Sodium alginate microspheres co-loaded with nanogold-producing probiotics and curcumin albumin nanoparticles and preparation and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a sodium alginate microsphere co-loaded with nanogold-producing probiotics and curcumin albumin nanoparticles as well as preparation and application of the sodium alginate microsphere. Probiotic escherichia coli Nissle 1917 is modified to synthesize nanogold, curcumin albumin nanoparticles are prepared by using a self-assembly principle, then sodium alginate microspheres co-loaded with nanogold probiotics and curcumin albumin nanoparticles are prepared by using a high-voltage electrostatic method, and the sodium alginate microspheres are used for treating inflammatory bowel diseases. Compared with the traditional preparation of nanogold, the invention provides a novel green synthesis method, and meanwhile, the activity of probiotics is kept. In order to solve the problems of poor water solubility and the like of curcumin, albumin is used for carrying drugs, curcumin albumin nanoparticles are prepared and then combined with sodium alginate microspheres, and the curcumin albumin nanoparticles have colon targeting property, improve the bioavailability of probiotics and curcumin and are effectively used for treating inflammatory bowel diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to sodium alginate microspheres co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles, and preparation and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is a chronic and recurrent gastrointestinal inflammatory disease. Studies have reported that the etiology and pathogenesis of IBD are related to the high imbalance of intestinal flora and the increase of reactive oxygen species (ROS). At present, clinical drug treatment faces problems such as difficulty in accurately targeting the inflammatory intestine and limited efficacy, and long-term medication will bring serious side effects to patients. Therefore, it is expected to fundamentally treat inflammatory bowel disease by combining the strategy of targeted removal of ROS with the regulation of intestinal flora balance to restore a healthy intestinal microenvironment.

[0003] In recent years, probiotics have become a new treatment for inflammatory bowel disease. Escherichia coli Nissle 1917 is a probiotic with simple genetic manipulation and good biosafety, making it the preferred chassis for constructing engineered bacteria. Escherichia coli Nissle 1917 has the general characteristics of probiotics and can improve intestinal function in a variety of ways, such as maintaining the intestinal mucosal barrier, secreting antimicrobial compounds, and activating the immune system. It has protective and repair functions on the intestinal mucosal barrier and is used in the treatment of inflammatory bowel disease. However, the therapeutic effect of single probiotic treatment is limited by its inability to resist the gastrointestinal environment, poor colonization ability, and short retention time.

[0004] Gold nanoparticles (AuNPs) refer to gold particles with a size at the nanometer level, usually less than 100nm. Due to its adjustable shape and size, easy synthesis and surface modification, excellent optical properties and good biocompatibility, it has been widely used in the biomedical field. At the same time, studies have shown that AuNPs have a variety of enzyme activities, including peroxidase, superoxide dismutase and glucose oxidase, so they have good ability to eliminate reactive oxygen and can be used for the treatment of inflammatory bowel disease. The traditional preparation methods of AuNPs mainly include chemical synthesis, physical synthesis, etc. Compared with the above methods, the biosynthesis of gold nanoparticles has the advantages of easy reduction at room temperature, environmental protection, low cost, and non-toxicity, and is receiving widespread attention. Among them, the synthesis of AuNPs using Escherichia coli Nissle 1917 has the characteristics of simple preparation process, environmental friendliness and good biosafety.

[0005] Curcumin is a natural polyphenol extracted from the rhizome of turmeric, which has anti-inflammatory, antioxidant and anti-cancer functions. Studies have shown that curcumin has a significant effect on improving inflammatory bowel disease, but its poor water solubility, low absorption rate and poor bioavailability limit its clinical application. Human serum albumin is widely used as a carrier for poorly soluble drugs because of its good biosafety, rich groups and high stability. In recent years, studies have shown that albumin, as a nanocarrier, can form nanocomplexes with hydrophobic drugs, reduce drug toxicity, increase drug bioavailability and improve drug targeting.

[0006] Sodium alginate microspheres refer to a type of microsphere material made with sodium alginate as the base material. Sodium alginate, also known as sodium alginate, is a natural polysaccharide that is widely used in the biomedical field because of its non-toxicity, biodegradability, good biocompatibility and pH sensitivity. In addition, sodium alginate microspheres have the characteristics of high drug loading, and can also control the release of nanoparticles in the intestine, playing a sustained-release role. And because the negative charge on its surface facilitates its enrichment in the inflammatory site, it is often used as an oral drug delivery system for the treatment of inflammatory bowel disease.

[0007] The invention firstly transforms the probiotic Escherichia coli Nissle 1917 to synthesize nano-gold by genetic engineering, and simultaneously prepares curcumin albumin nanoparticles by self-assembly, and finally prepares sodium alginate microspheres co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles by high-voltage electrostatic method. The microspheres can not only target the intestinal inflammation site by oral administration, but also regulate the intestinal redox homeostasis by removing active oxygen by nano-gold, and can also improve the bioavailability of curcumin by nano-drug preparation, and regulate the balance of intestinal flora by supplementing probiotics, so as to effectively treat inflammatory bowel disease. Summary of the invention

[0008] In view of this, the present invention proposes a sodium alginate microsphere co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles, and the preparation and application thereof.

[0009] To achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention provides a method for preparing sodium alginate microspheres co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles, which is carried out under sterile conditions according to the following steps: S1: Connect the human metallothionein 1E gene to the pET-28a plasmid to obtain a recombinant plasmid; transform the obtained recombinant plasmid into Escherichia coli Nissle 1917 by heat shock method to obtain an engineered probiotic; inoculate a single colony of the obtained engineered probiotic into LB liquid culture medium containing 50 mg / mL kanamycin, and culture at 37°C and 200 rpm until OD 600=0.6, adding IPTG, inducing at 18°C ​​and 200rpm for 14h to obtain an induced product; centrifuging the obtained induced product, discarding the supernatant, collecting the precipitate and washing it with sterile water, resuspending the washed precipitate in 50mL of chloroauric acid aqueous solution, culturing at 37°C for 3-5h, then centrifuging, discarding the supernatant, collecting the precipitate and washing it with sterile water to obtain nano-gold-producing probiotics; S2: dissolving human serum albumin in sterile water to obtain a human serum albumin solution, and dissolving curcumin in anhydrous ethanol to obtain a curcumin solution; adjusting the pH of the obtained human serum albumin solution to 8.0, and then dripping the obtained curcumin solution into the solution at a certain dripping speed under stirring, continuing to stir for 4 hours, and then adding glutaraldehyde aqueous solution, continuing to stir for 20 hours, centrifuging, discarding the supernatant, collecting the precipitate, and obtaining curcumin albumin nanoparticles; S3: Mix the nano-gold-producing probiotics and curcumin albumin nanoparticles in sterile water to obtain a mixed aqueous solution 1; stir and mix the obtained mixed aqueous solution 1 and the sodium alginate aqueous solution to obtain a mixed aqueous solution 2; add the obtained mixed aqueous solution 2 into a disposable syringe, use a medical 21G flat-head needle, connect an electrode on the syringe needle, the needle is the positive electrode, load voltage, and add the mixed aqueous solution 2 dropwise into the CaCl2 aqueous solution at a certain rate through a microinjection pump to perform a cross-linking reaction, and keep a certain distance between the needle and the liquid surface of the CaCl2 aqueous solution to obtain sodium alginate microspheres co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles.

[0010] Preferably, in the above step S1, the final concentration of IPTG is 0.1-0.3 mM; the concentration of the aqueous solution of chloroauric acid is 1-3 mM.

[0011] Preferably, in the above step S1, the centrifugal parameters are: temperature 4°C, rotation speed 4500-6000 rpm, time 4-10 min.

[0012] Preferably, in the above step S2, the concentration of the human serum albumin solution is 1-2 mg / mL; the concentration of the curcumin solution is 1-2 mg / mL; the concentration of the glutaraldehyde aqueous solution is 4wt%-8wt%; and the volume ratio of the human serum albumin solution: the curcumin solution: the glutaraldehyde aqueous solution is 1:4:0.02.

[0013] Preferably, in the above step S2, the stirring speed is 1200-1500 rpm; the dropping speed of the curcumin solution is 0.5-1 mL / min; and the centrifugal parameters are: temperature 20° C., speed 12000-13000 rpm, and time 15-20 min.

[0014] Preferably, in the above step S3, the concentration of the nano-gold-producing probiotics in the mixed aqueous solution 1 is 1×10 9CFU / mL; the concentration of curcumin albumin nanoparticles in the mixed aqueous solution 1 is 600 μg / mL; the concentration of the sodium alginate aqueous solution is 2wt%~4wt%; the concentration of the CaCl2 aqueous solution is 0.1wt%; the volume ratio of the mixed aqueous solution 1: sodium alginate aqueous solution is 1:1; the volume ratio of the mixed aqueous solution 2: CaCl2 aqueous solution is 1:20.

[0015] Preferably, in the above step S3, the voltage is 10-13 kV; the dripping acceleration rate of the mixed aqueous solution 2 is 0.1-0.3 mL / min; and the distance between the needle and the liquid surface of the CaCl2 aqueous solution is 9 cm.

[0016] The second aspect of the present invention provides a sodium alginate microsphere co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles, which is prepared by the above-mentioned preparation method.

[0017] The third aspect of the present invention provides the use of the above-mentioned sodium alginate microspheres in the preparation of a drug for treating inflammatory bowel disease.

[0018] The significant advantages of the present invention are: The present invention provides a sodium alginate microsphere co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles, and preparation and application thereof. The preparation method requires aseptic operation, comprising: introducing a plasmid containing human metallothionein 1E gene into Escherichia coli Nissle 1917 to construct an engineered probiotic, inoculating the engineered probiotic into LB medium (containing kanamycin) and culturing in a shaker until OD 600 =0.6, IPTG is added to induce the production of metallothionein. Metallothionein acts as a reducing agent and is reduced in a chloroauric acid solution to obtain gold nanoparticles; curcumin albumin nanoparticles are then prepared by self-assembly. Finally, the mixed aqueous solution of nanogold-producing probiotics and curcumin albumin is mixed with a sodium alginate aqueous solution, and a sodium alginate microsphere co-loaded with nanogold-producing probiotics and curcumin albumin nanoparticles is obtained by a high-voltage electrostatic method. The present invention proposes a new method for synthesizing nanogold using genetically engineered probiotic Escherichia coli Nissle 1917, which has the advantages of mild preparation conditions, green friendliness and good biosafety, while maintaining the activity of probiotics and giving probiotics the ability to remove active oxygen. The use of albumin to prepare nanoformulations improves the water solubility and bioavailability of curcumin. Finally, sodium alginate microspheres co-loaded with nano-gold probiotics and curcumin albumin nanoparticles were prepared by high-voltage electrostatic method. Oral administration can resist the gastrointestinal environment, target the sites of intestinal inflammation, remove reactive oxygen species in the sites of inflammation, and regulate the balance of intestinal flora, effectively treating inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is a SEM image of the nano-gold-producing probiotics prepared in Example 1 of the present invention.

[0021] Figure 2 This is a TEM image of the nano-gold-producing probiotics prepared in Example 1 of the present invention.

[0022] Figure 3 TEM image and particle size distribution diagram of nano-gold produced by the nano-gold-producing probiotics prepared in Example 1 of the present invention.

[0023] Figure 4 This is a scanning electron micrograph of the curcumin albumin nanoparticles prepared in Example 1 of the present invention.

[0024] Figure 5 This is a transmission electron microscopy image of the curcumin albumin nanoparticles prepared in Example 1 of the present invention.

[0025] Figure 6 This is a particle size distribution diagram of curcumin albumin nanoparticles prepared in Example 1 of the present invention.

[0026] Figure 7 This is the CLSM image of the sodium alginate microspheres prepared in Example 1 of the present invention.

[0027] Figure 8 This is a UV-visible absorption spectra of nano-gold produced by the nano-gold-producing probiotics prepared in Examples 1 to 3 of the present invention.

[0028] Fig. 9 The plate colony diagram and live bacteria statistics diagram of the nano-gold-producing probiotics prepared in Examples 1 to 3 of the present invention.

[0029] Fig.10 This is a diagram of the POD-like enzyme activity of nanogold produced by the nanogold-producing probiotics prepared in Example 1 of the present invention.

[0030] Fig.11 This is a graph showing the DPPH elimination ability of the curcumin albumin nanoparticles prepared in Example 1 of the present invention.

[0031] Fig.12 These are micrographs of the sodium alginate microspheres prepared in Example 1 of the present invention in different simulated digestive fluids.

[0032] Fig.13 The relative weights of the sodium alginate microspheres prepared in Example 1 of the present invention after incubation in different simulated digestive fluids.

[0033] Fig.14 This is a graph showing the weight changes of mice with inflammatory bowel disease treated with sodium alginate microspheres prepared in Example 1 of the present invention.

[0034] Fig.15This is a graph showing the colon length of mice with inflammatory bowel disease treated with sodium alginate microspheres prepared in Example 1 of the present invention.

[0035] Fig.16 This is a statistical graph of the colon length of mice with inflammatory bowel disease treated with sodium alginate microspheres prepared in Example 1 of the present invention.

[0036] Fig.17 This is an H&E pathological section image of inflammatory bowel disease treated with sodium alginate microspheres prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0037] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0038] The sequence number of the human metallothionein 1E gene involved in the present invention in NCBI is NM_175617.4.

[0039] Unless otherwise specified, the experimental methods involved in the present invention are all conventional methods.

[0040] Unless otherwise specified, the materials and reagents involved in the present invention can be obtained from commercial sources.

[0041] Embodiment 1: This embodiment provides a method for preparing sodium alginate microspheres co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles, which is carried out under sterile conditions according to the following steps: S1: Connect the human metallothionein 1E gene to the pET-28a plasmid to obtain a recombinant plasmid; transform the obtained recombinant plasmid into Escherichia coli Nissle 1917 by heat shock method to obtain an engineered probiotic; inoculate a single colony of the obtained engineered probiotic into LB liquid culture medium containing 50 mg / mL kanamycin, and culture at 37°C and 200 rpm until OD 600 =0.6, add IPTG with a final concentration of 3mM, induce at 18°C, 200rpm for 14h to obtain an induced product; the obtained induced product is centrifuged at 4°C, 5400rpm for 5min, the supernatant is discarded, the precipitate is collected and washed 3 times with sterile water, the washed precipitate is resuspended in 50mL2mM chloroauric acid aqueous solution, cultured at 37°C for 3h, and then centrifuged at 4°C, 5400rpm for 5min, the supernatant is discarded, the precipitate is collected and washed 3 times with sterile water to obtain nanogold-producing probiotics.

[0042] S2: Dissolve 1 mg human serum albumin in 1 mL sterile water to obtain a human serum albumin solution, and dissolve 6 mg curcumin in 4 mL anhydrous ethanol to obtain a curcumin solution; adjust the pH of the obtained human serum albumin solution to 8.0, and then drip the obtained curcumin solution into the solution at a dropping speed of 1 mg / min under stirring at 12000 rpm, continue stirring for 4 hours, then add 20 μL of 4 vol% glutaraldehyde aqueous solution, continue stirring for 20 hours, centrifuge at 20°C and 12000 rpm for 15 minutes, discard the supernatant, collect the precipitate, and obtain curcumin albumin nanoparticles.

[0043] S3: Mix the nanogold-producing probiotics and curcumin albumin nanoparticles in sterile water to obtain a mixed aqueous solution 1. The concentration of the nanogold-producing probiotics in the mixed aqueous solution 1 is 1×10 9 CFU / mL, the concentration of curcumin albumin nanoparticles is 600μg / mL; the obtained mixed aqueous solution 1 and the sodium alginate aqueous solution with a concentration of 4wt% are stirred and mixed evenly in a volume ratio of 1:1 to obtain a mixed aqueous solution 2; the obtained mixed aqueous solution 2 is added into a disposable syringe, a medical 21G flat-head needle is used, an electrode is connected to the syringe needle, the needle is the positive electrode, the voltage is loaded with 10kv, 10mL of the mixed aqueous solution 2 is added dropwise into 200mL of 0.1wt% CaCl2 aqueous solution at a rate of 100μL / min through a microinjection pump to carry out a cross-linking reaction, the distance between the needle and the liquid surface of the CaCl2 aqueous solution is 9cm, and sodium alginate microspheres co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles are obtained.

[0044] Embodiment 2: This embodiment provides a method for preparing sodium alginate microspheres co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles, which is carried out under sterile conditions according to the following steps: S1: Connect the human metallothionein 1E gene to the pET-28a plasmid to obtain a recombinant plasmid; transform the obtained recombinant plasmid into Escherichia coli Nissle 1917 by heat shock method to obtain an engineered probiotic; inoculate a single colony of the obtained engineered probiotic into LB liquid culture medium containing 50 mg / mL kanamycin, and culture at 37°C and 200 rpm until OD 600 =0.6, add IPTG with a final concentration of 3 mM, induce at 18 ° C, 200 rpm for 14 h to obtain an induced product; the obtained induced product is centrifuged at 4 ° C, 5400 rpm for 5 min, the supernatant is discarded, the precipitate is collected and washed 3 times with sterile water, the washed precipitate is resuspended in 50 mL of 2 mM chloroauric acid aqueous solution, cultured at 37 ° C for 4 h, and then centrifuged at 4 ° C, 5400 rpm for 5 min, the supernatant is discarded, the precipitate is collected and washed 3 times with sterile water to obtain nanogold-producing probiotics.

[0045] S2: Dissolve 1 mg human serum albumin in 1 mL sterile water to obtain a human serum albumin solution, and dissolve 6 mg curcumin in 4 mL anhydrous ethanol to obtain a curcumin solution; adjust the pH of the obtained human serum albumin solution to 8.0, and then drip the obtained curcumin solution into the solution at a dropping speed of 1 mg / min under stirring at 12000 rpm, continue stirring for 4 hours, then add 20 μL of 4 vol% glutaraldehyde aqueous solution, continue stirring for 20 hours, centrifuge at 20°C and 12000 rpm for 15 minutes, discard the supernatant, collect the precipitate, and obtain curcumin albumin nanoparticles.

[0046] S3: Mix the nanogold-producing probiotics and curcumin albumin nanoparticles in sterile water to obtain a mixed aqueous solution 1. The concentration of the nanogold-producing probiotics in the mixed aqueous solution 1 is 1×10 9 CFU / mL, the concentration of curcumin albumin nanoparticles is 600μg / mL; the obtained mixed aqueous solution 1 and the sodium alginate aqueous solution with a concentration of 4wt% are stirred and mixed evenly in a volume ratio of 1:1 to obtain a mixed aqueous solution 2; the obtained mixed aqueous solution 2 is added into a disposable syringe, a medical 21G flat-head needle is used, an electrode is connected to the syringe needle, the needle is the positive electrode, the voltage is loaded with 10kv, 10mL of the mixed aqueous solution 2 is added dropwise into 200mL of 0.1wt% CaCl2 aqueous solution at a rate of 100μL / min through a microinjection pump to carry out a cross-linking reaction, the distance between the needle and the liquid surface of the CaCl2 aqueous solution is 9cm, and sodium alginate microspheres co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles are obtained.

[0047] Embodiment 3: This embodiment provides a method for preparing sodium alginate microspheres co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles, which is carried out under sterile conditions according to the following steps: S1: Connect the human metallothionein 1E gene to the pET-28a plasmid to obtain a recombinant plasmid; transform the obtained recombinant plasmid into Escherichia coli Nissle 1917 by heat shock method to obtain an engineered probiotic; inoculate a single colony of the obtained engineered probiotic into LB liquid culture medium containing 50 mg / mL kanamycin, and culture at 37°C and 200 rpm until OD 600 =0.6, add IPTG with a final concentration of 3 mM, induce at 18 ° C, 200 rpm for 14 h to obtain an induced product; the obtained induced product is centrifuged at 4 ° C, 5400 rpm for 5 min, the supernatant is discarded, the precipitate is collected and washed 3 times with sterile water, the washed precipitate is resuspended in 50 mL of 2 mM chloroauric acid aqueous solution, cultured at 37 ° C for 5 h, and then centrifuged at 4 ° C, 5400 rpm for 5 min, the supernatant is discarded, the precipitate is collected and washed 3 times with sterile water to obtain nanogold-producing probiotics.

[0048] S2: Dissolve 1 mg human serum albumin in 1 mL sterile water to obtain a human serum albumin solution, and dissolve 6 mg curcumin in 4 mL anhydrous ethanol to obtain a curcumin solution; adjust the pH of the obtained human serum albumin solution to 8.0, and then drip the obtained curcumin solution into the solution at a dropping speed of 1 mg / min under stirring at 12000 rpm, continue stirring for 4 hours, then add 20 μL of 4 vol% glutaraldehyde aqueous solution, continue stirring for 20 hours, centrifuge at 20°C and 12000 rpm for 15 minutes, discard the supernatant, collect the precipitate, and obtain curcumin albumin nanoparticles.

[0049] S3: Mix the nanogold-producing probiotics and curcumin albumin nanoparticles in sterile water to obtain a mixed aqueous solution 1. The concentration of the nanogold-producing probiotics in the mixed aqueous solution 1 is 1×10 9 CFU / mL, the concentration of curcumin albumin nanoparticles is 600μg / mL; the obtained mixed aqueous solution 1 and the sodium alginate aqueous solution with a concentration of 4wt% are stirred and mixed evenly in a volume ratio of 1:1 to obtain a mixed aqueous solution 2; the obtained mixed aqueous solution 2 is added into a disposable syringe, a medical 21G flat-head needle is used, an electrode is connected to the syringe needle, the needle is the positive electrode, the voltage is loaded with 10kv, 10mL of the mixed aqueous solution 2 is added dropwise into 200mL of 0.1wt% CaCl2 aqueous solution at a rate of 100μL / min through a microinjection pump to carry out a cross-linking reaction, the distance between the needle and the liquid surface of the CaCl2 aqueous solution is 9cm, and sodium alginate microspheres co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles are obtained.

[0050] Performance Testing: (1) The nano-gold-producing probiotics prepared in Example 1 were characterized by scanning electron microscopy and transmission electron microscopy. The results are as follows: Figures 1-2 As shown in the figure, it can be seen that the gold nanoparticles are distributed on the surface and inside of the bacteria.

[0051] (2) The particle size of the nano-gold distributed on the nano-gold-producing probiotics in Example 1 was counted. The specific operation method was as follows: the nano-gold-producing probiotics were resuspended in 15 mL of PBS, and the bacteria were crushed using a cell crusher with the parameters set to 400 W and 30 min. After crushing, the cells were centrifuged at 4° C. and 12,000 rpm for 30 min, the supernatant was collected, a small amount of liquid was dropped on a copper mesh, and the particle size was counted using Nanom measurer software. The results are shown in FIG. Figure 3 As shown in the figure, the average particle size of the nano-gold distributed on the nano-gold-producing probiotics is 16nm.

[0052] (3) The morphology of the curcumin albumin nanoparticles prepared in Example 1 was characterized by scanning electron microscopy and transmission electron microscopy. Figures 4-5As shown in the figure, the curcumin albumin nanoparticles are uniform spherical structures, uniform in size, and well dispersed.

[0053] (4) The particle size of the curcumin albumin nanoparticles prepared in Example 1 was characterized by dynamic light scattering. The results are as follows Figure 6 As shown in the figure, the average particle size of curcumin albumin nanoparticles is 232nm, and the particle size distribution index is 0.1, indicating that the nanoparticles are uniformly distributed.

[0054] (5) The sodium alginate microspheres prepared in Example 1 were characterized by laser confocal microscopy. Specifically, the nanogold-producing probiotics were first labeled with Cy5.5-NHS, and then the sodium alginate microspheres were prepared according to the above steps. The sodium alginate microspheres were placed in a laser confocal dish and photographed under a laser confocal microscope. The results are shown in FIG. Figure 7 As shown in the figure, the sodium alginate microspheres present a uniform spherical structure, and the nano-gold-producing probiotics and curcumin albumin nanoparticles are evenly distributed inside the microspheres.

[0055] (6) The production of nanogold by the nanogold-producing probiotics of Examples 1 to 3 was characterized by ultraviolet spectrophotometry. The method was as follows: the nanogold-producing probiotics were resuspended in 15 mL of PBS, and the bacteria were crushed by a cell crusher with the parameters set at 400 W for 30 min. After crushing, the cells were centrifuged and the supernatant was collected. The centrifuge parameters were set at 4° C. and 12,000 rpm for 30 min. 2 mL of the supernatant was measured by ultraviolet spectrophotometer. The results were as follows: Figure 8 As shown in the figure, the typical absorption peak of nano-gold appears at around 520nm, and the supernatant shows the characteristic purple color of nano-gold, indicating the successful preparation of nano-gold.

[0056] (7) The activity of the nano-gold-producing probiotics in Examples 1 to 3 was counted using the viable plate counting method. The specific operation was as follows: 1 mL of the nano-gold-producing probiotic suspension was diluted 10 7 Then use a spreading stick to spread 100 μL of the diluted bacterial cells on the LB agar plate, and then culture at 37°C for 16 hours to calculate the colony forming unit (CFU). The number of viable bacteria per unit volume (CFU / mL) = the average number of colonies in the same dilution gradient × the dilution multiple. The results are as follows Fig. 9 As shown in the figure, it can be seen that the nano-gold-producing probiotics prepared under different conditions are still active while producing nano-gold.

[0057] (8) The ability of the nano-gold-producing probiotics in Example 1 to remove active oxygen was characterized, specifically as follows: first, the nano-gold-producing probiotics were resuspended in 15 mL of PBS, and the bacteria were crushed using a cell crusher, with the parameters set to 400 W and 30 min. After crushing, the supernatant was collected, and the centrifuge parameters were set to 4 ° C and 12000 rpm for 30 min to obtain AuNPs after the nano-gold-producing probiotics were crushed. Then, 3,3',5,5'-tetramethylbenzidine (TMB) was used as a substrate, and the peroxidase-like activity of the AuNPs after the nano-gold-producing probiotics were detected in a sodium acetate buffer solution containing H2O2 at room temperature. The reaction system includes: TMB (0.2mM), H2O2 (0.08mM), AuNPs after the nano-gold probiotics are crushed, and 200μL HAc-NaAc buffer (0.1M, pH=4.5). The reaction is carried out for 15min, and its ultraviolet absorption peak is measured at 200-800nm. We use the 3,3',5,5'-tetramethylbenzidine (TMB) method to measure. In the presence of peroxidase, TMB can be oxidized by hydrogen peroxide to form a blue substance, and a characteristic absorption peak appears at 655nm. The results are shown in Fig.10 As shown. Fig.10 It can be seen that only the system containing AuNPs, H2O2 and TMB after the nano-gold-producing probiotics were crushed turned blue [the color of oxidized TMB (oxTMB)], and the corresponding absorption spectrum showed a strong absorption peak at 652nm, indicating that the AuNPs after the nano-gold-producing probiotics were crushed had strong peroxidase activity.

[0058] (9) The ability of the curcumin albumin nanoparticles in Example 1 to scavenge active oxygen species was characterized. Specifically, DPPH was used to characterize the ability to eliminate active oxygen species. The principle is that there are non-shared electrons (N·) inside the DPPH molecule. When reacting with electrons and free radicals, it will be converted into a stable structure (NH). DPPH appears purple in aqueous solution, but when the free radicals are removed and become a covalent bond structure, the color will turn into light yellow. And the measurement was carried out at 517nm to determine the DPPH free radical scavenging ability. Specifically as follows: DPPH with a concentration of 60μM was prepared with anhydrous ethanol, and curcumin albumin nanoparticles of different concentrations were added to the DPPH solution. After reacting for 30 minutes, the ultraviolet absorption at 400-800nm ​​was measured. The results are as follows: Fig.11 As shown in the figure, as the concentration of curcumin albumin nanoparticles increases, the absorption at 517nm gradually decreases, indicating that curcumin albumin nanoparticles have a good ability to remove DPPH.

[0059] (8) The sodium alginate microspheres of Example 1 were characterized for their in vitro targeting properties. Specifically, 100 mg of sodium alginate microspheres were placed in 1 mL of simulated intestinal fluid and simulated gastric fluid, respectively. After incubation in a 37°C water bath for 3 h, 6 h, and 12 h, respectively, the digestion of the microspheres was observed under an optical microscope, and the weight of the microspheres was counted. The results are as follows: Figures 12-13 As shown in the figure, the sodium alginate microspheres did not rupture in the simulated gastric fluid, but ruptured in the simulated intestinal fluid after 3 hours, which further shows that the sodium alginate microspheres have targeting properties.

[0060] (9) The therapeutic effect of sodium alginate microspheres in Example 1 on inflammatory bowel disease was characterized. Specifically, an inflammatory bowel disease model was first established by inducing DSS for 7 consecutive days, and then sodium alginate microspheres were administered by gavage every other day. The treatment was terminated after four administrations. The condition, weight and feces of the mice were observed every day. After the treatment, the mice were dissected and their colons were taken for pathological analysis. The results are as follows: Figures 14 to 17 shown.

[0061] Days 0 to 7 represent the weight changes of mice when the IBD model was induced, and days 7 to 12 represent the weight changes during the treatment process. As can be seen from the figure, during the treatment process, the DSS group continued to lose weight, the mice were listless, and had diarrhea, while the weight of the mice in the healthy group was slowly increasing; compared with DSS, the weight of the mice in the drug-treated group increased slowly, and basically returned to the weight before modeling after the end of treatment.

[0062] The effect of treatment was evaluated by the colon length of mice. As can be seen from the figure, the colon length of the drug-treated group was significantly increased compared with the DSS group, and there was no significant difference compared with the healthy group, indicating that the sodium alginate microspheres-treated group had a good therapeutic effect.

[0063] H&E sections showed that the mice in the healthy group had abundant intestinal glands in the mucosal layer, intact epithelium, normal cell morphology, abundant goblet cells, intact crypts, and no obvious damage or inflammatory response. In the DSS group, the crypt structure was destroyed, goblet cells were lost, replaced by proliferating connective tissue, and accompanied by more inflammatory cell infiltration. In the drug-treated group, the mucosal layer was restored, the cell morphology returned to normal, the goblet cells recovered in abundance, and the crypt structure was restored, with no significant difference from the healthy group.

[0064] In summary, alginate microspheres co-loaded with nanogold-producing probiotics and curcumin-albumin nanoparticles have a strong anti-inflammatory effect and can treat DSS-induced colon tissue damage, showing excellent anti-inflammatory properties.

[0065] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for preparing sodium alginate microspheres co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles, characterized in that: Follow these steps under sterile conditions: S1: Connect the human metallothionein 1E gene to the pET-28a plasmid to obtain a recombinant plasmid; transform the obtained recombinant plasmid into Escherichia coli Nissle 1917 by heat shock method to obtain an engineered probiotic; inoculate a single colony of the obtained engineered probiotic into LB liquid culture medium containing 50 mg / mL kanamycin, and culture at 37°C and 200 rpm until OD 600 =0.6, add IPTG, induce at 18°C, 200rpm for 14h to obtain an induced product; centrifuge the obtained induced product, discard the supernatant, collect the precipitate and wash it with sterile water, resuspend the washed precipitate in 50mL of chloroauric acid aqueous solution, culture at 37°C for 3-5h, then centrifuge, discard the supernatant, collect the precipitate and wash it with sterile water to obtain nano-gold-producing probiotics; S2: dissolving human serum albumin in sterile water to obtain a human serum albumin solution, and dissolving curcumin in anhydrous ethanol to obtain a curcumin solution; The pH of the obtained human serum albumin solution was adjusted to 8.0, and then the obtained curcumin solution was dripped into it at a certain dripping speed under stirring, and the stirring was continued for 4 hours, and then the glutaraldehyde aqueous solution was added, and the stirring was continued for 20 hours, centrifuged, the supernatant was discarded, and the precipitate was collected to obtain curcumin albumin nanoparticles; S3: Mix the nano-gold-producing probiotics and curcumin albumin nanoparticles in sterile water to obtain a mixed aqueous solution 1; stir and mix the obtained mixed aqueous solution 1 and the sodium alginate aqueous solution to obtain a mixed aqueous solution 2; add the obtained mixed aqueous solution 2 into a disposable syringe, use a medical 21G flat-head needle, connect an electrode on the syringe needle, the needle is the positive electrode, load voltage, and add the mixed aqueous solution 2 dropwise into the CaCl2 aqueous solution at a certain rate through a microinjection pump to perform a cross-linking reaction, and keep a certain distance between the needle and the liquid surface of the CaCl2 aqueous solution to obtain sodium alginate microspheres co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles.

2. The preparation method according to claim 1, characterized in that: In step S1, the final concentration of IPTG is 0.1-0.3 mM; the concentration of the aqueous solution of chloroauric acid is 1-3 mM.

3. The preparation method according to claim 1, characterized in that: In step S1, the centrifugal parameters are: temperature 4°C, rotation speed 4500-6000 rpm, time 4-10 min.

4. The preparation method according to claim 1, characterized in that: In step S2, the concentration of human serum albumin solution is 1-2 mg / mL; the concentration of curcumin solution is 1-2 mg / mL; the concentration of glutaraldehyde aqueous solution is 4wt%-8wt%; and the volume ratio of human serum albumin solution: curcumin solution: glutaraldehyde aqueous solution is 1:4:0.

02.

5. The preparation method according to claim 1, characterized in that: In step S2, the stirring speed is 1200-1500 rpm; the dropping speed of the curcumin solution is 0.5-1 mL / min; the centrifugal parameters are: temperature 20° C., speed 12000-13000 rpm, time 15-20 min.

6. The preparation method according to claim 1, characterized in that: In step S3, the concentration of the nanogold-producing probiotics in the mixed aqueous solution 1 is 1×10 9 CFU / mL; the concentration of curcumin albumin nanoparticles in the mixed aqueous solution 1 is 600 μg / mL; the concentration of the sodium alginate aqueous solution is 2wt%~4wt%; the concentration of the CaCl2 aqueous solution is 0.1wt%; the volume ratio of the mixed aqueous solution 1: sodium alginate aqueous solution is 1:1; the volume ratio of the mixed aqueous solution 2: CaCl2 aqueous solution is 1:

20.

7. The preparation method according to claim 1, characterized in that: In step S3, the voltage is 10-13 kV; the dripping rate of the mixed aqueous solution 2 is 0.1-0.3 mL / min; and the distance between the needle and the liquid surface of the CaCl2 aqueous solution is 9 cm.

8. A sodium alginate microsphere co-loaded with nano-gold-producing probiotics and curcumin albumin nanoparticles, characterized in that: Prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the sodium alginate microspheres according to claim 8 in the preparation of a medicament for treating inflammatory bowel disease.

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