Preparation method and application of gastrointestinal microenvironment responsive nano-coated probiotics

Abstract

The application provides a preparation method and application of a gastrointestinal microenvironment responsive nano-coated probiotic, so as to improve the bioavailability of the probiotic and realize the purpose of synergistically treating intestinal diseases by combining with a therapeutic drug. The gastrointestinal microenvironment responsive nano-coated probiotic is a seaweed salt which forms a coating on the surface of a single probiotic by chelating with calcium ions, and can also coat small molecule anti-inflammatory drugs to protect the probiotic from the gastric environment. When reaching the intestinal neutral environment, the coating is dissolved, and the inhibited probiotic and therapeutic drug are synchronously released. The probiotic inhibits the growth of pathogenic bacteria, maintains the intestinal flora, and maintains the balance of the intestinal flora; meanwhile, the anti-inflammatory effect of the drug is combined, so that the effect of synergistically treating intestinal diseases is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a method for preparing probiotics coated with a gastrointestinal microenvironment-responsive nano-coating and its application. Background Technology

[0002] The gut microbiota plays a crucial role in maintaining gut homeostasis and regulating human health. Current research has confirmed that gut microbiota dysbiosis is associated with a variety of diseases, including inflammatory bowel disease, obesity, diabetes, depression, and cancer. Bacteria continuously produce key biomolecules in the gut, which can reprogram the gut microbiota. Therefore, bacteria-based therapies are a promising approach to treating gut microbiota dysbiosis.

[0003] Fecal microbiota transplantation (FMT) has been considered a viable treatment for ulcerative colitis, but its clinical application is limited due to potential risks of pathogen infection and invasive procedures. Currently, genetically engineered bacteria have been used to enhance the tolerance of therapeutic microorganisms to harsh gastrointestinal conditions; however, there are potential safety concerns regarding the transfer of drug-resistant genes into healthy bacteria in the gut. Furthermore, oral probiotics, with their non-invasive nature and good patient compliance, have been used as a probiotic treatment for colitis. Probiotics can attach to intestinal epithelial cells, colonize the gut, and reconstruct the intestinal microenvironment. However, the bioavailability and therapeutic efficiency of probiotics need further improvement due to the harsh environment of the gastrointestinal tract (such as low pH, digestive enzymes, and bile acids). Therefore, maintaining the growth and proliferation capacity of oral probiotics is crucial.

[0004] Oral probiotic dosage forms, including liquids, capsules, and pills, are widely used to protect the viability of probiotics. However, these formulations are still insufficient to prevent gastrointestinal environmental damage to oral probiotics and improve their survival rate. Furthermore, in the clinical treatment of intestinal diseases, oral probiotics are often used in combination with other therapeutic drugs; however, taking multiple drugs in different dosage forms may reduce the efficiency of combined treatment due to different drug release pathways, and repeatedly taking multiple drugs in different dosage forms daily can also reduce patient compliance. Therefore, it is necessary to explore a strategy that can not only improve the bioavailability and therapeutic efficiency of oral probiotics, but also, ideally, avoid reducing patient compliance. Summary of the Invention

[0005] The purpose of this invention is to solve the following problems:

[0006] I. Current probiotic encapsulation strategies suffer from low probiotic bioavailability and unsatisfactory therapeutic effects;

[0007] Second, when probiotics are combined with drug therapy, it can reduce patient compliance.

[0008] Therefore, a method for preparing probiotics with a gastrointestinal microenvironment-responsive nano-coating and its application are provided.

[0009] The concept of this invention:

[0010] The research team of this invention conducted in-depth research into the shortcomings of existing technologies and believes that the main reasons for the above problems are the diversity of probiotic coating forms, coating states, and oral dosage forms when combined with drug therapy. To solve these problems, the research team of this invention believes that:

[0011] First, the coating form of probiotics is changed so that the outer coating layer can not only resist the strong acid and digestive enzymes in gastric juice, but also be successfully degraded in the intestine, restoring the biological activity of the coated probiotics, thereby improving bioavailability and therapeutic effect. Alginate (Alg), as a food additive extracted from brown algae polysaccharides, has good pH responsiveness and high biocompatibility. The research team intends to use it as a gastrointestinal microenvironment responsive and protective coating material in the technical solution of this invention.

[0012] Secondly, the coating state of probiotics should be changed to avoid coating multiple or a large number of bacteria in the same coating layer at the same time. This is to avoid the risk that even if they can resist the strong acid and digestive enzymes in gastric juice, the different environments of each probiotic in the same coating layer will prevent them from being able to carry out treatment synchronously when the release conditions are met, thus failing to reach the dosage required for treatment. There is still a risk of reduced bioavailability and treatment effect.

[0013] Finally, avoid diversity in oral dosage forms during combination therapy to ensure patient compliance and standardize the release pathways of probiotics and drugs, thereby reducing the impact of different release pathways on treatment efficiency.

[0014] Based on the above inventive concept, and to achieve the above-mentioned inventive objectives, the specific technical solution provided by this invention is as follows:

[0015] A method for preparing probiotics coated with a gastrointestinal microenvironment-responsive nano-coating, characterized by comprising the following steps:

[0016] 1) Cultivate probiotics to the logarithmic growth phase and wash them multiple times with a phosphate buffer solution with a pH of 7.2-7.4 (ensure thorough washing and no culture medium residue) to obtain probiotics;

[0017] 2) Resuspend the probiotics obtained in step 1) in a pre-cooled calcium chloride phosphate buffer solution and vortex; the pre-cooling here is mainly to prevent the temperature from becoming too high during subsequent vortexing, which would affect the state of the probiotics;

[0018] 3) Add alginate solution to the solution obtained in step 2) and vortex to obtain a probiotic suspension;

[0019] 4) Adjust the pH of the probiotic suspension obtained in step 3) to 3.0-4.0, and wash it multiple times with a phosphate buffer solution with a pH of 3.0-4.0. Centrifuge to obtain probiotics coated with a gastrointestinal microenvironment-responsive nano-coating. In this pH 3.0-4.0 environment, alginate and Ca... 2+ The formation of a gel allows for the encapsulation of individual probiotics.

[0020] Furthermore, in cases of combined drug therapy, in step 2), after resuspending the probiotics obtained in step 1) in a calcium chloride phosphate buffer solution pre-cooled at 2-6°C (preferably 4°C), the intestinal treatment drug can be added and vortexed.

[0021] Further, in step 2), the concentration of the calcium chloride phosphate buffer solution is 13.9 mM, the vortex rotation speed is 2000-3000 rpm, and the vortex time is 5-10 min.

[0022] Furthermore, in step 3), the concentration of the alginate solution is 20 mg / mL, the vortex rotation speed is 2000-3000 rpm, and the vortex time is 5-10 min; using this concentration of alginate solution can achieve the coating effect without affecting the normal growth and proliferation of probiotics.

[0023] Furthermore, in step 4), the pH of the probiotic suspension is adjusted using hydrochloric acid at a concentration of 0.1M.

[0024] Further, in step 3), the alginate is sodium alginate.

[0025] Furthermore, the probiotic is Escherichia coli Nissle 1917;

[0026] The therapeutic drug is 5-aminosalicylic acid.

[0027] Meanwhile, the present invention also provides a gastrointestinal microenvironment-responsive nano-coated probiotic, which is special in that: each probiotic cell is independently coated or simultaneously coated with several intestinal therapeutic drugs by the above method, that is, including a single probiotic cell including a nano-coated shell and the coated portion, or including a single probiotic cell including a nano-coated shell and the coated portion and several intestinal therapeutic drugs.

[0028] Furthermore, the application of the gastrointestinal microenvironment-responsive nano-coated probiotics prepared by the above method in the preparation of biomaterials for the prevention or treatment of intestinal diseases, such as the treatment of intestinal flora imbalance and colitis.

[0029] A biomaterial for the prevention or treatment of intestinal diseases, characterized in that: the active ingredient is a probiotic coated with a gastrointestinal microenvironment responsive nano-coating prepared by the above method.

[0030] The principle of this invention:

[0031] The surface of probiotics carries a negative charge. Upon the addition of calcium chloride, calcium ions bind to the surface of each individual probiotic cell through electrostatic interactions. The addition of sodium alginate further chelates the polysaccharide structure of the alginate with the calcium ions, forming a coating on the surface of each individual probiotic cell. In combination therapy, this coating can also simultaneously encapsulate several small-molecule therapeutic drugs, protecting both the probiotics and the therapeutic drugs in gastric acid. Upon reaching the neutral intestinal environment, the alginate coating dissolves, releasing the inhibited probiotics and therapeutic drugs. The probiotics inhibit the growth of pathogenic bacteria, maintaining the intestinal flora and preserving intestinal flora balance. Simultaneously, the anti-inflammatory effects of the drugs are combined to achieve a synergistic therapeutic effect on intestinal diseases. This invention is also suitable for encapsulating different clinical drugs and various microorganisms to enhance the treatment of intestinal diseases.

[0032] Advantages of this invention:

[0033] 1. The method of this invention can form a gastrointestinal microenvironment-responsive nano-coating on the surface of a single probiotic. When the release conditions are met, each probiotic is released synchronously, achieving synchronous and precise treatment, improving bioavailability and therapeutic effect.

[0034] 2. The method of the present invention can also utilize functional nano-coatings to simultaneously encapsulate individual probiotics and several drugs. When the release conditions are met, the probiotics and drugs are released simultaneously, i.e., through a unified release pathway, achieving synergistic therapeutic effects while ensuring patient compliance.

[0035] 3. This method is simple, safe, and widely applicable. Alginate is a food additive extracted from brown algae polysaccharides and has high biocompatibility. Furthermore, this method is suitable for encapsulating various clinical drugs and microorganisms, achieving a synergistic therapeutic effect on intestinal diseases by addressing gut microbiota imbalance. Attached Figure Description

[0036] Figure 1 Sodium alginate and Ca at different pH values ​​in Example 1 of this invention 2+ Images of the complex;

[0037] Figure 2 These are scanning electron microscope images of EcN and EcN@Alg in Embodiment 2 of the present invention;

[0038] Figure 3 The growth curves of EcN and EcN@Alg in acidic LB liquid medium (pH 4.0) in Example 2 of this invention are shown.

[0039] Figure 4 The growth curves of EcN and EcN@Alg in neutral LB liquid medium (pH 7.4) in Example 2 of this invention are shown.

[0040] Figure 5 The colony counts of EcN and EcN@Alg after incubation in simulated gastric fluid for different times in Example 3 of this invention;

[0041] Figure 6 The image shows a scanning electron microscope image of EcN and EcN@Alg after incubation in simulated gastric fluid for 0.5 h in Example 3 of the present invention.

[0042] Figure 7 The colony counts of EcN and EcN@Alg after incubation in simulated intestinal fluid for different times in Example 3 of this invention;

[0043] Figure 8 These are scanning electron microscope images of EcN@Alg after incubation in simulated intestinal fluid for different times in Example 3 of this invention;

[0044] Figure 9 A schematic diagram of gastrointestinal microenvironment-responsive alginate coating encapsulating EcN and 5-ASA;

[0045] Figure 10 This is a schematic diagram of the experimental design for the synergistic treatment of DSS-induced acute colitis in mice by EcN / 5-ASA@Alg in Example 5 of the present invention.

[0046] Figure 11 This is the body weight change curve of mice in Example 5 of the present invention from day 0 to day 9;

[0047] Figure 12 This refers to the disease activity index of mice in Example 5 of the present invention during days 0-9;

[0048] Figure 13 These are representative images of the colons of mice in different treatment groups in Example 5 of this invention;

[0049] Figure 14 The content of IL-6 in the mouse colon tissue in Example 5 of this invention;

[0050] Figure 15 The content of TNF-α in the mouse colon tissue in Example 5 of this invention;

[0051] Figure 16 The histological score of the mouse colon tissue in Example 5 of the present invention. Detailed Implementation

[0052] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments:

[0053] Example 1: pH sensitivity test of calcium alginate gel

[0054] 20 mg of sodium alginate was dissolved in 1 mL of ultrapure water, followed by the addition of 9 mL of 13.9 mM calcium chloride solution to obtain calcium alginate gel. The calcium alginate gel was centrifuged at 1000 × g for 5 min, washed twice with 0.1 M hydrochloric acid, and finally resuspended in 10 mL of 0.1 M hydrochloric acid. 0.1 M sodium hydroxide solution was added dropwise (20 μL), and the state of the calcium alginate gel was observed and the pH change was recorded. Figure 1 As shown, a gel is clearly formed under acidic conditions (pH 1.63), but the gel gradually dissolves when the pH reaches 5.27.

[0055] It is evident that calcium alginate can protect the encapsulated substances in an acidic environment through a gel coating, while it can dissolve in a neutral environment, releasing the encapsulated substances. It exhibits pH-responsive properties and therefore can be applied to the construction of gastrointestinal microenvironment responsive nanocoatings.

[0056] Example 2: Test on the effect of calcium alginate coating on the growth and proliferation of probiotics under different pH conditions

[0057] A single colony of *Escherichia coli* Nissle 1917 (EcN) was picked from a plate and inoculated into 4 mL of LB broth. The culture was incubated at 37°C with shaking at 220 rpm for 10 h. The bacterial culture was centrifuged at 4000 × g for 5 min and washed three times with phosphate-buffered saline (PBS, 10 mM, pH 7.2–7.4) to obtain the bacterial cells. The cells were resuspended 5 × 10⁻⁹ μL in 4°C pre-chilled PBS containing 13.9 mM calcium chloride. 8 CFU of bacteria was added and vortexed for 5 min (2800 rpm). Then, 100 μL of 20 mg / mL sodium alginate solution was added and vortexed for 5 min (2800 rpm). Next, the pH of the EcN suspension was adjusted to 3.0-4.0 with 0.1 M hydrochloric acid, and the bacteria were washed three times with PBS (10 mM, pH 3.0-4.0). After centrifugation, calcium alginate-coated EcN (i.e., EcN@Alg) was obtained.

[0058] EcN and EcN@Alg were dropped onto silicon wafers and allowed to air dry. Afterward, they were fixed with 4% paraformaldehyde for 4 hours and then dehydrated using a gradient of ethanol concentrations. After further air drying, the samples were sputter-coated with gold, and the morphology of the EcN and EcN@Alg was observed using scanning electron microscopy (SEM). Figure 2As shown, SEM results indicate that the EcN@Alg surface exhibits a mesh-like and wrinkled structure compared to the smooth surface of EcN, suggesting that a coating has formed on the surface of individual probiotics.

[0059] EcN and EcN@Alg were cultured in LB liquid medium at different pH values ​​at 37°C with shaking at 220 rpm, and the growth curves of EcN and EcN@Alg under different culture conditions were measured using a microplate reader. Figure 3 As shown, in acidic LB (pH 3.0-4.0), EcN entered the logarithmic growth phase after 0.5 h of culture and the stationary phase after 8 h. Because the surface of EcN is covered with a layer of calcium alginate, which restricts the physical space for material exchange and probiotic growth, no significant proliferation of EcN@Alg was observed within 10 h of culture. Figure 4 As shown, in neutral LB (pH 7.2-7.4), both EcN and EcN@Alg exhibited typical logarithmic growth and stationary phases. The results indicate that the probiotics are only temporarily inactivated after encapsulation with calcium alginate. In a neutral environment, EcN@Alg regains its activity as the coating dissolves. That is, the probiotics encapsulated in the nano-coating of this invention are temporarily inactivated in acidic culture media, but released and regain their activity in neutral culture media.

[0060] Example 3: In vitro evaluation of the protective effect of calcium alginate coating on probiotics in the gastrointestinal environment

[0061] EcN and EcN@Alg were cultured in simulated gastric fluid (SGF, pH 2.0) at 37°C with shaking at 220 rpm. After 0.5 h, 1 h, 1.5 h, and 2 h of culture, appropriate amounts of the bacterial suspension were serially diluted and plated. After incubation at 37°C for 12 h, plate counting was performed to determine the colony counts of EcN and EcN@Alg at different incubation times. Figure 5 As shown, a large number of EcN@Alg cells survived after 2 hours of incubation in SGF, but none of the EcN cells survived. These results indicate that EcN@Alg has a good protective effect within 2 hours. It takes approximately 2 hours for probiotics to pass through the stomach and enter the intestines; therefore, the coating can protect probiotics from the harmful effects of the gastric environment.

[0062] In addition, after SGF culture for 0.5 h, 1 h, 1.5 h, and 2 h, appropriate amounts of bacterial culture were taken, centrifuged at 5000 × g for 5 min to obtain bacterial cells, and washed three times with PBS of the corresponding pH. Then, EcN and EcN@Alg were dropped onto silicon wafers, allowed to air dry, fixed with 4% paraformaldehyde for 4 h, and dehydrated using a gradient of ethanol at different concentrations. After air drying, the samples were sputter-coated with gold, and the morphology of EcN and EcN@Alg after different SGF culture times was observed using SEM. Figure 6As shown, SEM results indicate that after incubation in SGF for 0.5 h, the surface of EcN showed obvious depressions and collapses, but the morphology of EcN@Alg did not change significantly.

[0063] EcN and EcN@Alg were cultured in simulated intestinal fluid (SIF, pH 6.8) at 37°C with shaking at 220 rpm. After 1 h, 2 h, 3 h, and 4 h of culture, appropriate amounts of the bacterial suspension were serially diluted and plated. After incubation at 37°C for 12 h, plate counting was performed to determine the colony counts of EcN and EcN@Alg at different incubation times. Figure 7 As shown, due to the protective effect of calcium alginate, the number of EcN@Alg was slightly lower than that of EcN during the first 1-2 hours of cultivation. However, after the coating dissolved at 3-4 hours of cultivation, the number of EcN@Alg was not significantly different from that of EcN.

[0064] In addition, after culturing in SIF for 1 h, 2 h, 3 h, and 4 h, appropriate amounts of bacterial culture were taken, centrifuged at 5000×g for 5 min to obtain bacterial cells, and washed three times with PBS of the corresponding pH. Then, EcN@Alg was dropped onto a silicon wafer, allowed to air dry, fixed with 4% paraformaldehyde for 4 h, and dehydrated using a gradient of ethanol at different concentrations. After air drying, the samples were sputter-coated with gold, and the morphology of EcN and EcN@Alg after different SIF culturing times was observed using SEM. Figure 8 As shown, SEM results indicate that the EcN@Alg surface was rough after 1 hour of incubation in SIF. However, after 4 hours of incubation, the EcN@Alg surface returned to a smooth state due to the dissolution of the calcium alginate coating.

[0065] It is evident that the coating method of the present invention, compared with the coating method of the prior art, can ensure the survival rate and activity of probiotics in the gastrointestinal environment after oral administration, can safely reach the intestine, and achieve synchronous release, thereby improving bioavailability and efficacy.

[0066] Example 4: Preparation of EcN / 5-ASA@Alg

[0067] Single colonies of EcN were picked from a plate and inoculated into 4 mL of LB liquid medium. The culture was incubated at 37°C with shaking at 220 rpm for 10 h. The bacterial culture was centrifuged at 4000 × g for 5 min and washed three times with PBS (10 mM, pH 7.2–7.4) to obtain the bacterial cells. The cells were resuspended 5 × 10⁻⁹ μL in PBS pre-chilled at 4°C containing 13.9 mM calcium chloride. 8CFU of bacteria were added, and mg of 5-aminosalicylic acid (5-ASA) was added, followed by vortexing for 5 min (2800 rpm). Then, 100 μL of 20 mg / mL sodium alginate solution was added, and the mixture was vortexed for 5 min (2800 rpm). Next, the pH of the EcN suspension was adjusted to 3.0-4.0 using 0.1 M hydrochloric acid, and the bacteria were washed three times with PBS (10 mM, pH 3.0-4.0). The mixture was then centrifuged to obtain EcN / 5-ASA@Alg. Figure 9 The diagram shows a gastrointestinal microenvironment-responsive calcium alginate coating encapsulating probiotics and 5-aminosalicylic acid. The method of this invention can independently encapsulate individual probiotic cells, and if combined with therapeutic drugs, it can simultaneously and uniformly encapsulate several drugs.

[0068] Example 5: EcN / 5-ASA@Alg synergistic treatment of DSS-induced acute colitis in mice

[0069] In this embodiment, a synergistic scheme was designed to examine the synergistic ability. Gastrointestinal microenvironment-responsive calcium alginate was simultaneously coated with probiotic EcN and the first-line drug 5-ASA with anti-inflammatory effects for the treatment of intestinal diseases.

[0070] Thirty female C57BL / 6 mice were randomly divided into six groups (n=5 per group): Control group, DSS (sodium dextran sulfate) group, EcN group, EcN@Alg group, 5-ASA group, and EcN / 5-ASA@Alg group. Figure 10 As shown, the treatments for each group of mice were as follows: (1) Control group: free access to deionized water; (2) DSS group: free access to deionized water containing 3.5% (w / v) DSS from day 0 to day 7; (3) EcN group: free access to deionized water containing 3.5% (w / v) DSS from day 0 to day 7, and oral administration of 10 mg / L of deionized water by gavage from day 3 to day 9. 9 CFU EcN / animal / day; (4) EcN@Alg group: Free drinking of deionized water containing 3.5% (w / v) DSS on days 0-7, and gavage of 10 oz on days 3-9. 9 CFUEcN@Alg / mouse / day; (5) 5-ASA group: Mice were allowed to drink deionized water containing 3.5% (w / v) DSS freely from day 0 to 7, and were given 1.6 mg 5-ASA / mouse / day by gavage from day 3 to 9; (6) EcN / 5-ASA@Alg group: Mice were given deionized water containing 3.5% (w / v) DSS freely from day 0 to 7, and were given 10 mg 5-ASA / mouse / day by gavage from day 3 to 9. 9 CFU EcN / 1.6mg 5-ASA@Alg / mouse / day. All mice were sacrificed on day 9.

[0071] Record the weight change, fecal viscosity, and fecal occult blood status for each mouse from day 0 to day 9. The Disease Activity Index (DAI) was calculated by combining the mouse weight loss index (0-4; 0: ≤1%; 1: 1-5%; 2: 5-10%; 3: 10-15%; 4: >15%), fecal viscosity (0-4; 0: normal; 1: soft but formed; 2: soft; 3: very soft; 4: watery diarrhea), and fecal occult blood status (0, 2, 4; 0: negative; 2: positive; 4: visible blood in stool). The DAI score was calculated as (weight loss score + fecal viscosity score + fecal occult blood score) / 3.

[0072] like Figure 11 As shown, mice in the DSS treatment group experienced a significant decrease in body weight from day 0 to day 9, indicating the successful establishment of the colitis animal model. No significant changes were observed in the EcN treatment group, while weight loss was inhibited in the EcN@Alg group, the 5-ASA group, and the EcN / 5-ASA@Alg group.

[0073] like Figure 12 As shown, the DAI score of mice in the DSS treatment group increased significantly from day 0 to day 9, further demonstrating the successful establishment of the colitis animal model. No significant changes were observed in the EcN treatment group, suggesting that probiotic treatment alone has limited efficacy. In the EcN@Alg group, 5-ASA group, and EcN / 5-ASA@Alg group, the DAI score significantly decreased due to the restoration of the gastrointestinal microenvironment caused by probiotic and drug treatment. Furthermore, on day 9, the DAI score of mice in the EcN / 5-ASA@Alg group was significantly lower than that in the EcN@Alg group and the 5-ASA group, indicating that EcN / 5-ASA@Alg has a synergistic therapeutic effect on colitis.

[0074] After each mouse was sacrificed, the length of its colon was measured. Figure 13 As shown, the colon length of mice in the DSS treatment group was significantly shortened. Under the treatment of probiotics and drugs, the EcN / 5-ASA@Alg group had the longest colon length compared with the EcN, EcN@Alg, and 5-ASA groups.

[0075] A segment of colon was collected and homogenized (10% w / v) in pre-cooled PBS (10.0 mM, pH 7.2-7.4) using an electric homogenizer. The colon homogenate was centrifuged at 5000 × g for 10 min at 4°C. The supernatant was diluted 2-fold and used to determine the levels of interleukin (IL-6) and tumor necrosis factor (TNF-α) in the colon tissue using an enzyme-linked immunosorbent assay (ELISA) kit. The levels of IL-6 and TNF-α in the same weight of colon from each group were analyzed. Figure 14 and Figure 15As shown, compared with the Control group, the expression levels of IL-6 and TNF-α in the DSS group increased by 5.22±2.11 times and 12.26±2.24 times, respectively. After treatment with EcN, EcN@Alg, 5-ASA, and EcN / 5-ASA@Alg, the levels of IL-6 and TNF-α significantly decreased. Furthermore, the IL-6 level in the EcN / 5-ASA@Alg treatment group was significantly lower than that in the 5-ASA group, suggesting that EcN / 5-ASA@Alg treatment is more effective than clinical drugs.

[0076] Colon, heart, liver, spleen, lung, and kidney of each mouse were fixed in 4% paraformaldehyde for 48 h and then embedded in paraffin. The tissues were sectioned into 5 μm sections using a paraffin microtome and stained with hematoxylin and eosin (H&E). The colonic histological scoring system combines the severity of inflammation (0-4 grades; 0: normal; 1: mild inflammation; 2: moderate inflammation; 3: severe inflammation; 4: extremely severe inflammation), lesion depth (0-3 grades; 0: normal; 1: submucosa; 2: muscularis propria; 3: serosa), degree of crypt damage (0-4 grades; 0: normal; 1: 1 / 3 crypt damage; 2: 2 / 3 crypt damage; 3: complete crypt damage, intact epithelial cells; 4: complete crypt damage, destroyed epithelial cells), and lesion area (0-4 grades; 0: ≤1%; 1: 1-25%; 2: 25-50%; 3: 50-75%; 4: >75%) to score the colon histologically. Histological score = (inflammation score + lesion depth score + crypt damage score) × lesion area score. Figure 16 As shown, compared with the control group mice, the colonic histological score of the DSS-treated group mice was significantly increased. After EcN treatment, the histological score did not change significantly, further demonstrating that the effect of using probiotics alone to treat colitis is limited. In the EcN@Alg group, the 5-ASA group, and the EcN / 5-ASA@Alg group, the histological scores were significantly reduced. Furthermore, the histological score of the EcN / 5-ASA@Alg group was significantly lower than that of the EcN@Alg group and the 5-ASA group, further demonstrating that EcN / 5-ASA@Alg has a synergistic therapeutic effect on colitis.

[0077] Therefore, it is evident that the gastrointestinal microenvironment-responsive nanocoating provided in this application significantly improves the bioavailability of oral probiotic preparations that simultaneously encapsulate individual probiotic cells and other therapeutic drugs, which is of great significance.

[0078] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the scope of the technology disclosed in the present invention, and such modifications or substitutions should all be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a gastrointestinal microenvironment-responsive nanocoating- coated probiotic, characterized in that, The method comprises the following steps: 1) culturing probiotics to logarithmic growth phase, and washing the probiotics multiple times with phosphate buffer solution with pH of 7.2-7.4; 2) resuspending the probiotics obtained in step 1) with pre-cooled calcium chloride phosphate buffer solution and vortexing; 3) adding alginate solution to the solution obtained in step 2) and vortexing to obtain a probiotic suspension; 4) adjusting the pH of the probiotic suspension obtained in step 3) to 3.0-4.0, washing multiple times with phosphate buffer solution with pH of 3.0-4.0, and centrifuging to obtain gastrointestinal microenvironment-responsive nanocoating coated probiotics.

2. The method for preparing gastrointestinal microenvironment-responsive nanocoating coated probiotics according to claim 1, wherein in step 2), after resuspending the probiotics obtained in step 1) with pre-cooled calcium chloride phosphate buffer solution, an intestinal therapeutic drug is added and vortexed.

3. The method for preparing gastrointestinal microenvironment-responsive nanocoating coated probiotics according to claim 1 or 2, wherein in step 2), the concentration of calcium chloride in the phosphate buffer solution is 13.9 mM, the vortexing rotation speed is 2000-3000 rpm, and the vortexing time is 5-10 min.

4. The method for preparing gastrointestinal microenvironment-responsive nanocoating coated probiotics according to claim 3, wherein in step 3), the concentration of alginate solution is 20 mg / mL, the vortexing rotation speed is 2000-3000 rpm, and the vortexing time is 5-10 min.

5. The method for preparing gastrointestinal microenvironment-responsive nanocoating coated probiotics according to claim 4, wherein in step 4), hydrochloric acid is used to adjust the pH of the probiotic suspension.

6. The method for preparing gastrointestinal microenvironment-responsive nanocoating coated probiotics according to claim 5, wherein in step 3), the alginate is sodium alginate.

7. The method for preparing gastrointestinal microenvironment-responsive nanocoating coated probiotics according to claim 2, wherein the Escherichia coli is Escherichia coli Nissle 1917.

8. The method for preparing gastrointestinal microenvironment-responsive nanocoating coated probiotics according to claim 2, wherein the intestinal therapeutic drug is 5-aminosalicylic acid. The probiotics obtained by the method of any one of claims 1-7 are independently coated or simultaneously coated with several intestinal therapeutic drugs.

9. Use of the gastrointestinal microenvironment-responsive nanocoating coated probiotics prepared by the method of any one of claims 1-7 in the preparation of biomaterials for preventing or treating intestinal diseases. The effective component is the gastrointestinal microenvironment-responsive nanocoating coated probiotics prepared by the method of any one of claims 1-7. ​ The probiotic is ​ Nissle 1917; ​ 8. A probiotic coated with a gastrointestinal microenvironment-responsive nanocoating, characterized in that: ​ ​ 10. A biomaterial for preventing or treating intestinal diseases, characterized by: ​

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