Bacteria mineralized metal nanoparticles for treatment of tumors

Abstract

A pharmaceutical composition, a radiotherapy sensitizer, an immunopotentiator or an immunostimulatory agent comprising a metallosome comprising metal nanoparticles and a bacterial component attached thereto, and uses thereof in immunoregulation and tumor treatment are provided.

Description

Bacteria-mineralized metal nanoparticles for tumor treatment Technical Field

[0001] The present invention relates to tumor treatment, in particular to metallobodies produced by bacterial mineralization of metals for tumor treatment. Background Art

[0002] Some complex structures naturally secreted by bacteria, such as bacterial outer membrane vesicles (OMVs), are nanosized particles produced by budding from the bacterial cell membrane and are commonly used for the exchange of substances and information between bacteria and the outside world. Therefore, these nanostructures not only contain cytoskeletal components such as lipoproteins and lipopolysaccharides (LPS), but may also contain nucleic acid molecules and metabolites, and have potential immunostimulatory abilities [Kulp A et al., Biological Functions and Biogenesis of Secreted Bacterial Outer Membrane Vesicles. Annu rev ​​microbiol: 2010, 64: 163-184; Toyofuku M et al., Types and origins of bacterial membrane vesicles. Nature Rev Microbiol: 2019, 17: 13-24 and Haurat MF et al. Selective sorting of cargo proteins into bacterial membrane vesicles. J Biol Chem: 2011, 286: 1269-1276]. Microbial-derived components, including LPS, CpG, poly(I:C), and bacterial outer membrane vesicles, have long been used as immunoadjuvants, effectively activating and modulating the immune response. Recently, bacterial-derived nanomembrane structures have been shown to activate the immune system, triggering IFN-γ-based antitumor effects and mediating LPS-induced pyroptosis [Kim OY et al., Bacterial outer membrane vesicles suppress tumor by interferon-gamma-mediated antitumor response. Nat Commun: 2017, 8: 626 and Vanaja SK et al., Bacterial outer membrane vesicles mediate cytosolic localization of LPS and caspase-11 activation. Cell: 2016, 165: 1106-1119], representing potent multivalent immunostimulants.Moreover, bacterial membranes can express modified antigens, cytokines, etc. through genetic engineering and other means, and are also good carriers with intrinsic adjuvant properties [Chen DJ et al., Delivery of foreign antigens by engineered outer membrane vesicle vaccines. Proc Natl Acad Sci USA: 2010, 107: 3099-3104 and Kesty NC et al., Incorporation of heterologous outer membrane and periplasmic proteins into escherichia coli outer membrane vesicles. J Biol Chem: 2004, 279: 2069-2076].

[0003] Radiotherapy is a common clinical tumor treatment method, especially for solid tumors. However, its therapeutic effect is often limited by factors such as tumor radiotherapy tolerance and insufficient irradiation dose. High atomic number metal nanomaterials, such as gold nanoparticles, can efficiently absorb high-energy rays and deposit irradiation energy. They can be used as radiotherapy sensitizers to enhance the killing effect of high-energy rays on tumor cells. Although the radiotherapy combined with immunotherapy model has broad prospects, not all patients can benefit from it. Due to tumor heterogeneity and the complexity of the immune system, the single-target regulatory effect may be difficult to show the same efficacy in all patients. Therefore, there is a need for agents and methods that can enhance the therapeutic effect of diseases, especially tumors. [Pitroda SP et al., Integration of radiotherapy and immunotherapy for treatment of oligometastases. Lancet Oncol. 2019; 20(8): e434-e442. and Brooks, ED et al., Time to abandon single-site irradiation for inducing abscopal effects. Nat Rev Clin Oncol., 2019; 16(2), 123-135.]

[0004] Since Roentgen accidentally discovered X-rays in 1895, various ionizing radiations represented by X-rays have been widely used in tumor treatment. Ionizing radiation kills tumor cells mainly by damaging DNA. Radiotherapy mainly causes damage to tissues through two mechanisms, directly damaging DNA and indirectly damaging tissues through the formation of reactive oxygen species (ROS). The immunological effects induced by ionizing radiation include immunostimulatory and immunosuppressive effects. When cell death caused by radiotherapy-induced DNA damage activates cGAS-STING pathway signaling, leading to the production of a series of damage-associated molecular patterns (DAMPs), including calretinin, ATP and HMGB1 proteins, the immunostimulatory effect is initiated. Calretinin binds to dendritic cells (DCs) and generates an "eat me" signal, leading to antigen processing and maturation of dendritic cells and other antigen-presenting cells (APCs). APCs then present these antigens to CD8 + Cytotoxic T cells are recruited through the interferon type I pathway, leading to antigen-specific T cell killing. Their immunosuppressive effects are mediated by factors such as reactive oxygen species (ROS), HIF1α, and PD-L1. ROS lead to the release of TGFβ, which inhibits antigen-specific T cell killing. This is also true for other immune cells such as regulatory T cells (Tregs), cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs). [Price JM et al., Predicting tumour radiosensitivity to deliver precision radiotherapy. Nat Rev Clin Oncol. 2023; 20(2): 83-98.]

[0005] Tumor hyperthermia is also a strategy to regulate the local immune environment of the tumor. Hyperthermia can promote the expression and secretion of heat shock proteins (HSPs) by tumor cells, transmit signals to various immune cells, promote their differentiation into immune phenotypes, and secrete a large number of proinflammatory cytokines [Lee S et al., Immunogenic effect of hyperthermia on enhancing radiotherapeutic efficacy. Int J Mol Sci: 2018, 19: 2795; Wust P et al., Hyperthermia in combined treatment of cancer. Lancet Oncol: 2002, 3: 487-497 and Zhang HG et al., Hyperthermia on immune regulation: A temperature's story. Cancer Lett: 2008, 271: 191-204]. At the same time, excessive heat can also accelerate blood flow, enlarge the endothelial space, promote blood tissue perfusion, and increase the tissue infiltration of immunomodulators and effector cells [Kong G et al., Hyperthermia enables tumor-specific nanoparticle delivery: Effect of particle size. Cancer Res: 2000, 60: 4440-4445 and Zhao R et al., Photothermal effect enhanced cascade targeting strategy for improved pancreatic cancer therapy by gold nanoshell@mesoporous silica nanorod. ACS Nano: 2017, 11: 8103-8113].

[0006] Gold nanoparticles, due to their good biosafety and surface plasmon optical resonance characteristics, can convert light energy into heat energy and are often used in tumor photothermal therapy research [Liu Y et al., Gold nanoparticles-mediated photothermal therapy and immunotherapy. Immunotherapy: 2018, 10: 1175-1188; Wei P et al., Dendrimer-stabilized gold nanostars as a multifunctional theranostic nanoplatform for CT imaging, photothermal therapy, and gene silencing of tumors. Adv Health Mater: 2016, 5: 3203-3213 and Zhang D et al., Intracellularly generated immunological gold nanoparticles for combinatorial photothermal therapy and immunotherapy against tumor. Nano Lett: 2019, 19: 6635-6646]. Gold also has a high atomic number (Z = 79). The mechanism of radiosensitization by gold nanomaterials is generally believed to be that gold atoms increase the cross-section of the tissue or cell's reaction with radiation, improving the effective deposition of high-energy radiation energy. This is because the X-ray absorption coefficient μ is related to the incident X-ray energy E and the atomic coefficient Z as follows: μ = ρZ4 / (AE3), where ρ is the density and A is the atomic mass. Therefore, changes in the atomic coefficient Z significantly alter the absorption coefficient μ. Therefore, materials containing elements with high atomic coefficients exhibit better X-ray energy attenuation. Normally, radiation photons can directly damage cellular DNA or indirectly react with water to generate free radicals, further damaging DNA. When gold nanoparticles accumulate in tumor areas, they effectively absorb X-ray energy and interact with the radiation to emit secondary electrons such as photoelectrons, Auger electrons, and Compton electrons. These secondary electrons not only interact directly with DNA but also react with water to increase free radical production, further increasing tumor cell sensitivity to radiation. This process is a physical sensitization mechanism.[Price JM et al., Predicting tumour radiosensitivity to deliver precision radiotherapy. Nat Rev Clin Oncol. 2023;20(2):83-98.; Qin X et al., Cell-Derived Biogenetic Gold Nanoparticles for Sensitizing Radiotherapy and Boosting Immune Response against Cancer. Small. 2021 Dec;17(50):e2103984. and Sun W et al., Aggregation-Induced Emission Gold Clustoluminogens for Enhanced Low-Dose X-ray-Induced Photodynamic Therapy. Angew Chem Int Ed Engl. 2020 Jun 15;59(25):9914-9921.].

[0007] Bacterial biomineralization is the process of reducing metal ions to elemental metals within cells or cell extracts using redox reactions in biological processes. It is a very mild and environmentally friendly metal reduction method [Jung JH et al., In vivo synthesis of nanocomposites using the recombinant escherichia coli. Small: 2018, 14 and Reith F et al., Biomineralization of gold: Biofilms on bacterioform gold. Science: 2006, 313: 233-236]. At present, people have explored the application of various bacteria in the reduction of different metal ions, including iron, zinc, cadmium, silver, gold, etc., mainly for environmental purification and heavy metal recovery [Gadd GM, Metals, minerals and microbes: Geomicrobiology and bioremediation. Microbiology: 2010, 156: 609-643 and Das SK et al., A green chemical approach for the synthesis of gold nanoparticles: Characterization and mechanistic aspect. Rev Environ Sci Bio-Tech: 2010, 9: 199-204].

[0008] During the development of tumor tissue, in order to evade recognition and elimination by the immune system, tumor cells suppress the body's anti-tumor immunity by blocking the immune response or activating negative immune regulatory pathways, including hindering the tumor tissue infiltration and functional activity of effector cells, upregulating the expression of immune checkpoints, and promoting antigen presentation towards immune tolerance [Whiteside TL, Immune suppression in cancer: Effects on immune cells, mechanisms and future therapeutic intervention. Semin Cancer Biol: 2006, 16: 3-15; Marigo I et al., Tumor-induced tolerance and immune suppression by myeloid-derived suppressor cells. Immunol Rev: 2008, 222: 162-179; Vinay DS et al., Immune evasion in cancer: Mechanistic basis and therapeutic strategies. Semin Cancer Biol: 2015, 35: S185-S198 and Beatty GL et al., Immune escape mechanisms as a guide for cancer immunotherapy. Clin Cancer Res:2015,21:687-692]. In these processes of immune activation and immunosuppression, a large number of molecular patterns, cytokines, and metabolites are involved. These act as important signaling molecules to participate in immune responses, promote communication between immune cells, and regulate immune direction. Cytokines, as the main signaling molecules of the immune system, can directly act on various immune cells. For example, IL-2 can promote the proliferation of effector immune cells such as natural killer (NK) cells and T cells; interferon-α (INF-α) plays an important role in the maturation of dendritic cells (DCs) and anti-tumor angiogenesis; and multiple immune checkpoint blockades, mainly CTLA-4 and PD-1, have been shown to effectively promote the generation of effector immune cells and their recognition and killing of tumor cells. However, due to the single target of these immunomodulators, their clinical response rate is relatively low, accompanied by significant toxic side effects, and their therapeutic efficacy still needs to be improved. Therefore, it is necessary to continue to explore new immunomodulators that, through rational design and simultaneous action on multiple targets, promote immune system activation or alleviate the tumor immunosuppressive environment.

[0009] Summary of the Invention

[0010] This article uses different species of bacteria (such as Escherichia coli) to mineralize and reduce gold ions, which are then wrapped by the bacterial membrane and secreted outside the cell through budding, producing gold bodies (Ausomes) with gold nanoparticles coated on the surface of the bacterial membrane. Because Ausomes contain a large number of bacteria-derived molecules, they have potential immunostimulatory capabilities and can systemically activate the immune system to produce activated immune cells and effector cells. At the same time, gold nanoparticles also give Ausomes photothermal conversion capabilities. Nanosized Ausomes can be enriched in tumor sites, achieving local hyperthermia in the tumor, increasing tissue blood perfusion, promoting tumor infiltration of effector cells, and alleviating the tumor's immunosuppressive microenvironment. In addition, Ausomes with high atomic numbers can effectively absorb and deposit high-energy radiation energy, thereby increasing the killing of in situ tumor cells by radiotherapy or reducing the irradiation dose, avoiding damage to the mucosa or other normal tissues. The research in this article shows that Ausomes can stimulate a strong systemic immune response, secrete a large number of pro-inflammatory cytokines, and produce effector cells such as CD8+ T cells and NK cells; Ausomes-mediated local tumor hyperthermia can increase blood perfusion, further promote tissue infiltration of effector cells, and improve the immune level in the tumor; and Ausomes can deposit radiation energy at the tumor site, enhance the therapeutic effect of radiotherapy, and induce the occurrence of "remote effect".

[0011] In one aspect, the present invention provides a pharmaceutical composition comprising metallosomes and optionally a pharmaceutically acceptable carrier, wherein the metallosomes comprise metal nanoparticles and bacterial components attached thereto, preferably, the bacterial components are selected from intracellular membranes, extracellular membranes, proteins, carbohydrates, nucleic acids, phospholipids and any combination thereof.

[0012] In one aspect, the present invention provides a use of a metallobody in the preparation of a medicament for treating a tumor in a subject, wherein the metallobody comprises metal nanoparticles and bacterial components attached thereto, preferably, the bacterial components are selected from intracellular membranes, extracellular membranes, proteins, carbohydrates, nucleic acids, phospholipids and any combination thereof.

[0013] In one aspect, the present invention provides an immunopotentiator or immunostimulator, comprising a metallobody and optionally a pharmaceutically acceptable carrier, wherein the metallobody comprises metal nanoparticles and bacterial components attached thereto, preferably, the bacterial components are selected from intracellular membranes, extracellular membranes, proteins, carbohydrates, nucleic acids, phospholipids and any combination thereof.

[0014] In one aspect, the present invention provides the use of metallobodies in the preparation of immunopotentiators or immunostimulants, wherein the metallobodies comprise metal nanoparticles and bacterial components attached thereto, preferably, the bacterial components are selected from bacterial inner membranes, bacterial outer membranes, proteins, carbohydrates, nucleic acids, phospholipids and combinations thereof.

[0015] In one aspect, the present invention provides a radiosensitizer comprising a metallobody and optionally a pharmaceutically acceptable carrier, wherein the metallobody comprises metal nanoparticles and bacterial components attached thereto, preferably, the bacterial components are selected from intracellular membranes, extracellular membranes, proteins, carbohydrates, nucleic acids, phospholipids and any combination thereof.

[0016] In one aspect, the present invention provides the use of metallobodies in the preparation of radiosensitizers, wherein the metallobodies comprise metal nanoparticles and bacterial components attached thereto, preferably, the bacterial components are selected from intracellular membranes, extracellular membranes, proteins, carbohydrates, nucleic acids, phospholipids and any combination thereof.

[0017] In one aspect, the present invention provides a method for separating and purifying membrane vesicles, wherein the membrane vesicles comprise metal nanoparticles and bacterial components attached to the membrane vesicles, preferably, the bacterial components are selected from intracellular membranes, extracellular membranes, proteins, carbohydrates, nucleic acids, phospholipids, and any combination thereof, wherein the metal is selected from gold, silver, manganese, titanium, and iron, preferably gold, and the method comprises:

[0018] (i) culturing bacteria in a solution containing metal ions, wherein the bacteria are preferably selected from the genus Escherichia, preferably Escherichia coli, such as DH5α or BL21 strains,

[0019] (ii) harvesting the culture fluid obtained in (i),

[0020] (iii) harvesting membrane vesicles from the culture supernatant of the culture fluid harvested in (ii), and optionally purifying the membrane vesicles.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1: E. coli converts Au into 3+ The mineralization process of gold nanoparticles. Escherichia coli and Au 3+ Co-incubation, SEM imaging of bacteria at different time points (A), scale: 4 μm / 500 nm; TEM imaging (B), scale: 1 μm / 200 nm; and the state of the bacterial solution (C).

[0023] Figure 2: Distribution of mineralized gold nanoparticles within bacteria. (A) STEM image of an ultrathin section of bacteria that have mineralized gold nanoparticles. (B) SEM and (C) TEM images of E. coli containing gold nanoparticles that have been synthesized but not yet secreted. Scale bar: 50 nm.

[0024] Figure 3: Ausome extraction and yield: (A) TEM images of Ausomes collected under different centrifugation conditions. (B) Curve of Ausome yield over time; scale bar: 200 nm.

[0025] Figure 4: Characterization of the physicochemical properties of Ausomes: (A) TEM and DLS characterization of the particle size and dispersion of Ausomes; scale bar: 100 nm. (B) TEM image of the morphology and structure of Ausomes; scale bar: 10 nm. (C) High-resolution TEM imaging of Ausomes; scale bar: 5 nm. (D) Selected area electron diffraction rings of Ausomes. TEM imaging of AuNPs (E); scale bar: 200 nm, and DLS size determination (F).

[0026] Figure 5: Lipid components of Ausomes.

[0027] Figure 6: Characterization of the photothermal properties of Ausomes: (A) UV-Vis absorption spectrum of Ausomes. (B) Temperature changes in Ausome solution concentration and laser power density. (C) UV-Vis absorption spectrum of AuNPs. (D) Photothermal conversion effect of AuNPs.

[0028] Figure 7: Cellular uptake of Ausomes: Two-photon imaging (A) and quantitative ICP-MS analysis (B) of 4T1 uptake of Ausomes or AuNPs at different incubation times. Two-photon imaging (C) and quantitative ICP-MS analysis (D) of BMDC uptake of Ausomes or AuNPs at different incubation times.

[0029] Figure 8: Ausomes stimulate DC maturation in vitro: (A, B) Flow cytometric analysis of the proportion of CD80+CD86+ mature DCs after incubation of BMDCs with Ausomes at different doses for 6 or 24 hours. (C, D) Flow cytometric analysis of the proportion of CD80+CD86+ mature DCs after incubation of BMDCs with the same doses of Ausomes, AuNPs, and laser-irradiated Ausomes for 24 hours.

[0030] Figure 9: Possible toxic side effects of Ausomes: (A) Hemolysis rate of Ausomes at different doses. (B, C) Changes in serum cytokine levels after intravenous injection of Ausomes at different doses.

[0031] Figure 10: Tumor inhibition effects of Ausomes at different doses: (A) Schematic diagram of the anti-tumor treatment process using Ausomes. (B) Tumor growth curve. (C) Changes in mouse body weight during treatment.

[0032] Figure 11: Safety evaluation of Ausomes for tumor therapy: (A) In vivo distribution of Ausomes 6 or 24 hours after intravenous injection. Blood biochemical analysis (B) and H&E staining of different organs (C) of mice treated according to the anti-tumor therapy immunization schedule; Scale bar: 50 μm.

[0033] Figure 12: Ausomes Tumor Accumulation: (A, B) Cy5.5-labeled Ausomes were injected intravenously, and in vivo imaging was performed at different time points to analyze tumor fluorescence accumulation intensity. (C) Ausomes were injected intravenously, and the gold content in the tumor was quantified by ICP-MS at different time points.

[0034] Figure 13: Ausome-mediated localized tumor hyperthermia: (A) Infrared thermal imaging and tumor temperature curve of tumor-bearing mice under laser injection (660 nm, 1.2 W / cm²) after intravenous injection of Ausomes or AuNPs. (C) Real-time MSOT imaging of mouse tumors before and after hyperthermia. (D) Confocal laser 3D reconstruction of FITC-dextran-labeled mouse blood vessels before and after hyperthermia.

[0035] Figure 14: In vivo immune response to Ausomes combined with localized hyperthermia in tumors: (A) Schematic diagram of Ausomes and Ausomes combined with hyperthermia immunization of tumor-bearing mice. (B) Fold change in the levels of various cytokines in serum and tumor tissues of immunized mice. Fold changes greater than 3-fold were treated as 3-fold.

[0036] Figure 15: Tumor infiltration of immune cells: 6 hours and 24 hours after tumor-bearing mice were treated according to the immunization schedule shown in Figure 14A, the infiltration of various immune cells in tumor tissues was analyzed by flow cytometry.

[0037] Figure 16: Antitumor efficacy and safety evaluation of Ausomes combined with localized tumor hyperthermia: (A) Schematic diagram of the antitumor immune program. (B) Tumor growth curve and comparison of tumor size after treatment. (C) Body weight changes of mice during treatment. Blood biochemical analysis of mice after treatment (D) and H&E-stained tissue sections of major organs (E); Scale bar: 50 μm.

[0038] FIG17 shows the microscopic morphology of Pseudomonas mineralized Ausomes.

[0039] FIG18 shows the microscopic morphology of Escherichia coli mineralized Ausomes.

[0040] FIG19 shows the microscopic morphology of Salmonella mineralized Ausomes.

[0041] FIG20 is the microscopic morphology of Fusobacterium mineralized Ausomes.

[0042] FIG21 is the microscopic morphology of Acinetobacter mineralized Ausomes.

[0043] FIG22 shows the microscopic morphology of Staphylococcus mineralized Ausomes.

[0044] FIG23 shows the microscopic morphology of mineralized Ausomes of Streptococcus.

[0045] FIG24 shows the microscopic morphology of Bacillus mineralized Ausomes.

[0046] FIG25 is the microscopic morphology of Lactobacillus mineralized Ausomes.

[0047] FIG26 shows the microscopic morphology of Bifidobacterium mineralized Ausomes.

[0048] FIG27 is a diagram showing the effect of the radiotherapy-only group in the plate clonal colony formation experiment.

[0049] Figure 28 shows the radiotherapy sensitization effect of different Ausomes in the plate cloning colony formation experiment, wherein Figure A shows the radiotherapy sensitization effect of Bifidobacterium mineralized Ausomes, Figure B shows the radiotherapy sensitization effect of Lactobacillus mineralized Ausomes, Figure C shows the radiotherapy sensitization effect of Staphylococcus mineralized Ausomes, Figure D shows the radiotherapy sensitization effect of Streptococcus mineralized Ausomes, Figure E shows the radiotherapy sensitization effect of Bacillus mineralized Ausomes, Figure F shows the radiotherapy sensitization effect of Acinetobacter mineralized Ausomes, Figure G shows the radiotherapy sensitization effect of Fusobacterium mineralized Ausomes, Figure H shows the radiotherapy sensitization effect of Salmonella mineralized Ausomes, Figure I shows the radiotherapy sensitization effect of Escherichia mineralized Ausomes, and Figure J shows the radiotherapy sensitization effect of Pseudomonas mineralized Ausomes.

[0050] Figure 29 shows the immunological effects of Pseudomonas mineralized Ausomes, where Panel A shows the results of tumor tissue-infiltrating effector CD4+ T cells, Panel B shows the results of tumor tissue-infiltrating effector CD8+ T cells, Panel C shows the results of tumor tissue-infiltrating dendritic cells, Panel D shows the results of lymph node antigen-specific CD4+ T cells, and Panel E shows the results of lymph node antigen-specific CD8+ T cells. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0051] Figure 30 shows the immunological effects of Escherichia coli mineralized Ausomes, wherein Panel A shows the results of tumor tissue-infiltrating effector CD4+ T cells, Panel B shows the results of tumor tissue-infiltrating effector CD8+ T cells, Panel C shows the results of tumor tissue-infiltrating dendritic cells, Panel D shows the results of lymph node antigen-specific CD4+ T cells, and Panel E shows the results of lymph node antigen-specific CD8+ T cells. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0052] Figure 31 shows the immunological effects of Salmonella mineralized Ausomes, where Panel A shows the results of tumor tissue-infiltrating effector CD4+ T cells, Panel B shows the results of tumor tissue-infiltrating effector CD8+ T cells, Panel C shows the results of tumor tissue-infiltrating dendritic cells, Panel D shows the results of lymph node antigen-specific CD4+ T cells, and Panel E shows the results of lymph node antigen-specific CD8+ T cells. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0053] Figure 32 shows the immunological effects of Fusobacterium mineralized Ausomes, where Panel A shows the results of tumor tissue-infiltrating effector CD4+ T cells, Panel B shows the results of tumor tissue-infiltrating effector CD8+ T cells, Panel C shows the results of tumor tissue-infiltrating dendritic cells, Panel D shows the results of lymph node antigen-specific CD4+ T cells, and Panel E shows the results of lymph node antigen-specific CD8+ T cells. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0054] Figure 33 shows the immunological effects of Acinetobacter mineralized Ausomes, where Panel A shows the results of tumor tissue-infiltrating effector CD4+ T cells, Panel B shows the results of tumor tissue-infiltrating effector CD8+ T cells, Panel C shows the results of tumor tissue-infiltrating dendritic cells, Panel D shows the results of lymph node antigen-specific CD4+ T cells, and Panel E shows the results of lymph node antigen-specific CD8+ T cells. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0055] Figure 34 shows the immunological effects of Staphylococcus mineralized Ausomes, where Panel A shows the results of tumor tissue-infiltrating effector CD4+ T cells, Panel B shows the results of tumor tissue-infiltrating effector CD8+ T cells, Panel C shows the results of tumor tissue-infiltrating dendritic cells, Panel D shows the results of lymph node antigen-specific CD4+ T cells, and Panel E shows the results of lymph node antigen-specific CD8+ T cells. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0056] Figure 35 shows the immunological effects of Streptococcus mineralized Ausomes, where Panel A shows the results of tumor tissue-infiltrating effector CD4+ T cells, Panel B shows the results of tumor tissue-infiltrating effector CD8+ T cells, Panel C shows the results of tumor tissue-infiltrating dendritic cells, Panel D shows the results of lymph node antigen-specific CD4+ T cells, and Panel E shows the results of lymph node antigen-specific CD8+ T cells. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0057] Figure 36 shows the immunological effects of Bacillus mineralized Ausomes, where Panel A shows the results of tumor tissue-infiltrating effector CD4+ T cells, Panel B shows the results of tumor tissue-infiltrating effector CD8+ T cells, Panel C shows the results of tumor tissue-infiltrating dendritic cells, Panel D shows the results of lymph node antigen-specific CD4+ T cells, and Panel E shows the results of lymph node antigen-specific CD8+ T cells. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0058] Figure 37 shows the immunological effects of Lactobacillus mineralized Ausomes, where Panel A shows the results of tumor tissue-infiltrating effector CD4+ T cells, Panel B shows the results of tumor tissue-infiltrating effector CD8+ T cells, Panel C shows the results of tumor tissue-infiltrating dendritic cells, Panel D shows the results of lymph node antigen-specific CD4+ T cells, and Panel E shows the results of lymph node antigen-specific CD8+ T cells. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0059] Figure 38 shows the immunological effects of Bifidobacterium-mineralized Ausomes, where Panel A shows the results of tumor tissue-infiltrating effector CD4+ T cells, Panel B shows the results of tumor tissue-infiltrating effector CD8+ T cells, Panel C shows the results of tumor tissue-infiltrating dendritic cells, Panel D shows the results of lymph node antigen-specific CD4+ T cells, and Panel E shows the results of lymph node antigen-specific CD8+ T cells. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: gold Ausomes combined with radiotherapy group.

[0060] Figure 39 shows the anti-tumor effect of Pseudomonas mineralized Ausomes, where Panel A shows the growth curve of the orthotopic tumor after radiotherapy, and Panel B shows the growth curve of the distal tumor. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0061] Figure 40 shows the anti-tumor effect of Escherichia coli mineralized Ausomes, where Panel A shows the growth curve of the orthotopic tumor after radiotherapy, and Panel B shows the growth curve of the distal tumor. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0062] Figure 41 shows the anti-tumor effect of Salmonella mineralized Ausomes, where Panel A shows the growth curve of the orthotopic tumor after radiotherapy, and Panel B shows the growth curve of the distal tumor. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0063] Figure 42 shows the anti-tumor effect of Fusobacterium mineralized Ausomes, where Panel A shows the growth curve of the orthotopic tumor after radiotherapy, and Panel B shows the growth curve of the distal tumor. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0064] Figure 43 shows the anti-tumor effect of Acinetobacter mineralized Ausomes, where Panel A shows the growth curve of the orthotopic tumor after radiotherapy, and Panel B shows the growth curve of the distal tumor. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0065] Figure 44 shows the anti-tumor effect of Staphylococcus mineralized Ausomes, where Panel A shows the growth curve of the orthotopic tumor after radiotherapy, and Panel B shows the growth curve of the distal tumor. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0066] Figure 45 shows the anti-tumor effect of Streptococcus mineralized Ausomes, where Panel A shows the growth curve of the orthotopic tumor after radiotherapy, and Panel B shows the growth curve of the distal tumor. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0067] Figure 46 shows the anti-tumor effect of Bacillus mineralized Ausomes, where Panel A shows the growth curve of the orthotopic tumor after radiotherapy, and Panel B shows the growth curve of the distal tumor. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0068] Figure 47 shows the anti-tumor effect of Lactobacillus mineralized Ausomes, where Panel A shows the growth curve of the orthotopic tumor after radiotherapy, and Panel B shows the growth curve of the distal tumor. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0069] Figure 48 shows the anti-tumor effect of Bifidobacterium-mineralized Ausomes, where Panel A shows the growth curve of the orthotopic tumor after radiotherapy, and Panel B shows the growth curve of the distal tumor. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0070] Figure 49 shows the effects of Escherichia coli mineralized Ausomes on promoting radiotherapy against glioma, osteosarcoma, breast cancer, pancreatic cancer, and melanoma. Panel A shows the effects of promoting radiotherapy against glioma, Panel B shows the effects of promoting radiotherapy against osteosarcoma, Panel C shows the effects of promoting radiotherapy against breast cancer, Panel D shows the effects of promoting radiotherapy against pancreatic cancer, and Panel E shows the effects of promoting radiotherapy against melanoma. Sal: saline group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0071] Figure 50 illustrates the effects of Escherichia coli mineralized Ausomes on promoting radiotherapy against liver cancer, gastric cancer, cervical cancer, lung cancer, and head and neck cancer. Panel A shows the effects of radiotherapy against liver cancer, Panel B shows the effects of radiotherapy against gastric cancer, Panel C shows the effects of radiotherapy against cervical cancer, Panel D shows the effects of radiotherapy against lung cancer, and Panel E shows the effects of radiotherapy against head and neck cancer. Sal: saline solution group; RT: radiotherapy alone group; AS: Ausomes injection only group; AS+RT: Ausomes combined with radiotherapy group.

[0072] Figure 51 shows the morphologies of Ausomes obtained by different preparation methods, where Figure A is prepared using a bacterial culture medium with a pH of 7, Figure B is prepared using a bacterial culture medium with a pH of 4, Figure C is prepared using a PBS buffer with a pH of 7, and Figure D is prepared using a PBS buffer with a pH of 4. DETAILED DESCRIPTION

[0073] Unless otherwise noted, the scientific and technical terms used herein should have the meanings commonly known to those skilled in the art. In addition, unless otherwise required, singular terms should include plural terms, and plural terms should include singular terms. The aforementioned techniques and methods are generally carried out according to conventional methods well known in the art and described in the references cited in this specification. See, for example, Sambrook et al.Molecular Cloning:A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)) and Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J.Wiley & Sons (New York, NY 1994), which are incorporated by reference; Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). All references cited herein, including patents, patent applications, articles, textbooks, etc., and the references cited therein, are hereby incorporated by reference in their entirety.

[0074] The experimental results of this paper show that different species of bacteria (such as Escherichia coli) can reduce gold ions into gold nanoparticles, which are secreted outside the bacteria to form gold nanoparticles with bacterial components on the surface, namely Ausomes. The metal bodies described in this paper, such as Ausomes, have bacterial outer membrane components and can exert similar immunological effects as OMVs. At the same time, they also contain metal nanoparticles such as gold nanoparticle cores. Metal nanoparticles give metal bodies two advantages: (1) In the extraction of nanostructures used as immune adjuvants from other bacterial sources, such as OMVs, due to their light weight, ultracentrifugation is generally required to achieve purification and collection, making industrialization difficult. Metal nanoparticles greatly increase the weight of metal bodies and can be centrifuged and precipitated at a speed of about 5000-15000g, such as about 11000g, which is of great significance to industrial production; (2) Metal nanoparticles have a variety of special properties, including photothermal conversion ability, radiotherapy sensitization ability, etc., which give metal bodies more functions. The combination of these functions can promote the intensity of immune response in many aspects.

[0075] In this invention, the photothermal conversion capacity of gold nanoparticles is combined to generate localized hyperthermia in the tumor, promoting blood perfusion of the tumor tissue, significantly enhancing the systemic immune stimulation effect of Ausomes, and at the same time enhancing the immune response in the tumor tissue and promoting the tissue infiltration of effector cells, thereby producing a stronger tumor inhibitory effect. Under the immune stimulation of Ausomes, the body mobilizes a large number of cytokines of different types and functions, indicating that Ausomes may activate multiple immune pathways and have strong immune stimulation capabilities; at the same time, the upregulation of pro-inflammatory cytokines and the downregulation of anti-inflammatory cytokines in tumor tissue also indicate the alleviation of the tumor's immunosuppressive environment.

[0076] In the present invention, the radiotherapy sensitization properties of gold nanoparticles are combined to improve the deposition of radiation in tumor tissue, enhance the tumor cell killing effect, and release a large amount of tumor antigens at the same time; at the same time, the bacterial-derived components in the mineralized products can mediate multi-target immune activation, trigger multiple immune responses, promote the presentation of tumor antigens, and regulate the tumor immune status; ultimately, radiotherapy kills in situ tumor cells, and enhanced secondary systemic immune responses eliminate distal or residual tumor cells, thereby achieving a multi-level anti-tumor effect.

[0077] Ausomes, nanoimmunomodulators with immunostimulatory, photothermal, and radiosensitizing properties, can stimulate systemic immune responses while also alleviating the tumor's immunosuppressive microenvironment, exerting a potent tumor-suppressing effect. In addition to their favorable biological effects, their structural composition also allows Ausomes to be rationally designed as a multifunctional platform for the development of better tumor treatment strategies.

[0078] In a first aspect, the present invention provides a pharmaceutical composition comprising metallobodies and optionally a pharmaceutically acceptable carrier, wherein the metallobodies comprise metal nanoparticles and bacterial components attached thereto.

[0079] As used herein, metallosomes refer to particles composed of metal nanoparticles and bacterial components attached thereto.

[0080] In one embodiment, the metal may be selected from gold, silver, manganese, titanium and iron, preferably gold.

[0081] In one embodiment, the metal nanoparticles are metal crystalline nanoparticles, such as gold crystals.

[0082] As used herein, metal nanoparticles are nanometer-sized particles formed of metal (preferably metal atoms or elemental metals). The metal nanoparticles can be of any suitable size known in the art, for example, a particle size between a few nanometers and several hundred nanometers.

[0083] In one embodiment, the metal nanoparticles have a particle size of between about X nanometers and Y nanometers, wherein X is an integer selected from 1-50, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and Y is an integer selected from 50-500, such as 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500.

[0084] In one embodiment, the metal nanoparticles have a particle size of about 5-500, 5-450, 5-400, 5-350, 5-300, 5-250, 5-240, 5-230, 5-220, 5-210, 5-200, 10-500, 10-450, 10-400, 10-350, 10-300, 10-250, 10-240, 10-230, 10-220, 10-210, 10-200, 20-500, 20-450, 20-400, 20-350, 20-300, 20-250, 20-240, 20-230, 20-220, 20-210, 20- 30-200, 30-500, 30-450, 30-400, 30-350, 30-300, 30-250, 30-240, 30-230, 30-220, 30-210, 30-200, 40-500, 40-450, 40-400, 40-350, 40-300, 40-250, 40-240, 40-230, 40-220, 40-210, 40-200, 50-500, 50-450, 50-400, 50-350, 50-300, 50-250, 50-240, 50-230, 50-220, 50-210 or 50-200 nm. In one embodiment, the metal nanoparticles have a particle size of about 10-200 nm, such as about 10-40 nm, about 10-30 nm, or about 20-30 nm, specifically about 30 nm.

[0085] As used herein, a metal is any suitable metal that can be used for in vivo administration, provided that it can be used for photothermal therapy or radiotherapy. The metal herein can be selected from gold, silver, manganese, titanium and iron, preferably gold.

[0086] In one embodiment, the metal nanoparticles are metal crystals.

[0087] As used herein, a metal crystal is a structure in which metal atoms interact with each other through metallic bonds and are arranged at lattice nodes. Metal crystals are well known in the art. For example, high-resolution lattice images and selected electron diffraction patterns can be found in the American Mineralogist Crystal Structure Database (arizona.edu).

[0088] In one embodiment, the gold nanoparticles are gold crystals.

[0089] As used herein, gold crystal is the crystalline state of elemental gold. The unit cell of a gold crystal is a face-centered cube, with a gold atom at each of the eight vertices of the cube, a gold atom at the center of each face, and each gold atom shared by adjacent unit cells.

[0090] In one embodiment, the bacterial component is selected from the group consisting of intracellular membranes, extracellular membranes, proteins, carbohydrates, nucleic acids, phospholipids, and any combination thereof. In one embodiment, the intracellular membranes, extracellular membranes, proteins, carbohydrates, nucleic acids, and phospholipids attached to the metal nanoparticles are derived from bacteria (e.g., bacteria that perform metal mineralization) and are immunogenic. For example, the bacterial component may be coated on the surface of the metal nanoparticles during bacterial mineralization, or obtained by extracting or crushing bacterial cells and then attaching them to the surface of the metal nanoparticles. Various techniques and means for attaching desired molecules or compounds to the surface of metal nanoparticles are known in the art.

[0091] In one embodiment, the bacterial component is directly or indirectly linked to the metal nanoparticles by covalent or non-covalent means, or the bacterial component is located in or on a surface organic layer that completely or partially covers the metal nanoparticles.

[0092] As described herein, completely coating the metal nanoparticles means that the organic layer completely surrounds the nanoparticles to form a closed spherical structure; partially coating the metal nanoparticles means that the organic layer only coats part of the outer surface of the nanoparticles, and the rest may be exposed or coated by other substances.

[0093] The organic layer can be any substance known in the art that is suitable for pharmaceutical use and can be attached to the surface of metal nanoparticles, such as a lipid membrane. The bacterial components described herein can be attached to the metal nanoparticles by connecting to or inserting into the membrane.

[0094] In one embodiment, each of said metallic bodies comprises a metallic nanoparticle.

[0095] In a preferred embodiment, the metalloids are produced by bacterial mineralization of metals. Bacterial mineralization is a process in which bacteria are brought into contact with a solution containing metal ions, thereby reducing the metal ions to elemental metals.

[0096] In a preferred embodiment, the bacteria used for metal mineralization are selected from the genera Neisseria, Bordetella, Escherichia and Salmonella, Pseudomonas, Fusobacterium, Acinetobacter, Staphylococcus, Streptococcus, Bacillus Cohn, Lactobacillus Beijerinck and Bifidobacterium. For example, the genus Neisseria can be selected from Neisseria meningitidis, Neisseria gonorrhoeae and Neisseria lactamica, and / or the genus Bordetella can be selected from Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Preferably, the gram-negative bacteria are selected from Neisseria meningitidis, Bordetella pertussis, Escherichia coli and Salmonella enterica, most preferably Escherichia coli, such as DH5α or BL21 strains.

[0097] In one embodiment, the bacteria used for metal mineralization are selected from Pseudomonas aeruginosa (e.g., BNCC337889), Escherichia coli (e.g., BNCC336454), Salmonella typhimurium (e.g., ATCC 14028), Fusobacterium nucleatum (e.g., BNCC280188), Acinetobacter baumannii (e.g., BNCC337173), Staphylococcus epidermidis (e.g., BNCC102555), Streptococcus salivarius (e.g., BNCC337521), Bacillus licheniformis (e.g., BNCC336463), Lactobacillus reuteri (e.g., BNCC337174), Staphylococcus epidermidis (e.g., BNCC102555), Streptococcus salivarius (e.g., BNCC337521), Bacillus licheniformis (e.g., BNCC336463), Lactobacillus reuteri (e.g., BNCC337173), and Bacillus licheniformis (e.g., BNCC336463). reuteri) (e.g., BNCC254476) or Bifidobacterium longum (e.g., BNCC185354).

[0098] In a preferred embodiment, the metallosomes are produced by bacteria during the process of metal mineralization through membrane vesicle encapsulation and secretion, wherein the membrane vesicles contain the metal nanoparticles and the membrane vesicles contain bacterial components.

[0099] As used herein, membrane vesicles are phospholipid bilayer structures, typically spherical with a diameter in the range of about 5-500 nm. Typically, membrane vesicles are non-replicating structures released by bacteria (e.g., Gram-negative bacteria) and are primarily composed of lipids, LPS, and outer membrane proteins. Membrane vesicles may include bacterial surface components, such as phospholipids (PL) inside the membrane and lipopolysaccharides (LPS) and PL outside. The cavity of the membrane vesicle may include compounds from the periplasm or cytoplasm, such as proteins, RNA / DNA, and peptidoglycan (PG), but the membrane vesicle lacks self-replication ability.

[0100] In one embodiment, the membrane of the membrane vesicle comprises one or more of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid (PA), diacylglycerol (DG), triglyceride (TG), ceramide (Cer), lipid A, galactosylceramide (GalCer), monostearate (MG), and sphingomyelin (SM), preferably all of them, more preferably, CL is a negative ion and / or lipid A is a positive ion.

[0101] In one embodiment, the membrane of the membrane vesicle comprises one or more of outer membrane protein C, outer membrane protein A, major outer membrane lipoprotein Lpp, elongation factor Tu2 / Tu1, D-methionine binding lipoprotein MetQ, permeation-inducing protein Y, possible lipoprotein YiaD, maltoporin, outer membrane protein assembly factor BamB, fructose bisphosphate aldolase class 2, chaperone protein DnaK, outer membrane lipoprotein RcsF, possible phospholipid binding protein MlaC, multidrug resistance protein MdtE, long-chain fatty acid transport protein, chaperone protein HtpG, outer membrane protein assembly factor BamA, and protein export membrane protein SecG, preferably all of them.

[0102] In one embodiment, the membrane vesicles have a particle size of about 5-500 nm, for example, a particle size of about 5-500, 5-450, 5-400, 5-350, 5-300, 5-250, 5-240, 5-230, 5-220, 5-210, 5-200, 10-500, 10-450, 10-400, 10-350, 10-300, 10-250, 10-240, 10-230, 10-220, 10-210, 10-200, 20-500, 20-450, 20-400, 20-350, 20-300, 20-250, 20-240, 20-230, 20-220, 20-2 30-200, 30-500, 30-450, 30-400, 30-350, 30-300, 30-250, 30-240, 30-230, 30-220, 30-210, 30-200, 40-500, 40-450, 40-400, 40-350, 40-300, 40-250, 40-240, 40-230, 40-220, 40-210, 40-200, 50-500, 50-450, 50-400, 50-350, 50-300, 50-250, 50-240, 50-230, 50-220, 50-210, or 50-200 nm. In one embodiment, the membrane vesicles have a particle size of about 10-200 nm.

[0103] In one embodiment, the membrane vesicle has a diameter of less than about 450, 440, 430, 420, 410, 400, 350, 300, 250, 220, 210, 200 nm or less. In one embodiment, the membrane vesicle has a diameter of less than 450 nm. In one embodiment, the membrane vesicle has a diameter of less than 220 nm.

[0104] In one embodiment, the membrane vesicles have a particle size of about 10-200 nm, such as about 10-150, 10-100, 10-50, 10-40, 10-30, 20-150, 20-100, 20-50, 20-40, 20-30 nm.

[0105] The membrane vesicles containing metal nanoparticles described herein can be formed by any suitable method known in the art, such as contacting a solution containing metal ions with bacteria that can produce membrane vesicles (such as Gram-negative bacteria), and producing membrane vesicles containing metal nanoparticles through bacterial mineralization.

[0106] The membrane vesicles described herein can be isolated or obtained by any suitable method known in the art, such as extraction from cells with detergents (e.g., deoxycholate) or naturally produced by bacteria that can produce membrane vesicles (e.g., bacteria budding to form membrane vesicles and shedding them into the medium).

[0107] In one embodiment, the membrane vesicles described herein are produced by Gram-negative bacteria or Gram-positive bacteria.

[0108] In one embodiment, the membrane vesicles described herein are natural membrane vesicles spontaneously produced by Gram-negative bacteria.

[0109] In one embodiment, the membrane vesicles described herein are obtained from gram-negative bacteria or gram-positive bacteria, for example, selected from Neisseria, Bordetella, Escherichia and Salmonella, Pseudomonas, Fusobacterium, Acinetobacter, Staphylococcus, Streptococcus, Bacillus, Lactobacillus and Bifidobacterium. For example, Neisseria can be selected from Neisseria meningitidis, Neisseria gonorrhoeae and Neisseria lactosus, and / or Bordetella can be selected from Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Preferably, the gram-negative bacteria are selected from Neisseria meningitidis, Bordetella pertussis, Escherichia coli and Salmonella enterica, most preferably Escherichia coli.

[0110] In one embodiment, the membrane vesicles described herein are obtained from Pseudomonas aeruginosa (e.g., BNCC337889), Escherichia coli (e.g., BNCC336454), Salmonella typhimurium (e.g., ATCC 14028), Fusobacterium nucleatum (e.g., BNCC280188), Acinetobacter baumannii (e.g., BNCC337173), Staphylococcus epidermidis (e.g., BNCC102555), Streptococcus salivarius (e.g., BNCC337521), Bacillus licheniformis (e.g., BNCC336463), Lactobacillus reuteri (e.g., BNCC254476), or Bifidobacterium longum (e.g., BNCC185354).

[0111] In particular, the bacteria that produce the membrane vesicles described herein can have one or more genetic modifications, for example, to remove factors such as proteins or toxins such as endotoxins that may have toxic or other adverse effects on the subject to which the membrane vesicles are administered, or to facilitate the production of membrane vesicles.

[0112] In particular, the pharmaceutical composition is used for photothermal therapy or radiotherapy of tumors. In one embodiment, the tumor is preferably a solid tumor, for example, selected from gastric cancer, liver cancer, biliary tract cancer, gallbladder cancer, colon cancer, lung cancer, bladder cancer, cervical cancer, ovarian cancer, breast cancer, melanoma, pancreatic cancer, kidney cancer, renal cell carcinoma, glioma, osteosarcoma, head and neck cancer, esophageal cancer and prostate cancer.

[0113] As used herein, "pharmaceutically acceptable carrier" refers to a substance that facilitates administration and absorption of an active substance into a subject and can be included in the compositions of the present invention without causing significant toxic side effects in the patient. Pharmaceutically acceptable carriers suitable for use in the present invention are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, ed. Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st edition (2005) describes compositions and formulations suitable for drug delivery.

[0114] Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline, sucrose, glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, salt solutions, alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone and coloring agents, etc. Those skilled in the art will appreciate that other pharmaceutical carriers may be used in the present invention.

[0115] As used herein, a "pharmaceutical composition" refers to a pharmaceutical product formulated to meet specific dosage requirements for the treatment or prevention of a disease and intended for use by a patient. The pharmaceutical composition may contain the metal bodies described herein, as well as other pharmaceutical excipients and tools.

[0116] The "excipients" described herein are pharmaceutical excipients, which refer to substances used in the production of drugs and the preparation of prescriptions, in addition to the active ingredients, that have been reasonably evaluated in terms of safety and are contained in pharmaceutical preparations. In addition to their purpose of excipient, acting as a carrier or improving stability, they can also have important functions such as solubilization, solubilization, and sustained-release. The drugs or pharmaceutical compositions herein can be formulated into dosage forms suitable for any suitable route of administration, such as intravenous, subcutaneous, parenteral, oral, intraperitoneal, intramuscular, intranasal, intraarterial or intralesional, such as tablets, powders, solutions, etc. In one embodiment, the drugs or pharmaceutical compositions described herein can be formulated into a form suitable for intravenous or subcutaneous administration.

[0117] Since metal nanoparticles have photothermal conversion properties and radiation sensitization properties, they can be used in photothermal therapy and radiotherapy. In one embodiment, the pharmaceutical composition is used for tumor photothermal therapy and radiotherapy.

[0118] In a second aspect, the present invention provides a use of a metallobody in the preparation of a medicament for treating a tumor in a subject, wherein the metallobody comprises metal nanoparticles and a bacterial component attached thereto, wherein the metallobody, metal nanoparticles, and bacterial component are as described or defined above in the first aspect.

[0119] In one embodiment, the drug is in a dosage form suitable for treating tumors by photothermal or radiotherapy.

[0120] In one embodiment, the tumor is preferably a solid tumor, for example selected from gastric cancer, liver cancer, biliary tract cancer, gallbladder cancer, colon cancer, lung cancer, bladder cancer, cervical cancer, ovarian cancer, breast cancer, melanoma, pancreatic cancer, kidney cancer, renal cell carcinoma, glioma, osteosarcoma, head and neck cancer, esophageal cancer and prostate cancer.

[0121] In one embodiment, the subject is selected from humans and other mammals such as cows, rats, mice, dogs, monkeys, goats, sheep, cows and deer, preferably humans.

[0122] In a third aspect, the present invention provides an immunopotentiator, immunostimulant, or radiosensitizer, comprising a metalloid and optionally a pharmaceutically acceptable carrier, wherein the metalloid comprises metal nanoparticles and a bacterial component attached thereto. In a fourth aspect, the present invention provides use of the metalloid in preparing an immunopotentiator, immunostimulant, or radiosensitizer, wherein the metalloid comprises metal nanoparticles and a bacterial component attached thereto. The metalloid, metal nanoparticles, and bacterial component are as described or defined in the first aspect.

[0123] As used herein, an immunopotentiator or immunostimulant refers to an agent that has the ability to stimulate the immune system, can systemically activate the immune system, secrete large amounts of proinflammatory cytokines, and produce activated immune cells such as antigen-presenting cells, CD8+ T cells, and NK cells. A radiosensitizer is a chemical or pharmaceutical preparation that, when used simultaneously with radiotherapy, can alter the responsiveness of tumor cells to radiation, thereby increasing the killing effect on tumor cells; radiosensitization refers to the process of increasing the sensitivity of tumor cells to radiation using drugs or physical methods in order to enhance the killing effect of radiation on tumor cells and improve the control and cure rates of tumors. Immunopotentiators, immunostimulants, or radiosensitizers can be administered in combination with other drugs to enhance the therapeutic effect. For example, an immunopotentiator can be administered together with a tumor therapeutic agent to enhance the body's immune activity and achieve better tumor treatment results.

[0124] The metallobodies described in this article not only contain multiple bacterial components but also contain a metal nanoparticle core, which can be used for photothermal therapy of tumors. Photothermal therapy involves gathering materials with high photothermal conversion efficiency near tumor tissue and, under the irradiation of an external light source (such as near-infrared light), converting light energy into heat energy to kill cancer cells.

[0125] For example, metal bodies containing metal nanoparticles can be enriched at the tumor site. When properly irradiated (for example, when irradiating gold nanoparticles with a 660nm laser), they can achieve local hyperthermia in the tumor, increase tissue blood perfusion, promote tumor infiltration of effector cells, and alleviate the tumor's immunosuppressive microenvironment. The hyperthermia effect refers to a strategy for regulating the local immune environment of the tumor. Hyperthermia can promote the expression and secretion of heat shock proteins by tumor cells, transmit signals to various immune cells, promote their differentiation into immune phenotypes, and secrete large amounts of proinflammatory cytokines. At the same time, hyperthermia also accelerates blood flow, increases the endothelial space between blood vessels, promotes tissue perfusion of blood, and increases the tissue infiltration of immunomodulators and effector cells.

[0126] The metallobodies described in this article not only contain multiple bacterial components but also a metal nanoparticle core, and can be used for tumor radiotherapy. Tumor radiotherapy is a localized treatment method that uses radiation to treat tumors. It uses radiation, such as alpha, beta, and gamma rays produced by radioisotopes, and x-rays, electron beams, proton beams, and other particle beams produced by various x-ray therapy machines or accelerators, to treat malignant tumors. Radiation is a beam of particles or waves carrying energy that can damage genes (DNA) and certain molecules in cells. Genes control cell growth and differentiation, and radiation damages the genes of cancer cells, preventing them from growing and dividing.

[0127] For example, metallobodies containing metal nanoparticles can be enriched at the tumor site and, when properly irradiated (for example, when gold nanoparticles are irradiated with X-rays), enhance the tumor cell killing effect while releasing a large amount of tumor antigens, inducing local infiltration of effector immune cells into the tumor, and mediating an enhanced systemic immune response.

[0128] In one embodiment, the present invention provides the use of the metallobody in the preparation of an immunopotentiator, immunostimulant, or radiosensitizer for photothermal therapy or radiotherapy of tumors, or as an immunopotentiator, immunostimulant, or radiosensitizer for photothermal therapy or radiotherapy of tumors.

[0129] In one embodiment, the present invention provides an immunopotentiator or immunostimulatory agent for tumor radiotherapy, comprising the metallobody according to the first aspect of the present invention.

[0130] In one embodiment, the present invention provides a radiosensitizer for tumor radiotherapy, comprising the metallobody according to the first aspect of the present invention.

[0131] In one embodiment, the present invention provides an immunopotentiator or immunostimulatory agent for photothermal therapy of tumors, comprising the metallobody according to the first aspect of the present invention.

[0132] Here, a suitable external light source can be selected for irradiation according to the metal nanoparticles contained in the metal bodies. For example, for gold, a light source with an emission wavelength of about 500-850 nm, such as about 660 nm, can be used for irradiation.

[0133] In a fifth aspect, the present invention provides a method for producing a metal body, wherein the metal body comprises the metal nanoparticles and membrane vesicles coating the metal nanoparticles, and bacterial components attached to the membrane vesicles (the metal nanoparticles, membrane vesicles, and bacterial components attached to the membrane vesicles are as described or defined above), the method comprising:

[0134] (i) culturing bacteria capable of metal mineralization in a solution containing metal ions,

[0135] (ii) harvesting the culture fluid obtained in (i),

[0136] (iii) isolating and optionally purifying the culture supernatant of the culture solution of (ii) to harvest the metallobodies in the supernatant.

[0137] The bacterium that can carry out metal mineralization known in the art includes those bacterium, and the bacterium can be gram-negative bacteria or gram-positive bacteria.In one embodiment, the bacterium that can carry out metal mineralization includes but is not limited to Neisseria, Bordetella, Escherichia and Salmonella, Pseudomonas, Fusobacterium, Acinetobacter, Staphylococcus, Streptococcus, Bacillus, Lactobacillus and Bifidobacterium, such as Neisseria can be selected from Neisseria meningitidis, Neisseria gonorrhoeae and Neisseria lactosus, and / or Bordetella can be selected from Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica.Preferably, the bacterium is selected from Neisseria meningitidis, Bordetella pertussis, Escherichia coli and Salmonella enterica, most preferably Escherichia coli, such as DH5α or BL21 strain.

[0138] Herein, the bacteria may be cultured in a suitable medium to a suitable state, for example, to a logarithmic growth phase, before being cultured in a solution containing metal ions. Suitable culture and collection conditions, such as temperature, time, culture medium, etc., are known in the art for any bacteria.

[0139] Herein, a solution containing metal ions refers to a solution containing metal ions that can be mineralized by bacteria and thus be coated in membrane vesicles. Metal ions that can be mineralized by bacteria are known in the art. In one embodiment, the metal can be selected from gold, silver, manganese, titanium and iron, preferably gold. In one embodiment, the solution containing gold ions can be selected from chloroauric acid (HAuCl4) solution. In one embodiment, the mass volume concentration of the chloroauric acid solution is about 1-5% (i.e. 1-5g chloroauric acid / 100mL), for example 1%. Herein, when referring to the concentration of chloroauric acid solution, unless otherwise specified, it refers to mass volume concentration, i.e. g / 100ml, for example 1% refers to 1g chloroauric acid / 100ml.

[0140] In one embodiment, the solution containing metal ions is a buffer solution containing metal ions such as PBS (main components of which are Na2HPO4, KH2PO4, NaCl and KCl).

[0141] In one embodiment, the bacterium is cultivated in the solution containing metal ions for at least about 0.5 hour, at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days. In one embodiment, the bacterium is cultivated in the solution containing metal ions for 1-10,1-9,1-8,1-7,1-6,1-5,2-10,2-9,2-8,2-7,2-6,2-5,3-10,3-9,3-8,3-7,3-6,3-5 days. Optionally, the cultivation can be carried out under shaking conditions, and / or can be carried out at suitable temperature such as room temperature.

[0142] In one embodiment, the culturing is carried out in a solution at about pH 6-8, such as 6-7 or 7-8, such as about 7.0.

[0143] In one embodiment, the culture is at room temperature, eg, about 25-37°C, with shaking.

[0144] Herein, harvesting the culture fluid can be performed using any suitable method or technique. In particular, impurities such as cells contained in the culture fluid can be removed, for example, by centrifugation (provided that the centrifugation speed does not precipitate a significant amount of metal bodies) or filtration (for example, using a sterile filter membrane, provided that the pore size of the filter membrane is smaller than the cells and larger than the particle size of the desired metal bodies, for example, using a filter membrane with a pore size of about 0.45 μm and 0.22 μm) to obtain a culture supernatant containing the metal bodies described herein. In particular, the culture fluid is centrifuged at about 5000-15000 g, for example, about 11000 g, for about at least 10-30 minutes, for example, at least 10 minutes, 20 minutes, or 30 minutes to remove cells from the culture fluid and obtain a culture supernatant.

[0145] Herein, metallobodies can be obtained from the culture supernatant using any suitable method or means known in the art, such as separation or filtration.

[0146] In particular, since the metallobodies described herein comprise metal nanoparticles, ultracentrifugation may not be used when harvesting the metallobodies from the culture supernatant by centrifugation. For example, centrifugation at about 5000-15000 g, such as about 11000 g, may be sufficient.

[0147] In one embodiment, the culture supernatant is centrifuged, for example at about 5000-15000 g, such as about 11000 g, for at least 10 minutes, 20 minutes, or 30 minutes.

[0148] In one embodiment, the metallobodies described herein are produced by the following method:

[0149] - adding Escherichia coli, preferably Escherichia coli in logarithmic growth phase, to a solution containing gold ions, such as chloroauric acid, and culturing with shaking at room temperature for, for example, about 0.5, 1, 2, 4, 6, 8, 10, 12, 18, 24, 30, 36, 48, 60, 72, 96, 120 hours, 6 days, 7 days, 8 days, 9 days, 10 days, preferably at least 12 hours, at least 48 hours, more preferably at least 72 hours,

[0150] - centrifuging the culture medium to remove cells and obtaining a supernatant,

[0151] - filtering the supernatant, for example, using a sterile filter membrane with a pore size of about 0.45 μm and / or 0.22 μm,

[0152] - centrifuging the filtered supernatant at at least about 5000-15000 g, such as 11000 g, for at least 10-30 minutes,

[0153] - Harvest the precipitate to obtain metallobodies.

[0154] In a sixth aspect, the present invention provides a method for treating a tumor in a subject by photothermal therapy or radiotherapy, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of the first aspect, the immunopotentiator or immunostimulatory agent or radiosensitizer of the third aspect, or the metal body prepared by the method of the fifth aspect, and irradiating the subject, such as the tumor site, optionally including administering other tumor therapeutic drugs such as tumor antigens or immune checkpoint inhibitors. In one embodiment, the amount of the metal body administered to the subject is no more than 20 mg / kg body weight, for example, about 5-15 or 10-15 mg / kg body weight, preferably about 15 mg / kg body weight.

[0155] In one embodiment, the present invention provides a method for enhancing the efficacy of radiotherapy, comprising administering to a subject an effective amount of a radiosensitizer or metal body as described herein before radiotherapy.

[0156] The administration can be by, but is not limited to, oral, nasal, parenteral, intravenous, intramuscular, intradermal, subcutaneous, buccal, inhalation or intramucosal. In one embodiment, the administration is by intravenous administration or subcutaneous.

[0157] As used herein, the pharmaceutical compositions, immunopotentiators / immunostimulants / radiosensitizers, metallobodies, and other tumor therapeutic agents of the present invention can be administered to a subject in need thereof by any suitable route, including but not limited to intravenous, subcutaneous, parenteral, oral, intraperitoneal, intramuscular, intranasal, intraarterial, or intralesional routes. In one embodiment, the pharmaceutical compositions, immunopotentiators / immunostimulants / radiosensitizers, and metallobodies are administered intravenously or subcutaneously.

[0158] As used herein, the irradiation is the irradiation of a tumor in a subject using a suitable source. Since the metal bodies described herein, such as the gold bodies, accumulate at the tumor site, they can mediate local hyperthermia or radiosensitization of the tumor.

[0159] In one embodiment, a laser of about 500-850 nm, for example about 660 nm, is used at 1.2 W / cm 2 The irradiation was carried out at a power density of 100 nm for 30 min.

[0160] In one embodiment, radiation such as X-rays is irradiated at a dose of about 1 to 10 Gy / min for 1 to 10 minutes.

[0161] The "other tumor therapeutic drugs" described herein refer to any substance or compound used for anti-tumor treatment, including any substance that can alleviate, reduce, alleviate or prevent the remission of clinical symptoms or diagnostic markers associated with neoplastic diseases, tumors and cancers when used alone or in combination with other compounds or treatments, and can be administered in combination with the metallobodies described herein or used in the compositions herein, including but not limited to toxins, alkylating agents, antibiotics, cytokines, hormones, photosensitizers, signal transduction regulators, antibodies, doxorubicin, paclitaxel, doxorubicin, gemcitabine, mitomycin, actinomycin D, cyclophosphamide, 5-fluorouracil, methotrexate, tumor antigens and immune checkpoint inhibitors and combinations thereof.

[0162] As used herein, "tumor antigen" refers to an antigen (e.g., polypeptide) that is uniquely or differentially expressed on tumor cells compared to normal or non-tumor cells. For example, tumor antigens include, but are not limited to, carcinoembryonic antigen (CEA), mucin 1 (MUC1), prostate-specific antigen (PSA), NEU proto-oncogene, prostate-specific membrane antigen (PSMA), telomerase-associated protein 2, prostatic acid phosphatase (PAP), E-cadherin, folate receptor α, or c-Met.

[0163] In some embodiments, the tumor antigen can be conjugated to the metallosome of this invention. The conjugation of the antigen to the bacterial membrane component on the metallosome can be carried out using any conventional method known in the art, for example, by an antimicrobial peptide. For example, the antimicrobial peptide can be connected to the antigen (covalently or non-covalently bound), and then the antigen compound connected to the peptide can be connected to the antimicrobial peptide by a polypeptide or protein on the bacterial membrane. This is within the technical knowledge and ability of those skilled in the art.

[0164] Immune checkpoints are a class of immunosuppressive molecules that can regulate the intensity and breadth of immune responses, thereby avoiding damage and destruction of normal tissues. During the occurrence and development of tumors, immune checkpoints become one of the main causes of immune tolerance. The "immune checkpoint inhibitors" described herein refer to any substance or agent suitable for targeting immune checkpoint molecules. Through immune checkpoint inhibitors, T cell activity and immune responses are promoted. In particular, immune checkpoints include but are not limited to PD-1 (programmed cell death protein 1), CTLA-4 (cytotoxic T lymphocyte-associated protein 4), PD-L1 (programmed cell death ligand 1), PD-L2 (programmed cell death ligand 2), etc. Immune checkpoint inhibitors include antibodies targeting immune checkpoints, such as anti-PD-1, anti-PD-L1, anti-PD-L2 or anti-CTLA-4 antibodies.

[0165] The administration of other tumor therapeutic drugs and irradiation can be performed sequentially or simultaneously, for example, first administering other tumor therapeutic drugs and then irradiation, or first irradiation and then administering other tumor therapeutic drugs, or administering other tumor therapeutic drugs and irradiation simultaneously, which can be determined by the clinician according to the treatment plan and / or the specific situation of the patient.

[0166] As used herein, "therapeutically effective amount" refers to the amount of a medicament, compound, or material in a dosage formulation that is at least sufficient to produce a therapeutic effect in a subject. The exact amount depends on the purpose of the treatment and can be determined by one skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0167] As used herein, the tumor is any tumor that can benefit from hyperthermia or radiation therapy, preferably a solid tumor, for example, selected from gastric cancer, liver cancer, biliary tract cancer, gallbladder cancer, colon cancer, lung cancer, bladder cancer, cervical cancer, ovarian cancer, breast cancer, melanoma, pancreatic cancer, kidney cancer, renal cell carcinoma, glioma, osteosarcoma, head and neck cancer, esophageal cancer and prostate cancer. In one embodiment, the tumor is selected from breast cancer, colon cancer, liver cancer, gastric cancer, renal cell carcinoma, pancreatic cancer, ovarian cancer or esophageal cancer.

[0168] As used herein, a "subject" refers to an organism having a tumor that can be treated by administering a metallobody, immunopotentiator / immunostimulator / radiosensitizer, metallobody, or pharmaceutical composition as provided herein. Non-limiting examples include humans and other mammals (e.g., non-human mammals) such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows, and deer. In some embodiments, the subject is a human.

[0169] As used herein, "treating" a subject having a tumor means that the subject's tumor is partially or completely eliminated, or remains stable and does not progress or progresses at a reduced rate after treatment.

[0170] As used herein, the drug or pharmaceutical composition comprises a metallobody as defined herein and a pharmaceutically acceptable excipient. The composition preferably comprises a pharmaceutically acceptable carrier, medium or delivery vehicle, as conventionally known in the art (see, for example, "Handbook of Pharmaceutical Excipients", Rowe et al., eds. 7th ed., 2012, www.pharmpress.com).

[0171] The word "or" is intended to include "and" unless the context indicates otherwise.

[0172] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, an optionally included step means that the step exists or does not exist.

[0173] As used herein, the term "about" refers to a range of values ​​that include the specific value and that one skilled in the art would reasonably consider to be similar to the specific value. In certain embodiments, the term "about" refers to within the standard error of measurement using commonly accepted methods in the art. For example, in certain embodiments, about refers to + / - 10% or 5% of the specific value.

[0174] Ranges disclosed herein should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, description of a range from 1 to 6 should be considered to have specifically disclosed subranges from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0175] Example

[0176] The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or embodiment of the present invention. The scope of the present invention is defined by the appended claims. In combination with this specification and common knowledge in the art, a person of ordinary skill in the art can clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, those skilled in the art may make any modifications or changes to the technical solutions of the present invention, and such modifications and changes are also included in the scope of the present invention. The methods used in the following examples are all conventional methods unless otherwise specified. The experimental materials used can be easily obtained from commercial companies unless otherwise specified.

[0177] Materials and Methods

[0178] 1. Reagents

[0179] Chloroauric acid (HAuCl4) (Cat. No. 10010711) was purchased from Shanghai Hushi Chemical Co., Ltd. (China). RPMI 1640 medium, fetal bovine serum (FBS), penicillin / streptomycin solution, phosphate-buffered saline (PBS), and red blood cell lysis buffer were all purchased from Wisent (Canada). L-glutamine and β-mercaptoethanol were purchased from Gibco (USA). IL-4 and GM-CSF cytokines were purchased from ProSpec (Israel). Fluorescent molecule-labeled antibodies, including fluorescein isothiocyanate (FITC)-anti-mouse-CD11c, phycoerythrin-cyanine-7 (PE-Cy-7)-anti-mouse CD80, allophycocyanin (APC)-anti-mouse CD86, FITC-anti-mouse CD3, PE-anti-mouse CD4, PE-Cy7-anti-mouse CD8, APC-anti-mouse CD19, FITC-anti-mouse CD49, PE-Cy7-anti-mouse CD69, PE-Cy7-anti-mouse CD25, Alexa Fluor 647-anti-mouse Foxp 3, FITC-anti-mouse F4 / 80, PE-anti-mouse CD11b, and Alexa Fluor 595-anti-mouse Gr1, were purchased from Biolegend (USA). Cytokine quantification kit, LEGENDPlex helper T cell factor multifactor detection kit (catalog number: 740005), LEGENDPlex inflammatory factor multifactor detection kit (catalog number: 740446), and LEGENDPlex pro-inflammatory chemokine multifactor detection kit (catalog number: 740451) were all purchased from Biolegend (USA).

[0180] 2. Instruments

[0181] Table 1: List of instruments used in the experiment

[0182] 3. Cells and Animals

[0183] 4T1 mouse breast cancer cells were purchased from ATCC (USA) and cultured in RPMI-1640 medium supplemented with 10% FBS and 100 μg / mL of the dual-antibody at 37°C in an atmosphere containing 5% carbon dioxide. Bone marrow-derived DCs were isolated and cultured. Six- to eight-week-old female Balb / c mice were purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd. (China) and maintained in a pathogen-free environment with continuous food and water. All animal-related experimental procedures were performed in accordance with procedures and protocols approved by the Animal Care and Welfare Committee of the National Center for Nanoscience and Technology.

[0184] Example 1: Synthesis of Ausomes by E. coli

[0185] 1.1: The process of E. coli synthesizing Ausomes

[0186] Escherichia coli strain DH5α was cultured to the logarithmic growth phase, collected by centrifugation, and resuspended in PBS. 1% chloroauric acid solution was added and cultured at 37°C with shaking at 200 rpm.

[0187] In order to track the process of Escherichia coli synthesizing Ausomes and secreting them outside the cell, after adding chloroauric acid, a small amount of bacterial culture solution was taken at 0.5, 2, 4, 8, 12 hours, and 1, 2, 3, 4, 5, 6, 7, 8, 10 days, and centrifuged at 6000rpm for 10 minutes to collect the precipitate. The bacteria were fixed overnight with 2.5% glutaraldehyde, and dropped onto a copper mesh and silicon wafer coated with a carbon support film. After standing and drying at room temperature, they were observed using a transmission electron microscope (TEM) and a scanning electron microscope (SEM). The bacteria were also prepared into ultrathin sections to observe the generation of Ausomes inside the bacteria. Take a small amount of bacterial culture solution, centrifuge at 6000rpm for 10 minutes, and collect 2-3mm 3 Immediately add cell clusters of different sizes to 2.5% glutaraldehyde fixative and fix overnight at 4°C. Wash three times with PBS for 10 minutes each time, then fix with 0.1% osmium acid at room temperature for 2 hours. After thorough washing with PBS, dehydrate with a gradient of 50%, 70%, 80%, 90%, and 100% ethanol for 10 minutes at each concentration, and dehydrate three times with 100% ethanol. Then replace with 100% acetone twice for 10 minutes each time. Then soak the sample with embedding agent. After acetone and embedding agent are prepared in a volume ratio of 1:1 and 1:2, add them to the sample, soak for 1 hour at room temperature, and add pure embedding agent overnight. Then pick out the sample and place it in an embedding plate, place it at 37°C overnight, and polymerize it at 60°C for 48 hours until hardened. The embedded sample was sliced ​​into 70 nm thick sections using an ultramicrotome, placed on a copper grid coated with a carbon support film, and stained with uranyl acetate for 15-30 minutes. After three rinses and drying, the sections were stained with lead citrate for 5-10 minutes, rinsed thoroughly, and dried. The sections were then observed using a scanning electron microscope (STEM) mode.

[0188] 1.2: Ausomes extraction conditions

[0189] After 72 hours of co-culture of chloroauric acid and Escherichia coli, the bacterial culture solution was taken and centrifuged at 6000 rpm for 30 minutes to separate the bacteria. The supernatant solution was filtered with sterile filter membranes with pore sizes of 0.45 μm and 0.22 μm respectively to further remove the residual Escherichia coli in the supernatant. Then the solution was centrifuged at 8000 rpm, 10000 rpm, 12000 rpm, and 13000 rpm for 10 minutes or 30 minutes respectively. A small amount of Ausomes solution was added dropwise to a copper mesh coated with a carbon support film. After the water evaporated completely, the morphology of Ausomes was observed under TEM. The overall particle size and uniformity of Ausomes collected under 8 centrifugal conditions were evaluated.

[0190] 1.3: Changes in Ausomes yield with incubation time

[0191] Next, we measured the rate of gold nanoparticle formation. At the aforementioned time points (0.5, 2, 4, 8, and 12 hours, and 1, 2, 3, 4, 5, 6, 7, 8, and 10 days), 1 ml of bacterial culture was collected and centrifuged at 6,000 rpm for 10 minutes, after which the bacteria were discarded. The supernatant was then filtered through sterile filters with 0.45 μm and then 0.22 μm pore sizes to further remove any residual E. coli. The supernatant was then centrifuged at 13,000 rpm for 30 minutes, and any remaining unreduced gold ions were discarded. The precipitate was collected and quantitatively analyzed using inductively coupled plasma mass spectrometry (ICP-MS) to analyze the changes in gold nanoparticle formation over time.

[0192] 1.4: Extraction and characterization of Ausomes

[0193] 1.4.1 Morphology and structure of Ausomes

[0194] The extracted Ausomes were dispersed in PBS, and the hydrated particle size of Ausomes was analyzed by dynamic light scattering (DLS).

[0195] A small amount of the Ausome solution was dripped onto a copper grid coated with a carbon support film. After the water evaporated completely, the morphology of the Ausomes was observed under a TEM. The crystal structure and lattice constant of the Ausomes were analyzed using high-resolution imaging and selected area electron diffraction (SAED).

[0196] 1.4.2 Composition analysis of the organic layer on the surface of Ausomes

[0197] We analyzed the protein and lipid components of Ausomes by proteomics and lipidomics.

[0198] During proteomic analysis, the proteins contained in the ausomes are first extracted and their concentrations quantified. After undergoing reductive alkylation and FASP enzymatic digestion, the peptide segments are analyzed and detected using a liquid phase tandem mass spectrometer. The mass spectrometry results are analyzed using the MaxQuant software package to generate the proteomic data for the ausomes.

[0199] Ausomes lipid qualitative analysis involves precipitating proteins with a 3:10 volume ratio of methanol and methyl tert-butyl ether. After centrifugation at 12,000 rpm for 10 minutes, the supernatant is evaporated to dryness. The lipids are then re-dissolved in a 1:1 mixture of isopropanol and acetonitrile and analyzed using ultra-fast liquid chromatography-mass spectrometry. Mass spectrometry data are acquired using XCalibur and analyzed using Progenesis QI software and the Lipidmaps database to generate the qualitative lipidomics of Ausomes.

[0200] 1.4.3 Characterization of the Photothermal Properties of Ausomes

[0201] The Ausomes solution was placed in a double-sided transparent quartz cuvette, and the absorption of Ausomes in the wavelength range of 400-900 nm was detected using a UV-visible spectrophotometer.

[0202] 200 μL of 250 μg / mL Ausomes solution (the gold concentration detected by ICP-MS was used as the standard to measure the amount of Ausomes used in each experiment in this study) was placed in the wells of a 96-well ELISA plate with a detachable strip. A 660 nm laser was used at 0.5 W / cm 2 , 1.0W / cm 2 , 1.5W / cm 2 , 2.5W / cm 2 The sample was irradiated at a laser power density of 100 nm. The laser power density was calculated by measuring the actual laser power and the area of ​​the spot. An infrared thermal imager was then used to monitor the solution temperature, recording its changes with laser irradiation duration.

[0203] The power density of the 660 nm laser was fixed at 1.5 W / cm 2 Ausomes solutions of different concentrations (500 μg / mL, 250 μg / mL, 125 μg / mL, 63 μg / mL, 31 μg / mL, 15 μg / mL) were placed in an ELISA plate, irradiated with a laser, and the solution temperature was monitored with an infrared thermal imager, and its changes with the laser irradiation time were recorded.

[0204] 1.4.4 Synthesis and Characterization of Ausomes Reference Gold Nanoparticles

[0205] Add 1 mL of 1% chloroauric acid solution to 100 mL of water, heat in an oil bath at 120°C, and stir at 400 rpm for 5 minutes. Then, add 1 mL of 10 mg / mL trisodium citrate solution, heat in an oil bath at 120°C, and stir at 400 rpm for 15 minutes. Transfer the solution to room temperature and cool it down by stirring at 1100 rpm for 20 minutes. Then, add 10 mg of HS-polyethylene glycol (PEG(3500)-NH2) and stir overnight. Centrifuge at 13,000 rpm, rinse twice with water, and finally disperse in PBS and store at 4°C in the dark.

[0206] The particle size and morphology of gold nanoparticles (AuNPs) were characterized by DLS and TEM. Their UV-visible absorption spectra were measured, and the photothermal properties related to laser power density and gold nanoparticle concentration were characterized according to the above steps.

[0207] result:

[0208] Escherichia coli is an FDA-approved protein expression system with good safety and is widely used in the biopharmaceutical industry [Marisch K et al., Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level sod protein production. Microb Cell Fact: 2013, 12: 58]. In addition, Escherichia coli has been shown to be able to express trivalent gold ions (Au 3+) is reduced to zero-valent gold nanoparticles, and there are reports that Escherichia coli OMVs are used to activate the immune system in vivo to treat tumors [Kim OY et al., Bacterial outer membrane vesicles suppress tumor by interferon-gamma-mediated antitumor response. Nat Commun: 2017, 8: 626; Narayanan KB et al., Biological synthesis of metal nanoparticles by microbes. Adv Colloid Interface Sci: 2010, 156: 1-13 and Du L et al., Biosynthesis of gold nanoparticles assisted by escherichia coli DH5 alpha and its application on direct electrochemistry of hemoglobin. Electrochem Commun: 2007, 9: 1165-1170]. Therefore, Escherichia coli was selected as our biofactory to synthesize and produce gold nanoparticles with intrinsic immune stimulating ability. First, DH5α E. coli was cultured to the logarithmic growth phase, then the culture medium was discarded, the cells were dispersed in PBS, and incubated with 1mM chloroauric acid at 37°C. In order to monitor the synthesis process, the morphology and state of the bacteria were observed by TEM and SEM at different time points. As can be seen from Figure 1A-C, without the addition of Au, 3+ Previously, the bacterial structure was complete and the surface was smooth, and the clear capsule layer and pili structure could be clearly observed. 3+ After 30 minutes of co-incubation, the capsule layer of E. coli became fuzzy, and many ultra-small nanoparticles appeared in it. This phenomenon is consistent with the early protection mechanism of bacteria under external heavy metal ion pressure. 3+First, it is adsorbed to the extracellular matrix (EPS) layer, and then reduced to ultra-small gold nanoparticles by reducing substances in it, such as some enzymes or special protein structures, and embedded in the EPS [Kang F et al., Extracellular saccharide-mediated reduction of Au3+ to gold nanoparticles: New insights for heavy metals biomineralization on microbial surfaces. Environ Sci Tech: 2017, 51: 2776-2785]. 3+ After 4 hours of co-culture, we found 30nm nanoparticles inside the bacteria. From the contrast, we believe that they are gold nanoparticles. Then at 12 hours, more nanoparticles were observed at the edge of the bacteria. 3+ The pale yellow color of the culture medium changed to a light pink, and then the red color gradually deepened with the extension of the incubation time. Since the color of the 30nm gold nanoparticle solution is red, this also indicates that more and more gold nanoparticles are being generated in the culture medium. After incubation for 48 hours, a large number of gold nanoparticles accumulated at the edge of the bacteria and showed a tendency to be secreted outward, and the bacterial culture medium also turned a deeper red.

[0209] In with Au 3+ During the incubation, we observed the destruction of the bacterial capsule layer, which may be due to the Au 3+ It provides a pathway for Au to diffuse into cells and be reduced to gold nanoparticles. 3+After incubation for 48 hours, the bacteria were sliced ​​into 70 nm ultrathin sections and negatively stained using STEM to observe the internal structure and distribution of the gold nanoparticles. As shown in Figure 2A, a small amount of synthesized gold nanoparticles were distributed at the edges of the E. coli, in the periplasmic space between the inner and outer membranes. This is consistent with the conclusions of some studies on the mechanism of metal ion reduction in vivo [Deplanche K et al., Biorecovery of gold by escherichia coli and desulfovibrio desulfuricans. Biotechnol Bioeng: 2008, 99: 1055-1064]. The 30 nm gold nanoparticles embedded in the periplasmic space caused expansion of the space, deforming both the inner and outer membranes. This is similar to one of the mechanisms of OMV formation: local pressure changes caused by protein aggregation in the periplasmic space lead to changes in the curvature of the bacterial outer membrane, thereby triggering the formation of OMVs [Kulp A et al., Biological Functions and Biogenesis of Secreted Bacterial Outer Membrane Vesicles. Annu rev ​​microbiol: 2010, 64: 163-184]. Therefore, we hypothesize that the formation of gold nanoparticles promotes the formation of OMVs. From SEM imaging of E. coli, we can see that after 12 hours or longer incubation time, the bacterial surface becomes more wrinkled (Figure 1A), providing evidence that the formation of gold nanoparticles in the periplasmic space leads to bacterial outer membrane budding. At the same time, in TEM imaging of E. coli at 24 and 48 hours, we observed scattered nanoparticles in the extracellular region, indicating that the gold nanoparticles are secreted outside the cell after synthesis in the periplasm. To further demonstrate the secretion mechanism of gold nanoparticles, we used TEM and SEM imaging at the 48-hour incubation timepoint to identify some E. coli cells in the process of secreting gold nanoparticles, as shown in Figures 2B and 2C. The gold nanoparticles distributed at the edges of the bacteria had clearly protruded outward but had not yet completely detached from the cell. A distinct thin layer was observed on the surface of the protrusions, which, based on the TEM contrast, indicated organic matter. Based on these experimental observations, it is reasonable to hypothesize that the gold nanoparticles produced by reduction in the periplasm of E. coli may be encapsulated in the bacterial membrane and secreted outside the cell through the formation of membrane vesicles, forming gold nanoparticles coated with the bacterial outer membrane, which we named Ausomes.

[0210] We then evaluated the yield of Ausomes synthesized by Escherichia coli under different incubation times. We extracted extracellular free Ausomes using a two-step centrifugation procedure. First, the bacteria were separated from the culture supernatant by low-speed centrifugation; then the gold nanoparticles were separated from the proteins, bacterial vesicles and unreacted gold ions in the culture medium by high-speed centrifugation. The precipitate was washed with PBS and collected by centrifugation to obtain Ausomes. In the second centrifugation step, we used four different centrifugal speeds for 10 minutes or 30 minutes to optimize the uniformity of the final collected product. As shown in Figure 3A, relatively pure Ausomes were obtained under all eight centrifugation conditions, and no other organic impurities were observed. By comparison, it can be seen that the greater the centrifugal speed and the longer the time, the more small-sized Ausomes (10-20nm) in the collected product, among which the centrifugal speed has a greater effect on the product size. The average particle size of Ausomes collected by centrifugation at 8000 rpm for 10 minutes or 30 minutes was larger and more uniform; however, a little red remained in the supernatant after centrifugation, indicating that some Ausomes products would be lost at this centrifugation rate. 3+ Ausomes of all sizes were collected by centrifugation at 13,000 rpm for 30 minutes at different reaction times, and the yield of ausomes was measured by ICP-MS. As shown in Figure 3B, the yield of ausomes gradually increased with the extension of reaction time, and the output rate remained relatively stable.

[0211] We used TEM and DLS to characterize the size and dispersibility of Ausomes. As shown in Figure 4A, Ausomes are uniform in size and well dispersed, with no aggregation. DLS characterizes the average hydrated particle size to be around 60 nm. By magnifying the TEM, it can be clearly observed that Ausomes are a core-shell structure consisting of a high-contrast inorganic core and a low-contrast surface organic layer. The thickness of the organic layer is about 3-4 nm under TEM (Figure 4B). High-resolution imaging shows multiple crystal planes of the core, as shown in Figure 4C; through software measurement, we identified the spacing between two of the stripes as approximately 0.204 nm and 0.235 nm, which is consistent with the lattice constants of the d(111) and d(200) crystal planes of gold crystals. The diffraction rings shown by selected area electron diffraction of Ausomes are also consistent with (111), (200), (220), and (311) of gold crystals (Figure 4D), further verifying that the core of Ausomes is gold crystals. As a control, we also chemically synthesized surface-modified PEG-coated gold nanoparticles (AuNPs) with a similar particle size to Ausomes, as shown in Figure 4E,F.

[0212] We then investigated the composition of the surface organic layer of ausomes through proteomics and lipidomics. Lipidomic analysis using both positive and negative point spray ionization modes revealed that ausomes contain a large number of lipids. Table 2 and Figure 5 summarize the most abundant phospholipids and lipids without phosphate groups, which are important components of the phospholipid bilayer, providing evidence that ausomes possess a membrane structure. In addition to the major lipids of the E. coli cell membrane, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL), phosphatidic acid (PA), and diacylglycerol (DG) were also detected at high abundance. The relative proportions of PE, PG, and CL differed from those in normal E. coli. This abnormal lipid composition is associated with bacterial autophagy and membrane rearrangements, possibly due to the extremely nutrient-deficient culture environment, heavy metal ion stress, and the occurrence of numerous membrane vesicles. Furthermore, in positive point spray mode, Lipid A was detected in Ausomes. Lipid A is the phospholipid portion of lipopolysaccharide (LPS), a key component of the bacterial outer membrane. Its primary function is to anchor LPS to the outer membrane, suggesting the presence of an outer membrane in Ausomes. These data confirm our hypothesis that gold nanoparticles synthesized by bacteria are secreted extracellularly through membrane vesicle formation, forming gold nanoparticles with the outer membrane attached to their surfaces. Furthermore, we analyzed the protein composition of Ausomes using proteomics. Table 3 summarizes some of the proteins identified as highly abundant or unique. Ausomes demonstrate the presence of numerous proteins localized to the outer membrane and those residing in the inner membrane. These bacterial outer membrane proteins (OMPs) or lipoproteins suggest the immunostimulatory potential of Ausomes. The protein transporter inner membrane protein SecG and the outer membrane protein assembly protein BamA are localized to the inner and outer membranes of bacteria, respectively, and have previously been used to distinguish between inner and outer membranes. Proteomic analysis revealed that the presence of BamA and the absence of SecG in Ausomes further provided evidence for the outer membrane covering of the Ausome surface.

[0213] Table 2: Lipids with higher abundance in Ausomes according to lipidomics analysis

[0214] Table 3: Proteins contained in some Ausomes identified by proteomics

[0215] Based on the surface plasmon resonance phenomenon, gold nanoparticles can convert the absorbed light energy into various physical and chemical effects, such as photothermal, photoacoustic, photoelectric, etc. Photothermal conversion is one of the main directions of biological applications of gold nanomaterials, so we also verified the photothermal conversion ability of Ausomes. The UV-visible absorption spectrum shows that the maximum absorption peak of Ausomes is at 535nm (Figure 6A). In order to ensure good tissue penetration, we chose a laser wavelength (660nm) that is more red but can detect obvious absorption to excite Ausomes. As shown in Figure 6B, Ausomes solutions of different concentrations, after 1.5W / cm 2 Irradiation with a 660nm laser of varying power density resulted in rapid temperature increases, reaching a maximum temperature in approximately 3 minutes and maintaining a plateau temperature. The plateau temperature of each solution was positively correlated with the Ausome concentration. Similarly, we fixed the solution concentration at 250 μg / mL and irradiated Ausome solutions with varying power densities. The temperature of the plateau temperature was positively correlated with the power density. UV-visible spectrophotometry and photothermal conversion experiments confirmed that chemically synthesized AuNPs exhibited similar absorption peaks and photothermal conversion capabilities to Ausomes (Figures 6C, D), and can be used as a control for Ausomes in related experiments. These results suggest that Ausomes can convert absorbed light energy into heat. While the photothermal conversion efficiency is relatively low due to the spherical morphology and small size of gold nanoparticles, the converted heat energy is sufficient to reach the hyperthermic temperature range (39-42°C). Therefore, we anticipate that Ausomes can induce localized hyperthermia-mediated immunomodulatory effects at the tumor site.

[0216] Example 2: Cellular uptake and in vitro immune stimulation studies of Ausomes

[0217] 2.1 Cellular Uptake of Ausomes

[0218] 4T1 tumor cells and BMDCs were seeded into confocal microscopy dishes and cultured in RPMI 1640 medium supplemented with 10% FBS at 37°C in an atmosphere of 5% CO. After 24 hours, 50 μg of Ausomes were added to the cells for co-incubation. After 6 and 24 hours of incubation, the supernatant was discarded, and the cells were washed three times with PBS. The gold signal in the cells was detected using a two-photon confocal microscope.

[0219] 4T1 tumor cells and BMDCs were seeded into 24-well plates and cultured for 24 hours. 50 μg of Ausomes were then added and incubated with the cells. At 6 and 24 hours, the supernatant was discarded, and the cells were washed three times with PBS. The cells were then digested to remove organic matter, and gold was quantitatively detected using ICP-MS.

[0220] 2.2 Ausomes stimulate BMDC maturation

[0221] 100,000 BMDCs were cultured in RPMI 1640 medium supplemented with 10% FBS and co-incubated with a series of Ausomes at different concentrations (0.3 μg / mL, 0.6 μg / mL, 1.3 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL). The proportion of CD80+CD86+ mature DCs was then detected by flow cytometry at 6 and 24 hours.

[0222] 100,000 BMDCs were cultured in RPMI 1640 medium supplemented with 10% FBS and incubated with 5 μg / mL of AuNPs, 5 μg / mL of Ausomes, and 5 μg / mL of AuNPs after irradiation with 660 nm laser at 1.5 W / cm 2 Ausomes were irradiated at a power density of 100 nm for 30 min and incubated for 24 h, and then the proportion of CD80+CD86+ mature DCs was detected by flow cytometer.

[0223] result:

[0224] To investigate the immunostimulatory capacity of Ausomes, we first examined the interaction between Ausomes and cells. Ausomes were co-incubated with BMDCs or 4T1 tumor cells, then imaged using two-photon laser scanning microscopy, and the cellular uptake of Ausomes was quantitatively detected using ICP-MS. As shown in Figures 7A and 7B, both Ausomes and AuNPs were taken up by tumor cells at approximately 15% of the feed amount, without exhibiting significant selectivity. Extending the incubation time did not further promote tumor cell enrichment of Ausomes or AuNPs. In BMDCs incubated with Ausomes or AuNPs for 6 hours (Figures 7C and 7D), significant two-photon luminescence signals of gold nanoparticles were detected, and approximately 30% of the Ausomes were taken up. As the incubation time increased, at 24 hours, stronger TPL signals of Ausomes were detected in BMDCs, and the uptake increased by approximately 40%. In contrast, the uptake of AuNPs by BMDCs was about 60% less and did not increase further with incubation time, indicating that BMDCs can selectively recognize Ausomes, suggesting their good immunogenicity.

[0225] DCs are among the first immune cells to respond to mutations or invasion by foreign pathogens. After recognizing and ingesting stimuli, they differentiate into a mature phenotype, promoting antigen presentation and cytokine secretion, initiating both innate and adaptive immune responses. Therefore, we evaluated the immunostimulatory capacity of DCs by examining Ausome-mediated maturation. After incubation with varying doses of Ausomes, mature DCs expressing high levels of the costimulatory factors CD80 and CD86 were screened by flow cytometry. As shown in Figures 8A and 8B, even a low dose of Ausomes stimulated for 6 hours to generate a significant number of mature DCs. Increasing the dose or duration of stimulation further increased the proportion of mature DCs. 10 μg of Ausomes stimulated for 24 hours resulted in over 85% mature DCs. The rapid and efficient immune response induced by this low dose demonstrates the excellent immunostimulatory capacity of Ausomes. In addition, we also evaluated the immunostimulatory ability of Ausomes after laser irradiation. As can be seen from Figure 8C and D, laser irradiation or heating did not affect the stimulation of BMDCs maturation by Ausomes.

[0226] Example 3: Study on the in vivo dosage of Ausomes

[0227] 3.1 Ausomes trigger cytokine secretion in vivo

[0228] Different doses of Ausomes (5 mg / kg, 10 mg / kg, 15 mg / kg, and 20 mg / kg) were intravenously injected into Balb / c mice. Six hours later, mouse serum was collected and cytokine levels were measured using a multifactorial detection kit. Microspheres modified with each cytokine capture antibody were mixed with a standard sample and serum sample in a 1.5 mL centrifuge tube. The mixture was shaken at 1000 rpm and incubated at room temperature in the dark for 2 hours. The tube was then centrifuged at 1000 g for 5 minutes, the supernatant discarded, and washed once with 300 μL of wash buffer. The detection antibody was then added, shaken at 1000 rpm, and incubated at room temperature in the dark for 1 hour. PE-labeled streptavidin was added to the reaction system, shaken at 1000 rpm, incubated at room temperature in the dark for 30 minutes, washed once with wash buffer, and resuspended before analysis by flow cytometry. The flow cytometry data were then processed using LEGENDplex v8.0 software to calculate the serum concentration of each cytokine.

[0229] 3.2 Tumor therapeutic effects of different doses of Ausomes

[0230] 4T1 breast cancer cells were inoculated into the mammary fat pad of 6-8 week old female Balb / c mice and the cells were grown until the tumors grew to 50 mm. 3 Ausomes were injected intravenously at doses of 5 mg / kg, 10 mg / kg, 15 mg / kg, and 20 mg / kg, respectively, once every three days. The tumor volume and body weight of the mice were measured every other day. The tumor volume was calculated using the formula: V = ab 2 / 2, a and b represent the long axis and short axis of the mouse tumor, respectively. When the tumor volume reaches 1000mm 3 Terminate the experiment.

[0231] 3.3 Distribution of Ausomes in vivo

[0232] 4T1 tumor cells were inoculated into the mammary fat pad of 6-8 week old female Balb / c mice and the tumors were grown to 200 mm. 3 Around 6 and 24 hours after intravenous injection of 250 μg of Ausomes or AuNPs, the heart, liver, spleen, lungs, kidneys, thymus, lymph nodes, and intestines of the mice were removed. After weighing, the organs were heated with hydrogen peroxide and concentrated nitric acid to remove organic molecules, and the gold content in each organ was determined by ICP-MS.

[0233] 3.4 Evaluation of Ausomes’ Toxic and Side Effects in Vivo

[0234] Female Balb / c mice aged 6-8 weeks without tumor burden were intravenously injected with Ausomes at doses of 5 mg / kg, 10 mg / kg, 15 mg / kg, and 20 mg / kg, once every three days, for a total of three immunizations. The heart, liver, spleen, lung, and kidney of the mice were then removed and made into tissue sections and stained with hematoxylin-eosin (H&E) to analyze whether the major organs had pathological damage. At the same time, the serum levels of biochemical indicators such as ALT (alanine aminotransferase), AST (aspartate aminotransferase), BUN (urea nitrogen), and CREA (creatinine) were measured to evaluate the liver and kidney functions of the mice at different Ausomes injection doses.

[0235] result:

[0236] When we consider applying Ausomes in vivo to stimulate an immune response in a complete immune system, the large number of bacterial-derived molecules contained in Ausomes suggests potential safety risks. Therefore, we studied the in vivo application dose of Ausomes to ensure the immune stimulatory effect without causing significant toxic side effects. The first consideration for intravenous administration is the potential hemolysis caused by the drug. When Ausomes of different doses were co-incubated with mouse whole blood, it was found that Ausomes of various concentrations did not damage blood cells (Figure 9A). The large number of bacterial-derived molecules in Ausomes is the molecular mechanism of their immune stimulatory ability, but it may also trigger a cytokine storm in the immune system to damage the body. Therefore, we intravenously injected different doses of Ausomes, 5 mg / kg, 10 mg / kg, 15 mg / kg, and 20 mg / kg, into healthy mice, and then measured the levels of multiple cytokines in the serum using a multifactorial kit to evaluate the extent of the systemic immune response. Figure 9B shows the multiple increase in the mouse serum concentration of each cytokine compared to the untreated control group. It can be seen that at all doses, Ausomes had no significant effect on the serum concentrations of IL-10, IL-1α, IL-12, and IFN-β; significantly upregulated cytokines included IFN-γ, TNF-α, and IL-6. As shown in Figure 9C, Ausomes at all doses stimulated the body to produce a large amount of IFN-γ, TNF-α, and IL-6. There was no obvious correlation between their blood concentrations and the dose, which may be due to the complex regulatory mechanism of the immune system. However, there were significant differences between the different dose treatment groups, indicating that the degree of immune response stimulated by Ausomes can be controlled by adjusting the dose.

[0237] We then evaluated the anti-tumor effects of Ausomes at different doses to provide guidance for their in vivo dosage. 4T1 tumor cells were orthotopically inoculated into the mammary fat pad of female Balb / c mice. When the tumor volume of the mice reached 50 mm 3 At the same time, different doses of Ausomes (5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg) were intravenously injected to treat mouse tumors, once every three days, as shown in Figure 10A. The weight and tumor volume of the mice were recorded every other day during immunization. Since the intravenous injection of Ausomes caused a decrease in the weight of the mice (Figure 10B), we controlled the number of administrations to 3 times. On the 18th day, the tumor volume of the mice in the control group without any treatment exceeded 1000mm 3 The experiment was terminated. The tumor growth curve of the mice showed that the 15 mg / kg dose induced the best tumor inhibition effect ( FIG10C ).

[0238] The weight loss of mice suggested that Ausomes caused some side effects, so we conducted some experiments to determine whether different doses of Ausomes would cause damage to other organs during tumor treatment. First, we used ICP-MS to quantitatively analyze the distribution of Ausomes in various organs or tissues after intravenous injection. As shown in Figure 11A, 6 hours after administration, high gold content was detected in the liver, spleen and lymph nodes. They are all organs or tissues directly related to the immune system or have inherent immune properties, indicating that the body has made a systemic immune response to exogenous nanoparticles. After 24 hours, the aggregation of Ausomes in the liver and spleen further increased. At the same time, we also observed that compared with AuNPs, more Ausomes accumulated in the lungs, which may be related to the interaction of the lung mucosal immunity with Ausomes with higher immunogenicity. We then injected healthy mice with different doses of Ausomes according to the immune program for tumor treatment, and evaluated the liver and kidney functions of mice treated with different doses of Ausomes by blood biochemical analysis. As shown in Figure 11B, at a dose of 20 mg / kg, the blood level of AST increased significantly, which may be related to the local inflammation caused by the large accumulation of Ausomes in the liver. Other doses of Ausomes did not cause significant changes in these indicators, indicating that there was no effect on liver and kidney function. Since Ausomes accumulated in large quantities in the liver and spleen, we analyzed the damage to the organs through H&E-stained tissue sections. As can be seen from Figure 11C, all doses of Ausomes did not cause obvious pathological changes in the liver and spleen, and there was no obvious tissue damage in the heart, lungs, and kidneys where other Ausomes accumulated relatively less. Combined with the safety of different doses and the tumor treatment effect, 15 mg / kg was selected as the dosage for in vivo studies.

[0239] Example 4: Study on Local Hyperthermia of Ausomes Tumors

[0240] 4.1 Enrichment of Ausomes at Tumor Sites

[0241] The Cy5.5-NHS fluorescent molecule was added to the Ausomes solution, the pH was adjusted to 7.2, and the reaction was allowed to proceed overnight at room temperature. The solution was then centrifuged at 13,000 rpm for 30 minutes, the supernatant discarded, and the solution washed once with PBS. The Cy5.5 fluorescent molecule-labeled Ausomes were then resuspended and injected intravenously into 4T1 tumor-bearing mice (250 μg / mouse). The mice were then imaged at different time points using an in vivo spectrum imaging system (IVIS), and the fluorescence intensity of the tumor sites was quantitatively analyzed.

[0242] In addition, AuNPs and Ausomes were injected into the veins of tumor-bearing mice, respectively. The tumors were removed at different time points, and the gold content in the tumors was detected by ICP-MS after digestion.

[0243] 4.2 Ausomes-mediated local tumor hyperthermia

[0244] Ausomes and AuNPs were intravenously injected into 4T1 tumor-bearing mice at a dose of 15 mg / kg. Six hours later, the AuNPs were irradiated with a 660 nm laser at 1.2 W / cm 2 The mouse tumor was irradiated at a power density of 100 nm and the tumor temperature was measured and imaged using an infrared thermal imager; and the tumor temperature was recorded at 30-second intervals.

[0245] 4.3 Local hyperthermia promotes tumor blood perfusion

[0246] 4T1 tumor cells were inoculated into the axilla of 6-8 week old female Balb / c mice. Two weeks later, Ausomes and AuNPs were intravenously injected into the mice at a dose of 15 mg / kg. Six hours later, the tumors were illuminated with a 660 nm laser at 1.2 W / cm 2 The cells were irradiated at a power density of 100 nm for 30 minutes, and the signals of oxygenated hemoglobin and hemoglobin were detected by real-time multispectral photoacoustic tomography (MSOT) to evaluate tumor blood flow.

[0247] Tumor vascular fluorescence imaging has also been used to evaluate the effects of hyperthermia on blood vessels. 6-8 week old female Balb / c mice were inoculated with 4T1 tumor cells under the mammary fat pad. Two weeks later, Ausomes and AuNPs were intravenously injected into the mice at a dose of 15 mg / kg. A 660 nm laser was used at 1.2 W / cm 2 The mice were irradiated at a power density of 100 nm for 30 minutes, followed by intravenous injection of FITC-labeled dextran solution (3 mg / mouse) to label blood vessels. Fifteen minutes later, the mice were euthanized, the tumors removed, and the surface blood was carefully washed with PBS before being fixed overnight in a tissue fixative. Laser confocal microscopy was used to scan the tumor tissue with a 5 μm step size, and three-dimensional reconstruction of the tumor vasculature was performed.

[0248] 4.4 Local hyperthermia promotes the immune stimulatory effect of Ausomes

[0249] 4.4.1 Local hyperthermia promotes cytokine secretion

[0250] 4T1 tumor cells were inoculated into the mammary fat pad of 6-8 week old female Balb / c mice and the cells were grown until the tumors grew to 100 mm. 3 15 mg / kg of Ausomes or AuNPs were injected intravenously into mice; 6 hours later, the tumors were irradiated with a 660 nm laser at 1.2 W / cm 2 Then, 6 days after the first immunization, Ausomes and AuNPs were intravenously injected into mice at a dose of 15 mg / kg respectively; 6 hours later, the mice were irradiated with a 660 nm laser at a power density of 1.2 W / cm 2 Tumors were irradiated at a power density of 100 nm for 30 minutes. Six hours after irradiation, mouse serum and tumor samples were collected. A small amount of tumor tissue fragments were added to RIPA lysis buffer containing 1% protease inhibitors and ground using a cryo-tissue grinder. The tissue was then centrifuged at 12,000 rpm for 15 minutes, and the supernatant was collected. The levels of various cytokines and chemokines in the tissue lysate and serum were quantified using a multifactorial assay kit as described above.

[0251] 4.4.2 Local hyperthermia promotes immune cell infiltration

[0252] Tumor tissues 6 and 24 hours after treatment were ground using a 70 μm pore filter to form a single-cell suspension. Fluorescently labeled antibodies were then added for staining at 4°C for 20 minutes. The cells were washed once with RPMI1640 medium containing 2% FBS and resuspended. The tumor tissue levels of various immune cells, including CD4+ T cells (anti-CD3 antibody + anti-CD4 antibody), CD8+ T cells (anti-CD3 antibody + anti-CD8 antibody), B cells (anti-CD19 antibody), NK cells (anti-CD49 antibody), activated NK cells (anti-CD49 antibody + anti-CD69 antibody), macrophages (anti-F4 / 80 antibody), DCs (anti-CD11c antibody), Tregs (anti-CD4 antibody + anti-C25 antibody + anti-Foxp3 antibody), and myeloid-derived suppressor cells (MDSCs; anti-CD11b antibody + anti-Gr1 antibody), were analyzed by flow cytometry.

[0253] 4.5 Local hyperthermia promotes the tumor therapeutic effect of Ausomes

[0254] 4T1 tumor cells were inoculated into the mammary fat pad of 6-8 week old female Balb / c mice and the cells were grown until the tumors grew to 50 mm. 3 15 mg / kg of Ausomes or AuNPs were injected intravenously into mice; 6 hours later, the tumors were irradiated with a 660 nm laser at 1.2 W / cm 2 The mice were irradiated at a power density of 100 nm for 30 minutes, and the immune / hyperthermia treatment was performed three times at intervals of 3 days. The tumor volume of the mice was measured every other day. The tumor volume calculation formula is the same as above: V = ab 2 / 2, a and b represent the long axis and short axis of the mouse tumor, respectively. When the tumor volume reaches 1000mm 3 Terminate the experiment.

[0255] 4.6 In vivo safety evaluation of Ausomes immunostimulation combined with local hyperthermia

[0256] During tumor treatment, mice were weighed every other day, and weight changes were recorded. Following the experiment, mice were euthanized, and their hearts, livers, spleens, lungs, and kidneys were removed, sliced, and stained with H&E to analyze whether the various treatments caused pathological damage to major organs. Serum was also collected and levels of biochemical markers such as ALT, AST, BUN, and CREA were measured to evaluate liver and kidney function.

[0257] result:

[0258] Having verified the photothermal conversion capability of Ausomes, we next examined Ausome-mediated localized tumor hyperthermia. We first tested the time point at which Ausomes accumulate at the tumor site. 250 μg of Ausomes labeled with the fluorescent Cy5.5 molecule were intravenously injected into mice and fluorescence accumulation in the tumor site was observed using IVIS at different time points. As shown in Figures 12A and 12B, the Cy5.5 signal in the tumor accumulated over time, reaching a peak 12 hours after injection, then slowly decayed. Cy5.5 fluorescence was still observed in the tumor site 72 hours after injection. To quantitatively measure gold accumulation in the tumor site, we intravenously injected 250 μg of Ausomes or gold nanoparticles into 4T1 tumor-bearing mice. Tumors were isolated and digested into gold ions at different time points, and then quantitatively analyzed by ICP-MS. As shown in Figure 12C, high levels of Ausomes accumulated in the tumor site between 2 and 12 hours after injection, then slowly decreased, a trend similar to that observed with in vivo fluorescence labeling. Unlike Ausomes, AuNPs reached maximum aggregation 12 hours after intravenous injection and maintained it until 24 hours, and maintained a relatively high level until 72 hours, which may be due to the higher immunogenicity of Ausomes promoting the uptake and clearance of antigen-presenting cells in the tumor site.

[0259] Based on the above results, after intravenous injection of 15 mg / kg Ausomes into orthotopic tumor-bearing mice, the tumors were concentrated for 6 hours with a 660 nm laser at 1.2 W / cm 2 The tumor was irradiated with a power density of 100 nm and the tumor temperature was measured by an infrared thermal imager. As shown in Figure 13A, B, under laser irradiation, the tumor heated up rapidly, reached a plateau in about 2 minutes, and the maximum temperature reached about 41°C. The tumors of mice injected intravenously with AuNPs also had similar local hyperthermia effects, but the tumors in the PBS-treated group did not show obvious temperature changes under laser irradiation. We then used real-time multispectral photoacoustic tomography (MSOT) to detect the tissue distribution of hemoglobin (HbO2) and hypoxic hemoglobin (Hb) in the tumor before and after laser irradiation as a marker of blood perfusion. 4T1 tumor cells were inoculated into the armpits of mice. Two weeks later, 15 mg / kg of Ausomes or AuNPs were injected intravenously. Six hours later, a 660 nm laser was used to irradiate the tumor at 1.2 W / cm 2The tumor was irradiated with a power density of 100 nm for 30 minutes, and then a tomography scan was performed using MSOT. As shown in Figure 13C, compared with the ctrl control group without any treatment and the Ausomes-treated group without laser irradiation, we observed HbO2 and Hb signals in the deeper parts of the tumor tissue in the AuNPs group and the Ausomes group after laser irradiation, indicating that after excessive heat treatment, the blood contents can better infiltrate into the tumor tissue. In addition, we used FITC-labeled dextran to fluorescently label the blood vessels and visually observe the changes in tumor blood vessels by laser confocal imaging. 15 mg / kg of Ausomes or AuNPs were intravenously injected into mice bearing tumors in situ, and laser irradiation was performed under the above conditions 6 hours later. Then 3 mg of FITC-dextran was injected intravenously, and the tumor was removed and fixed 10 minutes later for imaging. As shown in Figure 13D, compared to tumors not irradiated by laser, the fluorescence signals in the blood vessels of the hyperthermia-treated tumors were more numerous and stronger, indicating increased blood flow in the tumor site. Significant fluorescence signals were also observed in the tissue outside the blood vessels, indicating that dextran had leaked from the tumor blood vessels into the tumor tissue. These results demonstrate that intravenously injected Ausomes can mediate localized hyperthermia in the tumor under laser irradiation, promoting blood perfusion in the tumor site and tumor tissue infiltration of blood contents.

[0260] Ausomes, which are composed of a gold nanoparticle core and a bacterial membrane shell, are multifunctional nanoimmunomodulators that have both photothermal conversion and immune stimulation. We have previously verified the immune activation effect of Ausomes and their ability to mediate local hyperthermia in tumors. Next, we tested the immune response and anti-tumor effect of the two functional stimulations of Ausomes. First, we constructed an in situ breast cancer model by inoculating 4T1 cells in the mammary fat pad of female Balb / c mice. In order to simultaneously detect the innate immune response and the adaptive immune response, tumor-bearing mice were immunized twice, as shown in Figure 14A. When the tumor volume reached 100mm 3 When 15 mg / kg of Ausomes or AuNPs were injected intravenously, 6 hours later, a 660 nm laser was used at 1.2 W / cm 2The mice were irradiated at a power density of 100 nm for 30 minutes. Seven days later, the same immunohistochemistry protocol was performed. Six hours after laser irradiation, tumors and blood were collected from the mice, and the concentrations of various cytokines in tissue and serum were measured using a Luminex microsphere-based multifactor detection kit. As shown in Figure 14B, the heat map represents the fold increase in cytokines and chemokines in each treatment group compared to the untreated Ctrl control group. Increases of 3-fold or more were counted as 3-fold. Mice immunostimulated with Ausomes showed increased serum levels of a large number of pro-inflammatory cytokines, such as IFN-γ, TNF-α, IL-17F, IL-17A, IL-22, IL-13, IL-21, IL-2, and IL-9, as well as chemokines involved in the recruitment of effector cells such as T cells and NK cells, such as CCL3, CXCL9, CXCL10, CCL20, CCL5, and CCL4, indicating the activation of a systemic immune response. Combined with local hyperthermia in the tumor, the blood concentrations of these cytokines and chemokines were further increased. AuNPs-mediated local hyperthermia in tumors promoted IL-6 expression and downregulated the expression of cytokines such as CM-CSF, IL-1α, IL-1β, IL-5, IL-27, and CCL22. These immunosuppressive cytokines were also downregulated in the Ausomes-treated group and the Ausomes-combined local hyperthermia group, indicating that the negative immune regulatory mechanism was suppressed to a certain extent. In tumor tissue, AuNPs-mediated local hyperthermia alone also stimulated a certain degree of immune response, with tissue levels of IL-2 and IL-6 both upregulated. Ausome stimulation alone triggered upregulation of TNF-α, IL-6, IL-2, and IL-1α in tumor tissue. Combined with local hyperthermia, in addition to further promoting the secretion of these cytokines, Ausomes also significantly increased levels of pro-inflammatory cytokines such as IL-22, IL-1β, IL-17A, IL-17F, IL-23, IL-27, and IFN-β. Furthermore, we observed elevated expression of the anti-inflammatory cytokine IL-10, potentially reflecting a negative feedback mechanism triggered by an overly robust immune response. Furthermore, Ausome stimulation increased the concentrations of chemokines involved in T cell recruitment, such as CCL3, CCL4, and CCL20, in tumor tissue, while also decreasing the expression of CCL22 and CCL17, which are associated with Tregs and MDSCs. Combined with local hyperthermia, these effects, which enhance the immune response in tumor tissue, were further enhanced. These experimental observations demonstrate that Ausomes have the ability to induce systemic immune responses, and that their mediated local hyperthermia can further enhance both systemic and local immune responses.

[0261] Tumor-bearing mice were immunized according to the above immunization protocol. Six and 24 hours after laser irradiation, the tumors were disaggregated into single cells and stained with fluorescently labeled antibodies. Flow cytometry was then used to analyze the infiltration of various immune cells in the tumor tissue. As shown in Figure 15, AuNPs-mediated local hyperthermia alone led to an increase in F4 / 80+ macrophages in the tumor tissue. In mice immunized with Ausomes, a significant increase in CD3+CD8+ T cells, CD49+ NK cells, and CD49+CD69+ activated NK cells was detected in the tumor tissue. Combined local hyperthermia further promoted the infiltration of CD3+CD4+ T cells, CD49+ NK cells, and CD49+CD69+ activated NK cells, with the number of infiltrating cells increasing over time. The levels of other immune cells, such as B cells, DCs, Tregs, and MDSCs, did not change significantly.

[0262] Next, we verified the tumor suppressive effects of hyperthermia, Ausomes, and Ausomes combined with hyperthermia in a 4T1 breast cancer orthotopic model. 3 When 15 mg / kg of Ausomes or AuNPs were injected subcutaneously, 6 hours later, a 660 nm laser was used at 1.2 W / cm 2 The tumor volume was measured every other day and the tumor size reached 1000 mm. 3 , the experiment was terminated. As can be seen from the tumor growth curve and the size of the tumor at the end of the experiment, hyperthermia further enhanced the inhibitory effect on the basis of Ausomes' tumor treatment (Figure 16B). During the tumor treatment process, the weight of the mice was measured and recorded every other day. As can be seen from Figure 16C, Ausomes caused a certain degree of weight loss, but the additional hyperthermia treatment did not further promote the weight loss of the mice. After the end of the experiment, the serum of the mice in each treatment group was collected for blood biochemical tests to evaluate liver and kidney function; and the main organ sections were stained with H&E for pathological analysis. As shown in Figures 16D and E, Ausomes combined with hyperthermia caused an increase in AST, suggesting the occurrence of liver inflammation, but there were no obvious pathological changes in the liver tissue morphology. No obvious damage was observed in other treatment groups and organs, indicating that at the dose and immunization procedure used in the experiment, Ausomes or Ausomes combined with hyperthermia did not cause obvious toxic side effects.

[0263] Example 5: Preparation of Microbial Mineralized Gold Nanoparticle Radiotherapy Sensitizers (Ausomes)

[0264] As described in Example 1, Pseudomonas aeruginosa (e.g., BNCC337889), Escherichia coli (e.g., BNCC336454), Salmonella typhimurium (e.g., ATCC 14028), Fusobacterium nucleatum (e.g., BNCC280188), Acinetobacter baumannii (e.g., BNCC337173), Staphylococcus epidermidis (e.g., BNCC102555), Streptococcus salivarius (e.g., BNCC337521), Bacillus licheniformis (e.g., BNCC336463), Lactobacillus reuteri (e.g., BNCC254476), or Bifidobacterium longum (e.g., BNCC185354) were cultured in the corresponding liquid culture medium and placed at 37°C with shaking at 200 rpm for overnight culture until the logarithmic growth phase. The bacteria were collected by centrifugation and the culture medium was discarded. The bacteria were resuspended in PBS at pH 7.4, 1% chloroauric acid solution was added, and the culture was shaken at 200 rpm at 37°C.

[0265] After 8 days of co-culture with chloroauric acid and bacteria, the culture medium was centrifuged at 5000g for 30 minutes to separate the bacteria. The supernatant was filtered through sterile membranes with 0.45μm and then 0.22μm pore sizes. The supernatant was then centrifuged at 15000g for 30 minutes in a high-speed centrifuge. The supernatant was discarded, and the resulting pellet was resuspended to obtain Ausomes.

[0266] The microscopic morphology of the obtained Ausomes is shown in Figures 17-26.

[0267] Example 6: Radiotherapy physical sensitization effect

[0268] Select tumor cells growing in the logarithmic phase and prepare the cell suspension to the required concentration. Plate 500 cells per well of a 6-well plate. After adding the cells, shake well and observe under a microscope to confirm that the cells are evenly distributed. After culturing for 24 hours, add Ausomes (final concentration is 20μg / mL). After the cells are incubated with Ausomes for 6 hours, replace with fresh culture medium and irradiate with a biological irradiator at a dose of 6Gy. After irradiation, fresh culture medium can be replaced every 3 days. After 2 weeks, terminate when each cell colony is visible to the naked eye. Under a microscope, when the number of cells in each cell colony is greater than 50, it can be counted as 1 colony. After about 1-2 weeks, depending on the morphology and size of the colony, discard the culture medium, wash it with pre-cooled PBS, add 1ml of methanol / 4% paraformaldehyde, fix it on ice for 10 minutes, then add PBS to wash it again, add crystal violet, and stain it at room temperature for 10 minutes. After recovering the crystal violet, continue to wash it with PBS 3 times and dry it. After complete drying, take pictures and count the cells. The results are shown in Figures 27-28. In the presence of Ausomes, the number of cell clones is significantly reduced, indicating a good enhancement of the radiation-killing effect.

[0269] Example 7: Immune Effects

[0270] C57BL6 mice were subcutaneously inoculated with 5 × 10 6 MC38 colon cancer cells were cultured in an SPF environment. When the tumor size grew to about 200 mm 3 At the same time, the mice were divided into 4 groups, namely, saline group (Sal), radiotherapy group (RT), Ausomes injection group (AS) and Ausomes combined with radiotherapy group (AS+RT). For the PBS group, 50uL of PBS solution was injected adjacent to the tumor, and for the Ausomes treatment group, 50uL of Ausomes solution (2mg / mL) was injected adjacent to the tumor. After 24 hours of injection, irradiation was performed. For the radiotherapy group, the tumor area was irradiated with 6Gy in a biological irradiator for a total of 1 time. After 48 hours of irradiation, Ausomes were injected adjacent to the tumor again, and thereafter, 50uL of Ausomes solution was injected adjacent to the tumor every 3 days for a total of 2 times. 24 hours after the last injection, the spleen and tumor tissues of the mice were taken to analyze the composition of effector immune cells in the tissues, including CD4+T cells, CD8+T cells and DC cells (CD11c+). The results are shown in Figures 29-38. After the use of Ausomes, the proportion of antigen-specific T cells in the spleen increased significantly, indicating that Ausomes combined with radiotherapy effectively activated the systemic immune response; the infiltration levels of effector T cells and DC cells in tumor tissues increased, indicating that Ausomes effectively improved the immune environment of the tumor site after radiotherapy.

[0271] Example 8: Promoting the anti-tumor effect of radiotherapy

[0272] C57BL6 mice were subcutaneously inoculated with 5 × 10 6 MC38 colon cancer cells were cultured in an SPF environment. When the tumor size grew to about 200 mm 3 At 3 pm, 5 × 10 6 MC38 colon cancer cells were used to construct a multiple or distant metastatic tumor model. The mice were divided into 4 groups, namely, normal saline group (Sal), radiotherapy group (RT), Ausomes injection group (AS) and Ausomes combined with radiotherapy group (AS+RT). For the PBS group, 50uL of PBS solution was injected adjacent to the tumor. For the Ausomes treatment group, 50uL of Ausomes solution was injected adjacent to the tumor. After 24 hours of injection, irradiation was performed. For the radiotherapy group, the tumor area was irradiated with 6Gy in a biological irradiator for a total of 1 time. Ausomes were injected adjacent to the tumor again 48 hours after irradiation. Thereafter, 50uL of Ausomes were injected adjacent to the tumor every 3 days for a total of 2 times. The long and short side dimensions of the mouse tumor were measured every other day.

[0273] The results are shown in Figures 39-48. It can be seen that compared with radiotherapy alone, the combination of Ausomes further promoted the inhibitory effect of radiotherapy on in situ tumors and enhanced the regression of distal tumors, while the administration of Ausomes alone did not cause similar effects, indicating that Ausomes are good radiotherapy sensitizers and can trigger a systemic response to eliminate small distal lesions.

[0274] Example 9: Application as a radiosensitizer in other tumor types

[0275] 5×10 6 Mouse tumor cells (glioma cells, osteosarcoma cells, melanoma cells, breast cancer cells, pancreatic cancer cells, liver cancer cells, gastric cancer cells, head and neck cancer cells, cervical cancer cells or lung cancer cells) were cultured in an SPF environment. 3 When, the mice were divided into 4 groups, namely normal saline group (Sal), radiotherapy group alone (RT), Ausomes group (AS) and Ausomes combined with radiotherapy group (AS+RT). In this embodiment, Escherichia coli mineralized Ausomes are taken as an example. For the PBS group, 50uL of PBS solution was injected adjacent to the tumor. For the Ausomes treatment group, 50uL of Ausomes solution was injected adjacent to the tumor (Day 0). After 24 hours of injection, irradiation treatment was performed (Day 1). For the radiotherapy group, the tumor area was irradiated with 6Gy in a biological irradiator for a total of 1 time. After 48 hours of irradiation, Ausomes were injected adjacent to the tumor again. Thereafter, 50uL of Ausomes solution was injected adjacent to the tumor every 3 days for a total of 2 times. The long and short side sizes of the mouse tumors were measured every other day.

[0276] The experimental results are shown in Figures 49-50. It can be seen that compared with radiotherapy alone, Ausomes further promoted the inhibitory effect of radiotherapy on in situ tumors, indicating that Ausomes are good radiotherapy sensitizers.

[0277] Example 10: Ausomes obtained by different preparation methods

[0278] (1) Using bacterial culture medium with a pH of 7

[0279] Escherichia coli DH5α was cultured in the appropriate liquid medium at 37°C with shaking at 200 rpm overnight until the logarithmic growth phase. The bacteria were harvested by centrifugation and the medium discarded. The bacteria were resuspended in a pH 7 bacterial culture medium, and 1% chloroauric acid solution was added. The culture was then shaken at 200 rpm at 37°C.

[0280] After 8 days of co-culture with chloroauric acid and bacteria, the bacterial culture was centrifuged at 5000g for 30 minutes to separate the bacteria. The supernatant was filtered through sterile membranes with 0.45μm and then 0.22μm pore sizes to further remove any residual bacterial cells. The supernatant was then centrifuged at 15000g for 30 minutes in a high-speed centrifuge. The supernatant was discarded, and the resulting pellet was resuspended to obtain Ausomes and stored at -80°C or in liquid nitrogen. The microscopic morphology is shown in Figure 51, Panel A.

[0281] (2) Using a bacterial culture medium with a pH of 4

[0282] Escherichia coli DH5α was cultured in the appropriate liquid culture medium at 37°C with shaking at 200 rpm overnight until the logarithmic growth phase. The bacteria were harvested by centrifugation and the culture medium discarded. The bacteria were resuspended in a pH 4 bacterial culture medium, and 1% chloroauric acid solution was added. The culture was then shaken at 200 rpm at 37°C.

[0283] After 8 days of co-culture with chloroauric acid and bacteria, the bacterial culture was collected and centrifuged at 5000g for 30 minutes to separate the bacteria. The supernatant was filtered through sterile membranes with 0.45μm and then 0.22μm pore sizes to further remove any residual bacterial cells. The supernatant was then centrifuged at 15000g for 30 minutes in a high-speed centrifuge. The supernatant was discarded, and the resulting pellet was resuspended to obtain Ausomes and stored at -80°C or in liquid nitrogen. The microscopic morphology is shown in Figure 51, Panel B.

[0284] (3) Using PBS buffer with a pH of 7

[0285] Escherichia coli DH5α was cultured in the appropriate liquid medium at 37°C with shaking at 200 rpm overnight until the logarithmic growth phase. The bacteria were harvested by centrifugation and the medium discarded. The bacteria were resuspended in PBS buffer (pH 7), 1% chloroauric acid solution was added, and the culture was shaken at 200 rpm at 37°C.

[0286] After 8 days of co-culture with chloroauric acid and bacteria, the bacterial culture was centrifuged at 5000g for 30 minutes to separate the bacteria. The supernatant was filtered through sterile membranes with 0.45μm and then 0.22μm pore sizes to further remove any residual bacterial cells. The supernatant was then centrifuged at 15000g for 30 minutes in a high-speed centrifuge. The supernatant was discarded, and the resulting pellet was resuspended to obtain Ausomes and stored at -80°C or in liquid nitrogen. The microscopic morphology is shown in Figure 51, Panel C.

[0287] (4) Using PBS buffer with a pH of 4

[0288] Escherichia coli DH5α was cultured in the appropriate liquid medium at 37°C with shaking at 200 rpm overnight until the logarithmic growth phase. The bacteria were harvested by centrifugation and the medium discarded. The bacteria were resuspended in PBS buffer (pH 4) and 1% chloroauric acid solution was added. The culture was then shaken at 200 rpm at 37°C.

[0289] After 8 days of co-culture with chloroauric acid and bacteria, the bacterial culture was collected and centrifuged at 5000g for 30 minutes to separate the bacteria. The supernatant was filtered through sterile membranes with 0.45μm and then 0.22μm pore sizes to further remove any residual bacterial cells. The supernatant was then centrifuged at 15000g for 30 minutes in a high-speed centrifuge. The supernatant was discarded, and the resulting pellet was resuspended to obtain Ausomes and stored at -80°C or in liquid nitrogen. The microscopic morphology is shown in Figure 51, Panel D.

[0290] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the preparation method of a multifunctional metal body (radiotherapy sensitizer, immunostimulant, or immunopotentiator), its product, and its application. However, the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments for implementation. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

[0291] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0292] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A pharmaceutical composition comprising a metal body and optionally a pharmaceutically acceptable carrier, wherein the metal body comprises metal nanoparticles and bacterial components attached thereto, preferably, the bacterial components are selected from intracellular membranes, extracellular membranes, proteins, carbohydrates, nucleic acids, phospholipids and any combination thereof.

2. The pharmaceutical composition of claim 1, wherein: - the metal nanoparticles are metal crystals, and / or the metal is selected from gold, silver, manganese, titanium and iron, preferably gold; and / or - the bacterial component is directly or indirectly connected to the metal nanoparticles by covalent or non-covalent means, or the bacterial component is located in or on an organic layer that completely or partially covers the metal nanoparticles; and / or - the particle size of the metal nanoparticles is about 5-500 nm, for example about 10-200 nm; and / or - the metal bodies are metal nanoparticles completely coated with bacterial-derived components; and / or -The pharmaceutical composition is used for photothermal therapy or radiotherapy of tumors. Optionally, the pharmaceutical composition also contains other tumor therapeutic drugs such as immune checkpoint inhibitors.

3. The pharmaceutical composition of claim 1 or 2, wherein the metallosomes are produced by mineralization of metals by bacteria, wherein the bacteria are preferably selected from the genus Neisseria, Bordetella, Escherichia, Salmonella, Pseudomonas, Fusobacterium, Acinetobacter, Staphylococcus, Streptococcus, Bacillus, Lactobacillus and Bifidobacterium, for example selected from Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, Fusobacterium nucleatum, Acinetobacter baumannii, baumannii, Staphylococcus epidermidis, Streptococcus salivarius, Bacillus licheniformis, Lactobacillus reuteri or Bifidobacterium longum, preferably Escherichia, more preferably Escherichia coli, such as DH5α or BL21 strains.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the pharmaceutical composition is used to treat a tumor in a subject, preferably a solid tumor, for example selected from gastric cancer, liver cancer, biliary tract cancer, gallbladder cancer, colon cancer, lung cancer, bladder cancer, cervical cancer, ovarian cancer, breast cancer, melanoma, pancreatic cancer, kidney cancer, renal cell carcinoma, glioma, osteosarcoma, head and neck cancer, esophageal cancer and prostate cancer, and / or, the subject is selected from humans and other mammals such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows and deer, preferably humans.

5. Use of metallobodies in the preparation of a drug for treating a tumor in a subject, wherein the metallobodies comprise metal nanoparticles and bacterial components attached thereto, preferably, the bacterial components are selected from intracellular membranes, extracellular membranes, proteins, carbohydrates, nucleic acids, phospholipids and any combination thereof.

6. The use of claim 5, wherein: - the metal nanoparticles are metal crystals, and / or the metal is selected from gold, silver, manganese, titanium and iron, preferably Gold; and / or - the bacterial component is directly or indirectly connected to the metal nanoparticles by covalent or non-covalent means, or the bacterial component is located in or on an organic layer that completely or partially covers the metal nanoparticles; and / or - the particle size of the metal nanoparticles is about 5-500 nm, for example about 10-200 nm; and / or - the metal bodies are metal nanoparticles completely coated with bacteria-derived components, wherein the bacteria-derived components are located on the organic layer coating the metal nanoparticles; and / or - The drug is used for photothermal therapy or radiotherapy of tumors. Optionally, the drug also contains other tumor therapeutic drugs such as immune checkpoint inhibitors.

7. The method of claim 5 or 6, wherein the metallobodies are produced by mineralizing metals from bacteria, wherein the bacteria are preferably selected from the genera Neisseria, Bordetella, Escherichia, Salmonella, Pseudomonas, Fusobacterium, Acinetobacter, Staphylococcus, Streptococcus, Bacillus, Lactobacillus and Bifidobacterium, for example selected from Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, Fusobacterium nucleatum, Acinetobacter baumannii, Staphylococcus epidermidis, Streptococcus salivarius, Bacillus licheniformis, Lactobacillus reuteri or Bifidobacterium longum, preferably Escherichia, more preferably Escherichia coli, such as DH5α or BL21 strains.

8. The use of any one of claims 5 to 7, wherein the medicament is used to treat a tumor in a subject, preferably a solid tumor, for example selected from gastric cancer, liver cancer, biliary tract cancer, gallbladder cancer, colon cancer, lung cancer, bladder cancer, cervical cancer, ovarian cancer, breast cancer, melanoma, pancreatic cancer, kidney cancer, renal cell carcinoma, glioma, osteosarcoma, head and neck cancer, esophageal cancer and prostate cancer, and / or, the subject is selected from humans and other mammals such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows and deer, preferably humans.

9. A radiosensitizer, immunopotentiator or immunostimulator, comprising a metal body and optionally a pharmaceutically acceptable carrier, wherein the metal body comprises metal nanoparticles and bacterial components attached thereto, preferably, the bacterial components are selected from intracellular membranes, extracellular membranes, proteins, carbohydrates, nucleic acids, phospholipids and any combination thereof.

10. The radiosensitizer, immunopotentiator or immunostimulatory agent of claim 9, wherein: - the metal nanoparticles are metal crystals, and / or the metal is selected from gold, silver, manganese, titanium and iron, preferably gold; and / or - the bacterial component is directly or indirectly connected to the metal nanoparticles by covalent or non-covalent means, or the bacterial component is located in or on an organic layer that completely or partially covers the metal nanoparticles; and / or - the particle size of the metal nanoparticles is about 5-500 nm, for example about 10-200 nm; and / or - the metal bodies are metal nanoparticles completely coated with bacterial-derived components; and / or - The immunopotentiator or immunostimulator is used for tumor photothermal therapy or radiotherapy, and / or the radiotherapy sensitizer is used for radiotherapy.

11. The radiosensitizer, immunopotentiator or immunostimulator of claim 9 or 10, wherein the metallosomes are produced by bacterial mineralization of metals, wherein the bacteria are preferably selected from the genera Neisseria, Bordetella, Escherichia, Salmonella, Pseudomonas, Fusobacterium, Acinetobacter, Staphylococcus, Streptococcus, Bacillus, Lactobacillus and Bifidobacterium, for example selected from Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, Fusobacterium nucleatum, Acinetobacter baumannii, Staphylococcus epidermidis, Streptococcus salivarius, Bacillus licheniformis, Lactobacillus reuteri or Bifidobacterium longum, preferably Escherichia, more preferably Escherichia coli, such as DH5α or BL21 strains.

12. The radiosensitizer, immunopotentiator or immunostimulatory agent of any one of claims 9 to 11, wherein the radiosensitizer, immunopotentiator or immunostimulatory agent is used for tumor treatment in a subject, preferably, the tumor is a solid tumor, for example, selected from gastric cancer, liver cancer, biliary tract cancer, gallbladder cancer, colon cancer, lung cancer, bladder cancer, cervical cancer, ovarian cancer, breast cancer, melanoma, pancreatic cancer, kidney cancer, renal cell carcinoma, glioma, osteosarcoma, head and neck cancer, esophageal cancer and prostate cancer, and / or, the subject is selected from humans and other mammals such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows and deer, preferably humans.

13. Use of metal bodies in the preparation of radiosensitizers, immunopotentiators or immunostimulants, wherein the metal bodies comprise metal nanoparticles and bacterial components attached thereto, preferably, the bacterial components are selected from bacterial inner membranes, bacterial outer membranes, proteins, carbohydrates, nucleic acids, phospholipids and combinations thereof.

14. The use of claim 13, wherein: - the metal nanoparticles are metal crystals, and / or the metal is selected from gold, silver, manganese, titanium and iron, preferably gold; and / or - the bacterial component is directly or indirectly connected to the metal nanoparticles by covalent or non-covalent means, or the bacterial component is located in or on an organic layer that completely or partially covers the metal nanoparticles; and / or - the particle size of the metal nanoparticles is about 5-500 nm, for example about 10-200 nm; and / or - the metal bodies are metal nanoparticles completely coated with bacterial-derived components; and / or - The immunopotentiator or immunostimulator is used for tumor photothermal therapy or radiotherapy, and / or the radiotherapy sensitizer is used for radiotherapy.

15. The method of claim 13 or 14, wherein the metallobodies are produced by bacterial mineralization of metals, wherein the bacteria are preferably selected from the genera Neisseria, Bordetella, Escherichia, Salmonella, Pseudomonas, Fusobacterium, Acinetobacter, Staphylococcus, Streptococcus, Bacillus, Lactobacillus and Bifidobacterium, for example selected from Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, Fusobacterium nucleatum, Acinetobacter baumannii, Staphylococcus epidermidis, Streptococcus salivarius, Bacillus licheniformis, Lactobacillus reuteri or Bifidobacterium longum, preferably Escherichia, more preferably Escherichia coli, such as DH5α or BL21 strains.

16. The use of any one of claims 13 to 15, wherein the radiosensitizer, immunopotentiator or immunostimulator is used for the treatment of tumors in a subject, preferably, the tumor is selected from solid tumors, for example, selected from gastric cancer, liver cancer, biliary tract cancer, gallbladder cancer, colon cancer, lung cancer, bladder cancer, cervical cancer, ovarian cancer, breast cancer, melanoma, pancreatic cancer, kidney cancer, renal cell carcinoma, glioma, osteosarcoma, head and neck cancer, esophageal cancer and prostate cancer, and / or, the subject is selected from humans and other mammals such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows and deer, preferably humans.

17. A method for preparing a metal body, comprising: (i) culturing bacteria capable of metal mineralization in a solution containing metal ions, (ii) harvesting the culture fluid obtained in (i), (iii) harvesting metallobodies from the culture supernatant of the culture fluid harvested in (ii), and optionally purifying them.

18. The method of claim 17, wherein: (a) the metal is gold, and optionally the solution containing metal ions is a chloroauric acid solution, preferably the concentration of the chloroauric acid solution is about 1-5%, such as 1%; and / or (b) the bacteria are cultured for at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 18, 24, 30, 36, 48, 60, 72, 96, 120 hours, 6 days, 7 days, 8 days, 9 days, 10 days; and / or (c) culturing at room temperature, e.g., about 25-37° C., optionally with shaking; and / or (d) obtaining a culture supernatant containing metal bodies from the culture solution, for example by centrifugation, and optionally, further filtering the culture supernatant, for example through a sterile filter membrane; and / or (e) centrifuging the culture supernatant containing the metal bodies, for example at about 5000-15000 g, such as about 11000 g, for at least 10 minutes, 20 minutes or 30 minutes.

19. The method of claim 17 or 18, wherein the bacterium is selected from the genera Neisseria, Bordetella, Escherichia, Salmonella, Pseudomonas, Fusobacterium, Acinetobacter, Staphylococcus, Streptococcus, Bacillus, Lactobacillus and Bifidobacterium, preferably Escherichia, more preferably Escherichia coli, such as DH5α or BL21 strains.

20. The method of any one of claims 17-19, wherein the metal bodies are prepared as follows: - adding a solution containing gold ions, such as chloroauric acid, to a solution containing bacteria, preferably bacteria in the logarithmic growth phase, and culturing with shaking at room temperature for at least 12 hours, preferably at least 48 hours, more preferably at least 72 hours, to obtain a culture solution, - Centrifuge the culture solution to obtain the supernatant containing metal bodies, - filtering the supernatant containing the metal bodies, for example, using a sterile filter membrane with a pore size of about 0.45 μm and / or 0.22 μm, - centrifuging the filtered supernatant at about 5000-15000 g, such as 11000 g, for at least 10 minutes, preferably at least 30 minutes, and - Harvest the precipitate to obtain metallobodies.

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