Leakage-free core-shell hydrogel microsphere delivery system for non-contact flora transplantation as well as construction method and application of leakage-free core-shell hydrogel microsphere delivery system

Abstract

The invention discloses a leak-free core-shell hydrogel microsphere delivery system for non-contact flora transplantation as well as a construction method and application of the leak-free core-shell hydrogel microsphere delivery system. According to the invention, bacteria are encapsulated in a core of a core-shell microsphere through a two-step photo-initiation emulsion polymerization method of methacrylic acid gelatin, and a non-contact microbiota transplantation system (NMTS) is constructed. The NMTS can be used for biological encapsulation of various strains, shows encapsulation efficiency close to 100%, and has little influence on bacterial activity at the same time. NMTS demonstrates: a shielding effect that protects sealed bacteria from low pH values in the gastrointestinal tract and bile acid disruption; sufficient mechanical strength ensures that bacterial leakage is prevented and the infection risk is eliminated in the whole gastrointestinal tract delivery process; and internal and external substance exchange is realized, and the intestinal microorganism structure is remodeled. The invention provides a unique bacterial cell microencapsulation method which is a non-contact, safe and effective micropopulation transplantation method.

Description

Technical Field

[0001] The invention relates to a leakage-free core-shell hydrogel microsphere delivery system for contactless flora transplantation and a construction method and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Microbes are ubiquitous in the human body, with trillions of bacteria present on all available surfaces of the human body, such as the skin, oral cavity, gastrointestinal (GI) tract, and urogenital tract, playing a key role in maintaining tissue homeostasis and microecological balance. Disruption of the balance of microbial communities may lead to metabolic and immune disorders, and even cause the occurrence and development of different diseases. For example, increasing evidence shows that intestinal flora imbalance is associated with the occurrence of inflammatory bowel disease, obesity, diabetes, arthritis, hypertension, heart disease, Alzheimer's disease, and cancer. Transplanting beneficial bacteria to the corresponding parts to intervene and treat diseases by reshaping the microbial composition has been widely explored. Among them, representative fecal microbiota transplantation has been used to eliminate Clostridium difficile infection, while vaginal microbiota transplantation has been successfully used to treat vaginitis and restore a healthy microbial balance. Unfortunately, the US Food and Drug Administration has terminated several fecal microbiota transplantation-related clinical trials due to uncontrollable pathogen colonization and even severe infection or death caused by the introduction of antibiotic-resistant bacteria. In addition, transplanting specific types or combinations of natural probiotics or genetically modified bacteria has been widely used to minimize side effects. For example, natural lactic acid bacteria have been transplanted to prevent recurrent urinary tract infections, while engineered Bdellovibrio bacteriovorus has been used to clean dental plaque biofilms and alleviate periodontitis without causing significant safety issues. Despite these achievements, previous transplantation methods have exposed exogenous bacteria to the in vivo environment, resulting in direct contact between the transplanted bacteria and the surrounding environment, which may induce unpredictable adverse effects through colonization competition and secretion of large amounts of invasive substances. At the same time, direct contact of transplanted bacteria in the body always has the problem of low bioavailability, because various biological damages inevitably impair the activity of bacteria. For example, after oral administration, bacteria are easily inactivated by the intestinal environment, especially gastric acid and bile acid. In the past few years, our group and other research groups have developed many engineering strategies to carefully endow bacteria with various advantages, such as improved bioavailability, enhanced disease site targeting and colonization ability, reduced toxicity, and improved efficacy. However, methods to avoid direct contact between implanted bacteria and the host to improve bioavailability and eliminate safety issues during transplantation have not been reported. Summary of the invention

[0003] The purpose of the present invention is to provide a leakage-free core-shell hydrogel microsphere delivery system for contactless microbiota transplantation, a construction method and application thereof, in view of the problems of low bioavailability and infection risk inevitably existing in the prior art when beneficial bacteria are transplanted into a specific microbiota to treat diseases by reshaping a healthy microbial structure due to direct contact of exogenous bacteria in the body.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] In a first aspect, the present invention provides a method for constructing a leakage-free core-shell hydrogel microsphere delivery system for contactless bacterial transplantation, wherein the method comprises the following steps: forming gelatin methacrylate core-shell hydrogel microspheres by two-step photo-initiated gelatin methacrylate (GelMA) free radical emulsion polymerization, and encapsulating probiotics in the core of the gelatin methacrylate core-shell hydrogel microspheres; and

[0006] Step 1: Disperse the live probiotics in a PBS solution containing a photoinitiator and methacrylate gelatin, then drop the solution into the oil phase, and initiate free radical emulsion polymerization under light conditions to obtain bacteria-loaded core hydrogel microspheres;

[0007] Step 2: The bacteria-loaded core hydrogel microspheres obtained in step 1 are dispersed in an aqueous solution containing a photoinitiator and methacrylate gelatin, and then added dropwise to the oil phase to further initiate free radical emulsion polymerization under light conditions to obtain a core-shell hydrogel microsphere delivery system loaded with probiotics.

[0008] Furthermore, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

[0009] Furthermore, the oil phase comprises mineral oil and Span 80.

[0010] Furthermore, the content of Span 80 in the oil phase is 0.5-2 wt %.

[0011] Furthermore, the colony count of the live probiotics is 1×10 8 CFUs.

[0012] Furthermore, the live probiotics include but are not limited to Escherichia coli.

[0013] Furthermore, the PBS solution in step 1 contains 0.1-1 wt % of photoinitiator and 5-15 wt % of GelMA.

[0014] Furthermore, the aqueous solution in step 2 is a PBS solution containing 0.1-1 wt% of a photoinitiator, 5-15 wt% of GelMA and bacteria-loaded core hydrogel microspheres.

[0015] In a second aspect, the present invention provides a core-shell hydrogel microsphere delivery system constructed by the construction method described in the first aspect, wherein the core-shell hydrogel microsphere delivery system is loaded with live probiotics.

[0016] In a third aspect, the present invention provides an application of a core-shell hydrogel microsphere delivery system constructed by the construction method described in the first aspect in transplanting probiotics for non-diagnostic and therapeutic purposes, preparing probiotic products, or preparing products for transplanting probiotics.

[0017] Furthermore, the transplantation of probiotics includes but is not limited to transplanting probiotics into the skin, oral cavity, gastrointestinal tract, urogenital tract, lungs and other parts.

[0018] In a fourth aspect, the present invention provides the use of a core-shell hydrogel microsphere delivery system constructed by the construction method described in the first aspect in the preparation of a product for treating inflammatory diseases and / or diseases related to intestinal flora imbalance.

[0019] Furthermore, the inflammatory diseases include inflammatory diseases of the skin, oral cavity, gastrointestinal tract and urogenital tract.

[0020] Furthermore, the diseases related to intestinal flora imbalance include but are not limited to diabetes, arthritis, hypertension, heart disease, Alzheimer's disease and related cancers caused by persistent gastrointestinal inflammation.

[0021] Furthermore, the gastrointestinal inflammatory disease includes but is not limited to colorectal inflammation.

[0022] In some embodiments of the present invention, a core-shell hydrogel microsphere delivery system is provided for use in preparing a product for treating colitis, wherein the core-shell hydrogel microsphere delivery system is loaded with Escherichia coli (EcN). The core-shell hydrogel microsphere delivery system exhibits good safety and effective therapeutic properties, and can still safely and effectively treat colitis in a clinical situation where a simulated transplanted microbiome is accidentally contaminated by pathogenic bacteria.

[0023] The present invention provides a method for constructing a leak-free core-shell hydrogel microsphere delivery system for contactless microbiota transplantation, and the construction of a leak-free, contactless microbiota transplantation system (NMTS) is achieved through core-shell microgels. Figure 1). With the help of two-step photo-initiated free radical emulsion polymerization of methacrylated gelatin (GelMA), the target bacteria can be simply encapsulated into the core of the formed core-shell microgel. NMTS has a nearly 100% encapsulation efficiency and can be used for bioconfinement and confinement of a variety of bacterial strains. With the network structure of gelatin hydrogel, NMTS has little effect on the viability of bacteria and has good permeability, which is beneficial for the absorption of nutrients and the excretion of metabolites. As a proof-of-principle test for transplanting probiotics into the intestinal microbiota, NMTS showed a protective effect, protecting the carried bacteria from the threat of low pH and bile acids in the gastrointestinal tract after oral administration. NMTS also has sufficient mechanical strength to maintain the integrity of the microgel, avoiding the leakage of bacteria during the passage through the digestive tract, thereby avoiding the risk of infection. When evaluated in antibiotic-induced mice, NMTS effectively restored the balanced composition of the intestinal microbiota. In a mouse colitis model, oral administration of NMTS was able to safely and effectively treat mice, prevent pathogen infection, and alleviate intestinal damage and inflammatory responses. Therefore, NMTS provides an attractive strategy for encapsulation of bacterial cells and offers a contactless platform for safe and effective microbiota transplantation.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1) With the help of two-step photo-initiated free radical emulsion polymerization of methacrylated gelatin (GelMA), the target bacteria can be simply encapsulated into the core of the formed core-shell microgel; NMTS has an encapsulation efficiency of nearly 100% and can be used for biocontainment of a variety of bacterial strains; after oral administration, NMTS exhibits a shielding effect to protect the encapsulated bacteria from intraluminal invasion; sufficient mechanical strength to prevent bacteria from leaking during the entire gastrointestinal tract and eliminate the risk of infection; good permeability to allow the exchange of internal and external substances and regulate the intestinal microbiota;

[0026] 2) The NMTS-encapsulated probiotics provided by the present invention show good therapeutic efficacy and safety: oral NMTS can safely and effectively treat colitis in mice. In clinical scenarios, when the microbial flora taken is contaminated by pathogens, NMTS can block pathogen infection and reduce intestinal inflammation and tissue damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1Schematic diagram of the design and preparation of NMTS for contactless microbiota transplantation: bacteria are encapsulated into the core of core-shell microgels through two-step photoinitiated emulsion polymerization of GelMA; after oral administration, NMTS exhibits a shielding effect, protecting the encapsulated bacteria from intraluminal invasion; sufficient mechanical strength to prevent bacterial leakage during passage through the entire gastrointestinal tract and eliminate the risk of infection; and good permeability to allow the exchange of internal and external substances and regulate the intestinal microbiota.

[0028] Figure 2 Characterization of NMTS and the results of biological activity detection of encapsulated bacteria; (af) Schematic diagrams and typical LSCM images of core microspheres encapsulating different bacteria in a single form: (a) Schematic diagram of core microspheres encapsulating EcN; (b) LSCM image of EcN expressing mCherry encapsulated in FITC-labeled GelMA microspheres, scale bar: 50μm; (c) Schematic diagram of core microspheres encapsulating VNPs; (d) LSCM image of VNPs expressing GFP encapsulated in RhB-labeled GelMA microspheres, scale bar: 50μm; (e) (f) LSCM image of core microspheres encapsulating EcN and VNPs; (g) Schematic diagram of core-shell microspheres with bacteria carried in the core; (h, i) Typical LSCM images of core-shell microspheres with bacteria carried in the core, scale bars: 50 μm (h), 100 μm (i); (j) Count of unencapsulated bacteria in all components collected during the preparation process; (k) Size distribution of core microspheres, obtained by Image J software for statistical analysis of approximately 100 microspheres; (l) Schematic diagram of NMTS maintaining bacterial viability and metabolic activity; (m) GFP fluorescence intensity of free EcN expressing GFP and NMTS; (n) Digital photo of indole test of tryptophan metabolism of bacteria after encapsulation, black arrows indicate positive purple indole rings; (o) Indole production was determined by HPLC; Data are expressed as mean ± standard deviation; Statistical analysis was performed using unpaired Student's t test; ns, no significant difference.

[0029] Figure 3The resistance of NMTS to simulated gastrointestinal environment in vitro is demonstrated; (a) Schematic diagram of NMTS resistance to gastrointestinal peristaltic contraction; (b) Schematic diagram of NMTS resistance to gastric acid and bile salts; (c) Rheological properties of core-shell hydrogel microspheres and (d) bulk hydrogels in frequency sweep mode at 37°C; (e) Compression stress-strain curve of bulk hydrogel, with the compression strain set to 60%; (f, g) GFP-expressing free EcN or NMTS after incubation with (f) SGF and (g) bile acid at different time points under stirring at 37°C. Fluorescence intensity; (h) Representative LSCM images of NMTs encapsulated with mCherryEcN after different treatments at 37°C with stirring; SGF: incubated in SGF for 15 min; SIF: incubated in SIF for 1 h; bile acid: incubated in bile acid for 1 h; SGF-SIF: incubated in SGF for 15 min and then incubated in SIF for 1 h; Scale bar: 50 μm; (i) The number of bacteria leaked from NMTs after different treatments; Results are expressed as mean ± SD; Statistical analysis was performed using unpaired Student's t-test, and p values ​​are given, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant difference.

[0030] Figure 4 The in vivo bioencapsulation and retention of NMTS are shown; (a) Schematic diagram of good bioencapsulation of NMTS in vivo; (b) Experimental design of in vivo bioencapsulation: mice were given 1×10 8 CFUs EcN ECM or NMTS, free EcN as control, gastrointestinal tract was obtained at the indicated time points, and the contents and tissues of the stomach, small intestine, cecum and colon were homogenized and used for bacterial plate counting; (ce) The number of bacteria leaked in the stomach, small intestine, cecum and colon after administration for (c) 4 hours, (d) 8 hours and (e) 24 hours; (f) Typical LSCM image of NMTS encapsulating EcN expressing mCherry present in the stomach 4 hours after administration, scale bar: 50μm; (g) NM obtained from feces Optical microscopy of TS, scale bar: 50 μm; (h) IVIS images of the gastrointestinal tract 4 and 18 hours after gavage of free EcN or NMTS expressing mCherry; (i) Quantitative results of gastrointestinal mCherry fluorescence intensity at different time points; error bars represent standard deviation (n=4); statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test, and p values ​​are given, **p<0.01, ***p<0.001, ****p<0.0001; ns, no significant difference.

[0031] Figure 5The ability of NMTS to eliminate pathogen infection is demonstrated; (a) Schematic diagram of the occurrence and severity of pathogen infection eliminated by NMTS; (b) Experimental design for NMTS safety evaluation: mice were treated with streptomycin on day -1, and then orally gavaged with 8×10 6 NMTS of CFUs STm, and samples were taken 2 days after infection, unencapsulated STm and PBS were used as controls; (c) Body weight changes of mice during the experiment; (d) Survival curves of mice after different treatments; (e) Spleen weights of mice after different treatments; (f) Numbers of STm in gastrointestinal segments, (g) major organs and (h) blood; (ik) Routine blood analysis, including (i) WBC, (j) RBC and (k) PLT counts; (l,m) Cytokine levels in serum, including (l) TNF-α and (m) IL-6, detected using ELISA kits; Representative colon (n) H&E and (o) MPO staining images, scale bar: 100 μm; error bars represent standard deviations (n ​​= 5); One-way ANOVA and Tukey's multiple comparison test and unpaired Student's Statistical analysis was performed using t-test, and p values ​​are given as *p<0.05, **p<0.01, ***p<0.001; ns, no significant difference.

[0032] Figure 6 The potential of NMTS-mediated intestinal flora regulation is demonstrated; (a) Schematic diagram of the effect of NMTS on regulating imbalanced intestinal flora; (b) Experimental design for evaluating the effect of NMTS: mice were fed with 0.5 mg / ml cefoperazone for 14 days to induce intestinal flora disturbance, and then 1×10 8 Figure 3 NMTS of CFUs EcN. The cecum was obtained the next day for 16S rRNA sequencing and analysis. Unencapsulated EcN and PBS were used as controls. (c, d) Bacterial taxonomic spectrum of intestinal microorganisms at the (c) phylum and (d) family levels in cecal contents of cefoperazone-infected mice treated with PBS, EcN or NMTS. (e) PCA results of intestinal microorganisms of infected mice at the phylum level. Short distances between groups indicate small differences between groups. (f) Relative abundance of Bacteroidetes in the intestinal microbiota. (g) Ratio of relative abundance of Proteobacteria to Bacteroidetes. Relative abundance of (h) Firmicutes and (i) Enterobacteriaceae in the intestinal microbiota. Error bars represent standard deviations (n ​​= 5). Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test. p values ​​are given, *p < 0.05, **p < 0.01; ns, no significant difference.

[0033] Figure 7Demonstrates the value of NMTS for safe treatment; (a) Schematic diagram of safe and effective treatment mediated by NMTS; (b) Experimental design for evaluating the safety and efficacy of NMTS: mice were pretreated with streptomycin on day -1 and then orally administered with 8×10 6 Colitis was induced by infection with CFUsSTm. 5×10 6 CFUs EcN and 5×10 3 NMTS of CFUs STm, and samples were taken on the 4th day. PBS and equal amount of free EcN+STm groups were used as controls, respectively. (c) Changes in mouse body weight during the experiment; (d) Survival curves of mice after different treatments; (e) Number of STm in stomach, (f) small intestine, (g) cecum and (h) colon; (i) Number of EcN in stomach, (j) small intestine, (k) cecum and (l) colon; (m) Spleen weight of mice after different treatments; (n,o) Serum Cytokine levels, (n) TNF-α and (o) IL-6; (p) H&E staining and (q) MPO staining images of colon, scale bar: 100 μm; error bars indicate standard deviation (n=4 or 5); statistical analysis was performed using unpaired Student's t test and one-way ANOVA with Tukey's multiple comparison test between two groups, and p values ​​are given, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0034] Figure 8 Schematic diagram of the synthesis route of GelMA and the photo-crosslinking process of GelMA hydrogel.

[0035] Fig. 9 For Gelatin and GelMA 1 H NMR spectrum.

[0036] Fig.10 The standard curve of indole concentration and integrated area determined by HPLC.

[0037] Fig.11 is the bacterial leakage rate of NMTS under different simulated gastrointestinal environment treatments.

[0038] Fig.12 : (a) Colony plate images of EcN leaked from core microspheres (ECM, middle) and core-shell microspheres (NMTS, right) in the stomach, small intestine, colon and cecum 4 hours after oral administration. Unencapsulated EcN was used as a control (left), and the image is the result of a 10-fold dilution; (b) The number of bacteria leaked in feces 4, 8 and 24 hours after administration; (c) Digital photo of hydrogel microspheres in the dissected stomach 4 hours after administration, yellow arrow: hydrogel microspheres.

[0039] Fig.13 Representative H&E staining images of mouse heart, liver, spleen, lung and kidney tissue sections after different treatments. Scale bar: 100 μm.

[0040] Fig.14 is the relative abundance of Proteobacteria in the gut microbiota.

[0041] Fig.15 Representative H&E staining images of the cecum. Black arrows: submucosal edema. Yellow arrows: disappearance of intestinal glands in the lamina propria. Scale bar: 100 μm.

[0042] Fig.16 Representative H&E staining images of mouse heart, liver, spleen, lung and kidney tissue sections after different treatments. Scale bar: 100 μm. DETAILED DESCRIPTION

[0043] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0044] Some of the experimental materials and methods involved in the embodiments of the present invention are as follows:

[0045] 1. Reagents and strains

[0046] Gelatin (240 g bloom) and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) were purchased from Aladdin. Methacrylic anhydride, mineral oil, and Tween 80 were provided by Sigma-Aldrich. PBS buffer (10×, pH 7.4) was provided by Sangon Biotech. PBS used in all experiments was diluted 10 times. Deionized water was used to prepare all aqueous solutions. Unless otherwise stated, other reagents and solvents were provided by Sigma-Aldrich and used without further purification. Escherichia coli Nissle 1917 (EcN), Salmonella strain VNP20009, Escherichia coli BL21, and Salmonella STm were purchased from China General Microbiological Culture Collection Center (CGMCC, China). Plasmids pBBR1MCS2-Tac-mCherry (kanamycin resistance) and pBBR1MCS2-Tac-eGFP (gentamicin resistance) were obtained from domestic suppliers and used as received. All bacteria were grown in LB medium at 37 °C supplemented with appropriate antibiotics.

[0047] 2. Synthesis and characterization of methacryloyl gelatin (GelMA)

[0048] GelMA was synthesized by a one-step amidation reaction of gelatin with methacrylic anhydride ( Figure 8). The specific synthesis method includes: completely dissolving 5g of gelatin in 40ml of PBS at 50°C. Under stirring at 50°C, a certain amount of methacrylic anhydride is added dropwise to react with gelatin for 3 hours. Then, preheated PBS (50°C, 200ml) is added to stop the reaction. After 15 minutes, dialyze at 50°C for 1 week using a dialysis bag (molecular weight cutoff: 12-14kDa) to remove salts and unreacted methacrylic anhydride. Finally, the solution was freeze-dried for 5 days to obtain a white porous foam, which was stored at -20°C for use. The degree of methacryloyl substitution (DMS) of GelMA is defined as the percentage of lysine side chain amino groups in gelatin replaced by methacryloyl groups, which is measured by hydrogen nuclear magnetic resonance spectroscopy ( 1 HNMR) (Bruker Avance NEO 400M spectrometer). GelMA and gelatin were dissolved in deuterated water for testing. The peak area of ​​aromatic amino acids (7.1-7.4ppm) in GelMA or gelatin in each spectrum was used as a reference. The peak area of ​​lysine methylene (2.8-2.95ppm) was used to calculate DMS, and the calculation formula is as follows:

[0049]

[0050] according to 1 H NMR ( Fig. 9 ) test results, the DMS of GelMA was calculated to be 81.9%.

[0051] 3. Preparation and characterization of NMTS

[0052] NMTS were prepared by two-step photoinitiated free radical emulsion polymerization of GelMA, and bacteria were encapsulated in the core of the obtained core-shell hydrogel microspheres. 8 CFUs of live bacteria were dispersed in 200 μl of 10 wt% GelMA / PBS precursor solution (neutral, pH 7.4) containing 0.5 wt% photoinitiator LAP. The mixture was added dropwise to an oil phase consisting of 1 ml mineral oil and 1% Span 80 under stirring. UV light at 365 nm (10 mW / cm 2) for 1 minute, and then the bacteria-loaded core hydrogel microspheres (referred to as ECM) were separated from the mixture by centrifugation at 2500rpm for 2 minutes, and washed twice with PBS. To prepare NMTS, the obtained bacteria-loaded core microspheres (about 200μl) were dispersed in 400μl GelMA / LAP / PBS precursor solution (the content of the precursor solution components is the same as above), and the core-shell structure was obtained through a similar procedure. Finally, the core-shell microspheres encapsulating bacteria were resuspended in PBS for later use. The morphology of NMTS was observed by laser confocal scanning microscopy (LSCM, Leica&TCS SP8, Germany). For visualization, EcN expressing mCherry or GFP, VNPs expressing GFP, and GelMA labeled with FITC, RhB or Cy5.5 were used as needed. The size distribution of core microspheres (about 100) was determined by statistical analysis of multiple representative LSCM images using ImageJ software. All fractions in the preparation were collected, diluted appropriately, and incubated overnight on LB plates at 37°C to count bacterial colonies. The encapsulation efficiency of bacteria in NMTS was calculated by plate counting of unencapsulated bacteria in these fractions.

[0053] 4. Viability of bacteria after encapsulation

[0054] 1×10 8 CFUs of GFP-expressing EcN were encapsulated in NMTS. The difference in GFP fluorescence intensity between encapsulated bacteria and an equal amount of free EcN was measured at a wavelength of 535 nm using a Biotek microplate reader to assess the survival of encapsulated bacteria.

[0055] 5. Metabolic capacity of encapsulated bacteria

[0056] The tryptophan metabolism indole test was used to analyze the metabolism of encapsulated bacteria. 8 CFUs of EcN and BL21 were encapsulated in NMTS. NMTS was incubated in 4 ml sterile peptone medium (pH 7.6, 1 g peptone and 0.5 g sodium chloride per 100 ml distilled water) at 37°C for 2 days. Then a few drops of ether were added and mixed. After a few minutes, two drops of Kovac reagent were added to the supernatant to qualitatively show the production of indole. The purple indole ring in the upper ether of the mixture indicated a positive result, indicating that the encapsulated bacteria can secrete tryptophanase to metabolize tryptophan to produce indole. Equal amounts of free EcN and BL21 were used as controls, and peptone medium was used as a blank. High performance liquid chromatography (HPLC, Agilent & 1260 Infinity II, USA) was used for quantitative analysis of indole production, and 3 parallel samples were tested in each group.

[0057] The following HPLC test conditions were used: Diamonsil C18 column (5 μm, 150×4.6 mm). The column temperature and detection wavelength were set to 45° C. and 260 nm, respectively. The mobile phase consisted of 0.1% formic acid in water (volume / volume) and 0.1% formic acid in acetonitrile (volume / volume). The injection volume and flow rate were 5 μl and 1.0 ml / min, respectively.

[0058] 6. Rheological properties

[0059] The rheological properties of core-shell hydrogel microspheres and block hydrogels were measured using a rotational rheometer (RL-MARS3, Thermo Hakke, USA). A parallel plate rotor was used with a plate diameter of 20 mm and a plate gap of 1 mm. The test temperature was 37 °C, the strain was set to 1%, and the scanning frequency range was 0.01 to 50 Hz in oscillation mode. Core-shell hydrogel microspheres and block hydrogels (circular sheet samples, 20×2 mm) were prepared using 10 wt % of the GelMA precursor solution. The storage modulus (G') and loss modulus (G") were obtained.

[0060] 7. Compression performance

[0061] The compression performance test of the block hydrogel was carried out on a universal material testing machine (Instron-3342, 50N sensor) and repeated three times. The strain rate was set to 5 mm / min, the size of the cylindrical sample was 5×3 mm, and the compression strain was set to 60%.

[0062] 8. Protective effect of NMTS on SGF and bile acid

[0063] The NMTS encapsulating EcN expressing GFP was immersed in 1 ml of SGF (simulated gastric fluid, pH 1.2, containing 0.2 g of sodium chloride, 0.32 g of pepsin and 700 μl of 36.5% hydrochloric acid per 100 ml of distilled water) and gently shaken at 37°C. At the predetermined time point, the precipitate in the solution was collected, washed and resuspended in PBS. The GFP fluorescence intensity from the surviving bacteria was recorded at a wavelength of 535 nm by a microplate reader. Equal amounts of free EcN (1×10 8 CFUs) were used as a control, and the experiment was repeated for three parallel samples.

[0064] Similarly, 1×10 8 CFUs of EcN and NMTS were assessed by resuspending each in 1 ml of PBS supplemented with bile salts (0.3 mg / ml).

[0065] 9. In vitro evaluation of bioencapsulation of NMTS in simulated gastrointestinal environment

[0066] 8×10 8CFUs of EcN expressing mCherry were encapsulated into the core of NMTS and incubated in a constant temperature shaker (37°C, 300 rpm) with SGF (simulated gastric fluid), SIF (simulated intestinal fluid), bile acid, and the sequential combination of SGF and SIF. After different treatments (SGF: incubated in SGF for 15 minutes; SIF: incubated in SIF for 1 hour; bile acid: incubated in bile acid for 1 hour; SGF-SIF: incubated in SGF for 15 minutes and then incubated in SIF for 1 hour), all precipitates were collected, washed, spread on solid LB plates containing 100 μg / ml kanamycin, and incubated overnight at 37°C to count the leaked bacteria. At the same time, a small amount of precipitate was resuspended in PBS for LSCM observation.

[0067] 10. Experimental Animals

[0068] Female Balb / c mice aged 6–8 weeks and female ICR mice aged 6–8 weeks were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd. All animal experiments were performed in accordance with the ethics committee guidelines evaluated and approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University (A2020033).

[0069] 11. In vivo evaluation of NMTS bioencapsulation

[0070] Experiments were performed in female ICR mice aged 6-8 weeks. Four mice were used per group at each time point. After fasting for 12 h, mice were orally administered with free EcN expressing mCherry, EcN-loaded core microspheres (ECM), and EcN-encapsulated core-shell microspheres (NMTS). The number of bacteria was equal between the three groups (1×10 8 CFUs). After 4, 8 and 24 hours of administration, the contents and tissues of the stomach, small intestine, cecum and colon were collected and homogenized in PBS. 50 μl of the homogenate was spread on a solid LB agar plate and cultured at 37°C for 24 hours for bacterial counts. At the same time, feces were collected and resuspended in sterile PBS (1 g feces in 2 ml PBS) for bacterial plate counts, and hydrogel microspheres were obtained from the feces by gradient centrifugation to observe integrity. In addition, the stomach of NMTS-treated mice was collected 4 hours after administration and cut transversely. The integrity of the core-shell microspheres encapsulated with EcN in the stomach was observed by LSCM.

[0071] 12. Retention of NMTS in the gastrointestinal tract

[0072] Female ICR mice aged 6-8 weeks were used. They were randomly divided into groups (n=4 per group). After fasting for 12 hours (no food), NMTS encapsulating EcN expressing mCherry was orally administered to the mice, and the gastrointestinal tract of the mice was harvested at a predetermined time point after administration for IVIS imaging (INIS Lumina II, Caliper). 5×10 7 CFUs of free EcN were used as control.

[0073] 13. Construction of a mouse enteritis model infected with Salmonella typhimurium STm

[0074] Female Balb / c mice aged 6-8 weeks were pretreated with 100 μl of 200 mg / ml streptomycin orally one day before infection. The mice were randomly divided into groups (n=5 per group) and then given PBS, free STm (8×10 6 CFUs) and NMTS (8×10 6 CFUs). Mice were recorded daily during the experiment and samples were collected on day 5. Blood was analyzed for WBC, RBC, and PLT counts. Spleen weight was recorded and blood samples were centrifuged at 4000 g for 5 min to collect serum, where TNF-α and IL-6 concentrations were measured using ELISA kits. Gastrointestinal tract and major organs were collected for STm counts and histochemical analysis.

[0075] 14. Construction of a mouse model of intestinal flora disturbance induced by cefoperazone

[0076] Female Balb / c mice aged 6-8 weeks were fed with sterile drinking water containing 0.5 mg / ml cefoperazone for 14 days. The mice were then randomly divided into groups (n=5 per group) and given free EcN (1×10 8 CFUs) or NMTS (1×10 8 CFUs) or PBS. The cecal contents were harvested the next day for 16s rRNA sequencing and analysis.

[0077] 15.16s rRNA gene sequencing and analysis

[0078] The test was conducted by Beijing Biomarker Biotechnology Co., Ltd. Total DNA was extracted from the cecal contents, and sequencing libraries were constructed by Illumina HiSeq 2500 for sequencing, and the results were analyzed on the BMKCloud database analysis platform. Beta diversity analysis was performed using PCA. Microbial composition analysis was performed at the phylum and family levels.

[0079] 16. In vivo evaluation of the safety and therapeutic potential of NMTS

[0080] Female Balb / c mice aged 6-8 weeks were pretreated with 100 μl of 200 mg / ml streptomycin solution orally one day before infection. The mice were randomly divided into three groups (n=5 in each group) and orally administered STm (8×10 6 CFUs) to induce colitis. Starting from the next day, PBS, free EcN (5×10 6 CFUs) and STm (5×10 3 CFUs) and encapsulated EcN (5×10 6 CFUs) and STm (5×10 3 CFUs) of NMTS. The weight of mice was recorded daily during the experiment, and samples were collected for analysis on the 4th day. Bacterial plate counts of EcN and STm were performed on samples from various sections of the gastrointestinal tract, spleen weight was recorded, and blood samples were centrifuged at 4000g for 5 minutes to obtain serum, and the concentrations of TNF-α and IL-6 in them were determined using ELISA kits. The cecum and organs were fixed in 4% paraformaldehyde and subsequently subjected to H&E staining for histological analysis.

[0081] 17. Statistical analysis

[0082] All statistical analyses were performed using GraphPad Prism 8.0. Results are presented as mean ± standard deviation. Statistical analysis was performed using unpaired Student's t test between two groups or one-way analysis of variance (ANOVA) and Tukey's multiple comparison test between three groups, and p values ​​are given. Significance was defined as p<0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Pearson's correlation coefficient was used to measure the linear association between two variables.

[0083] Example 1: Design, preparation and characterization of NMTS

[0084] Polymer hydrogels are composed of a three-dimensional cross-linked network and a large amount of water, similar to natural extracellular matrix. Due to their porous structure and permeability, hydrogels are often used as scaffolds for cell culture and encapsulation. Gelatin-based hydrogels have excellent biocompatibility and adjustable physical and chemical properties, and have been widely used in the fields of tissue engineering and drug delivery. In order to ensure the integrity of the hydrogel during its passage through the entire digestive tract, the present invention selects GelMA as a monomer for preparing NMTS, which can adjust the mechanical strength of the hydrogel by changing the cross-linking density of the gelatin network in a simple and feasible manner. In order to resist the harsh environment of GI (GastroInestinal), maintain the survival rate of bacteria, and inhibit the leakage of encapsulated bacteria after transplantation, the present invention designs and prepares a core-shell microstructure, and encapsulates the target bacteria into the core of the microgel.

[0085] The synthesis route and characterization of GelMA are as follows: Figure 8 and Fig. 9 As shown. In order to prepare NMTS, the GelMA / LAP / PBS prepolymer containing live bacteria is cross-linked by photoinitiated free radical emulsion polymerization to obtain bacteria-loaded core microspheres, which are then further dispersed in the prepolymer and cross-linked in the same manner to obtain a core-shell structure. The present invention first selects Escherichia coli Nissle1917 (EcN) to prepare the bacteria-loaded core of NMTS. Laser scanning confocal microscopy (LSCM) images show that gelatin microspheres labeled with fluorescein isothiocyanate (FITC) co-localize with EcN expressing the red fluorescent protein mCherry, indicating that the bacteria are encapsulated in the hydrogel network ( Figure 2 a and b). To verify the versatility of this approach, we used attenuated Salmonella enterica serovar VNP20009 (VNPs), and the fluorescence signals emitted by gelatin hydrogels labeled with rhodamine isothiocyanate B (RhB) and VNPs carrying green fluorescent protein (GFP) overlapped, demonstrating that bacteria were also successfully encapsulated ( Figure 2 c and d). In addition, EcN and VNPs can also be loaded into the microspheres simultaneously, as evidenced by the presence of both mCherry and GFP fluorescence signals in GelMA microgels labeled with carboxy-cyanine dye Cy5.5. These results indicate that the microspheres are capable of encapsulating different bacteria in a single or combined form ( Figure 2 e and f). Figure 2 g and h confirm the formation of core-shell microgels. LSCM shows that the core-shell microgels are spherical with clear double-layer ring boundaries. The FITC-labeled microsphere cores are co-localized with bacteria expressing fluorescent proteins, proving that the bacteria are distributed in the cores of the microspheres ( Figure 2 i). The number of unencapsulated bacteria in all other components collected during the preparation process was counted using the Luria-Bertani (LB) agar plate counting method, and the bacterial encapsulation efficiency was calculated to be 99.93% ( Figure 2 j). Figure 2 As shown in Figure k, several representative LSCM images of core microspheres were statistically analyzed using ImageJ software to obtain a histogram of the core microsphere diameter, indicating that the diameter of the core microspheres was mainly distributed between 30 and 70 μm. In summary, NMTS can be used as a simple and universal delivery platform to achieve nearly complete bioencapsulation of various bacteria.

[0086] Example 2: Activity detection of bacteria after encapsulation

[0087] The activity of encapsulated bacteria is crucial to ensure the effectiveness of microbial transplantation. In order to study whether core-shell microgel-based bioencapsulation affects the activity of bacteria, this example measured the activity and metabolic capacity of encapsulated bacteria ( Figure 2l). The fluorescence intensity of EcN expressing pBBR1MCS2-Tac-GFP fluorescent protein was measured to evaluate the viability of encapsulated bacteria. Figure 2 As shown in Figure 5, the GFP fluorescence intensity of the encapsulated bacteria was comparable to that of the same amount of free EcN, indicating that the materials and procedures involved in the preparation of NMTS had little effect on bacterial activity. In addition, tryptophan metabolism was selected to study the metabolic capacity of encapsulated bacteria. A variety of bacteria can metabolize tryptophan and produce specific metabolites, such as indole and its derivatives, D-tryptophan, and serotonin. These bioactive molecules play a variety of functions in the intestine, including intestinal immune regulation and intestinal barrier protection. The indole test is to determine the ability of encapsulated bacteria to degrade tryptophan into indole by secreting tryptophanase. Figure 2 As shown in Figure 1, encapsulated EcN and Escherichia coli BL21 (BL21) showed comparable positive results with the corresponding equal amounts of free bacteria, with the appearance of purple indole rings in the upper ether of the reaction mixture after the addition of Kovac's reagent. The concentration of indole produced was quantitatively analyzed by high-performance liquid chromatography (HPLC). Interestingly, there was no significant difference in indole production between free bacteria and encapsulated bacteria (including EcN and BL21), which verified that encapsulation of these core-shell microgels had no significant effect on bacterial tryptophan metabolism ( Figure 2 o and Fig.10 ). In short, the bacteria in NMTS remain alive and can exchange substances with the surrounding environment in a non-contact manner to maintain their activity and metabolic capacity.

[0088] Example 3: Resistance of NMTS to in vitro simulated gastrointestinal environment

[0089] Although previous studies have reported that encapsulated E. coli cannot escape due to the limited pore size of the hydrogel (5-50 nm), the complex in vivo environment may pose a potential challenge to the leakage-free bioencapsulation of bacteria during transplantation. After oral administration, NMTS will inevitably be affected by the dynamic environment of the gastrointestinal tract, such as continuous peristalsis ( Figure 3 a), gastric acid and bile salts ( Figure 3 b) In this example, the rheological and compressive properties of core-shell hydrogel microspheres and block hydrogels were characterized to understand whether the mechanical strength of NMTS can withstand the damage of the gastrointestinal environment. First, the rheological properties of NMTS were measured using the frequency sweep mode, which is widely used to evaluate the stability of three-dimensional cross-linked networks. Figure 3c and d show the corresponding frequency variation curves of the storage modulus (G′) and loss modulus (G″). Within the tested frequency range, the G′ of both the core-shell microgel and the block hydrogel is greater than G″, showing typical gel properties. In addition, the results at low frequencies show that NMTS has a uniform and stable cross-linked network and solid-like properties without damage. Differently, at higher frequencies, the G′ and G″ of these hydrogels increase, which may be due to the rearrangement failure of the highly cross-linked polymer chains within the time scale of the applied motion, resulting in the hardening of the polymer network. Figure 3 As shown in Figure e, the block hydrogel with a solid content of 10% has high mechanical strength, a compression modulus of 0.93±0.22MPa, and a compressive stress of 88.97±7.94kPa at a compression strain of 60%, which is much higher than the intragastric pressure (12.9mmHg during feeding) and intestinal pressure (20mmHg at night in the jejunum). The toughness of this gelatin hydrogel demonstrates the potential of NMTS to resist mechanical stirring of intestinal peristalsis in the digestive tract environment and lays the foundation for obtaining leak-free bacterial encapsulation.

[0090] To evaluate the protective effect of NMTS, the survival rate of encapsulated bacteria after treatment with simulated gastric fluid (SGF) and bile salts was tested. NMTS encapsulated with GFP-expressing EcN were incubated in simulated gastric fluid (SGF) (pH 1.2) and bile salts (0.3 mg / ml) at 37 °C for different time points. 8 Free EcN of CFUs was used as a control. After only 5 minutes of immersion in SGF, the fluorescence intensity of free bacteria dropped sharply, indicating that most of the EcN was inactivated due to its inability to resist the low pH value ( Figure 3 f). In contrast, the vast majority of EcN encapsulated in NMTS remained viable at 30 min, and a small fraction even survived 2 h after exposure to SGF. In addition, NMTS effectively protected EcN encapsulated therein from the effects of bile salts, as the fluorescence intensity of the bacteria barely decreased even after 6 h of treatment, whereas this was not the case for free EcN ( Figure 3 g). Resistance to SGF and bile salts was greatly enhanced, indicating that NMTS is sufficient to protect the entrained bacteria from environmental insults.

[0091] Further, this example evaluates the bioencapsulation behavior of NMTS in a simulated digestive tract environment. EcN-encapsulated NMTS were co-cultured with SGF, simulated intestinal fluid (SIF), bile salts, and a combination of SGF and SIF at 37°C. The shaking speed was 300 rpm to simulate the shear forces associated with the digestive tract. At predetermined time points, the precipitate was collected for confocal imaging and bacterial plate counts to study the integrity of the core-shell microspheres, the survival rate of encapsulated bacteria, and the ability of NMTS to prevent bacterial leakage. Figure 3 h shows the morphology of microgel NMTS encapsulating EcN after different treatments, indicating that almost all EcN expressing mCherry are well embedded in the core and emit red fluorescence. LSCM images show that very few EcN are observed outside the microgel, which proves that the bacterial leakage of NMTS is limited. The quantitative calculation of the number of leaked bacteria by plating further proves that NMTS almost completely encapsulates bacteria under these conditions. Figure 3 i and Fig.11 As shown, the ratio of leaked EcN to encapsulated EcN was calculated to be 0% in SGF, 0.0450% in SIF, 0.0011% in bile salts, and 0% in the SGF-SIF combination. These in vitro experiments indicate that NMTS not only achieves intact bioencapsulation of bacteria with negligible leakage, but also protects the encapsulated bacteria from digestive tract-related damage.

[0092] Example 4: Bioencapsulation and retention of NMTS in vivo

[0093] The satisfactory bioencapsulation effect of NMTS on bacteria may be attributed to the core-shell structure of the hydrogel microspheres. To verify this hypothesis, this example further compared the encapsulation capacity of core microspheres and core-shell microspheres encapsulating EcN in vivo. The number of EcN leaked in the stomach, small intestine, cecum, colon and feces at the specified time points was obtained by plate counting method to quantify the difference between the core microspheres (ECM) and core-shell microspheres encapsulating EcN in mice orally ( Figure 4 a and b). Equal amounts of free EcN (1×10 8 CFUs) were used as controls. Figure 4 c and Fig.12 As shown in a and b, a significant reduction in the number of bacteria was observed in all samples collected from mice treated with core microspheres or core-shell microspheres 4 hours after administration compared with unencapsulated EcN. All samples in the core-shell microsphere group showed negligible leakage compared with the free EcN and EcN-encapsulated core microsphere groups. In particular, in the cecum and colon, the number of bacteria leaked from mice taking core-shell microspheres was significantly reduced by 98.89% and 99.11%, respectively, compared with mice taking core microspheres. Similar trends of NMTS-encapsulated EcN were also found in the digestive tract and collected feces as the administration time was extended to 8 hours and 24 hours ( Figure 4 d, e and Fig.12 b).

[0094] In addition, 4 hours after the oral administration, the stomach was collected and cut open along the transverse axis. Fig.12c shows a photo of the stomach cavity, verifying that the core-shell microspheres are still in the state of solid gel. The morphology of NMTS in the stomach was further observed by LSCM. Consistent with the in vitro bioencapsulation results for SGF, NMTS is still a core-shell structure, and the EcN carried is completely sealed in the microspheres ( Figure 4 f) In addition, NMTS was obtained in mouse feces after passing through the digestive tract. Figure 4 The optical microscopy image of g shows a relatively intact spherical structure. These data strongly demonstrate that NMTS with a core-shell structure has sufficient bacterial containment in vivo.

[0095] Adequate retention in the intestine is essential for the transplanted bacteria to exert their biological functions. In this example, the retention of NMTS in the mouse intestine was tracked by an in vivo imaging system (IVIS). 7 NMTS containing CFUs of EcN expressing mCherry was administered by gavage, and the intestine was harvested at the indicated time points and observed by IVIS. Obviously, the mCherry fluorescence signal harvested from the gastrointestinal tract of mice taking NMTS was much stronger than that of mice taking the same amount of free EcN at 4 and 18 hours after gavage, indicating that the bioavailability of encapsulated bacteria was greatly improved ( Figure 4 h). The fluorescence intensity in the gastrointestinal tract was further quantified by IVIS and increased by 3.54-fold and 6.50-fold compared with free bacteria at 4 and 18 hours after administration, respectively ( Figure 4 i) The improved bioavailability may be due to the protective effect of NMTS, which not only reduces the inactivation of encapsulated bacteria when exposed to the harsh digestive tract environment, but also the specific recognition between the intrinsic binding domains in the gelatin network and the arginine-glycine-aspartic acid sequence in the intestinal mucosa.

[0096] Example 5: NMTS eliminates pathogen infection during transplantation

[0097] During the clinical implementation of fecal microbiota transplantation, inadvertent introduction of pathogens into the intestine is one of the main causes of severe infection and even death. Our findings on non-leakage encapsulation of bacteria prompted us to further evaluate whether NMTS can eliminate potential safety issues of transplanted pathogens. Salmonella typhimurium (STm) is a common foodborne digestive tract pathogen that can induce systemic infection or severe gastroenteritis in mammals, including humans. Given that reducing the incidence and severity of pathogenic bacterial infections is an effective safety verification method, we then evaluated the hazards of NMTS by orally encapsulating microspheres of STm ( Figure 5 a) The mice were pretreated with streptomycin on day -1 and then orally administered with 8×10 6 CFUsSTm NMTS for 4 consecutive days, and collect samples on the 5th day ( Figure 5 b). The mice were orally administered with equal amounts of free STm and PBS as a control.

[0098] As expected, the body weight of mice treated with free STm dropped dramatically on day 3, and 40% of the mice survived beyond day 5 ( Figure 5 c and d). Unlike free STm, the food intake and activity of mice treated with NMTS were normal, comparable to those of mice treated with PBS. We found that the body weight of mice in the NMTS group was stable, and no mortality was observed during the experiment. To further evaluate the safety of NMTS encapsulated with STm, we collected the main organs, digestive tract, and peripheral blood of mice. There was no significant difference in spleen weight between the NMTS group and the PBS group, while there was a significant increase in the free STm group compared with the PBS group ( Figure 5 e). Figure 5 f and g show the number of STm in each segment of the digestive tract and major organs collected, respectively. A significant decrease in the number of bacteria was observed in all samples collected from mice in the NMTS group compared with the free STm group. In particular, in the small intestine, cecum, colon, liver, spleen, and lungs, the number of STm in NMTS-treated mice was significantly reduced by 95.16%, 94.75%, 94.94%, 94.32%, 88.48%, and 84.95%, respectively, compared with mice treated with free STm. A similar trend was also found in the blood ( Figure 5 h).

[0099] Next, we evaluated the inflammation associated with bacterial infection in treated mice using blood routine and cytokine assays. As expected, mice treated with free STm had higher white blood cell (WBC) counts and lower red blood cell (RBC) and platelet (PLT) counts ( Figure 5 ik). However, NMTS-treated mice showed similar blood cell levels to those of PBS-treated mice. Meanwhile, NMTS-induced inflammatory responses were negligible, as reflected by the fact that the levels of inflammatory cytokines in the serum did not change much compared with those in the PBS group, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) ( Figure 5 l and m). However, oral administration of unencapsulated STm stimulated a significant upregulation of these cytokine levels. Histological analysis by hematoxylin and eosin (H&E) staining further confirmed that no histological damage such as severe submucosal edema and disappearance of intestinal glands in the lamina propria was observed in colon tissue sections of NMTS-treated mice ( Figure 5 n). In addition, myeloperoxidase (MPO) staining showed that brown MPO-positive cells were widely distributed in the colon of free STm-treated mice, while the NMTS group did not show such severe infiltration ( Figure 5o). In fact, histological inflammation was only present in mice treated with free STm. In addition, H&E staining of heart, liver, spleen, lung, and kidney tissues confirmed that NMTS treatment induced no detectable damage and inflammatory response ( Fig.13 ). Therefore, these results suggest that NMTS can eliminate the potential hazards of transplanted pathogens, which is crucial to ensure the safety of microbiota transplantation.

[0100] Example 6: NMTS-mediated regulation of intestinal flora

[0101] After confirming negligible leakage, improved survival, and safety of encapsulated bacteria, we further evaluated the efficacy of NMTS in regulating intestinal flora imbalance ( Figure 6 a). EcN has been used to treat various digestive and metabolic diseases. Therefore, we selected EcN as a representative of probiotics to evaluate whether NMTS would affect the efficacy of EcN in the cefoperazone-induced mouse intestinal flora imbalance model. Mice were fed with drinking water containing 0.5 mg / ml cefoperazone for 14 days, and then NMTS encapsulated with EcN was orally administered to the mice every day for 5 consecutive days. On the second day, cecal content samples were collected and the microbial structure was analyzed by 16s RNA sequencing ( Figure 6 b). Free EcN and PBS were used as control groups. Figure 6 The results of c and d showed that the microbial composition of mice in the NMTS group was closer to that of mice in the EcN group than to that of mice in the PBS group. Principal component analysis (PCA) also yielded similar results ( Figure 6 e). The distance between the NMTS group and the free EcN group was shorter, indicating that the composition of the microbiome was less different.

[0102] like Figure 6 fi and Fig.14 As shown, compared with the PBS group, NMTS treatment reduced the abundance of dominant bacterial phyla associated with intestinal inflammatory diseases, including Firmicutes and Proteobacteria. Importantly, NMTS treatment increased the abundance of Bacteroidetes, which is mainly composed of commensal bacteria. In addition, the relative abundance ratio of Proteobacteria to Bacteroidetes and the relative abundance of pathogenic Enterobacteriaceae decreased in the NMTS group compared with the PBS group. In addition, the NMTS group had a higher proportion of beneficial bacteria, such as Lactobacillaceae and Lachnospiraceae, compared with the free EcN group and the PBS group. The reduction in the number of pathogenic bacteria and the increase in the number of probiotic and commensal strains after treatment indicate that NMTS can bidirectionally regulate the disordered intestinal flora to restore a balanced microbial structure. In other words, NMTS enables the transplanted bacteria to actively communicate with the environment in the digestive tract in a non-contact manner.

[0103] Example 7: Potential for safe and effective treatment of NMTS in vivo

[0104] In this example, a mouse colitis model was constructed to evaluate the potential of oral NMTS for safe and effective treatment. Pathogenic contamination is a common safety issue in clinical practice of microbiome transplantation, which may lead to adverse side effects and reduced therapeutic efficacy. Given that NMTS is non-leaky and permeable, it can simultaneously avoid infection risks and regulate intestinal flora. We studied the advantages of NMTS by co-encapsulating with pathogenic STm, which simulates the clinical scenario ( Figure 7 a). Specifically, colitis mice were gavaged daily with 5×10 6 CFUs EcN and 5×10 3 CFUs STm (ratio to EcN was 1‰) of NMTS for 3 consecutive days, and samples were collected on the 4th day ( Figure 7 b). PBS and an equal amount of a mixture of free EcN and STm (called EcN+STm) were used as controls. As expected, the body weight of mice in the PBS group continued to decrease significantly ( Figure 7 c and d). It should be noted that, compared with the PBS group, the treatment with EcN mixed with 1‰STm not only failed to alleviate the weight loss, but accelerated the mortality rate, and the survival rate of the treated mice further dropped from 60% to 40% on the 4th day. Unlike the free EcN+STm group, the weight fluctuations of the mice in the NMTS group were negligible throughout the experiment, and no mortality was observed.

[0105] To determine the number of STm and EcN, various digestive tract segments were collected for bacterial plate counts. Figure 7 As shown in eh, the number of STm colonized in the stomach, small intestine, cecum, and colon of mice treated with free EcN+STm was 3.9-fold, 5.1-fold, 32.9-fold, and 26.3-fold higher than that of mice treated with PBS, respectively. Compared with the NMTS group, the number of STm in these locations increased by 9.5-fold, 167.6-fold, 68.3-fold, and 109.0-fold, respectively. Similar to STm, the number of EcN found in the digestive tract after NMTS treatment was limited, indicating that NMTS can effectively prevent the leakage of EcN and STm during transit throughout the digestive tract ( Figure 7 il). To evaluate the efficacy of NMTS, spleen, peripheral blood and major organs were also collected. Compared with the PBS group, the spleen weight of mice in the free EcN+STm group increased by 42.8%, while that in the NMTS group decreased by 12.8% ( Figure 7 m). Compared with PBS and EcN+STm treatment, NMTS treatment also reduced the levels of pro-inflammatory cytokines in serum, including TNF-α and IL-6 ( Figure 7 n and o). Figure 7p, q and Fig.15 As shown, the colon and cecum tissues of PBS-treated mice showed obvious damage, manifested by severe submucosal edema, disappearance of intestinal glands in the lamina propria, and infiltration of brown MPO-positive inflammatory cells. These symptoms were significantly alleviated after NMTS treatment but worsened after EcN+STm intervention. In addition, H&E staining of heart, liver, spleen, lung, and kidney tissues ensured that no detectable damage and inflammatory response were induced after NMTS treatment ( Fig.16 In conclusion, the results of this example demonstrate that NMTS can still safely and effectively treat colitis in a clinical setting where the transplanted microbiota is accidentally contaminated with pathogenic bacteria.

[0106] in conclusion:

[0107] The present invention proposes a non-leakage encapsulation strategy based on core-shell hydrogel microspheres for use in contactless microbiota transplantation. The target bacteria can be efficiently encapsulated into the core of the core-shell microspheres by a simple two-step photoinitiated free radical emulsion polymerization of GelMA. Due to the cross-linked network structure of gelatin, NMTS does not affect the activity of bacteria and has good permeability to ensure material exchange with the surrounding environment. To prove its principle, NMTS showed a shielding effect after oral administration, protecting the loaded bacteria from gastrointestinal tract-related irritation such as gastric acid and bile salts. NMTS also has sufficient mechanical strength to prevent bacteria from leaking during transport throughout the digestive tract, avoiding unnecessary risks of pathogen infection. In mice interfered with by antibiotics, NMTS showed permeability to exchange nutrients and metabolites and restore healthy intestinal flora structure. In terms of treatment, oral administration of NMTS can safely and effectively treat colitis mice. In clinical scenarios, when the microbiota taken is contaminated by pathogens, NMTS can block pathogen infection and reduce intestinal inflammation and tissue damage. The proposed non-leaking encapsulation based on core-shell hydrogel microspheres is expected to be useful for contactless microbiota transplantation in other sites, including the urogenital tract, lungs, and other sites.

[0108] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing a leak-free core-shell hydrogel microsphere delivery system for contactless bacterial transplantation, characterized in that: The construction method forms core-shell hydrogel microspheres by two-step photo-initiated methacrylated gelatin (GelMA) free radical emulsion polymerization, and probiotics are encapsulated in the core of the core-shell hydrogel microspheres; specifically comprises the following steps: Step 1: Disperse the live probiotics in a PBS solution containing a photoinitiator and methacrylate gelatin, then drop the solution into the oil phase, and initiate free radical emulsion polymerization under light conditions to obtain bacteria-loaded core hydrogel microspheres; Step 2: The bacteria-loaded core hydrogel microspheres obtained in step 1 are dispersed in a PBS solution containing a photoinitiator and gelatin methacrylate, and then added dropwise to the oil phase to further initiate free radical emulsion polymerization under light conditions to obtain a core-shell hydrogel microsphere delivery system loaded with probiotics.

2. The construction method according to claim 1, characterized in that: The photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

3. The construction method according to claim 1, characterized in that: The ingredients of the oil phase are mineral oil and Span 80.

4. The construction method according to claim 1, characterized in that: The PBS solution in step 1 contains 0.1-1 wt % of photoinitiator and 5-15 wt % of GelMA.

5. The construction method according to claim 1, characterized in that: The aqueous phase solution in step 2 is a PBS solution containing 0.1-1 wt% of a photoinitiator, 5-15 wt% of GelMA and bacteria-carrying core hydrogel microspheres.

6. A core-shell hydrogel microsphere delivery system constructed by the construction method according to any one of claims 1 to 5.

7. Use of the core-shell hydrogel microsphere delivery system constructed by the construction method according to any one of claims 1 to 5 in transplanting probiotics for non-diagnostic and non-therapeutic purposes, preparing probiotic products, or preparing products for transplanting probiotics.

8. Use of the core-shell hydrogel microsphere delivery system constructed by the construction method according to any one of claims 1 to 5 in the preparation of products for treating inflammatory diseases and / or diseases related to intestinal flora imbalance.

Images