Triggerable probiotic-drug conjugate as well as preparation method and application thereof

Abstract

The invention discloses a triggering probiotic-drug conjugate and a preparation method and application thereof, and belongs to the technical field of oral drug delivery systems, the triggering probiotic-drug conjugate comprises probiotics, an adhesive inner layer formed by coordination of tannic acid and Fe < 3 + >, and an outer layer formed by pterostilbene-phospholipid bridged by ROS-responsive thioether bonds and cholesterol; the inner layer coats the surfaces of the probiotics through coordination, and the outer layer coats the outer part of the inner layer through self-assembly. An outer lipid layer is decomposed under high-concentration ROS in an inflammation colon area, and probiotics coated with pterostilbene and a tannic acid layer are released. Pterostilbene relieves inflammation and rebuilds a pathological microenvironment, and catechol groups in a tannic acid layer and the surface of the intestinal tract generate multiple interactions to promote planting of probiotics. In UC mouse models, the conjugates exhibit enhanced therapeutic effects by inhibiting inflammation, restoring intestinal barrier function, and improving intestinal microbiota homeostasis. The invention has important transformation potential in clinical application of gastrointestinal diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of oral drug delivery system, and particularly relates to a triggerable probiotic-drug conjugate, a preparation method thereof and application thereof in treating ulcerative colitis (UC). BACKGROUND

[0002] Ulcerative colitis is a chronic intestinal inflammatory disease, and existing therapies have problems such as limited single mechanism efficacy, uncontrollable drug release, and non-specific distribution. Although oral probiotic therapy has broad prospects, conventional probiotics have low survival rate in the gastrointestinal environment and lack the ability to target and colonize lesions. In addition, there is severe inflammatory infiltration in the diseased area of the intestine, and a single probiotic therapy is difficult to achieve good efficacy.

[0003] Therefore, it is crucial to develop a delivery system that can simultaneously achieve probiotic lesion colonization and on-demand drug release. SUMMARY

[0004] In view of the defects of existing probiotic therapy, the present application designs and constructs an oral triggerable probiotic-drug conjugate. The conjugate comprises probiotics, an adhesive inner layer formed by coordination of tannic acid (TA) and Fe 3+ , a sulfide bond bridged purple heartwood tree-PSL and cholesterol outer layer responsive to reactive oxygen species (ROS), and an inner layer coated on the surface of probiotics through coordination, and an outer layer coated on the outside of the inner layer through self-assembly. This composite coating does not affect the survival and proliferation of probiotics. After oral administration, the composite coating effectively protects the probiotics from the harsh gastrointestinal environment and significantly improves the survival rate. Importantly, the outer lipid layer is decomposed under high concentration of ROS in the inflammatory colon area, releasing the purple heartwood tree and TA layer coated probiotics. Purple heartwood tree can reduce inflammation and remodel the pathological microenvironment, while the abundant catechol groups in the TA layer can interact with the intestinal surface to promote the colonization of probiotics. In a UC mouse model, the conjugate shows enhanced therapeutic effect by inhibiting inflammation, restoring intestinal barrier function and improving intestinal microbiota homeostasis. The present application has important translational potential in the clinical application of gastrointestinal diseases.

[0005] The present application achieves the above-mentioned purposes through the following technical solutions:

[0006] In the first aspect, the present application constructs a probiotic-drug conjugate mainly through self-assembly on the surface of probiotics.

[0007] As an optional mode, the probiotics are Escherichia coli Nissle 1917 (EcN); the inner layer is a metal polyphenol network formed by coordination of TA and Fe 3+ ; and the Fe 3+FeCl3 was selected as the providing source, and the mass ratio of FeCl3 to tannic acid in the inner layer was 1:1-1:3.

[0008] As an alternative, the PSL is obtained by chemical synthesis. The drug loading of the probiotic-drug conjugate is 1.97 mg / 10 9 CFU EcN.

[0009] In a second aspect, the probiotic-drug conjugate of the present application is prepared by the following method:

[0010] The preparation method of the probiotic-drug conjugate of the present application comprises the following steps:

[0011] (1) FeCl3 solution and TA solution are sequentially added to the EcN suspension washed with distilled water, and vortex mixed; then free FeCl3 and TA are removed by washing with distilled water; finally, centrifugation is performed to obtain EcN@TA;

[0012] (2) EcN@TA is resuspended in a PBS buffer containing Ca 2+ , PSL and cholesterol are added, and incubated; then free PSL and cholesterol are removed by washing with distilled water; finally, centrifugation is performed to obtain EcN@TA-PSL conjugate.

[0013] In the step (1), the specific operation is as follows: 10 μL of FeCl3 solution with a concentration of 10 mg / mL-30 mg / mL and 10 μL of TA solution with a concentration of 10 mg / mL-30 mg / mL (mass ratio of FeCl3 to TA 1:1-1:3) are sequentially added to 1 mL of EcN (1×10 9 CFU) to vortex for 30 s.

[0014] In the step (2), the specific operation is as follows: EcN@TA is resuspended in 1 mL of ice-cold PBS buffer containing 12.5 mM CaCl2 to form EcN@TA solution; PSL (0.01 mM) and cholesterol are dissolved in 5 mL of chloroform at a molar ratio of 4:1, and the lipid film is formed by evaporation at room temperature using a rotary evaporator, and then the lipid film is hydrated with 1 mL of EcN@TA solution at 37°C for 30 min.

[0015] In the step (2), the synthesis method of PSL is as follows: pterostilbene, thiohydroxy acetate anhydride and 4-dimethylaminopyridine (DMAP) are dissolved in dichloromethane. The reaction mixture is stirred at room temperature under a nitrogen atmosphere. After the reaction is completed, the mixture is concentrated under reduced pressure, purified to obtain an intermediate product. The intermediate product and palmitoyl lysophospholipid are dissolved in dichloromethane and stirred at room temperature, then 2,4,6-trichlorobenzoyl chloride and DMAP are added, and the reaction is stirred at room temperature under a nitrogen atmosphere.

[0016] In the step (2), dynamic light scattering, transmission electron microscopy, confocal laser microscopy and flow cytometry are used to verify the particle size, potential, morphology and coating coverage of the EcN@TA-PSL conjugate.

[0017] In a third aspect, the present invention provides use of the probiotic-drug conjugate described in the first aspect in a drug delivery system.

[0018] In a fourth aspect, the present invention provides use of the probiotic-drug conjugate described in the first aspect in the preparation of a drug for treating ulcerative colitis or colitis-related colon cancer.

[0019] In a fifth aspect, the present invention provides use of the probiotic-drug conjugate according to the first aspect in preparing a drug delivery system for oral administration.

[0020] The present invention has the following beneficial effects:

[0021] In this paper, EcN was used as a model probiotic, and FeCl3, TA, PSL, and cholesterol were used as raw materials. A probiotic-drug conjugate was constructed by self-assembly on the EcN surface layer. The formulation performance, gastrointestinal environment resistance, ROS response ability, intestinal adhesion effect in vivo and in vitro, and anti-UC effect of the conjugate were evaluated.

[0022] The preparation process of the present invention is simple, and the prepared probiotic-drug conjugate can effectively protect the probiotics from the gastrointestinal environment. It also responds to high concentrations of ROS in colonic lesions to release the drug and adhere to the coated probiotics, thereby achieving intelligent drug release and targeted colonization of the probiotics.

[0023] The probiotic-drug conjugate designed by the present invention achieves the treatment of UC. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the PSL in Example 1 of the present invention 1 H-NMR results;

[0025] Figure 2 is the PSL in Example 1 of the present invention 13 C-NMR results;

[0026] Figure 3 This is the high-resolution mass spectrometry result of PSL in Example 1 of the present invention;

[0027] Figure 4 HPLC purity results of PSL in Example 1 of the present invention;

[0028] Figure 5 is the PCL in Example 2 of the present invention1 H-NMR results;

[0029] Figure 6 is the PCL in Example 2 of the present invention 13 C-NMR results;

[0030] Figure 7 This is the high-resolution mass spectrometry result of PCL in Example 2 of the present invention;

[0031] Figure 8 This is the HPLC purity result of PCL in Example 2 of the present invention;

[0032] Figure 9 This is a graph showing the particle size of the probiotic-drug conjugate in Example 3 of the present invention;

[0033] Figure 10 This is the potential diagram of the probiotic-drug conjugate in Example 3 of the present invention;

[0034] Figure 11 This is a transmission electron micrograph of the probiotic-drug conjugate in Example 3 of the present invention;

[0035] Figure 12 This is a confocal image of the probiotic-drug conjugate in Example 3 of the present invention;

[0036] Figure 13 This is a plate coating diagram of the probiotic-drug conjugate in Example 4 of the present invention after being treated with different simulated gastrointestinal conditions;

[0037] Figure 14 This is a statistical graph of the counts of the probiotic-drug conjugate in Example 4 of the present invention after being treated under different simulated gastrointestinal conditions;

[0038] Figure 15 The drug release curves of the probiotic-drug conjugate in Example 5 of the present invention after being treated with different concentrations of H2O2;

[0039] Figure 16 This is a transmission electron micrograph of the probiotic-drug conjugate in Example 6 of the present invention after being treated with H2O2;

[0040] Figure 17 This is a confocal laser microscopy image of the probiotic-drug conjugate in Example 6 of the present invention after being treated with H2O2;

[0041] Figure 18 This is a flow cytometry image of the probiotic-drug conjugate in Example 6 of the present invention after being treated with H2O2;

[0042] Figure 19 Evaluation of the in vivo distribution of the probiotic-drug conjugate in Example 7 of the present invention;

[0043] Figure 20 Evaluation of the distribution of the probiotic-drug conjugate in the colon in Example 7 of the present invention;

[0044] Figure 21 Figure 8 shows the changes in body weight (A) and DAI score (B) of UC mice after treatment with the probiotic-drug conjugate in Example 8 of the present invention; G1 healthy group, G2 UC model group, G3 EcN combined with free pterostilbene group, G4 EcN@TA group, G5 EcN@TA-PCL group, and G6 EcN@TA-PSL group.

[0045] Figure 22 The changes in colon length of UC mice after treatment with the probiotic-drug conjugate in Example 8 of the present invention; G1 healthy group, G2 UC model group, G3 EcN combined with free pterostilbene group, G4 EcN@TA group, G5 EcN@TA-PCL group, G6 EcN@TA-PSL group;

[0046] Figure 23 The expression levels of inflammatory factors in UC mice after treatment with the probiotic-drug conjugate in Example 8 of the present invention are as follows: G1 healthy group, G2 UC model group, G3 EcN combined with free pterostilbene group, G4 EcN@TA group, G5 EcN@TA-PCL group, and G6 EcN@TA-PSL group.

[0047] Figure 24 HE images of the colon of UC mice after treatment with the probiotic-drug conjugate in Example 8 of the present invention; G1 healthy group, G2 UC model group, G3 EcN combined with free pterostilbene group, G4 EcN@TA group, G5 EcN@TA-PCL group, G6 EcN@TA-PSL group;

[0048] Figure 25 The changes in α and β diversity of the microbiota of UC mice after treatment with the probiotic-drug conjugate in Example 9 of the present invention;

[0049] Figure 26 The changes in the species and genus levels of the bacterial flora of UC mice after treatment with the probiotic-drug conjugate in Example 9 of the present invention;

[0050] Figure 27 These are the changes in the microbiome family level of UC mice after treatment with the probiotic-drug conjugate in Example 9 of the present invention. DETAILED DESCRIPTION

[0051] The above contents of the present invention are further described in detail through the following specific examples, but they do not limit the present invention to the examples.

[0052] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0053] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0054] Example 1: Synthesis of ROS-responsive PSL

[0055] Pterostilbene, thioglycolic anhydride (2eq) and DMAP (0.2eq) were dissolved in an appropriate amount of anhydrous dichloromethane (DCM). The reaction mixture was stirred at room temperature overnight under a nitrogen atmosphere. The reaction process was monitored by thin layer chromatography. After completion, the mixture was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (PE / EtOAc=1:1, 1‰ glacial acetic acid) to obtain an intermediate product. The intermediate product and palmitoyl lysolecithin (1eq) were then dissolved in an appropriate amount of anhydrous DCM and stirred at room temperature for 30 minutes. Subsequently, 2,4,6-trichlorobenzoyl chloride (3eq) and DMAP (3eq) were added, and the reaction mixture was stirred at room temperature overnight under a nitrogen atmosphere. The reaction process was monitored by thin layer chromatography. The target product was purified by preparative liquid chromatography using 95% methanol as the mobile phase. The synthesis of PSL was verified by hydrogen spectrum, carbon spectrum, high-resolution mass spectrometry and high-performance liquid chromatography, and the results were as shown below. Figure 1 、 Figure 2 、 Figure 3 and Figure 4 shown.

[0056] Example 2: Synthesis of PCL without ROS response

[0057] Pterostilbene, glutaric anhydride (2eq) and DMAP (0.2eq) were dissolved in an appropriate amount of anhydrous DCM. The reaction mixture was stirred at room temperature overnight under a nitrogen atmosphere. The reaction process was monitored by thin layer chromatography. After completion, the mixture was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (PE / EtOAc=1:1, 1‰ glacial acetic acid) to obtain an intermediate product. The intermediate product and palmitoyl lysolecithin (1eq) were then dissolved in an appropriate amount of anhydrous DCM and stirred at room temperature for 30 minutes. Subsequently, 2,4,6-trichlorobenzoyl chloride (3eq) and DMAP (3eq) were added, and the reaction mixture was stirred at room temperature overnight under a nitrogen atmosphere. The reaction process was monitored by thin layer chromatography. The target product was purified by preparative liquid chromatography using 95% methanol as the mobile phase. The synthesis of PCL was verified by hydrogen spectrum, carbon spectrum, high-resolution mass spectrometry and high-performance liquid chromatography, and the results are as shown below. Figure 5 、 Figure 6 、 Figure 7 and Figure 8 shown.

[0058] Example 3: Preparation and characterization of probiotic-drug conjugates

[0059] EcN cells were picked from the LB agar plate and cultured in LB medium at 37°C overnight. Then, the EcN cells were washed twice with deionized water and resuspended in 980 μL deionized water. Then, 1 mL of EcN suspension (1×10 9 To a 10 μL volume of 100 μL FeCl₃ (10 mg / mL) solution (CFU) was added and mixed thoroughly. Then, 10 μL TA (30 mg / mL) was added and vortexed for 30 seconds. The mixture was washed twice with deionized water to remove residual TA and FeCl₃ and collected by centrifugation to obtain EcN@TA.

[0060] The EcN@TA obtained by centrifugation was resuspended in 1 mL of ice-cold PBS buffer containing 12.5 mM CaCl2 to form an EcN@TA solution. PSL (0.01 mM) and cholesterol were dissolved in 5 mL of chloroform at a molar ratio of 4:1. The resulting solution was dried at room temperature using a rotary evaporator to obtain a lipid film. The resulting lipid film was hydrated in 1 mL of EcN@TA solution at 37°C for 30 min and then washed twice with deionized water to obtain an EcN@TA-PSL conjugate. The drug loading of pterostilbene in the prepared EcN@TA-PSL conjugate was 1.97 mg / 10 9 CFU EcN.

[0061] EcN@TA-PCL was prepared by the same method as above, except that PCL was used instead of PSL.

[0062] Dynamic light scattering, transmission electron microscopy, confocal laser microscopy and flow cytometry were used to verify the particle size, potential, morphology and coating coverage of EcN@TA-PCL and EcN@TA-PSL conjugates. Figure 9 、 Figure 10 、 Figure 11 and Figure 12 shown.

[0063] like Figure 9 As shown in Figure 2, the particle size of the EcN@TA-PCL conjugate is concentrated in the range of 1577±48nm, and the particle size of the EcN@TA-PSL conjugate is concentrated in the range of 1575±57nm, and the particle size distribution is concentrated. Figure 10 As shown in Figure 2, the Zeta potential of the EcN@TA-PCL conjugate is about -21 mV, and the Zeta potential of the EcN@TA-PSL conjugate is about -20 mV. Figure 11As shown in the figure, comparing EcN@TA with EcN@TA-PCL and EcN@TA-PSL, it can be seen that PCL and PSL further form a film / coat on the TA modified layer to form a "bacteria-TA-polymer" composite system, indicating that TA acts as a "bridge" (such as by virtue of its chemical activity and adhesion) to assist the subsequent modification of PCL and PSL. Figure 12 As shown, PCL / PSL was successfully modified on the surface of EcN@TA to form a "bacteria-TA-PCL / PSL" three-layer structure.

[0064] Example 4: Evaluation of the ability of probiotic-drug conjugates to resist the harsh environment of the gastrointestinal tract

[0065] Equal amounts of native EcN, EcN@TA, EcN@TA-PCL, and EcN@TA-PSL were incubated with 1 mL of simulated gastric fluid (SGF, pH 2, 30 min) or simulated intestinal fluid (SIF, pH 6.8, 4 h) or bile salt (0.3 mg / mL, 1 h) at 37°C with gentle shaking. At the designated time points, 100 μL samples were collected from each group, washed with PBS, serially diluted, and then spread on LB agar plates. Colonies were counted after incubation at 37°C for 24 h. The results are shown in Figure 2. Figure 13 and Figure 14 As shown in the results, the probiotic-drug conjugate significantly improved the survival rate of probiotics.

[0066] Example 5: ROS-responsive drug release and lipid layer lysis evaluation of probiotic-drug conjugates

[0067] Take 1 mL of EcN@TA-PCL and EcN@TA-PSL solutions (each solution contains an equal amount of pterostilbene) and put them into a dialysis bag (molecular cutoff value is 10 kilodaltons). The bag is immersed in 20 mL of PBS solution containing H2O2 (0.1mM, 1mM or 10mM) and 30% anhydrous ethanol, and the temperature is maintained at 37°C. The mixture is stirred continuously for 24 hours. At predetermined time points (0, 0.5, 1, 2, 4, 8, 12 and 24 hours), 1 mL of solution is collected and the released pterostilbene is quantified using high performance liquid chromatography. Figure 15 As shown in the Figure 3, the release rate of pterostilbene from the ROS-responsive EcN@TA-PSL accelerated with the increase of H2O2 concentration, while the non-ROS-responsive EcN@TA-PCL exhibited an extremely slow drug release rate even under the stimulation of 10 mM H2O2.

[0068] Example 6: Evaluation of ROS-responsive lipid layer lysis of probiotic-drug conjugates

[0069] The EcN, TA layer, and lipid membrane were labeled with Hoechst 33342, FITC, and Nile Red, respectively. Labeled EcN@TA-PCL and EcN@TA-PSL were incubated with 100 μM H2O2 for 12 h. After incubation, the samples were centrifuged, washed with PBS, and then analyzed for coating dissociation using transmission electron microscopy, confocal laser microscopy, and flow cytometry. Figure 16 、 Figure 17 、 Figure 18 As shown in Figure 3, the lipid layer in the ROS-responsive EcN@TA-PSL was almost completely lysed, while the lipid layer in the non-ROS-responsive EcN@TA-PCL was not significantly affected.

[0070] Example 7: Evaluation of the targeted colonization ability of probiotic-drug conjugates in vivo

[0071] To evaluate the specific colonization of EcN@TA-PSL in the inflammatory site of the colon, female Balb / c mice (6-8 weeks) were acclimated for 1 week and then given 3% dextran sodium sulfate (DSS) for 5 days to induce acute colitis. UC mice were orally treated with 1×10 8 CFU of Dir-labeled EcN, EcN@TA, EcN@TA-PCL, and EcN@TA-PSL were administered to healthy mice at the same time. 8 CFU of Dir-labeled EcN@TA-PSL was used as a control (EcN@TA-PSL-Con). Figure 19 As shown in the figure, at 2 hours, the fluorescence intensity of the bare EcN group was significantly lower than that of the other groups, demonstrating the protective ability of the coating for EcN. At 6 and 12 hours, the EcN@TA group showed the highest fluorescence intensity, highlighting the strong tissue adhesion ability of the TA layer. At 24 and 48 hours, the EcN@TA-PCL and EcN@TA-PSL-Con groups already showed extremely weak fluorescence signals, which may be due to the limited mucosal adhesion caused by the unexposed TA layer. However, strong fluorescence signals were still observed in the EcN@TA group and EcN@TA-PSL.

[0072] In order to observe the specific adhesion effect of EcN in the colon, we killed a certain number of mice at 6h and 48h and removed the colon for imaging. Figure 20As shown, at 6 hours, the EcN@TA-PSL group exhibited the highest fluorescence intensity, 1.9 times that of the EcN@TA group. This may be due to the fact that after oral administration, EcN@TA is distributed throughout the gastrointestinal tract and is unable to specifically adhere to the colon, while the responsive lipid membrane of the EcN@TA-PSL group provides ideal targeted adhesion. At 48 hours, almost no fluorescence signal was observed in the colon of the bare EcN group, and only sporadic fluorescence signals were observed in the non-ROS-responsive EcN@TA-PCL and EcN@TA-PSL-Con groups. In contrast, the EcN@TA-PSL group maintained high fluorescence intensity, 1.9, 9.7, 6.4, and 3.4 times that of the EcN@TA, EcN, EcN@TA-PCL, and EcN@TA-PSL-Con groups, respectively. This suggests that EcN@TA-PSL can release adhesively coated EcN in response to high ROS concentrations in colonic lesions, thereby achieving specific colonization.

[0073] Example 8: Evaluation of the efficacy of probiotic-drug conjugates in a DSS-induced UC mouse model

[0074] Six- to eight-week-old Balb / c female mice were randomly divided into six groups, with six mice in each group. One group received normal drinking water for seven days and PBS by oral gavage for five days. The remaining five groups received drinking water containing 3% DSS for seven consecutive days to induce colitis. Mice were then given normal drinking water and gavage daily with PBS, EcN combined with free pterostilbene, EcN@TA, EcN@TA-PCL, or EcN@TA-PSL (bacterial dose, 2×10 8 CFU (Cellular Fungus Units), the pterostilbene dose in the free pterostilbene group was comparable to that in the EcN@TA-PCL and EcN@TA-PSL groups (approximately 19.7 mg / kg), for 5 consecutive days. From the start of modeling to the end of treatment, fecal concentration, occult blood, and weight loss were recorded daily, and the disease activity index (DAI) score was calculated. One day after the last administration, mice were euthanized, and the colon was excised and measured in length. In addition, the distal colon segment was fixed in 4% paraformaldehyde for histopathological examination. The remaining colon tissue was homogenized and analyzed for myeloperoxidase (MPO) activity and cytokine levels (IL-10, TNF-α, IL-6, and IL-1β) using ELISA kits according to the manufacturer's protocol. G1 healthy group, G2 UC model group, G3 EcN combined with free pterostilbene group, G4 EcN@TA group, G5 EcN@TA-PCL group, and G6 EcN@TA-PSL group.

[0075] like Figure 21 As shown in the figure, after DSS treatment, the body weight of mice decreased sharply and the DAI score increased sharply; however, the mice treated with EcN@TA-PSL slowed down this trend and gained weight rapidly in the following days. The mice were killed on day 12 and the colon was isolated. Figure 22 As shown in Figure 3, EcN@TA-PSL prevented inflammation-induced colon shortening, and the colon length was significantly longer than that of the other treatment groups. In addition, we also detected the expression levels of pro-inflammatory and anti-inflammatory cytokines in colon tissue. Figure 23 As shown in Figure 2, EcN@TA-PSL treatment significantly reduced the expression levels of MPO and pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-6, and interleukin-1β), and increased the secretion of anti-inflammatory cytokines (interleukin-10). In addition, HE results showed that ( Figure 24 ), infiltration of inflammatory cells, damage to the mucosal layer, and disappearance of most goblet cells and crypt structures were observed in the model group, while the EcN@TA-PSL group could effectively reverse the colon damage caused by DSS, as shown by minimal inflammatory cell infiltration, intact epithelium, and regular finger-like crypt structures.

[0076] Example 9: Evaluation of the microbiota-regulating effect of probiotic-drug conjugates in a DSS-induced UC mouse model

[0077] Next, fecal samples were collected and 16S rRNA gene amplicon sequencing was performed to analyze the ability of EcN@TA-PSL to regulate intestinal flora. Figure 25 As shown in the results, the α-diversity and β-diversity of the UC model group decreased significantly, while EcN@TA-PSL treatment significantly limited the reduction of microbial abundance and diversity in colitis mice. The intestinal microbiota was then deeply analyzed. Figure 26As shown, the model group microbiota underwent dramatic changes in phylum relative abundance, primarily manifested by a decrease in the proportion of Firmicutes (which play a beneficial role by producing SCFAs, regulating immunity, and maintaining intestinal mucosal barrier function) and an increase in the proportion of Proteobacteria (which primarily participate in the development and progression of UC by promoting inflammation, disrupting the intestinal mucosal barrier, and exacerbating microbial imbalance). Treatment with EcN@TA-PSL significantly reversed this trend, and the microbial composition showed a high degree of similarity to that of the healthy control group. Furthermore, a ternary phase diagram was used to examine the differences in dominant species at the genus level between the EcN@TA-PSL and model groups. The results showed that Escherichia-Shigella (which primarily exacerbates UC by proinflammatory effects and disrupting microbial balance and is a key hallmark of intestinal dysbiosis in UC) was the dominant species in the model group. However, EcN@TA-PSL treatment reduced the abundance of Escherichia-Shigella and increased the proportion of Alloprevotella (a beneficial bacterium that produces short-chain fatty acids and maintains microbial balance). In addition, statistical analysis of several representative families further revealed the bacterial composition of treated colitis mice. EcN@TA-PSL treatment significantly increased the abundance of beneficial bacteria such as Prevotellaceae, Lachnospiraceae (producing short-chain fatty acids such as butyrate to maintain the integrity of the intestinal mucosal barrier), and Muribaculaceae (involved in mucus layer formation and immune regulation), and reduced the abundance of Bacteroidaceae. Figure 27 shown.

[0078] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the present invention's equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A triggerable probiotic-drug conjugate, characterized in that: Contains probiotics, tannic acid and Fe 3+ An adhesive inner layer formed by coordination and an outer layer composed of pterostilbene-phospholipid and cholesterol bridged by ROS-responsive thioether bonds; the inner layer coats the surface of the probiotics through coordination, and the outer layer is coated on the outside of the inner layer through self-assembly.

2. The triggerable probiotic-drug conjugate according to claim 1, characterized in that The probiotic is Escherichia coli Nissle 1917.

3. The triggerable probiotic-drug conjugate according to claim 1, characterized in that The Fe 3+ Ferric chloride is selected as the supply source; the mass ratio of ferric chloride to tannic acid in the inner layer is 1:1 to 1:

3.

4. The triggerable probiotic-drug conjugate according to claim 1, characterized in that The drug loading of pterostilbene in the triggerable probiotic-drug conjugate is 1.97 mg / 10 9 CFU EcN.

5. A method for preparing the triggerable probiotic-drug conjugate according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) FeCl3 solution and tannic acid solution were sequentially added to the EcN suspension washed with distilled water and vortexed to mix; then, free FeCl3 and tannic acid were removed by washing with distilled water; finally, the suspension was centrifuged to obtain EcN@TA; (2) Resuspend EcN@TA in a solution containing Ca 2+ ROS-responsive thioether-bridged pterostilbene-phospholipid and cholesterol were added to PBS buffer and incubated; free ROS-responsive thioether-bridged pterostilbene-phospholipid and cholesterol were then washed with distilled water to remove the free ROS-responsive thioether-bridged pterostilbene-phospholipid and cholesterol; and finally, the EcN@TA-PSL conjugate was obtained by centrifugation.

6. The method for preparing the triggerable probiotic-drug conjugate according to claim 5, characterized in that: In the step (2), the synthesis method of the ROS-responsive thioether-bridged pterostilbene-phospholipid is as follows: pterostilbene, thiohydroxyacetic anhydride, and 4-dimethylaminopyridine are dissolved in dichloromethane, the reaction mixture is stirred at room temperature under a nitrogen atmosphere, and after the reaction is completed, the mixture is concentrated under reduced pressure and purified to obtain an intermediate product; the intermediate product and palmitoyl lysolecithin are dissolved in dichloromethane, stirred at room temperature, 2,4,6-trichlorobenzoyl chloride and 4-dimethylaminopyridine are added, and the reaction is stirred at room temperature under a nitrogen atmosphere.

7. The method for preparing the triggerable probiotic-drug conjugate according to claim 6, characterized in that: In the step (1), FeCl3 solution and tannic acid solution are sequentially added to 1×10 9 CFU EcN suspension, vortex; In the step (2), EcN@TA is resuspended in ice-cold PBS buffer containing 12.5 mM CaCl2 to form an EcN@TA solution; ROS-responsive thioether-bridged pterostilbene-phospholipid and cholesterol are dissolved in chloroform at a molar ratio of 4:1, evaporated at room temperature to form a lipid film, and then the lipid film is hydrated with the EcN@TA solution at 37°C.

8. Use of the triggerable probiotic-drug conjugate according to any one of claims 1 to 4 in preparing a drug delivery system.

9. Use of the triggerable probiotic-drug conjugate according to any one of claims 1 to 4 in the preparation of a medicament for treating ulcerative colitis or colitis-associated colon cancer.

10. Use of the triggerable probiotic-drug conjugate according to any one of claims 1 to 4 in the preparation of a drug delivery system for oral administration.

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