Engineered probiotics with intracellular chimeric nanoszymes and preparation and application thereof

Abstract

The application provides an engineered probiotic with intracellular chimeric nanoscale enzyme, which is a probiotic modified with several antioxidant enzymes on an inner membrane; the antioxidant enzyme is composed of a metal oxide nanoscale enzyme with a particle size less than 10 nm and a targeting molecular structure and a saccharide molecular structure modified on the surface of the metal oxide nanoscale enzyme; the targeting molecular structure and the saccharide molecular structure are connected to the surface of the metal oxide nanoscale enzyme through a connecting group containing a metal ligand structure based on coordination and electrostatic interaction, and the antioxidant enzyme accounts for 5-20% of the mass of the engineered probiotic. The application also provides a method for preparing the engineered probiotic and application of the engineered probiotic in preparation of a medicine or food for preventing or treating antibiotic-associated diarrhea. The engineered probiotic can realize efficient elimination of hydroxyl radicals near a membrane induced by an antibiotic, so as to accurately inhibit a lipid peroxidation reaction and protect the integrity of a cell membrane.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a type of engineered probiotic with internally integrated nanozymes, its preparation method and its application in the treatment of antibiotic-associated diarrhea. Background Technology

[0002] Oral probiotics are currently a first-line clinical strategy for preventing and treating antibiotic-associated diarrhea (AAD), but their effectiveness is often significantly limited by the insufficient survival rate of probiotics under long-term antibiotic intervention. Therefore, improving the antibiotic tolerance of probiotics has become an important research direction for improving AAD treatment.

[0003] The bactericidal effect of antibiotics is closely related to the burst of hydroxyl radicals (·OH) they induce. After antibiotics act on bacteria, they can activate the electron transport chain and the tricarboxylic acid cycle, leading to a large consumption of reduced coenzyme I and disrupting cellular redox homeostasis. This process triggers the formation of superoxide anions (O2··OH). - The iron-sulfur clusters dissociate, releasing free iron ions; these ions react with hydrogen peroxide (H₂O₂) via the Fenton reaction to generate highly reactive ·OH radicals. These free radicals can cause irreversible damage such as DNA breakage, protein denaturation, and lipid peroxidation, constituting the core mechanism by which antibiotics kill bacteria. Therefore, intervening in ·OH-mediated oxidative damage is considered a feasible way to enhance probiotic tolerance.

[0004] Bacterial ·OH exhibits a distinct spatial distribution and functional duality: in the cytoplasm, low levels of ·OH participate in regulating energy metabolism remodeling and biosynthesis, playing a positive role in maintaining normal physiological activities; however, when located near the cell membrane, ·OH readily attacks the exposed unsaturated fatty acids in the phospholipid bilayer, triggering an autocatalytic lipid peroxidation chain reaction, leading to loss of membrane integrity and cell death. While traditional broad-spectrum antioxidant strategies can mitigate oxidative damage, they also interfere with intracellular ·OH-dependent signal transduction and metabolic regulation, thus failing to simultaneously address the dual needs of "protecting membrane structure" and "maintaining physiological function."

[0005] To address this contradiction, it is necessary to propose a method that can effectively block lipid peroxidation without affecting the normal intracellular redox homeostasis, thereby improving the survival ability of probiotics in an antibiotic environment. Summary of the Invention

[0006] To improve the tolerance of probiotics in antibiotic environments, this invention constructs an engineered probiotic with an intestinal membrane-integrated nanozyme based on a tolerance enhancement strategy that inhibits probiotic intestinal membrane-specific lipid peroxidation. This probiotic can precisely inhibit intracellular lipid peroxidation caused by antibiotics.

[0007] To achieve the above objectives, the materials of this invention adopt the following technical solution:

[0008] In a first aspect, the present invention provides a metal oxide nanocomposite material, which is composed of metal oxide nanoparticles with a particle size of less than 10 nm (M... x The structure consists of a target molecular structure (NT) and a carbohydrate molecular structure (Sac) modified with O; the NT and Sac are respectively linked to the M via a linker containing a metal ligand structure (Lig). x The O surface is connected based on coordination and electrostatic interactions.

[0009] In the metal oxide nanocomposite material of the present invention, the M x O can be selected from any one or more ultra-small oxide nanoparticles with a size not exceeding 10 nanometers, where M represents a metal atom and can be selected from any element selected from Fe, Cu, Zn, Mn, Mo, Pd, Sn, Ta, or Ir; x represents the number of metal atoms bonded to each oxygen atom. x Specifically, O can be selected from any of the following ultra-small oxide nanoparticles with a size not exceeding 10 nm: iridium oxide (Ir) x O, molybdenum oxide (Mo) x O, Tin oxide (Sn) x O, tantalum oxide (Ta) x O or iron oxide Fe x O; Ir oxide is the preferred option. x O.

[0010] In the metal oxide nanocomposite material of the present invention, the NT is a targeting molecule comprising the structure shown in the following formula (NT):

[0011] (NT), where n is an integer from 2 to 4; preferably 2 or 3.

[0012] In a preferred embodiment of the present invention, the NT is selected from molecules comprising any one of the following formulas (NT-1), (NT-2), or (NT-3); in the most preferred embodiment, the NT is a molecule comprising the structure of formula NT-1.

[0013] (NT-1)

[0014] (NT-2)

[0015] or

[0016] (NT-3).

[0017] In the metal oxide nanocomposite material of the present invention, the Sac can be derived from glucose, galactose, lactose, ribose or maltodextrin; preferably from maltodextrin.

[0018] In the metal oxide nanocomposite material of the present invention, the Lig in the linker containing the metal ligand structure (Lig) is located at the end of the linker that is not connected to NT or Sac, and can be a variety of existing functional groups that can interact with metal ions through coordination, chelation or electrostatic adsorption; preferably, the functional group is selected from DOTA, NOA, -SH, -COOH, EDTA, -NH3, etc.; the most preferred functional group is DOTA or NOA.

[0019] In a further preferred embodiment, the linker containing the metal ligand structure (Lig) further comprises a polyethylene glycol (PEG) molecular chain segment; the PEG molecular chain segment may be a polyethylene glycol molecular chain segment with a polymeric unit number of 20-230 (i.e., an average molecular weight of approximately 1000-10000 Da); most preferably, it is a polyethylene glycol molecular chain segment with a polymeric unit number of 40-50 (i.e., an average molecular weight of approximately 2000 Da).

[0020] Prior to this invention, the inventors first discovered that a series of molecules containing phenolic hydroxyl groups, as shown in formula (NT), possess intracellular membrane targeting properties. Based on this, this invention, in the aforementioned M... x Surface modification with NT and Sac can enable nanocomposites to target microorganisms into cells and the inner membrane.

[0021] Based on this, the present invention further provides an engineered probiotic with intracellular chimeric nanoenzymes, which is a probiotic with several antioxidant enzymes modified on its inner membrane; the antioxidant enzymes are the metal oxide nanocomposite materials of the present invention.

[0022] In the engineered probiotics of the present invention, each of the antioxidant enzymes is connected to the probiotic inner membrane through its contained NT structure.

[0023] The engineered probiotics described in this invention can be any one or more clinically used probiotics, including but not limited to: Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus bulgaricus, Lactobacillus johnsonii, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium infantis, Bacillus coagulans, Propionibacterium freundii, Enterococcus faecalis, Lactococcus lactis, or Streptococcus thermophilus, or a combination of two of these. In a preferred embodiment, the probiotic is any one of Lactobacillus rhamnosus (LGG), Lactobacillus plantarum, or Bifidobacterium longum.

[0024] In the preferred engineered probiotics of this invention, the antioxidant enzyme accounts for 5-20% of the mass of the engineered probiotics.

[0025] In a second aspect, the present invention also provides a method for preparing engineered probiotics containing the intracellular chimeric nanozyme described in the first aspect, comprising the following steps:

[0026] 1) Ultrasmall metal oxide nanozymes with a particle size of less than 10 nm were prepared by metal chloride under the action of a reducing agent and differential centrifugation, denoted as "M". x O”; where “M” x In "O", M represents a metallic element, and x represents the number of metallic atoms bonded to each oxygen atom.

[0027] 2) NT-SH and Sac-SH were reacted with maleimide functionalized modifiers with one end linked to Lig in a solvent at room temperature for 4-6 hours, with the pH of the reaction system controlled at 5-8.5. Covalent linkage was achieved through click reaction between thiol and maleimide bond, resulting in target molecules with Lig end groups and sugar molecules with Lig end groups, respectively.

[0028] The NT-SH mentioned is a targeting molecule with the following structure:

[0029] (NT-SH), where n is an integer from 2 to 4; the optimal choice is 2 or 3.

[0030] In a preferred embodiment, the NT-SH can be any of the following:

[0031] (NT-SH-1)

[0032] (NT-SH-2)

[0033] or

[0034] (NT-SH-3).

[0035] The optimal choice is NT-SH-1;

[0036] The Sac-SH mentioned refers to thiolated sugar molecules, which can be selected from any one or a combination of two or more of the following: thiolated glucose, thiolated galactose, thiolated lactose, thiolated ribose, or thiolated maltodextrin; thiolated maltodextrin is preferred.

[0037] The maleimide-functionalized modifier with one end connected to Lig is a functional group that can interact with metal ions through coordination, chelation, or electrostatic adsorption. Preferably, the functional group is selected from any one of DOTA, NOTA, -SH, -COOH, EDTA, and -NH3; most preferably, the functional group is DOTA or NOTA. In a further preferred embodiment, the maleimide-functionalized modifier with one end connected to Lig also contains polyethylene glycol segments with a polymerization unit number of 20-230 (preferably 40-50) as connecting arms.

[0038] 3) Take the M obtained in 1) x The target molecule with a Lig end group obtained from O and 2) and the functionalized sugar molecule with a Lig end group are mixed together at room temperature for at least 10 hours to obtain a composite metal oxide nanoparticle with a surface-modified inner membrane target molecule and a sugar molecule, denoted as "M". x O@SN", where "M" x In “O”, M represents a metal element, x represents the number of metal atoms bonded to each oxygen atom, S represents a carbohydrate molecule structure, and N represents an inner membrane targeting molecule structure.

[0039] 4) The nanoparticles M obtained in 3) x By co-incubating O@SN with probiotics (Probio) and washing, an engineered probiotic containing intracellular chimeric nanozymes can be obtained, denoted as "Probio@SN@M". x O".

[0040] In the preparation method of the present invention, the metal chloride solution in 1) can be selected from any one of FeCl3, MnCl2, SnCl4, TaCl3, and IrCl3; preferably IrCl3.

[0041] In the preparation method of the present invention, the molar ratio of the maleimide functionalized modifier with one end connected to Lig and the NT-SH in step 2) is 1~1.5:1~2, more preferably 1~1.2:1~1.5, and even more preferably 1.1:1.25.

[0042] In the preparation method of the present invention, the molar ratio of the maleimide functionalized modifier with one end connected to Lig in step 2) to the Sac-SH is 1:1~2; preferably 1:1~1.5; most preferably 1:1.2~1.3.

[0043] In the preparation method described in this invention, M in 3) xThe mass ratio of the target molecule with a Lig terminal group to the functionalized sugar molecule with a Lig terminal group is 1:0.1~2:0.1~2, preferably 1:0.1~2:0.1~1; more preferably 1:0.1-2:0.1-0.2; most preferably 1:0.1-0.5:0.2 or 1:1-2:0.1.

[0044] In the preparation method described in this invention, the probiotic Probio mentioned in step 4) and the M x The ratio of O@SN is 10. 8 CFU: 0.05~0.2mg; more preferably 10mg. 8 CFU: 0.05~0.1 mg; more preferably 10 mg. 8 CFU: 0.05 mg.

[0045] Thirdly, the present invention also provides the use of the engineered probiotics described in the first aspect in the preparation of drugs for the prevention or treatment of antibiotic-associated diarrhea.

[0046] In the application described in this invention, the antibiotic-associated diarrhea can be a symptom caused by any of the following antibiotics: (1) quinolone antibiotics, such as ciprofloxacin, levofloxacin and toloxacin; (2) β-amide antibiotics, such as penicillin G, amoxicillin and ampicillin; (3) aminoglycoside antibiotics, such as streptomycin, gentamicin and amikacin.

[0047] This invention is based on a probiotic tolerance enhancement strategy that inhibits endometrial-specific lipid peroxidation. By modifying (e.g., chimeric) probiotic endometrial membranes with nanozymes possessing antioxidant functions, it achieves precise scavenging of antibiotic-induced ·OH in the membrane periphery. A prerequisite for achieving the above objectives of this invention is the inventors' discovery that a specific class of compound molecules containing phenolic hydroxyl groups (whose structure includes the NT structure described in this invention) possesses bacterial endometrial targeting properties. Based on this discovery, this invention utilizes M… x The O surface was simultaneously modified with both this type of NT structure and the Sac structure of carbohydrate molecules to obtain M. x In O@SN, the Sac structure can be transported into the bacterial interior by ABC transporters on the surface of probiotics, thereby driving M... x O@SN enters the bacterial interior, while the NT structure, which has endomembrane targeting, can promote M x O@SN binds stably to the bacterial inner membrane, thereby enabling the antioxidant nanozyme to precisely remove ·OH from the antibiotic-induced membrane periphery without interfering with intracellular ·OH-dependent signal transduction and metabolic regulation. Compared to existing technologies, the probiotics described in this invention can block lipid peroxidation without affecting normal intracellular redox homeostasis, and their survival ability in antibiotic environments is significantly improved. Attached Figure Description

[0048] Figure 1 This is the Ir prepared in step 1) of Example 1. 3.2 Transmission electron microscopy image of O.

[0049] Figure 2 This is the 1H NMR spectrum of the thiolized targeting molecule NT-SH-1 prepared in step 2) of Example 1.

[0050] Figure 3 This demonstrates the bacterial endometrial targeting of the thiolized targeting molecule NT-SH-1 prepared in step 2) of Example 1.

[0051] Figure 4 This is the 1H NMR spectrum of DOTA-PEG2000-NT-1 prepared in step 3) of Example 1.

[0052] Figure 5 This is the 1H NMR spectrum of DOTA-PEG2000-MD prepared in step 4 of Example 1.

[0053] Figure 6 The Ir prepared in step 5 of Example 1 3.2 Transmission electron micrograph of O@SN.

[0054] Figure 7 These are schematic diagrams of the structures of a series of engineered probiotics prepared in Examples 15-22.

[0055] Figure 8 This demonstrates the proliferation ability of different probiotic materials in AMP in Experiment Example 1.

[0056] Figure 9 This demonstrates the proliferation ability of different probiotic materials in NOR in Experiment Example 1.

[0057] Figure 10 This demonstrates the proliferation ability of different probiotic materials in KAN in Experiment Example 1.

[0058] Figure 11 This reflects the different methods used to modify Ir in Experiment Example 2. 3.2 Comparison of the distribution of O in engineered probiotics before and after cell wall removal.

[0059] Figure 12 This reflects the bacterial survival rate of engineered probiotics prepared with different feed ratios in Experiment Example 4.

[0060] Figure 13 This shows the curves showing the change in the body weight of each group of mice over time in Experiment Example 5.

[0061] Figure 14The values ​​represent the body weight of each group of mice on the last day of the experiment in Example 5, where * represents p<0.05, ** represents p<0.01, and *** represents p<0.001.

[0062] Figure 15 The images depict typical fecal photographs of each group of mice in Experiment Example 5.

[0063] Figure 16 This reflects the fecal water content of each group of mice in Experiment 5 on the last day of the experiment. * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001. Detailed Implementation

[0064] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as they can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the appended claims.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0066] In this invention, unless the context otherwise requires, throughout the specification and the following claims, the word “comprising” and its variations such as “including” or “containing” will be understood to implicitly include the said integer or step, or group of integers or steps, but not exclude any other integer or step, or group of integers or steps.

[0067] In the following paragraphs, different aspects of the invention will be defined in more detail. Each aspect thus defined may be combined with any other one or more aspects unless expressly indicated to the contrary. In particular, any of the optional, preferred, or advantageous features may be combined with any other optional, preferred, or advantageous feature.

[0068] Several documents are referenced throughout this specification. Each document referenced herein (including all patents, patent applications, scientific publications, manufacturers' specifications, operating instructions, etc.) is incorporated herein by reference in its entirety, both above and below. Nothing herein shall be construed as an admission that the invention does not preclude any prior disclosure of such invention. In the event of any conflict between the definitions or teachings in such incorporated references and those set forth in this specification, the text of this specification shall prevail.

[0069] 1) Synthesis of ultrasmall metal oxide nanozymes: Under magnetic stirring, an aqueous solution of L-ascorbic acid was slowly added to a metal chloride solution. The pH of the mixture was then adjusted to approximately 8.5 using NaOH solution. The reaction was terminated by heating the mixture at 80°C for 24 hours. Subsequently, nanoparticles larger than 10 nm were removed by high-speed centrifugation, and free small molecules were removed by dialysis, thus obtaining ultrasmall metal oxide nanozyme materials. The purified nanozymes were concentrated by ultrafiltration. By changing the reaction time and the concentration of L-ascorbic acid, nanozymes of different sizes below 10 nm could be obtained. x O.

[0070] The metal chloride solution can be selected from any one of FeCl3, MoCl2, SnCl4, TaCl3, and IrCl3; IrCl3 is preferred. The prepared ultrasmall metal oxide nanozyme is iron oxide Fe with a particle size of less than 10 nm. x O, molybdenum oxide (Mo) x O, Tin oxide (Sn) x O, tantalum oxide (Ta) x O or Ir oxide x O; where x represents the number of metal atoms bonded to each oxygen atom.

[0071] 2) Synthesis of the functionalized endometrial targeting molecule (Lig-PEG2000-NT): A maleimide-based functionalized modifier, Lig-PEG2000-Mal, containing a PEG2000 segment linked to a Lig group at one end, and a thiolized endometrial targeting molecule, NT-SH, were dissolved in anhydrous DMSO or PBS buffer, respectively. The two were then mixed and reacted at 25°C with stirring for 4–6 hours. The pH of the reaction system was maintained at 7.4 during the reaction to ensure reaction efficiency. After the reaction was complete, a small amount of ethanolamine was added to terminate the reaction, and the product was purified by precipitation or dialysis. Finally, the target product (Lig-PEG2000-NT) was obtained by freeze-drying.

[0072] The structure of the endometrial targeting molecule NT-SH is as follows: , where n is an integer from 2 to 4; preferably 2 or 3.

[0073] In a preferred embodiment, the NT-SH can be (NT-SH-1) (NT-SH-2), or (NT-SH-3); NT-SH-1 is the preferred option.

[0074] 3) Synthesis of functionalized maltodextrin molecules (Lig-PEG2000-MD): A maleimide functionalizing agent, Lig-PEG2000-Mal, containing a PEG2000 segment and linked to a Lig group at one end, and a thiolized maltodextrin molecule (MD-SH) were dissolved separately in anhydrous DMSO or PBS buffer. The two were then mixed and reacted at 25°C with stirring for 4–6 hours, maintaining the pH of the reaction system at 6.5–7.5 throughout the reaction. After the reaction was complete, a small amount of ethanolamine was added to terminate the reaction, and the product was purified by precipitation or dialysis. Finally, Lig-PEG2000-MD was obtained by freeze-drying.

[0075] In other embodiments of the present invention, the thiolated maltodextrin molecule MD-SH can also be replaced with any one or more combinations of thiolated glucose Glc-SH, thiolated galactose Gal-SH, thiolated lactose Lac-SH or thiolated ribose Rib-SH, which can be used to synthesize functionalized sugar molecules Lig-PEG2000-Glc, Lig-PEG2000-Gal, Lig-PEG2000-Lac or Lig-PEG2000-Rib.

[0076] The Lig in the functionalized modifier is a functional group that can interact with metal ions through coordination, chelation, or electrostatic adsorption; preferably, the functional group is selected from DOTA, NOTA, -SH, -COOH, EDTA, -NH3, etc.; most preferably, the functional group is DOTA or NOTA.

[0077] In other embodiments of the present invention, PEG2000 in the functionalized modifier may be replaced with any one of PEG1000, PEG3400, PEG5000 or PEG10000.

[0078] 4) Synthesis of ultrasmall nanoparticles (M) with surface-modified inner membrane targeting molecules and carbohydrate molecules. x O@SN): Any M synthesized from 1) x O nanozyme was mixed with any one of the synthesized Lig-PEG2000-NT and 3) synthesized Lig-PEG2000-MD or other functionalized sugar molecules at room temperature for at least 10 hours. The resulting solution was then purified by dialysis (MWCO 8000-14000 Da) to prepare M. x O@SN.

[0079] 5) Engineered probiotics that synthesize intracellular chimeric nanozymes (Probio@SN@M) xO): After centrifuging and washing the probiotics (Probio), resuspend them in PBS. Centrifuge 1 mL of the bacterial culture and disperse it in M ​​containing (4) prepared in PBS. x In physiological saline, centrifuge and wash three times, then resuspend in physiological saline to construct Probio@SN@M. x O.

[0080] In a specific embodiment of the present invention, the mass ratio of metal chloride to L-ascorbic acid in 1) is 1:0.1~5, more preferably 1:0.5~2, and even more preferably 1:1~1.2.

[0081] In a specific embodiment of the present invention, in step 2), Lig-PEG2000-Mal and NT-SH are mixed in a molar ratio of 1~1.5:1~2 (more preferably 1~1.2:1~1.5, and even more preferably 1.1:1.25).

[0082] In the specific embodiments described in this invention, the molar ratio of the mixture of Lig-PEG2000-Mal and MD-SH thiolated sugar molecules in 3) is 1:1~2 (preferably 1:1~1.5; most preferably 1:1.2~1.3).

[0083] In the specific embodiments described in this invention, M in 4) x The mass ratio of functionalized sugar molecules such as O, Lig-PEG2000-NT, and Lig-PEG2000-MD is 1:0.1~2:0.1~2, preferably 1:0.1~2:0.1~1; more preferably 1:0.1-2:0.1-0.2; and most preferably 1:0.1-0.5:0.2 or 1:1-2:0.1.

[0084] In the specific embodiments described in this invention, Probio and M in 5) x The ratio of O@SN is 10. 8 CFU: 0.01~2mg; more preferably 10mg. 8 CFU: 0.05~1 mg; more preferably 10 mg. 8 CFU: 0.05~0.2 mg.

[0085] Based on the above specific embodiments, the present invention provides the following preferred embodiments and comparative examples for reference, to illustrate in detail the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0086] Example 1. Preparation of iridium oxide nanocomposites

[0087] First, an ultra-small iridium oxide nanozyme material was prepared. The specific raw materials and steps are as follows:

[0088] 1) Under magnetic stirring, 50 mL of L-ascorbic acid aqueous solution (100 mM) was slowly added to 50 mL of iridium chloride solution (10 mM). The pH of the mixture was then adjusted to approximately 8.5 using NaOH solution (1 M). The reaction was stopped after heating the mixture at 80 °C for 24 hours. Subsequently, larger nanoparticles were removed by high-speed centrifugation (12000 rpm, 12 min), and free small molecules were removed by dialysis (MWCO 8000-14000 Da), yielding ultra-small iridium oxide nanozymes (Ir) with a hydrated particle size of 6 nm. x A solution of O (X=3.2), Ir 3.2 The surface potential of O is -13 mV, and its microstructure is as follows: Figure 1 The transmission electron microscope image is shown. Experiments have verified that the ·OH scavenging ability of this ultra-small iridium oxide nanozyme solution increases with increasing concentration and time.

[0089] 2) Preparation of the thiol-targeting molecule NT-SH-1:

[0090]

[0091] (1) Under dry conditions, dopamine (compound 1) (1.0 eq) and 3-methyl-1H-pyrrole-2-carboxaldehyde (compound 2) (1.0 eq) were dissolved in methanol or acetonitrile (total concentration about 0.1-0.2 M), and stirred at room temperature under an inert atmosphere (such as nitrogen) for 30-60 min to form an imine; then 1-[(1-isocyano-3-methylbutyl)sulfonyl]-4-methylbenzene (compound 3) (1.0-1.2 eq) and 4-methyl-3-oxopentanoic acid (compound 4) (1.0-1.2 eq) were added sequentially, and the mixture was stirred at room temperature to carry out a four-component condensation reaction (Ugi reaction) for 12-24 h to obtain the target product of the Ugi reaction (intermediate 1).

[0092] (2) Dissolve intermediate 1 in glacial acetic acid (AcOH) or a mixed solvent of AcOH / ethanol (volume ratio approximately 1:1) (substrate concentration approximately 0.05-0.1 M), stir at slightly below room temperature, and add zinc powder (5-10 eq) in portions under an inert atmosphere (N2); then raise the reaction system to room temperature and continue stirring for 2-6 h, monitoring the reaction progress by LC-MS until the sulfonyl substrate is essentially eliminated, generating the target product, the thiolated targeting molecule NT-SH-1. This thiolated targeting molecule NT-SH-1 is named N-(3,4-dihydroxyphenylethyl)-N-(2-((1-mercapto-3-methylbutyl)amino)-1-(3-methyl-1H-pyrrolo-2-yl)-2-oxoethyl)-4-methyl-3-oxopentanamide, with the molecular formula C 26 H 37 N3O5S; molecular weight 503.2454; successful synthesis confirmed by 1H NMR (its NMR characterization results are as follows). Figure 2 (As shown).

[0093] NT-SH-1 and maleimide-fluorescein isothiocyanate (Mal-FITC, 1 mg / mL) were mixed and the mixture was shaken at 37°C for 12 hours to prepare fluorescently labeled NT-SH-1 (NT-SH-1). FITC ). 200 μL of NT-SH-1 FITC The bacterial suspension was mixed with 1 mg / mL LGG (OD600), and after 24 hours, the mixture was centrifuged (5000 rpm, 5 min) and washed three times. Fluorescence distribution within the bacteria was observed using a confocal microscope. Lysozyme (1 mg / mL) was then added to the bacterial suspension, and the cell wall was removed by incubation at 37°C for 30 min. Fluorescence distribution within the removed cell wall was observed using a confocal microscope.

[0094] The above confocal microscopy observation results are as follows: Figure 3 As shown, the green fluorescence in the bacteria before cell wall removal was mainly distributed at the edge, suggesting that NT-SH-1 particles may be distributed in the cell wall or inner membrane. After further removal of the cell wall, leaving only the inner membrane, the green fluorescence was still distributed at the edge, indicating that NT-SH-1 does indeed have an inner membrane target function.

[0095] 3) Dissolve DOTA-PEG2000-Mal and NT-SH-1 prepared in 2) separately in anhydrous DMSO or PBS buffer (pH 6.5) to prepare two solutions of 5 mg / mL each. Then, mix the two solutions, maintaining a molar ratio of DOTA-PEG2000-Mal to NT-SH-1 of 1:1.2 in the mixture. Stir the mixture at room temperature (25℃) for 4-6 hours, maintaining the pH around 6.5 during the reaction to ensure reaction efficiency. The reaction formula is as follows:

[0096]

[0097] After the reaction was complete, a small amount of ethanolamine was added to terminate the reaction, and the product was purified by precipitation or dialysis. Finally, the target product (DOTA-PEG2000-NT-1) was obtained by freeze-drying. Successful synthesis was confirmed by 1H NMR (see [link to original text]). Figure 4 ).

[0098] 4) Dissolve DOTA-PEG2000-Mal and thiolated maltodextrin (MD-SH) separately in anhydrous DMSO or PBS buffer (pH 6.5) to prepare 5 mg / mL solutions. Then, mix the two solutions, maintaining a 1:1.2 molar ratio of DOTA-PEG2000-Mal to MD-SH in the mixture. Stir the mixture at room temperature (25℃) for 4-6 hours, maintaining the pH around 6.5 during the reaction to ensure reaction efficiency. The reaction formula is as follows:

[0099]

[0100] After the reaction was complete, a small amount of ethanolamine was added to terminate the reaction, and the product was purified by precipitation or dialysis. Finally, the target product (DOTA-PEG2000-MD) was obtained by freeze-drying. Successful synthesis was confirmed by 1H NMR (see [link to original text]). Figure 5 ).

[0101] 5) The iridium oxide nanozyme Ir prepared in 1) 3.2 The solutions of O (1 mg / mL), DOTA-PEG2000-NT-1 (1 mg / mL) prepared in step 3), and DOTA-PEG2000-MD (0.1 mg / mL) prepared in step 4) were mixed in equal volumes and incubated at room temperature for at least 10 hours. The resulting solutions were then purified by dialysis to prepare iridium oxide nanocomposite materials, denoted as "Ir". 3.2 "O@MN1". Here, M represents the maltodextrin structure from MD-SH, and N1 represents the structure of the target molecule from NT-SH-1. In the composite material, Ir... 3.2The mass ratio of O, DOTA-PEG2000-NT-1, and DOTA-PEG2000-MD is 1:1:0.1. Upon testing, the Ir... 3.2 The hydrated particles of O@MN1 have a diameter of 8 nm and a surface potential of -23 mV. Their microstructure is as follows: Figure 6 The transmission electron microscope image is shown.

[0102] Example 2. Preparation of molybdenum oxide nanocomposites

[0103] The preparation method is largely the same as in Example 1, except that in step 1), the iridium chloride solution is replaced with a molybdenum chloride solution, which yields molybdenum oxide nanozymes (Mo) with a hydrated particle size of 8.5 nm. x Step 5) Finally, the molybdenum oxide nanocomposite material Mo can be obtained. x O@MN.

[0104] Example 3. Preparation of tin oxide nanocomposites

[0105] The preparation method is largely the same as in Example 1, except that in step 1), the iridium chloride solution is replaced with a tin chloride solution, which yields tin oxide nanozymes Sn with a hydrated particle size of 7.5 nm. x O. Step 5) Finally, the tin oxide nanocomposite material Sn is obtained. x O@MN.

[0106] Example 4. Preparation of tantalum oxide nanocomposites

[0107] The preparation method is largely the same as in Example 1, except that in step 1), the iridium chloride solution is replaced with a tantalum chloride solution, which can produce tantalum oxide nanozymes with a hydrated particle size of 3 nm. x Step 5) Finally, the tantalum oxide nanocomposite material Ta can be obtained. x O@MN.

[0108] Example 5. Preparation of iron oxide nanocomposites

[0109] The preparation method is largely the same as in Example 1, except that in step 1), the iridium chloride solution is replaced with a ferric chloride solution, which can produce molybdenum oxide nanozymes with a hydrated particle size of 4 nm. x O. Step 5) Finally, the iron oxide nanocomposite Fe can be obtained. x O@MN.

[0110] Example 6. Preparation of iridium oxide nanocomposites

[0111] The preparation method is largely the same as in Example 1, except that in step 5), iridium oxide nanozyme Ir is used. 3.2A solution of O (1 mg / mL), a solution of DOTA-PEG2000-NT-1 (2 mg / mL), and a solution of DOTA-PEG2000-MD (0.1 mg / mL) were mixed in equal volumes and incubated at room temperature for at least 10 hours. The resulting solution was then purified by dialysis to prepare an iridium oxide nanocomposite material, denoted as "Ir". 3.2 O@MN-2". In this embodiment, the composite material contains Ir. 3.2 The mass ratio of O, DOTA-PEG2000-NT-1 and DOTA-PEG2000-MD is 1:2:0.1.

[0112] Example 7. Preparation of iridium oxide nanocomposites

[0113] The preparation method is largely the same as in Example 1, except that in step 5), iridium oxide nanozyme Ir is used. 3.2 A solution of O (1 mg / mL), a solution of DOTA-PEG2000-NT-1 (0.1 mg / mL), and a solution of DOTA-PEG2000-MD (0.2 mg / mL) were mixed in equal volumes and incubated at room temperature for at least 10 hours. The resulting solution was then purified by dialysis to prepare an iridium oxide nanocomposite material, denoted as "Ir". 3.2 O@MN-3". In this embodiment, the composite material contains Ir. 3.2 The mass ratio of O, DOTA-PEG2000-NT-1 and DOTA-PEG2000-MD is 1:0.1:0.2.

[0114] Example 8. Preparation of iridium oxide nanocomposites

[0115] The preparation method is largely the same as in Example 1, except that in step 5), iridium oxide nanozyme Ir is used. 3.2 A solution of O (1 mg / mL), a solution of DOTA-PEG2000-NT-1 (0.2 mg / mL), and a solution of DOTA-PEG2000-MD (0.2 mg / mL) were mixed in equal volumes and incubated at room temperature for at least 10 hours. The resulting solution was then purified by dialysis to prepare an iridium oxide nanocomposite material, denoted as "Ir". 3.2 O@MN-4". In this embodiment, Ir... 3.2 The mass ratio of O, DOTA-PEG2000-NT-1 and DOTA-PEG2000-MD is 1:0.2:0.2.

[0116] Example 9. Preparation of iridium oxide nanocomposites

[0117] The preparation method is largely the same as in Example 1, except that in step 5), iridium oxide nanozyme Ir is used.3.2 A solution of O (1 mg / mL), a solution of DOTA-PEG2000-NT-1 (0.5 mg / mL), and a solution of DOTA-PEG2000-MD (0.2 mg / mL) were mixed in equal volumes and incubated at room temperature for at least 10 hours. The resulting solution was then purified by dialysis to prepare an iridium oxide nanocomposite material, denoted as "Ir". 3.2 O@MN-5". In this embodiment, Ir... 3.2 The mass ratio of O, DOTA-PEG2000-NT-1 and DOTA-PEG2000-MD is 1:0.5:0.2.

[0118] Example 10. Preparation of iridium oxide nanocomposites

[0119] The preparation method is largely the same as in Example 1, except that in step 5), iridium oxide nanozyme Ir is used. 3.2 An equal volume of solutions of O (1 mg / mL), DOTA-PEG2000-NT-1 (0.1 mg / mL), and DOTA-PEG2000-MD (0.1 mg / mL) was mixed, and the resulting solution was then purified by dialysis to prepare an iridium oxide nanocomposite material, denoted as "Ir". 3.2 O@MN-6". In this embodiment, Ir... 3.2 The mass ratio of O, DOTA-PEG2000-NT-1 and DOTA-PEG2000-MD is 1:0.1:0.1.

[0120] Example 11. Preparation of iridium oxide nanocomposites

[0121] The preparation method is largely the same as in Example 1, except that in step 5), iridium oxide nanozyme Ir is used. 3.2 A solution of O (1 mg / mL), a solution of DOTA-PEG2000-NT-1 (0.5 mg / mL), and a solution of DOTA-PEG2000-MD (1 mg / mL) were mixed in equal volumes and incubated at room temperature for at least 10 hours. The resulting solution was then purified by dialysis to prepare an iridium oxide nanocomposite material, denoted as "Ir". 3.2 O @MN-7". In this embodiment, Ir in the composite material 3.2 The mass ratio of O, DOTA-PEG2000-NT-1 and DOTA-PEG2000-MD is 1:0.5:1.

[0122] Example 12. Preparation of iridium oxide nanocomposites

[0123] The preparation method is largely the same as in Example 1, except that in step 5), iridium oxide nanozyme Ir is used. 3.2 A solution of O (1 mg / mL), a solution of DOTA-PEG2000-NT-1 (0.2 mg / mL), and a solution of DOTA-PEG2000-MD (2 mg / mL) were mixed in equal volumes and incubated at room temperature for at least 10 hours. The resulting solution was then purified by dialysis to prepare an iridium oxide nanocomposite material, denoted as "Ir". 3.2 O @MN-8". In this embodiment, Ir in the composite material 3.2 The mass ratio of O, DOTA-PEG2000-NT-1 and DOTA-PEG2000-MD is 1:0.2:2.

[0124] Example 13. Preparation of iridium oxide nanocomposites

[0125] The preparation method is largely the same as in Example 1, except that in step 2), compound 1 dopamine is replaced with 5-hydroxydopamine hydrochloride, while the other reactants remain unchanged. This yields the thiolized targeting molecule NT-SH-2, which also possesses intracellular membrane targeting properties. Its structure is as follows:

[0126] (NT-SH-2).

[0127] Following steps 3) through 5), the final iridium oxide nanocomposite material Ir can be obtained. 3.2 O@MN2.

[0128] Example 14:

[0129] The preparation method is largely the same as in Example 1, except that in step 2), compound 1 dopamine is replaced with 6-hydroxydopamine hydrobromide, while the other reactants remain unchanged. This yields the thiolized targeting molecule NT-SH-3, which also possesses intracellular membrane targeting properties. Its structure is as follows:

[0130] (NT-SH-3).

[0131] Following steps 3) through 5), the final iridium oxide nanocomposite material Ir can be obtained. 3.2 O@MN3.

[0132] Comparative Example 1. Preparation of copper oxide nanocomposites:

[0133] The preparation method is largely the same as in Example 1, except that in step 1), the iridium chloride solution is replaced with a copper chloride solution, which yields copper oxide nanozymes with a hydrated particle size of 14 nm. x O. Step 5) Finally, the copper oxide nanocomposite material Cu can be obtained. xO@MN1.

[0134] Comparative Example 2. Preparation of Zinc Oxide Nanocomposites

[0135] The preparation method is largely the same as in Example 1, except that in step 1), the iridium chloride solution is replaced with a zinc chloride solution, which yields zinc oxide nanozymes (Zn) with a hydrated particle size of 21 nm. x Step 5) Finally, the zinc oxide nanocomposite material Zn can be obtained. x O@MN1.

[0136] Comparative Example 3. Preparation of manganese oxide nanocomposites

[0137] The preparation method is largely the same as in Example 1, except that in step 1), the iridium chloride solution is replaced with a manganese chloride solution, which yields manganese oxide nanozymes (Mn) with a hydrated particle size of 15.5 nm. x O. Step 5) Finally, the manganese oxide nanocomposite material Mn can be obtained. x O@MN1.

[0138] Comparative Example 4. Preparation of Palladium Oxide Nanocomposites

[0139] The preparation method is largely the same as in Example 1, except that in step 1), the iridium chloride solution is replaced with a palladium chloride solution, which yields palladium oxide nanozymes Pd with a hydrated particle size of 17 nm. x Step 5) Finally, palladium oxide nanocomposite material Pd can be obtained. x O@MN1.

[0140] Comparative Example 5. Preparation of Iridium Oxide Nanocomposites

[0141] The preparation method is largely the same as in Example 1, except that in step 5), iridium oxide nanozyme Ir is used. 3.2 A solution of O (1 mg / mL), a solution of DOTA-PEG2000-NT-1 (5 mg / mL), and a solution of DOTA-PEG2000-MD (5 mg / mL) were mixed in equal volumes and incubated at room temperature for at least 10 hours. The resulting solution was then purified by dialysis to prepare an iridium oxide nanocomposite material, denoted as "Ir". 3.2 O@MN-D1". In the composite material of this comparative example, Ir 3.2 The mass ratio of O, DOTA-PEG2000-NT-1 and DOTA-PEG2000-MD is 1:5:5.

[0142] Comparative Example 6. Preparation of Iridium Oxide Nanocomposites

[0143] The preparation method is largely the same as in Example 1, except that in step 5), iridium oxide nanozyme Ir is used. 3.2 A solution of O (1 mg / mL), a solution of DOTA-PEG2000-NT-1 (5 mg / mL), and a solution of DOTA-PEG2000-MD (0.1 mg / mL) were mixed in equal volumes and incubated at room temperature for at least 10 hours. The resulting solution was then purified by dialysis to prepare an iridium oxide nanocomposite material, denoted as "Ir". 3.2 O @MN-D2". In the composite material of this comparative example, Ir 3.2 The mass ratio of O, DOTA-PEG2000-NT-1 and DOTA-PEG2000-MD is 1:5:0.1.

[0144] Comparative Example 7. Preparation of Iridium Oxide Nanocomposites

[0145] The preparation method is largely the same as in Example 1, except that in step 5), iridium oxide nanozyme Ir is used. 3.2 A solution of O (1 mg / mL), a solution of DOTA-PEG2000-NT-1 (5 mg / mL), and a solution of DOTA-PEG2000-MD (1 mg / mL) were mixed in equal volumes and incubated at room temperature for at least 10 hours. The resulting solution was then purified by dialysis to prepare an iridium oxide nanocomposite material, denoted as "Ir". 3.2 O @MN-D3". In the composite material of this comparative example, Ir 3.2 The mass ratio of O, DOTA-PEG2000-NT-1 and DOTA-PEG2000-MD is 1:5:1.

[0146] Comparative Example 8. Preparation of Iridium Oxide Nanocomposites

[0147] The preparation method is largely the same as in Example 1, except that in step 5), iridium oxide nanozyme Ir is used. 3.2 A solution of O (1 mg / mL), a solution of DOTA-PEG2000-NT-1 (5 mg / mL), and a solution of DOTA-PEG2000-MD (0.5 mg / mL) were mixed in equal volumes and incubated at room temperature for at least 10 hours. The resulting solution was then purified by dialysis to prepare an iridium oxide nanocomposite material, denoted as "Ir". 3.2 O @MN-D4". In the composite material of this comparative example, Ir 3.2 The mass ratio of O, DOTA-PEG2000-NT-1 and DOTA-PEG2000-MD is 1:5:0.5.

[0148] Comparative Example 9. Preparation of Iridium Oxide Nanocomposites

[0149] The preparation method is largely the same as in Example 1, except that in step 5), iridium oxide nanozyme Ir is used. 3.2 A solution of O (1 mg / mL), a solution of DOTA-PEG2000-NT-1 (0.5 mg / mL), and a solution of DOTA-PEG2000-MD (5 mg / mL) were mixed in equal volumes and incubated at room temperature for at least 10 hours. The resulting solution was then purified by dialysis to prepare an iridium oxide nanocomposite material, denoted as "Ir". 3.2 O@MN-D5". In this comparative example, the composite material contains Ir 3.2 The mass ratio of O, DOTA-PEG2000-NT-1 and DOTA-PEG2000-MD is 1:0.5:5.

[0150] Comparative Example 10. Preparation of Iridium Oxide Nanocomposites

[0151] The preparation method is largely the same as in Example 1, except that in step 5), iridium oxide nanozyme Ir is used. 3.2 A solution of O (1 mg / mL), a solution of DOTA-PEG2000-NT-1 (0.1 mg / mL), and a solution of DOTA-PEG2000-MD (5 mg / mL) were mixed in equal volumes and incubated at room temperature for at least 10 hours. The resulting solution was then purified by dialysis to prepare an iridium oxide nanocomposite material, denoted as "Ir". 3.2 O@MN-D6". In the composite material of this comparative example, Ir 3.2 The mass ratio of O, DOTA-PEG2000-NT-1 and DOTA-PEG2000-MD is 1:0.1:5.

[0152] Comparative Example 11. Preparation of Iridium Oxide Nanocomposites

[0153] The preparation method is largely the same as in Example 1, except that in step 5), iridium oxide nanozyme Ir is used. 3.2 A solution of O (1 mg / mL), a solution of DOTA-PEG2000-NT-1 (1 mg / mL), and a solution of DOTA-PEG2000-MD (5 mg / mL) were mixed in equal volumes and incubated at room temperature for at least 10 hours. The resulting solution was then purified by dialysis to prepare an iridium oxide nanocomposite material, denoted as "Ir". 3.2 O@MN-D7". In the composite material of this comparative example, Ir 3.2 The mass ratio of O, DOTA-PEG2000-NT-1 and DOTA-PEG2000-MD is 1:1:5.

[0154] Comparative Example 12. Preparation of Iridium Oxide Nanocomposites

[0155] The preparation method is largely the same as in Example 1, except that in step 5), iridium oxide nanozyme Ir is used. 3.2 A solution of O (1 mg / mL), a solution of DOTA-PEG2000-NT-1 (0.2 mg / mL), and a solution of DOTA-PEG2000-MD (5 mg / mL) were mixed in equal volumes and incubated at room temperature for at least 10 hours. The resulting solution was then purified by dialysis to prepare an iridium oxide nanocomposite material, denoted as "Ir". 3.2 O@MN-D8". In the composite material of this comparative example, Ir 3.2 The mass ratio of O, DOTA-PEG2000-NT-1 and DOTA-PEG2000-MD is 1:0.2:5.

[0156] Comparative Example 13. Preparation of Iridium Oxide Nanocomposites

[0157] The preparation method is largely the same as in Example 1, except that step 5) involves using the iridium oxide nanozyme Ir prepared in step 1). 3.2 The solution of O (1 mg / mL) and the solution of DOTA-PEG2000-MD prepared in step 4) (0.1 mg / mL) were mixed in equal volumes and incubated at room temperature for at least 10 hours. This yielded an iridium oxide nanocomposite material with its surface modified only by maltodextrin molecules and not by the inner membrane targeting NT molecule, named "Ir". 3.2 "O@M", where M represents the structure of maltodextrin.

[0158] Example 15. Preparation of engineered probiotics containing intracellular chimeric nanozymes

[0159] The probiotic LGG was cultured in 40 mL of MRS medium for 12 hours, centrifuged (5000 rpm, 5 minutes) and washed three times, then resuspended in PBS to obtain the bacterial suspension. 1 mL of the bacterial suspension (OD600 = 1, 10⁻⁶) was then... 9 CFU) was centrifuged and dispersed in 10 mL of Ir prepared in Example 1 containing 0.05 mg / mL. 3.2 O@MN1 was co-incubated with physiological saline, at which time LGG and Ir 3.2 The proportion of O@MN1 is approximately 10. 8 CFU: 0.05 mg. During co-incubation, after the initial dispersion, the bacteria were centrifuged again (5000 rpm, 5 minutes) 12 hours later and washed three times. Finally, they were resuspended in physiological saline to construct the engineered probiotic containing the intracellular chimeric nanozyme, denoted as "LGG@MN1@Ir". 3.2 For a schematic diagram of "O-1", please refer to [link / reference]. Figure 7 .

[0160] Example 16. Preparation of engineered probiotics containing intracellular chimeric nanozymes

[0161] The preparation method is largely the same as in Example 15, except that: 1 mL of bacterial suspension (OD600 = 1, 10) is used. 9 CFU was centrifuged and dispersed in 10 mL of Ir prepared in Example 1 containing 0.1 mg / mL. 3.2 O@MN1 was co-incubated in physiological saline; at this time, LGG and Ir 3.2 The proportion of O@MN1 is approximately 10. 8 CFU: 0.1 mg. This ultimately allows for the construction of an engineered probiotic containing an intracellular chimeric nanozyme, denoted as "LGG@MN1@Ir". 3.2 O-2", whose structural diagram can be found in [reference needed]. Figure 7 .

[0162] Example 17. Preparation of engineered probiotics containing intracellular chimeric nanozymes

[0163] The preparation method is largely the same as in Example 15, except that: 1 mL of bacterial suspension (OD600 = 1, 10) is used. 9 CFU was centrifuged and dispersed in 10 mL of Ir prepared in Example 1 at a concentration of 0.2 mg / mL. 3.2 O@MN1 was co-incubated in physiological saline; at this time, LGG and Ir 3.2 The proportion of O@MN1 is approximately 10. 8 CFU: 0.2 mg. This ultimately allows for the construction of an engineered probiotic containing an intracellular chimeric nanozyme, denoted as "LGG@MN1@Ir". 3.2 For a schematic diagram of "O-3", please refer to [link / reference]. Figure 7 .

[0164] Comparative Example 15. Preparation of engineered probiotics containing intracellular chimeric, unmodified target molecules of antioxidant enzymes

[0165] The preparation method is largely the same as in Example 15, except that: Ir is used in... 3.2 O@MN1 was replaced with Ir prepared in Comparative Example 13 3.2 The engineered probiotic material prepared was named LGG@M @Ir. 3.2 O.

[0166] Comparative Example 16. Preparation of a mixture of probiotics and antioxidant enzymes

[0167] The probiotic LGG was cultured in 40 mL of MRS medium for 12 hours, centrifuged (5000 rpm, 5 minutes) and washed three times, and then resuspended in PBS to obtain the bacterial culture. 1 mL of the bacterial culture (OD600=1) was centrifuged and dispersed in the Ir medium prepared in step 1) of Example 1. 3.2 Incubation with physiological saline of O yielded LGG and Ir. 3.2 A mixed solution of O is denoted as "LGG+Ir". 3.2 O".

[0168] Comparative Example 17. Preparation of engineered probiotics containing intracellular chimeric nanozymes

[0169] The preparation method is largely the same as in Example 15, except that: 1 mL of bacterial suspension (OD600 = 1, 10) is used. 9 CFU) was centrifuged and dispersed in 10 mL of Ir prepared in Example 1 containing 0.01 mg / mL. 3.2 O@MN1 was co-incubated in physiological saline; at this time, LGG and Ir 3.2 The proportion of O@MN1 is approximately 10. 8 CFU: 0.01 mg. The resulting engineered probiotic containing an intracellular chimeric nanozyme was constructed, denoted as "LGG@MN1@Ir". 3.2 O-D1".

[0170] Comparative Example 18. Preparation of engineered probiotics containing intracellular chimeric nanozymes

[0171] The preparation method is largely the same as in Example 15, except that: 1 mL of bacterial suspension (OD600 = 1, 10) is used. 9 CFU was centrifuged and dispersed in 10 mL of Ir prepared in Example 1 containing 0.02 mg / mL. 3.2 O@MN1 was co-incubated in physiological saline; at this time, LGG and Ir 3.2 The proportion of O@MN1 is approximately 10. 8 CFU: 0.02 mg. The resulting engineered probiotic containing an intracellular chimeric nanozyme was constructed, denoted as "LGG@MN1@Ir". 3.2 O-D2".

[0172] Comparative Example 19. Preparation of engineered probiotics containing intracellular chimeric nanozymes

[0173] The preparation method is largely the same as in Example 15, except that: 1 mL of bacterial suspension (OD600 = 1, 10) is used. 9 CFU was centrifuged and dispersed in 10 mL of Ir prepared in Example 1 containing 0.5 mg / mL. 3.2 O@MN1 was co-incubated in physiological saline; at this time, LGG and Ir 3.2The proportion of O@MN1 is approximately 10. 8 CFU: 0.5mg. This ultimately allows for the construction of an engineered probiotic containing an intracellular chimeric nanozyme, denoted as "LGG@MN1@Ir". 3.2 O-D3".

[0174] Comparative Example 20. Preparation of engineered probiotics containing intracellular chimeric nanozymes

[0175] The preparation method is largely the same as in Example 15, except that: 1 mL of bacterial suspension (OD600 = 1, 10) is used. 9 CFU) was centrifuged and dispersed in 10 mL of Ir prepared in Example 1 at a concentration of 1.0 mg / mL. 3.2 O@MN1 was co-incubated in physiological saline; at this time, LGG and Ir 3.2 The proportion of O@MN1 is approximately 10. 8 CFU: 1.0 mg. This ultimately allows for the construction of an engineered probiotic containing an intracellular chimeric nanozyme, denoted as "LGG@MN1@Ir". 3.2 O-D4".

[0176] Comparative Example 21. Preparation of engineered probiotics with intracellular chimeric nanozymes

[0177] The preparation method is largely the same as in Example 15, except that: 1 mL of bacterial suspension (OD600 = 1, 10) is used. 9 CFU was centrifuged and dispersed in 10 mL of Ir prepared in Example 1 at a concentration of 2.0 mg / mL. 3.2 O@MN1 was co-incubated in physiological saline; at this time, LGG and Ir 3.2 The proportion of O@MN1 is approximately 10. 8 CFU: 2.0 mg. This ultimately allows for the construction of an engineered probiotic containing an intracellular chimeric nanozyme, denoted as "LGG@MN1@Ir". 3.2 O-D5".

[0178] Experimental Example 1. Proliferation capacity of different probiotic materials in an antibiotic environment

[0179] LGG concentration of 10 8 Comparative Example 16, prepared with cfu / mL LGG+Ir 3.2 O, LGG@M@Ir prepared in Comparative Example 15 3.2 O, LGG@MN1@Ir prepared in Example 15 3.2O were placed in 5 mL of MRS medium containing different antibiotics (250 ng / mL norfloxacin, 5 μg / mL ampicillin, or 5 μg / mL kanamycin). The OD600 of the bacterial cultures was then measured at different time points (0, 2, 4, 6, 8, 10, 12, 18, 24, 36, and 48 hours) to assess antibiotic resistance in different bacteria. Results are as follows: Figures 8-10 As shown.

[0180] Depend on Figures 8-10 It can be seen that among the three materials containing probiotics, only the engineered probiotics with intracellular chimeric nanozymes prepared in Example 15 of this invention exhibit good and sustained proliferation ability. Simply mixing probiotics with metal oxide nanozymes, as in Comparative Example 16, does not effectively improve the proliferation ability of probiotics in antibiotics. Modifying only sugar molecules but not the inner membrane targeting molecule on the surface of the metal oxide nanozyme, as in Comparative Example 15, may temporarily and slightly improve the proliferation ability of probiotics in some antibiotics, but the significance and persistence of its effect are extremely significant compared to the engineered probiotics in Example 15 of this invention. This indicates that the targeting molecule NT modified on the surface of iridium oxide nanozymes is the decisive factor in precisely inhibiting bacterial inner membrane lipid peroxidation, thereby improving the antibiotic resistance of probiotics.

[0181] Experimental Example 2. Distribution of Metal Oxide Nanozymes in Different Probiotic Materials

[0182] 1 mL of Ir prepared from Comparative Example 13 3.2 O@M or Ir prepared in Example 1 3.2 O@MN1 (1 mg / mL) and 10 μL of N-hydroxysuccinimide-fluorescein isothiocyanate (NHS-FITC, 1 mg / mL) were mixed and the mixture was placed at 37°C and shaken for 12 hours to prepare fluorescently labeled Ir. 3.2 O@M and Ir 3.2 O@MN1. 200 μL of fluorescently labeled Ir... 3.2 O@M and Ir 3.2 O@MN1 (1 mg / mL) and 1 mL of LGG (OD600) were mixed, and after 24 hours, the mixture was centrifuged (5000 rpm, 5 min) and washed three times to obtain fluorescently labeled LGG@M@Ir. 3.2 O and fluorescently labeled LGG@MN1@Ir 3.2 O. LGG@M@Ir was observed using a confocal microscope. 3.2 O and LGG@MN1@Ir 3.2 Fluorescence distribution within O. Further towards LGG@M@Ir 3.2 O and LGG@MN1@Ir3.2 Lysozyme (1 mg / mL) was added to the O suspension and incubated at 37°C for 30 minutes to remove the cell wall. The cell wall was then observed using a confocal microscope. 3.2 O and dewall LGG@MN1@Ir 3.2 Fluorescence distribution within O.

[0183] The above confocal microscopy observation results are as follows: Figure 11 As shown, LGG@MN1@Ir before detachment 3.2 The green fluorescence in O is mainly distributed at the edges, suggesting that Ir 3.2 O@MN1 particles may be distributed in the cell wall or inner membrane. After detachment from the cell wall, only the inner membrane remains, and the green fluorescence is still distributed at the edge, further confirming Ir. 3.2 O@MN1 particles are indeed distributed in the inner membrane. In contrast, LGG@M@Ir particles before detachment... 3.2 O and LGG@M@Ir after dewalling 3.2 The green fluorescence in O was consistently distributed throughout the bacteria, indicating that Ir 3.2 O@M has no endometrial targeting effect. Based on the above results, it can be concluded that Ir in Example 1... 3.2 The target molecular structure N1 modified on the surface of O nanozymes is the decisive factor in anchoring them to the inner membrane.

[0184] Experimental Example 3. Effect of feed ratio on the free radical scavenging ability of metal oxide nanocomposites

[0185] The nanoparticles Ir prepared in Examples 1, 6-12, and Comparative Examples 5-12 were used. 3.2 O@MN1、Ir 3.2 O@MN-2、Ir 3.2 O@MN-3、Ir 3.2 O@MN-4、Ir 3.2 O@MN-5、Ir 3.2 O@MN-6、Ir 3.2 O@MN-7、Ir 3.2 O@MN-8、Ir 3.2 O@MN-D1、Ir 3.2 O@MN-D2、Ir 3.2 O@MN-D3、Ir 3.2 O@MN-D4、Ir 3.2 O@MN-D5、Ir 3.2 O@MN-D6、Ir 3.2 O@MN-D7、Ir 3.2 O@MN-D8 was used as the evaluation object.

[0186] To evaluate the scavenging ability of the above materials for hydroxyl radicals (·OH), 250 μM TMB (3,3′,5,5′-tetramethylbenzidine), 2 mM H2O2, 1 mM FeSO4, and the above nanoparticle dispersions (10 μg / mL) were mixed in 0.5 M acetate-acetate buffer (pH 4.5) and reacted in the dark for 5 minutes. The absorbance was then measured at 652 nm using a UV-Vis spectrophotometer, with the decrease in absorbance reflecting the scavenging efficiency of the material for ·OH. The results are shown in Table 1 below:

[0187] Table 1. Scavenging efficiency of metal oxide nanoparticles prepared with different feed ratios for ·OH

[0188] <![CDATA[Ir 3.2 O@MN1]]> 1:1:0.1 96.20 <![CDATA[Ir 3.2 O@MN-2]]> 1:2:0.1 91.79 <![CDATA[Ir 3.2 O@MN-3]]> 1:0.1:0.2 97.40 <![CDATA[Ir 3.2 O@MN-4]]> 1:0.2:0.2 88.31 <![CDATA[Ir 3.2 O@MN-5]]> 1:0.5:0.2 95.03 <![CDATA[Ir 3.2 O@MN-6]]> 1:0.1:0.1 93.81 <![CDATA[Ir 3.2 O@MN-7]]> 1:0.5:1 84.295 <![CDATA[Ir 3.2 O@MN-8]]> 1:0.2:2 84.96 <![CDATA[Ir 3.2 O@MN-D1]]> 1:5:5 38.48 <![CDATA[Ir 3.2 O@MN-D2]]> 1:5:0.1 34.38 <![CDATA[Ir 3.2 O@MN-D3]]> 1:5:1 31.15 <![CDATA[Ir 3.2 O@MN-D4]]> 1:5:0.5 44.74 <![CDATA[Ir 3.2 O@MN-D5]]> 1:0.5:5 51.02 <![CDATA[Ir 3.2 O@MN-D6]]> 1:0.1:5 57.06 <![CDATA[Ir 3.2 O@MN-D7]]> 1:1:5 43.85 <![CDATA[Ir 3.2 O@MN-D8]]> 1:0.2:5 40.78

[0189] As can be seen from Table 1, when Ir 3.2 When the feed ratio of O:DOTA-PEG2000-NT-1 is 1:0.1~2, the Ir prepared in Examples 1, 6-12 3.2 The catalytic activity of the O@MN material remains essentially unchanged, consistently exhibiting a scavenging capacity of over 84% for ·OH; while when Ir 3.2 When the feed ratio of O:DOTA-PEG2000-NT-1 was 1:2~5, Ir prepared in Comparative Examples 5-12 3.2 The catalytic activity of the O@MN material decreased significantly. When Ir 3.2 When the O:DOTA-PEG2000-MD feed ratio is 1:0.1~1, the Ir prepared in Examples 1, 6-12 3.2 The catalytic activity of the O@MN material remains essentially unchanged, consistently exhibiting a scavenging capacity of over 84% for ·OH; while when Ir 3.2 O: When the DOTA-PEG2000-MD feed ratio was 1:1~5, Ir prepared in Comparative Examples 5-12 3.2 The catalytic activity of the O@MN material decreased significantly.

[0190] Experiment Example 4. Comparison of drug resistance and survival ability of engineered probiotics prepared with different feed ratios

[0191] The bacterial survival rates of a series of engineered probiotics prepared in Examples 15-17 and Comparative Examples 17-21 after standing in PBS buffer and antibiotic buffer (5 μg / mL, kanamycin) for 12 hours were as follows: Figure 12 As shown, with Ir 3.2The concentration of O@MN1 gradually increased in the engineered probiotics, while the survival rate of the engineered probiotics in PBS buffer gradually decreased, indicating that the high concentrations (0.5~2 mg / mL) of nanozymes in Comparative Examples 19-21 have certain toxicity to bacteria. In the antibiotic-containing buffers, the survival rate of the engineered probiotics decreased with increasing Ir concentration. 3.2 The concentration of O@MN1 gradually increased, showing a trend of first increasing and then decreasing, indicating that the low concentration (0.01~0.02 mg / mL) of nanozyme in Comparative Examples 17-18 could not improve the drug resistance of probiotics. Based on the above results, when synthesizing the engineered probiotics described in this invention, the concentration of antioxidant nanozyme should be controlled at 0.05~0.2 mg / mL; the optimal concentration is 0.05 mg / mL.

[0192] Experimental Example 5. Efficacy Experiment of Engineered Probiotics in Treating Antibiotic-Related Diarrhea

[0193] Eight-week-old male rats were selected and randomly divided into 7 groups (n=5 per group): (1) healthy group; (2) NC group; (3) LGG group; (4) Ir 3.2 Group O; (5) LGG + Ir 3.2 Group O (6) LGG@M@Ir 3.2 Group O; (7) LGG@MN1@Ir 3.2 Group O. Among them, rats in group (1) were fed normally; while rats in groups (2)-(7) were fed drinking water containing amoxicillin (2 mg / mL) to induce intestinal flora imbalance. On days 4 to 10 of the experiment, rats in groups (3)-(7) were given LGG and Ir prepared in step 1) of Example 1 by gavage. 3.2 O, LGG+Ir prepared in Comparative Example 16 3.2 O, LGG@M @Ir prepared in Comparative Example 15 3.2 O, LGG@MN1@Ir prepared in Example 13 3.2 O; where (3), (5)-(7) each group ingested 10 mg of LGG. On day 10, antibiotic treatment was discontinued and administration was stopped. During the experiment, the weight changes of each rat were recorded. From day 4 to day 11, fecal samples were collected daily and counted on selective agar plates. Fecal photographs were taken on days 4, 9 and 11 and scored. On day 11, rats were sacrificed, and different parts of the intestine were collected; the contents were washed with PBS, and the tissue was homogenized and counted to determine the spatial distribution of bacteria in the intestine.

[0194] The results are as follows Figures 13-16 As shown, after 4 days of gavage, the engineered probiotic LGG@MN1@Ir prepared in Example 15 of this invention... 3.2O can more effectively inhibit the decrease in mouse body weight compared to other drugs. Figure 13 On the last day of the experiment (day 10), the mice recovered to normal weight indistinguishable from healthy mice, while the weight of mice in other experimental groups remained consistent with that of LGG@MN1@Ir. 3.2 There was a significant difference in group O ( Figure 14 From the typical fecal photographs and water content measurements of each group of mice, it can be seen that LGG@MN1@Ir 3.2 The fecal appearance of mice in group O was closest to that of healthy mice, while mice in other groups had varying degrees of soft or loose stools. Figure 15 The final measured fecal water content was also LGG@MN1@Ir 3.2 There was no significant difference between group O and the healthy group, while all other groups were significantly higher than LGG@MN1@Ir. 3.2 Group O ( Figure 16 This invention demonstrates that the engineered probiotics containing intracellular chimeric metal oxide nanozymes have a significant therapeutic effect on antibiotic-associated diarrhea.

Claims

1. An engineered probiotic containing an intracellular chimeric nanozyme, characterized in that: It is a probiotic with several antioxidant enzymes modified on its inner membrane; the antioxidant enzymes are composed of metal oxide nanozymes with a particle size of less than 10 nm and their surface-modified target molecular structures and sugar molecular structures; the target molecular structures and sugar molecular structures are respectively connected to the surface of the metal oxide nanozymes through linkers containing metal ligand structures based on coordination and electrostatic interactions. The metal oxide nanozyme is iridium oxide (Ir). 3.2 O; The linker containing the metal ligand structure includes a DOTA structure and a polyethylene glycol segment, wherein the DOTA structure is located at the end of the linker that is not connected to the target molecule structure or the sugar molecule structure. The aforementioned carbohydrate molecular structure is derived from maltodextrin; The target molecule structure is shown in the following formula (NT-1): (NT-1); Each of the antioxidant enzymes described herein is linked to the probiotic inner membrane via its contained target molecular structure; The probiotic mentioned is Lactobacillus rhamnosus; The antioxidant enzymes comprise 5-20% of the mass of the engineered probiotics.

2. A method for preparing engineered probiotics with intracellular chimeric nanozymes as described in claim 1, comprising the following steps: 1) Ultrasmall metal oxide nanozymes with a particle size of less than 10 nm were prepared by metal chloride under the action of reducing agent and differential centrifugation; the metal chloride solution was IrCl3 solution; 2) NT-SH-1 and Sac-SH were reacted with maleimide functionalized modifiers with metal ligands at one end in a solvent at room temperature for 4-6 hours, with the pH of the reaction system controlled at 5-8.

5. Covalent linkage was achieved through click reaction between thiol and maleimide bonds, resulting in target molecules and carbohydrate molecules with metal ligands at the end groups, respectively. The NT-SH-1 mentioned above is a targeting molecule with the following structure: (NT-SH-1); The Sac-SH mentioned above is thiolated maltodextrin; The maleimide-based functionalized modifier with a metal ligand at one end contains the metal ligand DOTA, and also contains polyethylene glycol segments with a polymerization unit number of 20-230 as connecting arms. The molar ratio of the maleimide functionalized modifier with a metal ligand at one end to the NT-SH-1 is 1~1.5: 1~2; The molar ratio of the maleimide functionalized modifier with a metal ligand at one end to the Sac-SH is 1:1~2; 3) The ultrasmall metal oxide nanozyme obtained in 1) is mixed with the target molecule and functionalized sugar molecule with metal ligands at the end group obtained in 2) at room temperature for at least 10 hours, and the mass ratio of ultrasmall metal oxide nanozyme, target molecule with metal ligands at the end group and functionalized sugar molecule with metal ligands at the end group is controlled to be 1:0.1~2:0.1~2; to obtain composite metal oxide nanoparticles with surface-modified inner membrane target molecule and sugar molecule. 4) The composite metal oxide nanoparticles obtained in 3) were co-incubated with Lactobacillus rhamnosus, and the ratio of Lactobacillus rhamnosus to composite metal oxide nanoparticles was controlled at 10:

1. 8 CFU: 0.01~2mg; thus, engineered probiotics with intracellular chimeric nanozymes are obtained.

3. A metal oxide nanocomposite material, characterized in that: It is composed of metal oxide nanozymes with a particle size of less than 10 nm and their surface-modified target molecular structures and carbohydrate molecular structures; the target molecular structures and carbohydrate molecular structures are respectively connected to the surface of the metal oxide nanozymes through linkers containing metal ligand structures based on coordination and electrostatic interactions. The metal oxide nanozyme is iridium oxide (Ir). 3.2 O; The linker containing the metal ligand structure includes a DOTA structure and a polyethylene glycol segment, wherein the metal ligand DOTA structure is located at the end of the linker that is not connected to the target molecule structure or the sugar molecule structure. The aforementioned carbohydrate molecular structure is derived from maltodextrin; The target molecule structure is shown in the following formula (NT-1): (NT-1)。 4. The metal oxide nanocomposite material according to claim 3, characterized in that: The number of polyethylene glycol molecular chain segment polymer units is between 20 and 230.

5. The use of the engineered probiotics of claim 1 or the engineered probiotics prepared by the method of claim 2 in the preparation of a drug for the prevention or treatment of amoxicillin-induced diarrhea.

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