Construction method and application of cerium oxide nanoszyme engineering bacteria hybrid system

Abstract

The application discloses a construction method of a cerium oxide nano-enzyme engineering bacteria hybrid system and application thereof, and particularly relates to the field of biological materials. The hybrid system is constructed by integrating metabolic regulation of engineering bacteria, nano-enzyme antioxidant and targeted modification, and has important clinical application value. The arginine-producing engineering bacteria EcN Arg is used to enhance the arginine secretion capacity, regulate HSCs metabolic phenotype through the arginine-NO axis, improve LSECs function and promote ECM degradation; the engineering bacteria are surface-modified to reduce excessive colonization capacity in normal tissues, reduce the risk of systemic toxicity and improve treatment safety; CeO2-HA is loaded on the surface of the modified engineering bacteria to remove excess ROS in the liver by using the antioxidant enzyme simulation activity, relieve oxidative stress and inflammatory response, and improve the hepatic sinusoid microcirculation in cooperation with NO generated by arginine metabolism, so that a multi-dimensional synergistic effect of "metabolic regulation-ROS removal-microenvironment remodeling" is formed.

Description

Technical Field

[0001] This invention relates to the field of biomaterials, specifically to a method for constructing a hybrid system of cerium oxide nanozymes engineered bacteria and its application. Background Technology

[0002] Liver fibrosis is a pathological process caused by various chronic liver injuries, including viral infections, alcohol abuse, and non-alcoholic fatty liver disease. Its core characteristic is the excessive accumulation of extracellular matrix (ECM) in the liver. Without effective intervention, it can gradually progress to cirrhosis, liver failure, and even hepatocellular carcinoma (HCC), seriously threatening global public health. This pathological process involves a complex network of cellular and molecular regulations: the transformation of hepatic stellate cells (HSCs) from a resting to an activated state and the subsequent secretion of large amounts of ECM is a key step. Simultaneously, activation of inflammatory macrophages, the synergistic effects of endoplasmic reticulum stress (ERS) and oxidative stress (OS), biomechanical microenvironmental imbalance, and gut microbiota-liver axis dysregulation all participate. Currently, there is still a lack of specific anti-fibrotic drugs in clinical practice, and traditional therapies suffer from drawbacks such as poor targeting, limited efficacy against single targets, and short drug half-lives.

[0003] Arginine plays a crucial role in regulating cellular metabolism and signaling pathways via the nitric oxide (NO) pathway. It can inhibit collagen deposition and improve the function of hepatic sinusoidal endothelial cells (LSECs). However, direct delivery of arginine is characterized by rapid metabolism and low utilization. HSC activation is accompanied by a shift in arginine metabolism: in the resting state, inducible nitric oxide synthase (iNOS) dominates the conversion of arginine to NO. After activation, iNOS is downregulated while arginase 1 (Arg1) increases more than fivefold. Arg1 promotes collagen synthesis by converting ornithine to proline, and specific inhibition of Arg1 can reduce collagen deposition by more than 40%. Meanwhile, NO derived from endothelial nitric oxide synthase (eNOS) is essential for maintaining the function of hepatic sinusoidal endothelial cells (LSECs). The binding of the TAZ-binding motif (a transcriptional coactivator with a PDZ-binding motif) to Krüppel-like factor 2 (KLF2) can enhance eNOS transcription and maintain the fenestration structure of LSECs. TAZ deficiency leads to a more than 40% downregulation of eNOS expression, accelerating fibrosis. Arginine, as a NOS substrate, exhibits excellent therapeutic potential. In a carbon tetrachloride (CCl4) model, it can restore eNOS activity, increase intrahepatic physiological NO levels, and improve hepatic blood perfusion. Its effect in reducing collagen deposition is significantly superior to the iNOS inhibitor SMT. Simultaneously, it can regulate the balance of matrix metalloproteinases (MMPs) / tissue metalloproteinase inhibitors (TIMPs) and promote ECM degradation. Cerium oxide nanoparticles (CeO2NPs) possess the advantages of Ce... 3+ / Ce 4+The dynamic valence state switching characteristics of the bacteria, which mimic superoxide dismutase (SOD) and catalase (CAT) activities, can continuously clear excess ROS in the liver, breaking the vicious cycle of "oxidative stress-inflammation-HSC activation." However, its liver accumulation efficiency is insufficient. Targeted delivery systems, such as HA modification, can further enhance its targeting efficacy. Engineered bacteria, as live biopharmaceuticals, can continuously secrete active substances, but they pose risks such as systemic toxicity and high immunogenicity. Currently, there is a lack of an integrated treatment system that simultaneously addresses targeted delivery, synergistic efficacy, and enhanced safety. Therefore, this invention is proposed. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to provide a method for constructing a cerium oxide nanozyme engineered bacteria hybrid system and its application in anti-liver fibrosis.

[0005] The present invention solves the above-mentioned technical problems through the following technical means:

[0006] This invention proposes a method for constructing a cerium oxide nanozyme engineered bacteria hybrid system, comprising the following steps: (1) Preparation of C-EcN Arg : The activated arginine-producing engineered bacteria (EcN) Arg After culturing to the logarithmic growth phase, the culture was resuspended to obtain a bacterial suspension; a dimethyl sulfoxide (DMSO) solution containing N-hydroxysuccinimide acrylate (NAS) was added to obtain EcN. Arg -NAS bacterial culture; then acrylamide (AAM), N-(3-aminopropyl)methacrylamide hydrochloride (APM), 3-methacrylamide phenylboronic acid (3-MAPBA), N,N'-methylenebisacrylamide (BIS), ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) were added sequentially. After the reaction, C-EcN was obtained. Arg ; (2) Preparation of CeO2-HA: a. Preparation of CeO2 nanozymes: Xylene was mixed with cerium(III) hydrated acetate, oleylamine, and water to obtain CeO2 nanozymes; b. Surface modification: CeO2 nanozyme was ultrasonically dispersed, and then added to an aqueous hyaluronic acid solution; dialyzed; CeO2-HA was obtained; (3) Preparation of RC-EcN Arg @CeO2: Combine the CeO2-HA prepared in step (2) with the C-EcN prepared in step (1). Arg Mix; incubate; purify, and you will get it.

[0007] This invention constructs a cerium dioxide nanoenzyme-in-situ polymerized engineered bacteria hybrid system (RC-EcN). Arg @CeO2), via EcN ArgThe engineered bacteria can continuously secrete arginine, regulating the arginine-NO axis to inhibit collagen deposition; CeO2 nanozymes, after being modified with hyaluronic acid (HA), enhance their targeting of activated hepatic stellate cells (HSCs) and clear excess ROS; the engineered bacteria, after in-situ polymerization modification, reduce immunogenicity and pathogenicity, achieving multi-dimensional synergistic anti-liver fibrosis through "metabolic regulation-ROS clearance-targeted enrichment", thereby improving treatment efficacy and safety.

[0008] Preferably, in step (1), the arginine-producing engineered bacteria (EcN) Arg The strain is a known strain, described in Canale FP, et al. Metabolic modulation of tumors with engineered bacteria for immunotherapy. Nature. 2021 Oct;598(7882):662-666. doi: 10.1038 / s41586-021-04003-2. It can be repeatedly obtained or constructed by those skilled in the art according to the method described in the literature. It was provided and deposited by Professor Liu Qi of Anhui Medical University.

[0009] Preferably, in step (1), the concentration of the bacterial suspension is 1×10⁻⁶. 8 CFU / mL.

[0010] Preferably, in step (1), EcN Arg The final concentration of NAS in the NAS bacterial culture is 0.005-0.02 mg / mL.

[0011] Preferably, in step (1), the molar ratio of acrylamide (AAM), N-(3-aminopropyl)methacrylamide hydrochloride (APM), 3-methacrylamide phenylboronic acid (3-MAPBA), N,N'-methylenebisacrylamide (BIS), ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) is (1815-1820):(28-32):(1-3):33.2:4.5:68.4; more preferably, it is 1819.2:28.7:1.0:33.2:4.5:68.4.

[0012] The CAS number of the acrylamide (AAM) is 79-06-1.

[0013] The CAS number of the N-(3-aminopropyl)methacrylamide hydrochloride (APM) is 72607-53-5.

[0014] The CAS number of the ammonium persulfate (APS) is 7727-54-0.

[0015] The CAS number of the 3-methylacrylamide phenylboronic acid (3-MAPBA) is 48150-45-4.

[0016] The CAS number of the N,N'-methylenebisacrylamide (BIS) is 110-26-9.

[0017] Preferably, in step (1), the reaction conditions are: reaction at 35-37°C in a nitrogen atmosphere, away from light.

[0018] Preferably, in step (a), the ratio of xylene to cerium acetate hydrate (III), oleylamine, and water is 10-15 mL: 0.4-0.5 g: 3-4 g: 1-5 mL, and more preferably 15 mL: 0.4 g: 3.2 g: 1 mL.

[0019] The CAS number of the oleylamine is 112-90-3.

[0020] The CAS number of the hydrated cerium acetate (III) is 206996-60-3.

[0021] Preferably, in step (b), the ultrasonic dispersion conditions are an ultrasonic power of 280-300 W, a frequency of 40-45 kHz, and an ultrasonic dispersion time of 20-30 min.

[0022] Preferably, in step (b), the concentration of hyaluronic acid in the hyaluronic acid aqueous solution is 2-4 mg / mL.

[0023] Preferably, in step (b), the molecular weight cutoff for dialysis is 3500 Da.

[0024] Preferably, in step (3), CeO2-HA and C-EcN Arg The volume ratio is 2-1:1, and more preferably 1:1.

[0025] Preferably, in step (3), the incubation method is to incubate in a constant temperature shaker at 35-37℃ with light-proof shaking at 180-200 rpm for 2-3 hours.

[0026] During this process, C-EcN Arg The phenylboronic acid groups (from 3-MAPBA monomers) in the polymer layer on the bacterial surface undergo a complexation reaction with the cis-vicinal diol structure in the hyaluronic acid molecules on the nanozyme surface to form stable cyclic borate ester bonds, thereby firmly anchoring the nanozyme to the bacterial surface.

[0027] Preferably, in step (3), the purification method is to first centrifuge and discard the supernatant, and then wash the precipitate twice with PBS.

[0028] This invention also proposes a cerium oxide nanozyme engineered bacteria hybrid system (RC-EcN) obtained by the above construction method. Arg @CeO2).

[0029] This invention also proposes the application of the cerium oxide nanozyme engineered bacteria hybrid system obtained by the above construction method in the preparation of drugs for the treatment / prevention of liver fibrosis.

[0030] This invention also proposes a drug for treating / preventing liver fibrosis, the main components of which include the above-mentioned cerium oxide nanozyme engineered bacteria hybrid system.

[0031] The liver fibrosis includes chemically induced liver fibrosis.

[0032] The drug can be formulated with medically acceptable excipients in a cerium oxide nanozyme engineered bacteria hybrid system; or the cerium oxide nanozyme engineered bacteria hybrid system can be mixed with other drugs that have preventive or therapeutic effects on liver fibrosis, with or without the addition of medically acceptable excipients.

[0033] Preferably, the drug is an oral liquid or an injection.

[0034] The RC-EcN prepared by this invention Arg The @CeO2-HA hybrid system can be targeted and enriched at sites of liver fibrosis. The arginine continuously secreted by engineered bacteria activates the arginine-NO axis, inhibiting hepatic stellate cell activation and collagen deposition, repairing hepatic sinusoidal endothelial cell function, and promoting extracellular matrix degradation. Cerium dioxide nanozymes, through CeO2... 3+ / Ce 4+ Valence state switching clears excess ROS in the liver, alleviates oxidative damage to hepatocytes, and breaks the vicious cycle of oxidative stress-inflammation-activation of hepatic stellate cells; Simultaneously, in-situ polymerization modification reduces the immunogenicity of engineered bacteria and inhibits their excessive proliferation, synergistically reshaping the normal physiological microenvironment in the liver and reversing the process of hepatocellular damage-induced liver fibrosis.

[0035] The beneficial effects of this invention are as follows: 1. This invention utilizes engineered arginine-producing bacteria EcN Arg Surface modification was performed, and CeO2-HA was loaded onto the surface of the engineered bacteria. Its antioxidant enzyme activity was used to clear excess ROS in the liver, alleviate oxidative stress and inflammatory response, and at the same time, NO produced by arginine metabolism improved hepatic sinusoidal microcirculation, forming a multi-dimensional synergistic effect of "metabolic regulation-ROS clearance-microenvironment remodeling". It also intervened in the two core pathological links of collagen deposition and oxidative stress, breaking through the limitations of single therapy. 2. The cerium oxide nanozyme engineered bacteria hybrid system (RC-EcN) constructed in this invention Arg@CeO2 can secrete arginine, eliminating the need for repeated administration. In-situ polymerization modification reduces its immunogenicity and pathogenicity, improving treatment safety. 3. CeO2 nanozymes, after being modified with HA, enhance their targeting of activated HSCs, improve intrahepatic enrichment efficiency using engineered bacteria as carriers, and exhibit excellent biocompatibility. 4. The preparation method is simple and controllable, easy to scale up production, and has broad clinical application prospects.

[0036] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0037] Figure 1 EcN in Embodiments 1 and 5 of the present invention Arg C-EcN Arg and RC-EcN Arg Transmission electron microscopy (TEM) image of @CeO2; scale bar: 1 μm; Figure 2 RC-EcN in Embodiment 5 of the present invention Arg Scanning electron microscopy (SEM) morphology and elemental analysis of @CeO2; scale bar: 500 nm; Figure 3 EcN in Embodiments 1 and 5 of the present invention Arg C-EcN Arg and RC-EcN Arg @Particle size distribution of CeO2 sample (A) and Zeta potential variation (B); Figure 4 EcN in Embodiments 1 and 5 of the present invention Arg C-EcN Arg and RC-EcN Arg Growth curve of @CeO2 in LB liquid medium; Figure 5 RC-EcN in Embodiment 5 of the present invention Arg Statistical chart of the bioactivity of @CeO2 (CCK-8 assay); Figure 6 EcN in Embodiments 1 and 5 of the present invention Arg C-EcN Arg and RC-EcN Arg @Statistical chart of arginine (L-Arg) content released by CeO2; Figure 7 EcN labeled Cy7 in Embodiment 6 of the present invention Arg Biodistribution in a mouse model of liver fibrosis and fluorescence imaging of ex vivo organs; Figure 8 The C-EcN labeled Cy7 in Embodiment 6 of this invention Arg Biodistribution in a mouse model of liver fibrosis and fluorescence imaging of ex vivo organs; Figure 9 The Cy7-marked RC-EcN in Embodiment 6 of this invention Arg Biodistribution of @CeO2 in a mouse model of liver fibrosis and fluorescence imaging of ex vivo organs; Figure 10 This is a statistical chart showing the serum liver function biochemical indicators (ALT and AST) levels of mice in each group after treatment in Example 6 of the present invention; Figure 11 This is a graph showing the changes in body weight of mice in each group during treatment in Example 6 of the present invention; Figure 12 These are pathological sections of mouse liver tissue stained with hematoxylin and eosin (H&E) in each group in Example 6 of this invention; scale bar: 200 μm; Figure 13 Masson-stained pathological sections of mouse liver tissue from each group in Example 6 of this invention; Scale bar: 200 μm; Figure 14 ROS fluorescent staining sections of mouse liver tissue from each group in Example 6 of this invention; Scale bar: 200 μm; Figure 15 This is a statistical chart showing the NO content in the supernatant of mouse liver tissue in each group in Example 6 of the present invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0039] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0040] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0041] Example 1: This embodiment proposes a bacterial (C-EcN) encased in a thin polymer shell. Arg The preparation method of ) includes the following steps: 1. Bacterial activation: The cryopreserved engineered arginine-producing bacteria (EcN) are activated... Arg Streak the bacteria onto LB agar plates and incubate overnight at 37°C. Pick single colonies and inoculate into liquid LB agar plates, incubating at 37°C with shaking at 220 rpm until the logarithmic growth phase. Centrifuge (7000 rpm, 5 min) to collect the cells, wash three times with sterile PBS buffer, resuspend in sterile PBS, and adjust the concentration to 1×10⁻⁶. 8 CFU / mL.

[0042] 2. Bacterial surface functionalization: Take 10 mL of the above bacterial suspension (1×10⁻⁶) 8 Add DMSO solution containing N-hydroxysuccinimide acrylate (NAS) (final NAS concentration 0.01 mg / mL), and stir for 2 h at 4°C in the dark to modify the bacterial surface with acrylyl groups. After the reaction, centrifuge (7000 rpm, 5 min), wash three times to remove unreacted NAS, and resuspend in 10 mL of sterile ice-cold PBS to obtain acrylated bacteria EcN. Arg -NAS; 3. In-situ polymerization: Take the EcN obtained from the bacterial surface functionalization in step two above. Arg - 1 mL of NAS bacterial culture was centrifuged at 7000 rpm for 5 min, the supernatant was removed, and the mixture was resuspended in ultrapure water. The final volume was 1 mL, containing 1 × 10⁻⁶ substances. 8 EcN Arg -NAS bacteria were sequentially treated with monomers acrylamide (AAM), N-(3-aminopropyl)methacrylamide hydrochloride (APM), 3-methacrylamide phenylboronic acid (3-MAPBA), and crosslinking agent N,N'-methylenebisacrylamide (BIS). After purging with nitrogen for 15 min to remove oxygen, ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) were added as initiators. The molar ratio of monomers n(AAM):n(APM):n(3-MAPBA):n(BIS):n(APS):n(TEMED) was 1819.2:28.7:1.0:33.2:4.5:68.4. The reaction was carried out at 37°C with stirring in the dark for 4 h.

[0043] 4. Purification: After the reaction was complete, centrifuge at 7000 rpm for 5 min, discard the supernatant, and wash the precipitate three times with sterile PBS to obtain bacteria (C-EcN) encapsulated in a thin polymer shell. Arg Store at 4℃ for later use.

[0044] Example 2: This embodiment proposes a bacterial (C-EcN) encased in a thin polymer shell. Arg The preparation method of ) differs from that of Example 1 in that: In step (2), the final concentration of NAS is 0.02 mg / mL.

[0045] In step (3), the monomer molar ratio n(AAM):n(APM):n(3-MAPBA):n(BIS):n(APS):n(TEMED) = 1817.2:28.7:1.0:33.2:4.5:68.4 (increasing the 3-MAPBA molar ratio). The rest is the same as in Example 1.

[0046] Example 3: This embodiment proposes a bacterial (C-EcN) encased in a thin polymer shell. Arg The preparation method of ) differs from that of Example 1 in that: In step (2), the final concentration of NAS is 0.005 mg / mL.

[0047] In step (3), the monomer molar ratio n(AAM):n(APM):n(3-MAPBA):n(BIS):n(APS):n(TEMED) = 1819.2:30.7:3.0:33.2:4.5:68.4 (reducing the 3-MAPBA molar ratio). The rest is the same as in Example 1.

[0048] Example 4: This embodiment proposes a method for preparing hyaluronic acid-modified CeO2 nanozyme (CeO2-HA), including the following steps: (1) Preparation of CeO2 nanozymes: CeO2 nanoparticles were prepared by mixing xylene solution with cerium acetate hydrate (III) and oleylamine, respectively. The specific process is as follows: 0.4 g of cerium acetate hydrate (III) and 3.2 g of oleylamine were weighed, and 15 mL of xylene was added. After stirring for 2 h, 1 mL of deionized water was added, and the mixture was stirred for 3 h under an inert gas environment at 90 °C. Finally, the precipitate was resuspended with acetone for 1 h, and then the precipitate was washed with acetone pre-cooled at -20 °C. The CeO2 nanozymes were obtained by centrifugation.

[0049] (2) Surface modification: Prepare an aqueous solution of 2 mg / mL hyaluronic acid (HA, MW=10-20 kDa). Disperse 10 mg of CeO2 nanozyme in 10 mL of deionized water and sonicate for 30 min (ultrasonic power 300 W, frequency 40 kHz) to ensure uniform dispersion. Add the HA solution dropwise to the CeO2 dispersion and stir at room temperature in the dark for 12 h.

[0050] (3) Purification: The reaction mixture was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water for 24 h, with the water changed 4-6 times during the process to remove unadsorbed HA. The dialysate was freeze-dried to obtain CeO2-HA nanozyme with a negatively charged surface and abundant carboxyl groups.

[0051] Example 5: This embodiment proposes a cerium oxide nanozyme engineered bacteria hybrid system (RC-EcN). Arg The method for constructing @CeO2 utilizes the specific recognition of phenylboronic acid-diol. It includes the following steps: (1) Raw material preparation: The hyaluronic acid-modified CeO2 nanozyme (CeO2-HA) prepared in Example 4 was prepared into a 1 mg / mL dispersion using PBS buffer (pH 7.4). The polymer-modified bacteria (C-EcN) containing phenylboronic acid groups prepared in Example 1 were also prepared. Arg Adjust the concentration to 1×10 8 CFU / mL.

[0052] (2) Self-assembly reaction: The above CeO2-HA dispersion was reacted with C-EcN Arg The bacterial suspensions were mixed at a volume ratio of 1:1.

[0053] (3) Incubation: The mixture was placed in a constant temperature shaker at 37°C and incubated at 200 rpm in the dark for 2 hours. During this process, the phenylboronic acid groups (from 3-MAPBA monomer) in the polymer layer on the bacterial surface undergo a complexation reaction with the cis-vicinal diol structure in the hyaluronic acid molecules on the surface of the nanozyme to form a stable cyclic borate bond, thereby firmly anchoring the nanozyme to the bacterial surface.

[0054] (4) Purification: After the reaction, centrifuge at 7000 rpm for 5 min, discard the supernatant to remove unbound free nanozymes, wash the precipitate twice with PBS, and resuspend to obtain bacteria loaded with CeO2 nanozymes (RC-EcN). Arg @CeO2).

[0055] Example 6: Establishing a mouse model of liver fibrosis induced by intraperitoneal injection of CCl4 Observe RC-EcN Arg Application of @CeO2 hybrid system in enrichment of liver fibrosis sites: First, free bacteria (EcN) were labeled using Cy-7 (a near-infrared anthocyanin fluorescent dye). Arg As a control group, bacteria (C-EcN) encapsulated in Cy-7-labeled thin polymer shells were prepared according to the method in Example 1. Arg ) and the hybrid system of Example 5 (RC-EcN)Arg @CeO2) was used as the experimental group. Subsequently, a mouse liver fibrosis model was established by intraperitoneal injection of CCl4. When the mice showed obvious fibrosis characteristics, the control group and the experimental group were divided into two groups according to the ratio of 1×10⁻⁶. 8 100 μL of a CFU / mL solution was injected into the tail vein of mice. Mice were sacrificed 48 hours after injection, and their organs were removed and analyzed using a small animal in vivo imaging system to determine the enrichment of bacteria in each organ. Figure 6 As shown, engineered bacteria modified with cerium dioxide nanozyme-in-situ polymerization (RC-EcN) Arg Data on the continued secretion of arginine after @CeO2, such as... Figure 7-9 The results, shown in the figure from left to right, represent the results at 0 h, 1 h, 4 h, 8 h, 12 h, 24 h, and 48 h after drug administration, as well as the results in major organs. 24 h after intravenous injection of free bacteria, the fluorescence intensity in the liver gradually decreased, and by 48 h, the fluorescence had essentially disappeared, indicating that the bacteria were largely cleared by the body's immune system. Meanwhile, C-EcN... Arg and RC-EcN Arg The @CeO2 hybrid system maintained a high level of liver fluorescence signal 48 hours after intravenous injection. Figure 7-9 The results showed that bacteria modified by in-situ polymerization could evade the body's immune clearance, prolong blood circulation time, and accumulate more effectively in liver fibrosis sites.

[0056] Observe RC-EcN Arg The reversal effect of the CeO2 hybrid system on liver fibrosis: To investigate the therapeutic effect of hybrid systems on liver fibrosis, we established a CCl4-induced liver fibrosis mouse model. After successful model establishment, 42 mice were selected and divided into 7 groups of 6 mice each: G1: Control (healthy mice); G2: CCl4 (model group); G3: CeO2-HA; G4: C-EcN Arg G5: C-EcN Arg @CeO2 (non-ROS response); (6)G6:RC-EcN Arg@CeO2 (ROS response); G7: Pirfenidon (positive control group). G5 was the non-ROS response control group, prepared similarly to G6, except that G5 did not include 3-methacrylamide phenylboronic acid (3-MAPBA) during preparation. Instead, CeO2-HA was directly linked to the monomer N-(3-aminopropyl)methacrylamide hydrochloride (APM), serving as the non-ROS response group to control the ROS-responsive cleavage of borate ester bonds. All treatment regimens were administered via tail vein injection to mice every 3 days for 2 weeks. Mouse weight was monitored every two days during treatment. Mice were sacrificed after treatment, serum was collected to detect liver function indicators (ALT / AST), and liver tissue was sectioned and stained. Figure 10 As shown, serum biochemical analysis revealed that, compared with the PBS group and the single-therapy group, RC-EcN... Arg The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the @CeO2 group mice were significantly reduced and approached the levels of the healthy group, indicating that RC-EcN Arg The @CeO2 hybrid system can effectively reduce liver inflammation and damage and restore liver function.

[0057] like Figure 11 As shown, statistical data plotted based on monitored mouse weight indicates that during drug treatment, the weight changes in each group of mice were relatively stable, with no significant weight loss, indicating that RC-EcN... Arg The @CeO2 hybrid system exhibits good biocompatibility. Figure 11 The results showed that the treatment system had good biocompatibility.

[0058] We stained the extracted liver tissue with hematoxylin and eosin (H&E), such as... Figure 12 As shown, H&E staining revealed disordered liver tissue structure in both the PBS group and the control group, with extensive inflammatory cell infiltration and hepatocyte necrosis; conversely, RC-EcN... Arg The liver lobule structure in the CeO2 treatment group was basically restored to normal, the hepatocytes were neatly arranged, and the inflammatory infiltration and cell necrosis areas were significantly reduced, proving that the system has significant anti-inflammatory and hepatoprotective effects. Figure 12 The results show RC-EcN Arg The @CeO2 treatment group showed a significant reduction in liver inflammation, minimal collagen fiber deposition area, and effective reversal of fibrosis.

[0059] The degree of collagen deposition in liver tissue was detected by Masson's trichrome staining, such as Figure 13 As shown, Masson staining revealed extensive blue collagen deposition in the liver tissue of the PBS group, forming a distinct pseudolobular structure; while RC-EcN... ArgThe blue collagen area was significantly reduced in the @CeO2 group, and the percentage of fibrosis area was the lowest. Figure 13 The results show RC-EcN Arg The @CeO2 treatment group showed a significant reduction in liver inflammation, minimal collagen fiber deposition area, and effective reversal of fibrosis.

[0060] DHE fluorescence staining was used to detect the degree of ROS clearance in liver tissue. Results are as follows: Figure 14 As shown, the liver tissue of the healthy group (G1) showed only weak basal red fluorescence, indicating that the ROS level was at physiological homeostasis; the liver tissue of the model group (G2) showed extremely strong red fluorescence, indicating a large accumulation of ROS in the liver and severe oxidative stress damage; the single CeO2-HA group (G3) and the single C-EcN group... Arg The fluorescence intensity of group G4 decreased slightly compared to the model group, but significant ROS accumulation was still present; C-EcN Arg The fluorescence intensity of the @CeO2 (non-ROS responsive) group (G5) was significantly reduced, while the red fluorescence of the ROS responsive group (G6) was almost the same as that of the healthy group, indicating the best clearance effect. The ROS level in the positive control group pirfenidone (G7) decreased, but the effect was far weaker than that of RC-EcN. Arg @CeO2 hybrid system. The results confirmed that the intrahepatic ROS level was significantly lower than that of the model group, which fully demonstrates that this hybrid system can efficiently remove excess ROS in the liver and block the oxidative stress-driven fibrosis pathway.

[0061] To further verify RC-EcN Arg The @CeO2 system, by regulating NO production through arginine metabolism, was used to detect NO levels in the liver tissues of mice in each group. Liver tissue was homogenized, and NO metabolites (NO2) in the tissue were determined colorimetrically using a Griess kit. - / NO3 - The content of NO indirectly reflects the NO level in the liver. Results are as follows: Figure 15 As shown, compared with the model group, RC-EcN Arg The @CeO2-HA (ROS-responsive) group maintained liver NO levels within a near-normal physiological range, significantly superior to other single-treatment groups. This indicates that the system can efficiently scavenge ROS while precisely regulating the arginine-NO metabolic pathway, achieving a synergistic balance between oxidative stress and NO signaling. These results demonstrate that the prepared RC-EcN... Arg @CeO2 effectively inhibits excessive collagen deposition and successfully reverses the process of liver fibrosis through the synergistic effect of scavenging reactive oxygen species and regulating arginine metabolism.

[0062] Example 7: This embodiment proposes a bacterial (C-EcN) encased in a thin polymer shell. ArgThe preparation method of ) differs from that of Example 1 in that: In step (3), the monomer molar ratio n(AAM):n(APM):n(3-MAPBA):n(BIS):n(APS):n(TEMED) = 1815:28:2:33.2:4.5:68.4. The rest is the same as in Example 1, and the effect is similar to that in Example 1.

[0063] Example 8: This embodiment proposes a method for preparing hyaluronic acid-modified CeO2 nanozyme (CeO2-HA), which differs from Example 4 in that: In step (1), the ratio of xylene to cerium acetate hydrate (III), oleylamine, and deionized water is 10 mL: 0.5 g: 3 g: 5 mL.

[0064] In step (2), the concentration of hyaluronic acid in the aqueous solution was 4 mg / mL, and the ultrasonic dispersion conditions were 280 W ultrasonic power and 45 kHz frequency; the ultrasonic dispersion time was 20 min. The rest was the same as in Example 4, and the effect was similar to that in Example 4.

[0065] Example 9: This embodiment proposes a method for preparing hyaluronic acid-modified CeO2 nanozyme (CeO2-HA), which differs from Example 4 in that: In step (1), the ratio of xylene to cerium acetate hydrate (III), oleylamine, and deionized water is 15 mL: 0.4 g: 4 g: 1 mL.

[0066] In step (2), the concentration of hyaluronic acid in the aqueous solution is 3 mg / mL. The rest is the same as in Example 4, and the effect is similar to that in Example 4.

[0067] Example 10: This embodiment proposes a bacterium (RC-EcN) loaded with CeO2-HA nanozyme. Arg The preparation method of (@CeO2) differs from that in Example 5 in that: In step (2), the CeO2-HA dispersion and C-EcN Arg The bacterial suspensions were mixed at a volume ratio of 2:1.

[0068] In step (3), the incubation method is to incubate at 180 rpm in a constant temperature shaker at 35℃ for 3 h in the dark.

[0069] The rest is the same as in Example 5, and the effect is similar to that in Example 5.

[0070] Comparative Example 1: The difference between this comparative example and Example 1 is that the final concentration of NAS is 0.003 mg / mL.

[0071] Experimental results show that when the NAS concentration is too low, the required polymer shell, i.e., the capsule shell, cannot be formed well during the subsequent preparation of bacterial capsules.

[0072] Comparative Example 2: The difference between this comparative example and Example 1 is that the final concentration of NAS is 0.1 mg / mL.

[0073] Experimental results show that when the NAS concentration is too high, EcN Arg Bacterial activity is reduced.

[0074] Results analysis: The synthesized bacteria loaded with CeO2 nanozymes (i.e., RC-EcN) were analyzed by SEM and TEM. Arg Characterization was performed on the CeO2 hybrid system. like Figure 1 As shown, transmission electron microscopy (TEM) results reveal that the unmodified engineered bacteria (EcN) Arg The surface is smooth and it has a typical rod shape; the in-situ polymerized modified bacteria (C-EcN) in Example 1 Arg The surface is coated with a uniform polymer shell; while in Example 5 the hybrid system (RC-EcN) Arg The surface of @CeO2 is rough, with a large amount of particulate matter attached. This was confirmed by scanning electron microscopy (SEM) imaging (e.g., Figure 2 As shown in the figure, the bacterial surface is uniformly coated with a high electron density nanozyme layer without damaging the bacterial cell structure. This indicates that the specific binding of phenylboronic acid and hyaluronic acid can effectively and firmly connect the modified nanozyme with the engineered bacteria encapsulated by the in-situ polymerization reaction.

[0075] RC-EcN measured by DLS Arg @Particle size and potential of the CeO2 hybrid system: like Figure 3 As shown, dynamic light scattering reveals the hybrid system (RC-EcN) in Example 5. Arg The average hydrated particle size of @CeO2 is 1450±35 nm (e.g., Figure 3 As shown in Figure A), the Zeta potential remains around -12.5 mV (as shown in Figure A). Figure 3 (As shown in B). Compared to the bacteria (C-EcN) encased in a thin polymer shell in Example 1. Arg The positive potential, the potential reversal, and the moderate increase in particle size indicate that the system was successfully constructed and possesses good stability. The particle size varies within a reasonable range in the figure, and the potential reversal indicates the successful loading of the HA-modified CeO2 nanozyme onto the polymer shell surface.

[0076] The growth of bacteria was observed using a UV spectrophotometer. like Figure 4 As shown, the free engineered bacteria entered the logarithmic growth phase after 2 hours of culture, and the bacterial density reached its peak after 8 hours, while the bacteria (C-EcN) encased in the thin polymer shell in Example 1... Arg ) and the hybrid system (RC-EcN) in Example 5 Arg The growth of CeO2 was restricted, and the bacterial density did not change significantly. This indicates that the dense polymer shell effectively inhibits excessive bacterial proliferation, which helps reduce the potential risk of infection during in vivo application.

[0077] RC-EcN was determined by the CCK8 method. Arg @Bioactivity of CeO2 hybrid systems: like Figure 5 As shown, the bioactivity of bacteria loaded with CeO2 nanozymes on the surface of the thin polymer shell reached 90.5 ± 4.2% of the initial activity. First, free bacteria (EcN... Arg In Example 1, bacteria (C-EcN) encased in a thin polymer shell Arg ) and the hybrid system (RC-EcN) in Example 5 Arg @CeO2) was diluted with sterile PBS and the OD was adjusted using a UV spectrophotometer. 600 =1.0; then, 100 μL of bacterial culture was added to a 96-well plate, with three replicates per group, and 10 μL of CCK8 solution was added simultaneously to mix. The plates were incubated at 37°C in the dark for 30 min. The intensity of the orange-yellow formazan salt produced by the free bacterial group was observed and detected using a microplate reader with an excitation wavelength of 450 nm. The results confirmed that shell modification did not significantly affect the metabolic activity of the bacteria.

[0078] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a cerium oxide nanozyme engineered bacteria hybrid system, characterized in that, Includes the following steps: (1) Preparation of C-EcN Arg : After activation of EcN Arg , the engineering bacteria were cultured to logarithmic growth phase, and then resuspended to obtain bacterial suspension; N-hydroxysuccinimide acrylate dissolved in dimethyl sulfoxide solution was added to obtain EcN Arg -NAS bacterial solution; then acrylamide, N-(3-aminopropyl) methacrylamide hydrochloride, 3-methyl methacrylamide phenylboronic acid and N,N'-methylene bisacrylamide, ammonium persulfate and tetramethylethylenediamine were added in turn, and C-EcN Arg was obtained after reaction; (2) Preparation of CeO2-HA: a. Preparation of CeO2 nanozymes: Xylene was mixed with cerium acetate hydrate, oleylamine, and water to obtain CeO2 nanozymes; b. Surface modification: CeO2 nanozyme was ultrasonically dispersed, and then added to an aqueous hyaluronic acid solution; dialyzed; CeO2-HA was obtained; (3) Preparation of RC-EcN Arg @CeO2: CeO2-HA prepared in step (2) and C-EcN prepared in step (1) were mixed. Arg mixed; incubated; purified, and obtained.

2. The construction method according to claim 1, characterized in that, In step (1), the concentration of the bacterial suspension was 1 x 10 8 CFU / mL; EcN Arg The final concentration of NAS in the NAS bacterial solution was 0.005-0.02 mg / mL.

3. The construction method according to claim 1, characterized in that, In step (1), the molar ratio of acrylamide, N-(3-aminopropyl)methacrylamide hydrochloride, 3-methacrylamide phenylboronic acid, N,N'-methylenebisacrylamide, ammonium persulfate, and tetramethylethylenediamine is (1815-1820): (28-32): (1-3): 33.2: 4.5: 68.4; the reaction conditions are under a nitrogen atmosphere, at 35-37℃, in the dark.

4. The construction method according to claim 1, characterized in that, In step (a), the ratio of xylene to cerium acetate hydrate, oleylamine, and water is 10-15 mL: 0.4-0.5 g: 3-4 g: 1-5 mL.

5. The construction method according to claim 1, characterized in that, In step (b), the ultrasonic dispersion conditions are: ultrasonic power 280-300 W, frequency 40-45 kHz; ultrasonic dispersion time 20-30 min; concentration of hyaluronic acid in the hyaluronic acid aqueous solution 2-4 mg / mL; and dialysis molecular weight cutoff 3500 Da.

6. The construction method according to claim 1, characterized in that, In step (3), CeO2-HA and C-EcN Arg The volume ratio is 2-1:1; the incubation method is to incubate in a constant temperature shaker at 35-37℃ with light-proof shaking at 180-200 rpm for 2-3 hours.

7. The cerium oxide nanozyme engineered bacteria hybrid system obtained by the construction method according to any one of claims 1-6.

8. The application of the cerium oxide nanozyme engineered bacteria hybrid system according to claim 7 in the preparation of drugs for the treatment / prevention of liver fibrosis.

9. A drug for treating / preventing liver fibrosis, the main component of which includes the cerium oxide nanozyme engineered bacteria hybrid system as described in claim 7.

10. The medicament according to claim 9, characterized in that, The liver fibrosis includes chemically induced liver fibrosis; the drug may be formulated with medically acceptable excipients in a cerium oxide nanozyme engineered bacteria hybrid system; or the cerium oxide nanozyme engineered bacteria hybrid system may be mixed with other drugs that have preventive or therapeutic effects on liver fibrosis, with or without the addition of medically acceptable excipients; the drug may be an oral liquid or an injection.

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