A phosphorus-containing melanin-engineered bacterial preparation, its preparation method and its biomedical application

Abstract

This invention discloses a tumor-targeting engineered bacterial preparation based on phosphorus-containing melanin, its preparation method, and its uses, belonging to the field of biomedical technology. The preparation is an engineered bacterial preparation with phosphorus-containing melanin coated on its surface or internally, obtained by catalysis of a chassis microorganism expressing tyrosinase and a phosphorus-containing functional substrate peptide. This engineered bacterial preparation can target hypoxic areas of tumors, achieving highly efficient local photothermal ablation of the tumor under near-infrared irradiation. Through the triple synergistic effects of photothermal-induced immunogenic cell death, the engineered bacterial immune adjuvant effect, and substrate peptide immunomodulation, it inhibits the growth of primary tumors and distant metastasis, exhibiting good biosafety and long-term immune protection. This invention provides a biological agent integrating active targeting, in-situ biosynthesis, photothermal therapy, and immune activation, demonstrating great application potential in the comprehensive treatment of solid tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a tumor-targeting engineered bacterium based on phosphorus-containing melanin and its application in photothermal immunotherapy. This engineered bacterium can synthesize phosphorus-containing melanin with near-infrared absorption and immunomodulatory functions in situ in a culture medium, for use in photothermal ablation and immune activation of solid tumors. Background Technology

[0002] Photothermal immunotherapy is a significant breakthrough in the field of tumor treatment in recent years. It converts light energy into heat energy to locally ablate tumors and releases tumor antigens to activate the whole body's immune system, achieving synergistic treatment of primary and metastatic tumors. Due to its advantages such as non-invasiveness, strong controllability, and ability to stimulate immune memory, this technology has become a strong candidate to replace traditional radiotherapy and chemotherapy. However, despite its promising prospects, existing photothermal immunotherapy regimens still face a series of key technical bottlenecks in clinical translation, which seriously restrict their therapeutic effects and widespread application. Specifically, these include: (1) Low targeted delivery efficiency: Traditional photothermal nanoparticles mainly rely on passive targeting (such as the EPR effect) or receptor-mediated endocytosis to achieve tumor enrichment. However, due to physiological barriers such as abnormal tumor vascular system, dense extracellular matrix and high interstitial pressure, the accumulation of photothermal agents in the tumor site is insufficient and the penetration depth is limited; (2) The immune activation ability of existing photothermal materials is limited and it is difficult to reverse the tumor immunosuppressive microenvironment: the intensity of immunogenic cell death induced by simple photothermal therapy is limited and it is difficult to continuously reverse the tumor immunosuppressive microenvironment, and the inhibitory effect on tumor recurrence and distant metastasis is not ideal; (3) The material has a single function: most photothermal agents only have thermal ablation ability and lack synergistic immune regulation function, making it difficult to maximize the therapeutic effect.

[0003] To address the aforementioned issues, researchers have recently attempted to introduce engineered bacterial delivery systems to seek breakthroughs. Bacterial therapy, due to its natural chemotaxis and colonization ability in hypoxic tumor regions, is considered an ideal carrier for overcoming tumor delivery barriers. Engineered bacteria can not only actively penetrate deep into tumors, but their inherent immunostimulatory effects can also serve as natural adjuvants, enhancing anti-tumor immune responses. Chinese invention patent application CN 117462675 A provides an engineered bacterial melanin-loaded nanosystem, which achieves variable-size tumor retention and metal ion loading through modification of specific peptides, effectively activating immune pathways. Furthermore, Chinese invention patent application CN 116333949 A constructs engineered bacteria that express tyrosinase at a controlled temperature, enabling the in-situ synthesis of melanin in tumors under laser induction for photothermal-immunotherapy. However, these research systems still suffer from limitations such as a single melanin type and simple substrate function, restricting further improvements in their photothermal performance and immunomodulatory capabilities.

[0004] This invention, based on synthetic biology and peptide molecular engineering technology, innovatively proposes a phosphorus-containing melanin-functionalized engineered bacterium with the following outstanding advantages: 1) Utilizing the engineered bacterium's natural chemotaxis towards hypoxic tumor regions, it achieves active enrichment and uniform distribution of photothermal agents deep within the tumor; 2) By introducing phosphate groups, it enhances the photothermal conversion efficiency of melanin, giving it stronger absorption and more efficient heating capabilities in the near-infrared region; 3) It integrates the catalytic sequence and immunologically active peptides into the same substrate, achieving "in-situ synthesis". Photothermal therapy The invention provides a novel tumor therapeutic agent that integrates active targeting, efficient photothermal therapy, and immune activation, demonstrating clear clinical application value and market prospects. This agent exhibits three synergistic functions: 4) direct biosynthesis of phosphorus-containing melanin within bacteria, avoiding the use of complex nanocarriers and improving system stability and biosafety. Summary of the Invention

[0005] To address the shortcomings and drawbacks of the existing technologies, this invention provides a tumor-targeting engineered bacterial preparation based on phosphorus-containing melanin. It uses a phosphorus-containing functional peptide as a substrate, which possesses both the properties of conversion into phosphorus-containing melanin and the ability to exert immunomodulatory effects. By heterologously expressing oxidases such as tyrosinase in engineered bacteria as catalysts, and co-incubating with the substrate peptide, engineered bacteria capable of in-situ synthesis of functionalized phosphorus-containing melanin can be obtained. The phosphorus-containing melanin-functionalized engineered bacterial preparation provided by this invention is expected to solve key technical bottlenecks in targeted delivery, material properties, and immunomodulation in existing photothermal immunotherapy, and has promising clinical application prospects and industrialization value.

[0006] To achieve the above objectives, the specific technical solution of the present invention is as follows: In a first aspect, the present invention provides a tumor-targeting engineered bacterial preparation based on phosphorus-containing melanin, wherein the engineered bacterial preparation comprises engineered bacteria whose surface or interior is coated with phosphorus-containing melanin: The engineered bacteria whose surface or interior is coated with phosphorus-containing melanin are formed by engineered bacteria that can express tyrosinase catalyzing the formation of phosphorus-containing melanin with near-infrared absorption capability in the presence of phosphorus-containing functional substrate peptides.

[0007] The phosphorus-containing functional substrate peptide is shown in general formula (I): X1—Y—X2—Z, where the N-terminal X1—Y—X2 is a tyrosinase catalytic fragment containing at least one phosphorylation modification group (abbreviation: p); the C-terminal Z is an immunomodulatory peptide. X1 is a phosphorylated amino acid; X2 is a flexible amino acid selected from glycine (abbreviation: G), alanine (abbreviation: A), valine (abbreviation: V), isoleucine (abbreviation: I), and leucine (abbreviation: L); Y is tyrosine; The engineered bacteria are one of Escherichia coli, Bacillus megaterium, attenuated Salmonella, Bacillus subtilis, Lactobacillus rhamnosus, and Bifidobacterium, carrying a tyrosinase expression plasmid.

[0008] Preferably, X1 is phosphorylated serine (abbreviation: pS), phosphorylated threonine (abbreviation: pT), or phosphorylated tyrosine (abbreviation: pY); Y is tyrosine (abbreviation: Y); X2 is glycine (abbreviation: G) or alanine (abbreviation: A) or other small-volume flexible amino acids; for example: pSYG, pTYG, pYYG, pSYA, pTYA, pYYA, etc.

[0009] The C-terminal immunomodulatory peptide Z is preferably the prototype of Tuftsin, its analogue, or an anti-inflammatory short peptide, more preferably KPR, TKPK, TKPRR, TTPR, RKDVY, KPV, GHK, etc. Here, the letters represent individual amino acids: K: lysine, M: methionine, T: threonine, D: aspartic acid, V: valine, Y: tyrosine, H: histidine, G: glycine, R: arginine, P: proline.

[0010] As a preferred embodiment of the present invention, in a specific embodiment, the corresponding phosphorus-containing functional substrate peptide is any combination of the following: The engineered bacterial preparation exhibits continuous absorption in the 400–900 nm wavelength range. The phosphorus-containing melanin is a melanin derivative containing phosphate groups, and its photothermal conversion efficiency is higher than that of ordinary melanin.

[0011] Secondly, the present invention provides a method for preparing a synthetic phosphorus-containing melanin-functionalized engineered bacterium, comprising: (1) Prepare a buffer solution of phosphorus-containing functional substrate peptide as a stock solution and filter it; (2) The engineered chassis microorganisms expressing tyrosinase were revived in a culture medium and cultured to a certain concentration; (3) Add the solution from step (1) to the chassis microbial solution from step (2), and add a certain concentration of copper salt for auxiliary catalysis; (4) The microbial solution in the chassis of step (3) above is reacted for a certain period of time to obtain a phosphorus-containing melanin-functionalized engineered bacteria preparation.

[0012] In step (1) above, the buffer solution is a phosphate buffer solution with a pH of 5.0-8.0; the concentration of the mother liquor is 20-200 mg / mL.

[0013] In step (2), the chassis microorganism is a gene-editable prokaryote, preferably a virus-attenuated Salmonella, Bacillus megaterium, Escherichia coli, Bacillus subtilis, Lactobacillus rhamnosus, or Bifidobacterium. The process of engineering the chassis microorganism to express tyrosinase, i.e., the transduction process of the tyrosinase plasmid, is carried out according to the literature (ActaBiomaterialia, 2024, 181, 176-187.), or commercially available chassis microorganisms expressing tyrosinase are purchased.

[0014] The following conditions should be considered for the resuscitation and culture of chassis microorganisms: In step (2), the concentration is the range of OD value of the chassis microorganisms: 0.1-2; preferably, the range of OD value of the chassis microorganisms is 0.2-1; more preferably, the range of OD value of the chassis microorganisms is 0.4-0.8.

[0015] In step (3), the concentration of the phosphorus-containing functional substrate peptide is 0.1-10 mg / mL; preferably, the concentration is 1-5 mg / mL; the copper salt includes copper sulfate, copper chloride, copper phosphate, and copper acetate. The Cu... 2+ The final concentration is 0.1~10 mM; preferably, the final concentration is 1~2 mM.

[0016] The reaction time is 6-120 h; preferably, the reaction time is 12-96 h; more preferably, the reaction time is 24-72 h.

[0017] Thirdly, this invention provides applications of the aforementioned engineered bacterial preparations. These include, but are not limited to, applications in the fields of biology and medicine, including but not limited to, molecular pigment labeling, protein tracing, and drug formulation development. The phosphorus-containing melanin-functionalized engineered bacteria, as a drug for combined photothermal immunotherapy of tumors, are preferably used as a photothermal immunotherapy drug for colorectal cancer, melanoma, glioma, liver cancer, breast cancer, or pancreatic cancer.

[0018] Beneficial effects Compared with the prior art, the present invention has the following beneficial effects: (1) Strong tumor targeting and deep penetration capabilities By utilizing the natural chemotaxis and colonization ability of bacteria in the hypoxic microenvironment of tumors, it can actively penetrate the interior of solid tumors, break through the vascular and stromal barriers, and achieve the enrichment of phosphorus-containing melanin deep within the tumor. Compared with traditional nanocarriers that rely on the EPR effect, it significantly improves the utilization rate of photothermal agents.

[0019] (2) A single platform integrates the functions of "biofactory + photothermal agent + immune adjuvant". By combining "tyrosinase engineered bacteria + phosphorus-containing functional substrate peptides", phosphorus-containing melanin is pre-synthesized, which has better photothermal conversion efficiency than ordinary melanin, while retaining immune-active peptide fragments, enabling the carrier to have three functions: photothermal agent production, tumor-targeted delivery and immune regulation.

[0020] (3) Photothermal ablation is highly efficient and has good safety. The phosphorus-containing melanin synthesized by engineered bacteria has excellent absorption and photothermal conversion capabilities in the near-infrared region. It can achieve significant temperature rise with short-term irradiation, effectively inhibiting the growth of primary tumors. As a probiotic for clinical application, engineered bacteria are easily cleared by the body and have good safety.

[0021] (4) Synergistically remodeling the tumor immune microenvironment This invention utilizes photothermal-induced immunogenic cell death, the adjuvant effect of engineered bacteria, and the immunomodulatory effects of immunomodulatory peptides to increase the number of cytotoxic T cells and effector memory T cells, effectively alleviating the immunosuppressive microenvironment.

[0022] In summary, the tumor-targeted photothermal immunotherapy formulation based on phosphorus-containing melanin-functionalized engineered bacteria provided by this invention achieves the organic integration of targeted delivery, photothermal ablation, and immune activation on a single platform, providing a new technical approach for the comprehensive treatment of solid tumors. Attached Figure Description Figure 1 This is an experimental diagram of the plasmid structure of pUC-57-TYR in Example 1; Figure 2 This is a WB identification image of the engineered bacteria expressing tyrosinase in Example 1; Figure 3 This is a photograph of the phosphorus-containing melanin-functionalized engineered bacteria prepared in Example 2. Figure 4 The ultraviolet-visible absorption spectrum of the phosphorus-containing melanin-functionalized engineered bacteria prepared in Example 2; Figure 5 The infrared spectrum of the phosphorus-containing melanin-functionalized engineered bacteria prepared in Example 2; Figure 6 Images of the phosphorus-containing melanin-functionalized engineered bacteria prepared in Examples 3-11; Figure 7 The infrared spectrum of the phosphorus-containing melanin-functionalized engineered bacteria prepared in Example 3; Figure 8 The infrared spectrum of the phosphorus-containing melanin-functionalized engineered bacteria prepared in Example 4; Figure 9 The infrared spectrum of the phosphorus-containing melanin-functionalized engineered bacteria prepared in Example 5; Figure 10 The infrared spectrum of the phosphorus-containing melanin-functionalized engineered bacteria prepared in Example 6; Figure 11 The infrared spectrum of the phosphorus-containing melanin-functionalized engineered bacteria prepared in Example 7; Figure 12 The infrared spectrum of the phosphorus-containing melanin-functionalized engineered bacteria prepared in Example 8; Figure 13 The infrared spectrum of the phosphorus-containing melanin-functionalized engineered bacteria prepared in Example 9; Figure 14 The infrared spectrum of the phosphorus-containing melanin-functionalized engineered bacteria prepared in Example 10; Figure 15 The infrared spectrum of the phosphorus-containing melanin-functionalized engineered bacteria prepared in Example 10; Figure 16 This is a photograph of the melanin-functionalized engineered bacteria prepared in Example 12. Figure 17 The infrared spectrum of the phosphorus-containing melanin-functionalized engineered bacteria prepared in Example 3; Figure 18 The phosphorus-containing melanin prepared in Example 13 31 P-NMR; Figure 19 Transmission electron microscope image of the melanin-functionalized engineered bacteria prepared in Example 2; Figure 20 Transmission electron microscope image (A) and elemental mapping diagram (B and C) of the melanin-functionalized engineered bacteria prepared in Example 3. Figure 21 Transmission electron microscope image of the melanin-functionalized engineered bacteria prepared in Example 4; Figure 22 a represents the photothermal temperature rise curves of the phosphorus-containing melanin-functionalized engineered bacteria prepared in Examples 2-4 after irradiation; Figure 22 b is the photothermal temperature rise curve of the melanin-functionalized engineered bacteria prepared in Example 12 after irradiation; Figure 23 This is a diagram showing the distribution of the phosphorus-containing melanin-functionalized engineered bacteria in mice after intravenous injection in Example 19; Figure 24 Example 20: In vivo photothermal conversion performance of phosphorus-containing melanin-functionalized engineered bacteria; Figure 25 The tumor treatment effect of phosphorus-containing melanin-functionalized engineered bacteria combined with photothermal therapy in Example 21; Figure 26 The protective effect of phosphorus-containing melanin-functionalized engineered bacteria combined with photothermal therapy on lung metastases in Example 23; Figure 27 The change in the number of T cells in the spleen of mice in Example 23; Figure 28 The change in the number of effector memory T cells in the spleen of mice in Example 23. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, the implementation of the invention is not limited thereto. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0024] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels. Specifically, the engineered E. coli expressing tyrosinase was edited according to Example 1. The other engineered chassis microorganisms expressing tyrosinase, including Bacillus megaterium, attenuated Salmonella, Bacillus subtilis, Lactobacillus rhamnosus, and Bifidobacterium, were all purchased from Hangzhou Baosai Biotechnology Co., Ltd.

[0025] Example 1: Transduction of tyrosinase plasmid in Escherichia coli To obtain chassis microorganisms expressing tyrosinase, the chassis microorganisms were transduced with tyrosinase plasmids according to the following steps: (1) Add the Escherichia coli suspension to a centrifuge tube, place it on ice for 10 min, and centrifuge at 4°C for 10 min.

[0026] (2) Then remove the supernatant and use pre-cooled 0.05 mol L -1 Slowly mix the CaCl2 solution to resuspend the bacteria, place on ice for 20 min, and centrifuge at 4°C for 10 min.

[0027] (3) After removing the supernatant, use 4 mL of pre-cooled 0.05 mol L... -1 Slowly mix a CaCl2 solution (containing 15% glycerol) to resuspend the bacteria, and briefly place it on ice to obtain a competent chassis microbial suspension.

[0028] (4) The designed and synthesized tyrosinase plasmid ( Figure 1 They were transferred into competent chassis microorganisms to obtain engineered bacteria that express tyrosinase.

[0029] (5) The expression of tyrosinase was detected by Western blot (WB).

[0030] This embodiment demonstrates that the molecular weight of tyrosinase protein is approximately 38 kDa, and clear contrast bands are visible in the Western blot results, indicating that the chassis microorganisms can correctly express tyrosinase (…). Figure 2 ).

[0031] Example 2 A method for preparing phosphorus-containing melanin-functionalized engineered Escherichia coli includes the following steps: (1) Prepare a buffer solution containing pTYGTKPR: Add pTYGTKPR peptide to a phosphate buffer solution with pH 7.4 to a concentration of 100 mg / mL, and then filter and sterilize. (2) Escherichia coli expressing tyrosinase was cultured in a medium containing 100 μg mL -1 In LB liquid medium containing kanamycin sulfate.

[0032] (3) OD 600 When the concentration is 0.6, add the phosphorus-containing functional substrate peptide solution from step 1 to a final concentration of 10 mg / mL, and add a trace amount of CuSO4 solution to the Cu... 2+ The final concentration was 10 mM.

[0033] (4) Take pictures at 0 and 24 h after addition and test the UV-Vis absorption spectra of the bacterial solution at different time points. Zero the baseline of the UV spectrometer before each test.

[0034] (5) Collect phosphorus-containing melanin-functionalized engineered Escherichia coli by centrifugation and freeze-dry them, and test the infrared spectrum of the bacterial surface.

[0035] This embodiment demonstrates that as the incubation time increases, the color of the solution gradually deepens. Figure 3 The UV absorbance of the solution at 300-900 nm showed significant absorption at 600-900 nm. Figure 4 Infrared spectroscopy results showed that, compared with the infrared spectrum of Escherichia coli, the engineered bacteria exhibited differences in the infrared spectrum at 1100-950 cm⁻¹. -1 The new or enhanced peaks are attributed to P=O stretching vibrations, POC stretching vibrations, etc., indicating the successful formation of phosphorus-containing melanin at the bacterial interface. Figure 5 ) Example 3 A method for preparing phosphorus-containing melanin-functionalized engineered Escherichia coli includes the following steps: (1) Prepare a buffer solution containing pYYGTKPR: Add pYYGTKPR peptide to a phosphate buffer solution with pH 7.4 to a concentration of 100 mg / mL, and then filter and sterilize.

[0036] (2) Escherichia coli expressing tyrosinase was cultured in a medium containing 100 μg mL-1 In TSA liquid medium containing kanamycin sulfate.

[0037] (3) OD 600 When the concentration is 1.0, add the phosphorus-containing functional substrate peptide solution from step 1 to a final concentration of 5 mg / mL, and add a trace amount of copper chloride solution to the Cu concentration. 2+ The final concentration was 2 mM.

[0038] (4) The above-mentioned chassis microbial solution was placed in a shaker at 37°C and 220 rpm for 120 h to obtain a phosphorus-containing melanin-functionalized engineered bacteria preparation.

[0039] This example demonstrates that as the incubation time increases, the color of the culture medium turns black ( Figure 6 Infrared spectroscopy results showed that, compared with the infrared spectrum of Escherichia coli, the engineered bacteria exhibited differences in the 1100-950 cm⁻¹ range. -1 The new or enhanced peaks are attributed to P=O stretching vibrations, POC stretching vibrations, etc., indicating the successful formation of phosphorus-containing melanin at the bacterial interface. Figure 7 ) Example 4 A method for preparing phosphorus-containing melanin-functionalized engineered Escherichia coli includes the following steps: (1) Prepare a buffer solution containing pSYGTKPR: Add pSYGTKPR peptide to a phosphate buffer solution with pH 7.4 to a final concentration of 50 mg / mL, and then filter and sterilize.

[0040] (2) Escherichia coli expressing tyrosinase was cultured in a medium containing 100 μg mL -1 In TSB liquid medium containing kanamycin sulfate.

[0041] (3) OD 600 When the concentration is 0.4, add the phosphorus-containing functional substrate peptide solution from step 1 to a final concentration of 0.1 mg / mL, and add a trace amount of copper chloride solution to the Cu concentration. 2+ The final concentration was 1 mM.

[0042] (4) The above-mentioned chassis microbial solution was placed in a shaker at 37°C and 220 rpm for 120 h to obtain a phosphorus-containing melanin-functionalized engineered bacteria preparation.

[0043] This example demonstrates that the culture medium turns black after the substrate peptide is added. Figure 6 Infrared spectroscopy results showed that, compared with the infrared spectrum of Escherichia coli, the engineered bacteria exhibited differences in the 1100-950 cm⁻¹ range. -1 The new or enhanced peaks are attributed to P=O stretching vibrations, POC stretching vibrations, etc., indicating the successful formation of phosphorus-containing melanin at the bacterial interface. Figure 8) Example 5 A method for preparing Bacillus megaterium based on phosphorus-containing melanin-functionalized engineered bacteria includes the following steps: (1) Prepare a buffer solution containing pTYATKPR: Add pTYATKPR peptide to a phosphate buffer solution with pH 7.4 to a final concentration of 200 mg / mL, and then filter and sterilize.

[0044] (2) Bacillus megaterium expressing tyrosinase was cultured in a solution containing 100 μg mL of [unclear text - likely a tyrosinase-containing medium]. -1 In LB liquid medium containing kanamycin sulfate.

[0045] (3) OD 600 When the concentration is 2, add the phosphorus-containing functional substrate peptide solution from step 1 to a final concentration of 0.2 mg / mL, and add a trace amount of copper acetate solution to the Cu concentration. 2+ The final concentration was 2 mM.

[0046] (4) The above-mentioned chassis microbial solution was placed in a shaker at 37°C and 220 rpm for 120 h to obtain a phosphorus-containing melanin-functionalized engineered bacteria preparation.

[0047] This example demonstrates that the culture medium turns black after the substrate peptide is added. Figure 6 Infrared spectroscopy results showed that, compared with the infrared spectrum of Escherichia coli, the engineered bacteria exhibited differences in the 1100-950 cm⁻¹ range. -1 The new or enhanced peaks are attributed to P=O stretching vibrations, POC stretching vibrations, etc., indicating the successful formation of phosphorus-containing melanin at the bacterial interface. Figure 9 ) Example 6 A method for preparing attenuated Salmonella based on phosphorus-containing melanin-functionalized engineered strains includes the following steps: (1) Prepare a buffer solution containing pYYATKPR: Add pYYATKPR peptide to a phosphate buffer solution with pH 5.0 to a final concentration of 50 mg / mL, and then filter and sterilize.

[0048] (2) Attenuated Salmonella expressing tyrosinase was cultured in a solution containing 100 μg mL of [unclear text - likely a tyrosinase-containing medium]. -1 In LB liquid medium containing kanamycin sulfate.

[0049] (3) OD 600 When the concentration is 0.6, add the phosphorus-containing functional substrate peptide solution from step 1 to a final concentration of 0.5 mg / mL, and add a trace amount of copper acetate solution to the Cu concentration. 2+ The final concentration was 0.5 mM.

[0050] (4) The above-mentioned chassis microbial solution was placed in a shaker at 37°C and 220 rpm for 48 h to obtain a phosphorus-containing melanin-functionalized engineered bacteria preparation.

[0051] This example demonstrates that the culture medium turns black after the substrate peptide is added. Figure 6 Infrared spectroscopy results showed that, compared with the infrared spectrum of Escherichia coli, the engineered bacteria exhibited differences in the 1100-950 cm⁻¹ range. -1 The new or enhanced peaks are attributed to P=O stretching vibrations, POC stretching vibrations, etc., indicating the successful formation of phosphorus-containing melanin at the bacterial interface. Figure 10 ) Example 7 A method for preparing Bacillus subtilis based on phosphorus-containing melanin-functionalized engineered bacteria includes the following steps: (1) Prepare a buffer solution containing pSYATKPR: Add pSYATKPR peptide to a phosphate buffer solution with pH 7.4 to a final concentration of 200 mg / mL, and then filter and sterilize.

[0052] (2) Bacillus subtilis expressing tyrosinase was cultured in a solution containing 100 μg mL of [unclear text - likely a tyrosinase concentration]. -1 In NB liquid medium containing kanamycin sulfate.

[0053] (3) OD 600 When the concentration is 1.2, add the phosphorus-containing functional substrate peptide solution from step 1 to a final concentration of 10 mg / mL, and add a trace amount of copper acetate solution to the Cu concentration. 2+ The final concentration was 2 mM.

[0054] (4) The above-mentioned chassis microbial solution was placed in a shaker at 37°C and 220 rpm for 72 h to obtain a phosphorus-containing melanin-functionalized engineered bacteria preparation.

[0055] This example demonstrates that the culture medium turns black after the substrate peptide is added. Figure 6 Infrared spectroscopy results showed that, compared with the infrared spectrum of Escherichia coli, the engineered bacteria exhibited differences in the 1100-950 cm⁻¹ range. -1 The new or enhanced peaks are attributed to P=O stretching vibrations, POC stretching vibrations, etc., indicating the successful formation of phosphorus-containing melanin at the bacterial interface. Figure 11 ) Example 8 A method for preparing Lactobacillus rhamnosus based on phosphorus-containing melanin-functionalized engineered bacteria includes the following steps: (1) Prepare a buffer solution containing pTYGRKDVY: Add pTYGRKDVY peptide to a phosphate buffer solution with pH 8.0 to a final concentration of 100 mg / mL, and then filter and sterilize.

[0056] (2) Culture Lactobacillus rhamnosus expressing tyrosinase in a medium containing 100 μg mL -1 In kanamycin sulfate MRS broth.

[0057] (3) OD 600 When the concentration is 0.1, add the phosphorus-containing functional substrate peptide solution from step 1 to a final concentration of 5 mg / mL, and add a trace amount of copper sulfate solution to the Cu concentration. 2+ The final concentration was 10 mM.

[0058] (4) The above-mentioned chassis microbial solution was placed in a shaker at 37°C and 220 rpm for 48 h to obtain a phosphorus-containing melanin-functionalized engineered bacteria preparation.

[0059] This example demonstrates that the culture medium turns black after the substrate peptide is added. Figure 6 Infrared spectroscopy results showed that, compared with the infrared spectrum of Escherichia coli, the engineered bacteria exhibited differences in the 1100-950 cm⁻¹ range. -1 The new or enhanced peaks are attributed to P=O stretching vibrations, POC stretching vibrations, etc., indicating the successful formation of phosphorus-containing melanin at the bacterial interface. Figure 12 ) Example 9 A method for preparing Bifidobacterium based on phosphorus-containing melanin-functionalized engineered bacteria includes the following steps: (1) Prepare a buffer solution containing pYYGRKDVY: Add pYYGRKDVY peptide to a phosphate buffer solution with pH 8.0 to a final concentration of 20 mg / mL, and then filter and sterilize.

[0060] (2) Bifidobacteria expressing tyrosinase were cultured in a solution containing 100 μg mL of water. -1 In TPY liquid medium containing kanamycin sulfate.

[0061] (3) OD 600 When the concentration is 0.4, add the phosphorus-containing functional substrate peptide solution from step 1 to a final concentration of 0.1 mg / mL, and add a trace amount of copper sulfate solution to the Cu concentration. 2+ The final concentration was 10 mM.

[0062] (4) The above-mentioned chassis microbial solution was placed in an anaerobic incubator at 37°C and reacted for 6 h to obtain a phosphorus-containing melanin-functionalized engineered bacteria preparation.

[0063] This example demonstrates that the culture medium turns black after the substrate peptide is added. Figure 6 Infrared spectroscopy results showed that, compared with the infrared spectrum of Escherichia coli, the engineered bacteria exhibited differences in the 1100-950 cm⁻¹ range. -1The new or enhanced peaks are attributed to P=O stretching vibrations, POC stretching vibrations, etc., indicating the successful formation of phosphorus-containing melanin at the bacterial interface. Figure 13 ) Example 10 A method for preparing Bifidobacterium based on phosphorus-containing melanin-functionalized engineered bacteria includes the following steps: (1) Prepare a buffer solution containing pSYGRKDVY: Add pSYGRKDVY peptide to a phosphate buffer solution with pH 8.0 to a final concentration of 20 mg / mL, and then filter and sterilize.

[0064] (2) Bifidobacteria expressing tyrosinase were cultured in a solution containing 100 μg mL of water. -1 In TPY liquid medium containing kanamycin sulfate.

[0065] (3) OD 600 When the concentration is 0.4, add the phosphorus-containing functional substrate peptide solution from step 1 to a final concentration of 0.1 mg / mL, and add a trace amount of copper phosphate solution to the Cu concentration. 2+ The final concentration was 1 mM.

[0066] (4) The above-mentioned chassis microbial solution was placed in an anaerobic incubator at 37°C and reacted for 24 h to obtain a phosphorus-containing melanin-functionalized engineered bacteria preparation.

[0067] This example demonstrates that the culture medium turns black after the substrate peptide is added. Figure 6 Infrared spectroscopy results showed that, compared with the infrared spectrum of Escherichia coli, the engineered bacteria exhibited differences in the 1100-950 cm⁻¹ range. -1 The new or enhanced peaks are attributed to P=O stretching vibrations, POC stretching vibrations, etc., indicating the successful formation of phosphorus-containing melanin at the bacterial interface. Figure 14 ) Example 11 A method for preparing Bifidobacterium based on phosphorus-containing melanin-functionalized engineered bacteria includes the following steps: (1) Prepare a buffer solution containing pTYARKDVY: Add pTYARKDVY peptide to a phosphate buffer solution with pH 8.0 to a final concentration of 20 mg / mL, and then filter and sterilize.

[0068] (2) Bifidobacteria expressing tyrosinase were cultured in a solution containing 100 μg mL of water. -1 In MRS liquid medium containing kanamycin sulfate.

[0069] (3) OD 600 When the concentration is 0.4, add the phosphorus-containing functional substrate peptide solution from step 1 to a final concentration of 0.1 mg / mL, and add a trace amount of copper phosphate solution to the Cu concentration. 2+The final concentration was 0.1 mM.

[0070] (4) The above-mentioned chassis microbial solution was placed in an anaerobic incubator at 37°C and reacted for 24 h to obtain a phosphorus-containing melanin-functionalized engineered bacteria preparation.

[0071] This example demonstrates that the culture medium turns black after the substrate peptide is added. Figure 6 Infrared spectroscopy results showed that, compared with the infrared spectrum of Escherichia coli, the engineered bacteria exhibited differences in the 1100-950 cm⁻¹ range. -1 The new or enhanced peaks are attributed to P=O stretching vibrations, POC stretching vibrations, etc., indicating the successful formation of phosphorus-containing melanin at the bacterial interface. Figure 15 ) Example 12 A method for preparing melanin-functionalized engineered Escherichia coli includes the following steps: (1) Prepare a buffer solution containing tyrosine: Add tyrosine to a phosphate buffer solution with pH 5.0 to a final concentration of 100 mg / mL, and then filter and sterilize.

[0072] (2) Escherichia coli expressing tyrosinase was cultured in a medium containing 100 μg mL -1 In LB liquid medium containing kanamycin sulfate.

[0073] (3) OD 600 When the concentration is 1.0, add the tyrosine solution from step 1 to a final concentration of 1 mg / mL, and add a trace amount of copper acetate solution to the Cu concentration. 2+ The final concentration was 2 mM.

[0074] (4) The above-mentioned chassis microbial solution was placed in a shaker at 37°C and 220 rpm for 48 h to obtain a phosphorus-containing melanin-functionalized engineered bacteria preparation.

[0075] This example demonstrates that the culture medium turns black after the addition of tyrosine. Figure 16 Infrared spectroscopy results showed that, compared with the infrared spectrum of Escherichia coli, the engineered bacteria exhibited differences in the 1100-950 cm⁻¹ range. -1 The new or enhanced peaks are attributed to P=O stretching vibrations, POC stretching vibrations, etc., indicating the successful formation of phosphorus-containing melanin at the bacterial interface. Figure 17 ) Example 13 The extraction and NMR characterization of phosphorus-containing melanin from phosphorus-containing melanin-functionalized engineered Escherichia coli in Example 2 included the following steps: (1) Collect bacterial cells at 8,000–10,000×g for 10–15 min; resuspend the bacterial cells thoroughly in 1 M NaOH and incubate in a water bath at 80 °C for 1 h.

[0076] (2) 10,000×g, 10 min, collect the supernatant.

[0077] (3) Add 6 M HCl dropwise to adjust the pH to <2, and collect the precipitate by centrifugation. (4) Wash repeatedly with deionized water 2–5 times until the washing solution is close to neutral.

[0078] (5) Drying: Vacuum drying or freeze drying to obtain phosphorus-containing melanin powder.

[0079] (6) The phosphorus spectrum of phosphorus-containing melanin and substrate peptides was detected by nuclear magnetic resonance spectrometry.

[0080] This embodiment demonstrates that a new peak appeared in the phosphorus spectrum, indicating that the substrate peptide synthesized a newly generated phosphorus chemical form during the enzymatic reaction, demonstrating the successful generation of phosphorus-containing melanin (…). Figure 18 ).

[0081] Example 14 The morphological characterization of the phosphorus-containing melanin-functionalized engineered Escherichia coli in Example 2 included the following steps: (1) Add 10 μL of the bacteria to be tested onto the copper grid. After 15 min, use filter paper to absorb the excess liquid.

[0082] (2) Add deionized water to clean the sample on the copper grid. After cleaning, use filter paper to absorb the excess liquid to be tested.

[0083] (3) After the copper mesh sample is fully dried, observe it under TEM.

[0084] This embodiment demonstrates that engineered E. coli can catalytically oxidize phosphorylated peptide substrates to generate phosphorylated melanin (…). Figure 19 ).

[0085] Example 15 The morphological characterization of the phosphorus-containing melanin-functionalized engineered Escherichia coli in Example 3 included the following steps: (1) Add 10 μL of the bacteria to be tested onto the copper grid. After 15 min, use filter paper to absorb the excess liquid.

[0086] (2) Add deionized water to clean the sample on the copper grid. After cleaning, use filter paper to absorb the excess liquid to be tested.

[0087] (3) After the copper mesh sample is fully dried, observe it under TEM.

[0088] After the addition of the phosphorylated peptide substrate, particulate matter appeared at the interface of the engineered bacteria. The presence of phosphorus in the mapping diagram further illustrates that the engineered E. coli can catalytically oxidize the phosphorylated peptide substrate to produce phosphorus-containing melanin. Figure 20 ) Example 16 The morphological characterization of the phosphorus-containing melanin-functionalized engineered Escherichia coli in Example 4 included the following steps: (1) Add 10 μL of the bacteria to be tested onto the copper grid. After 15 min, use filter paper to absorb the excess liquid.

[0089] (2) Add deionized water to clean the sample on the copper grid. After cleaning, use filter paper to absorb the excess liquid to be tested.

[0090] (3) After the copper mesh sample is fully dried, observe it under TEM.

[0091] This embodiment demonstrates that engineered E. coli can catalytically oxidize phosphorylated peptide substrates to generate phosphorylated melanin (…). Figure 21 ).

[0092] Example 17 The in vitro photothermal performance evaluation of phosphorus-containing melanin-functionalized engineered bacteria in Examples 2-4 included the following steps: Samples of different concentrations were placed in 1 cm cuvettes, connected to an 808 nm laser device, and the laser power was set to 2 W cm⁻¹. -2 The cuvette containing the sample to be tested was continuously irradiated for 10 minutes, and the temperature of the solution was recorded every second using a digital thermometer.

[0093] The results showed that the phosphorus-containing melanin-functionalized engineered Escherichia coli had good heating ability, with the maximum temperature reaching 49.6℃. Figure 22 a).

[0094] Example 18 The in vitro photothermal performance evaluation of melanin-functionalized engineered bacteria in Example 12 included the following steps: Samples of different concentrations were placed in 1 cm cuvettes, connected to an 808 nm laser device, and the laser power was set to 2 W cm⁻¹. -2 The cuvette containing the sample to be tested was continuously irradiated for 10 minutes, and the temperature of the solution was recorded every second using a digital thermometer.

[0095] The results showed that melanin-functionalized engineered Escherichia coli had a certain ability to raise the temperature, but its photothermal conversion efficiency per unit time was lower than that of phosphorus-containing melanin-functionalized engineered Escherichia coli. Figure 22 b).

[0096] Example 19 The evaluation of the tumor-targeting effect of phosphorus-containing melanin-functionalized engineered Escherichia coli in Example 2 includes the following steps: (1) Establishment of a mouse subcutaneous colorectal cancer model: CT 26 cells in the logarithmic growth phase were collected by trypsin digestion, washed with PBS, and counted. 100 µL of 4T1 tumor cells were subcutaneously injected into the right hind limb of mice, with each mouse receiving 1×10⁻⁶ 4T1 cells. 7 One subcutaneous breast cancer model was established.

[0097] (2) Phosphorus-containing melanin-functionalized engineered Escherichia coli was intravenously injected into mice at a dose of 1×10⁻⁶. 7 / Only.

[0098] (3) 48 hours after injection, the mice were humanely euthanized, and the heart, liver, spleen, lung, kidney and tumor tissues of the mice were removed, weighed and homogenized in PBS and plated. The number of colonies on the plates was counted.

[0099] This embodiment demonstrates that 48 hours after injection, the number of bacteria in normal organs is significantly lower than that in tumor tissue, indicating that phosphorus-containing melanin-functionalized engineered Escherichia coli has significant targeting properties in tumor tissue and potential therapeutic applications. Figure 23 ).

[0100] Example 20 The in vivo photothermal effect of phosphorus-containing melanin-functionalized engineered Escherichia coli includes the following steps: (1) The method for establishing a mouse subcutaneous colorectal cancer model is described in Example 18.

[0101] (2) When the tumor volume in mice exceeds 100 mm 3 Mice were randomly divided into two groups: a control group and a group of engineered bacteria containing phosphorus melanin.

[0102] (3) The corresponding drugs were injected according to the above group. After 24 hours, the mouse tumor site was irradiated with an 808 nm laser. The mouse was photographed every 1 minute using an infrared imager to record the temperature of the mouse tumor site. Then, the average temperature of the mouse tumor site at different time points was analyzed on FLIR TOOLS software, and the temperature rise curve of the mouse tumor site was plotted.

[0103] This embodiment demonstrates that phosphorus-containing melanin-functionalized engineered bacteria possess excellent photothermal conversion performance in vivo, laying the foundation for subsequent in vivo photothermal immunotherapy for tumors. Figure 24 ).

[0104] Example 21 The therapeutic effect of phosphorus-containing melanin-functionalized engineered bacteria combined with photothermal therapy on tumors includes the following steps: (1) The method for establishing a mouse subcutaneous colorectal cancer model is described in Example 18.

[0105] (2) All mice were randomly divided into two groups: a control group and a group containing phosphorus-containing melanin-functionalized engineered bacteria. According to the above groups, different formulations were injected into the tail vein of the mice, with each mouse receiving 1×10⁻⁶ bacteria. 7 indivual.

[0106] (3) For the phosphorus-containing melanin-functionalized engineered bacterial group, 24 h after injection of the corresponding drug, the tumor sites of mice were irradiated with an 808 nm laser with a laser power of 1.5 W cm⁻¹. -2 The irradiation time is 10 minutes.

[0107] (4) Take pictures of the mouse tumors for observation.

[0108] This embodiment demonstrates that, compared to the PBS group, tumor growth was significantly inhibited in the phosphorus-containing melanin-functionalized engineered bacteria group, and significant tumor shrinkage or partial disappearance could be observed. Figure 25 ).

[0109] Example 22 The therapeutic effect of phosphorus-containing melanin-functionalized engineered bacteria combined with photothermal therapy on metastatic tumors includes the following steps: (1) The method for establishing a mouse subcutaneous colorectal cancer model is described in Example 18.

[0110] (2) Mice were randomly divided into three groups: Control group, Surgery group and phosphorus-containing melanin-functionalized engineered bacteria group.

[0111] (3) Mice in the Control group were fed normally. Mice in the Surgery group were treated as follows: After anesthetizing the mice, the tumor site was disinfected, and the tumor site was removed with surgical scissors and forceps. The wound was then disinfected and sutured. For mice in the phosphorus-containing melanin-functionalized engineered bacteria group, the phosphorus-containing melanin-functionalized engineered bacteria were injected intravenously, and the tumor site of the mice was irradiated with an 808 nm laser for 10 min 24 h after injection.

[0112] (4) After all groups of mice have grown for 50 days, 100 µL of CT26-luc cells were intravenously injected into the mice. The amount of CT26-luc cells injected into each mouse was 1×10⁻⁶. 5 indivual.

[0113] (5) Detect the survival status of mice and count the survival time of mice in each group.

[0114] This embodiment demonstrates that after initial treatment and control of the primary tumor, mice were subjected to a tumor re-attack experiment, for example, by intravenous injection of CT26-luc luminescent cells to simulate distant metastasis. The long-term protective effect was assessed using in vivo bioluminescence imaging and survival curves. The results showed that the luminescent signal in the lungs of mice treated with phosphorus-containing melanin-functionalized engineered bacteria was significantly reduced, and their survival time was significantly prolonged. Figure 26 ).

[0115] Example 23 The immunomodulatory effects of phosphorus-containing melanin-functionalized engineered bacteria include the following steps: (1) The method for establishing the mouse metastatic tumor model is described in Example 21.

[0116] (2) All mice were randomly divided into three groups: a control group, a surgical group, and a group of engineered bacteria containing phosphorus melanin. According to the above groups, different formulations were injected into the tail vein of the mice, with each mouse receiving 1×10⁻⁶ bacteria. 7 indivual.

[0117] (3) 24 hours after injection, the mice were irradiated with laser, and then the spleens of the mice were taken to analyze the changes in the number of T cells and the content of memory T cells.

[0118] This embodiment demonstrates that exposure to engineered bacteria and photothermal stimulation can effectively increase the number of spleen T cells and effector memory T cells in mice, resulting in a long-lasting immune protective effect. Figures 27-28 ).

[0119] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An engineered bacteria preparation containing a phosphorous melanin, characterized in that, The engineered bacterial preparation comprises engineered bacteria whose surface or interior is coated with phosphorus-containing melanin: The engineered bacteria whose surface or interior is coated with phosphorus-containing melanin are produced by chassis microorganisms that express tyrosinase in the presence of phosphorus-containing functional substrate peptides. The phosphorus-containing functional substrate peptide is shown in general formula (I): X1—Y—X2—Z, where the N-terminal X1—Y—X2 is a tyrosinase catalytic fragment, X1 is a phosphorylated amino acid; X2 is a flexible amino acid selected from glycine, alanine, valine, isoleucine and leucine; Y is tyrosine; and the C-terminal Z is an immunomodulatory peptide.

2. The phosphorous-containing melanin engineering bacteria preparation according to claim 1, characterized in that, The chassis microorganism is one of Escherichia coli, Bacillus megaterium, attenuated Salmonella, Bacillus subtilis, Lactobacillus rhamnosus, and Bifidobacterium, which overexpresses tyrosinase.

3. The phosphorus-containing melanin-producing engineered bacteria preparation according to claim 1, characterized in that, X1 is phosphorylated serine (pS), phosphorylated threonine (pT), or phosphorylated tyrosine (pY); preferably, the N-terminal tyrosinase catalytic fragment X1-Y-X2 is selected from one or more combinations of pSYG, pTYG, pYYG, pSYA, pTYA, and pYYA.

4. The phosphorus-containing melanin-producing engineered bacteria preparation according to any one of claims 1-3, characterized in that, C-end Z is selected from one or more combinations of KPR, TKPK, TKPRR, TTPR, RKDVY, KPV, and GHK.

5. The phosphorus-containing melanin-producing engineered bacteria preparation according to any one of claims 1-3, characterized in that, The engineered bacterial preparation exhibits continuous absorption in the 400–900 nm wavelength range.

6. The phosphorus-containing melanin-producing engineered bacteria preparation according to any one of claims 1-3, characterized in that, The phosphorus-containing melanin is a melanin derivative containing phosphate groups.

7. The method for preparing the phosphorus-containing melanin-producing engineered bacteria preparation according to any one of claims 1-6, characterized in that... Its preparation process includes the following steps: (1) Prepare a buffer solution of phosphorus-containing functional substrate peptide as a stock solution and filter it; (2) The engineered chassis microorganisms expressing tyrosinase were revived in a culture medium and cultured to a certain concentration; (3) Add the solution from step (1) to the chassis microbial solution from step (2), and add a certain concentration of copper salt for auxiliary catalysis; (4) The microbial solution in the chassis of step (3) above is reacted for a certain period of time to obtain a phosphorus-containing melanin-functionalized engineered bacteria preparation.

8. The method for preparing the phosphorus-containing melanin-producing engineered bacteria preparation according to claim 7, characterized in that, In step (1), the buffer solution is a phosphate buffer solution with pH of 5.0-8.0; the mother liquor concentration is 20-200 mg / mL; in step (3), the final concentration of the phosphorus-containing functional substrate peptide is 0.1-10 mg / mL; preferably, the final concentration is 1-5 mg / mL; the copper salt is selected from copper sulfate, copper chloride, copper phosphate, copper acetate; the Cu 2+ The final concentration is 0.1-10 mM; preferably, the final concentration is 1-2 mM.

9. The method for preparing the phosphorus-containing melanin-producing engineered bacteria preparation according to claims 7-8, characterized in that, In step (2), the concentration is in the range of OD value of the chassis microorganisms: 0.1-2; preferably, the OD value of the chassis microorganisms is in the range of 0.2-1; in step (3), the reaction time is 6-120 h; preferably, the reaction time is 12-96 h; more preferably, the reaction time is 24-72 h.

10. Use of the phosphorus-containing melanin-producing engineered bacteria preparation according to any one of claims 1-6 in the preparation of a medicament for photothermal immunotherapy of solid tumors and / or for inhibiting tumor recurrence or metastasis.

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