CN118649143A - A therapeutic nano-coating modified single probiotic and its preparation method and application - Google Patents

Abstract

The invention provides a therapeutic nano-coating modified Shan Yisheng bacterium and a preparation method and application thereof, wherein the preparation method of the therapeutic nano-coating modified Shan Yisheng bacterium comprises the following steps: 1) Preparing amphiphilic chitosan oligosaccharide micelle; 2) Mixing the amphipathic chitosan oligosaccharide micelle and curcumin in an organic solvent, removing the organic solvent, and redissolving in water to obtain a curcumin/chitosan oligosaccharide nano micelle solution; 3) Adding the curcumin/chitosan oligosaccharide nano micelle solution into the probiotic suspension to obtain a probiotic/curcumin/chitosan oligosaccharide solution; 4) And adding the polyanionic polymer solution into the probiotic bacteria/curcumin/chitosan oligosaccharide solution to obtain the therapeutic nano-coating modified Shan Yisheng bacteria. The chitosan oligosaccharide carrier is utilized to realize the simultaneous delivery of the antioxidant substance curcumin and the probiotics, so that the bioavailability of the curcumin is effectively improved, and the chitosan oligosaccharide carrier has more excellent synergistic treatment effect on ulcerative colitis.

Description

A therapeutic nano-coating modified single probiotic and its preparation method and application

Technical Field

The present invention belongs to the technical field of probiotic products, and in particular, relates to a therapeutic nano-coating modified single probiotic and a preparation method and application thereof.

Background Art

Ulcerative colitis is a chronic inflammatory disease of the gastrointestinal tract. Due to the multifactorial nature of its etiology, the incidence of colitis is on the rise worldwide, bringing a huge health burden to humans. In recent years, probiotics have played a key role in human health. They have the functions of maintaining the balance of intestinal flora, inhibiting the growth of pathogenic bacteria, and thus enhancing nutrition, regulating immunity, and preventing diseases. Oral probiotics for the treatment of colitis have received increasing attention. However, probiotics often cannot play their role because of the effects of gastric acid and bile salts, which reduce their activity or even disappear after entering the human intestine. Therefore, some methods must be used to protect the activity of probiotics so that they can reach the intestinal tract and play their role.

The emergence of microencapsulation technology has solved this problem well. Microencapsulation technology can be used to embed probiotics in the wall material solution, enhance the resistance of probiotics to adverse external environments, control the release time and location of probiotics, and thus improve the survival rate of probiotics.

At present, the encapsulation of probiotics in microcapsules and hydrogels has been explored to improve their survival rate and colonization in gastrointestinal tissues. However, these methods have some defects, such as the size of microcapsules is generally large and difficult to control, resulting in low mass transfer efficiency, low activity of probiotics embedded in the center, and low colonization efficiency in the body; the pores of hydrogels are large, which cannot effectively prevent hydrogen ions and bile salts from killing probiotics, and the encapsulation efficiency is low. In addition, there are also defects such as complex preparation process and harmful cross-linking agents.

Summary of the invention

The purpose of the present invention is to provide a therapeutic nano-coating modified single probiotic and its preparation method and application, so as to solve the technical problems that the existing encapsulation technology cannot effectively encapsulate or the encapsulated probiotics have low activity and low colonization efficiency in the body.

The present invention is achieved through the following technical solutions:

A therapeutic nano-coating modified single probiotic comprises a probiotic, wherein the surface of the probiotic is sequentially coated with curcumin/chitosan oligosaccharide nano-micelles and a polyanion polymer.

Preferably, the probiotic is Escherichia coli nissle1917, Lactobacillus acidophilus or Bacteroides fragilis.

Preferably, the polyanionic polymer is sodium alginate or sodium hyaluronate.

The preparation method of the therapeutic nano-coating modified single probiotic comprises the following steps:

1) reacting chitosan oligosaccharide with ursolic acid, 1-hydroxybenzotriazole and 1,3-diisopropylcarbodiimide to prepare amphiphilic chitosan oligosaccharide micelles;

2) mixing the amphiphilic chitosan oligosaccharide micelles and curcumin in an organic solvent, removing the organic solvent, and redissolving in water to obtain a curcumin/chitosan oligosaccharide nano-micelle solution;

3) adding the curcumin/chitosan oligosaccharide nano-micelle solution into the probiotic suspension under stirring, and obtaining a probiotic/curcumin/chitosan oligosaccharide solution through electrostatic self-assembly;

4) adding the polyanionic polymer solution to the probiotic/curcumin/chitosan oligosaccharide solution under stirring, and obtaining the therapeutic nano-coating modified single probiotic via electrostatic self-assembly.

Preferably, step 1) is specifically as follows: dissolving ursolic acid, 1-hydroxybenzotriazole and 1,3-diisopropylcarbodiimide in DMF to obtain an activated ester solution; dissolving chitosan oligosaccharide in DMF and stirring and mixing with the activated ester solution, washing and rotary evaporation to obtain amphiphilic chitosan oligosaccharide micelles.

Preferably, in step 1), the molar ratio of chitosan oligosaccharide to ursolic acid is 1:(0.2-0.8) based on the molar amount of amino groups on the chitosan oligosaccharide chain.

Preferably, in step 2), the mass ratio of the amphiphilic chitosan oligosaccharide micelles to curcumin is 10:(1-5).

Preferably, in step 3), the mass of curcumin/chitosan oligosaccharide nano-micelles added to every 2 milliliters of the probiotic suspension is 0.5-4 mg, and the OD600 value of the probiotic suspension is 0.4-0.8.

Preferably, in step 4), the mass ratio of the polyanionic polymer to the probiotics/curcumin/chitosan oligosaccharide is (0.1-0.5):1.

The application of the therapeutic nano-coating modified single probiotic in the preparation of medicine for treating ulcerative colitis.

Compared with the prior art, the present invention has the following beneficial effects:

The preparation method of the therapeutic nano-coating modified single probiotic of the present invention comprises the following steps: firstly, chitosan oligosaccharide is modified by ursolic acid to obtain amphiphilic chitosan oligosaccharide micelles, hydrophobic curcumin is wrapped in the micelles, and then the micelles are adsorbed on the surface of the probiotics by electrostatic self-assembly, and finally the outer layer of the curcumin/chitosan oligosaccharide nano-micelles is further wrapped with a polyanionic polymer protective layer by electrostatic force to obtain a therapeutic nano-coating modified single probiotic. The present invention uses chitosan oligosaccharide and polyanionic polymer electrostatic self-assembly to modify the single probiotic layer layer by layer, which has the advantages of simple process, short production cycle, low cost, and easy size control. Compared with the traditional microcapsule method, the therapeutic nano-coating of the present invention realizes the encapsulation of a single probiotic, has a small size, fast mass transfer, and low carrier porosity, and can better protect the activity of the probiotics in the gastric environment. At the same time, the polyanionic polymer can target the inflammatory area and enhance the colonization of the probiotics in the body. At the same time, compared with a single probiotic encapsulation system, the present invention utilizes a chitosan oligosaccharide carrier to achieve the simultaneous delivery of the antioxidant curcumin and probiotics, effectively improving the bioavailability of curcumin and having a more excellent synergistic therapeutic effect on ulcerative colitis.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG1 is a schematic diagram of the preparation method of a therapeutic nano-coating modified single probiotic in Example 1

FIG. 2 is a TEM image of a single probiotic modified with a therapeutic nano-coating in Example 1.

FIG. 3 is a diagram showing the Zeta potential of probiotics before and after encapsulation in Example 1.

FIG. 4 is a graph showing colony counts of probiotics before and after encapsulation in Example 1 after being cultured in a liquid culture medium for two hours.

FIG. 5 is a graph showing bacterial activity of probiotics before and after encapsulation in Example 1.

FIG. 6 is a graph showing the probiotic plate counts of probiotics before and after encapsulation in simulated gastric fluid in Example 1.

FIG. 7 is a graph showing changes in the disease activity index of the ulcerative colitis mouse model after treatment in each treatment group.

FIG8 shows the changes in colon length of the UC mouse model after treatment in each treatment group.

FIG. 9 is an evaluation of the inflammatory responsive adhesion performance of a single probiotic modified with a therapeutic nanocoating in Example 1.

DETAILED DESCRIPTION

The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

It should be noted that the process equipment or devices not specifically specified in the following embodiments are all conventional equipment or devices in the art.

It should be noted that the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the present invention without substantially changing the technical content.

In the present invention, the single bacteria with therapeutic nano coating is obtained by electrostatically adsorbing chitosan oligosaccharide nano-micelles loaded with curcumin on the surface of the probiotics, and then electrostatically adsorbing the polyanion polymer.

The method for preparing the therapeutic nano-coating modified single probiotic of the present invention comprises the following steps:

1) Ursolic acid, 1-hydroxybenzotriazole and 1,3-diisopropylcarbodiimide are fully dissolved in DMF at a molar ratio of (1:1:1) to obtain an activated ester solution, 5 kDa chitosan oligosaccharide is fully dissolved in the DMF solution, mixed with the activated ester solution, washed with ether and evaporated under negative pressure to obtain amphiphilic chitosan oligosaccharide micelles (COU).

2) The amphiphilic chitosan oligosaccharide micelles were fully dissolved in methanol, and curcumin was added and mixed, and the mixture was subjected to rotary evaporation and reflux in the dark to fully remove the methanol, and the mixture was redissolved in ultrapure water to obtain curcumin/chitosan oligosaccharide nano-micelles (COU@CUR).

3) preparing a probiotic suspension, adding curcumin/oligochitosan nano-micelles into the probiotic suspension under magnetic stirring, and obtaining curcumin/oligochitosan micelles modified single probiotics (probiotics/curcumin/oligochitosan) by electrostatic self-assembly.

4) The polyanion polymer solution is slowly added to the probiotics/curcumin/chitosan oligosaccharide solution obtained in the above step 3) under magnetic stirring to obtain polyanion-coated probiotics/curcumin/chitosan oligosaccharide by electrostatic self-assembly.

In some specific embodiments of the present invention, in step 1), the molar ratio of chitosan oligosaccharide to ursolic acid is 1:(0.2-0.8) based on the molar amount of amino groups on the chitosan oligosaccharide chain.

In some specific embodiments of the present invention, in step 2), the mass ratio of the amphiphilic chitosan oligosaccharide micelles to curcumin is 10:(1-5).

In some specific embodiments of the present invention, in step 2), the rotary evaporation is refluxed for 30 min-2 h in the dark.

In some specific embodiments of the present invention, in step 3), the probiotics are selected from Escherichia coli nissle1917, Lactobacillus acidophilus or Bacteroides fragilis.

In some specific embodiments of the present invention, in step 3), the OD600 value of the probiotic suspension is 0.4-0.8.

In some specific embodiments of the present invention, in step 3), the mass of curcumin/chitosan oligosaccharide nano-micelles added to every two milliliters of probiotic suspension (OD600 value is 0.4-0.8) is 0.5-4 mg.

In some specific embodiments of the present invention, in step 4), the polyanionic polymer is sodium alginate (SA) or sodium hyaluronate (HA).

In some specific embodiments of the present invention, in step 4), the mass ratio of the polyanionic polymer to the probiotics/curcumin/chitosan oligosaccharide is (0.1-0.5):1.

Taking chitosan oligosaccharide, curcumin, sodium alginate, and Escherichia coli nissle1917 as examples, the reaction process of the present invention is illustrated as shown in Figure 1. The modified chitosan oligosaccharide is fully dissolved in methanol, mixed with curcumin in a certain proportion, the organic solvent methanol is removed by rotary evaporation, and redissolved in ultrapure water to obtain curcumin/chitosan oligosaccharide nanomicelles (COU@CUR); the curcumin/chitosan oligosaccharide nanomicelles are added to the probiotic suspension in a certain proportion, stirred to obtain (CEcN), and then sodium alginate is fully mixed in a certain proportion to obtain the final therapeutic nano-coating modified single bacteria (ACEcN).

After oral administration of the therapeutic nanocoating-modified single bacteria, curcumin and Escherichia coli nissle1917 are protected when delivered to the highly acidic environment of the stomach. When delivered to the intestine, sodium alginate cross-links with Ca2 ⁺ at the inflammatory site, thereby increasing EcN colonization. Due to the change in pH environment, curcumin and Escherichia coli nissle1917 are released to exert their biological activity.

The present invention is further described in detail below with reference to the embodiments.

Example 1

1) Synthesis of amphiphilic chitosan oligosaccharide micelles (COU)

Ursolic acid (0.913 g, 2 mmol), 1-hydroxybenzotriazole (0.27 g, 2 mmol), 1,3-diisopropylcarbodiimide (0.313 mL, 2 mmol) were dissolved in DMF and stirred at 4 °C for 16 h at a stirring speed of 400 rpm. A certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to about 3-5 with acetic acid. 5 kDa molecular weight chitosan oligosaccharide (based on the molar amount of amino groups on the chitosan oligosaccharide sugar chain, the molar ratio of chitosan oligosaccharide to ursolic acid was 1:0.5) was added, and stirred at room temperature until no solid particles were visible to the naked eye. After the above two were fully mixed, an appropriate amount of ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain amphiphilic chitosan oligosaccharide nanomicelles (COU).

2) Preparation of curcumin/chitosan oligosaccharide nanomicelles (COU@CUR)

Take 1g COU and fully dissolve it in methanol, add curcumin at a mass ratio of COU to curcumin (CUR) of 10:1, reflux in the dark for 0.5h, then remove methanol by rotary evaporation, and redissolve it in ultrapure water to obtain COU@CUR.

3) Curcumin/chitosan oligosaccharide nanomicelles encapsulating Escherichia coli nissle1917 (EcN/COU@CUR, CEcN)

Escherichia coli nissle1917 (EcN) was grown overnight in LB medium at 37°C; the overnight culture was diluted into fresh medium and grown at 37°C for 4 h; the cells were collected by centrifugation at 4000 rpm for 5 min and resuspended in ice-cold phosphate buffer. The bacterial OD600 was measured by UV-VIS, and the bacterial suspension was diluted to OD600 = 0.7.

Under magnetic stirring (200r), 750 μL COU@CUR (2 mg/mL) was added to 2 mL bacterial suspension and stirred for 10 min to obtain EcN/COU@CUR through electrostatic self-assembly.

4) Preparation of therapeutic nanocoating modified single probiotics (EcN/COU@CUR/SA, ACEcN)

Under magnetic stirring (200r), 375 μl of sodium alginate (2 mg/mL) was slowly added to EcN/COU@CUR and stirred for 10 min to obtain EcN/COU@CUR/SA, which was then stored at 4°C for further characterization. Figure 1 is a schematic diagram of the preparation process.

Example 2

1) Synthesis of amphiphilic chitosan oligosaccharide micelles (COU)

Ursolic acid (0.913 g, 2 mmol), 1-hydroxybenzotriazole (0.27 g, 2 mmol), 1,3-diisopropylcarbodiimide (0.313 mL, 2 mmol) were dissolved in DMF and stirred at 4 °C for 16 h at a stirring speed of 400 rpm. A certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to about 3-5 with acetic acid. 5 kDa molecular weight chitosan oligosaccharide (based on the molar amount of amino groups on the chitosan oligosaccharide sugar chain, the molar ratio of chitosan oligosaccharide to ursolic acid was 1:0.4) was added, and stirred at room temperature until no solid particles were visible to the naked eye. After the above two were fully mixed, an appropriate amount of ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain amphiphilic chitosan oligosaccharide nanomicelles (COU).

2) Preparation of curcumin/chitosan oligosaccharide nanomicelles (COU@CUR)

Take 1g COU and fully dissolve it in methanol, add curcumin at a mass ratio of COU to curcumin (CUR) of 10:5, reflux in the dark for 1h, then remove methanol by rotary evaporation, and redissolve it in ultrapure water to obtain COU@CUR.

3) Curcumin/chitosan oligosaccharide nanomicelles encapsulating Escherichia coli nissle1917 (EcN/COU@CUR)

EcN was grown in LB medium at 37°C overnight; the overnight culture was diluted into fresh medium and grown at 37°C for 4 h; the cells were collected by centrifugation at 4000 rpm for 5 min and resuspended in ice-cold phosphate buffer. The bacterial OD600 was measured by UV-VIS, and the bacterial suspension was diluted to OD600 = 0.6.

Under magnetic stirring (200r), 500 μL COU@CUR (2 mg/mL) was added to 2 mL bacterial suspension and stirred for 10 min to obtain EcN/COU@CUR through electrostatic self-assembly.

4) Preparation of therapeutic nanocoating modified single probiotic (EcN/COU@CUR/HA)

Under magnetic stirring (200r), 200 μl of sodium hyaluronate (2 mg/mL) was slowly added to EcN/COU@CUR and stirred for 10 min to obtain EcN/COU@CUR/HA.

Example 3

1) Synthesis of amphiphilic chitosan oligosaccharide micelles (COU)

Ursolic acid (0.913 g, 2 mmol), 1-hydroxybenzotriazole (0.27 g, 2 mmol), 1,3-diisopropylcarbodiimide (0.313 mL, 2 mmol) were dissolved in DMF and stirred at 4 °C for 16 h at a stirring speed of 400 rpm. A certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to about 3-5 with acetic acid. 5 kDa molecular weight chitosan oligosaccharide (based on the molar amount of amino groups on the chitosan oligosaccharide sugar chain, the molar ratio of chitosan oligosaccharide to ursolic acid was 1:0.2) was added, and stirred at room temperature until no solid particles were visible to the naked eye. After the above two were fully mixed, an appropriate amount of ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain amphiphilic chitosan oligosaccharide nanomicelles (COU).

2) Preparation of curcumin/chitosan oligosaccharide nanomicelles (COU@CUR)

Take 1g COU and fully dissolve it in methanol, add curcumin at a mass ratio of COU to curcumin (CUR) of 10:3, reflux in the dark for 2h, then remove methanol by rotary evaporation, and redissolve it in ultrapure water to obtain COU@CUR.

3) Curcumin/chitosan oligosaccharide nanomicelles encapsulating Escherichia coli nissle1917 (EcN/COU@CUR)

EcN was grown in LB medium overnight at 37°C; the overnight culture was diluted into fresh medium and grown at 37°C for 4 h; the cells were collected by centrifugation at 4000 rpm for 5 min and resuspended in ice-cold phosphate buffer. The bacterial OD600 was measured by UV-VIS, and the bacterial suspension was diluted to OD600 = 0.5.

Under magnetic stirring (200r), 1 mL of COU@CUR (2 mg/mL) was added to 2 mL of bacterial suspension and stirred for 10 min to obtain EcN/COU@CUR through electrostatic self-assembly.

4) Preparation of therapeutic nanocoating modified single probiotic (EcN/COU@CUR/SA)

Under magnetic stirring (200 r), 200 μl of sodium alginate (2 mg/mL) was slowly added to EcN/COU@CUR and stirred for 10 min to obtain EcN/COU@CUR/SA.

Example 4

1) Synthesis of amphiphilic chitosan oligosaccharide micelles (COU)

Ursolic acid (0.913 g, 2 mmol), 1-hydroxybenzotriazole (0.27 g, 2 mmol), 1,3-diisopropylcarbodiimide (0.313 mL, 2 mmol) were dissolved in DMF and stirred at 4 °C for 16 h at a stirring speed of 400 rpm. A certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to about 3-5 with acetic acid. 5 kDa molecular weight chitosan oligosaccharide (based on the molar amount of amino groups on the chitosan oligosaccharide sugar chain, the molar ratio of chitosan oligosaccharide to ursolic acid was 1:0.8) was added, and stirred at room temperature until no solid particles were visible to the naked eye. After the above two were fully mixed, an appropriate amount of ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain amphiphilic chitosan oligosaccharide nanomicelles (COU).

2) Preparation of curcumin/chitosan oligosaccharide nanomicelles (COU@CUR)

Take 1g COU and fully dissolve it in methanol, add curcumin at a mass ratio of COU to curcumin (CUR) of 10:3, reflux in the dark for 1h, then remove methanol by rotary evaporation, and redissolve it in ultrapure water to obtain COU@CUR.

3) Curcumin/chitosan oligosaccharide nanomicelles encapsulating Bacteroides fragilis ZY-312 (BFZY-312/COU@CUR)

Bacteroides fragilis ZY-312 was grown in 95% TSB medium plus 5% fetal bovine serum under anaerobic conditions at 37°C overnight; the overnight culture was diluted into fresh medium and grown under anaerobic conditions at 37°C for 3.5 hours; the cells were collected by centrifugation at 4000 rpm for 5 minutes and resuspended in ice-cold phosphate buffer. The bacterial OD600 was measured by UV-VIS, and the bacterial suspension was diluted to OD600 = 0.8.

Under magnetic stirring (200r), 1.25mL COU@CUR (2mg/mL) was added to 2mL BFZY-312 bacterial suspension and stirred for 10min to obtain BFZY-312/COU@CUR through electrostatic self-assembly.

4) Preparation of therapeutic nanocoating modified single probiotic (BFZY-312/COU@CUR/SA)

Under magnetic stirring (200r), 125 μl of sodium alginate (2 mg/mL) was slowly added to BFZY-312/COU@CUR and stirred for 10 min to obtain BFZY-312/COU@CUR/SA.

Example 5

1) Synthesis of amphiphilic chitosan oligosaccharide micelles (COU)

Ursolic acid (0.913 g, 2 mmol), 1-hydroxybenzotriazole (0.27 g, 2 mmol), 1,3-diisopropylcarbodiimide (0.313 mL, 2 mmol) were dissolved in DMF and stirred at 4 °C for 16 h at a stirring speed of 400 rpm. A certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to about 3-5 with acetic acid. 5 kDa molecular weight chitosan oligosaccharide (based on the molar amount of amino groups on the chitosan oligosaccharide sugar chain, the molar ratio of chitosan oligosaccharide to ursolic acid was 1:0.3) was added, and stirred at room temperature until no solid particles were visible to the naked eye. After the above two were fully mixed, an appropriate amount of ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain amphiphilic chitosan oligosaccharide nanomicelles (COU).

2) Preparation of curcumin/chitosan oligosaccharide nanomicelles (COU@CUR)

Take 1g COU and fully dissolve it in methanol, add curcumin at a mass ratio of COU to curcumin (CUR) of 10:4, reflux in the dark for 0.5h, then remove methanol by rotary evaporation, and redissolve it in ultrapure water to obtain COU@CUR.

3) Curcumin/chitosan oligosaccharide nanomicelles encapsulating Bacteroides fragilis ZY-312 (BFZY-312/COU@CUR)

Bacteroides fragilis ZY-312 was grown in 95% TSB medium plus 5% fetal bovine serum under anaerobic conditions at 37°C overnight; the overnight culture was diluted into fresh medium and grown under anaerobic conditions at 37°C for 3.5 hours; the cells were collected by centrifugation at 4000 rpm for 5 minutes and resuspended in ice-cold phosphate buffer. The bacterial OD600 was measured by UV-VIS, and the bacterial suspension was diluted to OD600 = 0.4.

Under magnetic stirring (200r), 250μl COU@CUR (2mg/mL) was added to 2mL BFZY-312 bacterial suspension and stirred for 10min to obtain BFZY-312/COU@CUR through electrostatic self-assembly.

4) Preparation of therapeutic nanocoating modified single probiotic (BFZY-312/COU@CUR/HA)

Under magnetic stirring (200r), 100 μl of sodium hyaluronate (2 mg/mL) was slowly added to BFZY-312/COU@CUR and stirred for 10 min to obtain BFZY-312/COU@CUR/HA.

Example 6

1) Synthesis of amphiphilic chitosan oligosaccharide micelles (COU)

Ursolic acid (0.913 g, 2 mmol), 1-hydroxybenzotriazole (0.27 g, 2 mmol), 1,3-diisopropylcarbodiimide (0.313 mL, 2 mmol) were dissolved in DMF and stirred at 4 °C for 16 h at a stirring speed of 400 rpm. A certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to about 3-5 with acetic acid. 5 kDa molecular weight chitosan oligosaccharide (based on the molar amount of amino groups on the chitosan oligosaccharide sugar chain, the molar ratio of chitosan oligosaccharide to ursolic acid was 1:0.6) was added, and stirred at room temperature until no solid particles were visible to the naked eye. After the above two were fully mixed, an appropriate amount of ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain amphiphilic chitosan oligosaccharide nanomicelles (COU).

2) Preparation of curcumin/chitosan oligosaccharide nanomicelles (COU@CUR)

Take 1g COU and fully dissolve it in methanol, add curcumin at a mass ratio of COU to curcumin (CUR) of 10:2, reflux in the dark for 1.5h, then remove methanol by rotary evaporation, and redissolve it in ultrapure water to obtain COU@CUR.

3) Curcumin/chitosan oligosaccharide nanomicelles encapsulating Lactobacillus acidophilus (La/COU@CUR)

Lactobacillus acidophilus was grown in MRS medium under anaerobic conditions at 37°C overnight; the overnight culture was diluted into fresh medium and grown under anaerobic conditions at 37°C for 3.5 hours; the bacteria were collected by centrifugation at 4000 rpm for 5 minutes and resuspended in ice-cold phosphate buffer. The bacterial OD600 was measured by UV-VIS, and the bacterial suspension was diluted to OD600 = 0.7.

Under magnetic stirring (200r), 1.5mL COU@CUR (2mg/mL) was added to 2mL La bacterial suspension and stirred for 10min to obtain La/COU@CUR through electrostatic self-assembly.

4) Preparation of therapeutic nanocoating modified single probiotic (La/COU@CUR/SA)

Under magnetic stirring (200 r), 600 μl of sodium alginate (2 mg/mL) was slowly added to La/COU@CUR and stirred for 10 min to obtain La/COU@CUR/SA.

Example 7

1) Synthesis of amphiphilic chitosan oligosaccharide micelles (COU)

Ursolic acid (0.913 g, 2 mmol), 1-hydroxybenzotriazole (0.27 g, 2 mmol), 1,3-diisopropylcarbodiimide (0.313 mL, 2 mmol) were dissolved in DMF and stirred at 4 °C for 16 h at a stirring speed of 400 rpm. A certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to about 3-5 with acetic acid. 5 kDa molecular weight chitosan oligosaccharide (based on the molar amount of amino groups on the chitosan oligosaccharide sugar chain, the molar ratio of chitosan oligosaccharide to ursolic acid was 1:0.7) was added, and stirred at room temperature until no solid particles were visible to the naked eye. After the above two were fully mixed, an appropriate amount of ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain amphiphilic chitosan oligosaccharide nanomicelles (COU).

2) Preparation of curcumin/chitosan oligosaccharide nanomicelles (COU@CUR)

Take 1g COU and fully dissolve it in methanol, add curcumin at a mass ratio of COU to curcumin (CUR) of 10:1, reflux in the dark for 2h, then remove methanol by rotary evaporation, and redissolve it in ultrapure water to obtain COU@CUR.

3) Curcumin/chitosan oligosaccharide nanomicelles encapsulating Lactobacillus acidophilus (La/COU@CUR)

Lactobacillus acidophilus was grown in MRS medium under anaerobic conditions at 37°C overnight; the overnight culture was diluted into fresh medium and grown under anaerobic conditions at 37°C for 3.5 hours; the cells were collected by centrifugation at 4000 rpm for 5 minutes and resuspended in ice-cold phosphate buffer. The bacterial OD600 was measured by UV-VIS, and the bacterial suspension was diluted to OD600 = 0.8.

Under magnetic stirring (200r), 2 mL of COU@CUR (2 mg/mL) was added to 2 mL of La bacterial suspension and stirred for 10 min to obtain La/COU@CUR through electrostatic self-assembly.

4) Preparation of therapeutic nanocoating modified single probiotic (La/COU@CUR/HA)

Under magnetic stirring (200r), 400 μl of sodium hyaluronate (2 mg/mL) was slowly added to La/COU@CUR and stirred for 10 min to obtain La/COU@CUR/HA.

Embodiment 8 (taking embodiment 1 as an example)

Properties and characterization of single probiotic bacteria modified with therapeutic nanocoatings

1) Therapeutic nanocoating to modify the morphology and size of single probiotics

Figure 2 is a high-resolution transmission electron microscopy image of a single probiotic modified with a therapeutic nanocoating. The image shows that the surface of the probiotic presents a shell of about 200 nm thickness. The Zeta potential of the probiotic was detected by a dynamic light scattering instrument (DLS). Figure 3 is a graph showing the potential change of the probiotic before and after encapsulation. The EcN before encapsulation has a negative potential, the CEcN potential after encapsulating the curcumin/chitosan oligosaccharide nanomicelles is positive, and the ACEcN potential obtained after continuing to encapsulate the sodium alginate is negative, that is, as the materials are assembled layer by layer, the potential changes from negative to positive and then to negative, proving that the materials curcumin/chitosan oligosaccharide nanomicelles and sodium alginate are encapsulated on the surface of the probiotic, which is consistent with the high-resolution transmission electron microscopy image.

2) Effect of therapeutic nanocoating on probiotic activity

Escherichia coli nissle1917 before and after encapsulation was placed in liquid culture medium for two hours, and then coated on the plate after gradient dilution. Figure 4 shows the probiotic plate count. It can be seen that the number of probiotics in EcN, CEcN, and ACEcN increases successively. The results show that the therapeutic nanocoating can promote the growth of probiotics. By monitoring the OD600 values of probiotics at different time points before and after encapsulation, the probiotic growth curve was drawn, as shown in Figure 5. It was observed that the growth curve of the probiotics coated with the nanocoating was roughly consistent with the free probiotic curve, indicating that the nanocoating had no effect on the activity of the probiotics.

Embodiment 9 (taking embodiment 1 as an example)

Evaluation of the tolerance of probiotics in simulated gastrointestinal fluid

The survival of EcN was further evaluated in simulated gastric fluid (SGF, pH 1.2) supplemented with pepsin. EcN before and after encapsulation were placed in simulated gastric fluid and intestinal fluid, respectively, and samples were taken at different time points. After centrifugation to remove gastric fluid, the samples were resuspended in PBS and coated on the plate after gradient dilution. As shown in Figure 6, compared with unencapsulated probiotics, ACEcN showed significantly improved tolerance to simulated gastric fluid and simulated intestinal fluid.

According to reports that Escherichia coli nissle1917 and Lactobacillus acidophilus have therapeutic effects on ulcerative colitis, in order to evaluate whether nano-coated probiotics can be used as an enhanced therapy for ulcerative colitis, the role of nano-coated probiotics in the treatment of ulcerative colitis was studied.

Embodiment 10 (taking embodiment 1 as an example)

(1) In vivo therapeutic effect of single probiotics modified with therapeutic nanocoatings

In the ulcerative colitis treatment experiment, 6-8 week old C57BL/6J mice were selected to construct the ulcerative colitis model. The mice were randomly divided into 6 groups: Control group, DSS group, EcN group, EcN&CUR group, CEcN group and ACEcN group.

Dextran sulfate sodium (DSS) was used as a 3% (w/v) aqueous solution to give mice free drinking water for 7 days to induce an ulcerative colitis model. The treatment cycle was set to 13 days, and oral gavage treatment was started on the 6th day of DSS induction, and the drug was administered for 6 consecutive times.

During the entire treatment cycle, the mice were weighed every two days, the fecal status and blood in the stool were observed, and the disease activity index (DAI) was scored for each group of mice according to the scoring criteria (Table 1): DAI is one of the indicators for evaluating the degree of colitis inflammation. Figure 7 shows the changes in the disease activity index scores of each group of mice. As can be seen from the figure, the ACEcN group significantly reduced the disease activity index of the UC model compared with the EcN group, indicating that the chitosan oligosaccharide loaded with curcumin modified probiotics significantly improved the therapeutic effect. Compared with the EcN&CUR group, the ACEcN group significantly reduced the disease activity index of the UC model, indicating that chitosan oligosaccharide loaded with curcumin can improve the bioavailability of curcumin, reduce the level of ROS in the inflammatory area, and enable probiotics to better colonize in the intestine to play a role; in addition, the disease activity index score of the ACEcN group was significantly lower than that of the CEcN group, further indicating that SA can target the inflammatory area and prolong the retention time of probiotics in the body.

After the treatment, the mice were killed and dissected, and their colons were taken to measure their lengths and compared. Colon length is one of the important indicators for evaluating the degree of inflammation in ulcerative colitis. Figure 8 Comparison of colon lengths of mice in each group. It can be seen that the ACEcN group significantly increased the colon length of mice with ulcerative colitis compared with the EcN group and the EcN&CUR group, and improved the inflammation of the mice.

Table 1 DAI score table

(2) ACEcN colonization effect at the inflammatory site

After the mice were induced to obtain an ulcerative colitis model by DSS, they were orally gavaged with ACEcN-ICG (ICG is indocyanine green). After 6 hours, an inflammatory probe was injected and the mice were dissected after waiting for 5 minutes. The heart, liver, spleen, lung, kidney and colon tissues were taken and photographed by animal in vivo imaging (IVIS). The imaging results are shown in Figure 9, where a and b in Figure 9 are signal images of EcN-ICG and ACEcN-ICG, respectively, and c and d in Figure 9 are signal images of EcN and ACEcN inflammatory probe labeling, respectively. The signal of ACEcN-ICG overlaps with the inflammatory labeling area in a large area, indicating that ACEcN has efficient inflammation-responsive adhesion properties.

In the present invention, curcumin, probiotics and polyanionic polymers (such as sodium alginate) are loaded with amphiphilic chitosan oligosaccharide micelles as raw materials to explore and develop a therapeutic nano-coating modified single probiotic. Compared with the single probiotic delivery system, the combined curcumin delivery system can protect curcumin and probiotics during the process of being transported to the stomach after oral administration, until the structure dissociates and releases curcumin and probiotics due to the change of environmental pH in the intestinal environment, so that their biological activity can be exerted.

Compared with the healthy state, the environment under intestinal inflammation is more harsh. The nano coating intervened in the mouse ulcerative colitis model by co-loading curcumin and probiotics, and significantly improved the inflammatory condition of the mouse ulcerative colitis model, highlighting that it can achieve a synergistic therapeutic effect compared with a single probiotic delivery system, can improve the bioavailability of curcumin and the activity of probiotics, prolong its intestinal retention time, and has good biocompatibility. The present invention has huge market potential in the field of probiotic technology and has broad prospects for application and development in other new directions.

Claims (10)

1. A therapeutic nano-coating modified single probiotic, characterized in that it comprises a probiotic, wherein the surface of the probiotic is sequentially coated with curcumin/chitosan oligosaccharide nano-micelles and a polyanion polymer. 2. The therapeutic nano-coating modified single probiotic according to claim 1, characterized in that the probiotic is Escherichia coli nissle1917, Lactobacillus acidophilus or Bacteroides fragilis. 3. The therapeutic nano-coating modified single probiotic according to claim 1, characterized in that the polyanionic polymer is sodium alginate or sodium hyaluronate. 4. The method for preparing the therapeutic nano-coating modified single probiotic according to any one of claims 1 to 3, characterized in that it comprises the following steps: 1) reacting chitosan oligosaccharide with ursolic acid, 1-hydroxybenzotriazole and 1,3-diisopropylcarbodiimide to prepare amphiphilic chitosan oligosaccharide micelles; 2) mixing the amphiphilic chitosan oligosaccharide micelles and curcumin in an organic solvent, removing the organic solvent, and redissolving in water to obtain a curcumin/chitosan oligosaccharide nano-micelle solution; 3) adding the curcumin/chitosan oligosaccharide nano-micelle solution into the probiotic suspension under stirring, and obtaining a probiotic/curcumin/chitosan oligosaccharide solution through electrostatic self-assembly; 4) adding the polyanionic polymer solution to the probiotic/curcumin/chitosan oligosaccharide solution under stirring, and obtaining the therapeutic nano-coating modified single probiotic via electrostatic self-assembly. 5. The method for preparing a therapeutic nano-coating modified single probiotic according to claim 4 is characterized in that step 1) specifically comprises: dissolving ursolic acid, 1-hydroxybenzotriazole and 1,3-diisopropylcarbodiimide in DMF to obtain an activated ester solution; dissolving chitosan oligosaccharide in DMF and stirring and mixing with the activated ester solution, and obtaining amphiphilic chitosan oligosaccharide micelles after washing and rotary evaporation. 6. The method for preparing a therapeutic nano-coating modified single probiotic according to claim 4, characterized in that, in step 1), the molar ratio of chitosan oligosaccharide to ursolic acid is 1:(0.2-0.8) based on the molar amount of amino groups on the chitosan oligosaccharide chain. 7. The method for preparing a therapeutic nano-coating modified single probiotic according to claim 4, characterized in that in step 2), the mass ratio of the amphiphilic chitosan oligosaccharide micelles to curcumin is 10:(1-5). 8. The method for preparing a therapeutic nano-coating modified single probiotic according to claim 4, characterized in that, in step 3), the mass of curcumin/chitosan oligosaccharide nano-micelles added to every two milliliters of probiotic suspension is 0.5-4 mg, and the OD600 value of the probiotic suspension is 0.4-0.8. 9. The method for preparing a therapeutic nano-coating modified single probiotic according to claim 4, characterized in that in step 4), the mass ratio of the polyanionic polymer to the probiotic/curcumin/chitosan oligosaccharide is (0.1-0.5):1. 10. Use of the therapeutic nano-coating modified single probiotic according to any one of claims 1 to 3 in the preparation of a drug for treating ulcerative colitis.