Nanoparticles loaded with lactic acid bacteria and preparation method and application thereof

Abstract

The present invention discloses a nano-microsphere loaded with lactic acid bacteria and its preparation method and application. ROS-responsive blank nano-microspheres PEG2000-TK-PlGA2000 and polydopamine-modified lactic acid bacteria are mixed in a volume ratio of 1:2, incubated at 25°C for 30 minutes, and washed with PBS to obtain nano-microspheres loaded with lactic acid bacteria. When administered orally, the nano-microspheres can protect lactic acid bacteria from the harsh digestive environment of the upper digestive tract. Due to the high ROS content in the inflamed intestine, the microspheres loaded with lactic acid bacteria will be degraded and release lactic acid bacteria. Polydopamine-modified lactic acid bacteria can prolong the residence time of lactic acid bacteria in the inflammatory area, further produce antimicrobial peptides to destroy harmful bacteria in the inflammatory tissue, and restore intestinal stability. At the same time, it only stays in the inflammatory area of ​​the lesion without affecting the flora in other normal areas.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a nano-microsphere loaded with lactic acid bacteria and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a group of chronic nonspecific intestinal inflammatory diseases whose etiology and pathogenesis are not yet fully understood, mainly including ulcerative colitis (UC) and Crohn's disease (CD). In IBD patients, inflamed colon cells produce excessive reactive oxygen species (ROS), which not only directly damage intestinal epithelial cells, but also lead to increased intestinal permeability, making it easier for harmful substances and microorganisms in the intestine to enter the blood circulation, triggering a systemic inflammatory response. The interaction between oxidative stress and inflammation aggravates the course of the disease. Therefore, regulating oxidative stress may become a potential strategy for the treatment of IBD.

[0003] Glucocorticoids are a common choice for the treatment of IBD, which can effectively control inflammation and relieve symptoms. However, long-term use of these drugs may cause systemic side effects such as immunosuppression, osteoporosis, metabolic disorders, etc. In addition, due to their low bioavailability at the site of inflammation, they may not be able to effectively target inflammation.

[0004] Probiotics are gradually gaining attention in the treatment of IBD, especially lactic acid bacteria (LIa). LIa improves intestinal health through multiple mechanisms: ① Enhance intestinal barrier function: LIa can enhance the tight junctions of intestinal epithelial cells, reduce intestinal permeability, and prevent harmful substances from entering the blood. ② Regulate immune response: LIa can regulate immune response by promoting the production of anti-inflammatory cytokines (such as IL-10 and TGF-β), thereby reducing intestinal inflammation. ③ Fight against harmful microorganisms: LIa can produce antibacterial substances and metabolites (such as lactic acid), inhibit the growth of pathogenic microorganisms, and thus improve the balance of intestinal microbiota.

[0005] However, it is difficult for probiotics to self-colonize at the site of infection in the complex digestive tract, resulting in poor overall efficacy and possible side effects. PEG2000-tk-PLGA2000 is a ROS-responsive material that can break in inflammatory areas with high ROS and is a carrier material with good biocompatibility. Polydopamine (PDA) is a substance similar to melanin that has multiple properties such as antioxidant properties, photothermal conversion, adhesion, biocompatibility, and biodegradability. Modifying LIa with PDA is equivalent to applying a protective layer with navigation function, which resists erosion by gastric juice and bile while improving the ability to target lesions, which is beneficial to improving the effect of enteritis treatment.

[0006] However, there is no corresponding technical solution in the prior art on how to integrate the above technologies. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a nano-microsphere loaded with lactic acid bacteria and a preparation method and application thereof, which can colonize the infected site in the digestive system and respond to the high ROS environment in the inflammatory area by combining with ROS to destroy harmful bacteria in the inflammatory tissue and restore intestinal stability.

[0008] The present invention is achieved by a method for preparing nano-microspheres loaded with lactic acid bacteria, comprising the following steps:

[0009] (1) Dissolve the carrier material PEG2000-tk-PLGA2000 in chloroform, extrude it using a liposome extruder, and then filter and wash it to obtain blank nanospheres;

[0010] (2) Mixing polydopamine (PDA) and lactic acid bacteria (Lla) and stirring at room temperature to prepare Lla@PDA;

[0011] (3) Blank nanoparticles PEG2000-tk-PLGA2000 were incubated with Lla@PDA at room temperature and washed with PBS to obtain nanoparticles loaded with lactic acid bacteria (LPT).

[0012] The particle size of the LPT is 1200-1500 nm.

[0013] Incubate at room temperature for 0.5 hours, with the incubation temperature ranging from 20°C to 35°C.

[0014] The volume ratio of PEG2000-tk-PLGA2000 to Lla@PDA is 1:2 to 1:6.

[0015] The PDA and Lla are added to a tirs-HCl solution with a pH value of 8.5 at a volume ratio of 1:5 to 1:15, and stirred at room temperature for 0.5 to 1.5 hours to prepare Lla@PDA.

[0016] The lactic acid bacteria-loaded nanoparticles are prepared by the above-mentioned preparation method.

[0017] The application of the above-mentioned lactic acid bacteria-loaded nanoparticles in the preparation of drugs for inhibiting intestinal inflammation.

[0018] The beneficial effects of the present invention are as follows: the present invention utilizes PEG2000-tk-PLGA2000 blank nano-microspheres to load polydopamine (PDA)-modified lactic acid bacteria (LIa), which are named LPT, and can effectively target the intestinal inflammation site. After oral administration of LPT, it can resist the harsh environment of the gastrointestinal tract and reach the intestinal inflammation area intact. The high ROS in the inflammation area causes the nano-microspheres to degrade, thereby releasing lactic acid bacteria in the inflammation area; they can colonize in the inflammation area, and inhibit the growth of harmful bacteria in the inflammation area by releasing antimicrobial peptides, consume the inflammation area, and alleviate the symptoms of inflammation, thereby treating inflammatory bowel disease. This method has better biocompatibility and safety than commercially available IBD drugs. The technical solution of the present invention can significantly treat IBD and is expected to be developed as a new drug for the treatment of IBD. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the preparation process of the LPT nanoparticles of the present invention.

[0020] Figure 2 TEM images of nanoparticles before and after loading lactic acid bacteria.

[0021] Figure 3 This is a fluorescent image of the inflammatory green marker probe DCFH-DA after co-incubation of LPT and intestinal Caco-2 cells.

[0022] Figure 4 This is a graph of the number of bacteria surviving after LPT co-culture of Staphylococcus aureus, Clostridium difficile, and Escherichia coli (from left to right) in medium with different concentrations of hydrogen peroxide for 6 hours.

[0023] Figure 5 Co-culture of LPT with Staphylococcus aureus, Clostridium difficile and Escherichia coli before and after treatment with 200 mmol hydrogen peroxide, and the survival number of the latter.

[0024] Figure 6 It is a flow chart for establishing inflammation models in different sites (gastric ulcer, colitis, colorectal inflammation).

[0025] Figure 7 This is a graph showing the amount of Lla colonization in the small intestine in different inflammatory models.

[0026] Figure 8 This is the distribution map of Lla colonization area analyzed by small animal tissue imaging.

[0027] Fig. 9 It is a chart showing the number of strains cultured from different parts of the body by separating the smear plate.

[0028] Fig.10 Figure 3 is a graph of Lla colonization in LPT-treated DSS-induced intestinal inflammation analyzed by small animal tissue imaging.

[0029] Fig.11 These are immunofluorescence images of HE-stained Mfn-1, Mfn-2, and Opa-1 proteins (scale bar = 100 μm). DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail. It should be understood that the specific implementation methods described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] like Figure 1 As shown, a method for preparing nanoparticles loaded with lactic acid bacteria of the present invention comprises the following steps:

[0032] (1) Dissolve the carrier material PEG2000-tk-PLGA2000 in chloroform, extrude it using a liposome extruder, and then filter and wash it to obtain blank nanospheres;

[0033] (2) Mixing polydopamine (PDA) and lactic acid bacteria (Lla) and stirring at room temperature to prepare Lla@PDA;

[0034] (3) Blank nanoparticles PEG2000-tk-PLGA2000 were incubated with Lla@PDA at room temperature and washed with PBS to obtain nanoparticles loaded with lactic acid bacteria (LPT).

[0035] The particle size of the LPT is 1200-1500 nm.

[0036] Incubate at room temperature for 0.5 hours, with the incubation temperature ranging from 20°C to 35°C.

[0037] The volume ratio of PEG2000-tk-PLGA2000 to Lla@PDA is 1:2 to 1:6.

[0038] The PDA and Lla are added to a tirs-HCl solution with a pH value of 8.5 at a volume ratio of 1:5 to 1:15, and stirred at room temperature for 0.5 to 1.5 hours to prepare Lla@PDA.

[0039] The lactic acid bacteria-loaded nanoparticles are prepared by the above-mentioned preparation method.

[0040] The application of the above-mentioned lactic acid bacteria-loaded nanoparticles in the preparation of drugs for inhibiting intestinal inflammation.

[0041] Example 1

[0042] This embodiment provides a method for preparing nanoparticles loaded with lactic acid bacteria, and the specific steps are as follows:

[0043] (1) Place 1 mg / ml of the carrier material PEG2000-tk-PLGA2000 in a 50 ml beaker, add 5 mM chloroform, and stir for 10 minutes to fully dissolve it. Use a liposome extruder to extrude blank nanospheres, filter with a 0.22 μm filter membrane, and wash with PBS three times to obtain blank nanospheres.

[0044] (2) PDA and Lla were added to a tirs-HCl solution with a volume ratio of 1:5 and stirred at room temperature for 1 h to prepare Lla@PDA.

[0045] (3) Blank nanospheres PEG2000-tk-PLGA2000 and Lla@PDA were incubated at room temperature for 0.5 h at a volume ratio of 1:2 at 25°C, and washed twice with PBS to obtain LPT.

[0046] (4) The PEG2000-tk-PLGA2000 and LPT prepared in Example 1 were characterized by electron microscopy, and the particle size of the blank nanosphere PEG2000-tk-PLGA2000 was 1056 nm, and the particle size of the LPT loaded with lactic acid bacteria was 1360 nm, indicating that the lactic acid bacteria were successfully encapsulated. Figure 2 .

[0047] Example 2

[0048] The nanoparticles loaded with lactic acid bacteria in this embodiment can effectively inhibit inflammatory response, and the specific steps are as follows:

[0049] 2,7-Dichlorofluorescein diacetate (DCFH-DA) is a sensitive fluorescent dye used to monitor intracellular reactive oxygen species. Therefore, 2,7-Dichlorofluorescein diacetate (DCFH-DA) was used to detect ROS levels. Different concentrations of hydrogen peroxide culture medium were prepared. Staphylococcus aureus, Clostridium difficile and Escherichia coli cultured to logarithmic phase were resuspended with different concentrations of hydrogen peroxide, and an equal amount of LPT was added and incubated for 2 hours. The results showed that the inflammatory green marker probe DCFH-DA significantly reduced the level of ROS. Figure 3 As shown in the figure, the number of LPT strains treated with hydrogen peroxide and antibiotics decreased as the concentration increased. Figure 4 (left) As shown; the number of LPT strains after LPS treatment of cell supernatant was similar to the previous result. Figure 4 (Second from left) The number of strains in LPT after treating the cell supernatant with bacterial homogenate also showed similar results. Figure 4 (Third from left). This may be because thioredoxin is a specific chemotactic bond that breaks under ROS stimulation, so we believe that thioredoxin can achieve the ability to clear ROS and protect cells from ROS damage when interacting with ROS. It can protect cells from ROS-induced damage. In addition, at the cellular level, neither Lla induced an inflammatory response in the cell, indicating that at the cellular level, both have a certain cellular safety, and the probiotics and their shells have no effect on inducing cellular inflammation. It further confirmed that ROS at this cellular level is sufficient to stimulate an inflammatory response in LPT.

[0050] Example 3

[0051] The nanoparticles loaded with lactic acid bacteria in this embodiment can resist the harsh environment of the gastrointestinal tract and reach the intestinal inflammation area intact. The specific steps are as follows:

[0052] By preparing different concentrations of hydrogen peroxide culture medium, Staphylococcus aureus, Clostridium difficile and Escherichia coli that were cultured to the logarithmic phase were resuspended with different concentrations of hydrogen peroxide, and an equal amount of LPT was added and incubated for 2 hours. 50 ml of Staphylococcus aureus, 50 ml of Clostridium difficile, and 50 ml of Escherichia coli were centrifuged at 3000 rpm for 5 min, 10 ml of the original medium was added, and the ultrasonic crusher was crushed on ice for 20 min, then centrifuged and the supernatant passed through a 0.22μm membrane. The results are shown in the figure. Staphylococcus aureus, Clostridium difficile, and Escherichia coli (from left to right) did not significantly reduce the number of bacterial survival after being cultured in different concentrations of hydrogen peroxide culture medium for 6 hours, as shown in the figure. Figure 5 shown.

[0053] Example 4

[0054] The nanoparticles loaded with lactic acid bacteria in this embodiment can effectively improve the enteritis of mice. The specific steps are as follows:

[0055] First, a mouse enteritis model was constructed. Female BALB / c mice, 5-8 weeks old, were selected. The animals were fasted without water for 24 hours for modeling. Isoflurane inhalation anesthesia was used. A plastic tube with a diameter of 1.0 mm and a length of about 5 cm was gently inserted into the mouse anus to a depth of about 2-3 cm, and a certain concentration of ethanol solution TNBS was slowly injected within 1 min. After filling, the plastic tube was slowly withdrawn, the anus was pinched, the mouse's tail was pinched, and the rat was inverted for 3 min to avoid the outflow of the modeling agent, so that the modeling agent could completely penetrate into the intestinal cavity of the rat. The ethanol solution of TNBS (5% TNBS aqueous solution mixed with anhydrous ethanol in a volume ratio of 2:1) was ready for preparation and use. Mice were fed DSS (5% drinking water) for 7 consecutive days to induce ulcerative colitis. Thereafter, DSS mice received oral Lla, Lla@PDA or LPT as different treatments in the next 7 days. DSS mice received oral Lla, Lla@PDA or LPT as different treatments, as shown in the figure ( Figure 6 By isolating tissues from different sites, we found that the amount of this colonization was closely related to inflammation ( Figure 7 ), in a gastric ulcer model, the bacterial survival rate in the stomach was significantly increased, and similar results were found in colitis and colitis ( Figure 8 ). Mice treated with LPT and imaging of LPT showed higher fluorescence intensity relative to the other treatment groups, indicating that LPT had a better ability to colonize in the intestine ( Fig.11 ). Although the presence of gastric acid may affect bacterial activity, gastric ulcers may cause feeding resistance in mice with reduced gastric acid secretion, allowing bacteria to colonize the stomach. Real-time imaging showed that Nile Red-labeled bacteria colonized the stomach, colon, and colorectum in gastric ulcer, colitis, and colitis models, respectively ( Fig. 9 HE sections showed epithelial cell degeneration, loss, partial detachment, mucus and inflammatory exudate ( Fig.10 ).

[0056] In summary, the lactic acid bacteria-loaded nanoparticles LPT of the present invention can resist the harsh environment of the gastrointestinal tract and effectively colonize in the inflamed intestine. When reaching the site of inflammation, the local excess reactive oxygen species are activated and released, activating the bacteria to release antimicrobial peptides to kill harmful bacteria. At the same time, PDA-modified lactic acid bacteria can prolong the residence time and effective effect of probiotics in the colon. This biocompatible nanoparticle may be beneficial for probiotic delivery applications, and can be used as a targeted carrier for treating inflammation in various parts, and is expected to provide a treatment for intestinal inflammatory diseases.

[0057] Those skilled in the art can achieve the final preparation technology by appropriately changing the conditions and routes by referring to the contents of this article. Although the method and preparation technology of the present invention have been described by preferred embodiments, relevant technicians can obviously modify or recombine the methods and technical routes described herein without departing from the content, spirit and scope of the present invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the spirit, scope and content of the present invention.

Claims

1. A method for preparing nanoparticles loaded with lactic acid bacteria, characterized in that: The following steps are involved: (1) Dissolve the carrier material PEG2000-tk-PLGA2000 in chloroform, extrude it using a liposome extruder, and then filter and wash it to obtain blank nanospheres; (2) mixing polydopamine with lactic acid bacteria and stirring at room temperature to obtain lactic acid bacteria@polydopamine; (3) Blank nanoparticles PEG2000-tk-PLGA2000 were incubated with lactic acid bacteria@polydopamine at room temperature and washed with PBS to obtain nanoparticles loaded with lactic acid bacteria.

2. The method for preparing nanoparticles loaded with lactic acid bacteria according to claim 1, characterized in that: The particle size of the nano-microspheres loaded with lactic acid bacteria is 1200-1500 nm.

3. The method for preparing nanoparticles loaded with lactic acid bacteria according to claim 1, characterized in that: Incubate at room temperature for 0.5 hours, with the incubation temperature ranging from 20°C to 35°C.

4. Nanoparticles loaded with lactic acid bacteria obtained by the preparation method according to any one of claims 1 to 3.

5. Use of the nanoparticles loaded with lactic acid bacteria as claimed in claim 4 in the preparation of drugs for inhibiting intestinal inflammation.

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