Medical reagent based on cell membrane vesicles as well as preparation method and application of medical reagent

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a medical reagent based on cell membrane vesicles, and a preparation method and application thereof. Background Art

[0002] High-atomic-number metal-based nanomaterials can produce a range of physical effects (such as the photoelectric effect, Compton scattering, and Rayleigh scattering) when irradiated with X-rays and are commonly used in the preparation of medical reagents. Among them, the hafnium oxide (HfO2) nanoparticle-based NBTXR3 radiosensitizer has been extensively studied in the clinic, but its reliance on intratumoral injection limits its applicable cancer types and therapeutic efficiency. Therefore, the development of a new medical reagent is crucial. Summary of the Invention

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

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

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

[0006] The present invention also provides the use of the above-mentioned medical reagent or drug in combination with radiation irradiation.

[0007] The present invention also provides the use of the above-mentioned medical reagent or medicine.

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

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

[0010] The medical reagent according to the embodiment of the present invention has at least the following beneficial effects:

[0011] The medical reagent of the embodiment has good tumor targeting, can alleviate the hypoxic state of the tumor microenvironment, promote the maturation of dendritic cells (DCs), the infiltration and activation of cytotoxic T cells, can be used as a radiosensitizer and immune adjuvant to enhance radioimmunotherapy, effectively inhibit tumor growth, and is 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 of 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 of gadolinium, hafnium, tungsten, gold, bismuth, palladium, platinum, tantalum, gallium, tellurium, and zirconium.

[0016] According to some embodiments of the invention, the phenol comprises a polyphenol.

[0017] According to some embodiments of the invention, the polyphenols include compounds having a molecular chemical structure that conforms to the White-Bate-Smith-Swain-Haslam (WBSSH) definition.

[0018] According to some embodiments of the present invention, the polyphenols include tannic acid, catechin, dopamine and condensed deformation, tannin molecules, epicatechin, gallol catechin, epigallocatechin, anthocyanidins, anthraquinones, chalcones, lignans, naphthoquinones, neolignans, proanthocyanidins, pigments tannic acid, xanthones, condensates of polyphenols synthesized by plant biosynthesis or artificial fermentation, theaflavins, polymers of anthocyanidins, highly condensed tannins, and artificially synthesized molecules containing polyphenol groups, or a combination 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, 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 comprises at least one of a vesicle and a synthetic nanoparticle.

[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 cell includes at least one of a prokaryotic cell and a eukaryotic cell.

[0023] According to some embodiments of the present invention, the prokaryotic cell comprises at least one of cyanobacteria and bacteria. The bacteria comprises 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 may be derived from, but are not limited to, lemon, tobacco, Arabidopsis thaliana, or rice.

[0025] According to some embodiments of the present invention, the nanocarriers have an average diameter of 100 nm to 150 nm. The average diameter is obtained by nanoparticle tracking analysis (NTA). For example, the average diameter 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 50 nm to 200 nm, for example, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, or 200 nm.

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

[0028] According to some embodiments of the present invention, those skilled in the art can select methods known in the art to isolate and obtain the vesicles, including but not limited to differential centrifugation, density gradient centrifugation, ultrafiltration, size exclusion chromatography, immunoaffinity chromatography, microfluidics, or sonication separation.

[0029] According to some embodiments of the present invention, each 100 μg of nanocarriers in the medical agent contains 1 μg-25 μg of high atomic number metal. For example, every 100 μg of the nanocarrier in the medical agent can 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 the high atomic number metal.

[0030] According to some embodiments of the present invention, each 100 μg of nanocarriers in the medical agent contains 5 μg-25 μg of the 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 150 nm to 300 nm, for example, 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, 250 nm, 255 nm, 260 nm, 265 nm, 270 nm, 275 nm, 280 nm, 285 nm, 290 nm, 295 nm, or 300 nm.

[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, -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 nanocarriers modified with the metal-phenol network have an average diameter of 150 nm to 300 nm. The average diameter is determined by NTA analysis. For example, the average diameter 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 agent includes at least one of a sensitizer, an adjuvant, and a cytotoxic substance.

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

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

[0037] According to the method for preparing the medical reagent described in the first embodiment of the second embodiment of the present invention, the method comprises the following steps:

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

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

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

[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, 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, the mass ratio may 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°C-10°C. For example, it can be 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C.

[0046] According to some embodiments of the present invention, the reaction time is 3 min to 20 min, for example, 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 invention, the phenol comprises a polyphenol.

[0049] According to some embodiments of the invention, the polyphenols include compounds having a molecular chemical structure that conforms to the White-Bate-Smith-Swain-Haslam (WBSSH) definition.

[0050] According to some embodiments of the present invention, the polyphenols include tannic acid, catechin, dopamine and condensed deformation, tannin molecules, epicatechin, gallol catechin, epigallocatechin, anthocyanidins, anthraquinones, chalcones, lignans, naphthoquinones, neolignans, proanthocyanidins, pigments tannic acid, xanthones, condensates of polyphenols synthesized by plant biosynthesis or artificial fermentation, theaflavins, polymers of anthocyanidins, highly condensed tannins, and artificially synthesized molecules containing polyphenol groups, or a combination thereof.

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

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

[0053] According to some embodiments of the present invention, the compound containing the high atomic number metal includes at least one of a halide (including but not limited to chloride, bromide or iodide) and a sulfate.

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

[0055] The biological cells are contacted with a catalase inducer to react and obtain the nanocarrier.

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

[0057] According to some embodiments of the present invention, the prokaryotic cell comprises at least one of cyanobacteria and bacteria, and 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 may be derived from, but are not limited to, lemon, tobacco, Arabidopsis thaliana, or rice.

[0059] According to some embodiments of the present invention, the biological cell is Escherichia coli; the OD of the biological cell in the reaction system is 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 hydrogen peroxide is 5 μM-200 μM.

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

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

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

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

[0068] According to some embodiments of the present invention, the centrifugal speed is 4000×g-6000×g, for example, 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-30 min, for example, 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 embodiment of the present invention comprises the medical agent described in the first embodiment and a pharmaceutically acceptable excipient or auxiliary component. Because the medicament employs all of the technical solutions of the medical agent of the aforementioned embodiment, it at least has all of the beneficial effects brought about by the technical solutions of the aforementioned embodiment.

[0071] According to some embodiments of the present invention, the auxiliary component includes at least one of other tumor radiotherapy sensitizers and immune checkpoint inhibitors. The auxiliary component is preferably such that it does not affect the effect of the medical agent described in the embodiment of the first aspect.

[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] According to the fourth aspect of the present invention, use of the medical agent described in the first embodiment or the drug described in the third embodiment in combination with radiation irradiation in any one of A1) to A4):

[0076] A1) preparing a product for killing cells;

[0077] A2) preparing a product that induces cell apoptosis;

[0078] A3) preparing a product for inhibiting cell proliferation;

[0079] A4) Preparation of products that induce DNA damage in cells.

[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 brain glioma cells.

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

[0084] According to some embodiments of the present invention, a single dose of radiation irradiation is 1 Gy-8 Gy, for example, 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 agent or the drug is used 8 hours to 24 hours before radiation exposure, for example, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, 12.5 hours, 13 hours, 13.5 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.

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

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

[0088] B1) preparing drugs for improving tumor radiosensitivity;

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

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

[0091] C1) inhibit tumor growth;

[0092] C2) Increase the proportion of mature DCs and CD3 + CD45 + CD8 + T cell ratio, GranB + CD8 + At least one of the T cell ratio and memory T cell ratio;

[0093] C3) increasing at least one of the levels of TNF-α, IFN-γ, and IL-6 in the tumor.

[0094] According to some embodiments of the invention, the tumor comprises 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 tumor, gastric cancer, melanoma, non-small cell lung cancer, cervical cancer, liver cancer, nasopharyngeal carcinoma, and brain glioma.

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

[0097] According to some embodiments of the 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 radiation dose of a single radiation therapy is 1 Gy-8 Gy, for example, 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 agent or the drug is used within 72 hours before radiotherapy, for example, 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 present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 Schematic diagram of the use of metallophenol outer membrane vesicles (Hf-OMVs) for tumor radioimmunotherapy; (A) Schematic diagram of the preparation process of Hf-OMVs. (B) Schematic diagram of the principle of using Hf-OMVs for tumor radioimmunotherapy;

[0103] Figure 2 Preparation and characterization results of Hf-OMVs; (A) Schematic diagram of Escherichia coli secretion of OMVs, (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 OMVs particle size distribution, (D) UV-visible (UV-Vis) absorption spectra of TA, OMVs and OMVs@TA (the dotted 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, the dotted lines indicate 2925 cm-1 and 2950 cm-2, respectively) of TA, OMVs and Hf-OMVs. -1 and 1710cm -1 ) spectra, (G) X-ray photoelectron spectroscopy (XPS) spectra of OMVs and Hf-OMVs, (H) high-resolution XPS spectra of Hf 4f in Hf-OMVs, (I) SDS-PAGE analysis of OMVs and Hf-OMVs, (J) TEM images 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 of the particle size distribution of OMVs and Hf-OMVs, (L) characterization of the hydrated particle size of OMVs and Hf-OMVs, (M) Zeta potential characterization of OMVs and Hf-OMVs;

[0104] Figure 3 UV-Vis absorption spectra of TA and Hf-OMVs (the dotted lines indicate the absorption peaks at 280 nm and 307 nm, respectively);

[0105] Figure 4 is the XPS spectrum 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 E. coli with and without H2O2 stress;

[0108] Figure 7 Performance test results of different OMVs catalyzing H2O2 oxygen production; (A) Oxygen production curves of OMVs catalyzed by H2O2 under the presence or absence of H2O2 stress, (B) Statistical graph of O2 concentration after 5 min of reaction in Figure A, (C) O2 production curves of OMVs catalyzed by H2O2 at different concentrations, (D) Statistical graph of O2 concentration after 5 min of reaction in Figure C, (E) O2 production curves of OMVs catalyzed by H2O2 at different concentrations, (F) Statistical graph of O2 concentration after 5 min of reaction in Figure E;

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

[0110] Figure 9 Results of in vivo testing of the ability of OMVs prepared in Preparation Example 1 and Hf-OMVs prepared in Example 1 to alleviate tumor hypoxia; (A) Immunofluorescence images of HIF-1α in tumor tissues after different treatments (scale bar: 100 μm); (B) statistical analysis of the relative fluorescence intensity in Figure A;

[0111] Figure 10 The cellular uptake of Hf-OMV by 4T1 cells; (A) flow cytometry detection results, (B) ICP-OES analysis results; (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 are shown;

[0113] Figure 12The results of the in vitro radiotherapy (RT) sensitization test of Hf-OMVs prepared in Example 1; (A) Schematic diagram of the radiotherapy sensitization experiment, (B) IC of Hf-OMVs on 4T1 cells after combined radiotherapy 50 , (C) Relative viability of 4T1 cells after treatment with different concentrations of OMVs, Hf-TA, and Hf-OMVs combined with radiotherapy, (D) Representative flow cytometry images of cell apoptosis after different treatments, (E) Statistical results of cell apoptosis rate in Figure D, (F) Representative photos of cell clone formation experiment and statistical analysis results of relative clone area, (G) Fluorescence images of γ-H2AX in 4T1 cells after different treatments, (H) Statistical results of relative fluorescence intensity in Figure G;

[0114] Figure 13 is the hemolysis rate of Hf-OMVs on red blood cells at different concentrations;

[0115] Figure 14 Blood biochemistry and routine blood test 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 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 process; (B) Hf in tumor 4+ Content analysis results;

[0118] Figure 17 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) Photos of tumor tissues of mice in each group after the experiment, (E) Statistical results of tumor weights of mice in each group after the experiment, (F) Body 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 This is the statistical result of the tumor growth inhibition rate of each group of mice after the experiment;

[0120] Figure 19 The results of anti-tumor immune response detection of mice after different treatments; (A) DC maturation (CD80 + CD86 + ) flow cytometry representative diagram, (B) CD8 + GranB in T cells + Representative flow cytometry images of T cells, (C) Statistical analysis of mature DC cells in tumor tissues of each group, (D) CD3 + CD45 + CD8 in cells + Statistical analysis of T cells, (E) CD8 + GranB in cells + Statistical analysis of T cells, (F) tumor necrosis factor-α (TNF-α) levels in tumor tissues of each group, (G) interferon-γ (IFN-γ) levels in tumor tissues of each group, (H) interleukin-6 (IL-6) levels in tumor tissues of each group;

[0121] Figure 20 Results of the abscopal effect assay induced by Hf-OMVs combined with radiotherapy; (A) Schematic diagram of the experimental process for evaluating the abscopal effect, (B) average tumor size change curve of primary tumors, (C) average tumor size change curve of distal tumors, (D) statistical analysis results of distal tumor weight, (E) CD8 + Representative flow cytometry images of T cells, (F) memory T cells (CD44 + CD62L - ) Representative flow cytometry images of (G) mature DCs (CD80 + CD86 + ) Quantitative analysis results of cells, (H) Quantitative analysis results of CD8+ T cells in distal tumor tissues, (I) Quantitative analysis results of memory T cells in distal tumor tissues.

[0122] Figure 21 Statistical analysis results of primary tumor weight;

[0123] Figure 22 The body weight change curves 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 tumors and distal tumors in mice from different groups;

[0126] Figure 25 The figures are the statistical results of tumor inhibition rates in different groups of mice. DETAILED DESCRIPTION

[0127] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0128] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0129] In the description of the present invention, if there is a description of first, second, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[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 such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

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

[0132] Unless otherwise specified, "room temperature" in the present invention means (25±5)°C.

[0133] In the present invention, unless otherwise specified, the “particle size” in NTA analysis and dynamic light scattering analysis refers to “diameter”.

[0134] In this article, "Outer Membrane Vesicles" (OMVs) are spherical, lipid bilayer membrane structure vesicles (20-250 nanometers in diameter) secreted by Gram-negative bacteria, containing biologically active substances such as proteins, nucleic acids, and metabolites. As a carrier of intercellular communication, it participates in immune regulation, substance transfer, and disease occurrence, and shows potential in the biomedical field (such as drug delivery and vaccine development). Outer membrane vesicles are mainly released by Gram-negative bacteria through "budding" of the cell membrane, and eukaryotic cells (such as mammalian cells) can also secrete similar structures (such as extracellular vesicles). Its formation is affected by environmental pressure, gene regulation, etc., and it is one of the strategies for cells to actively adapt to external changes.

[0135] Unless otherwise specified, the "vesicle" herein may be an endogenous vesicle, a secreted exogenous vesicle, or an artificially constructed vesicle.

[0136] As used herein, "phenol" is an organic compound in which a hydroxyl group (-OH) is directly linked to an aromatic hydrocarbon nucleus (benzene ring or condensed 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 , yellow or brown powder.

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

[0139] Radiosensitizers can increase the radiation dose deposition at the tumor site at a safe radiation dose, providing an opportunity to enhance the immunogenic effect of radiotherapy. Among them, metal elements with high atomic numbers (high Z metals) and their nano-derivatives stand out due to their unique properties, including gold (Au), hafnium (Hf), bismuth (Bi), gadolinium (Gd), etc. In particular, the NBTXR3 radiosensitizer based on hafnium oxide (HfO2) has been widely studied in the clinic, sparking research interest in hafnium-based nanomaterials. However, HfO2 used for radiosensitization in clinical reports mainly relies on intratumoral injection, which greatly limits the types of cancers that can be treated and the efficiency of treatment. Therefore, Hf 4+ The delivery of hafnium-based nanomaterials to tumor sites to improve therapeutic efficiency has attracted widespread attention. OMVs, which are highly modifiable and have good tumor targeting ability, 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 have the potential to integrate OMVs and Hf 4+ MPNs have the potential to enhance cancer radioimmunotherapy. Due to the appropriate choice of metal ions and phenolic ligands, MPNs have unique functional properties. For example, MPNs prepared by chelating high-Z metal ions with phenolic reagents can be used as nanoradiosensitizers to enhance radiotherapy. In addition, polyphenols are considered to be promising drug carriers due to their multiple biological functions such as adhesion, antithrombosis and anti-tumor. For example, tannic acid (TA) is a natural polyphenol compound that has been approved as a safe substance by the US Food and Drug Administration (FDA) and is widely used in surface coating applications due to its rich phenolic hydroxyl groups. Importantly, MPNs are easy to prepare and have minimal cytotoxicity and can be deposited on biological matrices such as bacteria, yeast and mammalian cell surfaces to form biomimetic nanocloaks. Natural polyphenol nanocoatings that can be loaded with drugs provide bacteria with an editable therapeutic modality, thereby achieving multimodal therapy.

[0141] In some embodiments, the present invention designs metallo-phenol outer membrane vesicles (Hf-OMVs) by incorporating hafnium ions (Hf 4+ ) ligated with TA to enhance cancer radioimmunotherapy ( Figure 1 Figure A in the figure). Due to inheriting the characteristics and nanostructure of OMVs, Hf-OMVs can effectively accumulate at the tumor site. In addition, catalase deposited in the OMVs envelope can alleviate the hypoxia of the tumor microenvironment (TME) and amplify the immunomodulatory effect, thereby enhancing the accumulation of ionizing radiation caused by Hf at the tumor site, inducing tumor cell death and releasing tumor antigens. At the same time, OMVs components can promote the maturation of dendritic cells (DCs) and bind to the released tumor antigens, further promoting T cell activation, thereby triggering an effective immune response ( Figure 1 (B in Figure 2). In a mouse breast cancer model, Hf-OMVs combined with radiotherapy demonstrated effective inhibition of primary and distant tumor progression and induced a robust antitumor immune memory response. Thus, this invention provides a simple and effective nanoplatform that combines the advantages of natural OMVs and metallophenol networks for cancer radioimmunotherapy.

[0142] OMVs lack radiosensitization, while 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, tumor targeting can be synergistically improved, while also enhancing radiosensitization.

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

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

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

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

[0147] (2) Add 4 μL of hydrogen peroxide solution with concentrations of 0, 0.625, 1.25, 2.5, and 3.75 mM and 200 μL of color development working solution to a 96-well plate, incubate at 25°C for 15 min, and then measure the 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, and 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 minutes, 450 μL of catalase reaction termination solution was added to obtain a reaction termination solution. After mixing 40 μL of detection buffer with 10 μL of reaction termination solution, 10 μL was taken and placed in a 96-well plate, 200 μL of color development working solution was added, and A was measured after incubation at 25°C for 15 minutes. 520 nm , substituted into the standard curve in step (2) to calculate the catalase activity (catalase activity (U / mg) = [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-OMV precipitate) and transfer to a beaker. Then, add 2 mL of nitric acid for digestion. After digestion, bring the volume up to 10 mL and analyze using inductively coupled plasma optical emission spectrometry.

[0151] Unless otherwise specified, the steps for HIF-1α immunofluorescence staining are as follows:

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

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

[0154] Unless otherwise specified, all results are presented as mean ± standard deviation (SD) of independent experiments. All data were analyzed using Prism 8.0.1 software (GraphPad). Data from two groups were analyzed using the t-test, and data from more than two groups were analyzed using one-way (or two-way) analysis of variance. 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, the steps are as follows:

[0158] Escherichia coli DH5α (purchased from Vazyme) was inoculated into LB medium and cultured at 37°C and 220 rpm with shaking until the OD 600 The RI was approximately 1.3; H2O2 (final concentration 50 μM) was added to the culture medium and cultured for another 24 hours. The bacterial solution was centrifuged at 5000 × g for 30 minutes at 4°C, the supernatant was collected, and filtered through a 0.45 μm filter; ultracentrifugation was performed at 150,000 × g for 2 hours at 4°C, the precipitate was collected, washed twice with sterile PBS, and the OMVs were resuspended in sterile PBS to obtain an OMV suspension.

[0159] The results showed that the protein concentration of the OMVs prepared in this preparation example was 2 μg / μL, and the catalase activity was 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, with the only difference being that the H2O2 treatment is omitted.

[0162] The results showed that the protein concentration of the OMVs prepared in this preparation example was 2 μg / μL, and the catalase activity was 15 U / mg.

[0163] Example 1

[0164] This example provides a medical reagent, the preparation method of which is as follows:

[0165] To 25 μL of a 2 μg / μL OMV suspension, add 5 μL of a 30 mg / mL tannic acid solution (prepared by dissolving tannic acid in PBS) and vortex for 10 seconds. Then, add 5 μL of a 10 mg / mL HfCl₄ solution (prepared by dissolving HfCl₄ in PBS) to a final HfCl₄ concentration of 100 μg / mL. Vortex mix and sonicate in a waterbath for 5 minutes. The reaction mixture is centrifuged at 5000 × g for 15 minutes at 4°C. The supernatant is removed and the pellet is washed twice with sterile PBS to obtain Hf-OMV particles.

[0166] The temperature of the water bath ultrasonic treatment was 4°C and the power was 70W.

[0167] Example 2

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

[0169] Example 3

[0170] This example provides a medical reagent, and the preparation method is basically the same as that of Example 1, except that the concentration of the HfCl4 solution is different, and the final concentration of the HfCl4 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), the preparation method of which is basically the same as that of Example 1, except that the HfCl4 solution is replaced with an equal volume of PBS (the final HfCl4 reaction concentration is 0 μg / mL).

[0173] Comparative Example 2

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

[0175] Test 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 negatively stained with uranyl acetate for 60 seconds. After drying, the grid was examined using a 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 using 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 were measured using a UV-Vis spectrometer (Shimadzu Corporation) within the range of 200 nm to 400 nm. The amount of Hf grafted into the medical reagents prepared in Examples 1-3 and Comparative Example 1 (per 100 μg of OMVs) was analyzed using ICP-OES. 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 measured using an infrared spectrometer (ThermoFisher Scientific). XPS spectra and high-resolution XPS spectra of the OMVs prepared in Preparation Example 1 and Hf-OMVs prepared in Example 1 were measured using an X-ray photoelectron spectrometer (Thermo Fisher Scientific). The OMVs prepared in Preparation Example 1 and the Hf-OMVs prepared in Example 1 were treated with lysis buffer (Solabo, BC3710) for 30 minutes, and their protein concentrations were determined. After further treatment at 100°C for 10 minutes, the OMVs were loaded onto a 10% SDS-PAGE gel and run at 80 V for 30 minutes and 120 V for 1 hour. The gels were stained with Coomassie Brilliant Blue for 20 minutes, destained at room temperature, and the SDS-PAGE electrophoretograms were observed. The OMVs suspension prepared in Preparation Example 1 and the Hf-OMVs suspension 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). The Hf-OMVs suspension prepared in Example 1 was dropped onto an unstained carbon film and dried at room temperature. Data was then acquired using a TEM (FEI-TALOS-F200X) equipped with an energy dispersive spectrometer. A 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 Figure 2-Figure 5 shown.

[0178] The OMVs prepared in Preparation Example 1 showed 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, which indicated that the OMVs were successfully extracted ( Figure 2AC diagram in Figure 1). After preparation, OMVs@TA has the characteristic peaks of TA and shows similar absorption peaks to OMVs ( Figure 2 Figure D in the figure).

[0179] The Hf element was successfully modified on the surface of OMVs, and the content of Hf element modified on the surface of OMVs gradually increased with the increase of the final concentration of HfCl4 reaction. It reached saturation when the final concentration of HfCl4 reaction reached 100 μg / mL, and the amount of Hf grafted per 100 μg OMVs was 10 μg ( Figure 2 OMVs and Hf-OMVs contain similar elements, and OMVs do not contain Hf elements ( Figure 2 In the G graph, Figure 4 Compared with OMVs, the Hf4f high-resolution XPS spectrum of Hf-OMVs can be decomposed into two peaks at 17.7eV and 19.5eV, which further confirms the presence of Hf element in Hf-OMVs ( Figure 2 H in Figure ). And Hf-OMVs inherited the proteins of OMVs ( Figure 2 EDS elemental analysis showed that Hf-OMVs contained carbon (C), oxygen (O), nitrogen (N), phosphorus (P) and Hf ( Figure 5 ).

[0180] Hf-OMVs exhibited spherical lipid droplets similar to those of OMVs and had good homogeneity ( Figure 2 Figure J in Figure 1). 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, with a peak particle size of 197.6 nm, which was larger than that of OMVs (hydrated particle size of 165.8 nm, peak particle size of 146 nm). The Zeta potential of Hf-OMVs was -34.8 mV, which was lower than that of OMVs (Zeta potential of -30.8 mV), which may be attributed to the fact that polyphenol complexes are generally negatively charged ( Figure 2 (KM diagram in ).

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

[0182] Test Example 2 (Hypoxia Alleviation Effect of Hf-OMVs)

[0183] 1. The relative catalase activity of the OMVs prepared in Preparation Example 1 and Preparation Example 2 (Preparation Example 1 is recorded as: OMVs+H2O2; Preparation Example 2 is recorded as: OMVs) was analyzed.

[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 minutes, and monitor the changes in dissolved oxygen concentration in the reaction system.

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

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

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

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

[0189] 6. Nine BALB / c mice (5-week-old, female) were subcutaneously injected with 5×10 54T1 cells were randomly divided into three groups. In the OMVs group, mice were intravenously injected with OMVs (100 μg / mouse, 100 μL) on day 6. In the Hf-OMVs group, mice were intravenously injected with the Hf-OMVs described in Example 1 (Hf-OMVs in Test Examples 3-9 below refer to the Hf-OMVs described in Example 1) (100 μg / mouse, 100 μL) on day 6. In the PBS group, mice were injected with an equal volume of PBS on day 6. Two days later, mice were euthanized, and tumor tissues were harvested, embedded, sectioned, and stained for HIF-1α immunofluorescence.

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

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

[0192] The concentration of O2 generated by catalyzing H2O2 by the OMVs of Preparation Example 1 increased steadily over time, reached a peak at about 3 minutes, and remained stable thereafter in a dose-dependent manner, which also indicated that the catalase activity of the OMVs of Preparation Example 1 was stable ( Figure 7 CF graph).

[0193] The expression level of HIF-1α in 4T1 cells increased significantly after culture in an oxygen-deficient environment, and it could be inhibited by adding free catalase. After the addition of OMVs of Preparation Example 1 or Hf-OMVs of Example 1 to 4T1 cells in an oxygen-deficient state for 8 hours and 24 hours, the expression level of HIF-1α was significantly reduced ( Figure 8 ). This indicates that OMVs and Hf-OMVs can alleviate the hypoxia state of tumor cells in a long-term manner.

[0194] Compared with the PBS group, the expression level of HIF-1α in tumor tissues of the OMVs group and Hf-OMVs group was significantly decreased ( Figure 9 This suggests that OMVs and Hf-OMVs have the ability to alleviate tumor tissue hypoxia. Therefore, Hf-OMVs inherit the catalase activity of OMVs, effectively alleviating tumor hypoxia and contributing to radiotherapy sensitization.

[0195] Test Example 3 (Uptake of Hf-OMVs by 4T1 Cells)

[0196] 4T1 cells were seeded in 12-well cell culture plates and confocal microplates. DiI-labeled Hf-OMVs (prepared in Example 1 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 microplates at various time points. The final concentration of Hf-OMVs was 100 μg / mL. After incubation, cells in the 12-well plates were analyzed by flow cytometry (detecting DiI-positive signals) and ICP-OES (detecting Hf content; cellular uptake of Hf-OMVs (%) = mass of Hf detected in the cell sample / mass of Hf initially added). Cells in the confocal microplates were fixed with 4% paraformaldehyde, stained with DAPI, and observed using a CLSM (ZEISS LSM980). The uptake of Hf-OMVs by 4T1 cells was evaluated at 0, 2, 4, 8, 12, and 24 hours.

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

[0198] The fluorescence signal gradually increased with the extension of incubation time and reached saturation at 12 hours ( Figure 10 After 2 hours of co-incubation, 4T1 cells showed obvious intracellular fluorescence signals, which reached a peak after 12 hours ( Figure 10 This is consistent with the results of flow cytometry and ICP-OES analysis.

[0199] Test Example 4 (In vitro radiotherapy sensitization effect of Hf-OMVs)

[0200] 1. 4T1 cells were seeded in a 96-well plate and cultured 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 then added to the 96-well plate for co-culture. After 24 hours, CCK-8 reagent (Vazyme) was added to the cell culture medium and incubated at 37°C for 1 hour. The absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0201] 2. 4T1 cells were seeded in a 96-well plate and cultured overnight. Various concentrations of OMVs prepared in Preparation Example 1, Hf-TA prepared in Comparative Example 2, or Hf-OMVs prepared in Example 1 were then added to the 96-well plate for co-culture. After 12 hours, the 96-well plate was irradiated with a dose of 6 Gy of X-rays and then cultured for an additional 24 hours. Cell viability was calculated using the CCK-8 assay.

[0202] 3. Apoptosis Assay: 4T1 cells were seeded in 12-well cell culture plates. 24 hours later, DMEM medium containing OMVs prepared in Preparation 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 was the same as that of 80 μg / mL Hf-OMVs based on protein concentration; the final concentration of Hf-TA was the same as that of 80 μg / mL Hf-OMVs based on Hf concentration). The same volume of DMEM medium was added to the control group. 12 hours later, the 12-well plates were irradiated with a dose of 6 Gy of X-rays and cultured for an additional 24 hours. The plates were then trypsinized 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 allowed to stand for 10 minutes (Annexin V-FITC / PI Apoptosis Detection Kit, DOJINDO). Finally, the cells were resuspended in 300 μL of buffer and detected by flow cytometry (LSRFortessa SORP, Becton & Dickinson) to calculate the cell apoptosis rate.

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

[0204] 5. DNA damage assay: 4T1 cells were seeded in a confocal microplate culture dish and the DMEM medium containing OMVs prepared in Preparation 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 was the same as that of 100 μg / mL Hf-OMVs in terms of protein concentration; the final concentration of Hf-TA was the same as that of 100 μg / mL Hf-OMVs in terms of Hf concentration) was added to the 4T1 cells. After 12 hours of culture, the confocal microplate was exposed to a dose of 6 Gy of X-rays for irradiation. After 24 hours of culture, the cells were fixed with 4% paraformaldehyde and then incubated with 0.1% Triton-X100 for 5 minutes. BSA (10 mg / mL) was then added for 1 hour. γ-H2AX antibody (1:1000, Abcam) was then added and incubated at 4°C for 16 hours. Finally, Cy3 fluorescent secondary antibody (1:50, Proteintech) was added and incubated for 1 hour. Finally, cell nuclei were stained with DAPI and detected under CLSM (ZEISS LSM980).

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

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

[0207] The radiosensitization effect induced by Hf-OMVs was dose-dependent, and its IC 50 123 μg / mL ( Figure 12 Figure B). After 4T1 cells were treated with different concentrations of OMVs, Hf-TA, and Hf-OMVs and then irradiated with X-rays (6Gy), the relative cell viability of the three groups was significantly reduced, and there was a certain degree of dose-dependent effect ( Figure 12 Furthermore, the relative cell activity of the Hf-OMVs group was significantly lower than that of the OMVs group and the Hf-TA group.

[0208] The late apoptosis rate of cells induced by Hf-OMVs+RT group was about 23.1%, which was much higher than that of OMVs+RT group (the late apoptosis rate of cells was about 12.8%) and Hf-TA+RT group (the late apoptosis rate of cells was about 15.1%) ( Figure 12 This indicates that Hf-OMVs can significantly enhance radiotherapy-induced cell 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 (the relative clone area of ​​G1 was 1, and the relative clone area of ​​G2 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 (the relative clone area of ​​G3 was 0.44, and the relative clone area of ​​G4 was 0.25) ( Figure 12 (F) This indicates that Hf-OMVs+RT can significantly inhibit the proliferation of 4T1 cells.

[0210] DNA damage caused by DNA double-strand breaks is the main cause of cell death caused by radiotherapy, and γ-H2AX is often used as a marker of DNA damage. The group that did not receive X-ray irradiation had no obvious DNA damage, while the PBS+RT group and OMVs+RT group induced moderate DNA damage. Compared with the PBS+RT group and OMVs+RT group, the Hf-TA+RT group and Hf-OMVs+RT group significantly increased the DNA damage level of 4T1 cells. Among them, the Hf-OMVs+RT group induced the highest level of DNA damage ( Figure 12 GH diagram).

[0211] In summary, the surface modification of Hf-OMVs imparts unique physical properties, enabling them to deposit more ionizing radiation, thereby inducing a strong radiosensitization effect. Hf, when incorporated into the OMV surface via a metal-phenol network, synergistically enhances the radiosensitization effect, significantly reducing cancer cell survival, enhancing radiotherapy-induced apoptosis, inhibiting cancer cell proliferation, and inducing DNA damage.

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

[0213] 1. Collect whole blood from BALB / c mice and centrifuge at 1200rpm for 10 minutes to separate red blood cells (RBCs). RBCs were mixed with an equal volume of PBS containing different concentrations (0.1, 0.25, 0.5, 1, 1.5 and 2 μg / uL) 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 15000rpm for 5 minutes and photographed. The absorbance of the supernatant at 570nm was measured using an enzyme-linked microplate reader to calculate the hemolysis rate (hemolysis rate = (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 and injected with PBS containing Hf-OMVs (100 μg / mouse, 100 μL) or an equal amount of PBS through the tail vein. The administration was done every other day for a total of three times. After 16 days, the mice were euthanized, whole blood and serum were collected by eye bleeding, and the main organs (heart, liver, spleen, lungs and kidneys) were dissected. The blood in the anticoagulant tube was tested by an automatic blood cell analyzer. In addition, the whole blood was centrifuged at 3000 rpm for 10 minutes at 4°C, and the blood biochemical indicators were detected using a biochemical automatic analyzer. Different organs were fixed with 4% paraformaldehyde, then embedded, sectioned and stained with H&E.

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

[0216] In the presence of different concentrations of Hf-OMVs, no obvious erythrocyte lysis was observed ( Figure 13 Routine blood tests, blood biochemical analysis, and H&E sections all showed that Hf-OMVs had no significant in vivo toxicity, and no significant histological damage to major organs ( Figure 14 ).

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

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

[0219] 1. On day 1, BALB / c mice (5-6 weeks old, female, n=4) were injected subcutaneously with 5×10 5 4T1 cells. DiR dye was added to the OMVs of Preparation Example 1, incubated at 37°C for 30 minutes, and washed twice with PBS to obtain DiR-labeled OMVs. On the 8th day, the DiR-labeled OMVs prepared in Preparation 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. The mice were euthanized at 72 hours, and the main organs (heart, liver, spleen, lungs and kidneys) and tumor tissues were obtained and imaged using an IVIS imaging system. Subsequently, ICP-OES was used to analyze the tumor targeting effect of Hf-OMVs.

[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 5On 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; in terms of Hf, 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 the tumor tissues were obtained and digested for ICP-OES analysis to calculate the Hf content in the tumor tissues. 4+ content.

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

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

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

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

[0225] BALB / c mice (5-6 weeks old, female) were subcutaneously inoculated with 5×10 5 4T1 cells. When the tumor volume grows to about 100mm 3 At day 0 (denoted as day 0), the 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, 5 mice in each group).

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

[0227] (2) OMVs group (G2): OMVs prepared in Preparation Example 1 were intravenously injected with PBS (100 μg / mouse, 100 μL) on days 0 and 3, respectively;

[0228] (3) Hf-OMVs group (G3): Hf-OMVs prepared in Example 1 were intravenously injected in PBS (100 μg / mouse, 100 μL) on days 0 and 3, respectively;

[0229] (4) PBS+RT group (G4): 100 μL PBS was injected intravenously on days 0 and 3, and the tumor was locally irradiated with X-rays on days 2 and 5 (dose of 6 Gy, and the rest of the area was shielded with a lead cover);

[0230] (5) OMVs + RT group (G5): OMVs prepared in Example 1 were intravenously injected with PBS (100 μg / mouse, 100 μL) on days 0 and 3, respectively. The tumors were locally irradiated with X-rays (dose of 6 Gy, and the rest of the tumors were shielded with lead caps) on days 2 and 5.

[0231] (6) Hf-OMVs+RT group (G6): Hf-OMVs prepared in Example 1 were intravenously injected with PBS (100 μg / mouse, 100 μL) on days 0 and 3, and the tumors were locally irradiated with X-rays (dose of 6 Gy, and the rest of the tumors were shielded with lead caps) on days 2 and 5.

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

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

[0234] All mice survived during the entire treatment period, and there was no statistical difference in body weight among the groups ( Figure 17 Figure F). OMVs and Hf-OMVs alone had little effect on tumor growth. PBS+RT and OMVs+RT had 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 showed the most significant tumor suppression effect, with the most severe cell apoptosis, significantly inhibiting tumor cell proliferation, and causing severe cell nucleus atrophy ( Figure 17 This indicates that Hf-OMVs can significantly enhance the therapeutic efficacy of RT against solid tumors in vivo.

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

[0236] The experimental process was the same as that of Test Example 7. On the 14th day, the mice were euthanized and the tumors were removed. 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). The tumor tissue was 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 tissue was digested using an automatic tissue processor and then filtered with 70 μm and 40 μm filters to prepare a single cell suspension. Subsequently, the cells were first labeled with a live dead dye (Live Dead, FVS510; Biolegend). For analysis of mature DCs, cells were incubated in the dark with antibodies against specific markers (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 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), and CD8 (BV605; clone: ​​53-6.7; Becton & Dickinson)). The cells were then fixed and stained with GranB (APC; clone: ​​QA16A02; Biolegend). For 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 shown.

[0238] 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 suggests 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 DC maturation.

[0239] CD8 + The infiltration of cytotoxic T cells can effectively promote the anti-tumor effect. Flow cytometry analysis results showed that CD8 + 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. Among them, the infiltration of CD8+T cells in tumor tissues treated with Hf-OMVs+RT increased from 6.87% to 30.84%, and GranB + CD8 + The proportion of T cells increased from 2.9% to 13.4%, which was the most significant increase. The results of immunofluorescence analysis were consistent with the results of flow cytometry analysis. After Hf-OMVs+RT treatment, the levels of TNF-α, IFN-γ and IL-6 in tumor tissues increased significantly. This indicates that Hf-OMVs+RT treatment significantly promoted CD8 + T cell infiltration and CD8 + Activation of T cells.

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

[0241] Test Example 9 (Anti-tumor abscopal effect of Hf-OMVs combined with radiotherapy)

[0242] 5×10 5 4T1 cells were subcutaneously inoculated into the right skin (primary tumor) of BALB / c mice (5-6 weeks old, female). Five days later, 5×10 5 4T1 cells (distal tumor) were used to establish a bilateral tumor model. When the primary tumor volume grew to approximately 100 mm 3At day 0 (denoted as day 0), the 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, 5 mice in each group).

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

[0244] (2) OMVs group (G2): OMVs prepared in Preparation Example 1 were intravenously injected with PBS (100 μg / mouse, 100 μL) on days 0 and 3, respectively;

[0245] (3) Hf-OMVs group (G3): Hf-OMVs prepared in Example 1 were intravenously injected in PBS (100 μg / mouse, 100 μL) on days 0 and 3, respectively;

[0246] (4) PBS+RT group (G4): 100 μL PBS was injected intravenously on days 0 and 3, and the primary tumor was locally irradiated with X-rays on days 2 and 5 (dose of 6 Gy, and the rest of the site was shielded with a lead cover);

[0247] (5) OMVs + RT group (G5): OMVs prepared in Example 1 were intravenously injected with PBS (100 μg / mouse, 100 μL) on days 0 and 3, respectively. The primary tumor was locally irradiated with X-rays on days 2 and 5 (dose of 6 Gy, and the rest of the site was shielded with a lead cover);

[0248] (6) Hf-OMVs+RT group (G6): PBS containing Hf-OMVs prepared in Example 1 (100 μg / mouse, 100 μL) was intravenously injected on days 0 and 3, respectively. The primary tumor was locally irradiated with X-rays on days 2 and 5 (the dose was 6 Gy, and the rest of the area was shielded with a lead cover).

[0249] The size of primary and distal tumors and the weight of mice were monitored every other day. On day 14, mice were euthanized, and the primary and distal tumors were dissected, weighed, and photographed to calculate the tumor inhibition rate (tumor inhibition rate (%) = tumor weight of treatment group / tumor weight of PBS group × 100%). The expression of CD8 + T cells, memory T cells (CD44 + CD62L - ), mature DC (CD80 + CD86 + )Changes in cells.

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

[0251] OMVs or Hf-OMVs alone had only a weak inhibitory effect on primary and distal tumors. However, Hf-OMVs combined with radiotherapy significantly inhibited the growth of both primary and distal tumors, with an inhibition rate of 74.68% for distal tumors, far superior to RT or OMVs plus RT.

[0252] After Hf-OMVs+RT combined treatment, the proportion of mature DCs in distal tumors increased significantly to about 21.46%, which was much higher than that in other groups (G1: 6.68%; G2: 11.3%; G3: 11.66%; G4: 13.2%; G5: 15.52%); CD8 + The proportion of T cells was approximately 19.16%, which was much higher than that of other groups (G1: 5.9%; G2: 7.86%; G3: 7.38%; G4: 11.85%; G5: 13.16%); the proportion of memory T cells in distal tumors was approximately 19.74%, which was much higher than that of other groups (G1: 6.17%; G2: 9.29%; G3: 8.88%; G4: 11.34%; G5: 15.28%). Moreover, during the entire treatment period, the weight of mice in each group did not decrease significantly. This indicates that Hf-OMVs combined with radiotherapy can promote the proliferation of CD8 + The recruitment of T cells 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 are described in detail above in conjunction with the embodiments, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A medical reagent, characterized in that: The medical agent includes a nanocarrier modified with a metal-phenol network; The metal is a high atomic number metal.

2. The medical reagent according to claim 1, characterized in that The atomic number of the high atomic number metal is not less than 60; Preferably, the high atomic number metal includes at least one of gadolinium, hafnium, tungsten, gold, bismuth, palladium, platinum, tantalum, gallium, tellurium, and zirconium.

3. The medical reagent according to claim 1, characterized in that The catalase activity of the nanocarrier is not less than 15 U / mg; and / or, the nanocarrier comprises at least one of a vesicle and a synthetic nanoparticle; and / or, the hydrated particle size of the nanocarrier is 50 nm to 200 nm; and / or, each 100 μg of nanocarriers in the medical agent contains 1 μg-25 μg of the high atomic number metal; And / or, the hydrated particle size of the nanocarrier modified with the metal-phenol network is 150 nm to 300 nm.

4. The medical reagent according to claim 1, characterized in that The phenols include polyphenols; Preferably, the polyphenols include compounds that conform to the molecular chemical structure defined by White-Bate-Smith-Swain-Haslam; Preferably, the polyphenols include tannic acid, catechin, dopamine and condensed deformation, tannin molecules, epicatechin, gallol catechin, epigallocatechin, anthocyanidins, anthraquinones, chalcones, lignans, naphthoquinones, neolignans, proanthocyanidins, pigments tannic acid, xanthones, condensates of polyphenols synthesized by plant biosynthesis or artificial fermentation, theaflavins, polymers of anthocyanidins, highly condensed tannins, and artificially synthesized molecules containing polyphenol groups, or a combination thereof.

5. The method for preparing the medical reagent according to any one of claims 1 to 4, characterized in that: The following steps are involved: A mixture of the nanocarrier, phenol and a high atomic number metal ion source is prepared and reacted to obtain the medical reagent.

6. The preparation method according to claim 5, 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 a biological cell, the method for preparing the nanocarrier comprises: contacting the biological cell with a catalase inducer to react to obtain the nanocarrier; and / or, the mass ratio of the nanocarrier to the 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 reaction temperature is 1°C-10°C; And / or, the reaction time is 3 min-20 min; And / or, the ultrasonic power of the reaction is 50W-90W.

7. A drug, characterized in that The invention comprises the medical agent according to any one of claims 1 to 4 and pharmaceutically acceptable excipients or auxiliary components.

8. Use of the medical agent according to any one of claims 1 to 4 or the drug according to claim 7 in combination with radiation irradiation in any one of A1) to A4): A1) preparing a product for killing cells; A2) preparing a product that induces cell apoptosis; A3) preparing a product for inhibiting cell proliferation; A4) preparing a product that induces DNA damage in cells; Preferably, the cells in A1) to A4) include abnormal cells; More preferably, the abnormal cells include at least one of tumor cells and inflammatory cells.

9. Use of the medical agent according to any one of claims 1 to 4 or the medicament according to claim 7 in any one of B1) to B2): B1) preparing drugs for enhancing tumor radiosensitivity; B2) Prepare drugs to improve the effect of tumor radiotherapy.

10. The use according to claim 9, characterized in that The improved tumor radiotherapy effect in B2) includes at least one of C1) to C3): C1) inhibit tumor growth; C2) Increase the proportion of mature DCs and CD3 + CD45 + CD8 + T cell ratio, GranB + CD8 + At least one of the T cell ratio and memory T cell ratio; C3) increasing at least one of the levels of TNF-α, IFN-γ, and IL-6 in the tumor.

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