Nano photosensitizer modified by outer membrane vesicles of genetically engineered bacteria as well as preparation method and application of nano photosensitizer

Abstract

The invention provides a nano photosensitizer modified by outer membrane vesicles of genetically engineered bacteria as well as a preparation method and application of the nano photosensitizer. The nano photosensitizer is a nano material with the particle size of 80-120 nm, and the nano photosensitizer modified by the genetically engineered bacterium outer membrane vesicle comprises dipalmitic acid phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl lecithin (DOPC), cholesterol, a small molecule dye and CD47nb-OMV membrane protein. The chemical structure of the small molecule dye is shown as a formula I or a formula II. The nano photosensitizer OBDlp modified by outer membrane vesicles of genetically engineered bacteria serves as a novel nano photosensitizer, tumor targeting PTT is mediated through NIR II FL / PA imaging, tumor immunogenicity is improved, antigen presenting cells are activated, and infiltration and functionalization of CD8 + T cells are caused. And OBDlp and laser are combined to induce release of a large amount of DAMPs and trigger ICD to promote maturation of cDC1s. Meanwhile, a CD47 nano antibody carried by the nano photosensitizer induces polarization of M1 phenotype macrophages by blocking a CD47-SIRP alpha signal transduction axis, and an immunosuppression tumor microenvironment is reversed.

Description

Technical Field

[0001] The invention relates to the field of biological immune nanomaterials, and in particular to a nano photosensitizer modified by genetically engineered bacterial outer membrane vesicles, and a preparation method and application thereof. Background Art

[0002] Triple-negative breast cancer (TNBC) is a lethal female cancer with a high rate of metastasis and recurrence. The median overall survival of metastatic triple-negative breast cancer is approximately one year. Current chemotherapy options for TNBC are limited and have systemic toxic effects. Photothermal therapy (PTT) is an emerging ablative therapy. PTT destroys tumors without damaging surrounding normal tissues and has shown advantages in the treatment of localized solid tumors. It uses heat to induce apoptosis and leads to strong immunogenic cell death (ICD) and antigen release. However, breast cancer treated with localized PTT may still develop distant metastasis. This recurrence is due to the typical immunosuppressive tumor microenvironment (TME) of this disease, including insufficient immune cell infiltration, impaired immune cell function, and accumulation of immunosuppressive molecules. Cooling the TME promotes immune evasion and tumor metastasis. Therefore, immune reactivation is crucial to improve the efficacy of anti-tumor therapy after PTT.

[0003] The process of antigen presentation is crucial for anti-tumor immune responses. During this process, tumor-resident type I conventional dendritic cells (cDC1) bind to CD8 + Antigen cross-presentation by T cells is essential for a robust antitumor response. However, CD8 + T cell deficiency is an immunosuppressive feature of various solid tumors, including breast cancer. Therefore, activation of the antigen presentation cascade of cDC1s is an important mechanism to enhance CD8 + An ideal approach to T cell anti-tumor immunity. Endogenous CD8 + DC activation of T cells is a complex multistep immune cascade involving intratumoral antigen uptake, activation, and migration to tumor-draining lymph nodes (TdLN). Each of these steps is critical for triggering an adequate antitumor immune response. During activation, cDC1s are also prevented from maturing by immunosuppressive molecules in the TME, such as TGF-β and IL-10 secreted by M2 phenotype tumor-associated macrophages (TAMs). Therefore, the key to successfully reversing cDC1s dysfunction is the need to eliminate the tethering of immunosuppressive molecules in the TME, in addition to the need to absorb sufficient amounts of tumor-specific antigens.

[0004] PTT has been shown to induce ICD in tumor cells, but this strategy has limited effects on remodeling the TME and has difficulty in strongly activating tumor-resident cDC1s.

[0005] Therefore, it is necessary to develop nanophotosensitizers for immunotherapy. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a nano photosensitizer modified with genetically engineered bacterial outer membrane vesicles and a preparation method and application thereof.

[0007] To achieve the above object, the technical scheme adopted by the present invention is: a nano photosensitizer (OBDlp) modified by genetically engineered bacterial outer membrane vesicles, wherein the nano photosensitizer is a nano material with a particle size of 80-120 nm, and the nano photosensitizer modified by the genetically engineered bacterial outer membrane vesicles comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), cholesterol, small molecule dyes and CD47nb-OMV membrane protein; The chemical structure of the small molecule dye is shown in Formula I or Formula II. Formula (I); Formula (II); The molar ratio of dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine and cholesterol is (4.5-5.5): (2.5-3.5): (0.8-1.2): 1; The weight ratio of small molecule dye to CD47nb-OMV membrane protein is (1.5~10):1000; The nano photosensitizer is dispersed into a solution by various components and then extruded into a nano material.

[0008] Bacterial outer membrane vesicles (OMVs) have shown excellent anti-tumor potential. OMVs have similar functions to bacteria and can be delivered to tumor tissues in a targeted manner. Genetically engineered bacteria can stably prepare optimized OMVs with specific functional molecules, providing a new platform for tumor immunotherapy. OMVs have great prospects as a new type of biomaterial in improving the efficiency of cancer immunotherapy. Rational design of functional OMV nanoplatforms loaded with photosensitizers can improve drug delivery, reverse the immunosuppressive environment of solid tumors, induce specific tumor-killing immune effects, and prevent the formation of distant metastases. The above-mentioned genetically engineered bacterial outer membrane vesicle-modified nanophotosensitizer (OBDlp) first developed an engineered bacterial OMV (CD47nb-OMV), namely CD47nb-OMV membrane protein, which is an immune amplifier for encapsulation of a new NIR-II small molecule organic dye (BThDCMI) (chemical structure shown in Formula I), which can stimulate cDC1s-mediated antigen cross-presentation. The chemical structure of the small molecule dye is shown in Formula I or Formula II. The new molecular skeleton composed of dicyanomethyleneindene (DCMI) is used as a π-conjugated acceptor, which is connected to a triarylamine donor to regulate absorption. Among them, the maximum absorption rate of the embedded high quinone π bridge is 783nm (CH2Cl2), with an absorption far exceeding 700nm. Under the guidance of NIRII fluorescence / photoacoustic imaging, more specific imaging can be achieved without interference. Under the guidance of NIRII fluorescence / photoacoustic imaging, OBDlp-mediated targeted PTT induced ICD and M1 phenotype macrophage polarization, thereby effectively activating robust cDC1s / CD8 + T cell anti-tumor immune cascade, inhibiting the development of breast tumors (e.g. Figure 1 The combination of the genetically engineered bacterial outer membrane vesicle-modified nanophotosensitizer (OBDlp) for immune cascade reaction and PTT provides a new material and method for the treatment of TNBC, which is of great significance for advancing cancer immunotherapy and improving prognosis.

[0009] Preferably, the weight ratio of cholesterol to CD47nb-OMV membrane protein is (5-25):1000.

[0010] Preferably, the method for preparing CD47nb-OMV membrane protein comprises the following steps: culturing genetically engineered Escherichia coli MG1655, adding arabinose to induce the production of CD47nb-OMV membrane protein; transferring the genetically modified pBAD plasmid into the genetically engineered Escherichia coli MG1655; the method for preparing the genetically modified pBAD plasmid comprises: cloning the DNA fragment encoding the CD47nb-ClyA fusion protein into the pBAD plasmid by a one-step Gibson assembly method to obtain the genetically modified pBAD plasmid.

[0011] Preferably, the preparation method of the small molecule organic dye comprises the following steps: N, N-bis(4-methylphenyl)-3-(tributyltinyl)benzo[c]thiophene-1-amine or N, N-bis(4-methylphenyl)-3-(tributyltinyl)thiophene-1-amine reacts with 2-(3-chloro-1H-inden-1-ylidene)malononitrile in toluene under reflux under the catalysis of bis(triphenylphosphine)palladium dichloride to obtain a compound represented by formula (II) or formula (I).

[0012] Preferably, the preparation method of the genetically engineered bacterial outer membrane vesicle-modified nano photosensitizer comprises the following steps: (1) dissolving dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine and cholesterol in chloroform to obtain solution A, and mixing a tetrahydrofuran solution of a small molecule dye represented by formula I or formula II with solution A; (2) blowing the mixed solution of step (1) into the CD47nb-OMV membrane protein with an inert gas, drying to remove the solvent, and re-dissolving the solid product in a PBS buffer solution; (3) The solution obtained in step (2) is extruded in an extruder with a pore size of 80 to 120 nm for 8 to 15 cycles to obtain a nano photosensitizer modified with genetically engineered bacterial outer membrane vesicles.

[0013] Preferably, the concentration of the tetrahydrofuran solution of the small molecule dye represented by Formula I or Formula II is 1.5-5 mg / mL.

[0014] The present invention also provides a method for preparing any of the above genetically engineered bacterial outer membrane vesicle-modified nano photosensitizers, the method comprising the following steps: (1) dissolving dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine and cholesterol in chloroform to obtain solution A, and mixing a tetrahydrofuran solution of a small molecule dye represented by formula I or formula II with solution A; (2) blowing the mixed solution of step (1) into the CD47nb-OMV membrane protein with an inert gas, drying to remove the solvent, and re-dissolving the solid product in a PBS buffer solution; (3) The prepared solution obtained in step (2) is extruded in an extruder with a pore size of 80 to 120 nm for 8 to 15 cycles to obtain a nano photosensitizer modified with outer membrane vesicles of genetically engineered bacteria.

[0015] Preferably, the method for preparing the CD47nb-OMV membrane protein comprises the following steps: (I) synthesizing a DNA fragment encoding CD47nb-ClyA fusion protein, and cloning the DNA fragment into a pBAD plasmid using a one-step Gibson assembly method to obtain a genetically modified pBAD plasmid; (II) Transforming the genetically modified pBAD plasmid into Escherichia coli MG1655 to obtain genetically engineered Escherichia coli MG1655; (III) culturing the genetically engineered Escherichia coli MG1655 in LB medium supplemented with 20-30 μg / mL chloramphenicol at 36.5-37.5°C until the OD600 is greater than 0.45; (VI) Adding arabinose to induce genetically engineered Escherichia coli MG1655 to express CD47nb-OMV membrane protein; (V) After removing bacteria, CD47nb-OMV membrane proteins were collected by centrifugation.

[0016] Preferably, in step (VI), arabinose is added to a mass fraction of 0.45% to 0.55% to induce the genetically engineered Escherichia coli MG1655 to express CD47nb-OMV membrane protein; and the culture is carried out for 12 to 20 hours.

[0017] The present invention also provides the use of any of the above genetically engineered bacterial outer membrane vesicle-modified nano photosensitizers in promoting cDC1s-mediated anti-tumor immune cascade reactions.

[0018] like Figure 1 Preparation of OBD1p and OBD1p-mediated PPT to cDC1s / CD8 + Schematic diagram of the T cell anti-tumor immune cascade reaction. After OBDlp is combined with laser and delivered to the tumor tissue, it can induce tumor cell ICD and release DAMPs. The release of CD47nb causes M2 phenotype macrophages to repolarize into M1 phenotype macrophages, which will alleviate the tumor microenvironment's obstacles to cDC1 maturation. Therefore, cDC1s are successfully activated in the reshaped "hot" tumor microenvironment, triggering downstream CD8 + T cell anti-tumor immune cascade response to combat distant invasion and metastasis.

[0019] The beneficial effects of the present invention are as follows: the present invention provides a nanophotosensitizer modified by genetically engineered bacterial outer membrane vesicles, and a preparation method and application thereof. The nanophotosensitizer modified by genetically engineered bacterial outer membrane vesicles of the present invention first develops an engineered bacterial OMV (CD47nb-OMV), namely CD47nb-OMV membrane protein, which is an immune amplifier for encapsulation of a novel NIR-II small molecule organic dye (BThDCMI) (chemical structure as shown in Formula I), which can stimulate cDC1s-mediated antigen cross-presentation. The chemical structure is a small molecule dye as shown in Formula I or Formula II, and a new molecular skeleton composed of dicyanomethyleneindene (DCMI) is used as a π-conjugated receptor, which is connected to a triarylamine donor for regulating absorption. Among them, the maximum absorption rate of the embedded high quinone π bridge is 783nm (CH2Cl2), with an absorption far exceeding 700nm. Under the guidance of NIRII fluorescence / photoacoustic imaging, more specific imaging can be achieved without interference. Under the guidance of NIRII fluorescence / photoacoustic imaging, OBDlp-mediated targeted PTT induced ICD and M1 phenotype macrophage polarization, which in turn effectively activated robust cDC1s / CD8 + T cell anti-tumor immune cascade, inhibiting the development of breast tumors (e.g. Figure 1 The combination of the genetically engineered bacterial outer membrane vesicle-modified nanophotosensitizer (OBDlp) for immune cascade reaction and PTT provides a new material and method for the treatment of TNBC, which is of great significance for advancing cancer immunotherapy and improving prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Preparation of the nano photosensitizer OBD1p modified by genetically engineered bacterial outer membrane vesicles and OBD1p-mediated PPT for cDC1s / CD8 + Schematic diagram of T cell anti-tumor immune cascade reaction. Among them, a is the schematic diagram of the preparation of the nano photosensitizer OBDlp modified by genetically engineered bacterial outer membrane vesicles, and b is the PPT of the nano photosensitizer OBDlp modified by genetically engineered bacterial outer membrane vesicles on cDC1s / CD8 + Schematic diagram of the T cell anti-tumor immune cascade.

[0021] Figure 2Schematic diagram of the preparation and characterization results of the nanophotosensitizer OBDlp modified by the genetically engineered bacterial outer membrane vesicles of the embodiment of the present invention. (a) Schematic diagram of the preparation of PhDCMI, ThDCMI and BThDCMI. (b) Absorption result diagram of PhDCMI. (c) Absorption result diagram of ThDCMI. (d) Absorption result diagram of BThDCMI. (e) Plasmid schematic diagram of CD47nb-OMV and SDS-PAGE of engineered E. coli MG1655 and OMV. (f) CD47 antibody binding rate of 4T1 cells after different treatments. (g) In vitro phagocytosis of 4T1 cells by macrophages after different treatments. (h) Macrophage CD80 expression received different treatments. (i) Macrophage CD206 expression received different treatments. (j) TEM images of BDlp and OBDlp. Scale bar = 100 nm. (k) Size distribution of OMV, BDlp and OBDlp. (l) Absorption spectra of BThDCMI, BDlp and OBDlp. (m) Fluorescence spectra of BThDCMI, BDlp and OBDlp. (n) In vitro NIR II fluorescence imaging signal intensity of BDlp and OBDlp solutions.

[0022] Figure 3 The results of the in vitro photothermal therapy and DCs maturation experiment of the nano-photosensitizer OBDlp modified with genetically engineered bacterial outer membrane vesicles according to the present invention are shown in Figure 1. (a) The DCs were irradiated with laser (808 nm, 1 W / cm 2 ) Temperature changes of BDlp or OBDlp solutions at different times. (b) Live / dead cell staining of 4T1 cells receiving different treatments. Scale bar = 25 μm. (c) Cell viability of 4T1 cells treated with PBS, BThDCMI, BDlp, or OBDlp, with or without laser irradiation. (d) Immunofluorescence staining against CRT of 4T1 cells receiving different treatments. Scale bar = 25 μm. (e) HMGB1 levels of 4T1 cells receiving different treatments. (f) ATP levels of 4T1 cells receiving different treatments. (g) Maturation of DCs stimulated with medium derived from tumor cells receiving different treatments. (h) TNF-α levels of DCs stimulated with medium derived from tumor cells receiving different treatments. (i) IL-10 levels of DCs stimulated with medium derived from tumor cells receiving different treatments. (j) CD80 and CD86 expression levels of DCs stimulated with medium derived from tumor cells receiving different treatments.

[0023] Figure 4The results of the in vivo NIRII fluorescence / photoacoustic tumor imaging experiment of the nanophotosensitizer OBDlp modified with the genetically engineered bacterial outer membrane vesicles of the embodiment of the present invention are shown in FIG. (a) In vivo fluorescence imaging of tumor-bearing mice at different time points. (b) Ex vivo fluorescence imaging of organs and tumors. (c) NIR II fluorescence imaging of tumor-bearing mice at different time points. (d) Ex vivo near-infrared II fluorescence imaging of organs and tumors. (e) Quantitative analysis of FL signal intensity of tumor-bearing mice injected systemically at different time points. (f) Quantitative analysis of FL signal intensity of ex vivo organs and tumors. (g) Quantitative analysis of NIR II FL signal intensity of tumor-bearing mice at different time points. (h) Quantitative analysis of NIR II FL signal intensity of ex vivo organs and tumors. (i) In vivo NIR II photoacoustic imaging of tumor-bearing mice at different time points. (j) Quantitative analysis of photoacoustic imaging signal intensity of tumor-bearing mice at different time points.

[0024] Figure 5 The results of the PTT-activated M1 phenotype macrophage polarization and cDC1s maturation experiment mediated by the nanophotosensitizer OBDlp modified with the genetically engineered bacterial outer membrane vesicles of the present invention are shown in Figure 1. (a) Flow cytometry of M1 phenotype macrophages in tumors and spleen. (b) Quantitative analysis of M1 phenotype macrophages and the ratio of M1 macrophages to M2 macrophages in tumor tissues. (c) Quantitative analysis of M1 phenotype macrophages and the ratio of M1 macrophages to M2 macrophages in spleen. (d) Flow cytometry of cDC1s, cDC2s and TdLNs in tumors. (e) Quantitative analysis of total DCs, cDC1s and cDC1s / cDC2 ratios in tumors. (f) Flow cytometry and quantitative analysis of CD86 in tumor cDC1. (g) Quantitative analysis of the ratio of cDC1s / cDC2 in total DCs, cDC1 and TdLN. (h) Flow cytometric plots and quantitative analysis of CD86 in cDC1 in TdLN.

[0025] Figure 6Schematic diagram of the results of the PTT-induced T cell proliferation and activation experiment mediated by the nanophotosensitizer OBDlp modified with the genetically engineered bacterial outer membrane vesicles of the embodiment of the present invention. (a) Flow cytometry of functional CD8+T cells in tumors and TdLNs. (b) Quantitative analysis of CD4+T cells and CD8+T cells in tumors. (c) Quantitative analysis of proliferative CD8+T cells and exhausted CD8+T cells in tumors. (d) Quantitative analysis of functional CD4+T cells and CD8+T cells in tumors. (e) Quantitative analysis of CD4+T cells and CD8+T cells in TdLNs. (f) Quantitative analysis of proliferative CD8+T cells and exhausted CD8+T cells in TdLNs. (g) Quantitative analysis of functional CD4+T cells and CD8+T cells in TdLNs. (h) CD4+ / CD8+ immunofluorescence staining in tumors. Scale bar = 40μm. (i) Quantitative analysis of Treg and CD8+ T cell / Treg ratio in tumors. (j) Quantitative analysis of Treg and CD8+ T cell / Treg ratio in TdLNs. (k) TNF-α level in tumor or serum. (l) IFN-γ level in tumor or serum. (m) IL-6 level in tumor or serum. (n) TGF-β level in tumor or serum.

[0026] Figure 7 The results of in vivo anti-tumor treatment with the nanophotosensitizer OBDlp modified with the outer membrane vesicles of genetically engineered bacteria according to the embodiment of the present invention are shown in Figure 1. (a) Schematic diagram of the treatment procedure for 4T1 tumor-bearing mice. (b) Changes in tumor volume of treated mice. (c) Tumor weight of each group. (d) Photos of harvested mouse tumors. (e) Changes in tumor volume of each group. (f) Survival time of treated mice. (g) Changes in mouse weight during treatment. (h) TUNEL staining of tumors of mice in different groups. Scale bar = 200 μm.

[0027] Figure 8PTT treatment of distant tumor invasion and lung metastasis mediated by nanophotosensitizer OBDlp modified by genetically engineered bacterial outer membrane vesicles according to an embodiment of the present invention. (a) Schematic diagram of the treatment procedure for mice with bilateral tumors. (b) Changes in the volume of primary tumors in treated mice. (c) Changes in the volume of distant tumors in treated mice. (d) Changes in the volume of primary tumors and distant tumors in each group. (e) Weights of primary tumors and distant tumors in mice. (f) Survival time of treated mice. (g) Flow cytometry graphs and quantitative analysis of DCs in distant tumors. (h) Quantitative analysis of mature DCs, M1 phenotype macrophages, and M2 phenotype macrophages in distant tumors. (i) Flow cytometry graphs and quantitative analysis of CD8+T cells in distant tumors. (j) Quantitative analysis of CD4+T cells and the ratio of CD4+T cells to CD8+T cells in distant tumors. (k) Quantitative analysis of proliferative CD8+T cells and functional CD8+T cells in distant tumors. (l) Quantitative analysis of lung metastasis and lung weight in each group. (m) H&E staining.

[0028] Fig. 9 The results of the evaluation experiment of the inhibitory effect of the nanophotosensitizer cDC1s and CD8+T cells modified by the genetically engineered bacterial outer membrane vesicles of the present invention on the distant tumor. (a) Schematic diagram of the treatment procedure of the in vivo experiment. (b) Changes in the volume of distant tumors in treated mice. (c) Survival time of treated mice. (d) Weight of distant tumors in treated mice. (e) Photos of collected distant tumors. Scale bar = 1 cm. (f) Changes in the volume of distant tumors in each group. Fig.10 This is a process flow chart for preparing small molecule dyes in nano-photosensitizers modified with outer membrane vesicles of genetically engineered bacteria according to an embodiment of the present invention. DETAILED DESCRIPTION

[0029] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0030] Example 1 As a genetically engineered bacterial outer membrane vesicle modified nano photosensitizer (OBDlp) in an embodiment of the present invention, the nano photosensitizer is a nano material with a particle size of 100 nm, and the genetically engineered bacterial outer membrane vesicle modified nano photosensitizer includes dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), cholesterol, small molecule dyes and CD47nb-OMV membrane protein; The chemical structure of the small molecule dye is shown in Formula I. Formula (I); namely BThDCMI; The molar ratio of dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine and cholesterol is 5:3:1:1; The weight ratio of small molecule dye to CD47nb-OMV membrane protein was 5:1000; The nano photosensitizer is dispersed into a solution by various components and then extruded into a nano material.

[0031] Example 2 As a genetically engineered bacterial outer membrane vesicle-modified nano photosensitizer (OBDlp) in an embodiment of the present invention, the only difference between this embodiment and embodiment 1 is that the chemical structure of the small molecule dye is as shown in Formula II, Formula (II), namely ThDCMI.

[0032] Example 3 A method for preparing a genetically engineered bacterial outer membrane vesicle-modified nano photosensitizer (OBDlp) as an embodiment of the present invention comprises the following steps: (1) Preparing CD47nb-OMV membrane protein. The preparation method of CD47nb-OMV membrane protein comprises the following steps: (I) synthesizing a DNA fragment encoding CD47nb-ClyA fusion protein (synthesized by Shenzhen BGI Co., Ltd.), and cloning the DNA fragment into the pBAD plasmid using a one-step Gibson assembly method to obtain a genetically modified pBAD plasmid; (II) Transforming the genetically modified pBAD plasmid into Escherichia coli MG1655 to obtain genetically engineered Escherichia coli MG1655; (III) The genetically engineered Escherichia coli MG1655 was cultured in LB medium supplemented with 25 μg / mL chloramphenicol at 37°C with shaking at 200 rpm until the OD600 reached 0.5; (VI) adding arabinose to the LB medium at a mass concentration of 0.5%, and continuing to culture for 16 hours to induce the genetically engineered Escherichia coli MG1655 to express CD47nb-OMV membrane protein; (V) Collect the bacterial suspension, remove the bacteria by centrifugation through a 0.45 μm filter, and collect the CD47nb-OMV membrane protein by ultracentrifugation.

[0033] The preparation method of the small molecule dye represented by formula I or formula II is as follows: N, N-bis(4-methylphenyl)-3-(tributyltinyl)benzo[c]thiophene-1-amine or N, N-bis(4-methylphenyl)-3-(tributyltinyl)thiophene-1-amine reacts with 2-(3-chloro-1H-inden-1-ylidene)malononitrile in toluene under reflux under the catalysis of bis(triphenylphosphine)palladium dichloride to obtain a compound represented by formula (II) or formula (I).

[0034] Specific synthetic technology routes such as Fig.10 shown.

[0035] The preparation method of the nano photosensitizer modified by the genetically engineered bacterial outer membrane vesicles comprises the following steps: (1) Dissolving dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine and cholesterol in chloroform to obtain solution A, and mixing a tetrahydrofuran solution of a small molecule dye represented by formula I or formula II with solution A at a concentration of 2 mg / ml; (2) blowing nitrogen gas into the mixed solution of step (1) into the CD47nb-OMV membrane protein, and drying under vacuum to remove the solvent, and re-dissolving the solid product in a 20 mmol / L PBS buffer solution at pH 7.4; (3) The solution obtained in step (2) is extruded in an extruder with a pore size of 100 nm for 10 cycles to obtain a nano photosensitizer modified with outer membrane vesicles of genetically engineered bacteria, which is then subjected to dialysis purification.

[0036] Comparative Example 1 As a nano photosensitizer modified with outer membrane vesicles of genetically engineered bacteria in comparative example 1 of the present invention, the only difference between this comparative example and example 1 is: The chemical structure of the small molecule dye is shown in Formula VI, Formula (VI), namely PhDCMI.

[0037] The preparation method is the same as that of Example 1-3 except that the small molecule dye is replaced.

[0038] Comparative Example 2 As a nano photosensitizer modified by outer membrane vesicles of genetically engineered bacteria as comparative example 1 of the present invention, the only difference between this comparative example and example 1 is that the nano photosensitizer includes dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), cholesterol, and a small molecule dye.

[0039] That is, compared with Example 1, the nano photosensitizer of this comparative example does not include CD47nb-OMV membrane protein, which is recorded as BD1p.

[0040] An experimental method: (I) Evaluate the ability of CD47nb-OMV membrane protein to enhance macrophage phagocytosis.

[0041] RAW264.7 macrophages were seeded into 96-well plates at 5 × 10 cells per well. 4 cells. 4T1 cells were incubated with Dil stain (1,1'-dioctadecyl-3,3,3',3'-tetramethylcyanamide perchlorate) for 20 minutes. Dil-labeled 4T1 cells were pretreated with PBS, OMVs (bacterial outer membrane vesicles), or CD47nb-OMVs for 2 hours. Pretreated 4T1 cells were co-incubated with macrophages at a ratio of 2:1 at 37°C for 4 hours. After washing with PBS, flow cytometry was performed to study the ratio of Dil+ cells to F4 / 80+ cells to evaluate the phagocytosis of tumor cells by macrophages.

[0042] 2. Tumor cell uptake 4T1 cells were incubated overnight in a 24-well plate at 5 × 10 cells per well. 4 cells. Fresh culture medium containing BDlp (nano photosensitizer not modified with bacterial extracellular vesicles) or OBDlp (photosensitizers in Example 1 and Example 2) was replaced and incubated with 4T1 cells for different time periods. Cellular uptake of nanoparticles was observed by fluorescence microscopy and flow cytometry.

[0043] 3. In vitro cytotoxicity assay OBDlp or indocyanine green (ICG) solution (50 μg / ml) was added to a 96-well plate. The solution was observed by infrared thermal imaging under 808 nm laser irradiation (1 W / cm 2 ) temperature changes. 4T1 cells were seeded in 96-well plates with 10,000 cells per well. After 24 h, the medium was replaced with fresh medium containing BThDCMI, BDlp, or OBDlp, respectively. The cells were exposed to 808 nm laser irradiation (1 W / cm 2 , 10min). Then the cell viability was detected by CCK8 assay and live / dead cell staining.

[0044] 4. In vitro macrophage polarization 4T1 cells were seeded in 24-well plates and cultured overnight at 37°C. After the above different treatments, 4T1 cells were fixed with 4% paraformaldehyde. Cells were treated with blocking solution for 30 minutes and then stained with DAPI (4',6-diamidino-2-phenylindole) for 5 minutes. Cells were washed with PBS and incubated with anti-CRT antibody for 2 hours. FITC (fluorescein isothiocyanate)-labeled secondary antibody was added to the cell wells. CRT exposure of 4T1 cells was observed by fluorescence microscopy.

[0045] (V) In vivo NIR II fluorescence imaging and photoacoustic imaging of OBDlp Female Balb / c mice were used to establish a subcutaneous tumor-bearing mouse model. 4T1 cells (1×10 per mouse) were injected into the mouse 6 Once the tumors in the mice grew to about 200 mm 3 OBDlp or BDlp (5 mg / kg BThDCMI) was intravenously injected into tumor-bearing mice (n = 3). In vivo NIR II fluorescence / photoacoustic imaging was performed at different time points. Mice were euthanized 48 h after injection, and organs and tumors were collected for ex vivo imaging. BDlp and OBDlp were intravenously injected into tumor-bearing mice, and NIR II fluorescence imaging (excitation: 808 nm, emission: 1075 nm) was performed at different time points. Imaging signals were quantitatively analyzed by IVIS software and Image J software.

[0046] 6. Activation of cDC1 Tumor-bearing mice were randomly divided into 6 groups (n=4), including control group, laser group, BDlp group, OBDlp group, BDlp+laser group, and OBDlp+laser group. Seven days after receiving different treatments, the mice were euthanized, and tumors and tumor-draining lymph nodes (TdLN) were collected. The tissues were minced and processed by collagenase digestion to obtain single-cell suspensions. The single-cell suspensions were incubated with fluorescent-labeled antibodies at 4°C for 30 minutes and then analyzed by flow cytometry.

[0047] 7. Promoting polarization of M1 phenotype macrophages Tumor-bearing mice were randomly divided into 6 groups (n=4). The experimental groups were control group, laser group, BDlp group, OBDlp group, BDlp+laser group, and OBDlp+laser group. Seven days after treatment, the mice were euthanized, and the tumors and spleens were collected. The tissue suspensions were incubated with macrophage-related antibodies and then analyzed by flow cytometry.

[0048] 8. Improve T cell proliferation and activation The tumor-bearing mice were randomly divided into 6 groups (n=4): control group, laser group, BDlp group, OBDlp group, BDlp+laser group, and OBDlp+laser group. Seven days later, the tumors and TdLNs of the mice were collected. Flow cytometry was used to analyze various subtypes of T cells.

[0049] 9. Cytokine detection On the seventh day after administration of various treatments, tumor-bearing mice were euthanized and their peripheral blood and tumors were collected. The levels of various cytokines in peripheral blood and tumor tissues were then analyzed according to the instructions of the cytokine ELISA kit.

[0050] 10. Inhibiting distant invasion and lung metastasis of breast tumors 4T1 cells (1 × 10 per mouse) 6 1×10 cells per mouse) were injected subcutaneously into the right dorsal flank of Balb / c mice to establish primary tumors. Three days later, 4T1 cells (1×10 6 The mouse model of distant tumor-bearing mice was established by subcutaneously injecting 100 cells into the other dorsal flank of the mouse. 3 The tumor-bearing mice were divided into 6 groups (n=5): (i) control group, (ii) laser group, (iii) BDlp group, (iv) OBDlp group, (v) BDlp+laser group, and (vi) OBDlp+laser group. After intravenous injection of BDlp or OBDlp (5 mg / kg BThMCDI), the tumors of the mice were exposed to laser irradiation (808 nm, 1 W / cm 2 , 10 minutes). The bilateral tumor volume and body weight of the mice were continuously monitored after treatment. The mice were sacrificed on the 15th day after treatment, and the bilateral tumors were harvested to measure the tumor weight. The mouse lungs were collected to count the number of lung metastases.

[0051] XI. Evaluation of the effects of CD8 and MHC-I blockade on distant tumors 4T1 cells (1 × 10 per mouse) 6 1×10 cells per mouse) were injected subcutaneously into the right dorsal flank of Balb / c mice to establish primary tumors. Two days later, 4T1 cells (1×10 6 The distant tumor-bearing mouse model was established by subcutaneously injecting 100 cells into the other dorsal flank of the mouse. 3 Tumor-bearing mice were divided into 5 groups (n=5): (i) control group, (ii) OBDlp+laser, (iii) OBDlp+laser+isotype, (iv) OBDlp+laser+anti-MHC-I and (v) OBDlp+laser+anti-CD8. OBDlp (5 mg / kg BThMCDI) was injected intravenously, and 48 hours later, the primary tumors of mice were exposed to laser irradiation (808 nm, 1 W / cm 2 , 10 minutes). Anti-CD8 antibody, anti-MHC-I antibody and isotype antibody were injected intraperitoneally every 3 days. The distant tumor volume and body weight changes of mice after treatment were continuously monitored. Mice were sacrificed on the 15th day after treatment, and distant tumors were harvested to measure tumor weight. Mouse lungs were collected to count the number of lung metastases.

[0052] 2. Experimental Results 1. Preparation of genetically engineered bacterial outer membrane vesicles modified nanophotosensitizer (OBDlp) The nano photosensitizer (OBDlp) modified by genetically engineered bacterial outer membrane vesicles is a composite small molecule dye. The small molecule dye uses dicyanomethyleneindene (DCMI) as a new molecular skeleton and as a π-conjugated receptor. It is connected to three different triarylamine donors (Example 1, Example 2, and Comparative Example 1) to adjust the absorption ( Figure 2 a). All final compounds synthesized were confirmed by nuclear magnetic resonance (1H, 13C NMR) spectroscopy ( Figure 2 bd). Among them, the maximum absorption rate of the embedded high quinone π bridge is 783nm, which can be used for further research on phototherapy applications ( Figure 2 d).

[0053] To confirm the expression of CD47nb on OMVs, nonpathogenic Escherichia coli MG1655 ( Figure 2 e). The expression of CD47nb-ClyA protein in engineered bacteria and OMVs was verified by SDS-PAGE and Western blotting ( Figure 2 e). To detect the function of CD47nb on OMV, 4T1 breast cancer cells were incubated in OMV and CD47nb-OMV, and the binding rate of FITC-labeled CD47 antibody was detected by flow cytometry. When 4T1 cells were incubated with CD47nb-OMV, the binding of CD47 antibody gradually decreased, indicating that CD47nb on the surface of OMV can specifically bind to CD47 in 4T1 cells ( Figure 2 f).

[0054] The ability of CD47nb-OMV to enhance macrophage phagocytosis was then evaluated. DIL-labeled 4T1 cells were pretreated with PBS, OMV, or CD47nb-OMV and co-incubated with macrophages, and the ratio of Dil+ cells to F4 / 80+ cells was measured to evaluate the phagocytosis of macrophages on tumor cells. Compared with the PBS and OMV groups, CD47nb-OMV treatment resulted in a significant enhancement of macrophage phagocytosis ( Figure 2 g). OMVs alone also enhanced phagocytosis, suggesting that CD47nb-OMVs activated macrophages by combining CD47 blockade and bacterial stimulatory adjuvants ( Figure 2 g). In addition, CD47nb-OMVs had a significant stimulatory effect on macrophages, resulting in increased expression levels of CD80 and decreased expression levels of CD206 ( Figure 2 hi).

[0055] Transmission electron microscopy (TEM) showed that both BDlp and OBDlp exhibited spherical structures ( Figure 2j). Dynamic light scattering (DLS) analysis showed that the particle sizes of OMV, BDlp, and OBDlp were ~12 nm, ~91 nm, and ~122 nm, respectively ( Figure 2 k). The zeta potentials of OMV, BDlp, and OBDlp V were -8.58±0.65 mV, -3.48±0.65 mV, and -6.28±0.76 mV, respectively. The zeta potential of OBDlp was more negative than that of BDlp, indicating that CD47nb-OMV was successfully modified.

[0056] The results of UV absorption spectra showed that the characteristic peaks of BDlp and OBDlp were similar to those of BThDCMI, with the peak located at ~783 nm ( Figure 2 l). The fluorescence spectrum showed that OBDlp has a distinct characteristic peak between ~930 nm in the near-infrared II window, indicating that OBDlp has the potential to be used as a contrast agent to enhance optical imaging in the NIR II window ( Figure 2 m). We further studied the in vitro NIR II fluorescence imaging signals of BDlp or OBDlp solutions with different concentrations. The NIR II fluorescence imaging intensity of BDlp or OBDlp solutions increased with the increase of concentration from 1-80 μg / mL ( Figure 2 n).

[0057] (II) In vitro photothermal therapy experiment The tumor cell uptake of BDlp or OBDlp was evaluated, and it was found that the modification of OMVs improved the cellular uptake of the liposomes. The intensity of the fluorescence signal in 4T1 cells increased significantly with the incubation time of OBDlp. Flow cytometry results showed that the cell uptake of OBDlp was significantly better than that of BDlp 4 h after treatment. The photothermal stability of OBDlp was studied by recording the temperature changes of repeated heating cycles.

[0058] like Figure 3 As shown in a, after 4 irradiation cycles, the temperature drop of OBDlp was minimal, while the temperature decay of ICG was obvious under the same laser irradiation. The cytotoxicity of OBDlp on 4T1 cells in vitro was evaluated. Live / dead staining showed that OBDlp+laser was able to effectively kill most tumor cells. It is worth noting that treatment with OBDlp alone or BDlp alone had no significant cytotoxic effect (Figure 3b). The in vitro antitumor activity of OBDlp+laser was evaluated by cell counting kit-8 (CCK-8), and the results showed that the viability of 4T1 cells treated with OBDlp+laser was 0.35 times higher than that treated with OBDlp ( Figure 3 c).

[0059] PTT induces immunogenic cell death (ICD) of tumor cells and promotes tumor antigen release. It was investigated whether OBDlp+laser could trigger ICD and further promote DC maturation. Calreticulin (CRT), high-mobility group protein 1 (HMGB1), and ATP were used as damage-associated molecular pattern (DAMP) indicators that trigger DC phagocytosis to evaluate the ICD of 4T1 cells. As shown in the figure, the OBDlp+laser group showed more obvious CRT exposure in 4T1 cells compared with the other groups ( Figure 3 c). Low intracellular HGMB1 concentration and intracellular ATP level indicate that OBDlp+laser treatment promoted HGMB1 and ATP release ( Figure 3 ef). These results indicate that OBDlp-mediated PTT effectively triggered ICD and enhanced the immunogenicity of 4T1 cells.

[0060] Dendritic cells (DCs) will be stimulated to mature after absorbing DAMPs released by tumor cells. Subsequently, the potential of OBDlp+laser-induced ICD to promote the maturation of DCs was investigated. After co-incubation with culture medium of OBDlp+laser-treated tumor cells, the expression levels of CD80 and CD86 in DCs were analyzed by flow cytometry ( Figure 3 g, j). Compared with the other treatment groups, the DC maturation level in the OBDlp+laser group was significantly increased ( Figure 3 g, j). Under DAMPs stimulation, the TNF level of DCs increased significantly, while the IL-10 level decreased ( Figure 3 h, i).

[0061] 3. In vivo NIR II fluorescence / photoacoustic tumor imaging experiments The addition of OMVs is expected to enhance the hypoxia targeting ability of nanoparticles. BDlp or OBDlp were intravenously injected into mice, and in vivo fluorescence imaging was performed at the indicated time points. Compared with BDlp, OBDlp showed greater accumulation in the tumor area and reached a peak at 48 hours after injection, which may depend on the specific molecules of OMVs ( Figure 4 a, e). Tumors and organs were collected for in vitro imaging 48 hours after injection. Compared with the BDlp group, tumors in the OBDlp group showed stronger signal intensity, indicating that the modification of OMVs significantly enhanced the delivery of photosensitizers to tumors ( Figure 4 b, f).

[0062] BDlp and OBDlp were intravenously injected into tumor-bearing mice, and NIR II fluorescence imaging (excitation: 808 nm, emission: 1075 nm) was performed at different time points. The results showed that OBDlp was enriched in tumor tissue within 48 h, and the fluorescence signal was significantly stronger than that of the BDlp group ( Figure 4 c, g).

[0063] Compared with NIR I window imaging, NIR II window imaging has a significantly improved signal-to-noise ratio, which can accurately monitor the distribution of photosensitizers in tumor tissues. In vitro imaging results showed that OBDlp accumulated more in tumor tissues than BDlp ( Figure 4 d). In particular, the accumulation of OBDlp in the liver was significantly reduced compared with BDlp, indicating that it can avoid the side effects caused by the large accumulation of photosensitizers in the organ ( Figure 4 h).

[0064] OBDlp's excellent photothermal conversion performance makes it promising for enhancing NIR II photoacoustic imaging. As shown by in vivo tumor photoacoustic imaging, OBDlp exhibited superior microvascular morphology and density in tumor tissues compared with BDlp after injection ( Figure 4 i). At 48 h after injection, the tumor signal intensity in the OBDlp group was higher than that in the BDlp group ( Figure 4 j). The study showed that OBDlp has the potential to become a NIR II window fluorescence imaging / photoacoustic imaging contrast agent for tumor targeted imaging.

[0065] (IV) OBDlp-mediated PTT-induced potent immune response experiment To evaluate whether OBDlp-mediated PTT establishes an immunostimulatory tumor microenvironment, 4T1 tumor-bearing mice were used to test the effect of OBDlp+laser on macrophage repolarization. Figure 5 As shown in a, after OBDlp+laser treatment, the proportion of M1 phenotype macrophages in the tumor spleen increased, and the M1 / M2 ratio increased significantly. The M1 phenotype macrophages in the tumors of the OBDlp+laser group and the OBDlp group were significantly higher than those in the control group ( Figure 5 b). It is worth noting that OBDlp+laser treatment not only induces intratumoral macrophages, but also induces splenic macrophages to polarize to the M1 phenotype, which will effectively reverse the immunosuppressive microenvironment within the tumor and enhance systemic immunity ( Figure 5 c).

[0066] In addition, we evaluated the effects of different treatment strategies on the maturation of DCs in tumors and tumor-draining lymph nodes (TdLNs). Flow cytometric analysis showed that the proportions of cDC1s (CD103+CD11b-CD11c+) and total DCs (CD11c+MHCII+CD45+) in tumor tissues and TdLNs in the OBDlp+laser group were significantly increased ( Figure 5 d). In addition, the ratio of cDC1s / cDC2s was higher in the OBDlp+laser group compared with the other treatment groups ( Figure 5e, g). In particular, the level of CD86 in the cDC1s population was significantly increased, indicating that OBDlp+laser treatment significantly induced the maturation of cDC1s in tumors and TdLNs ( Figure 5 f, h). Disruption of the CD47-SIRPα signaling axis is considered a promising immunotherapeutic strategy. In vitro and in vivo results showed that OBDlp treatment effectively activated DCs and macrophages. This may be mainly attributed to the stimulation of macrophage phagocytosis by OBDlp+laser-mediated PPT-induced ICD and CD47-SIRPα axis blockade.

[0067] The maturation of cDC1 and polarization of macrophages to the M1 phenotype can further mediate downstream adaptive immune responses by regulating the proliferation and activation of T cells. Therefore, the number and type of CD4+ T cells and CD8+ T cells in TdLN and tumors were further evaluated. Figure 6 As shown in a, OBDlp+laser treatment resulted in a significant increase in the proportion of CD8+T cells in tumor-draining lymph nodes. However, the number of CD4+T cells in TdLNs did not show a statistically significant change ( Figure 6 b, e). In the T cell population, the number of proliferative T cells increased significantly, and the number of exhausted T cells decreased significantly ( Figure 6 c, f). In addition, OBDlp+laser treatment showed the most robust immune response, with significant activation of CD4+T cells and CD8+T cells, with CD4+T cells showing a two-fold and three-fold increase, respectively, compared with the BDlp group ( Figure 6 d, g). Compared with the other treatment groups, the number of regulatory T cells in the tumor-draining lymph nodes in the OBDlp+laser group appeared to show a significant decrease ( Figure 6 i, j).

[0068] OBDlp+laser treatment has been shown to stimulate the proliferation and activation of CD4+T cells and CD8+T cells in tumor-draining lymph nodes. However, the most critical aspect of this treatment is its ability to induce the infiltration of CD4+T cells and CD8+T cells in the reshaped tumor microenvironment. This is crucial for anti-tumor immune response. Flow cytometric analysis of tumor tissue samples showed that the number of CD4+T cells and CD8+T cells in the tumor tissue of the OBDlp+laser group was significantly higher than that of the other groups ( Figure 6 f, h). In particular, the proliferative CD8+T cells and functional CD8+T cells in the OBDlp+laser group were 1.57 times and 2.11 times those in the BDlp group, respectively ( Figure 5f, h). In addition, the number of exhausted T cells and Treg cells in tumor tissues decreased significantly after OBDlp+laser treatment, indicating that T cell suppression in the tumor microenvironment was enhanced, which may be beneficial for PD-1 / PD-L1 immune checkpoint inhibitor treatment ( Figure 5 f, h). Compared with the control group, immunofluorescence staining of tumor tissues in the OBDlp+laser group showed a significant increase in CD8+T cell and CD4+T cell infiltration ( Figure 6 h).

[0069] There was no significant difference in the levels of inflammatory cytokines (including IFN-γ, TNF-α, and IL-6) in the tumors of the control group, laser group, and BDlp group. In contrast, the secretion of IFN-γ, TNF-α, and IL-6 in the tumors of the OBDlp+laser group was significantly increased ( Figure 6 km). In addition, the levels of anti-inflammatory cytokines (such as TGF-β) were significantly reduced in the OBDlp+laser group ( Figure 6 n). Notably, similar phenomena were observed in the peripheral blood of mice, further indicating that adaptive immunity against tumors was activated.

[0070] In summary, compared with the other treatment groups, the group receiving OBDlp+laser treatment triggered a strong antitumor immune response in DCs, macrophages, and further activated T cell cytotoxicity, which is crucial for achieving superior antitumor therapeutic effects. In summary, OBDlp+laser treatment can achieve cascade-enhanced antitumor immunotherapy through the following three steps: (i) CD47 nanoantibodies carried by OBDlp can induce the polarization of M1 phenotype TAMs and reshape the immunosuppression of the tumor microenvironment. (ii) OBDlp-mediated PTT can enhance the immunogenicity of tumor cells by triggering ICD; (iii) DAMPs released by ICD and M1 phenotype TAMs synergistically activate cDC1; (iv) Activated cDC1 and improved tumor microenvironment reactivate the function of tumor-infiltrating CD8+T cells.

[0071] 5. In vivo anti-tumor therapy experiments OBDlp+Laser not only directly kills tumor cells through PTT, but also promotes DCs maturation and M1 phenotype macrophage activation by enhancing tumor cell immunogenicity and the release of CD47 nanoantibodies. 48 h after intravenous injection of nanophotosensitizer, tumor-bearing mice were irradiated with laser (808 nm, 1 W / cm 2 , 10 min), and the tumor growth of mice was continuously monitored ( Figure 7 a). Compared with the control group, the tumor growth in the BDlp+laser group was inhibited, and the therapeutic effect could be further significantly improved after CD47nb-OMW modification ( Figure 7 b).

[0072] The inhibitory effect of BDlp+laser group on tumor growth was xx%, while the inhibitory effect of OBDlp+laser group on tumor growth increased to xx% ( Figure 7 e). On the 15th day after treatment, the tumor weight of the OBDlp+laser group was xx g, which was xx times higher than that of the control group and the BDlp+laser group ( Figure 7 cd). As shown in the figure, OBDlp+laser treatment also significantly prolonged the survival time of mice ( Figure 7 f). In addition, the body weight of each group of mice did not show any significant difference during the treatment ( Figure 7 g). TUNEL immunofluorescence experiments further confirmed the killing effects of BDlp+laser and OBDlp+laser on 4T1 tumor cells ( Figure 7 h).

[0073] (VI) Experiment on inhibiting distant invasion and lung metastasis of breast tumors We then used a bilateral subcutaneous tumor-bearing mouse model to evaluate the therapeutic effect of OBDlp+laser therapy on primary and distant tumors ( Figure 8 a). Laser, BDlp, OBDlp, and BDlp+laser treatments had limited effects against distant tumors. In contrast, OBDlp+laser treatment not only inhibited primary tumors but also significantly suppressed the growth of distant tumors ( Figure 8 bd). On the 15th day after treatment, the weight of primary and distant tumors in the OBDlp+laser group were significantly smaller than those in the other treatment groups ( Figure 8 e). Due to the inhibition of distant tumor invasion, the median survival time of mice in the OBDlp+laser group was significantly prolonged compared with that in the BDlp+laser group ( Figure 8 f). Notably, we found that OBDlp+laser treatment significantly increased the maturation of DCs and M1 phenotype macrophage polarization in distant tumors, as manifested by an increase in the proportion of CD80+CD86+ DCs, an increase in the number of F4 / 80+CD80+ macrophages, and a decrease in the number of F4 / 80+CD206+ macrophages ( Figure 8 At the same time, due to the activation of antigen-presenting cells, the level of CD8+T cells in distant tumors increased significantly, and the percentages of proliferative CD8+T cells and functional CD8+T cells increased significantly ( Figure 8 ik). The results showed that OBDlp+laser had an excellent inhibitory effect on distant tumors, which was mainly due to the immune response secondary to the promotion of DCs maturation and tumor-associated macrophage repolarization.

[0074] 7. Evaluation of the inhibitory effect of cells on distant tumors In addition, we performed rescue experiments to evaluate the role of cDC1 and CD8+ T cells in immunotherapy against distant tumors ( Fig. 9 a). Injection of anti-MHC-I antibodies blocked cDC1 migration to TdLNs, and anti-CD8 antibodies achieved in vivo depletion of CD8+ T cells. In addition, OBDlp+laser treatment improved the immunosuppressive tumor microenvironment, which may be beneficial for anti-PD-L1 antibody treatment. We also tested the combination of anti-PD-L1 antibody and OBDlp+laser to further improve the treatment efficiency. The results showed that CD8+ T cell depletion or MHC-I blockade after OBDlp+laser treatment significantly promoted distant tumor growth ( Fig. 9 b). Most importantly, OBDlp+laser combined with anti-PD-L1 antibody treatment further inhibited the growth of distant tumors, and the tumor inhibition rate even exceeded that of the OBDlp+laser group ( Fig. 9 df). In addition, the survival time of mice treated with OBDlp+laser combined with anti-PD-L1 antibody was above 60% at 50 days after treatment ( Fig. 9 c). These results suggest that OBDlp+laser therapy is highly dependent on the adaptive antitumor immune response mediated by cDC1s and CD8+ T cells and can also be combined with anti-PD-L1 antibody therapy to achieve enhanced distant tumor suppression.

[0075] From the above experiments, it can be seen that the nanophotosensitizer OBDlp modified by genetically engineered bacterial outer membrane vesicles, as a new type of nanophotosensitizer, mediates tumor-targeted PTT through NIR II FL / PA imaging, improves tumor immunogenicity and activates antigen-presenting cells, leading to the infiltration and functionalization of CD8+ T cells. OBDlp combined with laser induces the release of a large number of DAMPs and triggers ICD to promote the maturation of cDC1s. At the same time, the CD47 nanoantibody carried by the nanophotosensitizer induces M1 phenotype macrophage polarization by blocking the CD47-SIRPα signal transduction axis, reversing the immunosuppressive tumor microenvironment. In addition, activated antigen-presenting cells will induce the proliferation and functionalization of CD8+T cells and CD4+T cells, laying a solid foundation for effective anti-tumor immunotherapy. Overall, under the guidance of NIR II FL / PA imaging, OBDlp-mediated photoimmunotherapy simultaneously increases the activation of DCs and macrophages, thereby effectively inhibiting tumor growth and metastasis.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A nano photosensitizer modified with outer membrane vesicles of genetically engineered bacteria, characterized in that: The nano photosensitizer is a nano material with a particle size of 80-120 nm. The nano photosensitizer modified by the genetically engineered bacterial outer membrane vesicles includes dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), cholesterol, small molecule dyes and CD47nb-OMV membrane protein; The chemical structure of the small molecule dye is shown in Formula I or Formula II. Formula (I); Formula (II); The molar ratio of dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine and cholesterol is (4.5-5.5): (2.5-3.5): (0.8-1.2): 1; The weight ratio of small molecule dye to CD47nb-OMV membrane protein is (1.5~10):1000; The nano photosensitizer is dispersed into a solution by various components and then extruded into a nano material.

2. The nano photosensitizer modified with outer membrane vesicles of genetically engineered bacteria according to claim 1, characterized in that: The weight ratio of cholesterol to CD47nb-OMV membrane protein is (5~25):1000.

3. The nano photosensitizer modified with outer membrane vesicles of genetically engineered bacteria according to claim 1, characterized in that: The preparation method of CD47nb-OMV membrane protein comprises the following steps: culturing genetically engineered Escherichia coli MG1655, adding arabinose to induce the production of CD47nb-OMV membrane protein; transferring a genetically modified pBAD plasmid into the genetically engineered Escherichia coli MG1655; the preparation method of the genetically modified pBAD plasmid comprises: cloning a DNA fragment encoding CD47nb-ClyA fusion protein into a pBAD plasmid using a one-step Gibson assembly method to obtain a genetically modified pBAD plasmid.

4. The nano photosensitizer modified with outer membrane vesicles of genetically engineered bacteria according to claim 1, characterized in that: The preparation method of small molecule organic dye comprises the following steps: N, N-bis(4-methylphenyl)-3-(tributyltinyl)benzo[c]thiophene-1-amine or N, N-bis(4-methylphenyl)-3-(tributyltinyl)thiophene-1-amine reacts with 2-(3-chloro-1H-inden-1-ylidene)malononitrile in toluene under reflux under the catalysis of bis(triphenylphosphine)palladium dichloride to obtain a compound represented by formula (II) or formula (I).

5. The nano photosensitizer modified with outer membrane vesicles of genetically engineered bacteria according to claim 1, characterized in that: The preparation method of the nano photosensitizer modified by the genetically engineered bacterial outer membrane vesicles comprises the following steps: (1) dissolving dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine and cholesterol in chloroform to obtain solution A, and mixing a tetrahydrofuran solution of a small molecule dye represented by formula I or formula II with solution A; (2) blowing the mixed solution of step (1) into the CD47nb-OMV membrane protein with an inert gas, drying to remove the solvent, and re-dissolving the solid product in a PBS buffer solution; (3) The solution obtained in step (2) is extruded in an extruder with a pore size of 80 to 120 nm for 8 to 15 cycles to obtain a nano photosensitizer modified with genetically engineered bacterial outer membrane vesicles.

6. The nano photosensitizer modified with outer membrane vesicles of genetically engineered bacteria according to claim 5, characterized in that: The concentration of the tetrahydrofuran solution of the small molecule dye represented by formula I or formula II is 1.5-5 mg / mL.

7. The method for preparing the nano photosensitizer modified by outer membrane vesicles of genetically engineered bacteria according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: (1) dissolving dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine and cholesterol in chloroform to obtain solution A, and mixing a tetrahydrofuran solution of a small molecule dye represented by formula I or formula II with solution A; (2) blowing the mixed solution of step (1) into the CD47nb-OMV membrane protein with an inert gas, drying to remove the solvent, and re-dissolving the solid product in a PBS buffer solution; (3) The prepared solution obtained in step (2) is extruded in an extruder with a pore size of 80 to 120 nm for 8 to 15 cycles to obtain a nano photosensitizer modified with outer membrane vesicles of genetically engineered bacteria.

8. The method for preparing the nano photosensitizer modified with outer membrane vesicles of genetically engineered bacteria according to claim 7, characterized in that: The preparation method of the CD47nb-OMV membrane protein comprises the following steps: (I) synthesizing a DNA fragment encoding CD47nb-ClyA fusion protein, and cloning the DNA fragment into a pBAD plasmid using a one-step Gibson assembly method to obtain a genetically modified pBAD plasmid; (II) Transforming the genetically modified pBAD plasmid into Escherichia coli MG1655 to obtain genetically engineered Escherichia coli MG1655; (III) culturing the genetically engineered Escherichia coli MG1655 in LB medium supplemented with 20-30 μg / mL chloramphenicol at 36.5-37.5°C until the OD600 is greater than 0.45; (VI) Adding arabinose to induce genetically engineered Escherichia coli MG1655 to express CD47nb-OMV membrane protein; (V) After removing bacteria, CD47nb-OMV membrane proteins were collected by centrifugation.

9. The method for preparing a nano photosensitizer modified with outer membrane vesicles of genetically engineered bacteria according to claim 8, wherein in step (VI), arabinose is added to a mass fraction of 0.45% to 0.55% to induce the genetically engineered Escherichia coli MG1655 to express CD47nb-OMV membrane protein; and culture is performed for 12 to 20 hours.

10. Use of the nano-photosensitizer modified with the outer membrane vesicles of genetically engineered bacteria as claimed in any one of claims 1 to 6 in promoting the anti-tumor immune cascade reaction mediated by cDC1s.

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