Preparation method of an engineered bacterium loaded with a functional melanin nanosystem complex and application thereof

Abstract

The present application relates to the preparation method and application of the complex of the engineered bacteria loaded with the functionalized melanin nanosystem, which can effectively solve the problems of activating the STING pathway, reversing T cell exhaustion, improving the effect of tumor immunotherapy and ensuring the effect of cancer treatment drugs, and the melanin is prepared into melanin nanoparticles, and the carboxylated melanin nanoparticles are formed through the thiomalic acid reaction, the polypeptide chain and 2-cyano-6-amino-benzothiazole are modified at the carboxyl terminal of the melanin nanoparticles through the amidation reaction, the metal ions are complexed on the surface of the functionalized melanin nanoparticles through the metal complexation, and finally the functionalized melanin nanosystem is adhered to the surface of the engineered escherichia coli through the adhesion, so as to obtain the complex of the engineered bacteria loaded with the functionalized melanin nanosystem; the present application is convenient to operate, the method is stable and reliable, has good biocompatibility and long retention time, can play the multiple effects of targeting tumor, activating the cGAS-STING pathway, blocking the PD-1 / PD-L1 axis and reversing T cell exhaustion.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to a method for preparing a composite of engineered bacteria-loaded functionalized melanin nanoparticles and its application. Background Technology

[0002] Activation of the cGAS-STING signaling pathway (an innate immune signaling pathway) can release type I interferon, induce the production of T cells (also known as T lymphocytes), and thus exert an anti-tumor immune response. However, activation of the cGAS-STING signaling pathway induces high expression of PD-L1 (Programmed cell death ligand 1) on the surface of tumor cells. This binds to PD-1 on the surface of T cells, leading to T cell dysfunction and exhaustion. Therefore, a strategy of activating the cGAS-STING signaling pathway in combination with blocking the PD-1 / PD-L1 axis holds promise for achieving a more potent anti-tumor immunotherapy effect.

[0003] Melanin nanoparticles are small-diameter nanoparticles prepared from melanin. They possess phenolic hydroxyl and amino groups that can complex with metal ions such as manganese, effectively activating the cGAS-STING signaling pathway. However, despite their high tumor permeability, these small-diameter melanin nanoparticles may flow back into the bloodstream or diffuse into surrounding tissues, leading to insufficient tumor accumulation. To address this, melanin nanoparticles were carboxylated and then modified with an amidation reaction of polypeptide chains (Ala-Ala-Asn-Cys-Lys) and 2-cyano-6-aminobenzothiazole to obtain a functionalized melanin nanosystem. Under the action of legumain enzyme (cysteine ​​protease), the polypeptide chains in this system expose amino and thiol groups, which can undergo a click cycloaddition reaction with the free cyano group on 2-cyano-6-aminobenzothiazole. This increases the particle size and prevents the functionalized melanin nanoparticles from flowing back into the bloodstream, thereby enhancing the retention effect of nano-formulations at the tumor site.

[0004] To achieve effective accumulation and blockade of the PD-1 / PD-L1 signaling axis at tumor sites, functionalized melanin nanosystems were adhered to the surface of *E. coli* bacteria infused with PD-1 or PD-L1 plasmids, resulting in nanocomposites that activate the cGAS-STING signaling pathway and block the PD-1 / PD-L1 axis. Due to the hypoxic targeting ability of *E. coli*, the nanocomposites accumulated at the tumor site. As the bacteria grew, the functionalized melanin nanosystems detached and aggregated into large particles, forming a metal reservoir, under the action of legumain enzymes. In the tumor microenvironment, the functionalized melanin nanosystems released metal ions, activating the cGAS-STING signaling pathway. Simultaneously, under 808 nm laser irradiation, the temperature at the tumor site rose to 42°C due to the photothermal conversion of the melanin nanoparticles, thereby inducing *E. coli* to secrete PD-1 or PD-L1. This blocks the PD-1 / PD-L1 axis and reverses T cell depletion. Therefore, studying a complex of engineered bacteria loaded with functionalized melanin nanosystems that activates the cGAS-STING signaling pathway and reverses T cell exhaustion is of great value and significance. Summary of the Invention

[0005] In view of the above situation and to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing a functionalized melanin nanosystem complex loaded with engineered bacteria and its application, which can effectively solve the problems of activating the STING pathway, reversing T cell exhaustion, improving the effect of tumor immunotherapy, and ensuring the efficacy of cancer treatment drugs.

[0006] This invention discloses a method for preparing a composite of engineered bacteria-loaded functionalized melanin nanoparticles. The method involves preparing melanin nanoparticles, forming carboxylated melanin nanoparticles via a thiomalic acid reaction, modifying the carboxyl terminus of the melanin nanoparticles with polypeptide chains and 2-cyano-6-amino-benzothiazole via an amidation reaction, complexing metal ions onto the surface of the functionalized melanin nanoparticles via metal complexation, and finally adhering the functionalized melanin nanoparticle system to the surface of engineered *E. coli* bacteria using adhesion, thus obtaining a composite of engineered bacteria-loaded functionalized melanin nanoparticles. The method specifically includes the following steps:

[0007] (1) Preparation of melanin nanoparticles (MNP): Dissolve 5-12 mg of melanin in 5-10 mL of 0.1 mol / L sodium hydroxide and sonicate in an ice bath for 0.5 h; add 0.1 mol / L dilute hydrochloric acid to adjust the pH to 6.5-7.5, dialyze with a dialysis bag with MWCO of 2500 Da for 6 h, and freeze dry to obtain melanin nanoparticles with a particle size of 20 nm to 60 nm;

[0008] (2) Preparation of carboxylated melanin nanoparticles: Take 5-12 mg of melanin nanoparticles from step (1) and dissolve them in 8-10 mL of solvent. Add 6-8 mg of thiomalic acid and stir at room temperature (18-25℃, the same below) for 6-8 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 48 h and freeze dry to obtain carboxylated melanin nanoparticles.

[0009] The solvent is one of ultrapure water, PBS buffer at pH 7.4, DMF (N,N-dimethylformamide), or Tris-HCl buffer at pH 8.0 (hereinafter the same);

[0010] (3) Preparation of peptide chain modified melanin nanoparticles (MNP-pep): Weigh 8-12 mg of carboxylated melanin nanoparticles from step (2) and dissolve them in a solvent. Add 8-12 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 6-10 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 3-5 h. Add 5-9 mg of peptide chain. Add 0.1 mol / L sodium hydroxide to adjust the pH to 7.5-8.0. Stir at nitrogen in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. After freeze drying, obtain peptide chain modified melanin nanoparticles.

[0011] The polypeptide chain is a polypeptide chain containing the Ala-Ala-Asn-Cys-Lys (alanine-alanine-aspartic acid-cysteine-lysine) sequence;

[0012] (4) Preparation of 2-cyano-6-amino-benzothiazole modified melanin nanoparticles (MNP-CABT): Weigh 8-12 mg of carboxylated melanin nanoparticles and dissolve them in a solvent. Add 8-12 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 6-10 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 3-5 h. Weigh 3-7 mg of 2-cyano-6-amino-benzothiazole and dissolve it in a solvent. Add it to the reaction system. Add 0.1 mol / L dilute hydrochloric acid to adjust the pH to 7.5-8.0. Stir under nitrogen in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. Freeze dry to obtain 2-cyano-6-amino-benzothiazole modified melanin nanoparticles.

[0013] (5) Preparation of melanin nanoparticles loaded with metal ions (MNP-p&C@Mn): Weigh 8-12 mg of the polypeptide chain modified melanin nanoparticles from step (3) and 1.0-2.5 mg of the metal salt, dissolve them in the solvent, and sonicate them. After mixing them evenly, react them at 40°C for 1 h, and then put them into a dialysis bag with MWCO of 12000 Da for dialysis for 48 h to obtain the polypeptide chain modified melanin nanoparticles loaded with metal ions.

[0014] Preparation of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions: Weigh 8-12 mg of the 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles from step (4) and 1.0-2.5 mg of the metal salt, dissolve them in solvents respectively, and dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h, and then put them into a dialysis bag with MWCO of 12000 Da for dialysis for 48 h to obtain 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions.

[0015] The metal ion is one of manganese ion and zinc ion;

[0016] (6) Preparation of engineered bacteria-loaded functionalized melanin nanoparticle complex (MNP-p&C@Mn-BP): Weigh 0.5-2 mg of the melanin nanoparticles modified with the polypeptide chain loaded with metal ions from step (5), or 0.5-2 mg of the melanin nanoparticles modified with 2-cyano-6-amino-benzothiazole loaded with metal ions, and disperse them in 0.5-2 mL of solvent. Then add 10 mL of engineered E. coli Tris-HCl buffer. 8 CFU / mL, stirred at room temperature for 3 h, centrifuged at 8000 rpm for 10 min, supernatant was discarded, and the precipitate was resuspended in sterile Tris-HCl buffer to obtain the complex of engineered bacteria-loaded functionalized melanin nanosystem.

[0017] The engineered Escherichia coli is one of the engineered bacteria introduced with PD-1 or PD-L1 plasmids.

[0018] Application of the engineered bacteria-loaded functionalized melanin nanosystem complex prepared by the method in the preparation of antitumor drug injections.

[0019] Application of the engineered bacteria-loaded functionalized melanin nanosystem complex prepared by the method in the preparation of drugs that enhance the cGAS-STING immune signaling pathway.

[0020] The engineered bacteria-loaded functionalized melanin nanosystem complex prepared by the method is used in the preparation of melanin nanoparticle tumor microenvironment drugs based on photothermal sensitivity and Legumain enzyme-responsive particle size transition.

[0021] Application of the engineered bacteria-loaded functionalized melanin nanosystem complex prepared by the method in the preparation of antitumor drugs.

[0022] This invention is convenient to operate, and the method is stable and reliable. The engineered bacteria-loaded functionalized melanin nanosystem complex has good biocompatibility and long retention time. In the field of tumor treatment, it can exert multiple effects such as targeting tumors, activating the cGAS-STING pathway, blocking the PD-1 / PD-L1 axis, and reversing T cell exhaustion. It is an innovation in tumor treatment drugs with huge economic and social benefits. Attached Figure Description

[0023] Figure 1 This is a transmission electron microscope image of the nanocomposite system of the present invention.

[0024] Figure 2 This is a graph showing the photothermal conversion capability of the nanocomposite of the present invention under laser irradiation.

[0025] Figure 3 The present invention provides a nanosystem complex ELISA kit for detecting the secretion of PD-L1.

[0026] Figure 4 The image shows the particle size curves of the nanocomposite of this invention after incubation with MNP-pep, MNP-CABT, and MNP-pep+MNP-CABT.

[0027] Figure 5 This is a tissue distribution diagram of the nanocomposite system of the present invention in tumor-bearing mice and in vitro.

[0028] Figure 6 This is a schematic diagram showing the residence time of the nanocomposite of the present invention in tumor tissue in vivo (A: MNP-p&C@Mn; B: MNP-p&C@Mn-BP).

[0029] Figure 7 This is a diagram showing the expression of cGAS-STING-related proteins in tumor tissues using Western blot technology to detect the nanosystem complex of the present invention. Detailed Implementation

[0030] The specific embodiments of the present invention will be described in detail below with reference to examples.

[0031] The present invention can be described in the following embodiments.

[0032] Example 1

[0033] This invention discloses a method for preparing a composite of engineered bacteria-loaded functionalized melanin nanoparticles, which specifically includes the following steps:

[0034] (1) Preparation of melanin nanoparticles: Dissolve 5 mg of melanin in 5 mL of 0.1 mol / L sodium hydroxide, sonicate in an ice bath for 0.5 h; add 0.1 mol / L dilute hydrochloric acid to adjust the pH to 6.5, dialyze with a dialysis bag with MWCO of 2500 Da for 6 h, freeze dry to obtain melanin nanoparticles.

[0035] (2) Preparation of carboxylated melanin nanoparticles: Take 5 mg of melanin nanoparticles from step (1) and dissolve them in 8 mL of Tris-HCl buffer solution with pH 8.0. Add 6 mg of thiomalic acid, stir at room temperature for 6 h, dialyze with a dialysis bag with MWCO of 12000 Da for 48 h, freeze dry, and obtain carboxylated melanin nanoparticles.

[0036] (3) Preparation of peptide chain modified melanin nanoparticles: Weigh 8 mg of carboxylated melanin nanoparticles from step (2) and dissolve them in ultrapure water. Add 8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 6 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 3 h. Add 5 mg of peptide chain and add 0.1 mol / L sodium hydroxide to adjust the pH to 7.5. Stir at nitrogen in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. After freeze drying, obtain peptide chain modified melanin nanoparticles.

[0037] (4) Preparation of 2-cyano-6-amino-benzothiazole modified melanin nanoparticles: 8 mg of carboxylated melanin nanoparticles were weighed and dissolved in ultrapure water. 8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 6 mg of N-hydroxysuccinimide were added. The mixture was stirred at room temperature in the dark for 3-5 h. 3 mg of 2-cyano-6-amino-benzothiazole was weighed and dissolved in DMF and added to the reaction system. 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 7.5. The mixture was stirred under nitrogen in the dark for 24 h. Dialyzed with a dialysis bag with a MWCO of 12000 Da for 24 h and lyophilized to obtain 2-cyano-6-amino-benzothiazole modified melanin nanoparticles.

[0038] (5) Preparation of melanin nanoparticles loaded with metal ions: Weigh 8 mg of the polypeptide chain modified melanin nanoparticles from step (3) and 1.0 mg of metal salt MnCl2·4H2O, dissolve them in ultrapure water, and dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h, and then put them into a dialysis bag with MWCO of 12000 Da for dialysis for 48 h to obtain the polypeptide chain modified melanin nanoparticles loaded with metal ions.

[0039] Preparation of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions: Weigh 8 mg of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles from step (4) and 1.0 mg of metal salt MnCl2·4H2O, respectively, and dissolve them in ultrapure water. Dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h. Then, put them into a dialysis bag with MWCO of 12000 Da and dialyze for 48 h to obtain 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions.

[0040] (6) Preparation of engineered bacteria-loaded functionalized melanin nanoparticle complex: Weigh 0.5 mg of the melanin nanoparticles modified with metal ion-loaded polypeptide chains from step (5), or 0.5 mg of the melanin nanoparticles modified with metal ion-loaded 2-cyano-6-amino-benzothiazole, and disperse them in 0.5 mL of Tris-HCl buffer at pH 8.0. Then add 10 mL of Tris-HCl buffer to PD-1 engineered E. coli. 8 The mixture was stirred at room temperature for 3 hours with CFU / mL, centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in sterile Tris-HCl buffer to obtain the complex of engineered bacteria-loaded functionalized melanin nanosystem.

[0041] Example 2

[0042] This invention discloses a method for preparing a composite of engineered bacteria-loaded functionalized melanin nanoparticles, which specifically includes the following steps:

[0043] (1) Preparation of melanin nanoparticles: Dissolve 7 mg of melanin in 8 mL of 0.1 mol / L sodium hydroxide and sonicate in an ice bath for 0.5 h; add 0.1 mol / L dilute hydrochloric acid to adjust the pH to 7.0, dialyze with a dialysis bag with MWCO of 2500 Da for 6 h, and freeze dry to obtain melanin nanoparticles.

[0044] (2) Preparation of carboxylated melanin nanoparticles: Take 7 mg of melanin nanoparticles from step (1) and dissolve them in 9 mL of Tris-HCl buffer solution with pH 8.0. Add 7 mg of thiomalic acid, stir at room temperature for 7 h, dialyze with a dialysis bag with MWCO of 12000 Da for 48 h, freeze dry, and obtain carboxylated melanin nanoparticles.

[0045] (3) Preparation of peptide chain modified melanin nanoparticles: Weigh 9 mg of carboxylated melanin nanoparticles from step (2) and dissolve them in ultrapure water. Add 9 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 7 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 4 h. Add 6 mg of peptide chain and add 0.1 mol / L sodium hydroxide to adjust the pH to 7.8. Stir at nitrogen in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. After freeze drying, obtain peptide chain modified melanin nanoparticles.

[0046] (4) Preparation of 2-cyano-6-amino-benzothiazole modified melanin nanoparticles: 9 mg of carboxylated melanin nanoparticles were weighed and dissolved in ultrapure water. 9 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 7 mg of N-hydroxysuccinimide were added. The mixture was stirred at room temperature in the dark for 4 h. 4 mg of 2-cyano-6-amino-benzothiazole was weighed and dissolved in DMF and added to the reaction system. 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 7.8. The mixture was stirred under nitrogen in the dark for 24 h. The mixture was dialyzed for 24 h using a dialysis bag with a MWCO of 12000 Da. The mixture was then freeze-dried to obtain 2-cyano-6-amino-benzothiazole modified melanin nanoparticles.

[0047] (5) Preparation of melanin nanoparticles loaded with metal ions: Weigh 9 mg of the polypeptide chain modified melanin nanoparticles from step (3) and 1.5 mg of metal salt ZnCl2·4H2O, dissolve them in ultrapure water, and dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h, and then put them into a dialysis bag with MWCO of 12000 Da for dialysis for 48 h to obtain the polypeptide chain modified melanin nanoparticles loaded with metal ions.

[0048] Preparation of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions: 9 mg of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles from step (4) and 1.5 mg of metal salt ZnCl2·4H2O were weighed and dissolved in ultrapure water. They were dissolved by sonication and mixed evenly. The mixture was reacted at 40°C for 1 h and then placed in a dialysis bag with MWCO of 12000 Da for dialysis for 48 h to obtain 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions.

[0049] (6) Preparation of engineered bacteria-loaded functionalized melanin nanoparticle complex: Weigh 1.0 mg of the melanin nanoparticles modified with metal ion-loaded polypeptide chains from step (5), or 1.0 mg of the melanin nanoparticles modified with metal ion-loaded 2-cyano-6-amino-benzothiazole, and disperse them in 2 mL of Tris-HCl buffer at pH 8.0. Then add 0.5 mL of E. coli Tris-HCl buffer containing PD-L1 plasmid.8 The mixture was stirred at room temperature for 3 hours with CFU / mL, centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in sterile Tris-HCl buffer to obtain the complex of engineered bacteria-loaded functionalized melanin nanosystem.

[0050] Example 3

[0051] This invention discloses a method for preparing a composite of engineered bacteria-loaded functionalized melanin nanoparticles, which specifically includes the following steps:

[0052] (1) Preparation of melanin nanoparticles: Dissolve 8 mg of melanin in 10 mL of 0.1 mol / L sodium hydroxide, sonicate in an ice bath for 0.5 h; add 0.1 mol / L dilute hydrochloric acid to adjust the pH to 7.5, dialyze with a dialysis bag with MWCO of 2500 Da for 6 h, freeze dry to obtain melanin nanoparticles.

[0053] (2) Preparation of carboxylated melanin nanoparticles: Take 8 mg of melanin nanoparticles from step (1) and dissolve them in 10 mL of Tris-HCl buffer solution with pH 8.0. Add 8 mg of thiomalic acid, stir at room temperature for 8 h, dialyze with a dialysis bag with MWCO of 12000 Da for 48 h, freeze dry, and obtain carboxylated melanin nanoparticles.

[0054] (3) Preparation of peptide chain modified melanin nanoparticles: Weigh 10 mg of carboxylated melanin nanoparticles from step (2) and dissolve them in ultrapure water. Add 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 8 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 5 h. Add 8 mg of peptide chain and add 0.1 mol / L sodium hydroxide to adjust the pH to 8.0. Stir at nitrogen in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. After freeze drying, obtain peptide chain modified melanin nanoparticles.

[0055] (4) Preparation of 2-cyano-6-amino-benzothiazole modified melanin nanoparticles: 10 mg of carboxylated melanin nanoparticles were weighed and dissolved in ultrapure water. 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 8 mg of N-hydroxysuccinimide were added. The mixture was stirred at room temperature in the dark for 5 h. 5 mg of 2-cyano-6-amino-benzothiazole was weighed and dissolved in DMF and added to the reaction system. 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 8.0. The mixture was stirred in the dark under nitrogen for 24 h. The mixture was dialyzed for 24 h using a dialysis bag with a MWCO of 12000 Da. The mixture was then freeze-dried to obtain 2-cyano-6-amino-benzothiazole modified melanin nanoparticles.

[0056] (5) Preparation of melanin nanoparticles loaded with metal ions: Weigh 10 mg of the polypeptide chain modified melanin nanoparticles from step (3) and 2 mg of metal salt MnCl2·4H2O, dissolve them in ultrapure water, and dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h, and then put them into a dialysis bag with MWCO of 12000 Da for dialysis for 48 h to obtain the polypeptide chain modified melanin nanoparticles loaded with metal ions.

[0057] Preparation of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions: Weigh 10 mg of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles from step (4) and 2 mg of metal salt MnCl2·4H2O, respectively, and dissolve them in ultrapure water. Dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h. Then, put them into a dialysis bag with MWCO of 12000 Da and dialyze for 48 h to obtain 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions.

[0058] (6) Preparation of engineered bacteria-loaded functionalized melanin nanoparticle complex: Weigh 2 mg of the melanin nanoparticles modified with metal ion-loaded polypeptide chains from step (5), or 2 mg of the melanin nanoparticles modified with metal ion-loaded 2-cyano-6-amino-benzothiazole, and disperse them in 2 mL of Tris-HCl buffer at pH 8.0. Then add 1.5 mL of E. coli Tris-HCl buffer containing PD-L1 plasmid. 8 The mixture was stirred at room temperature for 3 hours with CFU / mL, centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in sterile Tris-HCl buffer to obtain the complex of engineered bacteria-loaded functionalized melanin nanosystem.

[0059] Example 4

[0060] This invention discloses a method for preparing a composite of engineered bacteria-loaded functionalized melanin nanoparticles, which specifically includes the following steps:

[0061] (1) Preparation of melanin nanoparticles: 10 mg of melanin was dissolved in 9 mL of 0.1 mol / L sodium hydroxide and sonicated in an ice bath for 0.5 h; 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 6.8, and the mixture was dialyzed for 6 h using a dialysis bag with a MWCO of 2500 Da. The mixture was then freeze-dried to obtain melanin nanoparticles.

[0062] (2) Preparation of carboxylated melanin nanoparticles: Take 10 mg of melanin nanoparticles from step (1) and dissolve them in 10 mL of ultrapure water. Add 7.5 mg of thiomalic acid, stir at room temperature for 7.5 h, dialyze with a dialysis bag with MWCO of 12000 Da for 48 h, freeze dry, and obtain carboxylated melanin nanoparticles.

[0063] (3) Preparation of peptide-modified melanin nanoparticles: Weigh 11 mg of carboxylated melanin nanoparticles from step (2) and dissolve them in PBS buffer at pH 7.4. Add 11 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 9 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 3.5 h. Add 7 mg of peptide chain and add 0.1 mol / L sodium hydroxide to adjust the pH to 7.5. Stir at nitrogen in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. After freeze-drying, obtain peptide-modified melanin nanoparticles.

[0064] (4) Preparation of 2-cyano-6-amino-benzothiazole modified melanin nanoparticles: 11 mg of carboxylated melanin nanoparticles were weighed and dissolved in PBS buffer solution with pH 7.4. 11 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 9 mg of N-hydroxysuccinimide were added. The mixture was stirred at room temperature in the dark for 3.5 h. 6 mg of 2-cyano-6-amino-benzothiazole was weighed and dissolved in ultrapure water. The mixture was added to the reaction system. 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 7.6. The mixture was stirred under nitrogen atmosphere in the dark for 24 h. The mixture was dialyzed for 24 h using a dialysis bag with MWCO of 12000 Da. The mixture was then lyophilized to obtain 2-cyano-6-amino-benzothiazole modified melanin nanoparticles.

[0065] (5) Preparation of melanin nanoparticles loaded with metal ions: Weigh 11 mg of the polypeptide chain modified melanin nanoparticles from step (3) and 2.5 mg of metal salt ZnCl2·4H2O, respectively, and dissolve them in ultrapure water. Dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h. Then, put them into a dialysis bag with MWCO of 12000 Da and dialyze for 48 h to obtain the polypeptide chain modified melanin nanoparticles loaded with metal ions.

[0066] Preparation of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions: 11 mg of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles from step (4) and 2.5 mg of metal salt ZnCl2·4H2O were weighed and dissolved in ultrapure water. They were dissolved by sonication and mixed evenly. The mixture was reacted at 40°C for 1 h and then placed in a dialysis bag with MWCO of 12000 Da for dialysis for 48 h to obtain 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions.

[0067] (6) Preparation of engineered bacteria-loaded functionalized melanin nanoparticle complex: Weigh 1.5 mg of the melanin nanoparticles modified with metal ion-loaded polypeptide chains from step (5), or 1.5 mg of the melanin nanoparticles modified with metal ion-loaded 2-cyano-6-amino-benzothiazole, and disperse them in 1.5 mL of PBS buffer at pH 7.4. Then add 1 mL of E. coli Tris-HCl buffer containing PD-L plasmid. 8 The mixture was stirred at room temperature for 3 hours with CFU / mL, centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in sterile Tris-HCl buffer to obtain the complex of engineered bacteria-loaded functionalized melanin nanosystem.

[0068] Example 5

[0069] This invention discloses a method for preparing a composite of engineered bacteria-loaded functionalized melanin nanoparticles, which specifically includes the following steps:

[0070] (1) Preparation of melanin nanoparticles: Dissolve 6 mg of melanin in 6 mL of 0.1 mol / L sodium hydroxide and sonicate in an ice bath for 0.5 h; add 0.1 mol / L dilute hydrochloric acid to adjust the pH to 7.2, dialyze with a dialysis bag with MWCO of 2500 Da for 6 h, and freeze dry to obtain melanin nanoparticles.

[0071] (2) Preparation of carboxylated melanin nanoparticles: Take 6 mg of melanin nanoparticles from step (1) and dissolve them in 8 mL of solvent DMF. Add 6.5 mg of thiomalic acid, stir at room temperature for 6.5 h, dialyze with a dialysis bag with MWCO of 12000 Da for 48 h, freeze dry, and obtain carboxylated melanin nanoparticles.

[0072] (3) Preparation of peptide chain modified melanin nanoparticles: Weigh 8 mg of carboxylated melanin nanoparticles from step (2) and dissolve them in DMF solvent. Add 8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 6 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 4.5 h. Add 5 mg of peptide chain and add 0.1 mol / L sodium hydroxide to adjust the pH to 7.5. Stir at nitrogen in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. After freeze drying, obtain peptide chain modified melanin nanoparticles.

[0073] (4) Preparation of 2-cyano-6-amino-benzothiazole modified melanin nanoparticles: 8 mg of carboxylated melanin nanoparticles were weighed and dissolved in DMF solvent. 8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 6 mg of N-hydroxysuccinimide were added. The mixture was stirred at room temperature in the dark for 3 h. 3-7 mg of 2-cyano-6-amino-benzothiazole was weighed and dissolved in Tris-HCl buffer solution with pH 8.0. The solution was added to the reaction system. 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 7.8. The mixture was stirred under nitrogen in the dark for 24 h. The mixture was dialyzed for 24 h using a dialysis bag with MWCO of 12000 Da. The mixture was then lyophilized to obtain 2-cyano-6-amino-benzothiazole modified melanin nanoparticles.

[0074] (5) Preparation of melanin nanoparticles loaded with metal ions: Weigh 8 mg of the polypeptide chain modified melanin nanoparticles from step (3) and 1.2 mg of metal salt MnCl2·4H2O respectively and dissolve them in DMF. Dissolve them by sonication. After mixing them evenly, react at 40°C for 1 h. Then put them into a dialysis bag with MWCO of 12000 Da and dialyze for 48 h to obtain the polypeptide chain modified melanin nanoparticles loaded with metal ions.

[0075] Preparation of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions: Weigh 8 mg of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles from step (4) and 1.2 mg of metal salt MnCl2·4H2O respectively and dissolve them in DMF. Dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h. Then, put them into a dialysis bag with MWCO of 12000 Da and dialyze for 48 h to obtain 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions.

[0076] (6) Preparation of engineered bacteria-loaded functionalized melanin nanoparticle complex: Weigh 0.8 mg of the melanin nanoparticles modified with metal ion-loaded polypeptide chains from step (5), or 0.8 mg of the melanin nanoparticles modified with metal ion-loaded 2-cyano-6-amino-benzothiazole, and disperse them in 0.8 mL of ultrapure water. Then add 10 mL of E. coli Tris-HCl buffer containing PD-L1 plasmid. 8 The mixture was stirred at room temperature for 3 hours with CFU / mL, centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in sterile Tris-HCl buffer to obtain the complex of engineered bacteria-loaded functionalized melanin nanosystem.

[0077] Example 6

[0078] This invention discloses a method for preparing a composite of engineered bacteria-loaded functionalized melanin nanoparticles, which specifically includes the following steps:

[0079] (1) Preparation of melanin nanoparticles: 12 mg of melanin was dissolved in 10 mL of 0.1 mol / L sodium hydroxide and sonicated in an ice bath for 0.5 h; 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 7.5, and the mixture was dialyzed for 6 h using a dialysis bag with a MWCO of 2500 Da. The mixture was then freeze-dried to obtain melanin nanoparticles.

[0080] (2) Preparation of carboxylated melanin nanoparticles: Take 12 mg of melanin nanoparticles from step (1) and dissolve them in 10 mL of Tris-HCl buffer solution with pH 8.0. Add 8 mg of thiomalic acid, stir at room temperature for 8 h, dialyze with a dialysis bag with MWCO of 12000 Da for 48 h, freeze dry, and obtain carboxylated melanin nanoparticles.

[0081] (3) Preparation of peptide chain modified melanin nanoparticles: Weigh 12 mg of carboxylated melanin nanoparticles from step (2) and dissolve them in Tris-HCl buffer solution with pH 8.0. Add 12 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 5 h. Add 9 mg of peptide chain and add 0.1 mol / L sodium hydroxide to adjust the pH to 8.0. Stir at nitrogen in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. After freeze drying, obtain peptide chain modified melanin nanoparticles.

[0082] (4) Preparation of 2-cyano-6-amino-benzothiazole modified melanin nanoparticles: 12 mg of carboxylated melanin nanoparticles were weighed and dissolved in Tris-HCl buffer solution with pH 8.0. 12 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10 mg of N-hydroxysuccinimide were added. The mixture was stirred at room temperature in the dark for 5 h. 7 mg of 2-cyano-6-amino-benzothiazole was weighed and dissolved in PBS buffer solution with pH 7.4. The mixture was added to the reaction system. 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 8.0. The mixture was stirred under nitrogen atmosphere in the dark for 24 h. The mixture was dialyzed for 24 h using a dialysis bag with MWCO of 12000 Da. The mixture was then lyophilized to obtain 2-cyano-6-amino-benzothiazole modified melanin nanoparticles.

[0083] (5) Preparation of melanin nanoparticles loaded with metal ions: Weigh 12 mg of the polypeptide chain modified melanin nanoparticles from step (3) and 2.5 mg of metal salt ZnCl2·4H2O, respectively, and dissolve them in Tris-HCl buffer at pH 8.0. Dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h. Then, put them into a dialysis bag with MWCO of 12000 Da and dialyze for 48 h to obtain the polypeptide chain modified melanin nanoparticles loaded with metal ions.

[0084] Preparation of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions: Weigh 12 mg of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles from step (4) and 2.5 mg of metal salt ZnCl2·4H2O, respectively, and dissolve them in Tris-HCl buffer solution at pH 8.0. Dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h. Then, put them into a dialysis bag with MWCO of 12000 Da and dialyze for 48 h to obtain 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions.

[0085] (6) Preparation of engineered bacteria-loaded functionalized melanin nanoparticle complex: Weigh 2 mg of the melanin nanoparticles modified with metal ion-loaded polypeptide chains from step (5), or 2 mg of the melanin nanoparticles modified with metal ion-loaded 2-cyano-6-amino-benzothiazole, and disperse them in 2 mL of Tris-HCl buffer at pH 8.0. Then add 1.5 mL of E. coli Tris-HCl buffer containing PD-L1 plasmid. 8 The mixture was stirred at room temperature for 3 hours with CFU / mL, centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in sterile Tris-HCl buffer to obtain the complex of engineered bacteria-loaded functionalized melanin nanosystem.

[0086] The engineered bacteria-loaded functionalized melanin nanosystems prepared in Examples 1-6 above can exert multiple effects in tumor therapy, such as targeting tumors, activating the cGAS-STING pathway, blocking the PD-1 / PD-L1 axis, and reversing T cell exhaustion. They can be effectively used in the preparation of anti-tumor drugs, and can be applied in the preparation of anti-tumor drugs (injectables), in the preparation of drugs that enhance the cGAS-STING immune signaling pathway, and in the preparation of tumor microenvironment drugs based on photothermal-sensitive, Legumain enzyme-responsive particle size transition melanin nanoparticles.

[0087] This invention is convenient to operate, stable and reliable in method, and simple in synthesis. It utilizes the hypoxic targeting and accumulation of engineered bacteria at the tumor site. Under the action of legumain enzymes in the tumor microenvironment, the particle size of the melanin nanoparticle system increases, thereby promoting the retention of nanoparticles at the tumor site, forming a metal reservoir. It exhibits good biocompatibility and a retention time of up to 72 hours. Furthermore, due to the photothermal conversion properties of the melanin nanoparticles, the engineered bacteria release PD-1 or PD-L1, blocking the PD-1 / PD-L1 signaling axis. The prepared functionalized melanin nanoparticle system loaded with engineered bacteria complex can efficiently activate the STING pathway and reverse T cell exhaustion, thereby improving the efficacy of anti-tumor immunotherapy. This invention has achieved consistently good results through repeated experiments (taking Example 2 as an example, each experiment was repeated at least 3 times, and the average value was taken). Relevant experimental data are as follows:

[0088] I. Characterization of Functionalized Melanin Nanosystems

[0089] 1. Determination of manganese ion content in functionalized melanin nanosystems:

[0090] Mn was determined by ICP-MS 2+ The drug content was calculated using formula (1). The drug loading of the sample reached approximately 3.38%.

[0091]

[0092] 2. Experiment on the determination of particle size and potential of engineered bacteria-loaded functionalized melanin nanocomplex:

[0093] Appropriate amounts of functionalized melanin nanosystem and engineered bacteria-loaded functionalized melanin nanosystem complex were dispersed in water. The hydrated particle size and potential were measured by laser nanoparticle size analyzer to be ~60nm and ~4μm, respectively, and the potentials were ~-13mV and ~-20mV, respectively.

[0094] 3. Transmission electron microscopy characterization of the engineered bacteria-supported functionalized melanin nanocomposite:

[0095] The complex was dissolved in ultrapure water to prepare a 50 μg / mL solution. One drop was added to a conventional carbon support membrane, and after the liquid evaporated, the image was taken using a transmission electron microscope. Figure 1 As shown, the synthesized functionalized melanin nanosystems were successfully adhered to the surface of engineered bacteria.

[0096] II. In vitro photothermal conversion experiment and PD-L1 secretion ability experiment of engineered bacteria loaded with functionalized melanin nanosystem complex:

[0097] To investigate the photothermal properties of this composite, composites were prepared by incubating engineered bacteria with melanin nanoparticles of varying concentrations. Figure 2 AB analysis showed that the temperature of the complex increased significantly with prolonged laser irradiation time and increased concentration of the melanin nanosystem, indicating that the complex possesses significant photothermal conversion capability under 808 nm laser irradiation. Furthermore, ELISA detection revealed successful secretion of PD-L1 (…) after laser irradiation. Figure 3 ).

[0098] III. Legumin enzyme-induced in vitro aggregation experiment:

[0099] MNP-pep, MNP-CABT, and MNP-pep+MNP-CABT were used as controls and incubated in PBS buffer at pH 6.5. The particle size of the samples was measured using a laser particle size analyzer at 1h, 2h, 4h, 8h, 12h, and 24h. Figure 4 The results showed that only MNP-pep+MNP-CABT showed a significant increase in particle size after incubation with Legumain, while the particle size of other groups remained at around 60 nm, indicating that MNP-pep+MNP-CABT has superior Legumain enzyme-sensitive aggregation characteristics.

[0100] IV. Tumor-targeting experiments on engineered bacteria-loaded functionalized melanin nanosystem complexes:

[0101] To investigate whether engineered bacteria-loaded functionalized melanin nanocomposites could actively target tumor tissue, the fluorescent dye IR783 was added according to the method used to prepare engineered bacteria-loaded functionalized melanin nanocomposites. When the tumor volume in tumor-bearing mice reached 200 mm², the target was tested. 3 At the time of administration, mice were injected via tail vein with a complex of free IR783 and engineered bacteria-loaded functionalized melanin nanoparticles (IR783 dosage: 0.8 mg / kg). X-ray and fluorescence images of mice in each group were acquired at different time points: 1 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h post-injection. 72 h after administration, the mice were dissected, and heart, liver, spleen, lung, kidney, and tumor tissues were removed. Signal acquisition was performed on the isolated tissues, and the fluorescence intensity of each tissue was observed. Figure 5 The results showed that the engineered bacteria-loaded functionalized melanin nanosystem complex had superior tumor targeting properties.

[0102] V. Retention capacity of engineered bacteria-loaded functionalized melanin nanocomplex in tumor tissue:

[0103] To establish a tumor-bearing model in mice, when the tumor volume of the tumor-bearing mice reaches ~200 mm... 3 Mice were anesthetized by tail vein injection at 24, 48, and 72 hours post-administration. Tumor tissue was dissected, embedded in embedding medium, and rapidly frozen in liquid nitrogen. Sections were then prepared using a cryostat at 500, 1000, and 1500 μm from the tumor apex. The sections were fixed with 4% PFA, and the tumor tissue was circled with an immunohistochemical pen. After incubation with DAPI staining working solution at room temperature in the dark for 10 min, the slides were washed with PBS, covered with coverslips, and finally mounted with nail polish for observation under a fluorescence microscope. Figure 6 The results showed that the engineered bacteria-loaded functionalized melanin nanosystem complex remained in tumor tissue in vivo for up to 72 hours, thus fully exerting the drug's effect.

[0104] VI. Experiment to detect the expression of cGAS-STING-related proteins in tumor tissues using Western blot technology:

[0105] 4T1 breast cancer cells were cultured and subcutaneously inoculated into BALB / c mice until the tumor volume reached 100 mm². 3 The mice were treated with medication every other day, with laser irradiation performed on days 4, 7, and 10. Tumor tissue was extracted from tumor-bearing mice and lysed with lysis buffer to obtain tissue protein samples. The expression of relevant proteins was detected by Western blot. Figure 7 The results showed that the engineered bacteria-loaded functionalized melanin nanosystem complex could upregulate the expression of pTBK1 and pIRF3 proteins and effectively activate the cGAS-STING pathway.

[0106] VII. Flow cytometry analysis of the ability of engineered bacteria loaded with functionalized melanin nanoparticles to block the PD-1 / PD-L1 axis:

[0107] Tumor tissue was extracted from tumor-bearing mice, ground, and a single-cell suspension of the tumor tissue was obtained. The suspension was incubated with antibody working solution on ice, washed, and loaded into flow cytometry tubes for analysis. Results showed that the formulation group downregulated PD-L1 protein on the surface of tumor cells from 19.3% to 8.63%, and reduced CD8+... + The proportion of T increased from 16.0% to 60.5%.

[0108] While conducting experiments on Example 2 using the experimental methods described above, the present invention also conducted the same experiments on other examples, and obtained the same or similar results, which will not be listed here one by one.

[0109] Experiments show that, compared with the prior art, the present invention has the following outstanding beneficial technical effects:

[0110] (1) The engineered bacteria-loaded functionalized melanin nanosystem complex provided in this experiment has excellent biocompatibility and stability, and can complex metal ions to exert immunotherapy.

[0111] (2) The engineered bacteria-loaded functionalized melanin nanosystem complex provided by the present invention can produce a significant photothermal effect under the action of 808nm laser, promoting the expression of PD-L1.

[0112] (3) The polypeptide chain and 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles used in this invention can undergo polymerization under enzyme stimulation, prolong their residence time in tumor tissue, and form a metal reservoir, thereby effectively activating the STING pathway. In addition, engineered E. coli secretes PD-L1 under laser, blocking the PD-1 / PD-L1 axis and reversing T cell exhaustion.

[0113] (4) The engineered bacteria-loaded functionalized melanin nanoparticle complex prepared in this invention exhibits good biocompatibility and can increase in particle size under the action of legumain enzyme in the tumor microenvironment, thereby enhancing the accumulation of nanoparticles at the tumor site. Furthermore, under laser irradiation, the engineered bacteria secrete PD-L1, restoring the function of effector T cells and enhancing the anti-tumor immunotherapy effect, with a tumor inhibition rate as high as 80%. Therefore, it can be used to prepare anti-tumor drugs, realizing its application in the preparation of anti-tumor drugs.

[0114] In summary, this invention provides a method for preparing and applying a complex of engineered bacteria-loaded functionalized melanin nanoparticles. This method effectively addresses the retention problem of nanoparticles at tumor sites, improves the efficacy of anti-tumor immunotherapy, and ensures effective drug delivery for cancer treatment. The process involves preparing melanin nanoparticles, reacting them with sodium hydroxide to generate carboxylated melanin nanoparticles, and then modifying the nanoparticles with polypeptide chains and 2-cyano-6-amino-benzothiazole via amidation. Metal ions are then complexed onto the surface of the melanin nanoparticles using the amino and phenolic hydroxyl groups, resulting in a functionalized melanin nanoparticle system. This system is then adhered to the surface of engineered bacteria through adhesion, yielding a complex of engineered bacteria-loaded functionalized melanin nanoparticles. The preparation method is simple, stable, and reliable, effectively improving the retention effect of nanoparticles at tumor sites, efficiently activating anti-tumor immune responses, and enhancing drug utilization and efficacy. This represents an innovation in tumor immunotherapy drugs with significant economic and social benefits.

Claims

1. A method for preparing a composite system of engineered bacteria-loaded functionalized melanin nanoparticles, characterized in that, Melanin was prepared into melanin nanoparticles, and carboxylated melanin nanoparticles were formed through a thiomalic acid reaction. A polypeptide chain and 2-cyano-6-amino-benzothiazole were modified at the carboxyl terminus of the melanin nanoparticles through an amidation reaction. Metal ions were then complexed onto the surface of the functionalized melanin nanoparticles through metal complexation. Finally, the functionalized melanin nanoparticle system was adhered to the surface of engineered *E. coli* bacteria using adhesion, resulting in a complex of engineered bacteria loaded with functionalized melanin nanoparticles. The specific steps include: (1) Preparation of melanin nanoparticles (MNP): Dissolve 5-12 mg of melanin in 5-10 mL of 0.1 mol / L sodium hydroxide and sonicate in an ice bath for 0.5 h; add 0.1 mol / L dilute hydrochloric acid to adjust the pH to 6.5-7.5, dialyze with a dialysis bag with MWCO of 2500 Da for 6 h, freeze dry to obtain melanin nanoparticles with a particle size of 20 nm to 60 nm; (2) Preparation of carboxylated melanin nanoparticles: Take 5-12 mg of melanin nanoparticles from step (1) and dissolve them in 8-10 mL of solvent. Add 6-8 mg of thiomalic acid and stir at room temperature for 6-8 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 48 h and freeze dry to obtain carboxylated melanin nanoparticles. The solvent is one of ultrapure water, PBS buffer at pH 7.4, DMF, or Tris-HCl buffer at pH 8.0; (3) Preparation of peptide chain modified melanin nanoparticles (MNP-pep): Weigh 8-12 mg of carboxylated melanin nanoparticles from step (2) and dissolve them in a solvent. Add 8-12 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 6-10 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 3-5 h. Add 5-9 mg of peptide chain. Add 0.1 mol / L sodium hydroxide to adjust the pH to 7.5-8.

0. Stir at nitrogen in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. After freeze drying, obtain peptide chain modified melanin nanoparticles. The polypeptide chain is a polypeptide chain containing the Ala-Ala-Asn-Cys-Lys (alanine-alanine-aspartic acid-cysteine-lysine) sequence; (4) Preparation of 2-cyano-6-amino-benzothiazole modified melanin nanoparticles (MNP-CABT): Weigh 8-12 mg of carboxylated melanin nanoparticles and dissolve them in a solvent. Add 8-12 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 6-10 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 3-5 h. Weigh 3-7 mg of 2-cyano-6-amino-benzothiazole and dissolve it in a solvent. Add it to the reaction system. Add 0.1 mol / L dilute hydrochloric acid to adjust the pH to 7.5-8.

0. Stir at nitrogen atmosphere in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. Freeze dry to obtain 2-cyano-6-amino-benzothiazole modified melanin nanoparticles. (5) Preparation of melanin nanoparticles loaded with metal ions (MNP-p&C@Mn): Weigh 8-12 mg of the polypeptide chain modified melanin nanoparticles from step (3) and 1.0-2.5 mg of the metal salt, dissolve them in the solvent, and sonicate them. After mixing them evenly, react them at 40°C for 1 h, and then put them into a dialysis bag with MWCO of 12000 Da for dialysis for 48 h to obtain the polypeptide chain modified melanin nanoparticles loaded with metal ions. Preparation of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions: Weigh 8-12 mg of the 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles from step (4) and 1.0-2.5 mg of the metal salt, dissolve them in solvents respectively, and dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h, and then put them into a dialysis bag with MWCO of 12000 Da for dialysis for 48 h to obtain 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions. The metal ion is one of manganese ion and zinc ion; (6) Preparation of engineered bacteria-loaded functionalized melanin nanoparticle complex (MNP-p&C@Mn-BP): Weigh 0.5-2 mg of the melanin nanoparticles modified with the polypeptide chain loaded with metal ions from step (5) and 0.5-2 mg of the melanin nanoparticles modified with 2-cyano-6-amino-benzothiazole loaded with metal ions, disperse them in 0.5-2 mL of solvent, and then add 10 mL of engineered Escherichia coli Tris-HCl buffer. 8 CFU / mL, stirred at room temperature for 3 h, centrifuged at 8000 rpm for 10 min, supernatant was discarded, and the precipitate was resuspended in sterile Tris-HCl buffer to obtain the complex of engineered bacteria-loaded functionalized melanin nanosystem. The engineered Escherichia coli is one of the engineered bacteria introduced with PD-1 or PD-L1 plasmids.

2. The method for preparing the engineered bacteria-loaded functionalized melanin nanocomposite according to claim 1, characterized in that, Includes the following steps: (1) Preparation of melanin nanoparticles: Dissolve 5 mg of melanin in 5 mL of 0.1 mol / L sodium hydroxide, sonicate in an ice bath for 0.5 h; add 0.1 mol / L dilute hydrochloric acid to adjust the pH to 6.5, dialyze with a dialysis bag with MWCO of 2500 Da for 6 h, freeze dry to obtain melanin nanoparticles. (2) Preparation of carboxylated melanin nanoparticles: Take 5 mg of melanin nanoparticles from step (1) and dissolve them in 8 mL of Tris-HCl buffer solution with pH 8.

0. Add 6 mg of thiomalic acid, stir at room temperature for 6 h, dialyze with a dialysis bag with MWCO of 12000 Da for 48 h, freeze dry, and obtain carboxylated melanin nanoparticles. (3) Preparation of peptide chain modified melanin nanoparticles: Weigh 8 mg of carboxylated melanin nanoparticles from step (2) and dissolve them in ultrapure water. Add 8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 6 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 3 h. Add 5 mg of peptide chain and add 0.1 mol / L sodium hydroxide to adjust the pH to 7.

5. Stir at nitrogen in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. After freeze drying, obtain peptide chain modified melanin nanoparticles. (4) Preparation of 2-cyano-6-amino-benzothiazole modified melanin nanoparticles: 8 mg of carboxylated melanin nanoparticles were weighed and dissolved in ultrapure water. 8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 6 mg of N-hydroxysuccinimide were added. The mixture was stirred at room temperature in the dark for 3-5 h. 3 mg of 2-cyano-6-amino-benzothiazole was weighed and dissolved in DMF and added to the reaction system. 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 7.

5. The mixture was stirred under nitrogen in the dark for 24 h. The mixture was dialyzed for 24 h using a dialysis bag with a MWCO of 12000 Da. The mixture was then freeze-dried to obtain 2-cyano-6-amino-benzothiazole modified melanin nanoparticles. (5) Preparation of melanin nanoparticles loaded with metal ions: Weigh 8 mg of the polypeptide chain modified melanin nanoparticles from step (3) and 1.0 mg of metal salt MnCl2·4H2O, dissolve them in ultrapure water, and dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h, and then put them into a dialysis bag with MWCO of 12000 Da for dialysis for 48 h to obtain the polypeptide chain modified melanin nanoparticles loaded with metal ions. Preparation of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions: Weigh 8 mg of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles from step (4) and 1.0 mg of metal salt MnCl2·4H2O, respectively, and dissolve them in ultrapure water. Dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h. Then, put them into a dialysis bag with MWCO of 12000 Da and dialyze for 48 h to obtain 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions. (6) Preparation of engineered bacteria-loaded functionalized melanin nanoparticle complex: Weigh 0.5 mg of the melanin nanoparticles modified with metal ion-loaded polypeptide chains from step (5) and 0.5 mg of the melanin nanoparticles modified with metal ion-loaded 2-cyano-6-amino-benzothiazole, and disperse them in 0.5 mL of Tris-HCl buffer at pH 8.

0. Then add 10 mL of Tris-HCl buffer to PD-1 engineered E. coli. 8 The mixture was stirred at room temperature for 3 hours with CFU / mL, centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in sterile Tris-HCl buffer to obtain the complex of engineered bacteria-loaded functionalized melanin nanosystem.

3. The method for preparing the engineered bacteria-loaded functionalized melanin nanocomposite according to claim 1, characterized in that, Includes the following steps: (1) Preparation of melanin nanoparticles: Dissolve 7 mg of melanin in 8 mL of 0.1 mol / L sodium hydroxide, sonicate in an ice bath for 0.5 h; add 0.1 mol / L dilute hydrochloric acid to adjust the pH to 7.0, dialyze with a dialysis bag with MWCO of 2500 Da for 6 h, freeze dry to obtain melanin nanoparticles; (2) Preparation of carboxylated melanin nanoparticles: Take 7 mg of melanin nanoparticles from step (1) and dissolve them in 9 mL of Tris-HCl buffer solution with pH 8.

0. Add 7 mg of thiomalic acid, stir at room temperature for 7 h, dialyze with a dialysis bag with MWCO of 12000 Da for 48 h, freeze dry, and obtain carboxylated melanin nanoparticles. (3) Preparation of peptide chain modified melanin nanoparticles: Weigh 9 mg of carboxylated melanin nanoparticles from step (2) and dissolve them in ultrapure water. Add 9 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 7 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 4 h. Add 6 mg of peptide chain and add 0.1 mol / L sodium hydroxide to adjust the pH to 7.

8. Stir at nitrogen in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. After freeze drying, obtain peptide chain modified melanin nanoparticles. (4) Preparation of 2-cyano-6-amino-benzothiazole modified melanin nanoparticles: 9 mg of carboxylated melanin nanoparticles were weighed and dissolved in ultrapure water. 9 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 7 mg of N-hydroxysuccinimide were added. The mixture was stirred at room temperature in the dark for 4 h. 4 mg of 2-cyano-6-amino-benzothiazole was weighed and dissolved in DMF and added to the reaction system. 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 7.

8. The mixture was stirred under nitrogen in the dark for 24 h. The mixture was dialyzed for 24 h using a dialysis bag with a MWCO of 12000 Da. The mixture was then freeze-dried to obtain 2-cyano-6-amino-benzothiazole modified melanin nanoparticles. (5) Preparation of melanin nanoparticles loaded with metal ions: Weigh 9 mg of the polypeptide chain modified melanin nanoparticles from step (3) and 1.5 mg of metal salt ZnCl2·4H2O, dissolve them in ultrapure water, and dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h, and then put them into a dialysis bag with MWCO of 12000 Da for dialysis for 48 h to obtain the polypeptide chain modified melanin nanoparticles loaded with metal ions. Preparation of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions: 9 mg of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles from step (4) and 1.5 mg of metal salt ZnCl2·4H2O were weighed and dissolved in ultrapure water. They were dissolved by sonication. After mixing evenly, they were reacted at 40°C for 1 h. Then they were placed in a dialysis bag with MWCO of 12000 Da and dialyzed for 48 h to obtain 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions. (6) Preparation of engineered bacteria-loaded functionalized melanin nanoparticle complex: Weigh 1.0 mg of the melanin nanoparticles modified with the polypeptide chain loaded with metal ions from step (5) and 1.0 mg of the melanin nanoparticles modified with 2-cyano-6-amino-benzothiazole loaded with metal ions, and disperse them in 2 mL of Tris-HCl buffer at pH 8.

0. Then add 0.5 mL of E. coli Tris-HCl buffer containing PD-L1 plasmid. 8 The mixture was stirred at room temperature for 3 hours with CFU / mL, centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in sterile Tris-HCl buffer to obtain the complex of engineered bacteria-loaded functionalized melanin nanosystem.

4. The method for preparing the engineered bacteria-loaded functionalized melanin nanosystem complex according to claim 1, characterized in that, Includes the following steps: (1) Preparation of melanin nanoparticles: Dissolve 8 mg of melanin in 10 mL of 0.1 mol / L sodium hydroxide, sonicate in an ice bath for 0.5 h; add 0.1 mol / L dilute hydrochloric acid to adjust the pH to 7.5, dialyze with a dialysis bag with MWCO of 2500 Da for 6 h, freeze dry to obtain melanin nanoparticles. (2) Preparation of carboxylated melanin nanoparticles: Take 8 mg of melanin nanoparticles from step (1) and dissolve them in 10 mL of Tris-HCl buffer solution with pH 8.

0. Add 8 mg of thiomalic acid, stir at room temperature for 8 h, dialyze with a dialysis bag with MWCO of 12000 Da for 48 h, freeze dry, and obtain carboxylated melanin nanoparticles. (3) Preparation of peptide chain modified melanin nanoparticles: Weigh 10 mg of carboxylated melanin nanoparticles from step (2) and dissolve them in ultrapure water. Add 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 8 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 5 h. Add 8 mg of peptide chain and add 0.1 mol / L sodium hydroxide to adjust the pH to 8.

0. Stir at nitrogen in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. After freeze drying, obtain peptide chain modified melanin nanoparticles. (4) Preparation of 2-cyano-6-amino-benzothiazole modified melanin nanoparticles: 10 mg of carboxylated melanin nanoparticles were weighed and dissolved in ultrapure water. 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 8 mg of N-hydroxysuccinimide were added. The mixture was stirred at room temperature in the dark for 5 h. 5 mg of 2-cyano-6-amino-benzothiazole was weighed and dissolved in DMF and added to the reaction system. 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 8.

0. The mixture was stirred in the dark under nitrogen for 24 h. The mixture was dialyzed for 24 h using a dialysis bag with a MWCO of 12000 Da. The mixture was then freeze-dried to obtain 2-cyano-6-amino-benzothiazole modified melanin nanoparticles. (5) Preparation of melanin nanoparticles loaded with metal ions: Weigh 10 mg of the polypeptide chain modified melanin nanoparticles from step (3) and 2 mg of metal salt MnCl2·4H2O, dissolve them in ultrapure water, and dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h, and then put them into a dialysis bag with MWCO of 12000 Da for dialysis for 48 h to obtain the polypeptide chain modified melanin nanoparticles loaded with metal ions. Preparation of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions: Weigh 10 mg of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles from step (4) and 2 mg of metal salt MnCl2·4H2O, respectively, and dissolve them in ultrapure water. Dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h. Then, put them into a dialysis bag with MWCO of 12000 Da and dialyze them for 48 h to obtain 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions. (6) Preparation of engineered bacteria-loaded functionalized melanin nanoparticle complex: Weigh 2 mg of melanin nanoparticles modified with metal ion-loaded polypeptide chains from step (5) and 2 mg of melanin nanoparticles modified with metal ion-loaded 2-cyano-6-amino-benzothiazole, and disperse them in 2 mL of Tris-HCl buffer at pH 8.

0. Then add 1.5 mL of E. coli Tris-HCl buffer containing PD-L1 plasmid. 8 The mixture was stirred at room temperature for 3 hours with CFU / mL, centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in sterile Tris-HCl buffer to obtain the complex of engineered bacteria-loaded functionalized melanin nanosystem.

5. The method for preparing the engineered bacteria-loaded functionalized melanin nanocomposite according to claim 1, characterized in that, Includes the following steps: (1) Preparation of melanin nanoparticles: 10 mg of melanin was dissolved in 9 mL of 0.1 mol / L sodium hydroxide and sonicated in an ice bath for 0.5 h; 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 6.8, and the mixture was dialyzed for 6 h using a dialysis bag with MWCO of 2500 Da. The mixture was then freeze-dried to obtain melanin nanoparticles. (2) Preparation of carboxylated melanin nanoparticles: Take 10 mg of melanin nanoparticles from step (1) and dissolve them in 10 mL of ultrapure water. Add 7.5 mg of thiomalic acid and stir at room temperature for 7.5 h. Dialyze them with a dialysis bag with MWCO of 12000 Da for 48 h and freeze dry to obtain carboxylated melanin nanoparticles. (3) Preparation of peptide-modified melanin nanoparticles: Weigh 11 mg of carboxylated melanin nanoparticles from step (2) and dissolve them in PBS buffer at pH 7.

4. Add 11 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 9 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 3.5 h. Add 7 mg of peptide chain and add 0.1 mol / L sodium hydroxide to adjust the pH to 7.

5. Stir at nitrogen in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. After freeze-drying, obtain peptide-modified melanin nanoparticles. (4) Preparation of 2-cyano-6-amino-benzothiazole modified melanin nanoparticles: 11 mg of carboxylated melanin nanoparticles were weighed and dissolved in PBS buffer solution with pH 7.

4. 11 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 9 mg of N-hydroxysuccinimide were added. The mixture was stirred at room temperature in the dark for 3.5 h. 6 mg of 2-cyano-6-amino-benzothiazole was weighed and dissolved in ultrapure water. The mixture was added to the reaction system. 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 7.

6. The mixture was stirred under nitrogen atmosphere in the dark for 24 h. The mixture was dialyzed for 24 h using a dialysis bag with MWCO of 12000 Da. The mixture was then lyophilized to obtain 2-cyano-6-amino-benzothiazole modified melanin nanoparticles. (5) Preparation of melanin nanoparticles loaded with metal ions: Weigh 11 mg of the polypeptide chain modified melanin nanoparticles from step (3) and 2.5 mg of metal salt ZnCl2·4H2O, respectively, and dissolve them in ultrapure water. Dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h. Then, put them into a dialysis bag with MWCO of 12000 Da and dialyze for 48 h to obtain the polypeptide chain modified melanin nanoparticles loaded with metal ions. Preparation of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions: 11 mg of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles from step (4) and 2.5 mg of metal salt ZnCl2·4H2O were weighed and dissolved in ultrapure water. They were dissolved by sonication and mixed evenly. The mixture was reacted at 40°C for 1 h and then placed in a dialysis bag with MWCO of 12000 Da for dialysis for 48 h to obtain 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions. (6) Preparation of engineered bacteria-loaded functionalized melanin nanoparticle complex: Weigh 1.5 mg of the melanin nanoparticles modified with metal ion-loaded polypeptide chains from step (5) and 1.5 mg of the melanin nanoparticles modified with metal ion-loaded 2-cyano-6-amino-benzothiazole, and disperse them in 1.5 mL of PBS buffer at pH 7.

4. Then add 1 mL of E. coli Tris-HCl buffer containing PD-L plasmid. 8 The mixture was stirred at room temperature for 3 hours with CFU / mL, centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in sterile Tris-HCl buffer to obtain the complex of engineered bacteria-loaded functionalized melanin nanosystem.

6. The method for preparing the engineered bacteria-loaded functionalized melanin nanosystem complex according to claim 1, characterized in that, Includes the following steps: (1) Preparation of melanin nanoparticles: Dissolve 6 mg of melanin in 6 mL of 0.1 mol / L sodium hydroxide, sonicate in an ice bath for 0.5 h; add 0.1 mol / L dilute hydrochloric acid to adjust the pH to 7.2, dialyze with a dialysis bag with MWCO of 2500 Da for 6 h, freeze dry to obtain melanin nanoparticles; (2) Preparation of carboxylated melanin nanoparticles: Take 6 mg of melanin nanoparticles from step (1) and dissolve them in 8 mL of DMF solvent. Add 6.5 mg of thiomalic acid, stir at room temperature for 6.5 h, dialyze with a dialysis bag with MWCO of 12000 Da for 48 h, freeze dry, and obtain carboxylated melanin nanoparticles. (3) Preparation of peptide chain modified melanin nanoparticles: Weigh 8 mg of carboxylated melanin nanoparticles from step (2) and dissolve them in DMF solvent. Add 8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 6 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 4.5 h. Add 5 mg of peptide chain and add 0.1 mol / L sodium hydroxide to adjust the pH to 7.

5. Stir at nitrogen in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. After freeze drying, obtain peptide chain modified melanin nanoparticles. (4) Preparation of 2-cyano-6-amino-benzothiazole modified melanin nanoparticles: 8 mg of carboxylated melanin nanoparticles were weighed and dissolved in DMF solvent. 8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 6 mg of N-hydroxysuccinimide were added. The mixture was stirred at room temperature in the dark for 3 h. 3-7 mg of 2-cyano-6-amino-benzothiazole was weighed and dissolved in Tris-HCl buffer solution with pH 8.

0. The solution was added to the reaction system. 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 7.

8. The mixture was stirred under nitrogen in the dark for 24 h. The mixture was dialyzed for 24 h using a dialysis bag with MWCO of 12000 Da. The mixture was then lyophilized to obtain 2-cyano-6-amino-benzothiazole modified melanin nanoparticles. (5) Preparation of melanin nanoparticles loaded with metal ions: Weigh 8 mg of the polypeptide chain modified melanin nanoparticles from step (3) and 1.2 mg of metal salt MnCl2·4H2O respectively and dissolve them in DMF. Dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h. Then, put them into a dialysis bag with MWCO of 12000 Da and dialyze for 48 h to obtain the polypeptide chain modified melanin nanoparticles loaded with metal ions. Preparation of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions: Weigh 8 mg of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles from step (4) and 1.2 mg of metal salt MnCl2·4H2O respectively and dissolve them in DMF. Dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h. Then, put them into a dialysis bag with MWCO of 12000 Da and dialyze for 48 h to obtain 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions. (6) Preparation of engineered bacteria-loaded functionalized melanin nanoparticle complex: Weigh 0.8 mg of the melanin nanoparticles modified with the polypeptide chain loaded with metal ions from step (5) and 0.8 mg of the melanin nanoparticles modified with 2-cyano-6-amino-benzothiazole loaded with metal ions, disperse them in 0.8 mL of ultrapure water, and then add 10 mL of E. coli Tris-HCl buffer containing PD-L1 plasmid. 8 The mixture was stirred at room temperature for 3 hours with CFU / mL, centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in sterile Tris-HCl buffer to obtain the complex of engineered bacteria-loaded functionalized melanin nanosystem.

7. The method for preparing the engineered bacteria-loaded functionalized melanin nanosystem complex according to claim 1, characterized in that, Includes the following steps: (1) Preparation of melanin nanoparticles: 12 mg of melanin was dissolved in 10 mL of 0.1 mol / L sodium hydroxide and sonicated in an ice bath for 0.5 h; 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 7.5, and the mixture was dialyzed for 6 h using a dialysis bag with a MWCO of 2500 Da. The mixture was then freeze-dried to obtain melanin nanoparticles. (2) Preparation of carboxylated melanin nanoparticles: Take 12 mg of melanin nanoparticles from step (1) and dissolve them in 10 mL of Tris-HCl buffer solution with pH 8.

0. Add 8 mg of thiomalic acid, stir at room temperature for 8 h, dialyze with a dialysis bag with MWCO of 12000 Da for 48 h, freeze dry, and obtain carboxylated melanin nanoparticles. (3) Preparation of peptide chain modified melanin nanoparticles: Weigh 12 mg of carboxylated melanin nanoparticles from step (2) and dissolve them in Tris-HCl buffer solution with pH 8.

0. Add 12 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10 mg of N-hydroxysuccinimide. Stir at room temperature in the dark for 5 h. Add 9 mg of peptide chain and add 0.1 mol / L sodium hydroxide to adjust the pH to 8.

0. Stir at nitrogen in the dark for 24 h. Dialyze with a dialysis bag with MWCO of 12000 Da for 24 h. After freeze drying, obtain peptide chain modified melanin nanoparticles. (4) Preparation of 2-cyano-6-amino-benzothiazole modified melanin nanoparticles: 12 mg of carboxylated melanin nanoparticles were weighed and dissolved in Tris-HCl buffer solution with pH 8.

0. 12 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10 mg of N-hydroxysuccinimide were added. The mixture was stirred at room temperature in the dark for 5 h. 7 mg of 2-cyano-6-amino-benzothiazole was weighed and dissolved in PBS buffer solution with pH 7.

4. The mixture was added to the reaction system. 0.1 mol / L dilute hydrochloric acid was added to adjust the pH to 8.

0. The mixture was stirred under nitrogen atmosphere in the dark for 24 h. The mixture was dialyzed for 24 h using a dialysis bag with MWCO of 12000 Da. The mixture was then lyophilized to obtain 2-cyano-6-amino-benzothiazole modified melanin nanoparticles. (5) Preparation of melanin nanoparticles loaded with metal ions: Weigh 12 mg of the polypeptide chain modified melanin nanoparticles from step (3) and 2.5 mg of metal salt ZnCl2·4H2O, respectively, and dissolve them in Tris-HCl buffer at pH 8.

0. Dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h. Then, put them into a dialysis bag with MWCO of 12000 Da and dialyze for 48 h to obtain the polypeptide chain modified melanin nanoparticles loaded with metal ions. Preparation of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions: Weigh 12 mg of 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles from step (4) and 2.5 mg of metal salt ZnCl2·4H2O, respectively, and dissolve them in Tris-HCl buffer solution at pH 8.

0. Dissolve them by sonication. After mixing them evenly, react them at 40°C for 1 h. Then, put them into a dialysis bag with MWCO of 12000 Da and dialyze for 48 h to obtain 2-cyano-6-amino-benzothiazole-modified melanin nanoparticles loaded with metal ions. (6) Preparation of engineered bacteria-loaded functionalized melanin nanoparticle complex: Weigh 2 mg of melanin nanoparticles modified with metal ion-loaded polypeptide chains from step (5) and 2 mg of melanin nanoparticles modified with metal ion-loaded 2-cyano-6-amino-benzothiazole, and disperse them in 2 mL of Tris-HCl buffer at pH 8.

0. Then add 1.5 mL of E. coli Tris-HCl buffer containing PD-L1 plasmid. 8 The mixture was stirred at room temperature for 3 hours with CFU / mL, centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in sterile Tris-HCl buffer to obtain the complex of engineered bacteria-loaded functionalized melanin nanosystem.

8. The use of the engineered bacteria-loaded functionalized melanin nanosystem complex prepared by the method of any one of claims 1-7 in the preparation of antitumor drugs.

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