Light-operated genetically engineered bacterium backpack and application thereof

Abstract

The invention relates to a light-operated genetically engineered bacterium knapsack and application thereof, and belongs to the technical field of genetic engineering and biological medicine crossing, the genetically engineered bacterium knapsack comprises an engineered bacterium, and the engineered bacterium is prepared by transferring a recombinant plasmid expressing a chemotactic factor CCL21 and a recombinant plasmid expressing cleavage protein into an initial strain escherichia coli; the surface of the engineering bacterium is modified with bromelain and PGA-DA-Cys. The genetically engineered bacterium knapsack can be anchored on the surface of macrophages so as to target tumor tissues, and the genetically engineered bacterium knapsack can significantly activate immune response of tumor parts so as to achieve a tumor immunotherapy effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a light-controlled genetic engineering bacteria backpack and applications thereof. Background Art

[0002] With the development of medical technology, immunotherapy has attracted increasing attention. Recently, cell-based drug delivery systems have attracted attention as a method to enhance the safety and efficacy of immunotherapy. Cell-based drug delivery systems commonly use red blood cells, T cells, NK cells, macrophages, and neutrophils in tumor treatment.

[0003] Macrophages, as one of the most abundant immune cells in the tumor microenvironment, possess natural tumor homing and innate phagocytic abilities, making them highly attractive candidates for tumor-targeted drug delivery. Currently, research on macrophage-based drug delivery systems has made considerable progress. For example, one study utilized the biotin-avidin interaction to modify doxorubicin-loaded liposomes on the macrophage surface, constructing a macrophage-liposome (MA-Lip) composite system. This system not only improves the efficiency of doxorubicin accumulation at the tumor site but also promotes deep penetration of the drug into the tumor and enhances the anti-tumor immune response. Furthermore, another study designed an IFN-γ nanoparticle "backpack" that, by loading it onto the macrophage surface, allows the nanoparticles to continuously release IFN-γ, inducing macrophage polarization toward the M1 phenotype, thereby exerting a tumor-killing effect. However, existing nanocarriers still suffer from issues such as drug leakage, low protein factor loading efficiency, and insufficient stability, limiting their potential for clinical application. Therefore, the development of an efficient, stable, and controllable macrophage delivery system remains a key focus. Summary of the Invention

[0004] In order to obtain a new tumor treatment reagent, the present invention provides a light-controlled genetically engineered bacteria backpack that can anchor to the surface of macrophages and then target tumor tissue. The genetically engineered bacteria backpack can significantly activate the immune response at the tumor site and achieve a tumor immunotherapy effect.

[0005] The present invention also provides an application of a light-controlled genetically engineered bacteria backpack in the preparation of tumor prevention and treatment drugs.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a light-controlled genetically engineered bacteria backpack, which includes engineered bacteria. The engineered bacteria are prepared by transferring a recombinant plasmid pET32a-CCL21 expressing a chemokine CCL21 and a recombinant plasmid pBV220-φX174E-p15A expressing a lytic protein into a starting strain of Escherichia coli.

[0008] The maps of the pET32a-CCL21 and the pBV220-φX174E-p15A are as follows Figure 12 As shown;

[0009] The surface of the engineered bacteria is modified with bromelain and PGA-DA-Cys.

[0010] Based on the same inventive concept, the present invention provides an application of a light-controlled genetically engineered bacteria backpack in the preparation of tumor prevention and treatment drugs.

[0011] Based on the same inventive concept, the present invention provides a tumor-targeting agent, which includes macrophages and the above-mentioned light-controlled genetically engineered bacteria backpack, and the genetically engineered bacteria backpack is anchored on the surface of the macrophages.

[0012] Optionally, the macrophages include RAW264.7.

[0013] Based on the same inventive concept, the present invention provides a method for preparing a tumor targeting agent, the preparation method comprising:

[0014] 1) Constructing a recombinant plasmid pET32a-CCL21 expressing the chemokine CCL21 and a recombinant plasmid pBV220-φX174E-p15A expressing the lytic protein. The maps of the pET32a-CCL21 and the pBV220-φX174E-p15A are as follows: Figure 12 As shown;

[0015] 2) Transforming the pET32a-CCL21 and the pBV220-φX174E-p15A into Escherichia coli, and obtaining the engineered bacteria Bac-LC after screening;

[0016] 3) stirring the Bac-LC, bromelain, and PGA-DA-Cys at pH 8.5 to obtain a genetically engineered backpack strain Bac-LC-BD;

[0017] 4) The Bac-LC-BD is dispersed in a culture medium containing MAL-PEG-NHS, and then co-incubated with macrophages to obtain the tumor-targeting reagent Bac-LC-BD@M.

[0018] Based on the same inventive concept, the present invention provides an application of a tumor targeting agent in the preparation of tumor prevention and treatment drugs.

[0019] Based on the same inventive concept, the present invention provides a tumor-targeted therapeutic drug, the active ingredient of which comprises the above-mentioned light-controlled genetically engineered bacteria backpack.

[0020] Alternatively, the present invention provides a tumor-targeted therapeutic drug, wherein the active ingredient of the drug comprises the above-mentioned tumor-targeting agent.

[0021] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0022] 1. The present invention discloses a light-controlled genetically engineered bacterial backpack, comprising an engineered bacterium and surface-modified bromelain and PGA-DA-Cys. Escherichia coli is genetically engineered and recombinant plasmids expressing a bacterial lytic protein (protein E of phiX174 phage) and a chemokine, CCL21, are transferred into the bacterium. DA contained on the surface of the engineered bacterial backpack converts light energy into heat energy, causing a temperature rise. The resulting pBV220-φX174E-p15A plasmid in the engineered bacterium can be induced to express the bacterial lytic protein at 42°C, causing the engineered bacterium to lyse. The engineered bacterium lyses and releases the pET32a-CCL21 plasmid, which in turn induces the expression of synthesized CCL21 and its contents. The fragments and bacterial contents produced by bacterial lysis can effectively regulate the transformation of M2 macrophages to M1 macrophages. CCL21 recruits more immune cells to the tumor site, significantly activating the immune response at the tumor site to achieve a tumor immunotherapy effect. Furthermore, the temperature rises to the optimal temperature for bromelain, thereby better degrading the tumor extracellular matrix.

[0023] 2. The present invention provides a tumor-targeting agent comprising macrophages and a backpack (Bac-LC-BD) anchored on their surface. By anchoring genetically engineered bacteria on the surface of macrophages, a bacterial "backpack" macrophage delivery system (Bac-LC-BD@M) is constructed. This system fully utilizes the macrophages' ability to target tumor sites, achieving efficient delivery of the bacterial "backpack" and utilizing a controllable lysis-immune activation cascade reaction to reshape the tumor microenvironment for effective tumor treatment.

[0024] 3. The present invention provides a tumor-targeting agent comprising a macrophage and a bacterial backpack anchored on its surface. Bacteria are natural immune agents that can interact with pattern recognition receptors such as Toll-like receptors, leading to the activation of NF-κB, a major transcription factor driving inflammation, thereby stimulating an immune response and exerting an anti-tumor effect. Compared with traditional anti-tumor drugs, the engineered bacteria-macrophage system combines the tumor-targeting ability of macrophages with the immunogenicity of the bacteria themselves, can stimulate an immune response at the tumor site, repolarize M2 macrophages at the tumor site to M1 macrophages, reshape the immunosuppressive tumor microenvironment, and have better biosafety.

[0025] 4. The present invention relates to a tumor-targeting agent. The tumor microenvironment refers to the local microenvironment in which tumors thrive. Its hypoxia, low pH, and immunosuppressive properties play a crucial role in the development, progression, and metastasis of tumors. These characteristics not only inhibit the function of immune cells in the tumor site but also provide unique conditions for bacterial colonization. Once macrophages transport bacteria to the tumor site, they are unable to control their subsequent growth. Overgrowth of bacteria can easily produce side effects on the body, limiting the effectiveness of bacterial treatment for tumors. Improving the safety of bacterial therapy and enhancing bacterial accumulation and therapeutic efficacy at the tumor site remain key scientific challenges facing bacterial cancer treatment. After colonization in tumor tissue, the engineered bacterial backpack of the present invention can be photothermally induced to express bacterial lysis proteins, causing the backpack to lyse at the tumor site, releasing synthesized CCL21 and its contents. This significantly activates the immune response at the tumor site and kills bacteria, improving the biosafety of the tumor-targeting agent and mitigating toxic side effects in non-target organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 Schematic diagram of the construction of the bacterial "backpack" macrophage delivery system (Bac-LC-BD@M).

[0028] Figure 2 Characterization results of bacterial "backpacks": (A) BCA assay for protein content on the surface of engineered bacteria with different treatments; (B) Zeta potential on the surface of engineered bacteria with different treatments; (C) growth curve of engineered bacteria; (D) fluorescence image of the engineered bacteria backpack, red represents bacteria, green represents PGA-DA-FAM, scale bar: 25 μm.

[0029] Figure 3 Verification of the expression function of Bac-LC: (A) Growth curves of wild-type bacteria and engineered bacteria at 42°C (n=3); (B) SDS-PAGE analysis of Bac-LC, lane M: Marker; lane C: uninduced E. coli; lane 1: bacterial culture of dual-plasmid engineered bacteria after IPTG induction; lane 2: supernatant of Bac-LC after IPTG induction and heat induction.

[0030] Figure 4 SEM image of Bac-LC-BD@M, scale bar: 25 μm.

[0031] Figure 5 Bac-LC-BD@M targeting experiment: (A) Representative IVIS images of tumor tropism after inoculation of different macrophages in the 4T1 mouse model; (B) Fluorescence quantification of mouse tumor sites.

[0032] Figure 6 After tumor-bearing mice were injected with drugs, they were irradiated with laser (808nm, 3min) and the thermal images and temperature changes of the tumor site were taken.

[0033] Figure 7 Evaluation of the anti-tumor effect of Bac-LC-BD@M: (A) Summary of tumor growth curves after different treatments; (B) Photograph of 4T1 tumor after treatment.

[0034] Figure 8 The results of TNF-ɑ (A) and IL-6 (B) detection in the serum of mice after treatment.

[0035] Figure 9 Figure 2 Flow cytometric analysis of immune cells in tumor sites: (A) Flow cytometric analysis and ratio quantitative analysis of M2 macrophages in tumor tissue; (B) Flow cytometric analysis and ratio quantitative analysis of DC cells; (C) Flow cytometric detection of T cells in tumors and quantitative analysis of CD8+ T cell ratio.

[0036] Figure 10 Analysis of CCL21 protein in mice: (A) Western blot detection of CCL21 in the tumor site (T) and surrounding healthy tissue (SNT) of mice; (B) Western blot semi-quantification.

[0037] Figure 11 These are the blood biochemical indicators of mice after treatment.

[0038] Figure 12 The following are the maps of the recombinant plasmids pET32a-CCL21 and pBV220-φX174E-p15A. DETAILED DESCRIPTION

[0039] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0040] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0041] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0043] The light-controlled genetically engineered bacteria backpack and its application of the present invention will be described in detail below with reference to examples and experimental data.

[0044] Example 1

[0045] This embodiment provides a method for preparing a tumor targeting agent, which is as follows:

[0046] (1) Construction of recombinant plasmid pET32a-CCL21 expressing chemokine CCL21 and recombinant plasmid pBV220-φX174E-p15A expressing lytic protein:

[0047] Construction of pET32a-CCL21: The amino acid sequence was obtained from NCBI. Synonymous codons were optimized based on the codon translation preferences of E. coli to improve translation efficiency and increase the proportion of soluble protein expression. Finally, the protein was synthesized at Jinweizhi Biotechnology Co., Ltd. For the pBV220-φX174E-p15A plasmid, the f1ori on the laboratory-preserved plasmid was replaced with the p15A ori using seamless cloning technology.

[0048] (2) The two recombinant plasmids were sequentially transformed into Escherichia coli competent cells by chemical transformation, and positive colonies were screened by resistance genes to obtain the engineered bacteria Bac-LC;

[0049] (3) The Bac-LC, bromelain and PGA-DA-Cys were stirred at pH 8.5 to obtain the genetically engineered bacterial backpack Bac-LC-BD:

[0050] The engineered bacteria Bac-LC was taken out from -80°C, thawed on ice, and added to LB medium. When OD600 = 0.4-0.6, centrifugation was performed and the supernatant was discarded. The obtained bacteria, bromelain, and PGA-DA-Cys were added to Tris buffer at pH 8.5 and stirred.

[0051] Wherein, PGA-DA-Cys is prepared by the following method:

[0052] γ-PGA was dissolved in deionized water at a concentration of 1%. EDC was added to the reaction solution and stirred for 30 minutes. Dopamine hydrochloride and NHS were then added sequentially to the mixture. The pH was adjusted to 5, and the mixture was stirred under N2 for 24 hours. After completion of the reaction, the reaction mixture was dialyzed for 48 hours using a 14 kDa dialysis bag. The dialyzed liquid was lyophilized to obtain purified γ-PGA-DA. The resulting γ-PGA-DA was dissolved in MES buffer, EDC was added, and the mixture was stirred at room temperature for 30 minutes. L-cysteine ​​and NHS were then added to the mixture. The mixture was stirred under N2 for 24 hours. The resulting mixture was dialyzed for 48 hours using a 14 kDa dialysis bag, and the dialyzed liquid was lyophilized.

[0053] (4) Dispersing the Bac-LC-BD in a culture medium containing MAL-PEG-NHS to obtain the tumor targeting reagent Bac-LC-BD@M:

[0054] Using the cell suspension digested during passage, a certain amount of cells were aspirated and inoculated into a 6-well plate after cell counting. The density of RAW 264.7 macrophages was 5x10 5 cells / well. Mix well to evenly distribute the cells in the plate, and finally place them in the incubator for culture. When the cells have attached to the wall, perform the following operations:

[0055] 1) First, add the synthesized bacterial backpack cells into DMEM medium containing 1 mg / mL MAL-PEG-NHS, mix well, and let it stand for 1 hour.

[0056] 2) Aspirate the culture medium of RAW 264.7 and add the liquid prepared in step 1. Incubate for 1 hour. Aspirate the supernatant again and add new culture medium to obtain Bac-LC-BD@M.

[0057] The surface functionalization of the engineered bacteria is achieved by coating bromelain and a thiol-polyglutamic acid-dopamine complex (PGA-DA-Cys) through covalent coupling technology. The functionalized modification layer mediates the complex to specifically bind to macrophage surface proteins, forming a genetically engineered bacteria-macrophage backpack.

[0058] Example 2

[0059] In this example, Bac-LC-BD was characterized and its performance was tested.

[0060] 1. Experimental method.

[0061] 1.1. Construction of Bac-LC-BD engineered bacteria backpack

[0062] The genetically engineered bacterial backpack Bac-LC-BD was constructed by referring to steps (1) to (3) of Example 1. The bacterial surface protein content was detected using a BCA protein quantification kit.

[0063] 1.2. Construction of Bac-LC-BD@M

[0064] Referring to steps (1) to (4) of Example 1, Bac-LC-BD@M was constructed.

[0065] 1.3. Induced expression of Bac-LC engineered bacteria

[0066] The engineered bacteria Bac-LC (transfected with plasmid but not modified with bromelain and DA-PGA-Cys) was taken out of the -80℃ freezer, thawed on ice, and LB medium containing ampicillin (50 μg / mL) and chloramphenicol (20 μg / mL) was added. The OD 600 When the value is 0.6-0.8, add 0.5mM IPTG to induce the production of CCL21 for 5 hours. After induction, centrifuge the bacterial solution, discard the supernatant, add new culture medium (containing ampicillin and chloramphenicol) and transfer to 42℃ for incubation. Take the bacterial solution every hour to measure the OD 600 The bacterial growth was statistically observed. The supernatants of the uninduced, IPTG-induced, and heat-induced bacterial cultures were run on SDS-PAGE to observe protein expression.

[0067] 1.4 Characterization of Bac-LC-BD@M

[0068] Bac-LC-BD@M was observed using SEM. First, cells were plated at 5x10 5 The cells were inoculated on the cell slide at a concentration of 1 μg / mL. After the cells adhered to the wall, the supernatant was aspirated and washed three times with PBS. 1 mL of 2.5% glutaraldehyde fixative was added and fixed at 4°C for 1 hour. After fixation, the cells were washed three times with sterile water and treated with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol, three times for each gradient, and 5 minutes each time. Finally, after the ethanol treatment was completed, the supernatant was discarded and the cells were treated with tert-butanol three times for 5 minutes each time. After the tert-butanol treatment was completed, a small amount of tert-butanol remained, which was frozen at -20°C and freeze-dried using a freeze dryer to ensure the preservation of the structural integrity of the sample, and then observed by transmission electron microscopy.

[0069] 1.5. In vivo distribution experiments

[0070] The subcutaneous 4T1 tumor model was established by stabilizing 6-8 week-old female BALB / C mice and incubating 4T1 cell suspension at 2×10 6 The cells were inoculated subcutaneously at the hind legs of mice at a density of 1 / 100 and the tumor size was recorded every two days. V = 0.5 × L × W 2, where L and W are the longest and shortest lengths of the tumor, respectively) were randomly divided into three groups: group (normal RAW264.7 cells), LPS group (RAW264.7 cells treated with 500 ng / mL LPS for 24 h), Bac-LC-BD@M group (1×10 6 CFU). In vivo imaging was performed 2 h, 4 h, 8 h, and 12 h after injection, and the mice were sacrificed and the heart, liver, spleen, lung, kidney and other major organs were obtained for in vitro imaging.

[0071] 1.6. Photothermal heating experiment on mouse tumors

[0072] Mice with subcutaneous 4T1 tumor model were randomly divided into 5 groups: PBS group, group (normal RAW264.7 cells), LPS group (RAW264.7 cells treated with 500 ng / mL LPS for 24 h), group (RAW264.7 cells and engineered bacteria were mixed), and Bac-LC-BD@M group. After drug injection (1×10 6 CFU, injected once every two days for a total of four times). Based on the results of in vivo distribution experiments, mice were irradiated with an 808nm infrared laser for 3 minutes 4 hours after each injection. Temperature changes in the mice were observed using an infrared thermal imager, and infrared thermal images of the mice were taken every 30 seconds. The resulting photothermal temperature rise curves were then plotted.

[0073] 1.7. Establishment of subcutaneous tumor model

[0074] After 6-8 weeks old female BALB / C mice were stable, 4T1 cell suspension was cultured at 2×10 6 The cells were inoculated subcutaneously at the hind legs of mice at a density of 1 / 100 and the tumor size was recorded every two days. V = 0.5 × L × W 2 . L and W are the longest and shortest lengths of the tumor, respectively. The tumor-bearing mice were randomly divided into 4 groups: PBS group (intravenous injection of PBS), Bac-L-BD@ML group (intravenous injection of Bac-L-BD@M, irradiation), Bac-LC-BD@M-RT group (intravenous injection of Bac-LC-BD@M, no irradiation), Bac-LC-BD@ML group (intravenous injection of Bac-LC-BD@M, irradiation - the difference between this group and the previous "Bac-L-BD@ML group" is that the engineered bacteria used in the Bac-L-BD@M system are not transferred with the CCL21 expression recombinant plasmid, but only with the lytic protein recombinant plasmid). Each mouse was intravenously injected with 1×10 6CFU were injected once every three days for a total of four times. Based on the targeting effect, 808 nm light irradiation was performed for 3 minutes 4 hours after each injection. To evaluate the in vivo immunosuppressive TME remodeling effect mediated by Bac-LC-BD@M, tumor tissues were collected from mice after treatment and flow cytometry was performed to detect immune cells in the tumor site. Serum was collected from mice to measure TNF-α and IL-6 levels.

[0075] 2. Experimental Results

[0076] a. Design and Characterization of Engineered Bacteria Backpacks

[0077] We constructed a bacterial "backpack" macrophage delivery system (Bac-LC-BD@M), which targets the delivery of engineered bacteria through the natural tumor-tropism properties of macrophages and uses a controllable lysis-immune activation cascade reaction to reshape the tumor microenvironment and effectively treat tumors.

[0078] like Figure 1 As shown, first, the present invention transferred the recombinant plasmids expressing the lytic protein and the chemokine CCL21 into Escherichia coli in succession to construct the Bac-LC engineered bacteria. Subsequently, we used polymer surface modification technology to modify bromelain and PGA-DA-Cys onto the surface of the engineered bacteria, successfully preparing the engineered bacteria "backpack". Finally, the "backpack" was connected to the surface of macrophages to successfully construct Bac-LC-BD@M. To verify whether the bacterial backpack was successfully synthesized, we used the BCA protein detection kit to detect the proteins on the bacterial surface, as shown in the figure. Figure 2 As shown in A, the surface protein content of Bac-LC-BD is significantly higher than that of untreated bacterial Bac-LC, indicating that bromelain has been successfully modified onto the bacterial surface. Since γ-PGA carries a large amount of negative charge, the surface Zeta potential of the bacterial "backpack" (Bac-BD) is significantly lower than that of the untreated bacterial Bac-LC ( Figure 2 B). In addition, the bacterial growth curve shows that ( Figure 2 C), after a series of modifications, the normal physiological activities of the bacteria were not affected, ensuring that while it played a specific function, it would not affect the overall effect due to excessive interference with the survival state of the bacteria. Bacteria that can express mCherry fluorescent protein were used to synthesize bacterial backpacks, and PGA-DA-Cys was replaced with PGA-DA-FAM (the purpose of the replacement was: FAM is a green fluorescent substance used to mark PGA-DA, and mCherry fluorescent protein is a red fluorescent substance used to mark bacteria). The bacterial "backpack" was observed under an inverted fluorescence microscope. Red represents bacteria, and green represents PGA-DA-FAM. The red fluorescence completely overlapped with the green fluorescence, indicating that PGA-DA-FAM was successfully modified to the bacterial surface ( Figure 2D) The above data show that bromelain and PGA-DA-Cys were modified onto the surface of the engineered bacteria, and the engineered bacteria "backpack" was successfully prepared.

[0079] To prove the effect of the engineered bacteria, they were induced, and the lysis curve showed that compared with the Bac group, the OD of the Bac-L group and the Bac-LC group (both without bromelain and PGA-DA-Cys modification) after heat induction for 1 h was 600 The value no longer increases, and CCL21 can be detected in the bacterial supernatant, proving that after the pET32a-CCL21 plasmid is transferred, the Bac-LC engineered bacteria can still lyse normally and release the expressed CCL21 ( Figure 3 ). Finally, we observed the construction effect of Bac-LC-BD@M through SEM images. Figure 4 It shows the presence of engineered bacterial backpacks on the cell surface.

[0080] b. Tumor targeting experiment of Bac-LC-BD@M

[0081] To demonstrate the targeting effect of Bac-L-BD@M on tumors, the drug was injected into the tail vein of 4T1 tumor-bearing mice, and the drug distribution in vivo was observed by IVIS. Figure 5 As shown in the figure, mouse fluorescence imaging results show that 2 hours after intravenous injection, the drug is distributed throughout the body's tissues and organs through the circulatory system. Fluorescence at the tumor site is significantly stronger than at other sites, and the fluorescence intensity at the tumor site in the Bac-LC-BD@M group is greater than that in the other two groups, demonstrating that Bac-LC-BD@M has good tumor targeting ability. The fluorescence intensity reaches a maximum 4 hours after injection and remains at a relatively high level thereafter. These results demonstrate that Bac-LC-BD@M has strong tumor targeting ability, reaching a peak at the tumor site 4 hours after injection, providing an important reference for selecting the time point for subsequent in vivo photothermal therapy.

[0082] c. Photothermal experiment of Bac-LC-BD@M in mice

[0083] To evaluate the photothermal therapeutic effect of Bac-LC-BD@M in vivo, tumor-bearing mice were randomly divided into groups and given corresponding drugs (PBS, LPS, and Bac-LC-BD@M). 4 hours after administration, the tumor site was irradiated with 808 nm laser, and the temperature changes in the tumor area were monitored in real time using an infrared thermal imager. Figure 6 As shown, during laser irradiation, the PBS group, group, LPS group and While the tumor tissue temperature in the Bac-LC-BD@M group showed only a slight increase, the temperature at the tumor site in the Bac-LC-BD@M group increased significantly (the engineered bacteria backpack contains DA, which converts light energy into heat energy under 808nm laser irradiation), ultimately reaching 45°C. This result demonstrates that Bac-LC-BD@M can significantly increase the temperature at the tumor site under 808nm laser irradiation, demonstrating an excellent photothermal effect and providing important experimental evidence for further tumor treatment research.

[0084] d. Inhibitory effect of Bac-LC-BD@M on tumors

[0085] To evaluate the anti-tumor effect in vivo, we established a 4T1 subcutaneous tumor model using BALB / c mice. The tumor-bearing mice were randomly divided into four groups and injected into the tail vein according to the grouping and treatment plan. The group requiring light exposure was exposed to light 4 hours after injection. The tumor volume was measured every two days during the treatment period. Figure 7 As shown in A, compared with the PBS group, the Bac-L-BD@ML group and the Bac-LC-BD@M-RT group inhibited tumor growth. However, the tumor in the Bac-LC-BD@ML group grew the slowest, and the tumor size was basically 300 mm at the end of treatment. 3 The following proves that the Bac-LC-BD@ML group has the best tumor treatment effect. After the mice were euthanized, we removed the mouse tumor tissue and took pictures ( Figure 7 B) The results intuitively reflect this result. The above results all prove that Bac-LC-BD@ML has a significant anti-tumor effect.

[0086] e. Bac-LC-BD@M reshapes the tumor suppressive microenvironment

[0087] After the treatment was completed, the proportion of M2 macrophages in the tumor tissue was first examined by flow cytometry. Figure 9 As shown in the figure, the proportion of M2 macrophages in the Bac-L-BD@ML and Bac-LC-BD@M-RT groups was similar, and they had a certain inhibitory effect on tumors compared with the PBS group. The proportion of M2 macrophages in the Bac-LC-BD@ML group was the lowest, indicating that the Bac-LC-BD@ML group had the best anti-tumor effect. At the same time, we also detected the proportion of T cells and DC cells by flow cytometry, as shown in the figure. Figure 9As shown in B and C, the T cells and DC cells in the Bac-L-BD@ML, Bac-LC-BD@M-RT and Bac-LC-BD@ML groups were significantly increased compared with the PBS group, among which the Bac-LC-BD@ML group had the highest ratio, indicating that high concentrations of CCL21 in the tumor site can effectively recruit more immune cells and enhance their infiltration in the tumor site. In addition, the TNF-α and IL-6 ELISA results in mouse serum ( Figure 8 ) showed that serum TNF-α and IL-6 concentrations in mice in the Bac-LC-BD@ML group were higher than in the other groups. These results demonstrate that after Bac-LC-BD@M reaches tumor tissue, light irradiation lyses the bacteria, releasing the produced CCL21. This attracts more immune cells to the tumor tissue, enhancing the immune response at the tumor site and further strengthening the anti-tumor effect.

[0088] f. Biosafety of the Bac-LC-BD@M System

[0089] To prove the biosafety of Bac-LC-BD@M, we took tumor tissue and healthy tissue around the tumor for Western blot analysis after the treatment. Figure 10 As shown in the results, no CCL21 was detected in the tumor tissues and normal tissues surrounding the tumors in the PBS group, Bac-L-BD@ML group, and Bac-LC-BD@M-RT group. No CCL21 was detected in the normal tissues surrounding the tumors in the Bac-LC-BD@ML group. Only the tumor tissues in the Bac-LC-BD@ML group were detected with CCL21. This indicates that Bac-LC-BD@M can accumulate in tumor tissues and CCL21 is released only after light irradiation, without adverse effects on normal tissues. ALT, AST, BUN, CREA( Figure 11 ) had no significant difference from the control group, and the values ​​were all within the normal range, indicating that the liver and kidney functions of the mice were normal.

[0090] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0091] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0092] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A light-controlled genetically engineered bacteria backpack, characterized in that: The genetically engineered bacteria backpack includes engineered bacteria, which are prepared by transferring the recombinant plasmid pET32a-CCL21 expressing the chemokine CCL21 and the recombinant plasmid pBV220-φX174E-p15A expressing the lytic protein into the starting strain Escherichia coli; The maps of the pET32a-CCL21 and the pBV220-φX174E-p15A are shown in FIG12 ; The surface of the engineered bacteria is modified with bromelain and PGA-DA-Cys.

2. Use of a light-controlled genetically engineered bacteria backpack as claimed in claim 1 in the preparation of tumor prevention and treatment drugs.

3. A tumor targeting agent, characterized in that: The tumor targeting agent comprises macrophages and the light-controlled genetically engineered bacteria backpack according to claim 1, wherein the genetically engineered bacteria backpack is anchored on the surface of the macrophages.

4. The method for preparing a tumor targeting agent according to claim 3, wherein: The preparation method comprises: 1) Constructing a recombinant plasmid pET32a-CCL21 expressing the chemokine CCL21 and a recombinant plasmid pBV220-φX174E-p15A expressing the lytic protein. The maps of the pET32a-CCL21 and the pBV220-φX174E-p15A are shown in FIG12 ; 2) Transforming the pET32a-CCL21 and the pBV220-φX174E-p15A into Escherichia coli, and obtaining the engineered bacteria Bac-LC after screening; 3) stirring the Bac-LC, bromelain, and PGA-DA-Cys at pH 8.5 to obtain a genetically engineered backpack strain Bac-LC-BD; 4) The Bac-LC-BD is dispersed in a culture medium containing MAL-PEG-NHS, and then co-incubated with macrophages to obtain the tumor-targeting reagent Bac-LC-BD@M.

5. Use of a tumor targeting agent as claimed in claim 3 in the preparation of tumor prevention and treatment drugs.

6. A tumor targeted therapeutic drug, characterized in that: The active ingredient of the medicine comprises the light-controlled genetically engineered bacteria backpack described in claim 1.

7. A tumor targeted therapeutic drug, characterized in that: The active ingredient of the medicine comprises the tumor targeting agent according to claim 3.

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