A sodium alginate microsphere co-loading nano-gold producing probiotics and curcumin albumin nanoparticles and preparation and application thereof

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a sodium alginate microsphere co-loading nano-gold probiotics and curcumin albumin nanoparticles and preparation and application thereof. The present application synthesizes nano-gold by modifying probiotics Escherichia coli Nissle 1917, prepares curcumin albumin nanoparticles by using the principle of self-assembly, and then prepares the sodium alginate microsphere co-loading nano-gold probiotics and curcumin albumin nanoparticles by using the method of high-voltage electrostatic, which is used for the treatment of inflammatory bowel disease. Compared with the traditional preparation of nano-gold, the present application proposes a new green synthesis method, while keeping the activity of probiotics. In order to improve the problem of poor water solubility of curcumin, the present application uses albumin to load drugs, prepares curcumin albumin nanoparticles, and then combines with the sodium alginate microsphere, which has colon targeting, improves the bioavailability of probiotics and curcumin, and is effectively used for the treatment of inflammatory bowel disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a sodium alginate microsphere co-loaded with gold nanoparticles, probiotic nanoparticles, and its preparation and application. Background Technology

[0002] Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is a chronic and relapsing inflammatory disease of the gastrointestinal tract. Studies have reported that the etiology and pathogenesis of IBD are related to severe gut microbiota dysbiosis and elevated reactive oxygen species (ROS). Current clinical drug treatments face challenges such as difficulty in precisely targeting the inflamed gut, limited efficacy, and serious side effects with long-term use. Therefore, a strategy combining targeted ROS clearance with regulation of gut microbiota balance to restore a healthy gut microenvironment holds promise for fundamentally treating inflammatory bowel disease.

[0003] In recent years, probiotics have emerged as a novel treatment option for inflammatory bowel disease (IBD). *Escherichia coli* Nissle 1917 is a genetically simple probiotic with good biosafety, making it a preferred substrate for constructing engineered bacteria. *E. coli* Nissle 1917 possesses the general characteristics of probiotics, improving intestinal function through various mechanisms such as maintaining the intestinal mucosal barrier, secreting antimicrobial compounds, and activating the immune system. It has protective and repairing functions for the intestinal mucosal barrier and has been applied to the treatment of IBD. However, single-dose probiotic therapy has limited efficacy due to its inability to withstand the gastrointestinal environment, poor colonization ability, and short retention time.

[0004] Gold nanoparticles (AuNPs) refer to gold particles with sizes in the nanometer range, typically smaller than 100 nm. Due to their tunable shape and size, ease of synthesis and surface modification, excellent optical properties, and good biocompatibility, they have found wide application in the biomedical field. Furthermore, studies have shown that AuNPs possess various enzymatic activities, including peroxidase, superoxide dismutase, and glucose oxidase, thus exhibiting excellent reactive oxygen species elimination capabilities and potential applications in the treatment of inflammatory bowel disease. Traditional methods for preparing AuNPs mainly include chemical and physical synthesis. Compared to these methods, the biosynthesis of gold nanoparticles offers advantages such as easy reduction at room temperature, environmental friendliness, low cost, and non-toxicity, and is attracting widespread attention. Among these methods, the synthesis of AuNPs using *Escherichia coli* Nissle 1917 is characterized by its simple preparation process, environmental friendliness, and good biosafety.

[0005] Curcumin is a natural polyphenol extracted from the rhizome of turmeric, possessing anti-inflammatory, antioxidant, and anti-cancer properties. 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, due to its good biocompatibility, abundant functional groups, and high stability, is widely used as a carrier for poorly soluble drugs. Recent studies have shown that albumin, as a nanocarrier, can form nanocomplexes with hydrophobic drugs, reducing drug toxicity, increasing drug bioavailability, and improving drug targeting.

[0006] Sodium alginate microspheres are microsphere materials made with sodium alginate as the base material. Sodium alginate, also known as sodium brown alginate, is a natural polysaccharide. Due to its non-toxicity, biodegradability, good biocompatibility, and pH sensitivity, it is widely used in the biomedical field. Furthermore, sodium alginate microspheres have a high drug loading capacity and can control the release of nanoparticles in the intestine, thus achieving a sustained-release effect. Moreover, because their surface carries a negative charge, they are conducive to accumulation at sites of inflammation, and therefore are often used as oral drug delivery systems for the treatment of inflammatory bowel disease.

[0007] This invention first utilizes genetic engineering to modify the probiotic *Escherichia coli* Nissle 1917 to synthesize gold nanoparticles. Simultaneously, it prepares curcumin-albumin nanoparticles via self-assembly. Finally, it uses a high-voltage electrostatic method to prepare sodium alginate microspheres co-loaded with gold nanoparticles, probiotics, and curcumin-albumin nanoparticles. These microspheres can target intestinal inflammation sites orally, regulate intestinal redox homeostasis by scavenging reactive oxygen species with gold nanoparticles, enhance the bioavailability of curcumin through nanomedicine formulations, and supplement probiotics to regulate intestinal flora balance, effectively treating inflammatory bowel disease. Summary of the Invention

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

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The first aspect of this invention provides a method for preparing sodium alginate microspheres co-loaded with probiotic nanoparticles producing gold nanoparticles and curcumin albumin nanoparticles, which is carried out under aseptic conditions according to the following steps:

[0011] S1: The human metallothionein 1E gene was ligated into the pET-28a plasmid to obtain a recombinant plasmid; the obtained recombinant plasmid was transformed into Escherichia coli Nissle 1917 by heat shock to obtain engineered probiotics; single colonies of the obtained engineered probiotics were inoculated into LB liquid medium containing 50 mg / mL kanamycin and cultured at 37°C and 200 rpm until OD500.600 =0.6 After adding IPTG, induce at 18℃ and 200rpm for 14h to obtain the induced product; centrifuge the obtained induced product, discard the supernatant, collect the precipitate and wash with sterile water, resuspend the washed precipitate in 50mL chloroauric acid aqueous solution, incubate at 37℃ for 3-5h, then centrifuge, discard the supernatant, collect the precipitate and wash with sterile water to obtain the probiotics that produce nano-gold;

[0012] S2: Human serum albumin was dissolved in sterile water to obtain a human serum albumin solution, and curcumin was dissolved 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 added dropwise to it at a certain rate while stirring. Stirring was continued for 4 hours, then glutaraldehyde aqueous solution was added, and stirring was continued for 20 hours. After centrifugation, the supernatant was discarded, and the precipitate was collected to obtain curcumin albumin nanoparticles.

[0013] S3: Mix the gold-producing probiotics and curcumin albumin nanoparticles in sterile water to obtain mixed aqueous solution 1; stir the obtained mixed aqueous solution 1 and sodium alginate aqueous solution evenly to obtain mixed aqueous solution 2; add the obtained mixed aqueous solution 2 to a disposable syringe, use a medical 21G flat-tipped needle, attach an electrode to the syringe needle with the needle as the positive electrode, apply voltage, and use a micro-injection pump to drop mixed aqueous solution 2 into CaCl2 aqueous solution at a certain rate to carry out cross-linking reaction, keeping a certain distance between the needle and the surface of CaCl2 aqueous solution, to obtain sodium alginate microspheres co-loaded with gold-producing probiotics and curcumin albumin nanoparticles.

[0014] Preferably, in step S1 above, the final concentration of IPTG is 0.1–0.3 mM; and the concentration of the chloroauric acid aqueous solution is 1–3 mM.

[0015] Preferably, in step S1 above, the centrifugation parameters are: temperature 4℃, rotation speed 4500~6000rpm, and time 4~10min.

[0016] Preferably, in step S2 above, 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 4 wt%-8 wt%; and the volume ratio of human serum albumin solution: curcumin solution: glutaraldehyde aqueous solution is 1:4:0.02.

[0017] Preferably, in step S2 above, the stirring speed is 1200-1500 rpm; the dropping rate of curcumin solution is 0.5-1 mL / min; and the centrifugation parameters are: temperature 20℃, speed 12000-13000 rpm, and time 15-20 min.

[0018] Preferably, in step S3 above, the concentration of the probiotics producing nano-gold in the mixed aqueous solution 1 is 1×10⁻⁶. 9 CFU / mL; the concentration of curcumin albumin nanoparticles in mixed aqueous solution 1 is 600 μg / mL; the concentration of sodium alginate aqueous solution is 2wt%–4wt%; the concentration of CaCl2 aqueous solution is 0.1wt%; the volume ratio of mixed aqueous solution 1 to sodium alginate aqueous solution is 1:1; the volume ratio of mixed aqueous solution 2 to CaCl2 aqueous solution is 1:20.

[0019] Preferably, in step S3 above, the voltage is 10-13 kV; the dropping rate of the mixed aqueous solution 2 is 0.1-0.3 mL / min; and the distance between the needle and the surface of the CaCl2 aqueous solution is 9 cm.

[0020] A second aspect of the present invention provides sodium alginate microspheres co-loaded with probiotic nanoparticles of gold nanoparticles and curcumin albumin nanoparticles, which are prepared by the above-described preparation method.

[0021] A third aspect of the present invention provides the use of the aforementioned sodium alginate microspheres in the preparation of a medicament for treating inflammatory bowel disease.

[0022] The significant advantages of this invention are:

[0023] This invention provides sodium alginate microspheres co-loaded with probiotic nanoparticles containing gold nanoparticles and curcumin albumin nanoparticles, as well as their preparation and application. The preparation methods all require aseptic operation and include: introducing a plasmid containing the human metallothionein 1E gene into *Escherichia coli* Nissle 1917 to construct engineered probiotics; inoculating the engineered probiotics into LB medium (containing kanamycin) and culturing them in a shaker until OD... 600 IPTG was added at a concentration of 0.6 to induce the production of metallothionein. Metallothionein acted as a reducing agent, reducing gold nanoparticles in chloroauric acid solution. Curcumin-albumin nanoparticles were then prepared via self-assembly. Finally, a mixed aqueous solution of the gold-producing probiotic and curcumin-albumin was mixed with an aqueous solution of sodium alginate, and sodium alginate microspheres co-loaded with the gold-producing probiotic and curcumin-albumin nanoparticles were obtained using a high-voltage electrostatic method. This invention proposes a novel method for synthesizing gold nanoparticles using the genetically engineered probiotic *Escherichia coli* Nissle 1917. This method offers advantages such as mild preparation conditions, environmental friendliness, and good biosafety, maintaining the activity of the probiotics while endowing them with the ability to scavenge reactive oxygen species. The use of albumin in the preparation of nano-formulations improves the water solubility and bioavailability of curcumin. Finally, sodium alginate microspheres co-loaded with gold nanoparticles, probiotics, and curcumin albumin nanoparticles were prepared using a high-voltage electrostatic method. Oral administration can resist the gastrointestinal environment, target intestinal inflammation sites, clear reactive oxygen species at the inflammation sites, and regulate the balance of intestinal flora, effectively treating inflammatory bowel disease. Attached Figure Description

[0024] Figure 1 This is a SEM image of the gold nanoparticle-producing probiotics prepared in Example 1 of the present invention.

[0025] Figure 2 This is a TEM image of the gold-producing probiotic prepared in Example 1 of the present invention.

[0026] Figure 3 TEM image and particle size distribution diagram of gold nanoparticles produced by the gold nanoparticle-producing probiotics prepared in Example 1 of this invention.

[0027] Figure 4 This is a scanning electron microscope image of the curcumin albumin nanoparticles prepared in Example 1 of the present invention.

[0028] Figure 5 This is a transmission electron microscope image of the curcumin albumin nanoparticles prepared in Example 1 of the present invention.

[0029] Figure 6 This is a particle size distribution diagram of the curcumin albumin nanoparticles prepared in Example 1 of the present invention.

[0030] Figure 7 This is a CLSM image of the sodium alginate microspheres prepared in Example 1 of the present invention.

[0031] Figure 8 The UV-Vis absorption spectra of the gold nanoparticles produced by the gold nanoparticle-producing probiotics prepared in Examples 1-3 of this invention are shown.

[0032] Figure 9 The images show plate colony diagrams and live bacteria statistics of the gold nanoparticle-producing probiotics prepared in Examples 1-3 of this invention.

[0033] Figure 10 This is a diagram of the POD-like enzyme activity of gold nanoparticles produced by the gold nanoparticle-producing probiotics prepared in Example 1 of this invention.

[0034] Figure 11 This is a graph showing the DPPH elimination ability of the curcumin albumin nanoparticles prepared in Example 1 of the present invention.

[0035] Figure 12 These are micrographs of sodium alginate microspheres prepared in Example 1 of the present invention in different simulated digestive solutions.

[0036] Figure 13 The relative weights of the sodium alginate microspheres prepared in Example 1 of this invention after incubation in different simulated digestive solutions.

[0037] Figure 14 This is a graph showing the weight change in mice treated with sodium alginate microspheres prepared in Example 1 of this invention for inflammatory bowel disease.

[0038] Figure 15 This is a graph showing the colon length in mice treated with sodium alginate microspheres prepared in Example 1 of this invention for inflammatory bowel disease.

[0039] Figure 16 This is a statistical diagram of colon length in mice treated with sodium alginate microspheres prepared in Example 1 of the present invention for the treatment of inflammatory bowel disease.

[0040] Figure 17 The image shows a pathological section (H&E) of sodium alginate microspheres prepared in Example 1 of this invention for the treatment of inflammatory bowel disease. Detailed Implementation

[0041] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0042] The human metallothionein 1E gene involved in this invention has the NCBI sequence number NM_175617.4.

[0043] Unless otherwise specified, all experimental methods involved in this invention are conventional methods.

[0044] Unless otherwise specified, all materials and reagents involved in this invention are commercially available.

[0045] Example 1:

[0046] This embodiment provides a method for preparing sodium alginate microspheres co-loaded with gold nanoparticle probiotics and curcumin albumin nanoparticles, which is carried out under aseptic conditions according to the following steps:

[0047] S1: The human metallothionein 1E gene was ligated into the pET-28a plasmid to obtain a recombinant plasmid; the obtained recombinant plasmid was transformed into Escherichia coli Nissle 1917 by heat shock to obtain engineered probiotics; single colonies of the obtained engineered probiotics were inoculated into LB liquid medium containing 50 mg / mL kanamycin and cultured at 37°C and 200 rpm until OD500. 600 =0.6, then add IPTG to a final concentration of 3mM, and induce at 18℃ and 200rpm for 14h to obtain the induced product; centrifuge the obtained induced product at 4℃ and 5400rpm for 5min, discard the supernatant, collect the precipitate and wash it 3 times with sterile water, resuspend the washed precipitate in 50mL of 2mM chloroauric acid aqueous solution, incubate at 37℃ for 3h, then centrifuge at 4℃ and 5400rpm for 5min, discard the supernatant, collect the precipitate and wash it 3 times with sterile water to obtain the probiotics producing nano-gold.

[0048] S2: Dissolve 1 mg of human serum albumin in 1 mL of sterile water to obtain a human serum albumin solution, and dissolve 6 mg of curcumin in 4 mL of anhydrous ethanol to obtain a curcumin solution. Adjust the pH of the obtained human serum albumin solution to 8.0, and then add the obtained curcumin solution dropwise at a rate of 1 mg / min while stirring at 12000 rpm. Continue stirring for 4 h, then add 20 μL of 4 vol% glutaraldehyde aqueous solution, and continue stirring for 20 h. Centrifuge at 20 °C and 12000 rpm for 15 min, discard the supernatant, collect the precipitate, and obtain curcumin albumin nanoparticles.

[0049] S3: Mix the gold-producing probiotics and curcumin albumin nanoparticles in sterile water to obtain mixed aqueous solution 1. The concentration of the gold-producing probiotics in mixed aqueous solution 1 is 1×10⁻⁶. 9 The concentration of CFU / mL and curcumin albumin nanoparticles was 600 μg / mL. The resulting mixed aqueous solution 1 and a 4 wt% sodium alginate aqueous solution were mixed evenly at a volume ratio of 1:1 to obtain mixed aqueous solution 2. The mixed aqueous solution 2 was added to a disposable syringe. A medical 21G flat-tipped needle was used, with an electrode attached to the needle (positive electrode). A voltage of 10 kV was applied, and 10 mL of mixed aqueous solution 2 was added dropwise to 200 mL of 0.1 wt% CaCl2 aqueous solution at a rate of 100 μL / min using a micro-injection pump to carry out the cross-linking reaction. The distance between the needle and the surface of the CaCl2 aqueous solution was 9 cm. Sodium alginate microspheres co-loaded with gold nanoparticles, probiotics, and curcumin albumin nanoparticles were obtained.

[0050] Example 2:

[0051] This embodiment provides a method for preparing sodium alginate microspheres co-loaded with gold nanoparticle probiotics and curcumin albumin nanoparticles, which is carried out under aseptic conditions according to the following steps:

[0052] S1: The human metallothionein 1E gene was ligated into the pET-28a plasmid to obtain a recombinant plasmid; the obtained recombinant plasmid was transformed into Escherichia coli Nissle 1917 by heat shock to obtain engineered probiotics; single colonies of the obtained engineered probiotics were inoculated into LB liquid medium containing 50 mg / mL kanamycin and cultured at 37°C and 200 rpm until OD500. 600 =0.6, then add IPTG to a final concentration of 3mM, and induce at 18℃ and 200rpm for 14h to obtain the induced product; centrifuge the obtained induced product at 4℃ and 5400rpm for 5min, discard the supernatant, collect the precipitate and wash it 3 times with sterile water, resuspend the washed precipitate in 50mL of 2mM chloroauric acid aqueous solution, incubate at 37℃ for 4h, then centrifuge at 4℃ and 5400rpm for 5min, discard the supernatant, collect the precipitate and wash it 3 times with sterile water to obtain the probiotics producing nano-gold.

[0053] S2: Dissolve 1 mg of human serum albumin in 1 mL of sterile water to obtain a human serum albumin solution, and dissolve 6 mg of curcumin in 4 mL of anhydrous ethanol to obtain a curcumin solution. Adjust the pH of the obtained human serum albumin solution to 8.0, and then add the obtained curcumin solution dropwise at a rate of 1 mg / min while stirring at 12000 rpm. Continue stirring for 4 h, then add 20 μL of 4 vol% glutaraldehyde aqueous solution, and continue stirring for 20 h. Centrifuge at 20 °C and 12000 rpm for 15 min, discard the supernatant, collect the precipitate, and obtain curcumin albumin nanoparticles.

[0054] S3: Mix the gold-producing probiotics and curcumin albumin nanoparticles in sterile water to obtain mixed aqueous solution 1. The concentration of the gold-producing probiotics in mixed aqueous solution 1 is 1×10⁻⁶. 9 The concentration of CFU / mL and curcumin albumin nanoparticles was 600 μg / mL. The resulting mixed aqueous solution 1 and a 4 wt% sodium alginate aqueous solution were mixed evenly at a volume ratio of 1:1 to obtain mixed aqueous solution 2. The mixed aqueous solution 2 was added to a disposable syringe. A medical 21G flat-tipped needle was used, with an electrode attached to the needle (positive electrode). A voltage of 10 kV was applied, and 10 mL of mixed aqueous solution 2 was added dropwise to 200 mL of 0.1 wt% CaCl2 aqueous solution at a rate of 100 μL / min using a micro-injection pump to carry out the cross-linking reaction. The distance between the needle and the surface of the CaCl2 aqueous solution was 9 cm. Sodium alginate microspheres co-loaded with gold nanoparticles, probiotics, and curcumin albumin nanoparticles were obtained.

[0055] Example 3:

[0056] This embodiment provides a method for preparing sodium alginate microspheres co-loaded with gold nanoparticle probiotics and curcumin albumin nanoparticles, which is carried out under aseptic conditions according to the following steps:

[0057] S1: The human metallothionein 1E gene was ligated into the pET-28a plasmid to obtain a recombinant plasmid; the obtained recombinant plasmid was transformed into Escherichia coli Nissle 1917 by heat shock to obtain engineered probiotics; single colonies of the obtained engineered probiotics were inoculated into LB liquid medium containing 50 mg / mL kanamycin and cultured at 37°C and 200 rpm until OD500. 600=0.6, then add IPTG to a final concentration of 3mM, and induce at 18℃ and 200rpm for 14h to obtain the induced product; centrifuge the obtained induced product at 4℃ and 5400rpm for 5min, discard the supernatant, collect the precipitate and wash it 3 times with sterile water, resuspend the washed precipitate in 50mL of 2mM chloroauric acid aqueous solution, incubate at 37℃ for 5h, then centrifuge at 4℃ and 5400rpm for 5min, discard the supernatant, collect the precipitate and wash it 3 times with sterile water to obtain the probiotics producing nano-gold.

[0058] S2: Dissolve 1 mg of human serum albumin in 1 mL of sterile water to obtain a human serum albumin solution, and dissolve 6 mg of curcumin in 4 mL of anhydrous ethanol to obtain a curcumin solution. Adjust the pH of the obtained human serum albumin solution to 8.0, and then add the obtained curcumin solution dropwise at a rate of 1 mg / min while stirring at 12000 rpm. Continue stirring for 4 h, then add 20 μL of 4 vol% glutaraldehyde aqueous solution, and continue stirring for 20 h. Centrifuge at 20 °C and 12000 rpm for 15 min, discard the supernatant, collect the precipitate, and obtain curcumin albumin nanoparticles.

[0059] S3: Mix the gold-producing probiotics and curcumin albumin nanoparticles in sterile water to obtain mixed aqueous solution 1. The concentration of the gold-producing probiotics in mixed aqueous solution 1 is 1×10⁻⁶. 9 The concentration of CFU / mL and curcumin albumin nanoparticles was 600 μg / mL. The resulting mixed aqueous solution 1 and a 4 wt% sodium alginate aqueous solution were mixed evenly at a volume ratio of 1:1 to obtain mixed aqueous solution 2. The mixed aqueous solution 2 was added to a disposable syringe. A medical 21G flat-tipped needle was used, with an electrode attached to the needle (positive electrode). A voltage of 10 kV was applied, and 10 mL of mixed aqueous solution 2 was added dropwise to 200 mL of 0.1 wt% CaCl2 aqueous solution at a rate of 100 μL / min using a micro-injection pump to carry out the cross-linking reaction. The distance between the needle and the surface of the CaCl2 aqueous solution was 9 cm. Sodium alginate microspheres co-loaded with gold nanoparticles, probiotics, and curcumin albumin nanoparticles were obtained.

[0060] Performance testing:

[0061] (1) The morphological characterization of the gold nanoparticle-producing probiotics prepared in Example 1 was performed using scanning electron microscopy and transmission electron microscopy. The results are as follows: Figures 1-2 As shown in the figure, the gold nanoparticles are distributed on both the surface and inside the bacteria.

[0062] (2) The particle size of the gold nanoparticles distributed on the gold nanoparticle-producing probiotics in Example 1 was statistically analyzed. The specific procedure was as follows: the gold nanoparticle-producing probiotics were resuspended in 15 mL of PBS, and the bacteria were disrupted using a cell disruptor with parameters set to 400 W for 30 min. After disruption, the cells were centrifuged at 4 °C and 12000 rpm for 30 min. The supernatant was collected, and a small droplet was placed on a copper grid. The particle size was statistically analyzed using Nanom measurer software. The results are as follows: Figure 3 As shown in the figure, the average particle size of the gold nanoparticles distributed on the gold-producing probiotics is 16 nm.

[0063] (3) The morphology of the curcumin albumin nanoparticles prepared in Example 1 was characterized using scanning electron microscopy and transmission electron microscopy, and the results are as follows: Figures 4-5 As shown in the figure, the curcumin albumin nanoparticles have a uniform spherical structure, are of uniform size, and are well dispersed.

[0064] (4) The particle size of the curcumin albumin nanoparticles prepared in Example 1 was characterized by dynamic light scattering, and the results are as follows: Figure 6 As shown in the figure, the average particle size of the curcumin albumin nanoparticles is 232 nm, and the particle size distribution index is 0.1, indicating that the nanoparticles are uniformly distributed.

[0065] (5) The morphology of the sodium alginate microspheres prepared in Example 1 was characterized using laser confocal microscopy. Specifically, the probiotics producing gold nanoparticles were first labeled with Cy5.5-NHS, and then the sodium alginate microspheres were prepared according to the aforementioned steps. The sodium alginate microspheres were placed in a laser confocal dish and photographed under a laser confocal microscope. The results are as follows: Figure 7 As shown in the figure, the sodium alginate microspheres exhibit a uniform spherical structure, and the gold-producing probiotics and curcumin albumin nanoparticles are evenly distributed inside the microspheres.

[0066] (6) The production of gold nanoparticles in the gold-producing probiotics of Examples 1-3 was characterized using ultraviolet spectrophotometry. The method was as follows: the gold-producing probiotics were resuspended in 15 mL of PBS, and the bacteria were disrupted using a cell disruptor at 400 W for 30 min. After disruption, the bacteria were centrifuged, and the supernatant was collected at 4 °C and 12000 rpm for 30 min. 2 mL of the supernatant was then analyzed using an ultraviolet spectrophotometer. The results are as follows: Figure 8 As shown in the figure, typical absorption peaks of gold nanoparticles appear around 520 nm, and the supernatant exhibits the characteristic purple color of gold nanoparticles, indicating the successful preparation of gold nanoparticles.

[0067] (7) The activity of the probiotics producing nano-gold in Examples 1-3 was statistically analyzed using the viable bacteria plate count method. The specific procedure was as follows: 1 mL of the probiotic suspension producing nano-gold was diluted 10... 7The bacterial cells were then spread 100 μL onto LB agar plates using a spreader, and incubated at 37°C for 16 hours. Colony forming units (CFU) were calculated, where the number of viable cells per unit volume (CFU / mL) = the average number of colonies at the same dilution gradient × the dilution factor. Results are shown below. Figure 9 As shown in the figure, the probiotics that produce gold nanoparticles prepared under different conditions still retain their activity while producing gold nanoparticles.

[0068] (8) The ability of the gold nanoparticle-producing probiotics in Example 1 to scavenge reactive oxygen species was characterized. Specifically, the gold nanoparticle-producing probiotics were first resuspended in 15 mL of PBS, and the bacteria were disrupted using a cell disruptor at 400 W for 30 min. After disruption, the bacteria were centrifuged, and the supernatant was collected. The centrifuge parameters were set to 4 °C and 12000 rpm for 30 min to obtain AuNPs after disruption of the gold nanoparticle-producing probiotics. Then, using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate, the peroxidase-like activity of the AuNPs after disruption of the gold nanoparticle-producing probiotics was detected at room temperature in a sodium acetate buffer solution containing H2O2. The reaction system included: TMB (0.2 mM), H2O2 (0.08 mM), AuNPs (broken from gold nanoparticle-producing probiotics), and 200 μL HAc-NaAc buffer (0.1 M, pH = 4.5). The reaction was carried out for 15 min, and the UV absorption peaks were measured in the 200–800 nm range. We used the 3,3',5,5'-tetramethylbenzidine (TMB) method. In the presence of peroxidase, TMB was oxidized by hydrogen peroxide to form a blue substance, with a characteristic absorption peak at 655 nm. The results are as follows: Figure 10 As shown. Figure 10 It can be seen that only the system containing AuNPs after the breakdown of probiotics producing gold nanoparticles, H2O2, and TMB turns blue [the color of oxidized TMB (oxTMB)], and the corresponding absorption spectrum shows a strong absorption peak at 652 nm, indicating that the AuNPs after the breakdown of probiotics producing gold nanoparticles have strong peroxidase activity.

[0069] (9) The ability of curcumin albumin nanoparticles in Example 1 to scavenge reactive oxygen species was characterized. Specifically, DPPH was used to characterize the reactive oxygen species scavenging ability. The principle is that DPPH molecules contain non-shared electrons (N·), which, when reacting with electrons and free radicals, will transform into a stable structure (NH). DPPH appears purple in aqueous solution, but when free radicals are scavenged and a covalent bond structure is formed, the color changes to pale yellow. The free radical scavenging ability of DPPH was determined by measuring at 517 nm. Specifically, 60 μM DPPH was prepared with anhydrous ethanol. Curcumin albumin nanoparticles of different concentrations were added to the DPPH solution and reacted for 30 min. The ultraviolet absorption at 400–800 nm was then measured. The results are as follows: Figure 11 As shown in the figure, the absorption at 517 nm gradually decreases with the increase of curcumin albumin nanoparticle concentration, indicating that curcumin albumin nanoparticles have a good ability to scavenge DPPH.

[0070] (8) In vitro targeting characterization of sodium alginate microspheres from Example 1 was performed. Specifically, 100 mg of sodium alginate microspheres were placed in 1 mL of simulated intestinal fluid and simulated gastric fluid, respectively, and incubated 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, sodium alginate microspheres do not burst in simulated gastric juice, but burst after 3 hours in simulated intestinal juice, further demonstrating that sodium alginate microspheres have targeting properties.

[0071] (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 feeding for 7 consecutive days. Sodium alginate microspheres were then administered via gavage every other day for four administrations, ending the treatment. During this period, the mice's condition, weight, and fecal condition were observed daily. After treatment, the mice were dissected, and the colons were taken for pathological analysis. The results are as follows: Figures 14-17 As shown.

[0072] The figures show the weight changes of mice from 0 to 7 days during the induction of the IBD model, and the weight changes from 7 to 12 days during the treatment process. As can be seen from the figure, during the treatment process, the DSS group still showed a continuous decrease in weight, lethargy, and diarrhea, while the weight of mice in the healthy group increased slowly. Compared with DSS, the weight of mice in the drug treatment group increased slowly and basically returned to the pre-model weight after the treatment.

[0073] The treatment effect was assessed by measuring the colon length in mice. As shown in the figure, the colon length in the treatment group was significantly increased compared to the DSS group, but there was no significant difference compared to the healthy group, indicating that the treatment group treated with sodium alginate microspheres had a good therapeutic effect.

[0074] H&E sections revealed that the healthy mice had abundant intestinal glands in the mucosa, intact epithelium, normal cell morphology, abundant goblet cells, and intact crypts, with no obvious damage or inflammatory response. In contrast, the DSS group showed crypt structure destruction, loss of goblet cells, replacement by proliferating connective tissue, and extensive inflammatory cell infiltration. The drug-treated group showed recovery of the mucosa, normal cell morphology, restored goblet cell abundance, and restored crypt structure, with no significant difference from the healthy group.

[0075] In summary, sodium alginate microspheres co-loaded with gold nanoparticles, probiotics, and curcumin albumin nanoparticles exhibit strong anti-inflammatory effects, can treat colon tissue damage caused by DSS, and demonstrate excellent anti-inflammatory properties.

[0076] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for preparing sodium alginate microspheres co-loaded with gold nanoparticles, probiotics, and curcumin albumin nanoparticles, characterized in that: Perform the following steps under aseptic conditions: S1: The human metallothionein 1E gene (GenBank accession number NM_175617.4) was ligated into the pET-28a plasmid to obtain a recombinant plasmid; the recombinant plasmid was transformed into *E. coli* Nissle 1917 by heat shock to obtain engineered probiotics; single colonies of the engineered probiotics were inoculated into LB liquid medium containing 50 mg / mL kanamycin and cultured at 37°C and 200 rpm until OD500. 600 =0.6 After adding IPTG, induce at 18℃ and 200rpm for 14h to obtain the induced product; centrifuge the obtained induced product, discard the supernatant, collect the precipitate and wash with sterile water, resuspend the washed precipitate in 50mL chloroauric acid aqueous solution, incubate at 37℃ for 3~5h, then centrifuge, discard the supernatant, collect the precipitate and wash with sterile water to obtain the probiotics that produce nano-gold. S2: Dissolve human serum albumin in sterile water to obtain human serum albumin solution, and dissolve curcumin in anhydrous ethanol to obtain curcumin solution; The pH of the obtained human serum albumin solution was adjusted to 8.

0. Then, the obtained curcumin solution was added dropwise to it at a certain rate while stirring. Stirring was continued for 4 hours. Then, glutaraldehyde aqueous solution was added and stirring was continued for 20 hours. After centrifugation, the supernatant was discarded and the precipitate was collected to obtain curcumin albumin nanoparticles. S3: Mix the gold-producing probiotics and curcumin albumin nanoparticles in sterile water to obtain mixed aqueous solution 1; stir the obtained mixed aqueous solution 1 and sodium alginate aqueous solution evenly to obtain mixed aqueous solution 2; add the obtained mixed aqueous solution 2 to a disposable syringe, use a medical 21G flat-tipped needle, attach an electrode to the syringe needle with the needle as the positive electrode, apply voltage, and use a micro-injection pump to drop mixed aqueous solution 2 into CaCl2 aqueous solution at a certain rate to carry out cross-linking reaction, keeping a certain distance between the needle and the surface of CaCl2 aqueous solution, to obtain sodium alginate microspheres co-loaded with gold-producing probiotics and curcumin albumin nanoparticles.

2. The preparation method according to claim 1, characterized in that: In step S1, the centrifugation parameters are: temperature 4℃, rotation speed 4500~6000rpm, and time 4~10min.

3. 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.

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

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

20.

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

7. A sodium alginate microsphere co-loaded with gold nanoparticles, probiotics, and curcumin albumin nanoparticles, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 6.

8. The use of sodium alginate microspheres co-loaded with gold nanoparticles and curcumin albumin nanoparticles as described in claim 7 in the preparation of a medicament for treating inflammatory bowel disease.

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