A tumor immune remodeling biological hybrid system, a preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a tumor immune remodeling biological hybrid system, a preparation method and application thereof. The system is engineered by probiotics to colonize locally in the tumor and continuously secrete Flt3L, and utilizes the enrichment effect of outer membrane vesicles to realize local expansion of dendritic cell precursors and differentiation of cDC1 cells, thereby improving the problems of insufficient number and limited function of dendritic cells in colorectal cancer. At the same time, the system can organically combine Flt3L-driven dendritic cell expansion with immunogenic stimulation induced by sonodynamic therapy to form a self-enhanced anti-tumor immune cycle mediated by dendritic cells, and promote the transformation of an immunosuppressive tumor microenvironment to an immune-activated state. In a colorectal cancer mouse model, the biological hybrid system can prolong the survival of mice, improve the survival rate, and inhibit tumor progression. Therefore, the system is expected to be applied to the treatment of clinical colorectal cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a tumor immune remodeling biohybrid system, its preparation method, and its application. Background Technology

[0002] Cancer immunotherapy, by mobilizing the body's own immune system to recognize and eliminate malignant tumor cells, has demonstrated significant clinical value in the treatment of various cancers. However, solid tumors such as colorectal cancer are often accompanied by a complex immunosuppressive tumor microenvironment, characterized by insufficient infiltration of local effector immune cells, limited antigen presentation capacity, and a lack of immune activation signals, resulting in low response rates and unsustainable efficacy of immunotherapy.

[0003] Dendritic cells (DCs) are core antigen-presenting cells connecting innate and adaptive immunity. They can take up tumor-associated antigens, respond to danger signals, and further initiate T cell-mediated anti-tumor immune responses. However, in immunologically insensitive solid tumors, DCs are usually insufficient in number and functionally impaired, specifically exhibiting lower maturity, weaker antigen processing capacity, and susceptibility to regulation by suppressive cytokines and myeloid immunosuppressive cells. These deficiencies limit the initiation, expansion, and effector differentiation of tumor-specific T cells, becoming a significant bottleneck restricting the efficacy of tumor immunotherapy.

[0004] Currently, strategies to increase the quantity and function of dendritic cells (DCs) mainly include in vitro preparation of DC vaccines and exogenous administration of Flt3L. While DC vaccines can directly replenish antigen-presenting cells, their preparation process is complex, antigen loading uniformity is poor, and there are problems such as insufficient homing and retention efficiency at tumor sites. Although exogenous Flt3L can promote the expansion of DC progenitor cells, free Flt3L is easily and rapidly cleared in vivo, resulting in low local tumor enrichment efficiency and making it difficult to achieve continuous and precise intratumoral delivery. Furthermore, in the absence of sufficient tumor antigens and immunogenic stimulating signals, simply expanding DCs is still insufficient to effectively induce DC maturation and anti-tumor immune responses.

[0005] Sonodynamic therapy (SDT) is a treatment method that utilizes ultrasound to activate sonosensitizers and generate reactive oxygen species (ROS) at the tumor site. It offers advantages such as deep tissue penetration, strong spatiotemporal controllability, and minimal invasiveness. SDT can not only directly induce tumor cell damage but also promote immunogenic cell death (ICD) in tumor cells, stimulating the release of tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs), thereby providing necessary immune stimulation signals for dendritic (DC) maturation and antigen presentation. However, current SDT methods still suffer from problems such as insufficient tumor enrichment of sonosensitizers, limited ROS generation efficiency in hypoxic tumor regions, and difficulty in inducing a sustained anti-tumor immune response under conditions of insufficient DC numbers.

[0006] Therefore, how to simultaneously achieve continuous amplification of dendritic cells (DCs) and in situ generation of immunogenic stimulation signals in the tumor local area is a technical problem that urgently needs to be solved to further improve the therapeutic effect of immunotherapy for solid tumors. Summary of the Invention

[0007] This invention aims to at least solve one of the technical problems existing in related technologies. Therefore, the first objective of this invention is to provide a method for preparing a tumor immune remodeling biohybrid system; the second objective is to provide a tumor immune remodeling biohybrid system; and the third objective is to provide an application of this tumor immune remodeling biohybrid system.

[0008] To achieve the first objective, the technical solution adopted by this invention is as follows: A method for preparing a tumor immune remodeling biohybrid system includes: by nlpI Using the deletion-type engineered bacterium ΔEcN as the host strain, a prokaryotic expression plasmid containing the signal peptide-Flt3L fusion expression element was introduced to construct the engineered probiotic ΔEcN-F expressing Flt3L; Flt3L is an Fms-like tyrosine kinase 3 ligand; Titanium-based nano-sound-sensing agents were prepared using a titanium source that can provide titanium ions and carboxyporphyrin ligands as raw materials via hydrothermal coordination reaction. The surface of the titanium-based nano-acoustic sensor was functionalized by using a phospholipid derivative containing boric acid groups modified with polyethylene glycol to obtain a functionalized titanium-based nano-acoustic sensor. The functionalized titanium-based nano-acoustic sensitizer was coupled to the surface of the engineered probiotic ΔEcN-F to obtain a tumor immune remodeling biohybrid system.

[0009] Preferably, the signal peptide is selected from at least one of ClyA signal peptide and OmpA signal peptide; ClyA is cytolysin A, and OmpA is outer membrane protein A.

[0010] Preferably, the PEG-modified phospholipid derivative containing boric acid groups is DSPE-PEG2000-PBA, and DSPE-PEG2000-PBA is 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-PEG2000-phenylboronic acid.

[0011] Preferably, the prokaryotic expression plasmid contains a kanamycin resistance gene.

[0012] Preferably, the titanium source is any one or more of titanium tetrachloride, tetrabutyl titanate, and titanium isopropoxide; The carboxyporphyrin ligand is a porphyrin derivative with multiple carboxyl substitutions, which can coordinate with titanium ions provided by the titanium source to form a nanoscale titanium-based coordination framework structure.

[0013] Preferably, the polycarboxylated porphyrin derivative is 5,10,15,20-tetra(parabenzoic acid)porphyrin.

[0014] Preferably, the titanium-based nano-acoustic sensor has a particle size distribution range of 20–90 nm, and its X-ray diffraction pattern shows characteristic diffraction peaks in the range of 2θ between 24° and 26°.

[0015] To achieve the second objective, the technical solution adopted by this invention is as follows: A tumor immune remodeling biohybrid system, prepared using any of the above-described methods, comprises an engineered probiotic ΔEcN-F carrier and a boric acid-functionalized titanium-based nanoacoustic sensor coupled to the surface of the engineered probiotic ΔEcN-F carrier.

[0016] The ΔEcN-F engineered bacteria can colonize tumor sites and continuously release Flt3L-enriched outer membrane vesicles as local bioreaction units. The Flt3L-enriched outer membrane vesicles promote the expansion of dendritic cell precursors, the differentiation of conventional type 1 dendritic cells (cDC1) and dendritic cell-mediated antigen presentation. The titanium-based nano-sound sensitizer generates reactive oxygen species under ultrasound activation, induces immunogenic death of tumor cells, and provides immunogenic stimulation signals that promote dendritic cell maturation.

[0017] To achieve the third objective, the technical solution adopted by this invention is as follows: Application of a tumor immune remodeling biohybrid system, using the tumor immune remodeling biohybrid system to prepare anti-tumor products, wherein the tumor is colorectal cancer.

[0018] Furthermore, the antitumor product has one or more of the following effects: Promotes dendritic cell maturation; Enhances natural killer cell infiltration; Increase CD8 + T cell levels; Increase CD8 + IFN-γ + T cell levels; Promote CD8 + High expression of CD62L and CD44 in T cells.

[0019] Furthermore, the antitumor product is a pharmaceutical preparation used in conjunction with ultrasound irradiation.

[0020] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: This invention provides a tumor immune remodeling biohybrid system, its preparation method, and its application. This system utilizes engineered probiotics to colonize the tumor site and continuously secrete Flt3L. Through the enrichment of outer membrane vesicles, it achieves local expansion of dendritic cell precursors and cDC1 cell differentiation, thereby improving the problems of insufficient dendritic cell quantity and limited function in colorectal cancer.

[0021] This system couples titanium-based nano-acoustic sensitizers to the surface of engineered probiotics, leveraging the tumor tropism of the engineered probiotics to enhance the accumulation and penetration of titanium-based nano-acoustic sensitizers in tumor tissues; simultaneously, under the condition of adenosine triphosphate (ATP) enrichment in the tumor microenvironment, titanium-based nano-acoustic sensitizers can achieve responsive dissociation and release. Under ultrasound, the system can generate reactive oxygen species, induce ICD in tumor cells, and promote the release of TAAs and DAMPs, thereby providing the immunogenic signals required for the maturation and antigen presentation of newly expanded dendritic cells.

[0022] The system organically combines Flt3L-driven dendritic cell expansion with sonodynamic therapy-induced immunogenic stimulation, forming a dendritic cell-mediated self-enhancing anti-tumor immune cycle. This can increase the ratio of effector T cells to regulatory T cells and enhance CD8+. + T-cell and NK-cell-mediated anti-tumor immune responses promote the transformation of the immunosuppressive tumor microenvironment into an immune-activated state.

[0023] Experimental results show that in a mouse model of colorectal cancer, the biohybrid system provided by this invention can prolong the survival time of mice, improve the survival rate, and inhibit tumor progression. Therefore, this system holds promise for clinical application in the treatment of colorectal cancer.

[0024] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0025] Figure 1 These are the PCR verification results of the gene knockout strain ΔEcN and the wild-type strain EcN provided in Example 1 of this invention.

[0026] Figure 2 The above are the characterization results of outer membrane vesicles (OMVs) provided in Example 1 of the present invention; wherein, Figure A shows the detection results of the yield of OMVs from EcN and ΔEcN, Figure B shows the transmission electron microscope image of OMVs from ΔEcN, and Figure C shows the hydration particle size distribution of OMVs from ΔEcN.

[0027] Figure 3 The results are from a Western blot assay of the Flt3L fusion protein in ΔEcN, ΔEcN-ClyA-F and ΔEcN-OmpA-F provided in Example 2 of this invention.

[0028] Figure 4 The results are ELISA detection results of the Flt3L content in OMVs from the sources of ΔEcN, ΔEcN-ClyA-F and ΔEcN-OmpA-F provided in Embodiment 2 of the present invention.

[0029] Figure 5 This is a bar chart showing the flow cytometry detection results and statistical results of different groups provided in Embodiment 3 of the present invention; wherein, Figure A shows the flow cytometry detection results and statistical results of different groups of BMDCs towards CD11c. + CD103 + Representative flow cytometry images of cDC1 cell differentiation; Figure B shows different groups of CD11c. + CD103 + A bar chart showing the statistical results of the proportion of cDC1 cells.

[0030] Figure 6 The cDC1 cells obtained from different treatment groups provided in Example 3 of this invention promote CD8. + The ability of T cells to activate and proliferate; Figure A shows the CD8+ levels in different groups. + Representative flow cytometry images of CD69 expression in T cells; Figure B shows CD8 expression in each group. +CFSE - The bar chart shows the statistical results of T cell proportions. Figure C shows the CD8+ detection results using the CFSE dilution method. + A representative flow cytometry histogram of T cell proliferation.

[0031] Figure 7 The following are the characterization results of the Ti-TCPP titanium-based nano-sound sensitizer (NS) provided in Example 4 of this invention; wherein, Figure A is the transmission electron microscope image of NS, Figure B is the result of dynamic light scattering method to detect the hydrated particle size distribution of NS, Figure C is the X-ray diffraction pattern of NS powder, Figure D is the high-resolution X-ray photoelectron spectrum of Ti 2p in NS, Figure E is the result of using 1,3-diphenylisobenzofuran (DPBF) as a reactive oxygen probe to detect the acoustic dynamic activity of NS, Figure F is the relative intensity change of DPBF, methylene blue (MB), TCPP and Ti-TCPP after ultrasonic irradiation for different times, and Figure G is the electron paramagnetic resonance spectrum of superoxide radicals generated by NS under ultrasonic action detected by using 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) as a scavenger.

[0032] Figure 8 The following are the characterization results of the tumor immune remodeling biohybrid system (ΔEcN-F@NS) provided in Example 5 of this invention; wherein, Figure A shows the results of co-localization of NS and EcN (EcN-mCherry) labeled with cherry red fluorescent protein observed by confocal fluorescence microscopy; Figure B shows the results of hydration particle size distribution of ΔEcN-F, ΔEcN-F@NS and ΔEcN-F@NS (denoted as ΔEcN-F@NS+ATP) detected by dynamic light scattering method; Figure C shows the transmission electron microscopy images of ΔEcN-F, ΔEcN-F@NS and ΔEcN-F@NS+ATP; and Figure D shows the zeta potential detection results of ΔEcN-F, ΔEcN-F@NS and ΔEcN-F@NS+ATP.

[0033] Figure 9 This is a bar chart showing the CCK-8 test results of different groups with and without ultrasonic treatment provided in Example 1 of this invention.

[0034] Figure 10 Example 1 of this invention provides the method of detecting tumor cell killing by calcein methyl ester (AM) / propidium iodide (PI) live-dead cell staining, with a scale bar of 50 μm.

[0035] Figure 11 The graph shows the flow cytometry detection results and statistical results provided in Example 2 of this invention; wherein, Graph A shows the flow cytometry detection results of different groups, and Graph B is a bar chart showing the statistical results of the proportion of CD11c+CD80+CD86+mature DCs in different groups.

[0036] Figure 12 This is a confocal fluorescence microscopy image of immature bone marrow-derived dendritic cells (BMDCs) and splenic T lymphocytes labeled with carboxyfluorescein diacetate succinimide (CFSE) provided in Example 2 of this invention.

[0037] Figure 13 This invention verifies the tumor targeting distribution and tumor accumulation of ΔEcN-F@NS provided in Example 3 in tumor-bearing mice; wherein, Figure A is the result of in vivo fluorescence imaging, Figure B is the in vitro fluorescence imaging of heart, liver, spleen, lung, kidney and tumor tissue, and Figure C is a bar chart of the quantitative statistical results of the relative fluorescence intensity of major organs and tumor tissue in each group.

[0038] Figure 14 This is the result of the detection of the colonization ability of ΔEcN-F@NS in tumor-bearing mice provided in Example 3 of this invention.

[0039] Figure 15 This invention verifies the anti-tumor effect of ΔEcN-F@NS combined with ultrasound in the CT26 subcutaneous colorectal cancer model provided in Example 4. Among them, Figure A is a curve of the change in tumor volume in mice during treatment, Figure B is a display of tumors in different groups after treatment, and Figure C is a bar chart of the statistical results of tumor quality in different groups.

[0040] Figure 16 This is the result of detecting immune cell infiltration in the CT26 subcutaneous colorectal cancer model using ΔEcN-F@NS combined with ultrasound, as provided in Example 4 of this invention; wherein, Figure A shows the CD11c in the tumor-draining lymph nodes of mice in different groups. + CD103 + Representative flow cytometry images of cDC1 cells; Figure B shows CD8+ in tumor-draining lymph nodes of mice from different groups. + CD62L + Representative flow cytometry images of T cells; Figure C shows CD3 in tumor tissues from different groups of mice. + CD8 + A representative flow cytometry image of T cells.

[0041] Figure 17 This is a photograph of the isolated tumor tissue from a tumor-bearing mouse provided in Example 5 of this invention.

[0042] Figure 18 This is the tumor microenvironment detection result provided in Example 5 of the present invention; wherein, Figure A shows the CD11c in the mesenteric lymph nodes of mice. + CD80 + CD86 + Representative flow cytometry images of mature dendritic cells (DCs), Figure B shows CD11c in mouse tumor tissue. +CD80 + CD86 + Representative flow cytometry images of mature dendritic cells (DCs), Figure C shows CD3 in mouse tumor tissue. - CD49b + Representative flow cytometry images of NK cells; Figure D shows CD3 in mouse tumor tissue. + CD4 - CD8 + Representative flow cytometry images of T cells, Figure E shows CD3 in mouse tumor tissue. + CD8 + IFN-γ + Representative flow cytometry images of T cells; F plot shows CD8+ in mouse spleen. + Representative flow cytometry image of central memory T cells with high CD44 and high CD62L expression.

[0043] Figure 19 This is a survival curve diagram of mice with orthotopic colorectal cancer in each group provided in Example 5 of this invention. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0045] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0046] Example 1 I. Constructing an engineered probiotic ΔEcN with high OMV secretion.

[0047] Escherichia coli Nissle 1917 (EcN) was selected as the host strain. Following the construction method of the ΔEcN strain disclosed in patent CN119970669A, its genome was analyzed. nlpI The gene was knocked out, resulting in the gene knockout strain ΔEcN.

[0048] Genomic DNA was extracted from wild-type EcN and ΔEcN and verified by PCR amplification. The results are as follows: Figure 1 As shown, the amplified fragments from the ΔEcN group were smaller than those from the wild-type EcN group, suggesting... nlpI Gene knockout was successful; further sequencing of the PCR products confirmed the result. nlpI The gene was precisely deleted.

[0049] EcN and ΔEcN culture supernatants were collected separately, and OMVs were separated by ultracentrifugation. The procedure was as follows: the bacterial strain was cultured to the logarithmic growth phase, and the bacterial culture was collected and then centrifuged at low speed (5000×) at 4°C. g (10 min) to remove intact bacterial cells and larger cell debris, collect the supernatant; then filter through a 0.22 μm filter membrane to further remove residual bacteria and impurities, obtaining a sterile, cell-free culture supernatant. The filtered supernatant is then ultracentrifuged (100,000 × 10 ... g OMVs were precipitated at 4℃ for 2 hours (2 h). After discarding the supernatant, the OMV precipitate was resuspended in sterile PBS buffer and washed again by ultracentrifugation (to remove non-specific proteins and culture medium residues) to obtain further purified OMVs. The characterization results are as follows: Figure 2 As shown; Figure A shows the detection results of OMVs produced from EcN and ΔEcN. From this figure, it can be seen that the OMVs produced by ΔEcN are approximately 1.35 ± 0.05 mg·L⁻¹. -1 It was higher than that of wild-type EcN by 0.61 ± 0.07 mg·L⁻¹. -1 Confirmed nlpI Gene knockout can enhance EcN’s ability to secrete OMVs.

[0050] Figure B shows a transmission electron microscope image of ΔEcN-derived OMVs. From this image, it can be seen that ΔEcN-derived OMVs have a nanoscale spherical structure with a diameter of approximately 50–100 nm. Figure C shows the hydrated particle size distribution of OMVs from ΔEcN. From this figure, we can see that the average hydrated particle size of OMVs from ΔEcN is approximately 202.57 ± 35.49 nm.

[0051] Example 2 The engineered probiotic expressing Flt3L was constructed as follows: I. Constructing fusion and recombination expression plasmids.

[0052] Prokaryotic expression plasmids containing the Flt3L fusion expression element and the kanamycin resistance gene were constructed. To promote the binding of Flt3L to bacterial membrane structures and increase its enrichment level in engineered bacterial OMVs, Flt3L was fused with either ClyA or OmpA signal peptides to construct two fusion recombinant expression plasmids: ClyA-Flt3L and OmpA-Flt3L.

[0053] The amino acid sequence of Flt3L is shown in SEQ ID NO.1; the nucleotide sequence encoding the ClyA signal peptide is shown in SEQ ID NO.2; and the amino acid sequence of the OmpA signal peptide is shown in SEQ ID NO.3.

[0054] The amino acid sequence of SEQ ID NO.1 is shown below: GTPDCYFSHSPISSNFKVKFRELTDHLLKDYPVTVAVNLQDEKHCKALWSLFLAQRWIEQLKTVAGSKMQTLLEDVNTEIHFVTSCTFQPLPECLRFVQTNISHLLKDTCTQLLALKPCIGKACQNFSRCLEVQCQPDSSTLLPPRSPIALEATELPEPRPRQ.

[0055] The nucleotide sequence of SEQ ID NO.2 is shown below: ATGACTGAAATCGTTGCAGATAAAACGGTAGAAGTAGTTAAAAACGCAATCGAAACCGCAGATGGAGCATTAGATCTTTATAATAAATATCTCGATCAGGTCATCCCCTGGCAGACCTTTGATGAAACCATAAAAGAGTTAAGTCGCTTTAAACAGGAGTATTCACAGGCAGCCTCCGTTTTAGTCGGCGATATTAAAACCTTACTTATGGATAGCCAGGATAAGTATTTTGAAGCAACCCAAACAGTGTATGAATGGTGTGGTGTTGCGACGCAATTGCTCGCAGCGTATATTTTGCTATTTGATGAGTACAATGAGAAGAAAGCATCCGCCCAGAAAGACATTCTCATTAAGGTACTGGATGACGGCATCACGAAGCTGAATGAAGCGCAAAAATCCCTGCTGGTAAGCTCACAAAGTTTCAACAACGCTTCCGGGAAACTGCTGGCGTTAGATAGCCAGTTAACCAATGATTTTTCAGAAAAAAGCAGCTATTTCCAGTCACAGGTAGATAAAATCAGGAAGGAAGCATATGCCGGTGCCGCAGCCGGTGTCGTCGCCGGTCCATTTGGATTAATCATTTCCTATTCTATTGCTGCGGGCGTAGTTGAAGGAAAACTGATTCCAGAATTGAAGAACAAGTTAAAATCTGTGCAGAATTTCTTTACCACCCTGTCTAACACGGTTAAACAAGCGAATAAAGATATCGATGCCGCCAAATTGAAATTAACCACCGAAATAGCCGCCATCGGTGAGATAAAAACGGAAACTGAAACAACCAGATTCTACGTTGATTATGATGATTTAATGCTTTCTTTGCTAAAAGAAGCGGCCAAAAAAATGATTAACACCTGTAATGAGTATCAGAAAAGACACGGTAAAAAGACACTCTTTGAGGTACCTGAAGTCTGA.

[0056] The amino acid sequence of SEQ ID NO. 3 is specifically shown as follows: MKKTAIAIAVALAGFATVAQA.

[0057] 2. The two fusion recombinant expression plasmids were introduced into the ΔEcN strain obtained by the construction method in Example 1.

[0058] Logarithmically growing ΔEcN bacterial culture was centrifuged under ice bath conditions to collect bacterial cells. Competent cells were prepared using the calcium chloride method. Then, 5 ng of recombinant expression plasmid was added to 100 μL of competent ΔEcN cells, gently mixed, and incubated on ice for 20 min. Subsequently, the cells were heat-shocked in a 42℃ water bath for 60 s, immediately transferred to ice for 3 min of cooling, and then 1000 μL of antibiotic-free LB liquid medium was added. The cells were then revived and cultured at 37℃ and 200 rpm for 1 h. The revived bacterial culture was plated on LB solid medium plates containing 50 μg / mL kanamycin and cultured at 37℃ for 12 h. After the culture was completed, resistant single colonies were picked, and positive engineered bacterial clones carrying ClyA-Flt3L and OmpA signal peptide-Flt3L fusion expression plasmids were obtained by colony PCR and sequencing verification, respectively, and were designated as ΔEcN-ClyA-F and ΔEcN-OmpA-F.

[0059] The above-mentioned positive engineered bacterial clones were expanded and cultured, bacterial cells were collected and bacterial lysates were prepared, and the expression of the Flt3L fusion protein was detected by Western blot (with ΔEcN bacterial lysate as a control). The results are as follows: Figure 3 As shown in the figure, a characteristic band of the ClyA-Flt3L fusion protein of approximately 37 kDa can be detected in the ΔEcN-ClyA-F bacterial lysate, while no corresponding band is detected in the ΔEcN strain that has not been introduced with the recombinant expression plasmid, indicating that the ClyA-Flt3L fusion protein can be expressed in ΔEcN.

[0060] Furthermore, the culture supernatants of ΔEcN-ClyA-F and ΔEcN-OmpA-F were collected according to the OMVs collection method in Example 1, and the OMVs secreted by the engineered bacteria were isolated and purified. The Flt3L content in the OMVs was detected by ELISA, and the results are as follows. Figure 4 As shown in the figure, Flt3L can be detected in OMVs from both engineered bacteria sources. However, the Flt3L content in OMVs from ΔEcN-ClyA-F source is significantly higher than that in OMVs from ΔEcN-OmpA-F source.

[0061] Therefore, based on the expression status of the fusion protein and the enrichment level of Flt3L in OMVs, the preferred configuration of the ClyA-Flt3L fusion recombinant expression plasmid was selected, and the engineered bacteria carrying the ClyA-Flt3L fusion expression plasmid ΔEcN-ClyA-F was referred to as ΔEcN-F below.

[0062] Example 3 Detection of ΔEcN-F and its Flt3L enrichment OMVs promotes dendritic cell differentiation and CD8 + The ability to activate T cells.

[0063] The experiment included a PBS control group, a ΔEcN bacterial lysate group, a ΔEcN-derived OMVs group, and ΔEcN-F and ΔEcN-F-derived OMVs groups (labeled as PBS, ΔEcN, ΔEcN OMVs, ΔEcN-F and ΔEcN-F OMVs, respectively).

[0064] Equal amounts of ΔEcN and ΔEcN-F bacterial cells were used to prepare bacterial lysates using an ultrasonic disruption method (small probe ultrasound, working frequency of 20 kHz, amplitude of 30%, ice bath for 5 min). The total protein concentration of the lysates was determined using the BCA method. Equal amounts of total protein were added to each well of ΔEcN and ΔEcN-F bacterial lysates, with a final concentration of 20 μg / mL.

[0065] Following the method described in Example 1, OMVs were isolated from the culture supernatants of ΔEcN and ΔEcN-F, respectively, and the total protein concentration of OMVs was determined using the BCA method. OMVs from ΔEcN and OMVs from ΔEcN-F were added in equal amounts of total protein, 20 μg per well, resulting in a final concentration of 20 μg / mL.

[0066] Bone marrow-derived cells were isolated from the bone marrow of 6-8 week old C57BL / 6 mice as follows: Femurs and tibias were harvested under aseptic conditions. The bone marrow cavity was flushed with RPMI 1640 medium to collect bone marrow cells. After filtering through a cell filter and removing red blood cells, the resulting bone marrow-derived cells were resuspended in RPMI 1640 complete medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin, granulocyte-macrophage colony-stimulating factor (GM-CSF) at a final concentration of 20 ng / mL, and interleukin-4 (IL-4) at a final concentration of 10 ng / mL). The cells were induced and cultured at 37°C and 5% CO2 for 6 days. During the culture period, 50% of the culture medium was replaced every 2 days, and fresh medium containing the same concentrations of GM-CSF and IL-4 was added. After the culture, immature bone marrow-derived dendritic cells (BMDCs) were obtained. The immature BMDCs were then cultured at a density of 5 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 cells per well in a 12-well culture plate, with a total culture medium volume of 1 mL per well.

[0067] BMDCs from each group were co-incubated at 37℃ and 5% CO2 for 24 h. After incubation, cells were collected, washed with PBS, and resuspended in flow cytometry staining buffer. FITC-labeled anti-mouse CD11c antibody and Pacific Blue (PB)-labeled anti-mouse CD103 antibody were added to the cell suspension. After incubation at room temperature in the dark for 30 min, cells were washed with flow cytometry fluorescence sorting (FACS) buffer and analyzed by flow cytometry, using CD11c... + CD103 + Cell proportion assessment evaluates the differentiation of BMDCs into conventional type 1 dendritic cells (cDC1). The detection results are as follows: Figure 5 As shown, the ΔEcN-F treatment group can significantly promote the conversion of BMDCs to CD11c. + CD103 + cDC1 cell differentiation, and ΔEcN-F-derived OMVs can also promote DC expansion and differentiation; in contrast, ΔEcN and its OMVs that do not express Flt3L have a weaker promoting effect. Figure A shows the different groups of BMDCs directed towards CD11c. + CD103 + Representative flow cytometry images of cDC1 cell differentiation; Figure B shows different groups of CD11c. + CD103 + A bar chart showing the statistical results of the proportion of cDC1 cells.

[0068] Further evaluation of the promotion of CD8 by cDC1 cells obtained from the above different treatment groups +The activation and proliferation capacity of T cells were assessed as follows: BMDCs were collected after 24 hours of treatment with PBS, ΔEcN bacterial lysate, ΔEcN-F bacterial lysate, ΔEcN-derived OMVs, and ΔEcN-F-derived OMVs, respectively. ΔEcN and ΔEcN-F bacterial lysates and their corresponding OMVs were added at equal amounts of total protein, 20 μg per well, to achieve a final total protein concentration of 20 μg / mL. After treatment, the culture supernatant was discarded, and the cells were washed with PBS to remove residual bacterial lysates or OMVs. BMDCs from different treatment groups were then collected. Splenic lymphocytes were isolated from mouse spleens and pre-cultured for 2 days. Before co-culturing, the splenic lymphocytes were labeled with carboxyfluorescein diacetate succinimide (CFSE). Then, at a splenic lymphocyte to BMDC ratio of 20:1, CFSE-labeled splenic lymphocytes were seeded with BMDCs from different treatment groups in 12-well plates and co-cultured at 37°C and 5% CO2 for 24 hours. After culture, the cells were collected, resuspended in FACS buffer, and APC-labeled anti-mouse CD8 antibody and PE-labeled anti-mouse CD69 antibody were added. After incubation at room temperature in the dark for 30 minutes, the cells were washed, and CD8+ was detected by flow cytometry. + CD69 in T cells + Cell proportion to evaluate CD8 + The activation level of T cells was also detected; simultaneously, CD8 was examined. + CFSE fluorescence dilution of T cells to evaluate CD8 + The proliferative capacity of T cells, results as follows Figure 6 As shown; where Figure A shows CD8 in different groups. + Representative flow cytometry images of CD69 expression in T cells; Figure B shows CD8 expression in each group. + CFSE - The bar chart shows the statistical results of T cell proportions. Figure C shows the CD8+ detection results using the CFSE dilution method. + A representative flow cytometry histogram of T cell proliferation.

[0069] from Figure 6 It can be seen that, compared with the PBS group, ΔEcN, and ΔEcN OMVs, ΔEcN-F and ΔEcN-F OMVs can both increase CD8 levels. + CD69 in T cells + Cell percentage and enhanced CFSE dilution indicate that it can promote CD8 + T cell activation and proliferation. These results indicate that ΔEcN-F and its secreted Flt3L enrichment of OMVs can promote the differentiation of BMDCs into cDC1 cells and enhance downstream CD8 activation. + T cell activation and proliferation.

[0070] Example 4 Ti-TCPP titanium-based nano-sound sensor was prepared by using titanium source and carboxyporphyrin ligand as raw materials and by hydrothermal coordination reaction.

[0071] The specific type of titanium source is titanium tetrachloride bis(tetrahydrofuran) complex (TiCl4·2THF), and the carboxyporphyrin ligand is 5,10,15,20-tetra(terebenzoic acid)porphyrin (TCPP).

[0072] The preparation process of titanium-based nano-sound-sensing agents is as follows: Preparation of titanium source solution: Add N,N-dimethylformamide (1 mL) to TiCl4·2THF (4 mg), and stir or sonicate until uniform at room temperature.

[0073] Preparation of TCPP solution: Add N,N-dimethylformamide (2 mL) to TCPP (2 mg) and stir or sonicate until homogeneous at room temperature.

[0074] Under stirring conditions, the obtained TCPP solution was slowly added to the titanium source solution. After mixing thoroughly, glacial acetic acid (200 μL) was added as a reaction regulator. Stirring continued for 10 min after the addition was complete to obtain a reaction mixture. This reaction mixture was transferred to a reaction vessel and refluxed at 90 °C for 24 h (to allow the titanium ions to coordinate with the carboxyl groups of TCPP). After the reaction was complete, the mixture was allowed to cool naturally to room temperature. The resulting reaction solution was then subjected to a 10000× [temperature value missing]. g Centrifuge for 5 min, discard the supernatant, collect the precipitate, resuspend and wash the precipitate with ethanol, and then dilute at 10000× 10000 rpm. g Centrifuge for 5 min, and wash three times to remove unreacted raw materials, glacial acetic acid, and residual organic solvents. Add the washed precipitate to anhydrous ethanol, vortex mix, and ultrasonically disperse to obtain a uniformly dispersed Ti-TCPP titanium-based nano-sound sensor (denoted as NS) ethanol dispersion. Store the NS ethanol dispersion at 4°C in the dark, and redisperse it by vortexing or short-term ultrasonication before use.

[0075] The characterization results of NS, such as Figure 7 As shown; Figure A shows a transmission electron microscope image of NS, which reveals that NS consists of uniform spherical particles. Figure B shows the results of dynamic light scattering method for detecting the hydrated particle size distribution of NS. From this figure, it can be seen that the average particle size of NS is approximately 39.25 nm. Figure C shows the X-ray diffraction pattern of NS powder. From this figure, it can be seen that NS has a relatively broad diffraction peak at 2θ of about 25°, which shows the low crystallinity characteristics of the porphyrin-based coordination network. No characteristic diffraction peaks of crystalline TiO2 were detected, suggesting that the titanium species are dispersed in the organic framework. Figure D shows the high-resolution X-ray photoelectron spectrum of Ti 2p in NS. From this figure, we can see that the Ti 2p spectrum is at 458.9 eV (2p... 3 / 2 ) and 464.7eV (2p 1 / 2 Ti appears at ) 4+ The relevant characteristic peaks indicate the presence of Ti-O bonds; Figure E shows the acoustic dynamic activity of NS detected using DPBF as a reactive oxygen species probe. Figure F shows the relative intensity changes of DPBF, methylene blue, TCPP, and Ti-TCPP after ultrasonic irradiation for different times. The ultrasonic irradiation conditions were 1 MHz and 1.5 W·cm⁻¹. -2 With a duty cycle of 50%, these two figures show that the DPBF absorption peak gradually decreases with increasing ultrasonic irradiation time, indicating that NS can generate reactive oxygen species under ultrasonic irradiation. Compared with free TCPP and MB, NS exhibits a higher reactive oxygen species generation efficiency. Figure G shows the electron paramagnetic resonance spectrum of superoxide radicals generated by NS under ultrasound with DMPO as a trapping agent. From this figure, it can be seen that NS produces the characteristic signal of DMPO-•OOH under ultrasound stimulation, suggesting that NS can generate superoxide radicals through the type I electron transfer pathway.

[0076] Example 5 The surface of the titanium-based nano-acoustic sensor was functionalized by using a polyethylene glycol-modified phospholipid derivative containing boric acid groups, resulting in a functionalized titanium-based nano-acoustic sensor. Specifically, the NS prepared in Example 4 was surface functionalized with DSPE-PEG2000-PBA to obtain a phenylboronic acid-functionalized titanium-based nano-acoustic sensor.

[0077] Among them, DSPE-PEG2000-PBA is 1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000-phenylboronic acid.

[0078] The preparation process of phenylboronic acid-functionalized titanium-based nano-sound-sensing agent is as follows: Take NS (containing 10 mg) ethanol dispersion, add it to ethanol solutions of DSPE-PEG2000 (containing 6 mg) and DSPE-PEG2000-PBA (containing 4 mg), and then make up the volume with ethanol to a total volume of 10 mL, so that the final concentrations of NS, DSPE-PEG2000 and DSPE-PEG2000-PBA are 1 mg / mL, 0.6 mg / mL and 0.4 mg / mL, respectively.

[0079] The above mixture was placed in an ice-water bath and sonicated using a probe-type ultrasonic cell disruptor (rated power 150W, sonication time 2h) to coat the surface of NS with DSPE-PEG2000 and DSPE-PEG2000-PBA; after sonication, the resulting dispersion was subjected to 10000× g Centrifuge at 4℃ for 5 min, discard the supernatant, and collect the precipitate. Resuspend the precipitate in sterile PBS and wash by centrifugation under the same conditions. Repeat the washing three times to remove residual ethanol and unbound DSPE-PEG2000 and DSPE-PEG2000-PBA from the system. After washing, resuspend the precipitate in sterile PBS and adjust the concentration to 1 mg / mL to obtain the phenylboronic acid-functionalized titanium-based nano-sound sensor, denoted as PBA-NS.

[0080] Take the ΔEcN-F engineered bacterial culture in the logarithmic growth phase, centrifuge at 4℃ and 1000×g for 5 min, discard the culture supernatant, and collect the bacterial cells. Wash the bacterial cells three times with sterile PBS to remove residual culture medium and free metabolites, then resuspend in sterile PBS to adjust the engineered bacterial concentration to 10. 9 CFU / mL (or adjust bacterial culture OD) 600 Up to 1). According to every 10 9 CFU engineered bacteria were added at a ratio of 1 mg PBA-NS, and the PBA-NS dispersion was mixed with the ΔEcN-F engineered bacteria suspension to achieve a final concentration of engineered bacteria of 10 in the mixture. 9 The CFU / mL and PBA-NS final concentrations are 1 mg / mL, with a total volume of 1 mL.

[0081] The above mixture was placed in a constant temperature shaker at 37°C and gently shaken at 200 rpm for 1 hour to allow the phenylboronic acid groups on the surface of PBA-NS to undergo reversible covalent bonding with the diol groups on the surface of ΔEcN-F bacteria, thereby coupling PBA-NS to the surface of ΔEcN-F engineered bacteria.

[0082] After incubation, at 4℃, 1000× g Centrifuge for 5 min under the same conditions, discard the supernatant, resuspend the precipitate in sterile PBS, and wash three times under the same conditions to remove uncoupled PBA-NS. After washing, resuspend the precipitate in sterile PBS and adjust the engineered bacteria concentration to 10. 9 The tumor immune remodeling biohybrid system, denoted as ΔEcN-F@NS, was obtained by CFU / mL and stored at 4°C for use within 24 hours.

[0083] The characterization results of ΔEcN-F@NS are as follows: Figure 8 As shown; Figure A shows the results of co-localization of NS with EcN (EcN-mCherry) labeled with cherry red fluorescent protein observed by confocal fluorescence microscopy. The figure shows that NS and EcN-mCherry co-localize, indicating that NS is successfully coupled to the bacterial surface.

[0084] To evaluate the ATP-responsive dissociation performance of ΔEcN-F@NS, three groups of samples were set up: ΔEcN-F group, ΔEcN-F@NS group, and ΔEcN-F@NS group treated with 400 μM ATP (denoted as ΔEcN-F@NS+ATP). ΔEcN-F and ΔEcN-F@NS were resuspended in sterile PBS buffer, and the cell concentration was adjusted to 1×10⁻⁶. 9 CFU / mL, 1 mL of each group was used for later use. ATP solution was added to the ΔEcN-F@NS suspension to bring the final ATP concentration in the reaction system to 400 μM, maintaining a total reaction volume of 1 mL to obtain ΔEcN-F@NS+ATP. The mixture was placed in a 37℃ constant-temperature shaker and incubated with gentle shaking at 100 rpm for 1 h. The hydrated particle size distribution was detected using dynamic light scattering, and the results are shown in Figure B. This figure shows that the hydrated particle size of ΔEcN-F@NS is slightly increased compared to ΔEcN-F, while the particle size of ΔEcN-F@NS+ATP decreases. This result indicates that ΔEcN-F@NS exhibits ATP-responsive dissociation characteristics.

[0085] Transmission electron microscopy images of ΔEcN-F, ΔEcN-F@NS, and ΔEcN-F@NS+ATP are shown in Figure C. This figure reveals that in the ΔEcN-F@NS group, a large number of NS nanoparticles are attached to the bacterial surface, with a clearly visible coating layer, confirming that NS successfully modifies the bacterial surface. In the ΔEcN-F@NS+ATP group, a large number of nanoparticles detach from the bacterial surface, indicating that ATP can trigger the detachment and dissociation of the outer NS nanolayer.

[0086] Figure D shows the Zeta potential detection results for ΔEcN-F, ΔEcN-F@NS, and ΔEcN-F@NS+ATP. From this figure, it can be seen that the negative charge weakens (potential increases) after ΔEcN-F@NS coating, indicating that NS coating changes the overall charge state of the bacterial surface; ΔEcN-F@NS+ATP: the potential shifts back to the ΔEcN-F potential value, indicating that the intrinsic negative charge of the bacteria is restored after the NS nanolayer dissociates.

[0087] Test Example 1 To investigate the ability of ΔEcN-F@NS to kill tumor cells in vitro using sonodynamics and induce immunogenic cell death.

[0088] The cytotoxicity and sonodynamic efficacy of NS, ΔEcN, ΔEcN@NS, ΔEcN-F, and ΔEcN-F@NS were evaluated using mouse colorectal cancer CT26 cells. The procedure is as follows: First, the effect of each treatment group on CT26 cell viability was detected using the CCK-8 assay. CT26 cells were divided into groups of 1×10⁶ cells per well. 5 Cells were seeded in the lower chamber of a 24-well Transwell plate, with 500 μL of complete culture medium added to each well, and cultured at 37°C and 5% CO2 for 24 h. Subsequently, PBS, NS, ΔEcN, ΔEcN@NS, ΔEcN-F, or ΔEcN-F@NS were added to the upper chamber of the Transwell plate, with a total volume of 200 μL for each group.

[0089] In the NS group, the final concentration of NS was 100 μg / mL, with 20 μg of NS per well; in the ΔEcN and ΔEcN-F groups, the final concentration of bacterial cells was 1 × 10⁻⁶. 8 CFU / mL, with 2×10⁻⁶ bacteria per well. 7 The final cell concentration in both the CFU, ΔEcN@NS group and the ΔEcN-F@NS group was 1×10⁻⁶. 8 CFU / mL, with 2×10⁻⁶ bacteria per well. 7 CFU were added, and the NS equivalent was calculated based on the actual NS loading to ensure that each well contained 100 μg / mL of NS, i.e., 20 μg of NS per well; an equal volume of sterile PBS was added to the PBS group.

[0090] Without ultrasonic treatment: Each group was incubated at 37℃ and 5% CO2 for 12 hours; Ultrasonic treatment: Ultrasonic conditions were 1MHz and 1.5W·cm. -2 With a 50% duty cycle and an irradiation time of 5 minutes, each group was cultured for 12 hours after sonication. The viability of CT26 cells in both untreated and sonicated groups was detected using the CCK-8 assay. The results are as follows: Figure 9 As shown.

[0091] The tumor cell killing effect was detected by calcein methyl ester (AM) / propidium iodide (PI) live-dead cell staining, and the results are as follows: Figure 10 As shown in the figure, the scale bar is 50 μm, ns indicates no statistical significance, and *** indicates P < 0.001. From this figure, it can be seen that treatment with ΔEcN-F or ΔEcN-F@NS alone did not cause significant cell death; however, after ΔEcN-F@NS was combined with ultrasound treatment, a large number of tumor cells died, indicating that ΔEcN-F@NS has an ultrasound-activated sonodynamic killing effect.

[0092] The NS group was denoted as NS+US after ultrasonic treatment, and the ΔEcN-F@NS group was denoted as ΔEcN-F@NS+US after ultrasonic treatment.

[0093] Test Example 2 To investigate the effect of ΔEcN-F@NS combined with ultrasound on promoting dendritic cell maturation and T cell activation.

[0094] The grouping is the same as in Example 1 of the test.

[0095] CT26 tumor cells were treated differently and then placed in the upper chamber of a Transwell chamber, while immature BMDCs were placed in the lower chamber for co-culture. After incubation for 12 hours in different treatment groups, the CT26 cells were irradiated with ultrasound at 1 MHz and 1.5 W / cm². -2 With a 50% duty cycle and an irradiation time of 5 min, BMDCs and culture supernatant were collected 24 h after ultrasonic irradiation.

[0096] Flow cytometry was used to detect the maturation status of BMDCs. BMDCs were resuspended in FACS buffer, and antibodies against PE-CD86, PerCP-Cy5.5-CD80, and APC-CD11c were added. After incubation at room temperature for 30 min, flow cytometry was performed. The results are as follows: Figure 11 As shown in the figure, it can be seen that the ΔEcN-F@NS+US group can significantly improve CD11c + CD86 + CD80 + The proportion of mature DCs; Figure A shows the results of flow cytometry analysis of different groups, and Figure B shows the CD11c results of different groups. + CD80 + CD86 + A bar chart showing the statistical results of the proportion of mature DCs.

[0097] Confocal microscopy was used to observe whether ΔEcN-F@NS activated BMDCs could effectively induce and stimulate T cell responses. The results are as follows: Figure 12 As shown, the scale bars for each figure are 20 μm. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. From this figure, it can be seen that BMDCs in the ΔEcN-F@NS+US group form a tight aggregate structure with T cells, indicating enhanced intercellular interactions and possible formation of immune synapses.

[0098] Test Example 3 The process of examining the in vivo tumor targeting and biodistribution of ΔEcN-F@NS is as follows: Mice bearing CT26 subcutaneous tumors were randomly divided into groups and injected via tail vein into free TCPP, NS, and ΔEcN-F@NS (containing an equal amount of TCPP, with approximately 10% bacterial count). 7 CFU was used for in vivo fluorescence imaging of small animals at 6, 12, 24, 36, and 48 hours after injection. Mice were sacrificed 48 hours after injection, and their hearts, livers, spleens, lungs, kidneys, and tumor tissues were collected for ex vivo fluorescence imaging. The results are as follows: Figure 13 As shown in the figure; Figure A shows the results of in vivo fluorescence imaging, Figure B shows the in vitro fluorescence imaging of the heart, liver, spleen, lung, kidney, and tumor tissues, and Figure C is a bar chart showing the quantitative statistical results of the relative fluorescence intensity of the major organs and tumor tissues in each group. Simultaneously, tumor tissue homogenate was taken, spread on LB agar plates containing kanamycin, incubated at 37°C, and colony-forming units were counted. The results are as follows. Figure 14 As shown.

[0099] from Figure 13 It can be seen that, compared with the free TCPP or NS group, the fluorescence signal of ΔEcN-F@NS in the tumor site gradually increased over time, reaching a peak at 48h; the fluorescence intensity of ΔEcN-F@NS in the tumor tissue was 2.73 times and 2.32 times that of the free TCPP group and the NS group, respectively.

[0100] from Figure 14 It can be seen that a large number of colonies can be recovered from tumor tissue at both 24h and 48h after ΔEcN-F@NS administration.

[0101] Figure 13 and Figure 14 The results showed that ΔEcN-F@NS has efficient active tumor targeting and colonization capabilities, and can achieve preferential accumulation of nano-soundsensitizers at the tumor site.

[0102] Test Example 4 The antitumor effect of ΔEcN-F@NS in a subcutaneous colorectal cancer model was investigated as follows: A subcutaneous colorectal cancer model was established by subcutaneously inoculating CT26 cells into male 6-8 week old BALB / c mice. The number of CT26 cells inoculated into each mouse was 1×10⁻⁶. 6 Six days after inoculation, tumor-bearing mice were randomly divided into five groups: PBS group, ΔEcN-F group, ΔEcN-F@NS group, ΔEcN@NS+US group, and ΔEcN-F@NS+US group.

[0103] The bacteria were administered via tail vein injection on days 0, 3, and 6, with a bacterial dose of 1 × 10⁶ per mouse. 7 CFU; For the group receiving ultrasound therapy, the tumor site was irradiated with ultrasound 12 hours after each dose, with ultrasound conditions of 1MHz and 1.5W·cm. -2The irradiation time was 50% duty cycle and 5 min. Tumor volume and mouse weight were measured every 2 days during treatment. Tumor volume was calculated using the following formula: Tumor volume = (length × width) 2 )÷2; The results are as follows Figure 15 As shown in the figure, compared with the PBS group, the ΔEcN-F group and the ΔEcN-F@NS group can delay tumor progression, but are still insufficient to completely inhibit tumor growth; the ΔEcN@NS+US group shows a moderate tumor inhibition effect; and the ΔEcN-F@NS+US group has the most significant tumor inhibition effect. Figure A shows the curve of tumor volume change in mice during treatment; Figure B shows the actual tumors of different groups after treatment; and Figure C shows the bar chart of the statistical results of tumor quality in different groups.

[0104] Flow cytometry was used to detect the composition and functional status of immune cells in tumor draining lymph nodes (TDLNs), and the results are as follows: Figure 16 As shown in the figure, the ΔEcN-F@NS+US group significantly increased the infiltration ratio of cDC1s and mature DCs in TDLNs and significantly increased CD62L. + CD8 + The proportion of T cells and the tumor-infiltrating CD8 + T cells (CD3) + CD4 - CD8 + The proportion of ) has increased significantly; Figure A shows the CD11c concentration in the tumor-draining lymph nodes of mice from different groups. + CD103 + Representative flow cytometry images of cDC1 cells; Figure B shows CD8+ in tumor-draining lymph nodes of mice from different groups. + CD62L + Representative flow cytometry images of T cells; Figure C shows CD3 in tumor tissues from different groups of mice. + CD8 + A representative flow cytometry image of T cells.

[0105] Figure 15 and Figure 16 The results showed that ΔEcN-F@NS combined with ultrasound irradiation could effectively remodel the tumor immune microenvironment and induce a systemic anti-tumor immune response.

[0106] Test Example 5 The antitumor effect of ΔEcN-F@NS in an orthotopic colorectal cancer model was investigated as follows: An orthotopic colorectal cancer model was established using CT26-Luc cells. CT26-Luc cell suspension was injected into the cecal wall of mice, with each mouse receiving 2 × 10⁶ CT26-Luc cells. 6 One, forming an in situ colorectal cancer model.

[0107] Mice were randomly divided into three groups: PBS group, ΔEcN-F@NS group, and ΔEcN-F@NS+US group, with 5 mice in each group. ΔEcN-F@NS was administered orally by gavage on days 3, 6, and 9, with the bacterial dose being 1 × 10⁻⁶ per mouse. 8 The CFU and PBS groups received an equal volume of PBS buffer via oral gavage; for the groups receiving ultrasound therapy (ΔEcN-F@NS+US group), the tumor site was irradiated with ultrasound at 1 MHz and 1.5 W·cm² 12 hours after each administration. -2 With a 50% duty cycle and an irradiation time of 5 minutes, some mice were sacrificed on day 12, and mesenteric lymph nodes, tumor tissue, and spleen were collected for immunoassay.

[0108] Representative photographs of the resected colon tissue taken on day 12 after the start of treatment, such as... Figure 17 As shown in the figure, the tumor size in the ΔEcN-F@NS+US group was smaller than that in the PBS group and the ΔEcN-F@NS group, which confirms the inhibitory effect of the ΔEcN-F@NS+US group on tumor growth.

[0109] The detection results of the tumor immune microenvironment, such as Figure 18 As shown; Figure A shows CD11c in the mesenteric lymph nodes of mice. + CD80 + CD86 + Representative flow cytometry images of mature dendritic cells (DCs), Figure B shows CD11c in mouse tumor tissue. + CD80 + CD86 + Representative flow cytometry images of mature dendritic cells (DCs), Figure C shows CD3 in mouse tumor tissue. - CD49b + Representative flow cytometry images of NK cells; Figure D shows CD3 in mouse tumor tissue. + CD4 - CD8 + Representative flow cytometry images of T cells, Figure E shows CD3 in mouse tumor tissue. + CD8 + IFN-γ + Representative flow cytometry images of T cells; F plot shows CD8+ in mouse spleen. +Representative flow cytometry image of central memory T cells with high CD44 and high CD62L expression.

[0110] from Figure 18 It can be seen that ΔEcN-F@NS+US treatment can increase the proportion of mature DCs, enhance NK cell infiltration, and increase CD8 levels. + T cells and CD8 + IFN-γ + T cell levels and promote CD8 + High expression of CD44 and CD62L leads to the formation of T cells.

[0111] Mouse survival analysis results, such as Figure 19 As shown in the figure, the overall survival of mice in the ΔEcN-F@NS+US group was prolonged, with some mice still surviving 40 days after tumor inoculation, while mice in the PBS group died within 25 days due to tumor progression.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a tumor immune remodeling biohybrid system, characterized in that, include: by nlpI Using the deletion-type engineered bacterium ΔEcN as the host strain, a prokaryotic expression plasmid containing the signal peptide-Flt3L fusion expression element was introduced to construct the engineered probiotic ΔEcN-F expressing Flt3L; Flt3L is an Fms-like tyrosine kinase 3 ligand; Titanium-based nano-sound-sensing agents were prepared using a titanium source that can provide titanium ions and carboxyporphyrin ligands as raw materials via hydrothermal coordination reaction. The surface of the titanium-based nano-acoustic sensor was functionalized by using a phospholipid derivative containing boric acid groups modified with polyethylene glycol to obtain a functionalized titanium-based nano-acoustic sensor. The functionalized titanium-based nano-acoustic sensitizer was coupled to the surface of the engineered probiotic ΔEcN-F to obtain a tumor immune remodeling biohybrid system.

2. The method for preparing the tumor immune remodeling biohybrid system as described in claim 1, characterized in that, The signal peptide is selected from at least one of ClyA signal peptide and OmpA signal peptide; ClyA is cytolysin A, and OmpA is outer membrane protein A.

3. The method for preparing the tumor immune remodeling biohybrid system as described in claim 1, characterized in that, The PEG-modified phospholipid derivative containing boric acid groups is DSPE-PEG2000-PBA, and DSPE-PEG2000-PBA is 1,2-distearate-sn-glycerol-3-phosphoethanolamine-PEG2000-phenylboronic acid.

4. The method for preparing the tumor immune remodeling biohybrid system as described in claim 1, characterized in that, The titanium source is any one or more of titanium tetrachloride, tetrabutyl titanate, and titanium isopropoxide. The carboxyporphyrin ligand is a porphyrin derivative containing multiple carboxyl substitutions.

5. The method for preparing the tumor immune remodeling biohybrid system as described in claim 4, characterized in that, The porphyrin derivative containing polycarboxyl substitution is 5,10,15,20-tetra(parabenzoic acid)porphyrin.

6. The method for preparing the tumor immune remodeling biohybrid system as described in claim 1, characterized in that, The titanium-based nano-acoustic sensor has a particle size distribution range of 20–90 nm, and its X-ray diffraction pattern shows characteristic diffraction peaks in the range of 2θ between 24° and 26°.

7. A tumor immune remodeling biohybrid system, characterized in that, The tumor immune remodeling biohybrid system, prepared using the method described in any one of claims 1 to 6, comprises an engineered probiotic ΔEcN-F carrier and a boric acid-functionalized titanium-based nanoacoustic agent coupled to the surface of the engineered probiotic ΔEcN-F carrier.

8. The application of a tumor immune remodeling biohybrid system, characterized in that, An antitumor product is prepared using the tumor immune remodeling biohybrid system as described in claim 7, wherein the tumor is colorectal cancer.

9. The application of the tumor immune remodeling biohybrid system as described in claim 8, characterized in that, The antitumor product has one or more of the following effects: Promotes dendritic cell maturation; Enhances natural killer cell infiltration; Increase CD8 + T cell levels; Increase CD8 + IFN-γ + T cell levels; Promote CD8 + High expression of CD62L and CD44 in T cells.

10. The application of the tumor immune remodeling biohybrid system as described in claim 8, characterized in that, The antitumor product is a pharmaceutical preparation used in conjunction with ultrasound irradiation.

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