cell membrane budding; eukaryotic cells (such as mammalian cells) can also secrete similar structures (such as extracellular vesicles). Their formation is influenced by environmental stress and gene regulation, representing one of the strategies by which cells actively adapt to external changes.

[0135] Unless otherwise specified, the term "vesicle" in this article can refer to endogenous vesicles, secreted exogenous vesicles, or artificially constructed vesicles.

[0136] In this article, "phenol" refers to an organic compound formed by the direct attachment of a hydroxyl group (-OH) to an aromatic ring (benzene ring or fused benzene ring).

[0137] In this article, tannic acid (TA), also known as tannic acid, is an organic compound with the chemical formula C. 76 H 52 O 46 It is a yellow or brownish-yellow powder.

[0138] Outer membrane vesicles (OMVs) naturally secreted by Gram-negative bacteria inherit similar functions from their source bacteria, are rich in microbial-associated molecular patterns, and therefore possess strong immunogenicity. As natural immune adjuvants, OMVs can be actively recognized and absorbed by antigen-presenting cells (APCs), effectively activating the immune system and demonstrating great potential for developing anti-tumor drugs. Under hydrogen peroxide stress, most bacteria deposit catalase in their membranes and secrete catalase-containing OMVs, which can be used to alleviate the hypoxic state of the tumor microenvironment (TME), thereby enhancing radiotherapy. Radiotherapy can trigger the release of tumor antigens and damage-associated molecular patterns, increasing tumor immunogenicity and thus promoting immune system activation. However, due to radioresistance, the immunomodulatory effects induced by radiotherapy are usually limited, which weakens the synergistic anti-tumor effect of OMVs and radiotherapy.

[0139] Radiosensitizers can increase radiation dose deposition at tumor sites under safe radiation doses, providing an opportunity to enhance the immunogenicity of radiotherapy. Among these, high atomic number metals (high-Z metals) and their nanomaterial derivatives stand out due to their unique properties, including gold (Au), hafnium (Hf), bismuth (Bi), and gadolinium (Gd). In particular, the hafnium oxide-based NBTXR3 radiosensitizer has been extensively studied clinically, sparking research interest in hafnium-based nanomaterials. However, clinical reports on HfO2 used for radiosensitization primarily rely on intratumoral injection, which significantly limits the types of cancer that can be treated and the treatment efficiency. Therefore, the use of HfO2 for radiosensitization... 4+ The delivery of hafnium-based nanomaterials to tumor sites to improve therapeutic efficiency has attracted widespread attention. OMVs, with their high modifiability and good tumor targeting capabilities, have the potential to serve as nanocarriers to improve drug delivery and promote deposition at tumor sites.

[0140] Metal-phenol networks (MPNs) are generated by the self-assembly of metal ions and phenolic compounds, and possess the ability to integrate OMVs and Hf. 4+ To enhance the potential of radioimmunotherapy for cancer, polyphenols (MPNs) possess unique functional properties due to the appropriate selection of metal ions and phenolic ligands. For example, MPNs prepared by chelating high-Z metal ions with phenolic reagents can be used as nanoradiosensitizers to enhance radiotherapy. Furthermore, polyphenols are considered promising drug carriers due to their diverse biological functions, including adhesion, antithrombosis, and antitumor activity. For instance, tannic acid (TA), a natural polyphenol compound approved as a safe substance by the U.S. Food and Drug Administration (FDA), is widely used in surface coating applications due to its high phenolic hydroxyl content. Importantly, MPNs, which are easily prepared and have minimal cytotoxicity, can be deposited on biomatrices such as the surfaces of bacteria, yeast, and mammalian cells to form biomimetic nanocloaks. Drug-loadable natural polyphenol nanocoatings provide editable therapeutic modalities for bacteria, enabling multimodal therapy.

[0141] In some embodiments, the present invention designs metal-phenol outer membrane vesicles (Hf-OMVs) by using hafnium ions (Hf... 4+ Coordination with TA to enhance cancer radioimmunotherapy ( ) Figure 1 (See Figure A in the original text). Due to inheriting the properties and nanostructure of OMVs, Hf-OMVs can effectively accumulate at tumor sites. Furthermore, catalase deposited in the OMV capsule can alleviate the hypoxic condition of the tumor microenvironment (TME) and amplify immunomodulatory effects, thereby enhancing the accumulation of Hf-induced ionizing radiation at the tumor site, inducing tumor cell death and releasing tumor antigens. Simultaneously, OMV components can promote the maturation of dendritic cells (DCs) and bind to released tumor antigens, further promoting T cell activation, thereby triggering an effective immune response. Figure 1 (See Figure B in the original text). In a mouse breast cancer model, it was demonstrated that the combination of Hf-OMVs and radiotherapy effectively inhibited the progression of both primary and distant tumors and induced a robust anti-tumor immune memory effect. Therefore, this invention provides a simple yet effective nanoplatform that combines the advantages of natural OMVs and metallophenol networks for cancer radioimmunotherapy.

[0142] OMVs lack radiosensitization capabilities, and free high atomic number metal ions are non-targeting and easily cleared by the kidneys (half-life <1 hour). By grafting high atomic number metal Hf onto the surface of OMVs with high catalase activity, it is possible to synergistically improve tumor lesion targeting and enhance radiosensitization.

[0143] Unless otherwise specified, TA was purchased from Aladdin; phosphate-buffered saline (PBS; 1×), trypsin, and Dulbecco modified Eagle medium (DMEM) were all purchased from Gibco. 1,1'-octadecyl-3,3,3',3'-tetramethylindole tetracarboxylate iodide (DiR), 4',6-diamino-2-phenylindole (DAPI), and 1,1'-tetramethylindole tetracarboxylate hydrochloride (DiI) were all purchased from Thermo Fisher Scientific.

[0144] Unless otherwise specified, protein concentration is determined using the BCA protein assay kit (Thermo Fisher Scientific, A55865).

[0145] Unless otherwise specified, catalase activity should be determined using a catalase assay kit (Beyotime, S0051). The test steps are as follows:

[0146] (1) Dilute H2O2 100 times with detection buffer, and measure A using a UV spectrophotometer with cuvette holder (cuvette path length (b) is 1 cm). 240 nm Calculate the hydrogen peroxide concentration (hydrogen peroxide concentration = 22.94 × A). 240 nm / b). Based on the measured hydrogen peroxide concentration, hydrogen peroxide solutions with concentrations of 0, 0.625, 1.25, 2.5, 3.75 mM, 5 mM, and 250 mM were prepared using detection buffer.

[0147] (2) Add 4 μL of hydrogen peroxide solutions with concentrations of 0, 0.625, 1.25, 2.5, and 3.75 mM, respectively, and 200 μL of colorimetric working solution to a 96-well plate. After incubation at 25°C for 15 min, measure A. 520nm Calculate the standard curve.

[0148] (3) OMVs were lysed to prepare the test sample with a protein concentration of 0.5 mg / mL. 20 μL of the test sample was mixed with 20 μL of detection buffer, then mixed with 10 μL of 250 mM hydrogen peroxide solution (using the detection buffer as a blank control). After incubation at 25°C for 3 min, 450 μL of catalase reaction termination solution was added to obtain the reaction termination solution. 40 μL of detection buffer was mixed with 10 μL of reaction termination solution, and 10 μL was placed in a 96-well plate. 200 μL of chromogenic working solution was added, and after incubation at 25°C for 15 min, A was measured. 520 nm Substitute the standard curve from step (2) into the equation and calculate the catalase activity (catalase activity (U / mg) = [number of micromoles of hydrogen peroxide consumed] × [dilution factor] / ([reaction minutes] × [sample volume] × [protein concentration]).

[0149] Unless otherwise specified, the steps for analyzing Hf content by ICP-OES are as follows:

[0150] Add 3 mL of 3% nitric acid to the sample to be tested (e.g., Hf-OMVs precipitate), then transfer it to a beaker. Add another 2 mL of nitric acid for digestion. After digestion, bring the volume to 10 mL and analyze using inductively coupled plasma atomic emission spectrometry (ICP-AES).

[0151] Unless otherwise specified, the HIF-1α immunofluorescence staining procedure is as follows:

[0152] After fixing the samples with 4% formaldehyde for 15 minutes, they were incubated with Triton X-100 solution for 5 minutes, followed by incubation with BSA (10 mg / mL, Sigma) for 1 hour. Then, they were stained with HIF-1α antibody (1:1000, Cell Signaling Technology) and incubated overnight at 4°C; FITC-labeled secondary antibody (1:500, Proteintech) was added, and incubation was continued for 1 hour. After staining, the cell nuclei were stained with DAPI, and the cells were then observed under a CLSM (ZEISS LSM980).

[0153] All experiments involving animals were conducted in accordance with the regulations established by the Animal Experimentation Ethics Committee of Shenzhen Bay Laboratory (License No.: AERL202401).

[0154] Unless otherwise specified, all results are expressed as mean ± standard deviation (SD) of independent experiments. All data were analyzed using Prism 8.0.1 software (GraphPad). For data from two groups, t-tests were used; for data from more than two groups, one-way (or two-way) ANOVA was used. The significance level was set at 0.05, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference.

[0155] Extracellular vesicles can be derived from living or dead organisms, transplanted tissues or organs, prokaryotic or eukaryotic cells and / or cultured cells.

[0156] Preparation Example 1

[0157] This example provides a method for preparing OMVs, with the following steps:

[0158] Escherichia coli DH5α (purchased from Vazyme) was inoculated into LB medium and cultured with shaking at 37°C and 220 rpm until OD500 was reached. 600 The concentration was approximately 1.3. H2O2 (final concentration 50 μM) was added to the culture medium, and the culture was continued for 24 h. The bacterial culture was centrifuged at 5000 × g for 30 minutes at 4 °C, the supernatant was collected, and filtered through a 0.45 μm filter. The culture was then ultracentrifuged at 150000 × g for 2 h at 4 °C, the precipitate was collected, washed twice with sterile PBS, and then resuspended in sterile PBS to obtain an OMVs suspension.

[0159] The OMVs prepared in this example were found to have a protein concentration of 2 μg / μL and a catalase activity of 20.7 U / mg.

[0160] Preparation Example 2

[0161] This preparation example provides a method for preparing OMVs. The steps are basically the same as those in Preparation Example 1, except that H2O2 treatment is used by default.

[0162] The OMVs prepared in this example were tested and found to have a protein concentration of 2 μg / μL and a catalase activity of 15 U / mg.

[0163] Example 1

[0164] This example provides a medical reagent, prepared as follows:

[0165] Add 5 μL of 30 mg / mL tannic acid solution (obtained by dissolving tannic acid in PBS) to 25 μL of OMVs suspension with a concentration of 2 μg / μL, and vortex for 10 s. Then add 5 μL of 10 mg / mL HfCl4 solution (obtained by dissolving HfCl4 in PBS) to bring the final HfCl4 concentration to 100 μg / mL, vortex to mix, and sonicate in a water bath for 5 min. Centrifuge the reaction solution at 5000 × g for 15 min at 4 °C, discard the supernatant, and wash the precipitate twice with sterile PBS to obtain Hf-OMVs particles.

[0166] The water bath ultrasonic treatment was performed at a temperature of 4℃ and a power of 70W.

[0167] Example 2

[0168] This example provides a medical reagent, the preparation method of which is basically the same as that in Example 1, the only difference being that the concentration of HfCl4 solution is different, so that the final concentration of HfCl4 in the reaction is replaced by 50 μg / mL instead of 100 μg / mL.

[0169] Example 3

[0170] This example provides a medical reagent, the preparation method of which is basically the same as that in Example 1, the only difference being that the concentration of HfCl4 solution is different, so that the final concentration of HfCl4 in the reaction is replaced by 150 μg / mL instead of 100 μg / mL.

[0171] Comparative Example 1

[0172] This example provides a medical reagent (OMVs@TA), which is prepared in the same way as in Example 1, except that the HfCl4 solution is replaced with an equal volume of PBS (the final concentration of HfCl4 in the reaction is 0 μg / mL).

[0173] Comparative Example 2

[0174] This example provides a medical reagent (Hf-TA), which is prepared in the same way as in Example 1, except that the OMVs suspension is replaced with an equal volume of PBS.

[0175] Detection Example 1

[0176] 10 μL of the OMVs suspension prepared in Preparation Example 1 or the Hf-OMVs suspension prepared in Example 1 was dropped onto a copper grid for 60 seconds, then stained with uranyl acetate for 60 seconds, dried, and detected by TEM (Hitachi Limited). The particle size distribution of the OMVs prepared in Preparation Example 1 and the Hf-OMVs prepared in Example 1 was analyzed by NTA. The UV-Vis absorption spectra of TA, the OMVs prepared in Preparation Example 1, the OMVs@TA prepared in Comparative Example 1, and the Hf-OMVs prepared in Example 1 in the range of 200 nm to 400 nm were detected by UV-Vis spectrometry (Shimadzu Corporation). The amount of Hf grafting (relative to 100 μg OMVs) in the medical reagents prepared in Examples 1-3 and Comparative Example 1 was analyzed by ICP-OES. The FT-IR spectra of TA, Hf-TA prepared in Comparative Example 2, OMVs prepared in Preparation Example 1, and Hf-OMVs prepared in Example 1 were detected using an infrared spectrometer (Thermo Fisher Scientific). The XPS spectra and high-resolution XPS spectra of the OMVs prepared in Preparation Example 1 and the Hf-OMVs prepared in Example 1 were detected using an X-ray photoelectron spectroscopy (Thermo Fisher Scientific). The OMVs prepared in Preparation Example 1 and the Hf-OMVs prepared in Example 1 were treated with lysis buffer (Solepro, BC3710) for 30 minutes, and the protein concentration was measured. After further treatment at 100°C for 10 minutes, they were added to a 10% SDS-PAGE gel and run at 80V for 30 minutes, then at 120V for 1 hour. The gel was stained with Coomassie Brilliant Blue for 20 minutes, destained at room temperature, and the SDS-PAGE electrophoresis results were observed. The OMVs suspensions prepared in Example 1 and the Hf-OMVs suspensions prepared in Example 1 were appropriately diluted with deionized water, and their particle size distribution, hydrated particle size, and Zeta potential were measured using a dynamic light scattering (DLS, Malvern Instruments) instrument. The Hf-OMVs suspension prepared in Example 1 was dropped onto an unstained carbon film, dried at room temperature, and data were acquired using a TEM (FEI-TALOS-F200X) equipped with an energy-dispersive spectroscopy (EDS) instrument. The HAADF image and corresponding EDS elemental distribution map of the Hf-OMVs prepared in Example 1 were obtained using a scanning transmission electron microscope (STEM).

[0177] The results are as follows Figures 2-5 As shown.

[0178] The OMVs prepared in Example 1 exhibited a typical lipid bilayer vesicle structure and particle size distribution. The average diameter of the OMVs was 123 nm, and the peak particle size was 94 nm, indicating that the OMVs were successfully extracted. Figure 2(AC diagram in the image). After preparation, OMVs@TA exhibits the characteristic peaks of TA and shows similar absorption peaks to OMVs. Figure 2 (D diagram in the image).

[0179] Hf was successfully modified on the surface of OMVs, and the content of Hf on the surface of OMVs gradually increased with the increase of the final concentration of HfCl4. Saturation was reached when the final concentration of HfCl4 reached 100 μg / mL, and the Hf grafting amount per 100 μg OMVs was 10 μg. Figure 2 (EF diagram in the image). OMVs and Hf-OMVs contain similar elements, and OMVs do not contain Hf elements ( Figure 2 G-graph in the middle, Figure 4 Compared to OMVs, the high-resolution XPS spectra of Hf4f in Hf-OMVs can be decomposed into two peaks at 17.7 eV and 19.5 eV, which further confirms the presence of Hf in Hf-OMVs. Figure 2 (See the H diagram in the image). Furthermore, Hf-OMVs inherit the proteins of OMVs (…). Figure 2 Figure I in the diagram). EDS elemental analysis showed that Hf-OMVs contain carbon (C), oxygen (O), nitrogen (N), phosphorus (P), and Hf elements (…). Figure 5 ).

[0180] Hf-OMVs exhibit spherical lipid droplets similar to OMVs, demonstrating good homogeneity. Figure 2 (See Figure J in the figure). The average diameter of Hf-OMVs obtained by NTA analysis was 192.3 nm. The hydrated particle size of Hf-OMVs was 202.8 nm, and the peak particle size was 197.6 nm, which was larger than that of OMVs (hydrated particle size was 165.8 nm, and peak particle size was 146 nm). The Zeta potential of Hf-OMVs was -34.8 mV, which was lower than that of OMVs (Zeta potential was -30.8 mV). This may be attributed to the fact that polyphenol complexes are usually negatively charged. Figure 2 (KM diagram in the image).

[0181] In conclusion, Hf-OMVs were successfully prepared.

[0182] Example 2 (Hypoxia-relieving effect of Hf-OMVs)

[0183] 1. Analyze the relative catalase activity of the OMVs prepared in Preparation Example 1 and Preparation Example 2 (Referred to as OMVs+H2O2 in Preparation Example 1 and OMVs in Preparation Example 2).

[0184] 2. Add 100 μg of OMVs prepared in Preparation Example 1 or Preparation Example 2 to 4 mL of PBS containing 400 μM H2O2, stir at room temperature for 6 min, and monitor the change in dissolved oxygen concentration in the reaction system.

[0185] 3. Add 120 μg of the OMVs prepared in Preparation Example 1 to 4 mL of PBS containing 0, 100, 200, and 400 μM H2O2, respectively. Stir the reaction at room temperature for 6 min and monitor the changes in dissolved oxygen concentration in the reaction system.

[0186] 4. PBS containing H2O2 (400 μM H2O2) and OMVs (final concentrations of 5 μg / mL, 10 μg / mL, and 20 μg / mL) prepared in Preparation Example 1 was stirred at room temperature for 6 min, and the changes in dissolved oxygen concentration in the reaction system were monitored.

[0187] 5. Place 5 × 10 4 Four T1 cells were seeded into confocal culture dishes. Group G1 cells were cultured in a normoxic environment for 24 h, then treated with PBS, and cultured for another 8 h or 24 h in a normoxic environment at 37°C. Group G2 cells were cultured in a hypoxic environment for 24 h, then treated with PBS, and cultured for another 8 h or 24 h in a hypoxic environment at 37°C. Group G3 cells were cultured in a hypoxic environment for 24 h, then treated with 200 μL of DMEM medium containing 200 μg of OMVs prepared in Example 1 (OMVs in Examples 3-9 below refer to OMVs prepared in Example 1), and cultured for another 8 h or 24 h in a hypoxic environment at 37°C. Group G4 cells were cultured in a hypoxic environment for 24 h, then treated with 200 μL of DMEM medium containing 200 μg of Hf-OMVs prepared in Example 1, and cultured for another 8 h or 24 h in a hypoxic environment at 37°C. After culture, HIF-1α immunofluorescence staining was performed.

[0188] The normoxic environment consisted of 5% CO2 and 95% air; the hypoxic environment referred to placing the confocal culture dish in a sealed hypoxic culture box; and the H2O2 content of the DMEM medium was 10 × 10⁻⁶. -6 M.

[0189] 6. Nine BALB / c mice (5 weeks old, female) were subcutaneously injected with 5 × 10⁵ mg / L of the solution. 5Four T1 cells were randomly divided into three groups. The OMVs group: On day 6, OMVs were intravenously injected into mice (100 μg / mouse, 100 μL); the Hf-OMVs group: On day 6, Hf-OMVs from Example 1 (Hf-OMVs in Examples 3-9 below refer to the Hf-OMVs from Example 1) were intravenously injected into mice (100 μg / mouse, 100 μL); the PBS group: On day 6, mice were injected with an equal volume of PBS. Two days later, the mice were euthanized, and tumor tissue was collected, embedded, sectioned, and subjected to HIF-1α immunofluorescence staining.

[0190] The results are as follows Figures 6-9 As shown.

[0191] Under H2O2 stress, *E. coli* deposits catalase from its envelope and releases catalase-containing OMVs. Compared with OMVs extracted under normal culture conditions, OMVs extracted under H2O2 induction exhibit 1.42 times the catalase activity. Figure 6 At 5 min, the dissolved oxygen concentration produced by the OMVs catalysis in Preparation Example 1 was 1.38 times that in Preparation Example 2. Figure 7 (AB diagram). This indicates that OMVs generated under H2O2 stress conditions have higher catalase activity.

[0192] The concentration of O2 catalyzed by the OMVs of Preparation Example 1 for the production of O2 from H2O2 increased steadily over time, reaching a peak at approximately 3 minutes, and then remained stable in a dose-dependent manner. This also indicates that the catalase activity of the OMVs of Preparation Example 1 is stable. Figure 7 (CF diagram).

[0193] The expression level of HIF-1α in 4T1 cells significantly increased after culture in a hypoxic environment, and could be inhibited by adding free catalase. After treatment with OMVs from Example 1 or Hf-OMVs from Example 1 for 8 hours and 24 hours in hypoxic 4T1 cells, the expression level of HIF-1α was significantly reduced. Figure 8 This indicates that OMVs and Hf-OMVs can effectively alleviate the hypoxic state of tumor cells in the long term.

[0194] Compared with the PBS group, the expression level of HIF-1α in tumor tissues of the OMVs group and the Hf-OMVs group was significantly reduced. Figure 9 This indicates that OMVs and Hf-OMVs have the ability to alleviate tumor tissue hypoxia. Therefore, Hf-OMVs inherit the catalase properties of OMVs, which can effectively alleviate tumor hypoxia and contribute to the sensitization effect of radiotherapy.

[0195] Example 3 (Detection of Hf-OMVs uptake by 4T1 cells)

[0196] 4T1 cells were seeded in 12-well cell culture plates and confocal culture dishes. DiI-labeled Hf-OMVs prepared in Example 1 (obtained by incubating Hf-OMVs with DiI at 37°C for 30 minutes and washing twice with PBS) were added to the 12-well plates and confocal culture dishes at different time points, with a final Hf-OMVs concentration of 100 μg / mL. After incubation, cells in the 12-well plates were used for flow cytometry analysis (detection of DiI positive signals) and ICP-OES analysis (detection of Hf content; Hf-OMVs cell uptake (%) = mass of Hf detected in the cell sample / mass of initially added Hf). Cells in the confocal culture dishes were fixed with 4% paraformaldehyde, stained with DAPI, and observed under a CLSM (ZEISS LSM980). This was to evaluate the Hf-OMVs uptake of 4T1 cells at 0h, 2h, 4h, 8h, 12h, and 24h.

[0197] The results are as follows Figure 10 As shown.

[0198] The fluorescence signal gradually increased with increasing incubation time, reaching saturation at 12 hours. Figure 10 (AB diagram). After co-incubation for 2 hours, 4T1 cells showed significant intracellular fluorescence signals, which peaked after 12 hours. Figure 10 (Figure C), which is consistent with the results of flow cytometry and ICP-OES analysis.

[0199] Case 4 (Extracorporeal radiotherapy sensitization effect of Hf-OMVs)

[0200] 1. After seeding 4T1 cells into 96-well plates and culturing them overnight, different concentrations of OMVs prepared in Preparation Example 1, Hf-TA prepared in Comparative Example 2, and Hf-OMVs prepared in Example 1 were added to the 96-well plates for co-culturing. After 24 hours, CCK-8 reagent (Vazyme) was added to the cell culture medium, and the cells were incubated at 37°C for 1 hour. Cell viability was calculated by measuring the absorbance at 450 nm using a microplate reader.

[0201] 2. After seeding 4T1 cells into 96-well plates and culturing them overnight, different concentrations of OMVs prepared in Preparation Example 1, Hf-TA prepared in Comparative Example 2, or Hf-OMVs prepared in Example 1 were added to the 96-well plates for co-culturing. After 12 hours, the 96-well plates were exposed to X-rays at a dose of 6 Gy for irradiation treatment, followed by continued culturing for 24 hours. Cell viability was calculated using the CCK-8 assay.

[0202] 3. Apoptosis Assay: 4T1 cells were seeded into 12-well cell culture plates. After 24 hours, DMEM medium containing OMVs prepared in Example 1, Hf-TA prepared in Comparative Example 2, or Hf-OMVs prepared in Example 1 was added (the final concentration of Hf-OMVs was 80 μg / mL; the final concentration of OMVs based on protein concentration was the same as that of 80 μg / mL Hf-OMVs; the final concentration of Hf-TA based on Hf concentration was the same as that of 80 μg / mL Hf-OMVs). The control group was treated with the same volume of DMEM medium. After 12 hours, the 12-well plates were exposed to 6 Gy of X-rays for irradiation, followed by 24 hours of culture. The cells were then digested with trypsin and transferred to flow cytometry tubes. After washing the cells, 5 μL of Annexin-FITC and PI staining solution was added to each sample, and the plates were incubated for 10 minutes (Annexin V-FITC / PI Apoptosis Detection Kit, DOJINDO). Finally, the cells were resuspended in 300 μL buffer and analyzed by flow cytometry (LSRFortessa SORP, Becton & Dickinson) to determine the apoptosis rate.

[0203] 4. Cell colony formation assay: 4T1 cells were seeded into 6-well cell culture plates. After 24 hours, DMEM medium containing OMVs prepared in Example 1, Hf-TA prepared in Comparative Example 2, or Hf-OMVs prepared in Example 1 (the final concentration of Hf-OMVs was 100 μg / mL; the final concentration of OMVs based on protein concentration was the same as that of 100 μg / mL Hf-OMVs; the final concentration of Hf-TA based on Hf concentration was the same as that of 100 μg / mL Hf-OMVs) was added to the 4T1 cells. The control (G1) group was treated with the same volume of PBS. After 12 hours of culture, the 6-well plates were exposed to X-rays at a dose of 6 Gy for irradiation treatment, followed by 8 days of culture. The fixed cells were stained with crystal violet staining solution, then fixed with 4% paraformaldehyde, washed, dried, and photographed. Using ImageJ, the area of ​​the cell clone region and the total area of ​​the wells in each group were calculated. The percentage of the clone area was calculated (percentage of clone area (%) = area of ​​cell clone region / total area of ​​wells × 100%) and then normalized to group G1 to obtain the relative clone area.

[0204] 5. DNA Damage Assay: 4T1 cells were seeded in confocal culture dishes. DMEM medium containing OMVs prepared in Example 1, Hf-TA prepared in Comparative Example 2, or Hf-OMVs prepared in Example 1 (the final concentration of Hf-OMVs was 100 μg / mL; the final concentration of OMVs based on protein concentration was the same as that of 100 μg / mL Hf-OMVs; the final concentration of Hf-TA based on Hf concentration was the same as that of 100 μg / mL Hf-OMVs) was added to the 4T1 cells. After culturing for 12 hours, the confocal culture dishes were exposed to X-rays at a dose of 6 Gy for irradiation treatment. After culturing for another 24 hours, the cells were fixed with 4% paraformaldehyde, followed by incubation with 0.1% Triton-X100 for 5 minutes, and then incubation with BSA (10 mg / mL) for 1 hour. Subsequently, γ-H2AX antibody (1:1000, Abcam) was added, and incubation was carried out at 4°C for 16 hours. Finally, Cy3 fluorescent secondary antibody (1:50, Proteintech) was added and incubated for 1 hour. The cell nuclei were then stained with DAPI and detected using a CLSM (ZEISS LSM980).

[0205] The results are as follows Figure 11 , Figure 12 As shown.

[0206] 4T1 cells treated with different concentrations of OMVs, Hf-TA, and Hf-OMVs all showed good cell viability. Figure 11 ).

[0207] The radiosensitization effect induced by Hf-OMVs is dose-dependent, and its IC50 value is [missing information]. 50 123 μg / mL Figure 12 Figure B). After treating 4T1 cells with different concentrations of OMVs, Hf-TA, and Hf-OMVs, the cells were irradiated with X-rays (6 Gy). The relative cell viability of all three groups was significantly reduced, and all showed a certain degree of dose-dependent effect. Figure 12 (See Figure C). Furthermore, the relative cell viability of the Hf-OMVs group was significantly lower than that of the OMVs group and the Hf-TA group.

[0208] The late apoptosis rate induced by the Hf-OMVs+RT group was approximately 23.1%, significantly higher than that of the OMVs+RT group (approximately 12.8%) and the Hf-TA+RT group (approximately 15.1%). Figure 12 (DE diagram). This indicates that Hf-OMVs can significantly enhance radiotherapy-induced apoptosis.

[0209] After X-ray irradiation, when cell clones were clearly formed, the G1 group (PBS+RT) and the G2 group (OMVs+RT) had similar cell clone areas (G1's relative clone area was 1, and G2's was 0.94), while the cell clone areas of the G3 group (Hf-TA+RT) and the G4 group (Hf-OMVs+RT) were significantly reduced (G3's relative clone area was 0.44, and G4's was 0.25). Figure 12 (Figure F). This indicates that Hf-OMVs+RT can significantly inhibit the proliferation of 4T1 cells.

[0210] DNA damage caused by double-strand breaks is a major cause of radiotherapy-induced cell death, and γ-H2AX is often used as a marker of DNA damage. No significant DNA damage was observed in the untreated group, while the PBS+RT and OMVs+RT groups induced moderate DNA damage. Compared to the PBS+RT and OMVs+RT groups, the Hf-TA+RT and Hf-OMVs+RT groups significantly increased the level of DNA damage in 4T1 cells. Among them, the Hf-OMVs+RT group induced the highest level of DNA damage. Figure 12 (GH diagram).

[0211] In summary, surface modification of Hf-OMVs endows them with unique physical properties, enabling them to deposit more ionizing radiation and thus induce a strong radiosensitizing effect. Hf, after being incorporated into the surface of OMVs via a metal-phenol network, can synergistically enhance the radiosensitizing effect, significantly reduce cancer cell survival, enhance radiotherapy-induced apoptosis, inhibit cancer cell proliferation, and induce DNA damage.

[0212] Test Example 5 (Safety of Hf-OMVs)

[0213] 1. Whole blood was collected from BALB / c mice and centrifuged at 1200 rpm for 10 minutes to separate red blood cells (RBCs). The RBCs were mixed with an equal volume of PBS containing different concentrations (0.1, 0.25, 0.5, 1, 1.5, and 2 μg / μL) of Hf-OMVs prepared in Example 1, and allowed to stand for 5 hours. Equal volumes of deionized water and PBS were used as positive and negative controls, respectively. The mixture was then centrifuged at 15000 rpm for 5 minutes and photographed. The absorbance of the supernatant at 570 nm was measured using a microplate reader to calculate the hemolysis rate (hemolysis rate = (experimental group A) / (experimental group A)). 570 nm - Negative control group A 570 nm ) / (Positive control group A) 570 nm - Negative control group A 570 nm(×100%). Each group had 4 replicates.

[0214] 2. Six healthy BALB / c mice (5-6 weeks old, female) were randomly divided into two groups. One group received a tail vein injection of PBS containing Hf-OMVs (100 μg / mouse, 100 μL) or an equal volume of PBS, administered every other day for a total of three times. Sixteen days later, the mice were euthanized, and whole blood and serum were collected via ocular sampling. Major organs (heart, liver, spleen, lungs, and kidneys) were dissected. Blood from anticoagulant tubes was analyzed using an automated hematology analyzer. Additionally, whole blood was centrifuged at 3000 rpm for 10 minutes at 4°C, and blood biochemical parameters were analyzed using an automated biochemical analyzer. Different organs were fixed with 4% paraformaldehyde, then embedded, sectioned, and stained with H&E.

[0215] The results are as follows Figures 13-14 As shown.

[0216] No significant erythrocyte lysis was observed in the presence of different concentrations of Hf-OMVs. Figure 13 Complete blood count, blood biochemistry analysis, and H&E sections all indicated that Hf-OMVs had no significant in vivo toxicity and no obvious histological damage to major organs. Figure 14 ).

[0217] In summary, Hf-OMVs exhibit good biocompatibility and safety in vivo.

[0218] Case 6 (In vivo tumor targeting effect of Hf-OMVs)

[0219] 1. On day 1, BALB / c mice (5-6 weeks old, female, n=4) received a subcutaneous injection of 5×10 5 4T1 cells were used. DiR dye was added to the OMVs prepared in Example 1, and the cells were incubated at 37°C for 30 min, followed by washing twice with PBS to obtain DiR-labeled OMVs. On day 8, the DiR-labeled OMVs prepared in Example 1 were intravenously injected into 4T1 tumor-bearing mice (100 μg / mouse, 100 μL), and the mice were imaged at different time points (4, 12, 24, 48, and 72 hours) using an IVIS imaging system. Mice were euthanized at 72 hours, and major organs (heart, liver, spleen, lung, and kidney) and tumor tissue were harvested and imaged using an IVIS imaging system. Subsequently, the tumor-targeting effect of Hf-OMVs was analyzed using ICP-OES.

[0220] 2. Twelve BALB / c mice (5-6 weeks old, female) were randomly divided into three groups (n=4). On day 1, the mice received a subcutaneous injection of 5 × 10⁻⁶ mg / L phosphate rock solution. 54T1 cells were collected. On day 8, HfCl4, Hf-TA prepared in Comparative Example 2, or Hf-OMVs prepared in Example 1 were intravenously injected into 4T1 tumor-bearing mice (the dosage of Hf-OMVs was 100 μg / mouse, 100 μL; the Hf concentration of HfCl4 and Hf-TA was the same as that of 100 μg / mL Hf-OMVs). After 48 hours, the mice were euthanized, and tumor tissue was obtained, digested, and analyzed by ICP-OES to calculate the Hf concentration in the tumor tissue. 4+ content.

[0221] The results are as follows Figure 15 and Figure 16 As shown.

[0222] IVIS spectral imaging showed that DiR-labeled OMVs accumulated continuously in the tumor site over time, reaching their peak at 48 hours; OMVs mainly accumulated in the liver, spleen, and tumor tissue. The fluorescence intensity of the tumor was 1.6 times that of the lung and 2.3 times that of the heart, respectively. Figure 15 ).

[0223] Compared to HfCl4 and Hf-TA, Hf-OMVs accumulate at higher levels in tumor sites. This indicates that Hf-OMVs have better tumor targeting and can effectively accumulate Hf elements at tumor sites. Figure 16 ).

[0224] Case 7 (In vivo radiosensitization effect of Hf-OMVs)

[0225] BALB / c mice (5-6 weeks old, female) were subcutaneously injected with 5×10 5 4T1 cells. When the tumor volume grows to approximately 100 mm... 3 At day 0, mice were randomly divided into 6 groups (PBS group, OMVs group, Hf-OMVs group, PBS+RT group, OMVs+RT group and Hf-OMVs+RT group, with 5 mice in each group).

[0226] (1) PBS group (G1): 100 μL of PBS was injected intravenously on days 0 and 3 respectively;

[0227] (2) OMVs group (G2): On days 0 and 3, PBS containing OMVs prepared in Example 1 (100 μg / animal, 100 μL) was administered intravenously.

[0228] (3) Hf-OMVs group (G3): On days 0 and 3, PBS containing Hf-OMVs prepared in Example 1 (100 μg / animal, 100 μL) was injected intravenously.

[0229] (4) PBS+RT group (G4): 100 μL of PBS was injected intravenously on days 0 and 3, and the tumor was subjected to local X-ray irradiation on days 2 and 5 (dose of 6 Gy, and the remaining parts were shielded with lead caps).

[0230] (5) OMVs+RT group (G5): On days 0 and 3, PBS containing OMVs prepared in Example 1 (100 μg / animal, 100 μL) was injected intravenously. On days 2 and 5, the tumor was subjected to local X-ray irradiation (dose of 6 Gy, and the remaining parts were shielded with lead caps).

[0231] (6) Hf-OMVs+RT group (G6): On days 0 and 3, PBS containing Hf-OMVs prepared in Example 1 (100 μg / animal, 100 μL) was injected intravenously. On days 2 and 5, the tumor was subjected to local X-ray irradiation (dose of 6 Gy, and the remaining parts were shielded with lead caps).

[0232] Mice body weight and tumor size were recorded every two days. Mice were euthanized on day 14, and tumors were dissected, weighed, and photographed to calculate tumor growth inhibition rate (tumor growth inhibition rate = 100% × tumor weight in the treatment group / tumor weight in the PBS group). Tumor tissues were fixed and sectioned with 4% paraformaldehyde and further stained with TUNEL, H&E, and Ki67.

[0233] The results are as follows Figure 17 , Figure 18 As shown.

[0234] All mice survived the entire treatment period, and there was no statistically significant difference in body weight among the groups. Figure 17 (See Figure F). OMVs and Hf-OMVs alone had almost no effect on tumor growth. PBS+RT and OMVs+RT showed poor inhibitory effects on tumor growth. The tumor growth inhibition rates of the OMVs group, Hf-OMVs group, PBS+RT group, OMVs+RT group, and Hf-OMVs+RT group were 15.5%, 11.7%, 43.6%, 53.1%, and 78.2%, respectively. Figure 17 BE diagram, Figure 18 The Hf-OMVs+RT group exhibited the most significant tumor-suppressive effect, with the most severe apoptosis, significantly inhibiting tumor cell proliferation and causing severe nuclear atrophy. Figure 17 (G diagram). This indicates that Hf-OMVs can significantly improve the in vivo therapeutic effect of RT on solid tumors.

[0235] Example 8 (Radiation-induced immune response based on Hf-OMVs)

[0236] The experimental procedure was the same as in Example 7. Mice were euthanized on day 14, and tumors were dissected. The levels of TNF-α, IFN-γ, and IL-6 in the tumor tissues of each group were detected by enzyme-linked immunosorbent assay (ELISA). Tumor tissues were cut into small pieces and placed in a culture medium supplemented with collagenase (0.2 mg / mL), deoxyribonuclease I (25 U / mL), and hyaluronidase (0.1 mg / mL). After digestion at 37°C for 1 hour, the tumor tissues were digested using an automated tissue processor and then filtered through 70 μm and 40 μm filters to prepare single-cell suspensions. Subsequently, the cells were labeled with a live-dead cell dye (FVS510; Biolegend). For analysis of mature DCs, cells were incubated in the dark with specifically labeled antibodies (CD45 (APC-Cy7; clone: ​​30-F11; Becton & Dickinson), CD11b (FITC; clone: ​​M1 / 70; Becton & Dickinson), CD11c (BV605; clone: ​​N418; Biolegend), CD80 (BV421; clone: ​​16-10A1; Becton & Dickinson), and CD86 (PE; clone: ​​GL1; Biolegend)). For T cell analysis, cells were first incubated in the dark with specific antibodies (CD45 (APC-Cy7; clone: ​​30-F11; Becton & Dickinson), CD3 (BUV395; clone: ​​145-2C11; Becton & Dickinson), CD4 (BV650; clone: ​​RM4-5; Biolegend), CD8 (BV605; clone: ​​53-6.7; Becton & Dickinson)), then fixed and stained with GranB (APC; clone: ​​QA16A02; Biolegend). For the analysis of memory T cells in distant tumors, CD44 (BB700; clone: ​​IM7; Becton & Dickinson) and CD62L (PE-Cy7; clone: ​​GK1.5; Becton & Dickinson) were used. Finally, the cell suspension was analyzed using flow cytometry (LSRFortessa SORP, Becton & Dickinson).

[0237] The results are as follows Figure 19 As shown.

[0238] Dendritic cells (DCs) are the most representative antigen-presenting cells (APCs) and play a crucial role in activating the immune system. Compared to the PBS group, the proportion of mature DCs in tumor tissues treated with OMVs and Hf-OMVs increased. Furthermore, the proportion of mature DCs was highest in the Hf-OMVs+RT group. This indicates that OMVs and Hf-OMVs have the potential to activate the immune system in vivo, and that the combination of Hf-OMVs and RT can further promote the maturation of DCs.

[0239] CD8 + Infiltration of cytotoxic T cells can effectively promote anti-tumor effects. Flow cytometry analysis results show that CD8+ cells are present in tumor tissue after radiotherapy. + Increased T cell infiltration, GranB + CD8 + The proportion of T cells increased; after injection of OMVs and Hf-OMVs, CD8 + T cell infiltration, GranB + CD8 + The proportion of T cells further increased. Specifically, in tumor tissue treated with Hf-OMVs+RT, the infiltration of CD8+ T cells increased from 6.87% to 30.84%, and GranB... + CD8 + The proportion of T cells increased from 2.9% to 13.4%, showing the most significant increase. Immunofluorescence analysis results were consistent with flow cytometry analysis results. After Hf-OMVs+RT treatment, the levels of TNF-α, IFN-γ, and IL-6 in tumor tissue were significantly increased. This indicates that Hf-OMVs+RT treatment significantly promotes CD8+ cell proliferation. + T cell infiltration and CD8 + T cell activation.

[0240] In summary, Hf-OMV-based radiotherapy can effectively promote anti-tumor immune responses, thereby enhancing the efficacy of combined radiotherapy and immunotherapy.

[0241] Case 9 (Anti-tumor telescopic effect of Hf-OMVs combined with radiotherapy)

[0242] 5×10 5 4T1 cells were subcutaneously injected into the right skin (primary tumor) of BALB / c mice (5-6 weeks old, female). Five days later, 5 × 10⁵ cells were injected into the left skin. 5 4T1 cells (distal tumor) were used to establish a bilateral tumor model. When the primary tumor volume reached approximately 100 mm... 3At day 0, mice were randomly divided into 6 groups (PBS group, OMVs group, Hf-OMVs group, PBS+RT group, OMVs+RT group and Hf-OMVs+RT group, with 5 mice in each group).

[0243] (1) PBS group (G1): 100 μL of PBS was injected intravenously on days 0 and 3 respectively;

[0244] (2) OMVs group (G2): On days 0 and 3, PBS containing OMVs prepared in Example 1 (100 μg / animal, 100 μL) was administered intravenously.

[0245] (3) Hf-OMVs group (G3): On days 0 and 3, PBS containing Hf-OMVs prepared in Example 1 (100 μg / animal, 100 μL) was injected intravenously.

[0246] (4) PBS+RT group (G4): 100 μL of PBS was injected intravenously on days 0 and 3, and the primary tumor was subjected to local X-ray irradiation treatment on days 2 and 5 (dose of 6 Gy, and the rest of the site was shielded with lead caps).

[0247] (5) OMVs+RT group (G5): On days 0 and 3, PBS containing OMVs prepared in Example 1 (100 μg / animal, 100 μL) was injected intravenously. On days 2 and 5, the primary tumor was subjected to local X-ray irradiation (dose of 6 Gy, and the remaining parts were shielded with lead caps).

[0248] (6) Hf-OMVs+RT group (G6): On days 0 and 3, PBS containing Hf-OMVs prepared in Example 1 (100 μg / animal, 100 μL) was injected intravenously. On days 2 and 5, the primary tumor was subjected to local X-ray irradiation (dose of 6 Gy, and the remaining parts were shielded with lead caps).

[0249] The size of the primary and distal tumors, as well as the body weight of the mice, were monitored every other day. On day 14, the mice were euthanized, and the primary and distal tumors were dissected, weighed, and photographed. The tumor inhibition rate was calculated (tumor inhibition rate (%) = tumor weight in the treatment group / tumor weight in the PBS group × 100%). CD8+ levels in the distal tumor tissue were assessed by flow cytometry. + T cells, memory T cells (CD44) + CD62L - ), mature DC (CD80) + CD86 + Changes in cells.

[0250] The results are as follows Figures 20-25 As shown.

[0251] OMVs or Hf-OMVs alone have only a weak inhibitory effect on primary and distant tumors. Hf-OMVs combined with radiotherapy can significantly inhibit the growth of primary and distant tumors, with an inhibition rate of 74.68% for distant tumors, which is far superior to RT or OMVs+RT.

[0252] After Hf-OMVs+RT combined therapy, the proportion of mature dendritic cells (DCs) in distant tumors significantly increased to approximately 21.46%, far higher than other groups (G1: 6.68%; G2: 11.3%; G3: 11.66%; G4: 13.2%; G5: 15.52%); CD8+ in distant tumors... + The proportion of T cells was approximately 19.16%, significantly higher than in other groups (G1: 5.9%; G2: 7.86%; G3: 7.38%; G4: 11.85%; G5: 13.16%); the proportion of memory T cells in distant tumors was approximately 19.74%, significantly higher than in other groups (G1: 6.17%; G2: 9.29%; G3: 8.88%; G4: 11.34%; G5: 15.28%). Furthermore, there was no significant decrease in body weight in any group of mice throughout the treatment period. This indicates that Hf-OMVs combined with radiotherapy can promote CD8 cell growth. + T cell recruitment activates the body's immune memory effect and inhibits the growth of distant tumors, which helps prevent tumor recurrence and metastasis.

[0253] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A medical reagent, characterized in that, The medical reagent includes a nanocarrier modified with a metal-phenol network; The metal is a high atomic number metal; the high atomic number metal is hafnium; The phenols include polyphenols; the polyphenols are tannic acid; The catalase activity of the nanocarrier is not less than 15 U / mg; the nanocarrier is an outer membrane vesicle secreted by Escherichia coli treated with hydrogen peroxide.

2. The medical reagent according to claim 1, characterized in that, The hydrated particle size of the nanocarrier is 50 nm-200 nm; And / or, each 100 μg nanocarrier in the medical reagent contains 1 μg-25 μg of a high atomic number metal; And / or, the hydrated particle size of the nanocarrier modified with the metal-phenol network is 150 nm-300 nm.

3. The method for preparing the medical reagent according to any one of claims 1 to 2, characterized in that, Includes the following steps: A mixture of the nanocarrier, phenol, and a high atomic number metal ion source was prepared and reacted to obtain the medical reagent.

4. The preparation method according to claim 3, characterized in that, The high atomic number metal ion source includes a compound containing the high atomic number metal; And / or, when the nanocarrier is a vesicle derived from Escherichia coli, the method for preparing the nanocarrier includes: contacting Escherichia coli with hydrogen peroxide to react and obtain the nanocarrier; And / or, the mass ratio of the nanocarrier to phenol is 1:(2-4); And / or, the mass ratio of the phenol to the high atomic number metal ion source is (2-4):1; And / or, the temperature of the reaction is 1℃-10℃; And / or, the reaction time is 3 min-20 min; And / or, the ultrasonic power of the reaction is 50 W-90 W.

5. A drug, characterized in that, Includes the medical reagents and pharmaceutically acceptable excipients or auxiliary ingredients as described in any one of claims 1 to 2.

6. The use of the medical reagent according to any one of claims 1 to 2 or the drug according to claim 5 in combination with radiation irradiation in any one of A1) to A4): A1) Preparation of products that kill cells; A2) Prepare products that induce apoptosis; A3) Prepare products that inhibit cell proliferation; A4) Prepare products that induce cellular DNA damage; The cells in question are breast cancer cells.

7. The use of the medical reagent according to any one of claims 1 to 2 or the drug according to claim 5 in any one of B1) to B2): B1) Prepare drugs to improve the radiosensitivity of tumors; B2) Prepare drugs to improve the efficacy of tumor radiotherapy; The tumor is breast cancer.

8. The application according to claim 7, characterized in that, The improvement of tumor radiotherapy efficacy described in B2) includes at least one of C1) to C3): C1) Inhibits tumor growth; C2) Increase the proportion of mature dendritic cells (DCs) and CD3 in the tumor microenvironment. + CD45 + CD8 + T cell percentage, GranB + CD8 + At least one of the following: the proportion of T cells and the proportion of memory T cells; C3) Increases at least one of the following levels in tumors: TNF-α, IFN-γ, and IL-6.

Images