Envelope type active probiotic biological regulator for treating Crohn's disease and preparation method of envelope type active probiotic biological regulator

Abstract

The invention discloses an envelope type active probiotic biological regulator for treating Crohn's disease and a preparation method of the envelope type active probiotic biological regulator. Iron ions and natural antioxidants are used for coordinating and encapsulating an inhibitor GW4869 synthesized / released by exosome, so that a nano regulator with positive electricity on the surface is formed; the active probiotics are combined with bifidobacterium adolescentis with electronegativity on the surface through electrostatic interaction, and the active probiotics biological regulator is obtained. The surface of the active probiotic biological regulator is wrapped with the synthesized ROS-responsive intestinal tissue adhesion enhancing coating, and the envelope type active probiotic biological regulator is obtained; after oral administration, the envelope-type active probiotic biological regulator responds to and removes ROS (reactive oxygen species), enhances the adhesion performance of the envelope-type active probiotic biological regulator in intestinal inflammation parts, continuously releases natural antioxidants, and further removes ROS; meanwhile, an exosome synthesis inhibitor GW4869 is released, so that synthesis and release of the exosome carrying dsDNA are effectively inhibited, and generation and release of inflammatory factors are blocked.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine and pharmaceuticals, and in particular to an envelope-type active probiotic bioregulator for treating Crohn's disease and a preparation method thereof. Background Art

[0002] Inflammatory bowel disease includes ulcerative colitis and Crohn's disease. Among them, Crohn's disease is a chronic transmural, recurrent inflammatory bowel disease that is closely related to an increased risk of colorectal cancer. With the significant increase in the incidence of inflammatory bowel disease worldwide, Crohn's disease has become a major disease problem that cannot be ignored and seriously affects global human health. Because its pathogenesis is not fully understood and it is still incurable, it often accompanies patients throughout their lives, which not only seriously affects the quality of life of patients, but also brings huge economic burdens to patients. Therefore, it will be of great significance to develop an effective drug preparation to treat Crohn's disease.

[0003] The cGAS-STING signaling pathway is the main effector for sensing exogenous danger signals from viruses and bacteria as well as endogenous signals, such as abnormal double-stranded DNA (dsDNA) in the cytoplasm. It is one of the basic mechanisms of natural defense of the host and plays an extremely important role in mediating anti-tumor immune response. However, dsDNA, including mitochondrial DNA (mtDNA) and nuclear genomic DNA (nDNA), plays an important role in the development of Crohn's disease as important damage-associated molecular patterns (DAMPs) in the abnormal intestinal microenvironment. Current studies have shown that damaged intestinal epithelial cells in inflammatory bowel disease can lead to mitochondrial damage and cell apoptosis, promoting the release of dsDNA. These released dsDNAs are secreted in the intestinal microenvironment along with exosomes and then taken up by macrophages, which in turn activate the cGAS-STING signaling pathway, triggering an inflammatory response and releasing a series of inflammatory cytokines, such as IL-6, TNF-α and IFN-β, which further damage intestinal epithelial cells and aggravate intestinal damage and deterioration. In addition, excessive ROS in the intestinal tract of inflammatory bowel disease can cause intestinal endothelial cell damage by inducing lipid peroxidation and DNA mutation, damaging protein function, changing epithelial permeability, and destroying the intestinal epithelial barrier, aggravating the secretion and release of intestinal epithelial cell exosomes carrying dsDNA, further leading to the occurrence or deterioration of inflammatory bowel disease. Therefore, it is extremely important and necessary to effectively remove high levels of ROS in inflammatory sites and greatly inhibit the secretion of exosomes (sEVs) carrying dsDNA, blocking the abnormal activation of the cGAS-STING signaling pathway in macrophages and the massive release of inflammatory factors.

[0004] In addition, excessive ROS can also lead to dysregulated pro-inflammatory response species-sensitive pathways in immune cells and induce dysbiosis of the intestinal microbiota. In a healthy intestinal microenvironment, the microbiota provides the host with essential vitamins and short-chain fatty acids, and also protects the host from colonization and invasion of pathogens. In contrast, disordered intestinal flora can induce a chronic inflammatory state, increase the production of toxins, and disrupt the host's metabolism, thereby accelerating the occurrence and development of Crohn's disease. Probiotic therapy is widely used to regulate intestinal flora and improve colitis symptoms. However, the adverse environment of the intestinal site limits the survival and retention time of probiotics in the intestine, resulting in a decrease in the therapeutic effect. Therefore, improving the residence time of probiotics in the intestine is a necessary measure to improve the therapeutic effect of probiotics regulating flora. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for preparing an envelope-type active probiotic bioregulator for treating Crohn's disease. The present invention first uses iron ions to coordinate with natural antioxidants (curcumin and quercetin) while encapsulating the inhibitor of exosome synthesis / release GW4869 to form a nano-regulator (GFe) with a positive surface charge; and then combines it with Bifidobacterium adolescentis (Bifidobacterium adolescentis, referred to as BA) was combined through electrostatic action to obtain an active probiotic bioregulator (BA@GFe); then the synthesized ROS-responsiveness enhancing intestinal tissue adhesion coating was wrapped on the surface of the active probiotic bioregulator to obtain an envelope-type active probiotic bioregulator (BA@GFe@HDE); after oral administration, the envelope-type active probiotic bioregulator (BA@GFe@HDE) first responded to the scavenging of ROS and adhered to the inflammatory site, and continuously released natural antioxidants, curcumin and quercetin, to further scavenged ROS, and at the same time released the inhibitor of exosome synthesis / release GW4869, thereby effectively inhibiting the synthesis and release of exosomes carrying dsDNA, blocking the release of inflammatory factors, and synergizing with probiotics to effectively regulate the intestinal flora and restore the flora homeostasis, thereby accurately treating Crohn's disease.

[0006] In order to achieve the above-mentioned purpose, the specific technical scheme of the present invention is:

[0007] The present invention provides a method for preparing an envelope-type active probiotic bioregulator for treating Crohn's disease, comprising the following steps:

[0008] (1) Natural antioxidants are coordinated with trivalent iron ions and loaded with neutral sphingomyelinase inhibitors synthesized / released by exosomes to obtain nano-modulators with positive surface charge;

[0009] (2) combining the nano-regulator with positive surface charge and the active probiotics with negative surface charge through electrostatic action to obtain an active probiotics bioregulator;

[0010] (3) Phenylboronic acid and hyaluronic acid (HA) were modified on dopamine to obtain a coating with ROS-responsive adhesion to intestinal tissue;

[0011] (4) Wrapping the ROS-responsive intestinal tissue-adherent coating on the surface of the active probiotic bioregulator to obtain the envelope-type active probiotic bioregulator.

[0012] Specifically, in step (1), the natural antioxidants are curcumin and quercetin;

[0013] The specific steps of the preparation method of the surface positively charged nano-regulator are as follows:

[0014] (1.1) dissolving ferric chloride, neutral sphingomyelinase inhibitor GW4869, bis(2-ethylhexyl) sebacate and polyoxyethylene polyoxypropylene ether block copolymer in an organic solvent, and obtaining a uniform solution A after ultrasonic treatment;

[0015] (1.2) curcumin, quercetin, neutral sphingomyelinase inhibitor GW4869, bis(2-ethylhexyl) sebacate and polyoxyethylene polyoxypropylene ether block copolymer are dissolved in an organic solvent, and a uniform solution B is obtained after ultrasonic treatment;

[0016] (1.3) The solution A obtained in step (a) and the solution B obtained in step (b) are simultaneously dropped into water containing polyacrylamine hydrochloride under ultrasonic conditions and stirred to obtain the surface positively charged nano-regulator.

[0017] Preferably, in terms of g, mg and mL, in step (1.1), the amount of ferric chloride is 12.8-24.4 mg, the amount of neutral sphingomyelinase inhibitor GW4869 is 2.4-4.8 mg, the amount of bis(2-ethylhexyl) sebacate is 3.2-6.8 mg, the amount of polyoxyethylene polyoxypropylene ether block copolymer is 9.6-16.8 mg, the volume of the organic solvent (preferably methanol) is 2-4 mL, and the ultrasonic treatment time is 1-3 min;

[0018] In step (1.2), the amount of curcumin is 6.4-12.2 mg, the amount of quercetin is 3.2-6.1 mg, the amount of neutral sphingomyelinase inhibitor GW4869 is 2.4-4.8 mg, the amount of bis(2-ethylhexyl) sebacate is 3.2-6.8 mg, the amount of polyoxyethylene polyoxypropylene ether block copolymer is 9.6-16.8 mg, the volume of the organic solvent (preferably methanol) is 2-4 mL, and the ultrasonic treatment time is 2-5 min;

[0019] In step (1.3), the concentration of the aqueous solution of polyacrylamine hydrochloride is 0.6-1.2 wt %, and the volume ratio of solution A, solution B and water is 1:1:20.

[0020] The dripping time of the solution A and the solution B is 1-3 minutes.

[0021] The ultrasonic treatment time is 1-3 min.

[0022] The stirring time is 12-24h.

[0023] Specifically, in step (2), the active probiotic with negative surface charge is Bifidobacterium adolescentis;

[0024] The preparation method of the active probiotic bioregulator is specifically as follows:

[0025] (2.1) dispersing Bifidobacterium adolescentis in a mixed solution of PBS and NaCl to obtain a solution containing Bifidobacterium adolescentis with negative surface charge;

[0026] (2.2) Ultrasonic dispersion of the surface positively charged nano-regulator in a PBS solution, followed by addition of the surface negatively charged Bifidobacterium adolescentis solution obtained in step (2.1), mixing, centrifugation and washing to obtain the active probiotic bioregulator.

[0027] More specifically, in step (2.1), the volume ratio of PBS to NaCl in the mixed solution is 8 mL: (0.6-1.2) mL, the mass fraction of the NaCl solution is 0.89% to 0.91%, and the bacterial colony count of Bifidobacterium adolescentis is 10 9 CFU / mL;

[0028] In step (2.2), the mass volume ratio of the surface positively charged nano-regulator, the PBS solution and the surface negatively charged Bifidobacterium adolescentis solution is (5-10) mg: 6 mL: (0.6-1.8) mL.

[0029] Specifically, in step (3), the specific steps are:

[0030] (3.1) Under alkaline conditions, dopamine reacts with di-tert-butyl dicarbonate to form an amino compound protected by tert-butyloxycarbonyl, denoted as Boc-DOPA;

[0031] (3.2) Under alkaline conditions, Boc-DOPA reacts with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl bromide to produce the product Boc-DOPA-EN;

[0032] (3.3) Boc-DOPA-EN undergoes a deprotection reaction to remove the tert-butyloxycarbonyl group to generate the product DOPA-EN;

[0033] (3.4) The carboxyl group of hyaluronic acid is first subjected to a carboxyl activation reaction with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride to form an active ester intermediate; the active ester intermediate undergoes an amidation reaction with DOPA-EN to obtain a coating HA-DOPA-EN that has ROS responsiveness and adhesion to intestinal tissue.

[0034] More specifically, in step (3.1), the molar ratio of dopamine to di-tert-butyl dicarbonate is 3-6:7-15;

[0035] In step (3.2), the molar ratio of Boc-DOPA to 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)benzyl bromide is 4-8:8-16;

[0036] In step (3.3), Boc-DOPA-EN undergoes a deprotection reaction in a mixture of dichloromethane and trifluoroacetic acid, wherein the volume ratio of dichloromethane to trifluoroacetic acid is 0.8-1:2.4-3;

[0037] In step (3.4), the mass ratio of hyaluronic acid to DOPA-EN is 80-160:180-260.

[0038] That is, phenylboronic acid and hyaluronic acid are modified on dopamine to obtain a coating (HDE) with ROS responsiveness and adhesion to intestinal tissue, specifically comprising:

[0039] 1) Synthesis of Boc-DOPA: 3-6 mM dopamine (DOPA) was dissolved in 15-30 mL methanol, and 8-12 mM triethylamine was added to the solution at 0°C, and then 7-15 mM di-tert-butyl dicarbonate [(Boc)2O] was added dropwise to the solution, and the above reactions all took place in a nitrogen environment; then the mixture was stirred at room temperature for 2-6 h, and then the methanol was removed by rotary evaporation, and then the obtained solid was dissolved in chloroform and washed with water for 3-5 times, and dried over anhydrous MgSO4 to obtain the final white solid product (Boc-DOPA);

[0040] 2) Synthesis of Boc-DOPA-EN: 4-8 mM of the Boc-DOPA synthesized in 1) above was dissolved in 12-24 mL of anhydrous dimethylformamide (DMF) and cooled to 0°C, then 8-16 mM K2CO3 was added to the above solution and stirred for 10 min; then 3-6 mL of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl bromide (EN, 8-16 mM) dissolved in DMF was added to the above mixed solution; the mixed solution was stirred at room temperature for 24-36 h, and DMF was removed by rotary evaporation, then the solid was dissolved in ethyl acetate, washed 3 times with saturated NH4C1, washed 3 times with brine, and then dried over anhydrous MgSO4 to obtain a crude product which was further purified by silica gel chromatography;

[0041] 3) DOPA-EN synthesis: Boc-DOPA-EN (0.5-10 mM) synthesized in 2) above was dissolved in 12-24 mL of dichloromethane / trifluoroacetic acid (DCM / TFA), the mixed solution was stirred at room temperature for 2-6 h, and then the solvent was completely removed by rotary evaporation to obtain the product DOPA-EN; the volume ratio of DCM to TFA was (0.8-1): (2.4-3);

[0042] 4) Synthesis of HA-DOPA-EN: 80-160 mg HA and 17-56 mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were added to 10-20 mL DMSO and stirred at room temperature for 0.5-2 h; then 17-56 mg N-hydroxysuccinimide (NHS, a co-catalyst to promote the formation of amide bonds) and 180-260 mg DOPA-EN were added to the above solution and stirred at room temperature overnight under nitrogen protection; then the solution was gently added to 20-40 mL ultrapure water, and dialyzed with water / acetonitrile for 1-2 days, with a volume ratio of water to acetonitrile of 1:1; then dialyzed with ultrapure water for 2-3 days, and finally freeze-dried to obtain the ROS-responsive coating HA-DOPA-EN (HDE).

[0043] Specifically, in step (4), the step of wrapping the ROS-responsive intestinal tissue-adherent coating on the surface of the active probiotic bioregulator is as follows:

[0044] ultrasonically dispersing the ROS-responsive intestinal tissue-adherent coating in a PBS solution and a NaCl solution, and then adding the active probiotic bioregulator;

[0045] The mass volume ratio of the active probiotic bioregulator, PBS solution, NaCl solution and ROS responsive coating is 1.6-3.2 mL: 5 mL: 1 mL: 1.2-2.4 mg, and the mass fraction of the NaCl solution is 0.89%-0.91%.

[0046] The invention also provides an envelope-type active probiotic bioregulator prepared by the preparation method.

[0047] The present invention also provides the use of the envelope-type active probiotic bioregulator in the preparation of a drug for treating Crohn's disease.

[0048] Beneficial effects of the present invention:

[0049] 1. The present invention provides an envelope-type active probiotic bioregulator, whose surface is coated with a ROS-responsive adhesion coating HDE, which greatly protects the probiotics from external damage. At the same time, it can achieve ROS response removal, and expose the catechol group to the outside to enhance the performance of the active probiotic bioregulator to accurately adhere to the fixed part of the intestine, that is, it realizes the design characteristics of "ROS response removal envelope opening and enhanced adhesion performance"; it overcomes the shortcomings of traditional drug treatment mode, such as single, poor accuracy, and fast metabolism, improves the precise controlled release and long-term action time of natural antioxidants, and greatly improves the treatment efficiency of Crohn's disease;

[0050] 2. The present invention provides an envelope-type active probiotic bioregulator. After responding to ROS to achieve precise adhesion to the inflammatory site, the nanoformulation on the surface of the probiotic continuously releases natural antioxidants (curcumin and quercetin) and exosome synthesis / release inhibitor GW4869; the precise release of natural antioxidants and GW4869 at fixed sites further eliminates the excessive ROS level at the inflammatory site, and greatly avoids the secretion of inflammatory factors mediated by the activation of the cGAS-STING signaling pathway in macrophages by inhibiting the synthesis and secretion of exosomes carrying dsDNA; the therapeutic strategy of "clearing ROS-inhibiting the secretion and release of damaged dsDNA-reducing inflammatory factors" is realized when the ROS response at the inflammatory site is turned on, resulting in a highly efficient, three-dimensional, and cyclical therapeutic effect;

[0051] 3. The present invention provides an envelope-type active probiotic bioregulator, the protective layer on the surface of which greatly ensures the activity of probiotics, realizes efficient intestinal flora regulation, and enriches the types of intestinal flora;

[0052] 4. The envelope-type active probiotic bioregulator provided by the present invention realizes a multi-level, three-dimensional, all-round treatment mode of precise adhesion to the inflammatory site, precise and long-term release of the coated drug, targeted removal of ROS, effective reduction of inflammatory factors, and restoration of intestinal flora balance;

[0053] 5. The present invention provides an envelope-type active probiotic bioregulator, in which iron ions can not only be used to coordinate with natural antioxidants to encapsulate therapeutic drugs, but also achieve the effect of long-term iron supplementation, thereby alleviating the complication caused by inflammatory bowel disease - iron deficiency anemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is a TEM image of the surface positively charged nano-regulator GFe in Example 1;

[0055] Figure 2 This is the particle size distribution diagram of the nano-regulator GFe in Example 1;

[0056] Figure 3 is the surface potential of GFe, BA, BA@GFe and BA@GFe@HDE in Example 1;

[0057] Figure 4 TEM images of BA (left) and BA@GFe@HDE (right) in Example 1;

[0058] Figure 5 Activity evaluation of probiotics in BA and BA@GFe@HDE in Example 1;

[0059] Figure 6 The present invention is a preparation process of the envelope-type active probiotic bioregulator and a schematic diagram of the principle of treating Crohn's disease. DETAILED DESCRIPTION

[0060] The present invention will be further described below in conjunction with the embodiments.

[0061] Example 1

[0062] A method for preparing an envelope-type active probiotic bioregulator for treating Crohn's disease, such as Figure 6 As shown, the following steps are included:

[0063] (1) Combine natural antioxidants with iron ions (Fe 3+ ) Coordinate loading of the exosome synthesis / release inhibitor GW4869 to obtain a surface positively charged nano-modulator (GFe);

[0064] The nano-regulator (GFe) is synthesized by ultrasonic rate-controlled titration method, which specifically includes:

[0065] 1) 12.8 mg of ferric chloride (FeCl3), 2.4 mg of GW4869, 3.2 mg of bis(2-ethylhexyl) sebacate (DOS) and 9.6 mg of polyoxyethylene polyoxypropylene ether block copolymer (F-127) were simultaneously dissolved in 2 mL of methanol and ultrasonicated for 1 min to fully dissolve them to form a uniform solution A;

[0066] 2) 6.4 mg of curcumin, 3.2 mg of quercetin, 2.4 mg of GW4869, 3.2 mg of bis(2-ethylhexyl) sebacate (DOS) and 9.6 mg of polyoxyethylene polyoxypropylene ether block copolymer (F-127) were dissolved in 2 mL of methanol and ultrasonicated for 2 min to fully dissolve them to form a uniform solution B;

[0067] 3) solution A of step 1) and solution B of step 2) are simultaneously dropped into deionized water containing 0.8 wt % polyacrylamine hydrochloride (PAH) under ultrasound, and then the solution is stirred to form a nano-conditioner;

[0068] The volume ratio of solution A, solution B and deionized water is 1:1:20; the dripping time of solution A and solution B is 2 min; the ultrasonic (60W) treatment time is 2 min; the stirring time is 12 h;

[0069] (2) The surface positively charged nano-regulator (GFe) is combined with the surface negatively charged Bifidobacterium adolescentis through electrostatic interaction to obtain an active probiotic bioregulator (BA@GFe);

[0070] Specifically include:

[0071] 1) Dispersing Bifidobacterium adolescentis (ATCC 15703) in a mixed solution of PBS and NaCl to obtain a solution of Bifidobacterium adolescentis with negative surface charge; the volume ratio of PBS to NaCl in the mixed solution is 8 mL:0.8 mL, the mass fraction of the NaCl solution is 0.9%, and the number of bacterial colonies is 10 9 CFU / mL;

[0072] 2) The surface positively charged nano-regulator (GFe) was ultrasonically dispersed (60W) in a PBS solution, and then a surface negatively charged Bifidobacterium adolescentis solution was added, mixed at a vortex speed of 100 rpm, centrifuged at a centrifugal speed of 300 rpm, and washed with PBS for 3 times to obtain a probiotic bioregulator (BA@GFe);

[0073] The mass volume ratio of the surface positively charged nano-regulator (GFe), PBS solution and surface negatively charged Bifidobacterium adolescentis solution is: 8 mg: 6 mL: 1.2 mL;

[0074] (3) Phenylboronic acid and hyaluronic acid were modified on dopamine to obtain a coating (HDE) with ROS-responsive adhesion to intestinal tissue;

[0075] Specifically include:

[0076] 1) Synthesis of Boc-DOPA: 3 mM dopamine (DOPA) was dissolved in 15 mL methanol, and 8 mM triethylamine was added to the solution at 0°C, followed by dropwise addition of 7 mM di-tert-butyl dicarbonate [(Boc)2O] to the solution, all of which occurred in a nitrogen environment; the mixture was then stirred at room temperature for 2 h, and then the methanol was removed by rotary evaporation, and the resulting solid was then dissolved in chloroform and washed with water three times, and dried over anhydrous MgSO4 to obtain the final white solid product (Boc-DOPA);

[0077] 2) Synthesis of Boc-DOPA-EN: 4 mM of the Boc-DOPA synthesized in 1) above was dissolved in 12 mL of anhydrous dimethylformamide (DMF) and cooled to 0°C, then 8 mM K2CO3 was added to the above solution and stirred for 10 min; then 3 mL of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl bromide (EN, 8 mM) dissolved in DMF was added to the above mixed solution; the mixed solution was stirred at room temperature for 24 h, and DMF was removed by rotary evaporation, then the solid was dissolved in ethyl acetate, washed 3 times with saturated NH4Cl, washed 3 times with brine, and then dried over anhydrous MgSO4 to obtain a crude product which was further purified by silica gel chromatography;

[0078] 3) DOPA-EN synthesis: Boc-DOPA-EN (0.6 mM) synthesized in 2) above was dissolved in 12 mL of dichloromethane / trifluoroacetic acid (DCM / TFA), the mixed solution was stirred at room temperature for 2 h, and then the solvent was completely removed by rotary evaporation to obtain the product DOPA-EN; the volume ratio of DCM to TFA was 0.8:2.4;

[0079] 4) Synthesis of HA-DOPA-EN: 80 mg HA and 17 mg EDC were added to 10 mL DMSO and stirred at room temperature for 0.5 h; 17 mg NHS and 180 mg DOPA-EN were then added to the above solution and stirred at room temperature overnight under nitrogen protection; the solution was then gently added to 20 mL ultrapure water, and dialyzed with water / acetonitrile for 1 day, with a volume ratio of water to acetonitrile of 1:1; then dialyzed with ultrapure water for 2 days, and finally freeze-dried to obtain the ROS-responsive coating HA-DOPA-EN (HDE);

[0080] (4) Wrapping the ROS-responsive intestinal tissue adhesion coating on the surface of the active probiotic bioregulator to obtain the envelope-type active probiotic bioregulator (BA@GFe@HDE);

[0081] Specifically include:

[0082] The ROS-responsive adhesive coating HDE obtained above was dispersed in PBS and NaCl solution under 60W ultrasound to obtain a ROS-responsive adhesive coating HDE with a negatively charged surface; then an active probiotic bioregulator (BA@GFe) with a surface modified with a positively charged nanoagent GFe was added, mixed at a vortex speed of 100 rpm, centrifuged at a centrifugal speed of 300 rpm, and washed with PBS for 3 times to obtain an envelope-type active probiotic bioregulator (BA@GFe@HDE);

[0083] Among them, the mass volume ratio of active probiotic bioregulator (BA@GFe), PBS solution, NaCl solution and surface negatively charged ROS responsive adhesion coating HDE solution is: 1.6mL: 5mL: 1mL: 1.2mg, and the mass fraction of NaCl solution is 0.91%.

[0084] Example 2

[0085] A method for preparing an envelope-type active probiotic bioregulator for treating Crohn's disease comprises the following steps:

[0086] (1) Combine natural antioxidants with iron ions (Fe 3+ ) Coordinate loading of the exosome synthesis / release inhibitor GW4869 to obtain a surface positively charged nano-modulator (GFe);

[0087] The nano-regulator (GFe) is synthesized by ultrasonic rate-controlled titration method, which specifically includes:

[0088] 1) 24.4 mg of ferric chloride (FeCl3), 4.8 mg of GW4869, 6.8 mg of bis(2-ethylhexyl) sebacate (DOS) and 16.8 mg of polyoxyethylene polyoxypropylene ether block copolymer (F-127) were simultaneously dissolved in 4 mL of methanol, and ultrasonicated for 3 min to fully dissolve them to form a uniform solution A;

[0089] 2) 12.2 mg of curcumin, 6.1 mg of quercetin, 4.8 mg of GW4869, 6.8 mg of bis(2-ethylhexyl) sebacate (DOS) and 16.8 mg of polyoxyethylene polyoxypropylene ether block copolymer (F-127) were dissolved in 4 mL of methanol and ultrasonicated for 5 min to fully dissolve them to form a uniform solution B;

[0090] 3) solution A of step 1) and solution B of step 2) are simultaneously dropped into deionized water containing 1.2 wt % polyacrylamine hydrochloride (PAH) under ultrasound, and then the solution is stirred to form a nano-conditioner;

[0091] The volume ratio of solution A, solution B and deionized water is 1:1:20; the dripping time of solution A and solution B is 2 min; the ultrasonic (90W) treatment time is 2 min; the stirring time is 16 h;

[0092] (2) The surface positively charged nano-regulator (GFe) is combined with the surface negatively charged Bifidobacterium adolescentis through electrostatic interaction to obtain an active probiotic bioregulator (BA@GFe);

[0093] Specifically include:

[0094] 1) Dispersing Bifidobacterium adolescentis (ATCC 15703) in a mixed solution of PBS and NaCl to obtain a solution of Bifidobacterium adolescentis with negative surface charge; the volume ratio of PBS to NaCl in the mixed solution is 8 mL:1.2 mL, the mass fraction of the NaCl solution is 0.91%, and the number of bacterial colonies is 10 9 CFU / mL;

[0095] 2) The surface positively charged nano-regulator (GFe) was ultrasonically dispersed (90W) in a PBS solution, and then a surface negatively charged Bifidobacterium adolescentis solution was added, mixed at a vortex speed of 100 rpm, centrifuged at a centrifugal speed of 300 rpm, and washed with PBS for 3 times to obtain an active probiotic bioregulator (BA@GFe);

[0096] The mass volume ratio of the surface positively charged nano-regulator (GFe), PBS solution and surface negatively charged Bifidobacterium adolescentis solution is: 10 mg: 6 mL: 1.8 mL;

[0097] (3) Phenylboronic acid and hyaluronic acid were modified on dopamine to obtain a coating (HDE) with ROS-responsive adhesion to intestinal tissue;

[0098] Specifically include:

[0099] 1) Synthesis of Boc-DOPA: 6 mM dopamine (DOPA) was dissolved in 30 mL methanol, and 12 mM triethylamine was added to the solution at 0°C, followed by dropwise addition of 15 mM di-tert-butyl dicarbonate [(Boc)2O] to the solution, all of which occurred in a nitrogen environment; the mixture was then stirred at room temperature for 6 h, and then the methanol was removed by rotary evaporation, and the resulting solid was then dissolved in chloroform and washed with water 5 times, and dried over anhydrous MgSO4 to obtain the final white solid product (Boc-DOPA);

[0100] 2) Synthesis of Boc-DOPA-EN: 8 mM of the Boc-DOPA synthesized in 1) above was dissolved in 24 mL of anhydrous dimethylformamide (DMF) and cooled to 0°C, then 16 mM K2CO3 was added to the above solution and stirred for 10 min; then 6 mL of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl bromide (EN, 16 mM) dissolved in DMF was added to the above mixed solution; the mixed solution was stirred at room temperature for 36 h, and DMF was removed by rotary evaporation, then the solid was dissolved in ethyl acetate, washed 3 times with saturated NH4Cl, washed 3 times with brine, and then dried over anhydrous MgSO4 to obtain a crude product which was further purified by silica gel chromatography;

[0101] 3) DOPA-EN synthesis: The Boc-DOPA-EN (10 mM) synthesized in 2) above was dissolved in 24 mL of dichloromethane / trifluoroacetic acid (DCM / TFA), the mixed solution was stirred at room temperature for 6 h, and then the solvent was completely removed by rotary evaporation to obtain the product DOPA-EN;

[0102] Among them, the volume ratio of DCM and TFA is 1:3;

[0103] 4) Synthesis of HA-DOPA-EN: 160 mg HA and 56 mg EDC were added to 20 mL DMSO and stirred at room temperature for 2 h; 56 mg NHS and 260 mg DOPA-EN were then added to the above solution and stirred at room temperature overnight under nitrogen protection; the solution was then gently added to 40 mL ultrapure water and dialyzed with water / acetonitrile for 2 days, with a volume ratio of water to acetonitrile of 1:1; it was then dialyzed with ultrapure water for 3 days, and finally freeze-dried to obtain the ROS-responsive coating HA-DOPA-EN (HDE);

[0104] (4) Wrapping the ROS-responsive intestinal tissue adhesion coating on the surface of the active probiotic bioregulator to obtain the envelope-type active probiotic bioregulator (BA@GFe@HDE);

[0105] Specifically include:

[0106] The ROS-responsive adhesive coating HDE obtained above was dispersed in PBS and NaCl solution under 90W ultrasound to obtain a ROS-responsive adhesive coating HDE with a negative surface charge; then an active probiotic bioregulator (BA@GFe) with a surface modified with a positively charged nanoagent GFe was added, mixed at a vortex speed of 100 rpm, centrifuged at a centrifugal speed of 300 rpm, and washed with PBS for 3 times to obtain an envelope-type active probiotic bioregulator (BA@GFe@HDE);

[0107] Among them, the mass volume ratio of active probiotic bioregulator (BA@GFe), PBS solution, NaCl solution and surface negatively charged ROS responsive adhesion coating HDE solution is: 3.2mL: 5mL: 1mL: 2.4mg, and the mass fraction of NaCl solution is 0.91%.

[0108] Example 3

[0109] A method for preparing an envelope-type active probiotic bioregulator for treating Crohn's disease comprises the following steps:

[0110] (1) Combine natural antioxidants with iron ions (Fe 3+ ) Coordinate loading of the exosome synthesis / release inhibitor GW4869 to obtain a surface positively charged nano-modulator (GFe);

[0111] The nano-regulator (GFe) is synthesized by ultrasonic rate-controlled titration method, which specifically includes:

[0112] 1) 16.6 mg of ferric chloride (FeCl3), 3.6 mg of GW4869, 5.4 mg of bis(2-ethylhexyl) sebacate (DOS) and 12.8 mg of polyoxyethylene polyoxypropylene ether block copolymer (F-127) were simultaneously dissolved in 3 mL of methanol and ultrasonicated for 2 min to fully dissolve them to form a uniform solution A;

[0113] 2) 8.6 mg of curcumin, 4.5 mg of quercetin, 3.6 mg of GW4869, 5.4 mg of bis(2-ethylhexyl) sebacate (DOS) and 12.8 mg of polyoxyethylene polyoxypropylene ether block copolymer (F-127) were dissolved in 3 mL of methanol and ultrasonicated for 2 min to fully dissolve them to form a uniform solution B;

[0114] 3) solution A of step 1) and solution B of step 2) are simultaneously dropped into deionized water containing 0.8 wt % polyacrylamine hydrochloride (PAH) under ultrasound, and then the solution is stirred to form a nano-conditioner;

[0115] The volume ratio of solution A, solution B and deionized water is 1:1:20; the dripping time of solution A and solution B is 2 min; the ultrasonic (60W) treatment time is 2 min; the stirring time is 12 h;

[0116] (2) The surface positively charged nano-regulator (GFe) is combined with the surface negatively charged Bifidobacterium adolescentis through electrostatic interaction to obtain an active probiotic bioregulator (BA@GFe);

[0117] Specifically include:

[0118] 1) Dispersing Bifidobacterium adolescentis (ATCC 15703) in a mixed solution of PBS and NaCl to obtain a solution of Bifidobacterium adolescentis with negative surface charge; the volume ratio of PBS to NaCl in the mixed solution is 8 mL:0.8 mL, the mass fraction of the NaCl solution is 0.91%, and the number of bacterial colonies is 10 8 CFU / mL;

[0119] 2) Ultrasonic (60W) dispersion of positively charged nano-regulator (GFe) in PBS solution, followed by addition of negatively charged Bifidobacterium adolescentis solution, mixing at a vortex speed of 100 rpm, centrifugation at a centrifugal speed of 300 rpm, and washing with PBS for 3 times to obtain an active probiotic bioregulator (BA@GFe);

[0120] The mass volume ratio of the surface positively charged nano-regulator (GFe), PBS solution and surface negatively charged Bifidobacterium adolescentis solution is 8 mg: 6 mL: 1.2 mL;

[0121] (3) Phenylboronic acid and hyaluronic acid were modified on dopamine to obtain a coating (HDE) with ROS-responsive adhesion to intestinal tissue;

[0122] Specifically include:

[0123] 1) Synthesis of Boc-DOPA: 5 mM dopamine (DOPA) was dissolved in 20 mL methanol, and 10 mM triethylamine was added to the solution at 0°C, followed by dropwise addition of 12 mM di-tert-butyl dicarbonate [(Boc)2O] to the solution, all of which occurred in a nitrogen environment; the mixture was then stirred at room temperature for 4 h, and then the methanol was removed by rotary evaporation, and the resulting solid was then dissolved in chloroform and washed with water three times, and dried over anhydrous MgSO4 to obtain the final white solid product (Boc-DOPA);

[0124] 2) Synthesis of Boc-DOPA-EN: 6 mM of the Boc-DOPA synthesized in 1) above was dissolved in 16 mL of anhydrous dimethylformamide (DMF) and cooled to 0°C, then 12 mM K2CO3 was added to the above solution and stirred for 10 min; then 5 mL of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl bromide (EN, 12 mM) dissolved in DMF was added to the above mixed solution; the mixed solution was stirred at room temperature for 28 h, and DMF was removed by rotary evaporation, then the solid was dissolved in ethyl acetate, washed with saturated NH4Cl 3 times, washed with brine 3 times, and then dried with anhydrous MgSO4 to obtain a crude product which was further purified by silica gel chromatography;

[0125] 3) DOPA-EN synthesis: Boc-DOPA-EN (5.6 mM) synthesized in 2) above was dissolved in 16 mL of dichloromethane / trifluoroacetic acid (DCM / TFA), the mixed solution was stirred at room temperature for 4 h, and then the solvent was completely removed by rotary evaporation to obtain the product DOPA-EN;

[0126] The volume ratio of DCM and TFA was 0.9:2.7;

[0127] 4) Synthesis of HA-DOPA-EN: 120 mg HA and 36 mg EDC were added to 15 mL DMSO and stirred at room temperature for 1 h; 36 mg NHS and 240 mg DOPA-EN were then added to the above solution and stirred at room temperature overnight under nitrogen protection; the solution was then gently added to 30 mL ultrapure water and dialyzed against water / acetonitrile for 1.5 days, with a volume ratio of water to acetonitrile of 1:1; it was then dialyzed against ultrapure water for 2 days, and finally freeze-dried to obtain the ROS-responsive coating HA-DOPA-EN (HDE);

[0128] (4) Wrapping the ROS-responsive intestinal tissue adhesion coating layer on the surface of the active probiotic bioregulator to obtain an envelope-type active probiotic bioregulator (BA@GFe@HDE);

[0129] Specifically include:

[0130] The ROS-responsive adhesive coating HDE obtained above was dispersed in PBS and NaCl solution under 60W ultrasound to obtain a ROS-responsive adhesive coating HDE with a negatively charged surface; then an active probiotic bioregulator (BA@GFe) with a surface modified with a positively charged nanoagent GFe was added, mixed at a vortex speed of 100 rpm, centrifuged at a centrifugal speed of 300 rpm, and washed with PBS for 3 times to obtain an envelope-type active probiotic bioregulator (BA@GFe@HDE);

[0131] The mass volume ratio of active probiotic bioregulator (BA@GFe), PBS solution, NaCl solution and surface negatively charged ROS responsive adhesion coating HDE solution is: 2.4mL:5mL:1.8mg, and the mass fraction of NaCl solution is 0.91%.

[0132] Test Example 1

[0133] The surface positively charged nanometer iron carrier nanoparticles GFe prepared in Example 1 were observed by TEM. The results are as follows: Figure 1 As shown, natural polyphenols and Fe 3+ The coordination formed uniform spherical nanoparticles with a particle size distribution of 200-300nm. Figure 2As shown, DLS particle size test further proved that the particle size was 286.5 nm, which fully demonstrated the successful preparation of nano-modulator iron carrier nanoparticles.

[0134] The surface potentials of GFe, BA, BA@GFe and BA@GFe@HDE prepared in Example 1 were tested. The surface potential results are shown in Figure 3 As shown, the dynamic scattering instrument (DLS) was used to measure the potential changes of the prepared GFe, BA, BA@GFe and their BA@GFe@HDE after combination, so as to judge whether the construction of the envelope-type active probiotic bioregulator was successful; the surface of GFe was positively charged, and the surface of BA was obviously negatively charged. With the successful electrostatic binding of GFe on the surface of BA, the surface potential of BA@GFe became positively charged, which was conducive to further modifying the negatively charged ROS-responsive adhesion coating on its surface. The results showed that the surface of BA@GFe@HDE was negatively charged after the adhesion coating was modified, further proving that the active probiotic bioregulator was successfully prepared by electrostatic adsorption.

[0135] The TEM morphology of BA@GFe@HDE prepared in Test Example 1 and bare BA was characterized. Figure 4 As shown, the TEM results of BA (left) and BA@GFe@HDE (right) show that after layer-by-layer electrostatic modification, the bacterial surface has a large number of modified nanoparticles, further proving the successful preparation of the active probiotic bioregulator.

[0136] To test the activity of the probiotics in the BA and BA@GFe@HDE prepared in Example 1, 0.5 mL of bacterial culture was extracted from a single test tube under anaerobic conditions and cultured in an anaerobic incubator at 37°C for 24 hours until the bacteria reached the logarithmic growth phase. The cultured bacterial solution was diluted to 10∧8 CFU / mL with sterile saline, and sterile agar plates were prepared in a sterile environment. The required Bifidobacterium adolescentis culture medium was poured into the culture dish, cooled and solidified. Subsequently, a sterile pipette was used to take an appropriate amount of the diluted bacterial solution and evenly coated it on the surface of the culture medium to ensure uniform coverage. The coated culture dish was placed in an incubator and continued to be cultured at 37°C under anaerobic conditions for 24 hours. The bacterial growth was observed and the morphology and number of growing colonies were recorded. The evaluation results are as follows: Figure 5 As shown, the results show that the colony count of Bifidobacterium adolescentis did not decrease significantly before and after the layer-by-layer electrostatic action on the surface of Bifidobacterium adolescentis, proving that the activity of Bifidobacterium adolescentis will not be affected during the functionalization process, which further ensures the activity of biological regulation.

Claims

1. A method for preparing an envelope-type active probiotic bioregulator for treating Crohn's disease, characterized in that: The following steps are involved: (1) Natural antioxidants are coordinated with trivalent iron ions and loaded with neutral sphingomyelinase inhibitors synthesized / released by exosomes to obtain nano-modulators with positive surface charge; (2) combining the nano-regulator with positive surface charge and the active probiotics with negative surface charge through electrostatic action to obtain an active probiotics bioregulator; (3) Phenylboronic acid and hyaluronic acid were modified on dopamine to obtain a coating with ROS-responsive adhesion to intestinal tissue; (4) Wrapping the ROS-responsive intestinal tissue-adherent coating on the surface of the active probiotic bioregulator to obtain the envelope-type active probiotic bioregulator.

2. The method for preparing the envelope-type active probiotic bioregulator for treating Crohn's disease according to claim 1, characterized in that: In step (1), the natural antioxidants are curcumin and quercetin; The specific steps of the preparation method of the surface positively charged nano-regulator are as follows: (1.1) dissolving ferric chloride, neutral sphingomyelinase inhibitor GW4869, bis(2-ethylhexyl) sebacate and polyoxyethylene polyoxypropylene ether block copolymer in an organic solvent, and obtaining a uniform solution A after ultrasonic treatment; (1.2) curcumin, quercetin, neutral sphingomyelinase inhibitor GW4869, bis(2-ethylhexyl) sebacate and polyoxyethylene polyoxypropylene ether block copolymer are dissolved in an organic solvent, and a uniform solution B is obtained after ultrasonic treatment; (1.3) The solution A obtained in step (a) and the solution B obtained in step (b) are simultaneously dropped into water containing polyacrylamine hydrochloride under ultrasonic conditions and stirred to obtain the surface positively charged nano-regulator.

3. The method for preparing the envelope-type active probiotic bioregulator for treating Crohn's disease according to claim 2, characterized in that: In terms of g, mg and mL, in step (1.1), the amount of ferric chloride is 12.8-24.4 mg, the amount of neutral sphingomyelinase inhibitor GW4869 is 2.4-4.8 mg, the amount of bis(2-ethylhexyl) sebacate is 3.2-6.8 mg, the amount of polyoxyethylene polyoxypropylene ether block copolymer is 9.6-16.8 mg, the volume of the organic solvent is 2-4 mL, and the ultrasonic treatment time is 1-3 min; In step (1.2), the amount of curcumin is 6.4-12.2 mg, the amount of quercetin is 3.2-6.1 mg, the amount of neutral sphingomyelinase inhibitor GW4869 is 2.4-4.8 mg, the amount of bis(2-ethylhexyl) sebacate is 3.2-6.8 mg, the amount of polyoxyethylene polyoxypropylene ether block copolymer is 9.6-16.8 mg, the volume of the organic solvent is 2-4 mL, and the ultrasonic treatment time is 2-5 min; In step (1.3), the concentration of the aqueous solution of polyacrylamine hydrochloride is 0.6-1.2 wt %, and the volume ratio of solution A, solution B and water is 1:1:

20. The dripping time of the solution A and the solution B is 1-3 minutes. The ultrasonic treatment time is 1-3 min. The stirring time is 12-24h.

4. The method for preparing the envelope-type active probiotic bioregulator for treating Crohn's disease according to claim 1, characterized in that: In step (2), the active probiotic with negative surface charge is Bifidobacterium adolescentis; The preparation method of the active probiotic bioregulator is specifically as follows: (2.1) dispersing Bifidobacterium adolescentis in a mixed solution of PBS and NaCl to obtain a solution of Bifidobacterium adolescentis containing surface negative charges; (2.2) Ultrasonic dispersion of the surface positively charged nano-regulator in a PBS solution, followed by addition of the surface negatively charged Bifidobacterium adolescentis solution obtained in step (2.1), mixing, centrifugation and washing to obtain the active probiotic bioregulator.

5. The method for preparing the envelope-type active probiotic bioregulator for treating Crohn's disease according to claim 4, characterized in that: In step (2.1), the volume ratio of PBS to NaCl in the mixed solution is 8 mL: (0.6-1.2) mL, the mass fraction of the NaCl solution is 0.89% to 0.91%, and the bacterial colony count of Bifidobacterium adolescentis is 10 9 CFU / mL; In step (2.2), the mass volume ratio of the surface positively charged nano-regulator, the PBS solution and the surface negatively charged Bifidobacterium adolescentis solution is (5-10) mg:6 mL:(0.6-1.8) mL.

6. The method for preparing the envelope-type active probiotic bioregulator for treating Crohn's disease according to claim 1, characterized in that: In step (3), the specific steps are: (3.1) Under alkaline conditions, dopamine reacts with di-tert-butyl dicarbonate to form an amino compound protected by tert-butyloxycarbonyl, which is denoted as Boc-DOPA; (3.2) Under alkaline conditions, Boc-DOPA reacts with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl bromide to produce the product Boc-DOPA-EN; (3.3) Boc-DOPA-EN undergoes a deprotection reaction to remove the tert-butyloxycarbonyl group to generate the product DOPA-EN; (3.4) The carboxyl group of hyaluronic acid is first subjected to a carboxyl activation reaction with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride to form an active ester intermediate; the active ester intermediate undergoes an amidation reaction with DOPA-EN to obtain a coating HA-DOPA-EN that has ROS responsiveness and adhesion to intestinal tissue.

7. The method for preparing the envelope-type active probiotic bioregulator for treating Crohn's disease according to claim 6, characterized in that: In step (3.1), the molar ratio of dopamine to di-tert-butyl dicarbonate is 3-6:7-15; In step (3.2), the molar ratio of Boc-DOPA to 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)benzyl bromide is 4-8:8-16; In step (3.3), Boc-DOPA-EN undergoes a deprotection reaction in a mixture of dichloromethane and trifluoroacetic acid, wherein the volume ratio of dichloromethane to trifluoroacetic acid is 0.8-1:2.4-3; In step (3.4), the mass ratio of hyaluronic acid to DOPA-EN is 80-160:180-260.

8. The method for preparing the envelope-type active probiotic bioregulator for treating Crohn's disease according to claim 1, characterized in that: In step (4), the step of coating the ROS-responsive intestinal tissue-adherent coating on the surface of the active probiotic bioregulator is as follows: ultrasonically dispersing the ROS-responsive intestinal tissue-adherent coating in a PBS solution and a NaCl solution, and then adding the active probiotic bioregulator; The mass volume ratio of the active probiotic bioregulator, PBS solution, NaCl solution and ROS responsive coating is: 1.6-3.2 mL: 5 mL: 1 mL: 1.2-2.4 mg, and the mass fraction of the NaCl solution is 0.89%-0.91%.

9. An envelope-type active probiotic bioregulator prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the envelope-type active probiotic bioregulator according to claim 9 in the preparation of a medicament for treating Crohn's disease.

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