Nano preparation for breaking oxidative stress-inflammation vicious circle and regulating intestinal flora as well as preparation method and application of nano preparation

Abstract

The invention provides a nano preparation for breaking oxidative stress-inflammation vicious circle and regulating intestinal flora as well as a preparation method and application of the nano preparation. The preparation method of the preparation comprises the following steps: dissolving potassium ferricyanide and polyvinylpyrrolidone in a solution, slowly dropwise adding a zinc acetate dihydrate solution into the solution, stirring, standing, and repeatedly performing ultrafiltration centrifugal washing to obtain zinc-doped Prussian blue; crushing the yeast cells to obtain yeast cell walls; the yeast cell wall and the zinc-doped Prussian blue are repeatedly co-extruded through a liposome extruder to obtain the nano preparation. The ZnPBA in the nano material can efficiently remove active oxygen at an inflammation part and reduce secretion of inflammatory factors, and the oxidative stress-inflammatory vicious circle of ulcerative colitis is broken; meanwhile, the YCW can deliver the ZnPBA to an inflammation part in a targeted manner to kill pathogenic escherichia coli, and mannose components can promote growth of probiotics to realize bidirectional regulation of intestinal flora. The material is simple in preparation method, can be synthesized on a large scale and is used for treating ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a nano preparation for breaking the vicious cycle of oxidative stress-inflammation and regulating intestinal flora, and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is characterized by excessive production of reactive oxygen species (ROS) induced by proinflammatory factors such as TNF-α and IL-6. In turn, ROS activate the NF-κB signaling pathway, leading to increased secretion of proinflammatory factors and a vicious cycle of ROS-inflammation. Worse still, high levels of ROS can act as electron acceptors, promoting anaerobic respiration and the reproduction of facultative anaerobes such as Enterobacteriaceae, ultimately leading to intestinal microbiota dysbiosis. Therefore, designing simple biomaterials to simultaneously address the above pathological factors is of great significance for the treatment of IBD. Nanozymes are widely used in the treatment of ROS-related diseases due to their advantages such as multi-enzyme activity, good stability, and easy large-scale preparation. However, most studies only focus on the ROS scavenging and anti-inflammatory properties of nanozymes, and have not specifically designed nanozymes to regulate intestinal flora for the treatment of IBD.

[0003] Herein, we designed an enzyme-like biomimetic oral formulation, ZnPBA@YCW, which can eliminate Escherichia coli occupying the ecological niche of IBD mice, promote the growth of multiple probiotics, and simultaneously break the ROS-inflammation vicious cycle. Summary of the invention

[0004] In order to overcome the defects in the prior art, the present invention provides a nano preparation for breaking the vicious cycle of oxidative stress-inflammation and regulating intestinal flora, as well as a preparation method and application thereof.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention is to provide a method for preparing a nanoformulation for breaking the vicious cycle of oxidative stress-inflammation and regulating intestinal flora, which comprises the following steps:

[0007] Step 1, preparing zinc-doped Prussian blue: dissolving potassium ferrocyanide and polyvinyl pyrrolidone in a solution, slowly dropping zinc acetate dihydrate solution into the solution, stirring and then standing, and repeatedly ultrafiltration and centrifugation washing to obtain zinc-doped Prussian blue;

[0008] Step 2, extracting yeast cell wall (YCW): breaking yeast cells to obtain yeast cell wall;

[0009] Step 3, repeatedly co-extrude the yeast cell wall and zinc-doped Prussian blue through a liposome extruder to obtain the nano preparation.

[0010] Furthermore, the mass ratio of potassium ferrocyanide to polyvinyl pyrrolidone is 0.03-0.05:1, preferably 0.04-0.045:1; in the solution, the concentration of polyvinyl pyrrolidone is 2.5-3.6% (w / v).

[0011] Furthermore, the concentration of the zinc acetate dihydrate solution is 0.1-0.2% (w / v).

[0012] Furthermore, in step 1, the volume ratio of the solution to the zinc acetate dihydrate solution is 1:0.8-1.2, preferably 1:1.

[0013] Furthermore, the stirring is magnetic stirring for 3 to 8 minutes; and after stirring, the mixture is allowed to stand for 2 to 5 hours.

[0014] Furthermore, the yeast cell wall is obtained by ball milling for 20 to 50 minutes using a ball mill, followed by repeated centrifugation, vortexing, ball milling, and washing until no complete yeast cells can be observed under a microscope.

[0015] Furthermore, the rotation speed of the ball mill is 600-800 r / min.

[0016] Furthermore, in step three, zinc-doped Prussian blue and yeast cell wall are co-extruded through a liposome extruder at a concentration ratio of 3 to 6:1.

[0017] Furthermore, the liposome extruder is equipped with a 0.8 μm polycarbonate membrane.

[0018] The second aspect of the present invention is to provide a nanoformulation prepared by the above preparation method.

[0019] The third aspect of the present invention is to provide the use of the above nanoformulation in the preparation of drugs for treating ulcerative colitis.

[0020] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0021] The ZnPBA in the nanomaterial provided by the present invention can efficiently remove reactive oxygen species at the inflammation site and reduce the secretion of inflammatory factors, breaking the vicious cycle of oxidative stress-inflammation in ulcerative colitis; at the same time, YCW can deliver ZnPBA to the inflammation site in a targeted manner, killing pathogenic Escherichia coli, and the mannose component in YCW can promote the growth of probiotics, achieving two-way regulation of intestinal flora. In addition, the preparation method of the nanomaterial is simple, and it can be synthesized in large quantities and used to treat ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a flow chart of preparing a nanoformulation for breaking the vicious cycle of oxidative stress-inflammation and regulating intestinal flora in one embodiment of the present invention;

[0023] Figure 2 The efficacy of the nanoformulation provided in Example 1 is shown; wherein, Figure A is a CLSM image after incubation of ZnPBA@YCM with Escherichia coli; Figure BE shows that oral ZnPBA@YCW can target and kill Enterobacteriaceae (Figure B) while promoting the growth of probiotics (Figure CE shows the abundance of Akkermansia, Lachnospiraceae_NK4A136_group, and Lactobacillaceae, respectively). DETAILED DESCRIPTION

[0024] The present invention provides a nanoformulation ZnPBA@YCW for breaking the vicious cycle of oxidative stress-inflammation and regulating intestinal flora. The nanoformulation ZnPBA@YCW can adhere to Escherichia coli, and ZnPBA can remove reactive oxygen and resist inflammation at the site of inflammation, killing Escherichia coli; at the same time, YCW can promote the growth of probiotics and achieve the regulation of intestinal flora.

[0025] The present invention is described in detail and specifically below through specific embodiments and drawings to provide a better understanding of the present invention, but the following embodiments do not limit the scope of the present invention.

[0026] The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0027] Example 1

[0028] This embodiment provides a nanoformulation for breaking the vicious cycle of oxidative stress-inflammation and regulating intestinal flora, and the preparation method thereof comprises the following steps (refer to Figure 1 ):

[0029] 1. Synthesis of zinc-doped Prussian blue (ZnPBA):

[0030] (1) Dissolve 13.2 mg potassium ferrocyanide and 0.3 g polyvinyl pyrrolidone (K8) in 10 ml deionized water and stir magnetically at room temperature for 10 min to form solution A.

[0031] (2) Dissolve 13.2 mg of zinc acetate dihydrate in 10 ml of deionized water and stir magnetically at room temperature for 5 min to form solution B.

[0032] (3) Solution B was slowly added dropwise to solution A, magnetically stirred for 5 min, and allowed to stand for 4 h. The mixture was repeatedly ultrafiltered and centrifuged using a 100K ultrafiltration centrifuge tube to obtain zinc-doped Prussian blue.

[0033] 2. Extraction of yeast cell wall (YCW): Dissolve 10 g of fresh yeast in 10 ml of deionized water, and ball mill for 30 min (speed 750 r / min). Repeat centrifugation, vortexing, ball milling, and washing until no complete yeast cells can be observed under a microscope.

[0034] 3. 0.1 mg / ml YCW and 0.5 mg / ml ZnPBA were co-extruded through a liposome extruder (0.8 μm polycarbonate membrane) and extruded repeatedly for more than 10 times to obtain ZnPBA@YCW.

[0035] Example 2

[0036] This example verifies the efficacy of the nanoformulation provided in Example 1. The specific experimental steps and results are as follows:

[0037] (1) E. coli expressing GFP were incubated with ZnPBA@YCW for 2 hours. The adhesion of ZnPBA@YCW to E. coli was observed using a confocal microscope. Figure 2 As shown in A, YCW can adhere to Escherichia coli.

[0038] (2) Mice were housed under specific pathogen-free conditions with a 12-h light / dark cycle and free access to standard mouse chow. C57BL / 6 mice (female, 6 weeks old) were randomly divided into five groups (n = 5): healthy group (PBS), PBS group (DSS+PBS), DSS+YCW group, DSS+ZnPBA group, and DSS+ZnPBA@YCW group. Mice in the DSS group ingested 3% DSS in sterilized drinking water for 7 consecutive days to induce the colitis model. On days 7, 9, and 11, mice in the healthy group and PBS group received oral gavage with 200 μL PBS, and the other groups received oral gavage with YCW, ZnPBA, and ZnPBA@YCW, respectively. On day 12, feces of each mouse were collected in autoclaved test tubes and stored at -80°C. 16S gene sequencing and analysis were performed at Shanghai Applied Protein Technology Co., Ltd. The total genomic DNA of the samples was extracted using the CTAB / SDS method, and the sequencing library was constructed and sequenced on the Illumina HiSeq 6000 platform. Finally, data analysis was performed on APT-BioCloud.

[0039] according to Figure 2 As shown in BE, oral ZnPBA@YCW can target and kill Enterobacteriaceae, while YCW promotes the growth of probiotics. In vitro fluorescence confocal microscopy confirmed that bacteria can adhere to YCW. In vivo 16sDNA sequencing proved that Enterobacteriaceae can be restored to normal levels.

[0040] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modification and substitution of the present invention are also within the scope of the present invention. Therefore, the equalization and modification made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A method for preparing a nanoparticle preparation for breaking the vicious cycle of oxidative stress-inflammation and regulating intestinal flora, characterized in that: The steps include: Step 1, preparing zinc-doped Prussian blue: dissolving potassium ferrocyanide and polyvinyl pyrrolidone in a solution, slowly dropping zinc acetate dihydrate solution into the solution, stirring and then standing, and repeatedly ultrafiltration and centrifugation washing to obtain zinc-doped Prussian blue; Step 2, extracting yeast cell walls: breaking yeast cells to obtain yeast cell walls; Step 3, repeatedly co-extruding the yeast cell wall and zinc-doped Prussian blue through a liposome extruder to obtain the nano preparation.

2. The preparation method according to claim 1, characterized in that: The mass ratio of potassium ferrocyanide to polyvinyl pyrrolidone is 0.03-0.05:1, preferably 0.04-0.045:1; in the solution, the concentration of polyvinyl pyrrolidone is 2.5-3.6% (w / v).

3. The preparation method according to claim 1, characterized in that: The concentration of the zinc acetate dihydrate solution is 0.1-0.2% (w / v).

4. The preparation method according to claim 3, characterized in that: In step 1, the volume ratio of the solution to the zinc acetate dihydrate solution is 1:0.8-1.2, preferably 1:

1.

5. The preparation method according to claim 1, characterized in that: The stirring is magnetic stirring for 3 to 8 minutes; after stirring, let it stand for 2 to 5 hours.

6. The preparation method according to claim 1, characterized in that: The yeast cell wall is obtained by ball milling for 20 to 50 minutes using a ball mill, followed by repeated centrifugation, vortexing, ball milling, and washing until no complete yeast cells can be observed under a microscope.

7. The preparation method according to claim 6, characterized in that: The rotation speed of the ball mill is 600-800 r / min.

8. The preparation method according to claim 1, characterized in that: In step three, zinc-doped Prussian blue and yeast cell walls are co-extruded through a liposome extruder at a concentration ratio of 3 to 6:1, and the liposome extruder is preferably equipped with a 0.8 μm polycarbonate membrane.

9. The nanoformulation prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the nanoformulation according to claim 9 in preparing a drug for treating ulcerative colitis.

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