Nano-coating modified intestinal probiotics and application thereof in treating inflammatory bowel disease

Abstract

The invention discloses nano-coating modified intestinal probiotics and application thereof in treatment of inflammatory bowel disease, dopamine with excellent biocompatibility is adopted to synthesize mesoporous nanoparticles through a soft template method, and then tungsten ions are loaded through mesoporous adsorption of mesoporous polydopamine and ion exchange with tungstate ions, so that the nano-coating modified intestinal probiotics are obtained. And then forming a nano coating on the surface of the viable bacteria through the viscosity of dopamine. After oral administration, the mesoporous polydopamine nano coating wrapping the probiotics can well shield damage of severe environments such as high-level oxidative stress environment, pepsase and trypsin to the probiotics in an inflammatory environment, so that the probiotics still have relatively high biological activity after entering intestinal tracts; and meanwhile, the slowly released tungsten ions can effectively inhibit the breeding of enterobacteriaceae, so that the effects of relieving intestinal inflammation, regulating intestinal flora composition and further regulating the liver function metabolism condition are achieved.

Description

Technical Field

[0001] The present invention belongs to the field of nano material preparation and biomedicine, and specifically relates to a nano coating modified intestinal probiotic and its application in treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) includes two major chronic diseases, ulcerative colitis (UC) and Crohn's disease (CD). To date, increasing evidence has confirmed a strong correlation between inflammatory bowel disease (IBD) and chronic liver disease (CLD). Intestinal microbial metabolites and their macromolecules reach the liver through the portal vein, and the liver receives signals to transport bile acids and other bioactive molecules to the intestine through the bile duct. As the "second brain" of the human body, intestinal microorganisms play an important role in the development of various liver diseases (non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver disease, acute liver failure, etc.). Although the etiology and pathogenesis of IBD are still unclear, disordered flora can induce a chronic inflammatory state, increase endotoxin production, and disrupt host metabolism. For example, poor colony expansion of Escherichia coli (E. coli) can aggravate intestinal inflammation. These microbial products, especially microbial-associated molecular patterns (MAMPs) such as lipopolysaccharide (LPS), are directly transferred to the portal vein and then reach the liver tissue through the gut-liver axis. In this case, intestinal LPS is recognized by the immune system and poses a serious threat to the normal metabolic function of the liver. Therefore, regulating the composition of the intestinal flora and the homeostatic balance of the gut-liver axis is an important strategy in the treatment of IBD.

[0003] Although probiotics have been studied in the treatment of colitis, oral probiotics alone are not particularly suitable for the treatment of gastrointestinal-related diseases, including ulcerative colitis (UC). Considering that when administered via the oral route, probiotics must withstand gastrointestinal stress, including extremely low gastric pH and harsh digestive enzymes, which greatly reduces the survival rate of probiotics, in addition, the beneficial properties of probiotics depend on the strain and are often unique and insufficient to treat complex diseases. Although genetic modification can give probiotics a higher survival rate and special therapeutic functions, the potential risks of gene transfer to other intestinal bacteria cannot be ignored. Summary of the invention

[0004] Based on the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a nano-coating modified intestinal probiotics, which modifies the intestinal probiotics with a nano-coating with antioxidant and anti-inflammatory effects, thereby solving the problems of poor therapeutic effect and low survival rate of probiotics in the treatment of inflammatory bowel disease, and regulating the impact of enteritis on liver function.

[0005] In order to solve the technical problem, the present invention adopts the following technical solution:

[0006] The present invention first discloses a nano-coating modified intestinal probiotic, which is characterized in that: the nano-coating modified intestinal probiotic is formed by adhering and modifying the surface of the living intestinal probiotic with tungsten-loaded mesoporous polydopamine nanoparticles to form a nano-coating. The diameter of the tungsten-loaded mesoporous polydopamine nanoparticles is 150-300nm. The intestinal probiotic is one of Lactobacillus acidophilus, Escherichia coli and Bifidobacterium.

[0007] The method for preparing the nano-coating modified intestinal probiotics of the present invention comprises the following steps:

[0008] Step 1: Preparation of tungsten-loaded mesoporous polydopamine

[0009] 36 mg of poloxamer is dissolved in a mixture of 6-10 mL of anhydrous ethanol and 6-10 mL of ultrapure water, and then 0.3-0.6 mL of 1,3,5-trimethylbenzene is added and ultrasonicated until uniformly dispersed, and then 0.3-0.6 mL of a 120 mg / mL dopamine hydrochloride aqueous solution and 1-3 mL of a 20 mg / mL TRIS buffer solution are added, and the mixture is stirred at room temperature for 6-8 hours. The obtained product is centrifuged and washed (with ethanol and deionized water in sequence) to obtain mesoporous polydopamine; the obtained mesoporous polydopamine is dispersed in 10-20 mL of deionized water, and 120-360 mg of tungstate (at least one of sodium tungstate dihydrate, calcium tungstate and ferrous tungstate) is added, and the mixture is stirred at room temperature for 6-12 hours. The obtained product is centrifuged and washed (with deionized water) to obtain tungsten-loaded mesoporous polydopamine nanoparticles.

[0010] Step 2: Preparation of nano-coating modified intestinal probiotics

[0011] 25-200 μg of tungsten-loaded mesoporous polydopamine nanoparticles were added to 1 mL of 10 7 ~10 9 CFU / mL intestinal probiotics in PBS buffer, incubate with stirring at room temperature for 3 to 5 hours, centrifuge, collect the precipitate, obtain the nano-coating modified intestinal probiotics, and disperse it in PBS buffer for storage.

[0012] The nano-coating modified intestinal probiotics of the present invention have the function of treating inflammatory bowel disease, are non-toxic to mammalian cells, have good biodegradability and safety, and can reduce the impact of colitis on liver metabolic function. The implementation mechanism is: polydopamine (PDA), a catecholamine neurotransmitter, has a black insoluble biopolymer produced by self-oxidation, which has good anti-inflammatory effect, biocompatibility, and degradability. The mesoporous structure of mesoporous polydopamine can not only provide higher drug loading performance, but also has larger reactive sites, and can provide certain protection for intestinal probiotics such as Lactobacillus acidophilus in harsh gastric acid environment and intestinal inflammation environment. Tungsten-loaded mesoporous polydopamine wraps intestinal probiotics, and after oral administration to the intestinal inflammation site, mesoporous polydopamine can effectively remove ROS in the inflammation site to alleviate the high level of oxidative stress environment, and its slowly released tungsten ions can target molybdenum cofactor-dependent bacterial respiration and energy metabolism to inhibit the reproduction of Enterobacteriaceae, and reduce the impact of the production of harmful bacterial endotoxins on liver function. Live probiotics that reach the intestine can effectively and accurately regulate the intestinal microbiome, enhance the intestinal immune barrier, reduce intestinal damage, and reduce the impact of bacterial endotoxin leakage on the normal function of the liver. The increase in the abundance of intestinal probiotics promotes the production of metabolites, including short-chain fatty acids and indole derivatives, and has a significant preventive and therapeutic effect on acute and chronic colitis induced by dextran sulfate sodium (DSS). The present invention provides a new strategy for the treatment of IBD and maintaining normal liver metabolism closely related to its pathogenesis.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] 1. The present invention uses tungsten-loaded mesoporous polydopamine to encapsulate probiotics, thereby improving the survival ability of probiotics under gastric acid, intestinal fluid and high-level oxidative stress conditions.

[0015] 2. Tungsten-loaded mesoporous polydopamine has excellent antioxidant and anti-inflammatory effects. Its slowly released tungsten ions and probiotics can specifically inhibit the abnormal proliferation of Escherichia coli during colitis and promote the growth of probiotics, effectively and accurately regulating the intestinal microbiome, reducing the side effects of simple tungstate regulation of the flora and the insufficient therapeutic effect of simple probiotics.

[0016] 3. The nano-coating modified probiotics of the present invention have a good therapeutic effect on the mouse colitis model and are superior to the clinical drug 5-aminosalicylic acid, and can also regulate the synthesis of bile acid and the like in the liver.

[0017] 4. The preparation process of the nano-coating modified probiotics of the present invention is simple, the conditions are mild, and it has the potential for industrialization and commercialization.

[0018] 5. The dopamine and probiotics used in the present invention have excellent biocompatibility, have no direct or indirect toxic effects on the human body, and have no potential toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the structure and formation of LA@WMPDA obtained in Example 1.

[0020] Figure 2 This is a transmission electron microscopy image of the WMPDA nanoparticles prepared in Example 1.

[0021] Figure 3 This is the X-ray photoelectron spectrum of WMPDA nanoparticles prepared in Example 1, and the inset is the valence state fitting at the tungsten ion peak.

[0022] Figure 4 This is the particle size distribution diagram of WMPDA nanoparticles prepared in Example 1.

[0023] Figure 5 This is a scanning electron microscope image of LA@WMPDA prepared in Example 1.

[0024] Figure 6 Transmission electron microscopy images of LA encapsulated with WMPDA of different concentrations prepared in Example 1.

[0025] Figure 7 Zeta potential diagrams of MPDA, WMPDA, LA and LA@WMPDA prepared in Example 1.

[0026] Figure 8 This is the growth curve of LA and LA@WMPDA prepared in Example 1 within 36 hours.

[0027] Fig. 9 This is a graph showing the probiotic activity of LA@WMPDA prepared in Example 1 under active oxygen conditions.

[0028] Fig.10 LA prepared in Example 1 ( Fig.10 a) and WMPDA( Fig.10 b) Cytotoxicity assay diagram.

[0029] Fig.11 This is a data chart showing weight loss of mice in different groups in the inflammatory bowel disease model treated in Example 1.

[0030] Fig.12 Figure 1 shows the colon of mice in different groups in the inflammatory bowel disease model of Example 1 ( Fig.12 a) and colon length statistical index data diagram ( Fig.12 b).

[0031] Fig.13This is a heat map of the relative abundance of family-level classifications in different groups of mice in the inflammatory bowel disease model treated in Example 1.

[0032] Fig.14 This is a data graph of bile acid indexes in the blood of different groups of mice in the inflammatory bowel disease model treated in Example 1.

[0033] Fig.15 This is a data chart of LPS indicators in the blood of different groups of mice in the inflammatory bowel disease model treated in Example 1. DETAILED DESCRIPTION

[0034] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0035] Example 1

[0036] like Figure 1 As shown, this embodiment prepares nano-coating modified intestinal probiotics (LA@WMPDA) according to the following steps:

[0037] Step 1: Preparation of tungsten-loaded mesoporous polydopamine nanoparticles (WMPDA)

[0038] 36 mg of poloxamer was dissolved in a mixture of 6 mL of anhydrous ethanol and 6 mL of ultrapure water, and then 0.3 mL of 1,3,5-trimethylbenzene was added and ultrasonicated until uniformly dispersed, and then 0.3 mL of a 120 mg / mL dopamine hydrochloride (DA) aqueous solution and 1 mL of a 20 mg / mL TRIS buffer solution were added, and the mixture was stirred at room temperature for 8 h. The obtained product was centrifuged (12000 rpm, 10 min) and washed (with ethanol and deionized water in sequence) to obtain mesoporous polydopamine (denoted as MPDA); the obtained mesoporous polydopamine was dispersed in 10 mL of deionized water, 120 mg of sodium tungstate dihydrate was added, and the mixture was stirred at room temperature for 6 h. The obtained product was centrifuged (12000 rpm, 10 min) and washed with deionized water to obtain tungsten-loaded mesoporous polydopamine nanoparticles (denoted as WMPDA).

[0039] Step 2: Preparation of nano-coating modified intestinal probiotics

[0040] 100 μg of tungsten-loaded mesoporous polydopamine nanoparticles were added to 1 mL of 10 8 CFU / mL intestinal probiotics (LA) were added to PBS buffer, stirred and incubated at room temperature for 3 hours, and then centrifuged at 6000 rpm for 5 minutes. The precipitate was collected to obtain nano-coating modified intestinal probiotics (denoted as LA@WMPDA), which were dispersed in PBS buffer and stored at 4°C.

[0041] Figure 2 This is a transmission electron microscope image of the WMPDA nanoparticles synthesized in this example. From the image, it can be seen that the diameter of the nanoparticles is 150-300 nm.

[0042] Figure 3 This is the X-ray photoelectron spectrum of WMPDA nanoparticles prepared in this example. The inset is the valence state fitting at the tungsten peak. It can be seen that W 6+ Successfully loaded on nanoparticles.

[0043] Figure 4 This is the hydrated particle size distribution diagram of the WMPDA nanoparticles prepared in this example. From the figure, it can be seen that the diameter of the nanoparticles is mainly distributed between 150 and 300 nm, which is consistent with the transmission electron microscopy image.

[0044] Figure 5 This is a scanning electron microscope image of LA@WMPDA prepared in this embodiment. The characterization method is as follows: the LA@WMPDA material is fixed by centrifugation, the fixing solution is glutaraldehyde and methanol (the volume ratio of the two is 5%:95%), and the fixing time is 1-2 hours. During the fixing step, care should be taken to avoid the generation of bubbles to ensure that the sample is evenly soaked. After fixation, use an appropriate amount of PBS buffer to wash the fixed bacteria for 10-15 minutes. Then dehydrate: dehydrate the sample by gradient ethanol method, usually starting from 30% ethanol and gradually increasing to 100%. The treatment time for each concentration of ethanol is 10-15 minutes. Finally, dilute it with anhydrous ethanol to form a dispersion and add it dropwise to the silicon wafer, dry it, and observe it in a scanning electron microscope.

[0045] In order to compare the effect of WMPDA nanoparticle concentration on LA encapsulation effect, the amount of tungsten-loaded mesoporous polydopamine nanoparticles added to PBS buffer in step 2 was adjusted to adjust its concentration in the system. Figure 6 The transmission electron micrographs of LA encapsulated by WMPDA of different concentrations are shown in Figure 1. The synthesized LA@WMPDA material is centrifuged, fixed, washed and gradient dehydrated according to the scanning sample preparation method. Finally, it is diluted with anhydrous ethanol to form a dispersion dropwise onto the transmission electron microscope copper grid, dried, and placed in a transmission electron microscope for observation. It can be seen from the figure that the nanoparticles can be well adhered and encapsulated on the surface of the probiotics.

[0046] Figure 7The Zeta potential diagram of MPDA, WMPDA, LA and LA@WMPDA in this example shows that the Zeta potential value of pure mesoporous polydopamine nanoparticle MPDA is -28.4mV, and the Zeta potential value of WMPDA modified with tungsten particles increases to -16.8mV; when the WMPDA modified with tungsten particles is attached to the surface of bacteria, it can be seen that the Zeta potential value of the bacteria is reduced to -10.3mV. Together with the transmission results, it shows that the mesoporous polydopamine nanoparticles are successfully modified on the surface of probiotics.

[0047] Figure 8 The growth curves of LA and LA@WMPDA in this example within 36 hours were characterized by anaerobically culturing LA and LA@WMPDA in MRS medium at 37°C (the LA concentrations of the two were kept the same). The OD was measured at the predetermined time points using a microplate reader (BioTek). 600 , with the culture time as the horizontal axis and the OD measured by the bacteria 600 The growth curve was plotted as the ordinate. It can be seen that the loading of WMPDA nanoparticles did not affect the growth of probiotics.

[0048] In order to compare the probiotic activity of LA@WMPDA with different WMPDA loading amounts under active oxygen conditions, the following test was performed: different masses (0, 25, 50, 100, 150, 200 μg) of tungsten-loaded mesoporous polydopamine nanoparticles were added to 1 mL of 10 8 CFU / mL intestinal probiotics (LA) were added to PBS buffer, stirred and incubated at room temperature for 3 hours, and then centrifuged at 6000 rpm for 5 minutes to collect the precipitate and obtain nano-coated intestinal probiotics (respectively LA, LA@WMPDA 25 , LA@WMPDA 50 , LA@WMPDA 100 , LA@WMPDA 150 , LA@WMPDA 200 ), added to MRS broth containing 200 μM H2O2, incubated at 37°C for 2 h, and then the number of viable probiotics was detected. Fig. 9 As shown, coating LA with WMPDA could maintain the growth of LA in normal or high-level oxidative stress environments, while the activity of probiotics alone was reduced under 200 μM H2O2 conditions.

[0049] Fig.10 For different concentrations of probiotics (such as Fig.10 a) and tungsten-loaded mesoporous polydopamine nanoparticles (as shown in Fig.10 b) is a cytotoxicity test diagram, wherein the method is: NCM460 cells are inoculated at 1×10 4The cells were inoculated at a density of 100 μL / well in a 96-well plate, and 100 μL of serum-containing 1640 medium was added for 24 hours. Then, nanoparticles or probiotics were added to different concentrations. After 24 hours of continuous culture, the cells were washed twice with PBS, and then 20 μL of 5 mg / mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to culture the cells for 4 hours. The supernatant was replaced with dimethyl sulfoxide (150 μL). Finally, the absorbance was recorded using a microplate reader (BioTek, ELX800) to calculate the cell survival rate. It can be seen that different concentrations of tungsten-loaded mesoporous polydopamine and probiotics have good cell compatibility.

[0050] Evaluation of the therapeutic performance of LA@WMPDA in inflammatory bowel disease: BALB / c mice (female, 6-8 weeks, 18-22 g) were randomly divided into 8 groups (n=8): 1) PBS+water (Control group); 2) PBS+3%DSS; 3) 5ASA+3%DSS; 4) Na2WO4+3%DSS; 5) LA+3%DSS; 6) MPDA+3%DSS; 7) WMPDA+3%DSS; 8) LA@WMPDA+3%DSS. They were kept for one week before modeling to adapt to the environment. The day when groups 2) to 8) began to orally drink drinking water containing 3%DSS was day 0. On day 6, DSS water was replaced with normal drinking water. On days 7, 9, 11 and 13, PBS buffer or PBS buffer containing 5ASA, Na2WO4, LA, MPDA, WMPDA and LA@WMPDA (administered at 4 mg / Kg) was orally administered. The mice were euthanized on day 15. By detecting the changes in the weight of mice, the severity of the disease can be intuitively seen. Fig.11 As shown in the figure, the weight of all DSS group mice decreased to varying degrees after 8 days of treatment. On the 12th day, the other control group treatments were unable to effectively prevent the trend of weight loss, while the weight of DSS colitis mice treated with LA@WMPDA was significantly restored. The harmful inflammatory response caused by colitis can also lead to a decrease in colon length. Therefore, in this example, the colon of mice was isolated and the colon length was measured to further evaluate the colon damage (such as Fig.12 The average colon length of mice in the DSS-treated group was only 6.3 cm, while the average colon length of mice in the Na2WO4 and LA-treated groups was 6.88 cm and 7.02 cm, respectively. The average colon length of mice in the LA@WMPDA-treated group recovered to 7.7 cm, which was close to the 7.84 cm of normal mice. This further proves that LA@WMPDA has a good protective effect against colitis. Then, the bacterial flora in the LA@WMPDA treatment model was measured. Fig.13As shown in Figure 2, the V4 region in the fecal samples of mice collected from different experimental groups was analyzed using 16S ribosomal RNA (rRNA) gene sequencing. The results of gene sequencing data analysis showed that the intestinal microbiota characteristics of the LA@WMPDA group were significantly different compared with the DSS group and the Na2WO4 group.

[0051] Based on the good therapeutic effect of LA@WMPDA on UC model, this example further explored the regulatory effect of LA@WMPDA on liver function of UC model. Fig.14 ) and LPS( Fig.15 ) levels, it can be seen that oral administration of LA@WMPDA can reduce blood bile acid levels and the leakage of bacterial endotoxin LPS.

[0052] The above are merely exemplary embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A nano-coating modified intestinal probiotic, characterized in that: The nano-coating modified intestinal probiotics is that the surface of the living intestinal probiotics is modified with a tungsten-loaded mesoporous polydopamine nano-coating.

2. The nano-coating modified intestinal probiotics according to claim 1, characterized in that: The intestinal probiotics are one of Lactobacillus acidophilus, Escherichia coli and Bifidobacterium.

3. A method for preparing the nano-coating modified intestinal probiotics according to claim 1 or 2, characterized in that: The steps include: Step 1, dissolving 36 mg of poloxamer in a mixture of 6-10 mL of anhydrous ethanol and 6-10 mL of ultrapure water, then adding 0.3-0.6 mL of 1,3,5-trimethylbenzene and ultrasonically dispersing it evenly, then adding 0.3-0.6 mL of a 120 mg / mL dopamine hydrochloride aqueous solution and 1-3 mL of a 20 mg / mL TRIS buffer solution, stirring at room temperature for 6-8 hours, and the obtained product is centrifuged and washed to obtain mesoporous polydopamine; dispersing the obtained mesoporous polydopamine in 10-20 mL of deionized water, adding 120-360 mg of tungstate, stirring at room temperature for 6-12 hours, and the obtained product is centrifuged and washed to obtain tungsten-loaded mesoporous polydopamine nanoparticles; Step 2: Add 25-200 μg of tungsten-loaded mesoporous polydopamine nanoparticles to 1 mL of 10 7 ~10 9 CFU / mL intestinal probiotics in PBS buffer, incubate with stirring at room temperature for 3 to 5 hours, centrifuge, collect the precipitate, obtain the nano-coating modified intestinal probiotics, and disperse it in PBS buffer for storage.

4. The preparation method according to claim 3, characterized in that: The tungstate is at least one of sodium tungstate dihydrate, calcium tungstate and ferrous tungstate.

5. Use of the nano-coating modified intestinal probiotics according to claim 1 or 2 in the preparation of a medicine for treating inflammatory bowel disease.

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