A nano-photosensitizer modified by outer membrane vesicles of genetically engineered bacteria, and its preparation method and application

Abstract

The present invention provides a nano-photosensitizer modified by gene engineering bacterial outer membrane vesicles, a preparation method thereof and an application thereof. The nano-photosensitizer is a nano-material with a particle size of 80-120 nm. The nano-photosensitizer modified by gene engineering bacterial outer membrane vesicles comprises dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), cholesterol, a small molecule dye and CD47nb-OMV membrane protein; the chemical structure of the small molecule dye is shown in Formula I or Formula II. The nano-photosensitizer OBDlp modified by gene engineering bacterial outer membrane vesicles, as a novel nano-photosensitizer, mediates tumor-targeted PTT through NIR II FL / PA imaging, improves tumor immunogenicity and activates antigen-presenting cells, resulting in the infiltration and functionalization of CD8+ T cells. OBDlp combined with laser induces the release of a large amount of DAMPs and triggers ICD to promote the maturation of cDC1s. Meanwhile, the CD47 nanobody carried by the nano-photosensitizer induces the polarization of M1 phenotype macrophages by blocking the CD47-SIRPα signal transduction axis, reversing the immunosuppressive tumor microenvironment.

Description

Technical Field

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

[0002] Triple-negative breast cancer (TNBC) is a fatal female cancer with a very high metastasis and recurrence rate. The median overall survival of metastatic triple-negative breast cancer is about one year. Currently, the chemotherapy regimens for TNBC are limited and have systemic toxic effects. Photothermal therapy (PTT) is an emerging ablation therapy. PTT destroys tumors without damaging surrounding normal tissues and shows advantages in treating localized solid tumors. It induces apoptosis by using heat and causes strong immunogenic cell death (ICD) and antigen release. However, breast cancer treated with localized PTT may still develop distant metastases. This recurrence is due to the typical immunosuppressive tumor microenvironment (TME) of this disease, including insufficient infiltration of immune cells, impaired immune cell function, and accumulation of immunosuppressive molecules. Cooling of the TME promotes immune evasion and tumor metastasis. Therefore, immune reactivation is crucial for improving the efficacy of anti-tumor therapy after PTT.

[0003] The antigen presentation process is crucial for the anti-tumor immune response. During this process, tumor-resident conventional type I dendritic cells (cDC1) cross-present antigens to CD8 + T cell antigen cross-presentation by cDC1 is necessary for a strong anti-tumor response. However, CD8 + T cell deficiency caused by cDC1 dysfunction is an immunosuppressive feature of various solid tumors including breast cancer. Therefore, activating the antigen presentation cascade of cDC1s is an ideal method to enhance the anti-tumor immunity of CD8 + T cells in breast cancer. Endogenous CD8 + T cell DC activation is a complex multi-step immune cascade reaction, involving antigen uptake, activation, and migration to tumor-draining lymph nodes (TdLN) within the tumor. Each of these steps is crucial for triggering a sufficient anti-tumor 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 (TAM). Therefore, the key to successfully reversing cDC1s dysfunction is to eliminate the restraint of immunosuppressive molecules in the TME and, in addition, to absorb a sufficient amount 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 is difficult to strongly activate tumor-resident cDC1.

[0005] Therefore, it is necessary to develop nano-photosensitizers for immunotherapy. SUMMARY OF THE INVENTION

[0006] An object of the present invention is to overcome the deficiencies of the prior art and provide a nano-photosensitizer modified by genetic engineering bacterial outer membrane vesicles, a preparation method thereof, and an application thereof.

[0007] To achieve the above object, the technical solution adopted by the present invention is: a nano-photosensitizer (OBDlp) modified by genetic engineering bacterial outer membrane vesicles, the nano-photosensitizer is a nano-material with a particle size of 80 - 120 nm, and the nano-photosensitizer modified by genetic engineering bacterial outer membrane vesicles includes dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), cholesterol, a small molecule dye, and CD47nb-OMV membrane protein;

[0008] The chemical structure of the small molecule dye is shown in Formula I or Formula II,

[0009]

[0010]

[0011] The molar ratio of dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, and cholesterol is (4.5 - 5.5) : (2.5 - 3.5) : (0.8 - 1.2) : 1;

[0012] The weight ratio of the small molecule dye to the CD47nb-OMV membrane protein is (1.5 - 10) : 1000;

[0013] The nano-photosensitizer is extruded into a nano-material after each component is dispersed into a solution.

[0014] Bacterial outer membrane vesicles (OMVs) already possess excellent anti-tumor potential. OMVs have functions similar to those of bacteria and can be targeted and delivered to tumor tissues. Genetically engineered bacteria can stably prepare optimized OMVs with specific functional molecules, providing a new platform for tumor immunotherapy. As a novel biomaterial, OMVs have great prospects 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 the CD47nb-OMV membrane protein, which is an immune amplifier for encapsulating a novel NIR-II small molecule organic dye (BThDCMI) (chemical structure shown in Formula I), and can stimulate antigen cross-presentation mediated by cDC1s. The small molecule dyes shown in Formula I or Formula II consist of a new molecular backbone composed of dicyanomethylene indene (DCMI) as a π-conjugated acceptor, which is connected to a triarylamine donor for absorption regulation. Among them, the absorption rate of the embedded high-quinone π-bridge is maximally 783 nm (CH 2 Cl 2 ), with absorption far exceeding 700 nm. 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 induces ICD and M1 phenotype macrophage polarization, and then effectively activates a robust cDC1s / CD8 + T cell anti-tumor immune cascade reaction, inhibiting the development of breast tumors (as shown in Figure 1 ). The above-mentioned genetically engineered bacterial outer membrane vesicle-modified nanophotosensitizer (OBDlp) for the combination of immune cascade reaction and PTT provides a new material and method for the treatment of TNBC, which is of great significance for promoting cancer immunotherapy and improving prognosis.

[0015] Preferably, the weight ratio of cholesterol to CD47nb-OMV membrane protein is (5 - 25):1000.

[0016] Preferably, the preparation method of CD47nb-OMV membrane protein includes 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 includes: cloning the DNA fragment encoding the CD47nb-ClyA fusion protein into the pBAD plasmid by one-step Gibson assembly method to obtain the genetically modified pBAD plasmid.

[0017] Preferably, the preparation method of the small molecule organic dye comprises the following steps:

[0018] N,N-bis(4-methylphenyl)-3-(tributylstannyl)benzo[c]thiophen-1-amine or N,N-bis(4-methylphenyl)-3-(tributylstannyl)thiophen-1-amine reacts with 2-(3-chloro-1H-inden-1-ylidene)malononitrile under the catalysis of bis(triphenylphosphine)palladium dichloride and refluxes in toluene to obtain the compound shown in formula (II) or formula (I).

[0019] Preferably, the preparation method of the nano photosensitizer modified by the outer membrane vesicles of the genetically engineered bacteria comprises the following steps:

[0020] (1) Dissolve dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine and cholesterol in chloroform to obtain solution A, and mix the tetrahydrofuran solution of the small molecule dye shown in formula I or formula II with solution A;

[0021] (2) Blow the mixed solution in step (1) into the CD47nb-OMV membrane protein with inert gas, dry to remove the solvent, and redissolve the solid product in PBS buffer solution;

[0022] (3) Extrude the solution obtained in step (2) 8 - 15 cycles in an extruder with a pore size of 80 - 120 nm to obtain the nano photosensitizer modified by the outer membrane vesicles of the genetically engineered bacteria.

[0023] Preferably, the concentration of the tetrahydrofuran solution of the small molecule dye shown in formula I or formula II is 1.5 - 5 mg / mL.

[0024] The present invention also provides the preparation method of the nano photosensitizer modified by the outer membrane vesicles of the genetically engineered bacteria as described in any one of the above, and the method comprises the following steps:

[0025] (1) Dissolve dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine and cholesterol in chloroform to obtain solution A, and mix the tetrahydrofuran solution of the small molecule dye shown in formula I or formula II with solution A;

[0026] (2) Blow the mixed solution in step (1) into the CD47nb-OMV membrane protein with inert gas, dry to remove the solvent, and redissolve the solid product in PBS buffer solution;

[0027] (3) Extrude the obtained compliant solution in step (2) 8 - 15 cycles in an extruder with a pore size of 80 - 120 nm to obtain the nano photosensitizer modified by the outer membrane vesicles of the genetically engineered bacteria.

[0028] Preferably, the preparation method of the CD47nb-OMV membrane protein comprises the following steps:

[0029] (I) Synthesize a DNA fragment encoding the CD47nb-ClyA fusion protein, and clone the DNA fragment into the pBAD plasmid by one-step Gibson assembly method to obtain a genetically modified pBAD plasmid;

[0030] (II) Transfer the genetically modified pBAD plasmid into Escherichia coli MG1655 to obtain genetically engineered Escherichia coli MG1655;

[0031] (III) Culture 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;

[0032] (VI) Add arabinose to induce the genetically engineered Escherichia coli MG1655 to express the CD47nb-OMV membrane protein;

[0033] (V) Remove the bacteria and centrifuge to collect the CD47nb-OMV membrane protein.

[0034] Preferably, in step (VI), add arabinose to a mass fraction of 0.45% - 0.55% to induce the genetically engineered Escherichia coli MG1655 to express the CD47nb-OMV membrane protein; culture for 12 - 20 hours.

[0035] The present invention also provides the application of the above-mentioned genetically engineered bacterial outer membrane vesicle-modified nanophotosensitizer in promoting the anti-tumor immune cascade reaction mediated by cDC1s.

[0036] Such as Figure 1 For the preparation of OBDlp and the PPT mediated by OBDlp on cDC1s / CD8 + Schematic diagram of the anti-tumor immune cascade reaction of T cells. After OBDlp is combined with laser and delivered to the tumor tissue, it can induce ICD of tumor cells and release DAMPs. The release of CD47nb reprograms M2-phenotype macrophages into M1-phenotype macrophages, which will relieve the hindrance of the tumor microenvironment to the maturation of cDC1s. Therefore, cDC1s are successfully activated in the reshaped "hot" tumor microenvironment, triggering the downstream anti-tumor immune cascade reaction of CD8 + T cells to combat distant invasion and metastasis.

[0037] The beneficial effects of the present invention are as follows: The present invention provides a nano-photosensitizer modified with genetically engineered bacterial outer membrane vesicles, its preparation method and application. The nano-photosensitizer modified with genetically engineered bacterial outer membrane vesicles of the present invention first developed an engineered bacterium OMV (CD47nb-OMV), that is, the CD47nb-OMV membrane protein, which is an immune amplifier for encapsulating a novel NIR-II small molecule organic dye (BThDCMI) (chemical structure as shown in Formula I), and can stimulate antigen cross-presentation mediated by cDC1s. The small molecule dyes shown in Chemical Structures of Formula I or Formula II are composed of a new molecular backbone of dicyanomethylene indene (DCMI) as a π-conjugated acceptor, which is connected to a triarylamine donor to regulate absorption. Among them, the absorption rate of the embedded high-quinone π-bridge is maximally 783 nm (CH 2 Cl 2 ), with an absorption far exceeding 700 nm. 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 induces ICD and M1 phenotype macrophage polarization, and then effectively activates a robust cDC1s / CD8 + T cell anti-tumor immune cascade reaction, inhibiting the development of breast tumors (as Figure 1 shown). The above-mentioned nano-photosensitizer (OBDlp) modified with genetically engineered bacterial outer membrane vesicles for the combination of immune cascade reaction and PTT provides a new material and method for the treatment of TNBC, and is of great significance for promoting cancer immunotherapy and improving prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a schematic diagram of the preparation of the nano-photosensitizer OBDlp modified with genetically engineered bacterial outer membrane vesicles in the embodiment of the present invention and the PPT mediated by OBDlp on the cDC1s / CD8 + T cell anti-tumor immune cascade reaction. Among them, a is a schematic diagram of the preparation of the nano-photosensitizer OBDlp modified with genetically engineered bacterial outer membrane vesicles, and b is a schematic diagram of the PPT mediated by the nano-photosensitizer OBDlp modified with genetically engineered bacterial outer membrane vesicles on the cDC1s / CD8 + T cell anti-tumor immune cascade reaction.

[0039] Figure 2Schematic diagram of the preparation and characterization results of the gene engineering bacteria outer membrane vesicle-modified nano-photosensitizer OBDlp in the embodiments of the present invention. (a) Schematic diagram of the preparation of PhDCMI, ThDCMI, and BThDCMI. (b) Absorption result graph of PhDCMI. (c) Absorption result graph of ThDCMI. (d) Absorption result graph of BThDCMI. (e) Plasmid schematic diagram of engineered Escherichia coli MG1655 and OMV's CD47nb-OMV and SDS-PAGE. (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 after different treatments. (i) Macrophage CD206 expression after 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.

[0040] Figure 3 Experimental result graph of in vitro photothermal therapy of the gene engineering bacteria outer membrane vesicle-modified nano-photosensitizer OBDlp and DCs maturation in the embodiments of the present invention. (a) Temperature change of BDLp or OBDlp solution irradiated with a laser (808 nm, 1 W / cm 2 ) for different times. (b) Live / dead cell staining of 4T1 cells treated differently. 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 for CRT of 4T1 cells treated differently. Scale bar = 25 μm. (e) HMGB1 level of 4T1 cells treated differently. (f) ATP level of 4T1 cells treated differently. (g) DC maturation stimulated with medium from tumor cells, treated differently. (h) TNF-α level of DCs stimulated with medium from tumor cells, treated differently. (i) IL-10 level of DCs stimulated with medium from tumor cells, treated differently. (j) CD80 and CD86 expression levels of DCs stimulated with tumor cell-derived medium, treated differently.

[0041] Figure 4Experimental results of in vivo NIRII fluorescence / photoacoustic tumor imaging of the nano-photosensitizer OBDlp modified with genetically engineered bacterial outer membrane vesicles in the embodiments of the present invention. (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 the FL signal intensity of tumor-bearing mice injected systemically at different time points. (f) Quantitative analysis of the FL signal intensity of ex vivo organs and tumors. (g) Quantitative analysis of the NIR II FL signal intensity of tumor-bearing mice at different time points. (h) Quantitative analysis of the 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 the photoacoustic imaging signal intensity of tumor-bearing mice at different time points.

[0042] Figure 5 Experimental results of PTT-activated M1 phenotype macrophage polarization and cDC1s maturation mediated by the nano-photosensitizer OBDlp modified with genetically engineered bacterial outer membrane vesicles in the embodiments of the present invention. (a) Flow cytometry plots of M1 phenotype macrophages in tumors and spleens. (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 spleens. (d) Flow cytometry plots of cDC1s, cDC2s, and TdLNs in tumors. (e) Quantitative analysis of total DCs, cDC1s, and the cDC1s / cDC2 ratio in tumors. (f) Flow cytometry plots and quantitative analysis of CD86 in tumor cDC1s. (g) Quantitative analysis of the cDC1s / cDC2 ratio in total DCs, cDC1s, and TdLNs. (h) Flow cytometry plots and quantitative analysis of CD86 in cDC1s in TdLNs.

[0043] Figure 6Schematic diagram of the experimental results of PTT induced T cell proliferation and activation mediated by the nano-photosensitizer OBDlp modified with the outer membrane vesicles of genetically engineered bacteria in the embodiments of the present invention. (a) Flow cytometry plots 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 = 40 μm. (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 levels in tumors or sera. (l) IFN-γ levels in tumors or sera. (m) IL-6 levels in tumors or sera. (n) TGF-β levels in tumors or sera.

[0044] Figure 7 In vivo anti-tumor therapy result graph of the nano-photosensitizer OBDlp modified with the outer membrane vesicles of genetically engineered bacteria in the embodiments of the present invention. (a) Schematic diagram of the treatment procedure for 4T1 tumor-bearing mice. (b) Changes in tumor volume of the treated mice. (c) Tumor weights of each group. (d) Photos of the harvested mouse tumors. (e) Changes in tumor volume of each group. (f) Survival time of the treated mice. (g) Changes in body weight of the mice during the treatment. (h) TUNEL staining of tumors from different groups of mice. Scale bar = 200 μm.

[0045] Figure 8PTT therapy for distant tumor invasion and lung metastasis mediated by nano-photosensitizer OBDlp modified with gene engineering bacteria outer membrane vesicles in the embodiments of the present invention. (a) Schematic diagram of the treatment procedure for bilateral tumor mice. (b) Changes in the primary tumor volume of the treated mice. (c) Changes in the distant tumor volume of the treated mice. (d) Changes in the volume of primary tumors and distant tumors in each group. (e) Weights of the primary tumors and distant tumors of the mice. (f) Survival time of the treated mice. (g) Flow cytometry plots 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 plots 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.

[0046] Figure 9 Graph showing the experimental results of evaluating the inhibitory effect of nano-photosensitizer cDC1s and CD8+ T cells modified with gene engineering bacteria outer membrane vesicles on distant tumors in the embodiments of the present invention. (a) Schematic diagram of the treatment procedure for in vivo experiments. (b) Changes in the distant tumor volume of the treated mice. (c) Survival time of the treated mice. (d) Weight of the distant tumors of the treated mice. (e) Photos of the collected distant tumors. Scale bar = 1 cm. (f) Changes in the volume of distant tumors in each group.

[0047] Figure 10 Process flow chart for the preparation of small molecule dyes in the nano-photosensitizer modified with gene engineering bacteria outer membrane vesicles in the embodiments of the present invention. Detailed implementation manners

[0048] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0049] Example 1

[0050] As a nano-photosensitizer (OBDlp) modified with gene engineering bacteria outer membrane vesicles in the embodiments of the present invention, the nano-photosensitizer is a nanomaterial with a particle size of 100 nm. The nano-photosensitizer modified with gene engineering bacteria outer membrane vesicles includes dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), cholesterol, small molecule dyes, and CD47nb-OMV membrane proteins;

[0051] The chemical structure of the small molecule dye is shown in Formula I,

[0052] That is, BThDCMI;

[0053] The molar ratio of dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine and cholesterol is 5:3:1:1;

[0054] The weight ratio of the small molecule dye to the CD47nb-OMV membrane protein is 5:1000;

[0055] The nano photosensitizer is obtained by extruding the components dispersed in a solution into a nano material.

[0056] Example 2

[0057] As a nano photosensitizer (OBDlp) modified by a genetically engineered bacterial outer membrane vesicle according to an embodiment of the present invention, the only difference between this example and Example 1 is that the chemical structure of the small molecule dye is as shown in Formula II,

[0058] That is, ThDCMI.

[0059] Example 3

[0060] As a preparation method of a nano photosensitizer (OBDlp) modified by a genetically engineered bacterial outer membrane vesicle according to an embodiment of the present invention, it includes the following steps:

[0061] (1) Prepare CD47nb-OMV membrane protein, and the preparation method of CD47nb-OMV membrane protein includes the following steps:

[0062] (Ⅰ) Synthesize a DNA fragment encoding the CD47nb-ClyA fusion protein (synthesized by BGI-Shenzhen Co., Ltd.), and clone the DNA fragment into the pBAD plasmid by one-step Gibson assembly method to obtain a genetically modified pBAD plasmid;

[0063] (Ⅱ) Transfer the genetically modified pBAD plasmid into Escherichia coli MG1655 to obtain genetically engineered Escherichia coli MG1655;

[0064] (Ⅲ) Culture the genetically engineered Escherichia coli MG1655 in LB medium supplemented with 25 μg / mL chloramphenicol at 37 °C with shaking at 200 rpm until the OD600 reaches 0.5;

[0065] (Ⅵ) Add arabinose to the LB medium to a mass concentration of 0.5%, and continue to culture for 16 hours to induce the genetically engineered Escherichia coli MG1655 to express CD47nb-OMV membrane protein;

[0066] (Ⅴ) Collect the bacterial suspension, remove the bacteria by centrifugation through a 0.45 μm filter membrane, and collect the CD47nb-OMV membrane protein by ultracentrifugation.

[0067] The preparation method of the small molecule dye shown in Formula I or Formula II is as follows:

[0068] N,N-bis(4-methylphenyl)-3-(tributylstannyl)benzo[c]thiophen-1-amine or N,N-bis(4-methylphenyl)-3-(tributylstannyl)thiophen-1-amine reacts with 2-(3-chloro-1H-inden-1-ylidene)malononitrile under the catalysis of bis(triphenylphosphine)palladium dichloride and refluxes in toluene to obtain the compound shown in Formula (II) or Formula (I).

[0069] The specific synthetic technical route is as Figure 10 shown.

[0070] The preparation method of the nano photosensitizer modified by the outer membrane vesicles of the genetically engineered bacteria includes the following steps:

[0071] (1) Dissolve dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine and cholesterol in chloroform to obtain Solution A, and mix the tetrahydrofuran solution of the small molecule dye shown in Formula I or Formula II with Solution A respectively, with a concentration of 2 mg / ml;

[0072] (2) Blow the mixed solution in step (1) into the CD47nb-OMV membrane protein with nitrogen, and dry it under vacuum to remove the solvent, and redissolve the solid product in 20 mmol / L PBS buffer solution with pH 7.4;

[0073] (3) Extrude the obtained compliant solution in step (2) 10 cycles in an extruder with a pore size of 100 nm to obtain the nano photosensitizer modified by the outer membrane vesicles of the genetically engineered bacteria, and perform dialysis purification treatment.

[0074] Comparative Example 1

[0075] As a nano photosensitizer modified by the outer membrane vesicles of the genetically engineered bacteria in Comparative Example 1 of the present invention, the only difference between this comparative example and Example 1 is:

[0076] The chemical structure of the small molecule dye is shown in Formula VI,

[0077] that is, PhDCMI.

[0078] The preparation method is the same as that of Examples 1-3 except for replacing the small molecule dye.

[0079] Comparative Example 2

[0080] As a nano-photosensitizer modified by the outer membrane vesicles of a genetically engineered bacterium in Comparative Example 1 of the present invention, the only difference between this comparative example and Example 1 is that the nano-photosensitizer includes dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), cholesterol, and a small molecule dye.

[0081] That is, compared with the nano-photosensitizer in Example 1, the nano-photosensitizer in this comparative example does not include the CD47nb-OMV membrane protein. It is denoted as BDlp.

[0082] An experimental method:

[0083] (I). Evaluate the ability of the CD47nb-OMV membrane protein to enhance the phagocytosis of macrophages.

[0084] Seed RAW264.7 macrophages into a 96-well plate, with 5×10 4 cells per well. Incubate 4T1 cells with Dil stain (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine perchlorate) for 20 minutes. Pretreat Dil-stained 4T1 cells with PBS, OMV (outer membrane vesicles of bacteria), or CD47nb-OMV for 2 hours respectively. Co-incubate the pretreated 4T1 cells with macrophages at a ratio of 2:1 at 37 °C for 4 hours. After washing with PBS, perform flow cytometry to study the ratio of Dil+ cells to F4 / 80+ cells to evaluate the phagocytosis of tumor cells by macrophages.

[0085] (II) Tumor cell uptake

[0086] Incubate 4T1 cells in a 24-well plate overnight, with 5×10 4 cells per well. Replace the fresh medium containing BDlp (nano-photosensitizer not modified by bacterial outer vesicles) or OBDlp (photosensitizer of Example 1 and Example 2), and co-incubate with 4T1 cells for different times. Observe the cellular uptake of nanoparticles by fluorescence microscopy and flow cytometry.

[0087] (III) In vitro cytotoxicity assay

[0088] Add OBDlp or indocyanine green (ICG) solution (50 μg / ml) to a 96-well plate. Observe the temperature change of the solution under 808 nm laser irradiation (1 W / cm 2 ) using an infrared thermal imager. Seed 4T1 cells in a 96-well plate, with 10,000 cells per well. After 24 hours, replace the medium with fresh medium containing BThDCMI, BDlp, or OBDlp respectively. Expose the cells to 808 nm laser irradiation (1 W / cm 2, (10 min). Then, cell viability was detected by CCK8 assay and live / dead cell staining.

[0089] (IV) In vitro macrophage polarization

[0090] 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. The cells were treated with blocking solution for 30 min and then stained with DAPI (4',6-diamidino-2-phenylindole) for 5 min. The cells were washed with PBS and incubated with anti-CRT antibody for 2 h. FITC (fluorescein isothiocyanate)-labeled secondary antibody was added to the cell wells. The exposure of CRT in 4T1 cells was observed by fluorescence microscopy.

[0091] (V) In vivo NIR II fluorescence imaging and photoacoustic imaging of OBDlp

[0092] Female Balb / c mice were used to establish a subcutaneous tumor-bearing mouse model. 4T1 cells (1×10 6 cells per mouse) were subcutaneously injected into the back of the mice. Once the tumors in the mice grew to approximately 200 mm 3 , OBDlp or BDLp (5 mg / kg BThDCMI) was intravenously injected into the tumor-bearing mice (n = 3). In vivo NIR II fluorescence / photoacoustic imaging was performed at different time points. The mice were euthanized 48 h after injection, and the organs and tumors were collected for ex vivo imaging. BDLp and OBDlp were intravenously injected into the tumor-bearing mice, and NIR II fluorescence imaging (excitation: 808 nm, emission: 1075 nm) was performed at different time points. The imaging signals were quantitatively analyzed by IVIS software and Image J software.

[0093] (VI) Activation of cDC1

[0094] The tumor-bearing mice were randomly divided into 6 groups (n = 4), including a control group, a laser group, a BDLp group, an OBDlp group, a BDLp + laser group, and an OBDlp + laser group. After 7 days of receiving different treatments, the mice were euthanized, and the tumors and tumor-draining lymph nodes (TdLN) were collected. The tissues were minced and treated with collagenase digestion to obtain a single-cell suspension. The single-cell suspension was incubated with fluorescently labeled antibodies at 4 °C for 30 min and then analyzed by flow cytometry.

[0095] (VII) Promotion of M1 phenotype macrophage polarization

[0096] Tumor-bearing mice were randomly divided into 6 groups (n = 4). The experimental groups were the control group, the laser group, the BDlp group, the OBDlp group, the BDlp + laser group, and the 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.

[0097] (VIII) Improvement of T cell proliferation and activation

[0098] Tumor-bearing mice were randomly divided into 6 groups (n = 4): the control group, the laser group, the BDlp group, the OBDlp group, the BDlp + laser group, and the 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.

[0099] (IX) Cytokine detection

[0100] On the seventh day after various treatments, the tumor-bearing mice were euthanized, and their peripheral blood and tumors were collected. Then, according to the instructions of the cytokine ELISA kit, the levels of various cytokines in the peripheral blood and tumor tissues were analyzed.

[0101] (X) Inhibition of distant invasion and lung metastasis of breast tumors

[0102] 4T1 cells (1×10 6 cells per mouse) were subcutaneously injected into the right dorsal side of Balb / c mice to establish primary tumors. Three days later, 4T1 cells (1×10 6 cells per mouse) were subcutaneously injected into the other dorsal side of the mice to establish a distant tumor-bearing mouse model. When the volume of the primary tumor in the mice reached approximately 100 mm 3 , treatment was carried out. The tumor-bearing mice were divided into 6 groups (n = 5): (i) the control group, (ii) the laser group, (iii) the BDlp group, (iv) the OBDlp group, (v) the BDlp + laser group, and (vi) the OBDlp + laser group. BDlp or OBDlp (5 mg / kg BThMCDI) was intravenously injected. Forty-eight hours later, the tumors of the mice were exposed to laser irradiation (808 nm, 1 W / cm 2 , 10 minutes). The bilateral tumor volumes and body weight changes 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 weights. The lungs of the mice were collected to calculate the number of lung metastases.

[0103] (XI) Evaluation of the effect of CD8 and MHC-I blockade on distant tumors

[0104] 4T1 cells (1×10 6Individual cells) were subcutaneously injected into the right dorsal side of Balb / c mice to establish primary tumors. Two days later, 4T1 cells (1×10 6 cells) were subcutaneously injected into the other dorsal side of the mice to establish a distant tumor-bearing mouse model. When the volume of the primary tumor in the mice reached approximately 100 mm 3 , treatment was carried out. The 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 intravenously injected. 48 hours later, the primary tumors of the 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 intraperitoneally injected every 3 days. The changes in the volume of distant tumors and body weight of the mice were continuously monitored after treatment. The mice were sacrificed on the 15th day after treatment, and the distant tumors were harvested to measure the tumor weight. The lungs of the mice were collected to calculate the number of lung metastases.

[0105] II. Experimental Results

[0106] (I) Preparation of Nanophotosensitizer (OBDlp) Modified by Genetically Engineered Bacterial Outer Membrane Vesicles

[0107] The nanophotosensitizer (OBDlp) modified by genetically engineered bacterial outer membrane vesicles was complexed with a small molecule dye. The small molecule dye uses dicyanomethylene indene (DCMI) as a new molecular backbone. As a π-conjugated acceptor, it is connected to three different triarylamine donors (Examples 1, 2, and Comparative Example 1 respectively) for adjusting absorption ( Figure 2 a). The final compounds of all syntheses were confirmed by nuclear magnetic resonance (1H, 13C NMR) spectroscopy ( Figure 2 b - d). Among them, the absorption rate of the compound embedded with the high quinone π-bridge is maximally 783 nm, which can be used for further research on phototherapy applications ( Figure 2 d).

[0108] To confirm the expression of CD47nb on OMVs, non-pathogenic Escherichia coli MG1655 was transformed with a single plasmid encoding the tagged CD47nb-ClyA fusion protein ( Figure 2 e). The expression of CD47nb-ClyA protein in engineered bacteria and OMVs was verified by SDS-PAGE and Western blotting ( Figure 2e). To detect the function of CD47nb on OMV, 4T1 breast cancer cells were incubated in OMV and CD47nb-OMV respectively, 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 could specifically bind to CD47 in 4T1 cells( Figure 2 f).

[0109] Then, the ability of CD47nb-OMV to enhance macrophage phagocytosis was evaluated. Dil-labeled 4T1 cells were pretreated with PBS, OMV or CD47nb-OMV and co-incubated with macrophages. The ratio of Dil+ cells to F4 / 80+ cells was detected to evaluate the phagocytosis of tumor cells by macrophages. Compared with the PBS and OMV groups, CD47nb-OMV treatment led to a significant enhancement of macrophage phagocytosis( Figure 2 g). OMV alone also enhanced phagocytosis, indicating that CD47nb-OMV activates macrophages by binding CD47 blockade and bacterial stimulation adjuvant( Figure 2 g). In addition, CD47nb-OMV had a significant stimulating effect on macrophages, resulting in an increase in the expression level of CD80 and a decrease in the expression level of CD206( Figure 2 h-i).

[0110] Transmission electron microscopy (TEM) showed that both BDlp and OBDlp presented spherical structures( Figure 2 j). Dynamic light scattering (DLS) analysis showed that the particle sizes of OMV, BDlp and OBDlp were ∼12nm, ∼91nm and ∼122nm respectively( Figure 2 k). The Zeta potentials of OMV, BDlp and OBDlp V were -8.58±0.65mV, -3.48±0.65mV and -6.28±0.76mV respectively. The potential of OBDlp was more negative than that of BDlp, indicating successful modification of CD47nb-OMV.

[0111] The results of ultraviolet absorption spectroscopy showed that the characteristic peaks of BDlp and OBDlp were similar to those of BThDCMI, and the peaks were located at ∼783nm( Figure 2 l). Fluorescence spectroscopy showed that OBDlp had obvious characteristic peaks between ∼930nm in the near-infrared II window, indicating that OBDlp had the potential to be used as a contrast agent for enhancing optical imaging in the NIR II window( Figure 2 m). We further studied the in vitro NIR II fluorescence imaging signals of different concentrations of BDlp or OBDlp solutions. The NIR II fluorescence imaging intensity of BDlp or OBDlp solutions increased with the increase of concentration from 1-80 μg / mLFigure 2 n).

[0112] (II) In vitro photothermal therapy experiments

[0113] The tumor cell uptake of BDlp or OBDlp was evaluated, and it was found that the modification of OMV improved the cellular uptake of liposomes. With the prolongation of the incubation time of OBDlp, the intensity of the fluorescence signal in 4T1 cells increased significantly. Flow cytometry results showed that the uptake of OBDlp by cells was significantly better than that of BDlp 4 h after treatment. The photothermal stability of OBDlp was investigated by recording the temperature changes during repeated heating cycles.

[0114] As Figure 3 shown in a, after 4 irradiation cycles, the temperature drop of OBDlp was the smallest, while the temperature decay of ICG was obvious under the same laser irradiation. The cytotoxicity of OBDlp against 4T1 cells in vitro was evaluated. Live / dead staining showed that OBDlp + laser could effectively kill most tumor cells. Notably, there was no significant cytotoxic effect with OBDlp treatment alone or BDlp treatment alone ( Figure 3 b). The in vitro anti-tumor 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).

[0115] PTT induces immunogenic cell death (ICD) of tumor cells and promotes the release of tumor antigens. It was investigated whether OBDlp + laser could trigger ICD and further promote the maturation of DCs. Calreticulin (CRT), high-mobility group box 1 (HMGB1), and ATP were used as indicators of damage-associated molecular patterns (DAMPs) that trigger DC phagocytosis to evaluate the ICD of 4T1 cells. As shown in the figure, compared with other groups, the OBDlp + laser group showed more obvious CRT exposure in 4T1 cells ( Figure 3 c). Low intracellular HGMB1 concentration and intracellular ATP level indicated that OBDlp + laser treatment promoted the release of HGMB1 and ATP ( Figure 3 e-f). These results indicated that OBDlp-mediated PTT effectively triggered ICD and enhanced the immunogenicity of 4T1 cells.

[0116] 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 the culture medium of tumor cells treated with OBDlp + laser, the expression levels of CD80 and CD86 in DCs were analyzed by flow cytometry ( Figure 3g, j). Compared with other treatment groups, the DC maturation level in the OBDlp + laser group was significantly increased ( Figure 3 g, j). Under the stimulation of DAMPs, the TNF level of DCs increased significantly, while the IL-10 level decreased ( Figure 3 h, i).

[0117] (III) In vivo NIR II fluorescence / photoacoustic tumor imaging experiments

[0118] The addition of OMV is expected to enhance the hypoxia targeting ability of the nanoparticles. BDlp or OBDlp was intravenously injected into mice, and in vivo fluorescence imaging was performed at the specified time points. Compared with BDlp, OBDlp showed greater accumulation in the tumor region and reached the peak at 48 hours after injection, which may depend on the specific molecules of OMV ( Figure 4 a, e). Tumors and organs were collected at 48 hours after injection for ex vivo imaging. Compared with the BDlp group, the tumors in the OBDlp group showed stronger signal intensity, indicating that the modification of OMV significantly enhanced the delivery of the photosensitizer to the tumor ( Figure 4 b, f).

[0119] 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 tissues within 48 h, and the fluorescence signal was significantly stronger than that of the BDlp group ( Figure 4 c, g).

[0120] Compared with NIR I window imaging, the signal-to-noise ratio of NIR II window imaging was significantly improved, enabling the precise monitoring of the distribution of the photosensitizer in tumor tissues. The ex vivo imaging results showed that more OBDlp accumulated in tumor tissues than BDlp ( Figure 4 d). In particular, compared with BDlp, the accumulation of OBDlp in the liver was significantly reduced, indicating that it could avoid the side effects caused by the massive accumulation of the photosensitizer in organs ( Figure 4 h).

[0121] The excellent photothermal conversion performance of OBDlp makes it promising to enhance NIR II photoacoustic imaging. As shown by in vivo tumor photoacoustic imaging, OBDlp showed better microvascular morphology and density than BDlp in tumor tissues 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 research shows that OBDlp has the potential to become a NIR II window fluorescence imaging / photoacoustic imaging contrast agent for tumor-targeted imaging.

[0122] (IV) Experiment on the Potent Immune Response Induced by OBDlp-mediated PTT

[0123] To evaluate whether OBDlp-mediated PTT established an immune-stimulatory tumor microenvironment, 4T1 tumor-bearing mice were used to test the effect of OBDlp + laser on macrophage repolarization. As Figure 5 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 proportion of M1-phenotype macrophages in the tumors of the OBDlp + laser group and the OBDlp group was significantly higher than that in the control group( Figure 5 b). Notably, OBDlp + laser treatment not only induced intratumoral macrophages but also polarized splenic macrophages into the M1 phenotype, which would effectively reverse the immunosuppressive microenvironment in the tumor and enhance systemic immunity( Figure 5 c).

[0124] In addition, we also evaluated the effect of different treatment strategies on DCs maturation. In the tumors and tumor-draining lymph nodes (TdLNs). Flow cytometry analysis showed that the proportions of cDC1s (CD103+CD11b-CD11c+) and total DCs (CD11c+MHCII+CD45+) in the tumor tissues and TdLNs of the OBDlp + laser group were significantly increased( Figure 5 d). In addition, the cDC1s / cDC2s ratio in the OBDlp + laser group was higher than that in other treatment groups( Figure 5 e, g). In particular, the CD86 level in the cDC1s population was significantly increased, indicating that OBDlp + laser treatment significantly induced the maturation of cDC1s in the tumors and TdLNs( Figure 5 f, h). Disrupting the CD47-SIRPα signaling axis is considered a promising immunotherapy strategy. In vitro and in vivo results showed that OBDlp treatment effectively activated DCs and macrophages. This may be mainly attributed to the stimulatory effect of OBDlp + laser-mediated PPT-induced ICD and CD47-SIRPα axis blockade on macrophage phagocytosis.

[0125] The maturation of cDC1s and the polarization of M1-phenotype macrophages can further mediate the downstream adaptive immune response by regulating the proliferation and activation of T cells. Therefore, the numbers and types of CD4+ T cells and CD8+ T cells in the TdLNs and tumors were further evaluated. As Figure 6 shown in a, OBDlp + laser treatment led to a significant increase in the proportion of CD8+ T cells in the tumor-draining lymph nodes. However, the number of CD4+ T cells in the TdLNs did not show a statistically significant change( Figure 6 b, e). Among the T cell population, the number of proliferative T cells increased significantly, and the number of exhausted T cells decreased significantlyFigure 6 c, f). In addition, OBDlp+laser treatment exhibited the most robust immune response, with significant activation of CD4+ T cells and CD8+ T cells, showing a two-fold and three-fold increase in CD4+ T cells respectively compared to the BDlp group ( Figure 6 d, g). The number of regulatory T cells in the tumor-draining lymph nodes of the OBDlp+laser group seemed to show a significant decrease compared to other treatment groups ( Figure 6 i, j).

[0126] OBDlp+laser treatment has been shown to stimulate the proliferation and activation of CD4+ T cells and CD8+ T cells within the tumor-draining lymph nodes. However, the most crucial aspect of this treatment is its ability to induce the infiltration of CD4+ T cells and CD8+ T cells in the remodeled tumor microenvironment. This is essential for the anti-tumor immune response. Flow cytometry analysis of tumor tissue samples showed that the numbers of CD4+ T cells and CD8+ T cells within the tumor tissue of the OBDlp+laser group were significantly higher than those of other groups compared to 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 of the BDlp group respectively ( Figure 5 f, h). In addition, the numbers of exhausted T cells and Treg cells in the tumor tissue significantly decreased after OBDlp+laser treatment, indicating enhanced T cell inhibition in the tumor microenvironment, which may be beneficial for PD-1 / PD-L1 immune checkpoint inhibitor treatment ( Figure 5 f, h). Immunofluorescence staining of tumor tissue in the OBDlp+laser group showed a significant increase in the infiltration of CD8+ T cells and CD4+ T cells compared to the control group ( Figure 6 h).

[0127] There were no significant differences in the levels of intratumoral inflammatory cytokines (including IFN-γ, TNF-α, and IL-6) among the control group, laser group, and BDlp group. In contrast, the secretion of IFN-γ, TNF-α, and IL-6 in the tumor of the OBDlp+laser group was significantly increased ( Figure 6 k-m). In addition, the levels of anti-inflammatory cytokines (such as TGF-β) in the OBDlp+laser group were significantly reduced ( Figure 6 n). Notably, a similar phenomenon was also observed in the peripheral blood of mice, further indicating that the adaptive immunity against tumors was activated.

[0128] In summary, compared with other treatment groups, the group receiving OBDlp + laser treatment elicited a strong anti-tumor immune response in DCs and macrophages and further activated T cell cytotoxicity, which is crucial for achieving superior anti-tumor treatment effects. In summary, OBDlp + laser treatment can achieve cascading enhanced anti-tumor immunotherapy through the following three steps: (i) The CD47 nanobody carried by OBDlp can induce the polarization of M1 phenotype TAM 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 TAM synergistically activate cDC1; (iv) Activated cDC1 and the improved tumor microenvironment reactivate the function of tumor-infiltrating CD8+ T cells.

[0129] (V) In vivo anti-tumor treatment experiment

[0130] Since 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 nanobody. 48 h after intravenous injection of the nanosensitizer, laser irradiation (808 nm, 1 W / cm 2 , 10 min) was performed on the tumor-bearing mice, and the tumor growth of the mice was continuously monitored ( Figure 7 a). Compared with the control group, the tumor growth in the BDlp + laser group was inhibited, and the treatment effect could be further significantly improved after CD47nb-OMW modification ( Figure 7 b).

[0131] The BDlp + laser group had an inhibitory effect on tumor growth, while the inhibitory effect of the OBDlp + laser group on tumor growth was increased compared with the BDlp + laser group ( Figure 7 e). On the 15th day after treatment, the tumor weight in the OBDlp + laser group was significantly lower than that in the control group and the BDlp + laser group ( Figure 7 c-d). As shown in the figure, OBDlp + laser treatment also significantly prolonged the survival time of the mice ( Figure 7 f). In addition, the body weight of each group of mice did not show any significant difference during the treatment process ( Figure 7 g). The TUNEL immunofluorescence experiment further confirmed the killing effect of BDlp + laser and OBDlp + laser on 4T1 tumor cells ( Figure 7 h).

[0132] (VI) Experiment on inhibiting distant invasion and lung metastasis of breast tumors

[0133] Then, we further used a bilateral subcutaneous tumor-bearing mouse model to evaluate the treatment effect of OBDlp + laser treatment on primary and distant tumorsFigure 8 a). The anti-distant tumor effects of laser, BDlp, OBDlp, and BDlp + laser treatment were limited. In contrast, OBDlp + laser treatment not only inhibited the primary tumor but also significantly inhibited the growth of distant tumors ( Figure 8 b-d). On the 15th day after treatment, the weights of primary and distant tumors in the OBDlp + laser group were significantly smaller than those in 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 longer than that in the BDlp + laser group ( Figure 8 f). Notably, we found that OBDlp + laser treatment significantly increased the maturation of DCs and the polarization of M1-phenotype macrophages in distant tumors, which was manifested as 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 g-h). Meanwhile, due to the activation of antigen-presenting cells, the level of CD8+ T cells in distant tumors was significantly increased, and the percentages of proliferative CD8+ T cells and functional CD8+ T cells were significantly increased ( Figure 8 i-k). The results showed that OBDlp + laser had excellent inhibitory effects on distant tumors, which was mainly due to the immune response secondary to the promotion of DCs maturation and tumor-associated macrophage repolarization.

[0134] (VII) Evaluation of the inhibitory effect of cells on distant tumors

[0135] In addition, we conducted rescue experiments to evaluate the roles of cDC1 and CD8+ T cells in immunotherapy against distant tumors ( Figure 9 a). Injection of anti-MHC-I antibody could block the migration of cDC1 to TdLNs, and anti-CD8 antibody could achieve in vivo depletion of CD8+ T cells. In addition, OBDlp + laser treatment improved the immunosuppressive tumor microenvironment, which might 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 depletion of CD8+ T cells or MHC-I blockade after OBDlp + laser treatment significantly promoted the growth of distant tumors ( Figure 9 b). Most importantly, the combination of OBDlp + laser and 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 ( Figure 9 d-f). In addition, the survival time of mice treated with the combination of OBDlp + laser and anti-PD-L1 antibody was more than 60% at 50 days after treatment ( Figure 9c). These results indicate that OBDlp+ laser therapy highly depends on the adaptive anti-tumor immune response mediated by cDC1s and CD8+ T cells and can also be used in combination with anti-PD-L1 antibody therapy to enhance distant tumor suppression.

[0136] As can be seen from the above experiments, the nano-photosensitizer OBDlp modified by genetically engineered bacterial outer membrane vesicles, as a novel nano-photosensitizer, mediates tumor-targeted PTT through NIR II FL / PA imaging, improves tumor immunogenicity and activates antigen-presenting cells, resulting in the infiltration and functionalization of CD8+ T cells. OBDlp combined with laser induces the release of a large amount of DAMPs and triggers ICD to promote the maturation of cDC1s. At the same time, the CD47 nanobody carried by the nano-photosensitizer induces the polarization of M1 phenotype macrophages 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. Generally speaking, under the guidance of NIR II FL / PA imaging, OBDlp-mediated photoimmunotherapy simultaneously increases the activation of DCs and macrophages, thus effectively inhibiting tumor growth and metastasis.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions 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 to 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. 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 preparation method of the nano photosensitizer 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 CD47nb-OMV membrane protein with an inert gas, drying to remove the solvent, and redissolving the solid product in a PBS buffer solution; (3) extruding the solution obtained in step (2) 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.

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 is reacted with 2-(3-chloro-1H-inden-1-ylidene)malononitrile in toluene 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) Extruding the solution obtained in step (2) 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.

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) Extruding the prepared solution obtained in step (2) 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 genetically engineered Escherichia coli MG1655 to express CD47nb-OMV membrane protein; and culture is performed for 12 to 20 hours.

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