Nanometer enzyme coating modified probiotics as well as preparation method and application thereof

Abstract

The invention relates to the technical field of surgical biomedical materials, and provides nano-enzyme coating modified probiotics as well as a preparation method and application thereof, dopamine hydrochloride, metformin and nano-enzyme which are preferably limited in dosage range are used for preparing a precursor solution, and the precursor solution reacts and wraps the probiotics. On the basis that polydopamine is adopted to protect probiotics from being damaged by gastric acid and digestive enzymes in the prior art, on one hand, metformin is adopted to provide mild reaction conditions, and the activity of the probiotics is kept; on the other hand, after nano-enzyme represented by cerium dioxide is adopted to assist oxidative polymerization of dopamine, active oxygen is decomposed at the intestinal inflammation part, MET is utilized to promote transformation of intestinal macrophages to an anti-inflammatory M2 type, and other mechanisms are utilized, so that the inflammation targeted removal function of coating modified probiotics is realized. The preparation method is simple, convenient and efficient, the activity of the modified bacteria, the inflammation targeting performance and the anti-inflammatory performance are proved by experiments, and the preparation method is suitable for being popularized and used for treating ulcerative colitis.

Description

Technical Field

[0001] This invention relates to the field of surgical biomedical materials technology, and more specifically, to a nanoenzyme-coated modified probiotic, its preparation method, and its application. Background Technology

[0002] Probiotic therapy has emerged as an emerging strategy for treating ulcerative colitis. While oral probiotics offer significant advantages such as being non-invasive and having high patient compliance, they still face significant physiological barriers. For example, the gastrointestinal tract surface area can be as large as 300 m². 2 Before reaching the colitis lesion, probiotics must successively overcome challenges such as extreme pH levels, digestive enzymes, and the mucus barrier; moreover, most probiotics are anaerobic and struggle to maintain activity in the colitis microenvironment rich in reactive oxygen species (ROS). On the other hand, colitis lesions exhibit complex microenvironmental dysbiosis, including intestinal barrier damage, immune system imbalance, and gut microbiome disruption, which poses advanced functional requirements for traditional probiotic therapies.

[0003] In recent years, chemical methods such as polymer encapsulation and surface modification have demonstrated remarkable versatility in terms of simplicity and on-demand design. As a general surface modification strategy, the polymerization of dopamine and its derivatives has been widely used for advanced surface functionalization. However, the oxidative polymerization mechanism of dopamine presents a significant challenge due to the stringent survival conditions required by probiotics. Traditional reaction conditions, such as those involving strong oxidants or strong ultraviolet radiation, are not suitable for bacterial surface modification, making it difficult to balance advanced chemical functionalization with the maintenance of probiotic activity. Therefore, developing a simple, ultra-mild, and biocompatible chemical modification strategy to endow probiotics with precise inflammatory-targeting clearance properties is crucial for improving the treatment efficacy of ulcerative colitis. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a nanoenzyme-coated modified probiotic, its preparation method and application. The steps are simple and efficient. While endowing the probiotic with precise inflammation-targeting and clearance properties, it also takes into account the mild conditions to maintain the activity of the probiotic, thereby improving the treatment effect of ulcerative colitis.

[0005] To achieve the above objectives, the present invention provides a nanoenzyme-coated modified probiotic, comprising probiotics and polydopamine, metformin and nanoenzymes coated on the surface of the probiotics.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned nanoenzyme-coated modified probiotics, comprising the following steps: S1. Dissolve the probiotics in a solvent to obtain the initial solution; S2. Add dopamine hydrochloride, metformin (MET), and nanozyme to the initial solution to obtain the reaction precursor solution; S3. Place the precursor solution in a constant temperature shaker to react, centrifuge to collect the precipitate, and obtain the probiotic nanoenzyme coating.

[0007] On the one hand, this solution uses metformin to provide mild reaction conditions, maintaining the activity of probiotics. Building upon existing technologies that use polydopamine to protect probiotics from damage by stomach acid and digestive enzymes, this solution further adds metformin, utilizing its antioxidant effects to prevent damage to probiotics during the dopamine polymerization process. Its protective effect has been demonstrated in the embodiments of this invention.

[0008] On the other hand, this approach utilizes multiple mechanisms to synergistically achieve targeted inflammation clearance. In normal colonic mucosa, because ROS levels are at the physiological baseline (nM level), the threshold for activating nanozyme catalytic activity is not reached, and Raman signals of nanozymes such as cerium dioxide (CeO2) are not detected. Simultaneously, the intact glycocalyx structure of the normal mucus layer hinders direct contact between the PDA coating and epithelial cell surface receptors, resulting in the absence of CC / CH vibrational signals. Therefore, under this microenvironment-dependent signal difference, the probiotics encapsulated in the probiotic nanozyme coating do not remain in the normal colonic mucosa. However, in inflammatory sites with high ROS levels, ROS triggers a nanozyme cascade reaction, improving local hypoxia through in-situ oxygen supply and creating a suitable metabolic environment for EcN colonization. The dynamic structural reorganization of the PDA coating enhances multimodal molecular interactions with oxidatively damaged tissues. MET acts on intestinal macrophages, reducing the expression of the pro-inflammatory phenotype M1 and increasing the number of anti-inflammatory M2, thereby further enhancing the retention effect of the probiotics encapsulated in the probiotic nanozyme coating at inflammatory sites. This multi-mechanism synergistic targeting strategy enables the probiotics encapsulated in the probiotic nanoenzyme coating to intelligently distinguish between pathological and physiological microenvironments, thereby improving therapeutic effects while reducing ecological interference with normal flora.

[0009] Furthermore, the probiotic nanoenzyme coating is stored at 4°C in an anaerobic chamber.

[0010] Further, in step S1, the probiotics include EcN ( Escherichia coli Nissle 1917), Lactobacillus plantarum and / or Lactobacillus johnsonii.

[0011] Furthermore, the nanozyme comprises one or more of cerium dioxide, iron tetroxide, and iron single-atom nanozymes. Preferably, the nanozyme is cerium dioxide. The above components can mimic catalase and superoxide dismutase, eliminating reactive oxygen species at the site of inflammation.

[0012] Further, in step S1, dopamine hydrochloride is first dissolved in a solvent, then metformin is added, and finally nanozyme is added.

[0013] Furthermore, the concentration of dopamine hydrochloride in the precursor solution is 1-10 mg / mL. -1 Preferably, the concentration of dopamine hydrochloride in the precursor solution is 1-5 mg / mL. -1 In one or more embodiments of the present invention, a probiotic coating with better biocompatibility can be obtained within the preferred range.

[0014] Further, each 1 mL of the reaction precursor solution contains 50-100 μg of dopamine hydrochloride, 10-100 μg of metformin, and 10-100 μg of nanozyme. Preferably, the mass ratio of dopamine hydrochloride to metformin is (5-10):1, and 20-50 μg of metformin is added to each 1 mL of the reaction precursor solution.

[0015] Furthermore, in step S3, the areal loading of the precursor solution is 100~300 μL cm⁻¹. -2 The area loading directly affects the coating thickness and uniformity. Setting the above-mentioned range limits helps to ensure that the final coating is uniform, continuous, and of appropriate thickness, avoiding the formation of discontinuous films due to too low a loading and avoiding the flow or agglomeration due to too high a loading.

[0016] Further, in step S1, the ambient temperature during the dissolution process is 4℃~40℃, and the air humidity is 10%~100%. Preferably, the ambient temperature is 25℃ and the air humidity is 70%.

[0017] Further, in step S1, the solvent is physiological saline or PBS buffer solution, and the dissolution is carried out by stirring until completely mixed. In one or more embodiments of the present invention, the stirring time is not less than 3 minutes.

[0018] Furthermore, in step S3, the centrifugation time is 3 to 10 minutes, preferably 5 minutes.

[0019] Furthermore, in step S3, the reaction is carried out under aerobic conditions for 1-2 hours. Within a certain range, the encapsulation performance and biocompatibility of the probiotic coating will vary significantly with changes in the composition of the precursor solution and temperature conditions. Taking all factors into consideration, the present invention preferably uses a limited range of amounts of dopamine hydrochloride, metformin, and nanozyme to prepare the precursor solution, and then reacts and mixes it under aerobic conditions at 37°C for 1-2 hours to obtain a better probiotic nanozyme coating.

[0020] Another object of the present invention is to provide the application of the above-mentioned nanoenzyme-coated modified probiotics in the preparation of a medicament for treating ulcerative colitis.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a nanozyme-coated modified probiotic. A precursor solution is prepared using dopamine hydrochloride, metformin, and nanozymes in a preferably defined range of amounts, which then reacts and encapsulates the probiotics. Building upon existing technologies that use polydopamine to protect probiotics from damage by gastric acid and digestive enzymes, this invention utilizes metformin to provide mild reaction conditions, maintaining probiotic activity. Furthermore, it employs nanozymes, represented by cerium dioxide, to assist in the oxidative polymerization of dopamine, decomposing reactive oxygen species at sites of intestinal inflammation. It also utilizes multiple mechanisms, including MET-promoting the transformation of intestinal macrophages into the anti-inflammatory M2 type, to achieve the inflammatory-targeting clearance function of the coated modified probiotics. The above preparation method is simple and efficient, and the modified bacterial activity, inflammatory targeting performance, and anti-inflammatory properties have all been experimentally demonstrated, making it suitable for widespread use in the treatment of ulcerative colitis. Attached Figure Description Figure 1 SEM images of EcN and EcN@DMCe from Example 1, scale bar: 0.5 μm.

[0022] Figure 2 AC-TEM image and corresponding elemental map of EcN@DMCe in Example 1, showing the distribution of C(I), Ce(II), N(III) and O(IV) and a combined image (V) of all elements, scale bar: 1 μm.

[0023] Figure 3 The XRD patterns of CeO2, EcN, EcN@DM and EcN@DMCe in Example 2 are shown.

[0024] Figure 4 The growth curves of EcN and EcN@DMCe in Example 1 are shown.

[0025] Figure 5 This is a quantitative analysis of EcN bacterial colonies after 2 h of dopamine polymerization under different starting conditions, as described in Example 1.

[0026] Figure 6 The survival rates of EcN and EcN@DMCe from Example 1 after exposure to SGF solution are shown.

[0027] Figure 7 The ROS scavenging capabilities of EcN and EcN@DMC in Examples 1 (a) and (b) include ·OH scavenging capability (a) and H2O2 scavenging capability (b).

[0028] Figure 8The images show fluorescence images of mice from Example 1 at different time points after gavage administration of Cy5.5-labeled EcN or EcN@DMCe.

[0029] Figure 9 a) Schematic diagram of the treatment curve of DSS-induced acute colitis mice in Example 1; b, c) Changes in body weight (b) and DAI score (c) of mice from day 0 to day 10 in the healthy group, PBS, EcN, EcN@DM group and EcN@DMCe group; d) Statistical analysis of colon tissue length on day 10 in different treatment groups, including the Health, PBS, EcN, EcN@DM and EcN@DMCe groups.

[0030] Figure 10 HE staining and AB staining of colon tissue from different treatment groups on day 10 after treatment in Example 1, including Health, PBS, EcN, EcN@DM and EcN@DMCe groups; Scale bar: 50 μm; Gray: Healthy; Dark blue: PBS; Light blue: EcN; Green: EcN@DM; Red: EcN@DMCe.

[0031] Figure 11 For Example 1, the ELISA method was used to quantitatively analyze the levels of IL-6 (a), TNF-α (b), IL-10 (c), and IL-1β (d) in different treatment groups (including healthy group, PBS, EcN, EcN@DM, and EcN@DMCe group) on day 10; gray: healthy; dark blue: PBS; light blue: EcN; green: EcN@DM; red: EcN@DMCe. Detailed Implementation

[0032] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0033] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0034] The present invention will now be further illustrated with specific examples. The following embodiments are only for explaining the present invention and do not constitute a limitation thereof. The test samples and test procedures used in the following embodiments include the following (if the specific experimental conditions are not specified in the embodiments, they are usually performed according to conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments can be obtained commercially unless otherwise specified).

[0035] Example 1 This embodiment provides a nano-enzyme-coated modified probiotic, including the following steps: (1) Take 100 μL of EcN (1×10 9 CFU mL -1 Dissolve the contents in 900 μL of PBS buffer to obtain solution A; (2) Add 1 mg of dopamine hydrochloride, 0.1 mg of metformin, and 50 μg of cerium dioxide (Shanghai Maclean Biochemical Technology Co., Ltd., China) to solution A to obtain solution B; (3) Place solution B in a shaker at 37 ℃ and 120 rpm for 2 h; (4) After the reaction, the bacterial precipitate was collected by centrifuging at 4000 rpm for 5 min and then washed three times with PBS to purify it. The modified probiotic EcN@DMCe with coating was obtained and stored in an anaerobic box (Mitsubishi Gas Chemical Co., Ltd., Japan) at 4°C.

[0036] Example 2 This embodiment provides a nano-enzyme-coated modified probiotic, including the following steps: (1) Take 100 μL of EcN (1×10 9 CFU mL -1 Dissolve the contents in 900 μL of PBS buffer to obtain solution A; (2) Add 1 mg of dopamine hydrochloride, 0.2 mg of metformin, and 50 μg of cerium dioxide (Shanghai Maclean Biochemical Technology Co., Ltd., China) to solution A to obtain solution B; (3) Place solution B in a shaker at 37 ℃ and 120 rpm for 2 h; (4) After the reaction, the bacterial precipitate was collected by centrifuging at 4000 rpm for 5 min and then washed three times with PBS to purify it. The modified probiotic EcN@DMCe with coating was obtained and stored in an anaerobic box (Mitsubishi Gas Chemical Co., Ltd., Japan) at 4°C.

[0037] Example 3 This embodiment uses the preparation method of Example 1, the only difference being that the probiotic is Lactobacillus plantarum / Lactobacillus johnsonii.

[0038] Example 4 This embodiment uses the preparation method of Example 1, the only difference being that the cerium dioxide is replaced with iron tetroxide / iron single-atom nanozyme.

[0039] Example of effect 1 This example utilizes scanning electron microscopy (SEM) to determine the morphology of the probiotic nanoenzyme coating obtained in Example 1. The specific method is as follows: the morphology of the probiotics was observed using a scanning electron microscope (SEM, S-4800, Hitachi, Japan). First, sufficient 3% glutaraldehyde solution was added to fix the probiotics for 0.5 h, then they were dehydrated in a gradient of 30%, 50%, 75%, 90%, and 100% ethanol for 15 min each. Finally, the sample was vacuum-dried and coated with platinum before observation. The results are as follows: Figure 1 As shown, the probiotic nanoenzyme coating exhibits stable encapsulation performance.

[0040] Example 2 This example utilizes transmission electron microscopy to determine the morphology of the probiotic nanoenzyme coating obtained in Example 1. The specific procedure is as follows: after fixing with 3% glutaraldehyde solution for 0.5 hours, the bacterial solution was dropped onto a copper grid and dried. The results are as follows... Figure 2 As shown: the probiotic nanoenzyme coating has good encapsulation performance.

[0041] Example 3 This example utilizes X-ray diffraction (XRD) technology to analyze the crystal structure of the sample obtained in Example 2, in order to determine the crystal form of the active component. In the XRD test, a Cu Kα radiation source was used, with a voltage of 40 kV and a current of 26 mA, and the XRD patterns of the sample were recorded. The results are as follows: Figure 3 As shown, the probiotic nanoenzyme coating exhibits excellent encapsulation performance.

[0042] Example of effect 4 This example demonstrates the growth curve determination of the probiotic nanozyme coating obtained in Example 1 using an enzyme-linked immunosorbent assay (ELISA) reader. The specific implementation method is as follows: EcN and EcN@DMCe were suspended in LB medium to a concentration of 1 × 10⁻⁶. 6 CFU mL -1 The concentration was set and the samples were incubated in shake flasks at 37°C (120 rpm). Optical density (OD) values ​​were recorded at 600 nm at predetermined time points using a microplate reader (Thermo Fisher Scientific, USA). Results are as follows: Figure 4 As shown: the probiotic nanoenzyme coating does not affect the normal growth of probiotics.

[0043] Example 5 This example demonstrates the catalyst toxicity test of the probiotic nanoenzyme coating obtained in Example 1 using the plate coating method. The specific implementation method is as follows: EcN 1 × 10 8 The reaction solution of CFU and dopamine hydrochloride was prepared using different catalysts (MET, APS, CuSO4, Tris). After incubation at 37°C for 2 h in a shaker, 100 μL was spread onto LB agar plates. Colonies were counted after incubation at 37°C for 24 h. Results are as follows: Figure 5 As shown, the metformin-catalyzed dopamine hydrochloride reaction has no significant toxicity to probiotics.

[0044] Example 6 This example demonstrates the gastric juice resistance test of the probiotic nanoenzyme coating obtained in Example 1 using the plate coating method. The specific implementation method is as follows: EcN and EcN@DMCe 1 × 10 8 CFU (per sample) were resuspended with pepsin and 1 mL of simulated gastric juice (SGF) at pH 2. After incubation at 37°C in a shaker for the predetermined time points, all samples were washed with fresh PBS and spread onto LB agar plates. Colonies were counted after incubation at 37°C for 24 h. Results are as follows. Figure 6 As shown: The probiotic nanoenzyme coating can protect probiotics from gastric acid erosion.

[0045] Example 7 This example demonstrates the effectiveness of the probiotic nanoenzyme coating obtained in Example 1 in scavenging reactive oxygen species using a micro-oxygen detector and electron paramagnetic resonance spectrometry. Specifically, the catalase-like activity of the probiotics was determined through an oxygen production experiment. The probiotics (1 × 10⁻⁶) 8 CFU was added to 50 mL of 10 mM H2O2 solution, and oxygen production was monitored using a portable dissolved oxygen meter.

[0046] Electron paramagnetic resonance (EPR) experiments confirmed that EcN and EcN@DMCe were prepared using a TiO2 / UV system to produce •OH. EcN or EcN@DMCe (1 × 10⁻⁶) 8 CFU was added to 100 µL of titanium dioxide dispersion (10 mg / mL). -1 The solution was then added to a container, followed by 200 µL of 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO). After exposure to UV light (365 nm) for 5 min, the EPR spectrum was recorded. The results are as follows: Figure 7 As shown: the probiotic nanoenzyme coating can effectively remove hydrogen peroxide and hydroxyl free radicals.

[0047] Example 8 This example utilizes a small animal in vivo imaging system to determine the inflammatory targeting performance of the probiotic nanozyme coating obtained in Example 1. Specifically, to evaluate the colonization effect of EcN@DMCe in the colitis microenvironment, 6-8 week old C57BL / 6J mice were treated with 3% DSS for 7 days to establish an acute colitis mouse model. Mice were randomly divided into three groups: DSS+EcN group, DSS+EcN@DMCe group, and H2O+EcN@DMCe group. On the 4th day after using 3% DSS, each mouse was administered 100 μL (1×10⁻⁶) via gavage. 9 CFU mL -1 Cy5.5-labeled probiotics. Mice were photographed using IVIS (PerkinLemer) 8, 24, 48, and 168 h after gavage treatment. Results are as follows: Figure 8 As shown: The probiotic nanoenzyme coating can effectively assist probiotics in colonizing the site of colon inflammation.

[0048] Example 9 This example demonstrates the anti-inflammatory properties of the probiotic nanoenzyme coating obtained in Example 1 using in vivo experiments on mice. The specific method was as follows: An acute colitis model was established in mice using 3% DSS. Mice were randomly divided into 5 groups: healthy group, DSS+PBS group, DSS+EcN group, DSS+EcN@DM group, and DSS+EcN@DMCe group. Starting from day 4 after using 3% DSS, each mouse was administered 100 μL of probiotics (1 × 10⁻⁶) by gavage. 9 CFU mL -1 Mice were euthanized for 6 consecutive days. Body weight and disease activity index were assessed daily. On the last day, mice were euthanized, and the following samples were collected for testing: Fecal samples were collected for sequencing. After recording colon length, a partial colon specimen was taken, fixed in 4% paraformaldehyde, and subjected to blinded histopathological analysis. Serum samples were obtained from blood collected by centrifugation at 4000 rpm. All serum and tissue samples were stored at -80 °C. Results are as follows: Figure 9-11 As shown: Probiotic nanoenzyme coating can effectively relieve enteritis and improve colon length and DAI score.

[0049] Obviously, the above embodiments of the present invention are examples provided to clearly illustrate the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A nano-enzyme coating modified probiotic, characterized in that, The probiotics are coated with polydopamine, metformin and nanoscale enzyme on the surface of the probiotics.

2. The method for preparing a nano-enzyme coating modified probiotic bacteria according to claim 1, characterized in that, The method comprises the following steps: S1, dissolving the probiotics in a solvent to obtain an initial solution; S2, adding dopamine hydrochloride, metformin and nanoscale enzyme into the initial solution in sequence to obtain a reaction precursor solution; S3, placing the reaction precursor solution in a constant temperature shaker for reaction, centrifuging to obtain the nanoscale enzyme coating modified probiotics.

3. The method for preparing nanoenzyme-coated modified probiotics according to claim 2, characterized in that, In step S1, the probiotics comprise EcN, plant lactobacillus and / or about formula lactobacillus.

4. The method of claim 2, wherein the nano-enzyme coating-modified probiotics are prepared by the following steps: (1) preparing a nano-enzyme coating solution; (2) mixing the nano-enzyme coating solution with the probiotics; (3) drying the mixture to obtain the nano-enzyme coating-modified probiotics. The nanoscale enzyme comprises cerium dioxide, triiron tetroxide and / or iron monatomic nanoscale enzyme.

5. The method of claim 2-4, wherein the nanoscale enzyme-coated probiotic is prepared by, The concentration of the dopamine hydrochloride is 1 to 10 mg mL -1 .

6. The method for preparing nanoenzyme-coated modified probiotics according to any one of claims 2 to 4, characterized in that, Each 1 mL of the reaction precursor solution contains 50-100 μg of dopamine hydrochloride, 10-100 μg of metformin and 10-100 μg of nanoscale enzyme.

7. The method of claim 2-4, wherein the nano-enzyme coating modified probiotic is prepared by the following steps: (1) preparing a nano-enzyme coating modified probiotic by mixing the probiotic and the nano-enzyme coating; (2) drying the nano-enzyme coating modified probiotic; and (3) packaging the nano-enzyme coating modified probiotic. In step S3, the area loading of the reaction precursor solution is 100 to 300 μL cm -2 .

8. The method of claim 2-4, wherein the probiotic nanozyme coating is prepared by, In step S3, the reaction is carried out under aerobic conditions for 1-2 h.

9. The method of claim 2-4, wherein the nano-enzyme coating modified probiotic is prepared by the following steps: (1) preparing a nano-enzyme coating modified probiotic by mixing the probiotic and the nano-enzyme coating; (2) drying the nano-enzyme coating modified probiotic; and (3) packaging the nano-enzyme coating modified probiotic. In step S3, the centrifugation time is 3-10 min.

10. Use of the nanoscale enzyme coating modified probiotics according to claim 1 in the preparation of a drug for treating ulcerative colitis.

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