Oral probiotic-mediated drug delivery system as well as preparation method and application thereof

Abstract

The invention relates to an oral probiotic-mediated drug delivery system as well as a preparation method and application thereof. The preparation method comprises the following steps: preparing polydopamine nano coating bacteria from probiotics and dopamine; a process of preparing drug-loaded polydopamine nanoparticles from dopamine and a functional drug; and a process of preparing a drug delivery system from the polydopamine nano-coated bacteria and the drug-loaded polydopamine nanoparticles, the drug delivery system prepared by the method is applied to preparation of drugs for preventing or treating tumors, colitis, diarrhea, constipation, diabetes mellitus and obesity. The preparation method disclosed by the invention has the advantages of simple preparation, mildness and stable performance, the prepared drug delivery system adopts a polydopamine single bacterium coating method to encapsulate the probiotics, and the drug-loaded nanoparticles are loaded on the surface of the probiotics, so that the effects of efficient drug delivery and micro-ecological regulation functions are achieved, and a better treatment effect is achieved.

Description

Technical Field

[0001] This invention relates to the technical field of drug delivery systems, and in particular to an oral probiotic-mediated drug delivery system, its preparation method, and its application. Background Technology

[0002] The gut, a key organ for digestion and absorption, also constitutes the body's largest immune regulatory system and microbial symbiotic system. Recent studies have revealed a significant link between gut microbiota imbalance and various diseases, including inflammatory bowel disease (IBD, such as Crohn's disease and ulcerative colitis), infectious intestinal diseases (such as Clostridium difficile infection), irritable bowel syndrome (IBS), and metabolic diseases (such as diabetes and obesity). Infections with Clostridium difficile and Campylobacter can easily trigger inflammatory bowel disease, while diarrhea-associated pathogens such as enterotoxigenic Escherichia coli (EPEC) and enterocolitogenic Escherichia coli (EAEC) may exacerbate chronic inflammation in IBD patients, thereby significantly increasing the risk of colorectal cancer (the risk of colorectal cancer in IBD patients is 2-4 times higher than in healthy individuals). Other intestinal diseases are also closely related to the overgrowth of pathogenic bacteria. For example, the overgrowth of methanogens may lead to intestinal gas retention, inducing the characteristic abdominal pain of IBS; the increase in the abundance of Firmicutes and the decrease in Bacteroidetes can lead to an imbalance in the host's energy metabolism homeostasis, thereby aggravating the patient's obesity symptoms.

[0003] For diseases related to dysbiosis, traditional chemotherapy (such as aminosalicylic acid preparations and glucocorticoids) can suppress inflammatory responses in the short term, but it has side effects such as impaired mucosal repair and systemic immunosuppression. The use of broad-spectrum antibiotics may further disrupt the gut microbiota structure, exacerbate dysbiosis, and induce the proliferation of drug-resistant strains, forming a vicious cycle of "inflammation-microbiota dysbiosis-secondary infection." In contrast, probiotic therapy demonstrates unique advantages through multi-dimensional intervention: specific strains (such as Bifidobacterium and Lactobacillus rhamnosus) can competitively inhibit pathogen colonization and regulate the intestinal immune microenvironment, inhibiting the overexpression of pro-inflammatory cytokines (such as TNF-α, IL-6, and IL-17), thus exerting an anti-inflammatory effect; their metabolites, such as butyrate and other short-chain fatty acids, can not only significantly increase the expression of tight junction-related genes (such as Occludin (OCLN), Claudin-4 (CLDN4), and Claudin-15 (CLDN15)), upregulate tight junction protein genes, and enhance intestinal barrier function, but also inhibit the secretion of pro-inflammatory cytokines, exerting a therapeutic effect through this dual mechanism. Clinical studies have confirmed that probiotic combination regimens can effectively improve the clinical remission rate in IBD patients and significantly reduce the incidence of antibiotic-associated diarrhea. Furthermore, probiotics possess hypoxia tropism and chemotaxis; therefore, drug-loaded probiotic delivery systems hold promise for achieving effective drug accumulation in deep tumor layers, thereby enabling targeted therapy.

[0004] However, during oral delivery, probiotic drug delivery systems are often susceptible to inactivation or degradation of both probiotics and the delivered drug before reaching the target site due to gastrointestinal physiological barriers such as drastic pH changes and enzyme activity. Traditional multi-bacterial encapsulation strategies (such as microencapsulation) often result in reduced bacterial survival rates and fail to fully utilize the hypoxic and chemotactic activities of individual bacteria. Novel single-bacterial encapsulation technologies, by constructing a nanocoating on the surface of individual probiotics, can significantly improve the survival rate of probiotics in simulated gastrointestinal fluids and enhance bacterial adhesion, while preserving bacterial motility and chemotactic abilities. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the first objective of this invention is to provide a method for preparing an oral probiotic-mediated drug delivery system, which has the advantages of simple preparation, mildness, and stable performance.

[0006] The second objective of this invention is to provide an oral probiotic-mediated drug delivery system that uses a polydopamine monobacterial coating method to encapsulate probiotics and loads functional drugs on their surface, thereby achieving both efficient functional drug delivery and microecological regulation functions, resulting in better therapeutic effects.

[0007] The third objective of this invention is to provide an application of an oral probiotic-mediated drug delivery system, which has the advantages of increasing the targeting efficiency of functional drugs to achieve intestinal-targeted functional drug therapy that combines efficient delivery and microecological regulation.

[0008] To achieve the first objective mentioned above, the present invention provides the following technical solution: A method for preparing an oral probiotic-mediated drug delivery system, comprising, The process of preparing polydopamine-coated bacteria from probiotics and dopamine; The process of preparing drug-loaded polydopamine nanoparticles from dopamine and functional drugs; And the process of preparing a drug delivery system from the polydopamine nanocoated bacteria and the drug-loaded polydopamine nanoparticles; In this process, the dopamine undergoes oxidative self-polymerization to form polydopamine nanoparticles, which are then self-assembled on the surface of probiotics to form a nanocoating. The functional drug is loaded onto the surface of the polydopamine nanoparticles through covalent bonds, π-π stacking, or hydrogen bonding.

[0009] Furthermore, in the process of preparing polydopamine-coated bacteria, probiotic colonies are first resuspended and dispersed in Tris·HCl buffer to obtain a probiotic suspension. Then, dopamine powder is added to Tris·HCl buffer and filtered through a 0.22 μm filter membrane to obtain a dopamine solution. The probiotic suspension and dopamine solution are then cultured at a constant temperature with shaking. After the culture is completed, post-processing is performed to obtain polydopamine-coated bacteria.

[0010] Furthermore, in the process of preparing polydopamine-coated bacteria, the pH of the Tris·HCl buffer was controlled at 5-10, and the culture medium contained 1×10 9 The dopamine concentration corresponding to CFU / mL probiotics is 0.1~10.0 mg / mL, the culture temperature is 35~40℃, and the culture time is 20~120 min.

[0011] Furthermore, in the process of preparing polydopamine-coated bacteria, the pH of the Tris·HCl buffer was controlled at 8-9, and the concentration of the culture medium was controlled at 1×10⁻⁶. 9 The dopamine concentration corresponding to CFU / mL probiotics is 0.2~4.0 mg / mL, the culture temperature is controlled at 37℃, and the culture time is 30~60 min.

[0012] Furthermore, in the process of preparing drug-loaded polydopamine nanoparticles, dopamine / water solution is first added to a mixed solution of water / ethanol / ammonia for oxidative self-polymerization. After polymerization, post-treatment is performed to obtain polydopamine nanoparticles. Then, a functional drug / DMSO solution is added to the polydopamine nanoparticle / water solution for reaction. After the reaction is completed, post-treatment is performed to obtain drug-loaded polydopamine nanoparticles.

[0013] Furthermore, in the process of preparing drug-loaded polydopamine nanoparticles, the polymerization temperature is controlled at 25~35℃, the polymerization time is 0.5~72.0h, the mass ratio of functional drug to polydopamine is 1~10:10~1, the reaction temperature is 25~35℃, and the reaction time is 1.0~8.0h.

[0014] Furthermore, in the process of preparing drug-loaded polydopamine nanoparticles, the polymerization temperature is controlled at 30°C, the polymerization time is 12.0 h, the mass ratio of functional drug to polydopamine is 4:1, the reaction temperature is 30°C, and the reaction time is 4.0~6.0 h.

[0015] Furthermore, in the process of preparing the drug delivery system, drug-loaded polydopamine nanoparticles are resuspended and dispersed in an aqueous solution of polydopamine-coated bacteria and then incubated with shaking to obtain the drug delivery system.

[0016] Furthermore, in the process of preparing the drug delivery system, the concentration of the functional drug is controlled at 150~300 μg / mL, and the mixture is incubated at 200 rpm in a 37°C incubator.

[0017] To achieve the second objective mentioned above, the present invention provides the following technical solution: An oral probiotic-mediated drug delivery system is prepared by the above method.

[0018] Furthermore, the probiotics are one or more of the probiotics of the genera *Lactobacillus*, *Bifidobacterium*, *Clostridium*, *Lactococcus*, *Enterococcus*, and *Escherichia*.

[0019] Furthermore, the probiotics are one or more of the following: Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium lactis, Clostridium butyricum, Lactococcus lactis, Enterococcus faecalis, Enterococcus faecium, and Escherichia coli.

[0020] Furthermore, the dopamine is oxidized and self-polymerized in Tris·HCl buffer or ethanol / ammonia water to form polydopamine nanoparticles, which are then self-assembled on the surface of the probiotics to form a nanocoating.

[0021] Furthermore, the functional drug is an aromatic functional drug that can be loaded onto the surface of polydopamine nanoparticles through covalent bonds, π-π stacking, or hydrogen bonding.

[0022] Furthermore, the functional drug is one or a combination of several of the following: camptothecin, irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38), doxorubicin, curcumin, gemcitabine, mesalazine, tofacitinib, glibenclamide, sitagliptin, phentermine, and loperamide.

[0023] To achieve the third objective mentioned above, the present invention provides the following technical solution: Application of an oral probiotic-mediated drug delivery system: The drug delivery system prepared by the above method is used in the preparation of drugs for the prevention or treatment of tumors, colitis, diarrhea, constipation, diabetes, and obesity.

[0024] In summary, the beneficial technical effects of this invention are as follows: Based on biomimetic interface engineering technology, this invention nano-coats the surface of probiotics with polydopamine, and then loads functional drugs to form a drug delivery system. This polydopamine nano-coating layer not only significantly enhances the survival rate of probiotics in harsh gastrointestinal environments such as gastric acid and bile salts, but also endows the drug delivery system with good intestinal targeting and long-term adhesion functions. In addition, polydopamine can achieve efficient loading of functional drugs through π-π conjugation and hydrogen bonding, and responds to drug release in the presence of reactive oxygen species (ROS) or reduced glutathione (GSH) in the intestine. After drug release, its probiotic carrier also has immunomodulatory and intestinal microecological remodeling functions, showing important application value in the prevention and treatment of intestinal diseases such as tumors and intestinal inflammation, as well as in the synergistic treatment of diseases such as diabetes, obesity, diarrhea, and constipation. Attached Figure Description

[0025] Figure 1 This is a growth curve diagram of the probiotics in Example 1 of the present invention.

[0026] Figure 2 This is a transmission electron microscope (TEM) schematic diagram of the polydopamine nanocoated bacteria prepared according to the present invention 3.

[0027] Figure 3 The graph shows the number of probiotics that survived in a simulated gastric juice environment after the polydopamine nanocoated bacteria prepared in 3-7 of this invention were exposed to the environment.

[0028] Figure 4 The diagram shows the number of probiotics that survived in a simulated small intestinal fluid environment after the polydopamine nanocoated bacteria prepared in 3-7 of this invention were exposed to the present invention.

[0029] Figure 5 The graph shows the number of probiotics that survived in a simulated bile salt environment after the polydopamine nano-coated bacteria prepared in 3-7 of this invention were exposed to the environment.

[0030] Figure 6 The above are probiotic growth curves of the drug delivery system prepared in Examples 13-17 of this invention.

[0031] Figure 7 This is an in vitro cumulative release curve of the drug delivery system of Embodiment 18 of the present invention.

[0032] Figure 8 This is a graph showing the survival rate of HT-29 cells in Example 18 of the present invention.

[0033] Figure 9 This is a graph showing the cytotoxicity of the drug delivery system of Example 18 of the present invention against HT-29 cells.

[0034] Figure 10 This is an image of the drug delivery system prepared in Example 18 of the present invention being taken up in HT-29 cells.

[0035] Figure 11 This is a fluorescence imaging image of isolated colon tissue from Example 18 of the present invention.

[0036] Figure 12 This is a fluorescence imaging image of mouse intestinal tissue from Example 18 of the present invention.

[0037] Figure 13 This is a graph showing the weight changes of mice in Example 18 of the present invention during treatment with different formulation groups.

[0038] Figure 14 This is a graph showing the change in bioluminescence intensity of tumors in different groups of mice during the drug administration cycle in Example 18 of the present invention. Detailed Implementation

[0039] To make the technical means, creative features, objectives and effects of this invention clearer and easier to understand, the invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0040] Example 1: This invention discloses an oral probiotic-mediated drug delivery system, composed of probiotics, polydopamine nanoparticles, and functional drugs. Among them, The probiotics are one or more of the following genera: Lactobacillus, Bifidobacterium, Clostridium, Lactobacillus, Enterococcus, and Escherichia. Preferably, Lactobacillus probiotics are one or more of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus bulgaricus. Bifidobacterium probiotics are one or more of the following: Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium bifidum, and Bifidobacterium lactis. Clostridium probiotics include Clostridium butyricum; Lactobacillus probiotics are one or more of the lactococci (lactic acid bacteria) species. Enterococcus probiotics are one or more of Enterococcus faecalis and Enterococcus faecium; Escherichia coli is a probiotic strain of the genus Escherichia. Dopamine is oxidized and self-polymerized in Tris·HCl buffer or ethanol / ammonia water to form polydopamine nanoparticles (PDA), which are then self-assembled on the surface of probiotics to form a nanocoating. The functional drug is an aromatic functional drug that can be loaded onto the surface of polydopamine nanoparticles through covalent bonds, π-π stacking, or hydrogen bonding; preferably, it is a combination of one or more of camptothecin, irinotecan, 7-ethyl-10-hydroxycamptothecin (SN38), doxorubicin, curcumin, gemcitabine, mesalazine, tofacitinib, glibenclamide, sitagliptin, phentermine, and loperamide.

[0041] In the drug delivery system of this embodiment, the probiotic is preferably Escherichia coli Nissle 1917 (EcN), and the functional drug is preferably 7-ethyl-10-hydroxycamptothecin (SN38). Probiotic (EcN) culture: Take 10 mL of LB broth culture base into a 12 mL sterile shaking tube, sterilize it by burning with an alcohol lamp and cool it, scrape off the probiotic colonies with good morphology, insert them into the culture medium in the shaking tube, shake gently, seal the opening of the shaking tube with sealing film, and label the shaking tube. Determination of the growth curve of probiotics (EcN): 1 mL of overnight cultured EcN was added to 49 mL of broth medium, and then evenly distributed into 5 sterile shake tubes. These tubes were then incubated in a constant temperature shaking incubator. Every 1 hour, 3 mL of the culture was taken from each tube twice, and the absorbance at 600 nm was measured. The result was expressed as an OD value. 600 A growth curve was plotted with the vertical axis representing the growth time and the horizontal axis representing the growth period. The results are as follows: Figure 1 As shown; from Figure 1 It can be seen that probiotics (EcN) grow rapidly in the first 2-3 hours. During this period, the number of live bacteria on the growth curve increases linearly, indicating that the bacteria are in the logarithmic growth phase. Around 4 hours, the bacteria enter the plateau phase, reaching the maximum bacterial concentration, at which point the total number of bacteria is stable.

[0042] Example 2: This is a method for preparing an oral probiotic-mediated drug delivery system disclosed in this invention, which differs from Example 1 in that it includes... The process of preparing polydopamine nanocoated bacteria (EcN-PDA) from probiotics and dopamine; The process of preparing drug-loaded polydopamine nanoparticles (PDA-SN38) from dopamine and functional drugs; And the process of preparing a drug delivery system (EcN-PDA-SN38) from polydopamine nanocoated bacteria and drug-loaded polydopamine nanoparticles; In this process, dopamine undergoes oxidative self-polymerization to form polydopamine nanoparticles, which then self-assemble on the surface of probiotics to form a nanocoating. Functional drugs are loaded onto the surface of polydopamine nanoparticles through covalent bonds, π-π stacking, or hydrogen bonding.

[0043] Example 3: This is a method for preparing an oral probiotic-mediated drug delivery system disclosed in this invention. The difference from Example 2 is that the specific implementation of S1 is as follows: S11 Preparation of Tris·HCl buffer: Dissolve 10 mM tris(hydroxymethyl)aminomethane (Tris) in 950 mL of pure water with stirring. Add hydrochloric acid (HCl) dropwise at 37 °C and adjust the pH of the solution to 8.5. Add pure water to make up to 1 L to obtain Tris·HCl buffer. S12 Preparation of probiotic suspension: Take 1 mL of probiotic colonies that have been cultured overnight, collect them by centrifugation at 4000 rpm for 5 min, wash them three times with pure water, and then resuspend and disperse them with 1 mL of Tris·HCl buffer to obtain probiotic suspension. S13 Preparation of dopamine solution: Add dopamine powder to Tris·HCl buffer solution and filter through a 0.22μm filter membrane to obtain dopamine solution; Preparation of polydopamine-coated bacteria (S14): After filtration through a filter membrane, 4.5 mL of dopamine solution and 0.5 mL of probiotic suspension were added to a 20 mL sterile vial, and the concentration of the culture medium was controlled at 1×10⁻⁶. 9 The dopamine concentration corresponding to CFU / mL probiotics was 0.8 mg / mL. The mixture was cultured in a 37℃ incubator with shaking for 40 min. After the reaction was completed, the bacteria were collected by centrifugation at 4000 rpm for 5 min and washed three times with pure water to obtain polydopamine-coated bacteria.

[0044] Characterization of polydopamine nanocoated bacteria: The polydopamine-coated EcN-PDA bacteria prepared in Example 3 were resuspended to an appropriate concentration and dropped onto a copper grid. After drying at room temperature, their morphology and size were observed using transmission electron microscopy. The results are as follows: Figure 2 As shown.

[0045] from Figure 2 As can be seen, EcN-PDA(B) is completely and firmly encapsulated by a thin film anchored by a large number of nanoparticles. Their rough surfaces are polydopamine coating layers, which contrast sharply with the smooth edges of the uncoated probiotic Plain-EcN(A). TEM images prove that dopamine has successfully formed a complete and dense polydopamine nanocoating layer on the surface of EcN.

[0046] Examples 4-7: These are preparation methods of an oral probiotic-mediated drug delivery system disclosed in this invention. The difference from Example 3 is that, in S14, the concentration of 1×10⁻⁶ bacteria in the culture medium is controlled to be 1×10⁻⁶. 9 The dopamine concentrations corresponding to CFU / mL probiotics are 0.4, 0.6, 1.0, and 2.0 mg / mL.

[0047] The protective effect of polydopamine nanocoating on probiotics in simulated gastric juice (SGF) and small intestinal juice (SIF) environments: The initial input is 2×10 9 Uncoated CFU probiotic Plain-EcN and polydopamine-coated EcN-PDA prepared in Examples 3-7 were resuspended and dispersed in 1 mL of simulated gastric fluid (pH=1.5, containing 3.2 mg / mL pepsin), and then incubated in a 37°C shaking incubator. After incubation for 0.5 h, 1 h, 2 h, and 3 h, bacterial suspensions of different concentrations were taken, centrifuged to remove the simulated gastric fluid, washed, and resuspended and dispersed in 1 mL of PBS. Then, 0.1 mL of each concentration was serially diluted 1:10, and 0.1 mL of each dilution was evenly spread on sterile petri dishes, with 3 parallel controls for each dilution. After incubating the petri dishes at 37°C for 24 h, the colony count was counted (30-100 colonies per plate was preferred). The survival rate and number of EcN cells treated with simulated gastric fluid were calculated according to the dilution factor. The results are as follows: Figure 3 As shown.

[0048] The initial input is 2×10 9 Uncoated probiotic Plain-EcN (CFU) and polydopamine-coated EcN-PDA prepared in Examples 3-7 were resuspended and dispersed in 1 mL of simulated small intestinal fluid (PBS, pH 6.8, containing 10 mg / mL trypsin), and incubated in a 37°C shaking incubator. After incubation for 0.5, 1, 2, 3, 6, 8, and 12 h, the bacterial suspension was removed, centrifuged to remove the simulated intestinal fluid, washed, and resuspended and dispersed in 1 mL of PBS. Then, a 1:10 serial dilution was performed, and 0.1 mL of each dilution was evenly spread onto sterile petri dishes, with three parallel controls for each dilution. After incubating the petri dishes at 37°C for 24 h, the colony count was counted (30-100 colonies per plate was ideal). The survival rate and number of EcN cells treated with simulated intestinal fluid were calculated according to the dilution factor. The results are as follows: Figure 4 As shown.

[0049] from Figure 3 and Figure 4It can be seen that after incubation with simulated gastric acid, the viable bacterial count of EcN-PDA was significantly higher than that of Plain-EcN. At a low DA concentration (0.4 mg / mL), there was no significant difference in the number of probiotics between EcN-PDA and Plain-EcN after SGF treatment, indicating that low concentrations of PDA did not show significant protective ability against EcN. However, EcN-PDA encapsulated with high concentrations of DA (>1 mg / mL) showed a large number of probiotic deaths in SGF, indicating that excessively thick PDA encapsulation not only failed to protect probiotics from the invasion of gastric acid and intestinal fluid, but also reduced the viability of the probiotics themselves. Furthermore, after PDA encapsulation, EcN-PDA exhibited a higher bacterial count and viability compared to plain-EcN.

[0050] The protective effect of polydopamine nanocoating on probiotics in a simulated bile salt environment: The initial input is 2×10 9 Uncoated probiotic Plain-EcN from CFU and polydopamine-coated EcN-PDA prepared in Example 3 were resuspended and dispersed in 1 mL of bile salt solution (5 mg / mL) and incubated in a 37°C shaking incubator. After incubation for 1, 2, 4, 8, and 12 hours, the bacterial suspension was removed, centrifuged to remove the bile salt solution, washed, and resuspended and dispersed in 1 mL of PBS. Then, a 1:10 serial dilution was performed, and 0.1 mL of each dilution was evenly spread onto sterile petri dishes, with three parallel controls for each dilution. After incubating the petri dishes at 37°C for 24 hours, the colony count was counted (30-100 colonies per plate was ideal). The survival rate and number of EcN cells after bile salt treatment were calculated according to the dilution factor. The results are as follows: Figure 5 As shown.

[0051] from Figure 5 It can be seen that in a bile salt solution with a concentration of 5 mg / mL, EcN protected by PDA coating exhibited significantly higher bacterial survival rates and growth activity compared to uncoated EcN. Specifically, the survival rate of PDA-EcN increased by 51.6 times and 9.5 times compared to plain-EcN after bile salt treatment for 4 h and 12 h, respectively, indicating that the polydopamine nanocoating layer can significantly enhance the tolerance of EcN to bile salts.

[0052] Example 8: This is a method for preparing an oral probiotic-mediated drug delivery system disclosed in this invention. The difference from Example 2 is that the specific implementation of S1 is as follows: S11 Preparation of Tris·HCl buffer: Dissolve 10 mM tris(hydroxymethyl)aminomethane (Tris) in 950 mL of pure water with stirring. Add hydrochloric acid (HCl) dropwise at 37 °C and adjust the pH of the solution to 8. Add pure water to make up to 1 L to obtain Tris·HCl buffer. S12 Preparation of probiotic suspension: Take 1 mL of probiotic colonies that have been cultured overnight, collect them by centrifugation at 4000 rpm for 5 min, wash them three times with pure water, and then resuspend and disperse them with 1 mL of Tris·HCl buffer to obtain probiotic suspension. S13 Preparation of dopamine solution: Add dopamine powder to Tris·HCl buffer solution and filter through a 0.22μm filter membrane to obtain dopamine solution; Preparation of polydopamine-coated bacteria (S14): After filtration through a filter membrane, 4.5 mL of dopamine solution and 0.5 mL of probiotic suspension were added to a 20 mL sterile vial, and the concentration of the culture medium was controlled at 1×10⁻⁶. 9 The dopamine concentration corresponding to CFU / mL probiotics was 0.1 mg / mL. The mixture was cultured in a 37℃ incubator with shaking for 30 min. After the reaction was completed, the bacteria were collected by centrifugation at 4000 rpm for 5 min and washed three times with pure water to obtain polydopamine-coated bacteria.

[0053] Example 9: This is a method for preparing an oral probiotic-mediated drug delivery system disclosed in this invention. The difference from Example 2 is that the specific implementation of S1 is as follows: S11 Preparation of Tris·HCl buffer: Dissolve 10 mM tris(hydroxymethyl)aminomethane (Tris) in 950 mL of pure water with stirring. Add hydrochloric acid (HCl) dropwise at 37 °C and adjust the pH of the solution to 9. Add pure water to make up to 1 L to obtain Tris·HCl buffer. S12 Preparation of probiotic suspension: Take 1 mL of probiotic colonies that have been cultured overnight, collect them by centrifugation at 4000 rpm for 5 min, wash them three times with pure water, and then resuspend and disperse them with 1 mL of Tris·HCl buffer to obtain probiotic suspension. S13 Preparation of dopamine solution: Add dopamine powder to Tris·HCl buffer solution and filter through a 0.22μm filter membrane to obtain dopamine solution; Preparation of polydopamine-coated bacteria (S14): After filtration through a filter membrane, 4.5 mL of dopamine solution and 0.5 mL of probiotic suspension were added to a 20 mL sterile vial, and the concentration of the culture medium was controlled at 1×10⁻⁶. 9 The dopamine concentration corresponding to CFU / mL probiotics was 10.0 mg / mL. The mixture was cultured in a shaking incubator at 37℃ for 60 min. After the reaction was completed, the bacteria were collected by centrifugation at 4000 rpm for 5 min and washed three times with pure water to obtain polydopamine-coated bacteria.

[0054] Example 10: This is a method for preparing an oral probiotic-mediated drug delivery system disclosed in this invention. The difference from Example 3 is that the specific implementation of step S2 is as follows: S21 Preparation of polydopamine nanoparticles: In a mixed solution of 18 mL water, 8 mL ethanol and 0.6 mL ammonia, add 2 mL of aqueous solution containing 100 mg dopamine, stir at low speed at 30 °C for 12 h, collect by centrifugation at 10000 rpm for 10 min, wash three times with pure water to obtain polydopamine nanoparticles, and store in a refrigerator at 4 °C. S22 Preparation of functional drug solution: The functional drug is added to dimethyl sulfoxide (DMSO) and dissolved by ultrasonication to obtain the functional drug solution; S23 Preparation of drug-loaded polydopamine nanoparticles: 0.5 mL of functional drug solution was added to 5 mL of aqueous solution of polydopamine nanoparticles, and the mass ratio of functional drug to polydopamine was controlled at 4:1. The mixture was stirred at 30 °C for 5 h. After the reaction was completed, the unloaded functional drug was removed by centrifugation at 4000 rpm for 5 min. The supernatant was collected by centrifugation at 10000 rpm for 10 min, washed with pure water, and drug-loaded polydopamine nanoparticles were obtained.

[0055] Example 11: This invention discloses a method for preparing an oral probiotic-mediated drug delivery system. The difference from Example 8 is that S2 is specifically implemented as follows: S21 Preparation of polydopamine nanoparticles: In a mixed solution of 18 mL water, 8 mL ethanol and 0.6 mL ammonia, add 2 mL of aqueous solution containing 100 mg dopamine, stir at low speed at 30 °C for 0.5 h, collect by centrifugation at 10000 rpm for 10 min, wash with pure water 3 times to obtain polydopamine nanoparticles, and store in a refrigerator at 4 °C. S22 Preparation of functional drug solution: The functional drug is added to dimethyl sulfoxide (DMSO) and dissolved by ultrasonication to obtain the functional drug solution; S23 Preparation of drug-loaded polydopamine nanoparticles: 0.5 mL of functional drug solution was added to 5 mL of aqueous solution of polydopamine nanoparticles, and the mass ratio of functional drug to polydopamine was controlled at 1:10. The mixture was stirred at 30 °C for 1 h. After the reaction was completed, the unloaded functional drug was removed by centrifugation at 4000 rpm for 5 min. The supernatant was collected by centrifugation at 10000 rpm for 10 min, washed with pure water, and drug-loaded polydopamine nanoparticles were obtained.

[0056] Example 12: This is a method for preparing an oral probiotic-mediated drug delivery system disclosed in this invention. The difference from Example 9 is that the specific implementation of S2 is as follows: S21 Preparation of polydopamine nanoparticles: In a mixed solution of 18 mL water, 8 mL ethanol and 0.6 mL ammonia, add 2 mL of aqueous solution containing 100 mg dopamine, stir at low speed at 30 °C for 72.0 h, collect by centrifugation at 10000 rpm for 10 min, wash three times with pure water to obtain polydopamine nanoparticles, and store in a refrigerator at 4 °C. S22 Preparation of functional drug solution: The functional drug is added to dimethyl sulfoxide (DMSO) and dissolved by ultrasonication to obtain the functional drug solution; S23 Preparation of drug-loaded polydopamine nanoparticles: 0.5 mL of functional drug solution was added to 5 mL of aqueous solution of polydopamine nanoparticles, and the mass ratio of functional drug to polydopamine was controlled at 10:1. The mixture was stirred at 30 °C for 8 h. After the reaction was completed, the unloaded functional drug was removed by centrifugation at 4000 rpm for 5 min. The supernatant was collected by centrifugation at 10000 rpm for 10 min, washed with pure water, and drug-loaded polydopamine nanoparticles were obtained.

[0057] Example 13: This invention discloses a method for preparing an oral probiotic-mediated drug delivery system. The difference from Example 10 is that, in S3, drug-loaded polydopamine nanoparticles are resuspended and dispersed in 1 mL of an aqueous solution of polydopamine-coated bacteria, and the concentration of the functional drug is controlled at 150 μg / mL. The mixture is then incubated at 200 rpm in a 37°C incubator to obtain the oral probiotic-mediated drug delivery system.

[0058] Examples 14-17: This invention discloses a method for preparing an oral probiotic-mediated drug delivery system. The difference from Example 10 is that in S3, the concentration of the functional drug is controlled to be 0, 300, 600, or 1200 μg / mL.

[0059] The drug delivery systems EcN-PDA-SN38 prepared in Examples 13-17 were transferred to LB broth medium, and their growth curves were measured to investigate the activity of EcN under different PDA-SN38 dosages. The results are as follows: Figure 6 As shown.

[0060] from Figure 6 As can be seen from the comparison of the growth curves of EcN with five different dosages, as the dosage increases, the growth curve of EcN shifts to the right and the logarithmic growth phase is prolonged, indicating that its activity is affected to some extent. However, when the dosage of the functional drug is 150 and 300 μg, the growth curve of EcN is similar to that of naked bacteria and reaches a similar plateau phase, indicating that the activity of EcN is relatively good at this dosage. Example 18: This is an application of an oral probiotic-mediated drug delivery system disclosed in this invention. The difference from Example 13 is that the drug delivery system prepared by the above method is used in the preparation of medicines for the prevention or treatment of tumors, colitis, diarrhea, constipation, diabetes, and obesity.

[0061] 1. In vitro drug release study of the drug delivery system: To evaluate the pH-sensitive drug release capacity of the EcN-PDA-SN38 formulation, SN38 release experiments were performed in PBS buffer at pH 7.4, 6.5, and 5.0. 1 mL of EcN-PDA-SN38 suspension was placed in a dialysis bag (MWCO 3500 Da), which was then immersed in 90 mL of release medium. The bag was placed in a water bath shaker at a constant temperature (37°C). 3 mL of the release solution was removed at 1, 2, 4, 8, 12, 24, and 48 h, and then replaced with an equal volume of release medium for further incubation. The SN38 concentration in the release solution was determined using the aforementioned UV-Vis spectrophotometric method, and the cumulative release rate was calculated. The results are shown below. Figure 7 As shown.

[0062] like Figure 7 As shown, since SN38 is an antitumor active drug, EcN-PDA-SN38 exhibits the lowest release rate under physiological conditions (37℃, pH 7.4 release medium), while its release is enhanced under tumor microenvironment (e.g., 37℃, pH 6.5 release medium) and tumor cell lysosomal environment (e.g., 37℃, pH 5.0 release medium). The data indicate that EcN-PDA-SN38 exhibits pH-sensitive release characteristics, responding to acidic conditions in the tumor microenvironment to achieve pH-sensitive drug release.

[0063] 2. In vitro cell evaluation study of drug delivery system: (1) In vitro cytotoxicity The toxicity of blank carriers such as EcN, EcN-PDA, and PDA nanoparticles to HT-29 cells was evaluated using the MTT assay. HT-29 cells were cultured to the logarithmic growth phase, and the resulting cell suspension was collected. Cells were counted using a cell counting chamber, and then 100 μL of the cell suspension was added to each well of a 96-well plate at a cell density of 5000 cells / well. The cells were incubated in a CO2 incubator for 24 h to allow for cell adhesion. After 24 h, the old culture medium was aspirated, and the cells were washed with PBS. 100 μL of culture medium suspensions containing different concentrations of EcN, EcN-PDA, or different concentrations of PDA nanoparticles were added, with blank cells in complete culture medium serving as a control. The cells were then incubated in a CO2 incubator for 24 h. 24 hours after drug administration, discard the old culture medium, wash with PBS, add 10 μL of MTT solution (5 mg / mL) to each well, shake well, and incubate in a CO2 incubator for 4 hours. Then discard the old culture medium, immediately add 150 μL of DMSO, and after the formazan dissolves, measure the absorbance at 570 nm using a microplate reader. Three parallel controls were performed in each group, and the experiment was repeated three times. The average value and cell viability were calculated. The results are as follows: Figure 8 As shown.

[0064] like Figure 8 The results show the cell viability of HT-29 cells at different concentrations of EcN, EcN-PDA, and PDA nanoparticles. When the concentration of EcN was below 2 × 10⁷ CFU, the cell viability was greater than 80%, indicating good biocompatibility. However, significant cytotoxicity was observed when the concentration of EcN reached 2 × 10⁸ CFU. In contrast, the EcN carrier encapsulated with PDA showed lower cytotoxicity and better biocompatibility even at concentrations of 2 × 10⁸ CFU and 2 × 10⁹ CFU compared to EcN alone. (See graph for cell viability of HT-29 cells at different concentrations of PDA). Figure 8 B) indicates that within the studied concentration range of PDA (<500 μg / mL), PDA exhibited good biocompatibility and did not show significant cytotoxicity.

[0065] (2) In vitro antiproliferative activity study HT-29 cell suspensions in logarithmic growth phase were collected and counted using a cell counting chamber under an inverted microscope. 100 μL of cell suspension was added to each well of a 96-well plate at a density of 5000 cells / well, and the cells were incubated in a CO2 incubator for 24 h to allow for cell adhesion. After good cell growth, the old culture medium was aspirated, and the cells were washed three times with PBS. Then, 100 μL of culture medium suspensions with different drug concentrations were added, and the cells were incubated in a CO2 incubator for 24 h. The experimental groups were as follows: free SN38 (dissolved in DMSO first and then diluted with culture medium), PDA-SN38, and EcN-PDA-SN38 groups. The EcN group had approximately 2 × 10⁷ CFU of bacteria. 24 hours after drug administration, the old culture medium was aspirated, and the cells were washed three times with PBS. 10 μL MTT solution (5 mg / mL) was added to each well, and the cells were shaken well and incubated for 4 hours. The old culture medium was then aspirated, and 150 μL LDMSO was added immediately. After the formazan dissolved, the absorbance at 570 nm was measured using a microplate reader. Three parallel controls were performed per group, and the experiment was repeated three times. The mean value, cell viability, and half-maximal inhibitory concentration (IC50) were calculated. 50 The cytotoxicity of SN38, PDA-SN38, and EcN-PDA-SN38 was detected, and the results are as follows: Figure 9 As shown.

[0066] like Figure 9 As shown, the cytotoxicity of SN38 is dose-dependent. Loading SN38 onto the surface of PDA nanoparticles resulted in a certain degree of reduced cytotoxicity and a slight increase in cell viability compared to free SN38. This may be because the drug-loaded nanoparticles have a sustained-release effect, and the peak concentration of the released drug is lower than that of free SN38, thus exhibiting lower antitumor activity. However, when drug-loaded nanoparticles were adsorbed onto the surface of EcN (EcN-PDA-SN38), the cell viability was somewhat lower compared to drug-loaded nanoparticles, indicating that the EcN drug delivery system can enhance the antitumor cytotoxicity of drug-loaded nanoparticles.

[0067] (3) Cellular uptake research The ability of HT-29 cells to take up EcN-PDA-SN38 was observed using laser confocal microscopy. Specifically, FITC-labeled drug-loaded nanoparticles were incubated with PDA nanoparticles, and IR775 was incubated with EcN-PDA to label the drug-loaded nanoparticles. IR775 labeled bacteria, and multi-channel fluorescence co-localization was used to characterize the uptake of EcN-PDA-SN38 by HT-29 cells. First, 1 mL of logarithmic growth phase HT-29 cell suspension (1×10⁵ cells) was added to a glass-bottomed culture dish (d=4cm) and cultured overnight to allow cell adhesion. Then, EcN-PDA-SN38 suspension was added to the culture dish and incubated for 0, 0.5, 1, 4, and 8 h. The culture medium was then discarded, and after washing the cells with PBS, they were fixed with 4% paraformaldehyde solution for 30 min, followed by washing with PBS. Finally, nuclear counterstaining with DAPI solution for 5 min was performed, and the cells were observed and photographed using laser confocal microscopy. The results are shown below. Figure 10 As shown.

[0068] like Figure 10 As shown, blue represents DAPI fluorescence labeled in the cell nucleus, green represents FITC fluorescence labeled in PDA-SN38 nanoparticles, and red represents IR775 fluorescence labeled in EcN-PDA. At 0.5h and 1h, fluorescence was mainly concentrated on the cell membrane surface, indicating that the system has some adhesiveness. After incubation for 4–8h, certain fluorescence intensities appeared in the cytoplasm and cell nucleus, indicating that EcN-PDA-SN38 can be endocytosed into the cytoplasm and eventually reaches the cell nucleus to exert its antitumor effect. The cellular uptake imaging fully demonstrates that EcN-PDA-SN38 can be effectively taken up by HT-29 cells and further exert its cytotoxic effect.

[0069] 3. Adhesion properties of polydopamine-coated bacteria in mouse intestine: (1) Study on the adhesion properties of EcN-PDA in mouse colon tissue in vitro To investigate the adhesion ability of polydopamine nanocoatings, IR775-labeled EcN and EcN-PDA were incubated with freshly collected mouse colon tissue for 1 hour. The adhesion ability of the material in the colon tissue was evaluated by the fluorescence intensity of the remaining IR775 on the tissue after different washing cycles. The results are as follows: Figure 11 As shown.

[0070] like Figure 11As shown, free IR775 itself has no intestinal adhesion ability. After washing, the amount of IR775 remaining in the intestine decreases rapidly, and the fluorescence disappears completely after three washes. Compared with the IR775 group, EcN has a certain intestinal adsorption capacity. After three washes, a small amount of fluorescence remains in the intestinal tissue. Compared with the EcN group, the EcN-PDA group still has a high fluorescence intensity in the mouse intestine after three washes. This indicates that PDA encapsulation can effectively enhance the adhesion ability of EcN in the intestine. Probiotics modified with polydopamine nanoparticles show improved mucosal adhesion and mucus penetration ability.

[0071] (2) Study on the adhesion properties of EcN-PDA in mouse colon tissue after oral administration Free IR775, EcN, and EcN-PDA were administered to mice via oral gavage. Twenty-four hours later, intestinal tissue from the mice was dissected for fluorescence imaging. The results are as follows: Figure 12 As shown.

[0072] like Figure 12 As shown, after 24 hours of gavage administration and subsequent sacrifice of mice followed by intestinal dissection and fluorescence imaging, free IR775 was almost completely metabolized, with virtually no fluorescence signal observed. In contrast, after oral administration, EcN showed some fluorescence signals in the cecum and colon, indicating a certain amount of residue in these areas. EcN-PDA exhibited significantly enhanced fluorescence signals in the intestine, with fluorescence signals 38 and 30 times greater than those in the oral IR775 group and EcN group, respectively. This suggests that coating EcN with PDA effectively enhances its adhesion to the mouse intestine, thereby further enhancing the absorption and bioavailability of the functional drug system loaded with EcN in the intestine.

[0073] 4. In vivo antitumor efficacy studies of drug delivery systems: An orthotopic colorectal cancer model was established by injecting luciferase-labeled HT29 cells into the colonic wall of nude mice. D-luciferin was then injected intraperitoneally, and IVIS imaging was performed, selecting cells with a bioluminescence intensity ≈ 1 × 10⁻⁶. 6 p / sec / cm −2 In vivo efficacy experiments were conducted on nude mice. The nude mice were randomly divided into 5 groups as follows: saline group, irinotecan hydrochloride group, coated probiotics (EcN-PDA) group, drug-loaded nanoparticles (PDA-SN38) group, and drug-loaded polydopamine-coated bacteria (EcN-PDA-SN38) group.

[0074] Each group of nude mice was administered 150 μL of the drug via gavage or oral administration at a dose of 3.5 mg / kg for SN38 or irinotecan hydrochloride, 6 × 10⁻⁶. 9CFU was administered at the EcN dose. Dosing was repeated every three days for 18 days, with IVIS in vivo imaging performed two days after each administration to observe tumor growth. The weight of the nude mice was measured and recorded during this period.

[0075] (1) Changes in body weight of mice in each group during drug administration Changes in mouse body weight during treatment, such as Figure 13 As shown in the figure. During the treatment period, the nude mice in each group showed good growth and steady weight gain, with no significant differences, indicating that the mice had good tolerance to each formulation at this dose.

[0076] (2) In vivo pharmacodynamic studies of the drug delivery system (EcN-PDA-SN38) After intraperitoneal injection of D-luciferin potassium salt, it is taken up by tumor cells and oxidized to emit light under the action of luciferase and ATP. This bioluminescence can be used to observe tumor changes in different groups of mice during treatment, such as… Figure 14 As shown in the figure, during the 18-day treatment cycle, the biofluorescence intensity emitted by the tumor indicated that the first-line chemotherapy drug irinotecan, polydopamine nanoparticles loaded with SN38 (PDA-SN38), or probiotics encapsulated with polydopamine (EcN-PDA) all had a certain degree of inhibitory effect on tumor growth. However, compared with the oral probiotic delivery system (EcN-PDA-SN38), the tumor growth was significantly inhibited, with a tumor inhibition rate of up to 90%, achieving a significantly better in vivo anti-tumor effect.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing an oral probiotic-mediated drug delivery system, characterized in that: include, The process of preparing polydopamine-coated bacteria from probiotics and dopamine; The process of preparing drug-loaded polydopamine nanoparticles from dopamine and functional drugs; And the process of preparing a drug delivery system from the polydopamine nanocoated bacteria and the drug-loaded polydopamine nanoparticles; In this process, the dopamine undergoes oxidative self-polymerization to form polydopamine nanoparticles, which are then self-assembled on the surface of probiotics to form a nanocoating. The functional drug is loaded onto the surface of the polydopamine nanoparticles through covalent bonds, π-π stacking, or hydrogen bonding.

2. The preparation method of an oral probiotic-mediated drug delivery system according to claim 1, characterized in that: In the process of preparing polydopamine-coated bacteria, probiotic colonies were first resuspended and dispersed in Tris·HCl buffer to obtain a probiotic suspension. Then, dopamine powder was added to the Tris·HCl buffer and filtered through a 0.22 μm filter membrane to obtain a dopamine solution. The probiotic suspension and dopamine solution were then cultured at a constant temperature with shaking. After the culture was completed, post-processing was performed to obtain polydopamine-coated bacteria.

3. The preparation method of an oral probiotic-mediated drug delivery system according to claim 2, characterized in that: In the process of preparing polydopamine nanocoated bacteria, the pH of the Tris·HCl buffer was controlled at 5-10, and the culture medium contained 1×10 9 The dopamine concentration corresponding to CFU / mL probiotics is 0.1~10.0 mg / mL, the culture temperature is 35~40℃, and the culture time is 20~120 min.

4. The preparation method of an oral probiotic-mediated drug delivery system according to claim 1, characterized in that: In the process of preparing drug-loaded polydopamine nanoparticles, an aqueous solution of dopamine is first added to a mixed solution of water / ethanol / ammonia to carry out oxidative self-polymerization. After polymerization, the nanoparticles are obtained through post-treatment. Then, a functional drug solution is added to the polydopamine nanoparticle / aqueous solution for reaction. After reaction, the nanoparticles are obtained through post-treatment.

5. The preparation method of an oral probiotic-mediated drug delivery system according to claim 4, characterized in that: In the process of preparing drug-loaded polydopamine nanoparticles, the polymerization temperature is controlled at 25~35℃, the polymerization time is controlled at 0.5~72.0h, the mass ratio of functional drug to polydopamine is 1~10:10~1, the reaction temperature is controlled at 25~35℃, and the reaction time is controlled at 1.0~8.0h.

6. The method for preparing an oral probiotic-mediated drug delivery system according to claim 1, characterized in that: In the process of preparing the drug delivery system, drug-loaded polydopamine nanoparticles are resuspended and dispersed in an aqueous solution of polydopamine-coated bacteria and then incubated with shaking to obtain the drug delivery system.

7. An oral probiotic-mediated drug delivery system, characterized in that: It is prepared by the method according to any one of claims 1 to 6.

8. The oral probiotic-mediated drug delivery system according to claim 7, characterized in that: The probiotics are one or more of the following: Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium lactis, Clostridium butyricum, Lactococcus lactis, Enterococcus faecalis, Enterococcus faecium, and Escherichia coli.

9. The oral probiotic-mediated drug delivery system according to claim 7, characterized in that: The functional drug is one or a combination of several of the following: camptothecin, irinotecan, 7-ethyl-10-hydroxycamptothecin, doxorubicin, curcumin, gemcitabine, mesalazine, tofacitinib, glibenclamide, sitagliptin, phentermine, and loperamide.

10. The application of an oral probiotic-mediated drug delivery system, characterized in that: The use of the drug delivery system prepared by the method according to any one of claims 1 to 6 in the preparation of medicaments for the prevention or treatment of tumors, colitis, diarrhea, constipation, diabetes, and obesity.

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