Oral nanoparticles based on Bacillus mobilis and preparation method and application thereof

Abstract

The present invention discloses an oral nanoparticle based on Bacillus mobilis, and its preparation method and application, which belongs to the technical field of pharmaceutical preparations characterized by special physical shapes. The method comprises the following steps: (1) human serum albumin and chlorogenic acid are incubated in an aqueous solution environment in the presence of glutathione; after the incubation is completed, a desolventizing agent is added to the system, and the precipitate is collected and purified to obtain human serum albumin-chlorogenic acid nanoparticles; (2) human serum albumin-chlorogenic acid nanoparticles are mixed with MH22 bacterial liquid to complete the loading, and an active oxygen responsive nanomaterial, i.e., oral nanoparticles, is obtained. The method adopts a "one-pot method" to synthesize human serum albumin-chlorogenic acid nanoparticles, which has the advantages of simple process and mild conditions, and is conducive to large-scale production. The oral nanoparticles are designed for the intestinal environment, have low toxicity and side effects, have excellent anti-inflammatory effects and the function of regulating microecology, and have good application prospects in the prevention and treatment of ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations characterized by a special physical shape, and in particular to oral nanoparticles based on Bacillus mobilis, and a preparation method and application thereof. Background Art

[0002] Ulcerative colitis (UC) is an inflammatory disease driven by the immune system that affects the digestive system. The incidence of UC is increasing worldwide, and its chronic, recurring, and unpredictable nature leads to high medical costs and imposes a significant economic burden on society.

[0003] Symptomatic treatment with 5-aminosalicylic acid preparations, glucocorticoids, and immunosuppressants is the mainstay of UC management. In addition, targeted biologic therapies, such as TNF and IL-12 antibodies, can be used as adjuncts to enhance therapeutic efficacy. Currently, the lack of understanding of the pathological and physiological mechanisms of UC has led to the suboptimal efficacy of existing therapies. Oral probiotic therapy shows promise as an adjuvant in the treatment of IBD by acting on the intestine to actively regulate bacterial composition and promote intestinal mucosal repair. However, due to the lack of antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD), these strictly anaerobic probiotics are susceptible to destruction by reactive oxygen species (ROS) in IBD, which reduces efficacy and prolongs treatment duration. There is an unmet need for more effective and safer IBD treatments that effectively target the inflamed colon and rapidly restore a healthy immune microenvironment by suppressing inflammation, remodeling intestinal barrier function, and regulating the gut microbiome in infected tissues. Further research into the pathogenesis of UC and the development of new, effective treatments are urgently needed. Oral nano-delivery system is a promising therapeutic strategy with the following advantages: increasing drug concentration in the intestinal inflammation area to maximize the duration of pharmacological action; preventing or reducing drug degradation to ensure efficacy; prolonging the duration of action, reducing the number of doses, and minimizing toxic side effects.

[0004] Reactive oxygen species (ROS) are a class of oxygen-containing chemically active molecules, primarily including superoxide anions, hydroxyl radicals, and hydrogen peroxide. The dynamic balance of ROS production and metabolism is crucial for maintaining normal cellular and tissue function. However, abnormal increases in ROS can lead to oxidative stress and a series of cellular and tissue damage. Increasing evidence indicates that ROS-induced oxidative stress is a key factor in the development and progression of inflammation. ROS can act by directly damaging and exacerbating inflammatory responses, directly stimulating the production of proinflammatory cytokines, or triggering inflammatory cascades through activation of inflammasomes, thereby exacerbating inflammatory symptoms.

[0005] Proinflammatory macrophages are abundant at sites of inflammation, and targeting these cells can achieve targeted treatment. However, high expression of inflammatory factors at these sites can lead to the accumulation of immune cells and increased levels of reactive oxygen species, limiting the effectiveness of anti-inflammatory treatments. Therefore, inhibiting the production of inflammatory factors is also crucial for anti-inflammatory treatments.

[0006] Numerous studies have reported that probiotics can achieve good results in inducing UC remission, relapse, and preventing complications. For example, Chinese invention patent publication number CN116891818A discloses a strain of Bacillus mobilis and its applications. This probiotic was isolated from the intestinal contents of deep-sea bass by the present research team in previous research. Bacillus toyonensis ), named MH22. MH22 has inhibitory effects on common pathogens such as Escherichia coli, Staphylococcus aureus, and Salmonella Enteritidis, and its probiotic properties have been verified in chickens.

[0007] Currently, the research team has discovered that MH22 possesses enzyme-like activity, but its application in the prevention and treatment of enteritis is still limited by many factors, particularly the difficulty in preparing probiotic hybrid nanoparticles using MH22 as a live carrier. Therefore, building on previous research, the team sought to develop a novel reactive oxygen species (ROS)-responsive nanomaterial that could achieve the combined functions of regulating the intestinal microbiome, scavenging ROS, and providing anti-inflammatory benefits. Addressing these challenges is crucial for the prevention and treatment of inflammatory diseases of the digestive system, such as ulcerative colitis. Summary of the Invention

[0008] In view of the above-mentioned defects of the prior art, in a first aspect of the present invention, a method for preparing oral nanoparticles based on Bacillus mobilis with simple process and mild conditions is provided, comprising the following steps:

[0009] (1) Human serum albumin (HSA) and chlorogenic acid (CGA) are incubated in an aqueous solution in the presence of glutathione (GSH); after the incubation is completed, a desolventizing agent is added to the system, and the precipitate is collected and purified to obtain human serum albumin-chlorogenic acid nanoparticles;

[0010] (2) Human serum albumin-chlorogenic acid nanoparticles are mixed with MH22 bacterial solution to complete the loading and obtain active oxygen responsive nanomaterials, namely oral nanoparticles.

[0011] Preferably, in step (1), the concentration of human serum albumin is 20-80 mg / mL; the concentration of chlorogenic acid is 10-20 mg / mL; and the concentration of glutathione is 50-200 mM.

[0012] The concentration of the raw materials should be controlled within an appropriate range. For example, if the human serum albumin concentration is below 20 mg / mL, the dosage is insufficient, resulting in an inability to coat chlorogenic acid and causing nanoparticles to aggregate. Above 80 mg / mL, the reduction is incomplete, resulting in vacuoles and inhomogeneous nanoparticles. Higher concentrations lead to faster subsequent precipitation and slower stirring. Taking all factors into consideration, 40 mg / mL is the most suitable final concentration for the reaction system. Within the above human serum albumin concentration range, the dosage of chlorogenic acid is designed based on the desired coating capacity. A controlled concentration of 10-20 mg / mL ensures complete coating and reduces residual chlorogenic acid, minimizing material waste and purification difficulties. Within the specified glutathione concentration, higher concentrations shorten the reduction reaction time during incubation. The dosage should not exceed this range, as this will result in excessive residual amounts, compromising subsequent purification and preparation.

[0013] Preferably, in step (1), the incubation temperature is from room temperature to human body temperature, and the incubation time is 1 to 4 hours.

[0014] Preferably, in step (1), the desolventizing agent is ethanol; and the volume ratio of the desolventizing agent to the solution to be added is 1 to 5:1.

[0015] The desolventizer supersaturates the product and forms a precipitate, facilitating separation in subsequent steps. Ethanol is a suitable desolventizer due to its non-toxicity and immiscibility with the packaging material. A volume ratio of 1:5:1 between the desolventizer and the solution to be added to the preparation system achieves good separation results. Below this range, precipitation is incomplete. Further increases in the desolventizer volume increase costs and waste solvent without significantly improving precipitation.

[0016] Preferably, in step (1), the collection and purification process of the precipitate is as follows: centrifugation to separate the precipitate, redispersing the precipitate in water to form a suspension; the suspension is dialyzed against water at 4°C with a dialysis cutoff value of 12-14KD to remove excess desolvent, free chlorogenic acid and glutathione.

[0017] The binding of human serum albumin-chlorogenic acid nanoparticles to MH22 is achieved through electrostatic adsorption. As presented in one or more embodiments of the present invention, those skilled in the art can flexibly adjust the dosage of human serum albumin-chlorogenic acid nanoparticles and MH22 based on actual application requirements to form oral nanoparticles with different functions.

[0018] Preferably, in step (2), the load is completed by shaking at room temperature, and the shaking time is 10 to 12 minutes.

[0019] It is best if the nanoparticles and bacterial solution are shaken for 10 to 12 minutes without sedimentation. The shaking time should not be too long, otherwise they may combine and aggregate into flocs, which is not conducive to loading.

[0020] In a second aspect of the present invention, provided are oral nanoparticles having active oxygen responsiveness, good biocompatibility, and good anti-inflammatory effects, which are prepared using the method of the first aspect of the present invention.

[0021] Preferably, the oral nanoparticles are formed by combining human serum albumin-chlorogenic acid nanoparticles and MH22 by electrostatic adsorption; human serum albumin-chlorogenic acid nanoparticles are water-soluble particles formed by a human serum albumin shell coating a chlorogenic acid core.

[0022] In a third aspect of the present invention, there is provided a use of the oral nanoparticles according to the second aspect of the present invention, specifically a use as a drug in treating inflammatory diseases of the digestive system.

[0023] Preferably, the oral nanoparticles are used as a drug in the treatment of ulcerative colitis.

[0024] Based on the above technical solution, the design concept of the present invention is to first modify chlorogenic acid with human serum albumin before loading MH22. Human serum albumin has excellent biocompatibility and strong binding ability with various drug molecules. During the modification process, human serum albumin is reduced with glutathione to cleave its disulfide bonds and free sulfhydryl groups. It is then cross-linked with the hydrophobic substance chlorogenic acid to form human serum albumin-chlorogenic acid nanoparticles with a hydrophilic outer layer and a hydrophobic inner layer. These particles are water-soluble particles that can be combined with self-isolated probiotic MH22 through electrostatic adsorption to obtain combined probiotic nanoparticles with a large number of drug-loaded nanoparticles adhered to the surface, i.e., oral nanoparticles.

[0025] In an inflammatory microenvironment with high levels of reactive oxygen species, the probiotic MH22 in the oral nanoparticles can remove a large amount of reactive oxygen species, thereby treating inflammation. However, high levels of inflammatory factors can cause immune cells to aggregate and produce more reactive oxygen species, limiting the anti-inflammatory effect. Chlorogenic acid can effectively inhibit the production of inflammatory factors, but due to its poor water solubility, it will be rapidly metabolized once it enters the body. Therefore, the design of the present invention uses a carrier to solve this problem, loading chlorogenic acid, an anti-inflammatory drug that can effectively inhibit inflammatory factors, and utilizing its synchronous release to exert its effect. In addition, when the nanoparticles reach the site of action, they will separate from the carrier probiotics. The probiotics are still active after separation and can stay in the intestine, playing a role in regulating the microecology, achieving the effect of "killing two birds with one stone".

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] The present invention provides a method for preparing oral nanoparticles, which adopts a "one-pot method" to synthesize human serum albumin-chlorogenic acid nanoparticles and is based on Bacillus mobilis and is made by electrostatic adsorption; this method has the advantages of simple process and mild conditions, and is conducive to scale-up production.

[0028] The present invention provides an oral nanoparticle designed for the intestinal application environment. The nanoparticle is a combination of probiotic nanoparticles with a large number of drug-loaded nanoparticles adhered to the surface. It has low toxicity and side effects, excellent anti-inflammatory effects and the function of regulating the microecology of the affected area.

[0029] The present invention provides an application of oral nanoparticles, which has good application prospects in the prevention and treatment of ulcerative colitis as a therapeutic drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Microscopic morphology of HSA / CGA NPs (A) and HSA / CGA@MH22 (B);

[0031] Figure 2 This is the fluorescence staining result of HSA / CGA@MH22;

[0032] Figure 3 is the particle size distribution of HSA / CGA NPs;

[0033] Figure 4 is the Zeta potential table of HSA / CGA NPs;

[0034] Figure 5 is the relative release rate of HSA / CGA NPs and HSA / CGA@MH22 in simulated gastrointestinal fluid;

[0035] Figure 6 The results of drug loading stability test of HSA / CGA@MH22 are shown;

[0036] Figure 7 This is the relationship between different MH22 bacterial concentrations and CGA drug loading;

[0037] Figure 8 The results of the O2 scavenging ability test at different MH22 bacterial concentrations are shown;

[0038] Figure 9 The dissolved oxygen release capacity test results of different MH22 bacterial concentrations;

[0039] Figure 10 The antioxidant capacity test results of different components against O2· (A), H2O2 (B), and ·OH (C);

[0040] Figure 11The antioxidant capacity test results of the drug delivery system containing different concentrations of CGA;

[0041] Figure 12 The growth activity test results of MH22 in the drug delivery system with a CGA concentration of 150 μg / ml in the absence of H2O2 (A) and the presence of H2O2 (B);

[0042] Figure 13 The results of the cell compatibility test of the drug delivery system in HT-29 cells in the absence of H2O2 (A) and the presence of H2O2 (B);

[0043] Figure 14 is the test result of the drug delivery system relative to the ROS concentration;

[0044] Figure 15 Schematic diagram of the experimental process for mice in the treatment group;

[0045] Figure 16 Schematic diagram of the weight changes of mice in the treatment groups;

[0046] Figure 17 Schematic diagram of the changes in DAI scores of mice in the treatment groups;

[0047] Figure 18 The colon morphology photos of mice in the treatment group;

[0048] Figure 19 Schematic diagram of the preparation process of HSA / CGA@MH22. DETAILED DESCRIPTION

[0049] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0050] Example 1

[0051] The preparation method of oral nanoparticles comprises the following steps:

[0052] (1) Human serum albumin (80 mg) and chlorogenic acid (20 mg) were dissolved in deionized water (2 mL) containing glutathione (50 mM) and incubated at 37 °C for 1 h. Ethanol (2 mL) was then added to the resulting solution to precipitate the product. The mixture was stirred at room temperature for 30 min, and the nanoparticles were separated by centrifugation and redispersed in deionized water to obtain a suspension. The suspension was dialyzed against deionized water at 4 °C for 24 h to remove ethanol, free chlorogenic acid, and glutathione. Human serum albumin-chlorogenic acid nanoparticles were obtained and designated as HSA / CGA NPs.

[0053] (2) Human serum albumin-chlorogenic acid nanoparticles (50 mg) and MH22 bacterial solution (1 mL, 1 McFarland turbidity) were mixed and shaken at room temperature for 10 min. The loading was completed by electrostatic adsorption to obtain oral nanoparticles, which were recorded as HSA / CGA@MH22.

[0054] In this example, a series of characterizations were performed on HSA / CGA NPs and HSA / CGA@MH22 to study the preparation effect of the method of the present invention.

[0055] The micromorphology of HSA / CGA NPs was observed using transmission electron microscopy (TEM); the surface micromorphology of HSA / CGA@MH22 was observed using scanning electron microscopy (SEM). Figure 1 shown.

[0056] In the figure, TEM imaging of HSA / CGANPs ( Figure 1 A) shows that HSA / CGANPs are spherical with irregular pits, which are basically uniform in size and dispersed evenly; SEM imaging of HSA / CGA@MH22 ( Figure 1 B) HSA / CGA@MH22 is rod-shaped, smoother than MH22, and has a lower sensitivity.

[0057] The HSA / CGA@MH22 drug delivery system was tested for fluorescence staining. RhB-HSA and Cy5-CGA were used as raw materials to synthesize HSA / CGA@MH22. The operation process was the same as above. Immediately after the preparation, the distribution of HSA and CGA was observed under a laser confocal microscope using a light source with an appropriate excitation wavelength. The fluorescence staining results are shown in Figure 2. Figure 2 shown.

[0058] Figure 2 The dual fluorescence experimental photos in the figure intuitively show the distribution of HSA and CGA in HSA / CGA@MH22 on the MH22 bacteria. It can be seen that the green fluorescence (Cy5-labeled CGA) and red fluorescence (RhB-labeled HSA) are evenly distributed and consistent with the shape of the bacteria, and the overlap of the two labeled fluorescence is very high. It can be inferred that HSA and CGA form a nanoparticle complex and are evenly adsorbed on the MH22 surface.

[0059] The particle size distribution and PDI of HSA / CGANPs and the Zeta potential of HSA / CGA@MH22 were measured by DLS. All the measured values ​​were the average of three measurements at 25°C. Figure 3 As shown; Zeta potential of HSA / CGA NPs, the results are as follows Figure 4 shown.

[0060] Depend on Figure 3 It can be seen that the average particle size of HSA / CGANPs is 99.6 ± 3.8 nm, the dispersion coefficient is 0.69 ± 0.14, and the particle size distribution is uniform; Figure 4 The results showed that the Zeta potential of HSA / CGA@MH22 was -21.36 mV, and that of MH22 was -27.67 mV. After MH22 was adsorbed and combined with HSA / CGANPs, the potential increased and the number of negative charges decreased.

[0061] HSA / CGA NPs and HSA / CGA@MH22 were tested in simulated gastrointestinal fluid to investigate the in vitro release properties of CGA from HSA / CGA@MH22. The relative release rates of the two solutions were measured over time, and the data were plotted. PBS buffers (pH 1.2 and 6.8) were used to simulate gastric fluid (SGF) and intestinal fluid (SIF), respectively. 1 mL of HSA / CGA NPs and 1 mL of HSA / CGA@MH22 solutions were precisely measured and placed in 250 mL Erlenmeyer flasks. 100 mL of SGF was added and the flasks were incubated at 37°C in a water bath. Samples were taken at pre-set time points (10, 30, 60, 90, and 120 min) and the CGA content was determined by HPLC. The solutions were then transferred to SIF and the CGA content was measured at different time points (130, 150, 180, 210, 240, 300, and 360 min). The release rates were calculated, and the cumulative release rate was statistically analyzed to plot time-release curves. The release rate is calculated according to the following formula: Release (%) = m t / m0×100%; where m t is the drug release amount within the measurement time, m0 is the total drug amount, and the test results are as follows Figure 5 shown.

[0062] Figure 5 The difference in in vitro drug release between HSA / CGANPs and HSA / CGA@MH22 was intuitively demonstrated. After 2 h of release in SGF, the cumulative release rate of HSA / CGANPs was 23.52%, while that of HSA / CGA@MH22 was 9.21%. After 4 h of release in SIF, the cumulative release rate of HSA / CGANPs was 53.95%, while that of HSA / CGA@MH22 was 23.22%.

[0063] Example 2

[0064] This example investigated the drug loading stability of HSA / CGA@MH22. The encapsulation efficiency of CGA in HSA / CGA@MH22 was determined. The liquid phase assay, standard curve construction, and encapsulation efficiency determination methods for CGA were as follows: Accurately weigh 25 mg of the CGA stock sample (drug), dissolve it in a small amount of methanol, dilute to volume in a 10 mL volumetric flask, and filter to obtain a CGA control stock solution. Dispense 0.0, 1.0, 2.0, 4.0, 8.0, and 12.0 mL of this stock solution into a 25 mL volumetric flask and dilute to volume with 70% methanol to prepare standard solutions of varying concentrations. Add 1 mL of the prepared HSA / CGA@MH22 solution to a centrifuge tube, sonicate for 30 minutes, and centrifuge at 12,000 rpm for 10 minutes. Collect the supernatant, filter with filter paper, and precisely aspirate 200 μL of the filtrate. Add 800 μL of methanol, mix thoroughly, and filter through a 0.45 filter to obtain the treated test solution. Prepare a 20 mg / mL CGA untreated control solution and a control test solution using the same method. Prepare the standard solutions, treated test solutions, and control test solutions at each concentration. Inject samples according to the chromatographic conditions (Agilent ZORBAX RHC18 column, 50 mm × 2.1 mm, 1.8 μm; mobile phase: acetonitrile:0.3% glacial acetic acid = 2:8; detection wavelength: 326 nm; flow rate: 1 mL / min; column temperature: 30°C; injection volume: 20 μL) and the requirements, and calculate the peak areas. First, examine the linear relationship, then calculate the encapsulation efficiency. The encapsulation efficiency calculation formula is as follows: EE %=Area 处理 / Area 对照 , Area 处理 and Area 对照 Respectively represent the corresponding peak area (μVsecond).

[0065] The drug loading stability test results of HSA / CGA@MH22 are as follows Figure 6 HPLC linear method validation showed that CGA had a good linear relationship in the range of 100-1200 μg / mL (R 2 =1.000). Under this condition, the encapsulation efficiency of CGA in HSA / CGA@MH22 was measured to be 56.17%±7.96%.

[0066] Example 3

[0067] This example studies the relationship between different MH22 cell concentrations and CGA drug loading, and investigates the optimal ratio of cell concentration to CGA drug loading. The results are as follows: Figure 7 shown.

[0068] The inventors took 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0×10 71 mL of bacterial solution with a CFU / mL was thoroughly mixed with 1 mL of 2.5 mg / mL CGA NPs. The precipitate was collected after centrifugation and ultrasonically disrupted. The CGA concentration was determined by HPLC. 7 CFU / mL, the bacterial concentration is low and the drug loading is small. When the bacterial concentration exceeds 2×10 7 CFU / mL, the loading capacity of CGA is enhanced, but the high concentration of bacteria in the solution is prone to flocculent precipitation. If the bacterial concentration is too low, the loading capacity is low, and if the bacterial concentration is too high, the dispersion is poor. Considering the nanoparticle loading capacity and adsorption capacity, it is decided to choose 1.0×10 7 When the bacterial solution with CFU / mL is mixed with the drug, no flocs are produced and the drug loading capacity is sufficient.

[0069] Example 4

[0070] This example studies the O2 scavenging ability of different MH22 bacterial cell concentrations and examines the difference in O2 scavenging ability between different MH22 bacterial cell concentrations. The results are as follows: Figure 8 shown.

[0071] The inventors took fresh bacterial liquid after culture and prepared it into 10 4 , 10 5 , 10 6 , 10 7 and 10 8 CFU / mL of bacterial solution was prepared for standby use. Take 100 μL of each concentration of bacterial solution and measure its concentration according to the kit operation (White Shark Biological Superoxide Anion Content Determination Kit). Each sample was measured in parallel 3 times, and the data were recorded as the mean of the three times and plotted. Figure 8 As can be seen from the figure, the superoxide anion content of the bacterial solution increases with different concentrations, indicating that the bacterial solution itself has the ability to generate superoxide anions or can catalyze the generation of superoxide anions. In actual applications, those skilled in the art can prepare the bacterial solution according to the actual required concentration.

[0072] Example 5

[0073] This example studies the dissolved oxygen release capacity of different MH22 bacterial cell concentrations. The inventors first prepared fresh bacterial liquid after culture and measured it with a turbidimeter to obtain a concentration of 10 4 , 10 5 , 10 6 , 10 7 and 10 8CFU / mL of bacterial solution was prepared for standby. 100 μL of each concentration of bacterial solution was taken and mixed with 5 mL of fresh culture medium respectively. Fresh culture medium without bacterial solution was used as control. After 12 hours of co-culture and stabilization, a BDO-200A dissolved oxygen meter was used for direct measurement. Real-time data was read and recorded every 15 seconds until the end of the recording at 11 minutes. A curve was drawn with time as the horizontal axis and dissolved oxygen concentration as the vertical axis. The results are shown in the figure. Figure 9 shown.

[0074] The curve shows that the dissolved oxygen concentration in the control group did not increase during the measurement period. However, the dissolved oxygen concentration in the fermentation broths of bacteria with different initial concentrations increased during the culture process and was proportional to the initial concentration of the bacteria broth.

[0075] Example 6

[0076] In this example, the components involved in the preparation were used as research objects, and the antioxidant capacity of HSA, CGA, HSA / CGA NPs, MH22, and HSA / CGA@MH22 against O2·, H2O2, and ·OH was tested respectively. The results are shown in Figure 2. Figure 10 shown.

[0077] Figure 10 The researchers visually demonstrated the differences in the in vitro O2-, H2O2-, and ·OH scavenging abilities of HSA / CGA@MH22. The authors found that HSA / CGA@MH22 exhibited the highest O2·, H2O2, and ·OH scavenging abilities, surpassing both HSA / CGANPs and MH22. O2· scavenged by over 70%, H2O2 by nearly 30%, and ·OH by over 60%, demonstrating the superior free radical scavenging capacity of HSA / CGA@MH22.

[0078] Example 7

[0079] This example is based on the preparation method of Example 1. The dosage of chlorogenic acid was controlled to prepare oral nanoparticles with chlorogenic acid concentrations of 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 300 μg / mL. According to the concentration values, the drug delivery systems used in the experimental groups are recorded as HSA / CGA. 50 @MH22、HSA / CGA 100 @MH22、HSA / CGA 150 @MH22、HSA / CGA 200 @MH22、HSA / CGA 300 @MH22; For comparison, the blank group (Untreated) without oral nanoparticles and the control group with only MH22 added were used.

[0080] In order to compare and test whether nanoparticles with different chlorogenic acid loadings can survive in a high hydrogen peroxide state simulating tumor or inflammation, the inventors gave 200 nM H2O2 intervention for 2 h and 1×10 7 The bacterial concentration blank was used as negative control, and MH22, HSA / CGA were analyzed respectively. 50 @MH22、HSA / CGA 100 @MH22、HSA / CGA 150 @MH22、HSA / CGA 200 @MH22 and HSA / CGA 300 @MH22 bacterial survival rate. The calculation process and operation of bacterial survival rate are as follows: take samples from each treatment group and determine the number of existing bacteria in each sample by turbidimetry (the number of bacteria in the treatment group = the OD value of the treatment group). 600 / Negative control OD 600 ×1×10 7 ), the test results are as follows Figure 11 shown.

[0081] As can be seen from the figure, under the same bacterial concentration, the survival rate of MH22 in the nanoparticles was the highest when loaded with 150 μg / mL chlorogenic acid. In subsequent experiments, HSA / CGA was used. 150 @MH22 is the optimal concentration.

[0082] Example 8

[0083] Using HSA / CGA in Example 7 150 @MH22 is the research subject. This example investigates HSA / CGA 150 @MH22 is affected in vitro in normal state and in simulated inflammatory state in order to determine whether its proliferation is abnormal compared with that of MH22 alone. 5 The initial bacterial solution was shaken and cultured under the same culture conditions as above. Samples were taken after 12 h, 24 h, 36 h, and 48 h of culture, and the bacterial count was determined using the absorbance turbidimetry method. The calculation formula is the same as above. The test results of the MH22 growth activity under different conditions with a chlorogenic acid concentration of 150 μg / mL are shown in the figure. Figure 12 shown.

[0084] Depend on Figure 12 It can be seen that under normal circumstances without intervention, MH22 in HSA / CGA150@MH22 increased from 10 5 The order of magnitude is increased to 10 7The number of MH22 cells did not increase significantly over the next 36 hours and tended to stabilize. After 200 nM H2O2 treatment, the growth rate of MH22 was basically the same as that without treatment, indicating that the growth of MH22 in HSA / CGA150@MH22 was not affected.

[0085] Example 9

[0086] This example uses the HSA / CGA in Example 7 150 @MH22 was used as the research object to test its cytocompatibility in HT-29 cells under different conditions; and the blank group without oral nanoparticles (Untreated), only HSA, and only chlorogenic acid (CGA) at a concentration of 150 μg / mL were added. 150 ), human serum albumin-chlorogenic acid nanoparticles (HSA / CGA) with chlorogenic acid concentration of 150 μg / mL 150 ), the control group was added with only MH22.

[0087] HT-29 cells were seeded in 24-well plates at a cell number of 2 × 10 5 After 24 hours of culture, the medium containing the test drug (untreated cells served as negative controls) was replaced with a medium containing the test drug (untreated cells served as negative controls) and cultured for another 24 hours. The medium was then discarded and the cells were washed once with PBS. Cell viability was assessed using a CCK-8 (Dojindo Chemical, Japan) kit. The old medium was removed and 120 μL of fresh medium containing 10 μL of CCK-8 reagent was added. The cells were incubated at 37°C for 2 hours. The absorbance (OD) of each well in the 96-well plate was then measured at 450 nm. A blank control was used for each experimental batch. Six replicates were set for each concentration, and each experiment was repeated three times. The OD value of each well was measured at 450 nm using a microplate reader.

[0088] The formula for calculating cell viability is: cell viability = (OD1-OD3) / (OD2-OD3)×100%.

[0089] The test drugs were HSA 40 mg / mL, CGA 150 μg / mL, HSA / CGA 150 Nanoparticles, MH22 1×10 7 CFU / mL, HSA / CGA 150 @MH22 50 μg / mL, tested simultaneously under normal conditions and H2O2-simulated inflammatory conditions, the test results are as follows Figure 13 shown.

[0090] Depend on Figure 13It can be seen that under normal conditions, the HSA, CGA, MH22 and HSA / CGA used 150 Nanoparticles all inhibited the proliferation of HT-29 cells. 150 @MH22 has the best ability to resist H2O2 interference and restore cell vitality.

[0091] Example 10

[0092] This embodiment is the same as HSA / CGA 150 @MH22 was used as the research subject to test the relative ROS concentration in the drug delivery system. HT-29 cells were seeded in a 24-well plate with a cell number of 2×10 5 After the cells adhered to the wall, the blank group was replaced with fresh culture medium, and the other groups were replaced with fresh culture medium containing 200 nM H2O2 for 12 hours. After the treatment, the cells in each group were treated with corresponding drugs. The intervention method and concentration were consistent with Example 9. After the intervention, DCFH-DA was added to each group and duplicate wells at a concentration of 10 μmol / L. The cells were incubated at 37°C for 30 minutes and centrifuged at 1000 rpm to collect the cell pellet for fluorescence detection. The excitation wavelength of the fluorescence detection was set to 500 nm and the emission wavelength was set to 525 nm. The results are shown in Figure 5. Figure 14 shown.

[0093] Depend on Figure 14 It can be seen that the ROS level in the normally grown HT-29 cells (Untreated group) was significantly lower than that in the Control group, indicating that HT-29 cells produced a large amount of ROS under the action of 200 nM H2O2. After HSA intervention alone, the ROS concentration did not decrease significantly, while the other treatment groups all decreased to varying degrees. The effect of the HSA / CGA150@MH22 group was the most obvious, which was better than that of the CGA 150 , HSA / CGA 150 , and MH22-treated groups.

[0094] Example 11

[0095] In this example, mice were used as biological experimental subjects to test the therapeutic effect of oral administration of nanoparticles.

[0096] 60 7-week-old male C57BL / 6 mice were housed in the SPF-level experimental animal barrier system of the Experimental Animal Center of Shenzhen People's Hospital. The mice were first acclimated for 1 week and fed freely during the period. After the acclimation, the mice were randomly divided into 6 groups (random number table method), with 10 mice in each group. Each group of mice was housed in 5 ZC-M1 model combination cages, with 2 mice in each cage and independent diets. The weight of each mouse was recorded during the experiment. The experimental period was 15 days, with the first 7 days as the adaptation period and the last 8 days as the intervention period. The first 3 days of the intervention period were the modeling period and the last 5 days were the treatment period. The specific treatments are shown in Table 1, and the experimental process is as follows. Figure 15 shown.

[0097] Table 1: Mouse treatment methods

[0098]

[0099] After the experiment, the mice were weighed and recorded. They were placed in a supine position with their head and tail lowered, euthanized by cervical dislocation, and fixed on a dissecting table. The entire intestine was removed from the stomach to the anus. The colonic contents were removed, and the intestine was flushed with PBS buffer before photographing. The mice were evaluated based on the weight record, stool characteristics, and the presence of blood in the stool (blood in the stool was determined using a fecal occult blood test kit). Scoring was performed according to the criteria shown in Table 2, and the DAI was calculated using the following formula: DAI = (S1 + S2 + S3) / 3 (where S1 is the weight loss rate score, S2 is the stool condition score, and S3 is the stool bleeding score).

[0100] Table 2: Mouse scoring criteria

[0101]

[0102] The changes in body weight of mice in each treatment group were as follows Figure 16 As shown in Figure 2, the changes in DAI scores of mice in each treatment group were as follows: Figure 17 The colon morphology of mice in each treatment group was as shown in Figure 18 As shown in the above experimental results, it can be seen that oral administration of HSA / CGA 150 @MH22 can significantly alleviate a series of clinical pathological manifestations in mice with DSS-induced intestinal inflammation, specifically increased body weight, reduced fecal water content, decreased DAI index, and increased colon length.

[0103] Based on the above examples, taking the preparation of HSA / CGA@MH22 as an example, Figure 19As shown, before loading MH22, chlorogenic acid is first modified with human serum albumin; during the modification process, glutathione is used to reduce human serum albumin to cleave its disulfide bonds and free sulfhydryl groups, and then cross-linked with the hydrophobic substance chlorogenic acid to form HSA / CGA with a hydrophilic outer layer and a hydrophobic inner layer; the water-soluble particles can be combined with the self-isolated probiotic MH22 by electrostatic adsorption to obtain HSA / CGA@MH22; this method has the advantages of simple process and mild conditions.

[0104] The test results of the above examples show that the probiotic MH22 in the oral nanoparticles can remove a large amount of reactive oxygen species in an inflammatory microenvironment with a high level of reactive oxygen species, thereby achieving the effect of treating inflammation. The present invention uses a carrier to solve the technical problem that chlorogenic acid has poor water solubility and is quickly metabolized after entering the body, thereby effectively inhibiting the production of inflammatory factors. When the nanoparticles reach the site of action, they will separate from the carrier probiotics. The probiotics are still active after separation and can stay in the intestine, playing a role in regulating the microecology. Therefore, the oral nanoparticles of the present invention have good application prospects in the prevention and treatment of ulcerative colitis.

[0105] The above describes in detail the preferred embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solutions that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A method for preparing oral nanoparticles, characterized in that: The steps include: (1) Human serum albumin and chlorogenic acid are incubated in an aqueous solution in the presence of glutathione; after the incubation is completed, a desolventizing agent is added to the system, and the precipitate is collected and purified to obtain human serum albumin-chlorogenic acid nanoparticles; The concentration of human serum albumin is 20-80 mg / mL; the concentration of chlorogenic acid is 10-20 mg / mL; the concentration of glutathione is 50-200 mM; and the desolvent is ethanol; (2) Human serum albumin-chlorogenic acid nanoparticles are mixed with MH22 bacterial solution to complete the loading process, thereby obtaining reactive oxygen species-responsive nanomaterials, i.e., oral nanoparticles; The MH22 is Bacillus mobilis Bacillus toyonensis ; The oral nanoparticles are formed by combining human serum albumin-chlorogenic acid nanoparticles and MH22 by electrostatic adsorption; the human serum albumin-chlorogenic acid nanoparticles are water-soluble particles formed by a human serum albumin shell coating a chlorogenic acid core.

2. The method according to claim 1, wherein: In the step (1), the incubation temperature is 37° C. and the incubation time is 1 to 4 h.

3. The method according to claim 1, wherein: In the step (1), the volume ratio of the desolventizing agent to the solution to be added is 1 to 5:

1.

4. The method according to claim 1, wherein In the step (1), the collection and purification process of the precipitate is as follows: the precipitate is separated by centrifugation, and the precipitate is redispersed in water to form a suspension; the suspension is dialyzed against water at 4°C, with a dialysis cutoff value of 12~14KD, to remove excess desolvent, free chlorogenic acid and glutathione.

5. The method according to claim 1, wherein: In the step (2), the load is completed by shaking at room temperature, and the shaking time is 10 to 12 minutes.

6. An oral nanoparticle, characterized in that: The method according to any one of claims 1 to 5 is used for preparation.

7. Use of the oral nanoparticles according to claim 6 in the preparation of a medicament for treating ulcerative colitis.

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