Prebiotics-loaded intestinal targeting exosome as well as preparation method and application thereof

Abstract

The invention discloses a prebiotic-loaded intestinal targeting exosome and a preparation method and application thereof.The preparation method comprises the steps that firstly, plasmids containing intestinal cell targeting peptide sequences are constructed through a gene editing technology, the plasmids are transfected and introduced into cells, the transfected cells are screened with antibiotics, and cell strains stably expressing intestinal cell targeting peptides are obtained; then culturing and adding prebiotics, and continuously culturing to enable the cells to take in the prebiotics and release the exosomes loaded with the prebiotics; and finally, collecting cell culture supernate, and centrifuging and resuspending for multiple times under a low-temperature condition to obtain the prebiotics-loaded intestinal targeting exosome. According to the preparation method, extraction of the exosome, loading of the prebiotics and an intestinal targeting modification process are optimized, so that the prepared intestinal targeting exosome loaded with the prebiotics can be used for remarkably improving the loading efficiency and intestinal targeting of the prebiotics and enhancing the stability of the exosome in gastrointestinal tracts; therefore, the delivery efficiency and the treatment effect of the prebiotics in the intestinal tract are improved.

Description

Technical Field

[0001] The present invention belongs to the fields of biomedicine and functional food, and specifically relates to a prebiotic-loaded intestinal-targeted exosome, a preparation method, and an application thereof. Background Art

[0002] Prebiotics are a class of non-digestible food ingredients that selectively stimulate the growth and reproduction of beneficial intestinal bacteria (such as bifidobacteria and lactic acid bacteria), thereby improving the balance of the intestinal microecology, enhancing intestinal barrier function, regulating the immune system, and positively impacting host health. Common prebiotics include oligofructose (FOS), inulin, and oligoxylose (XOS). Despite their numerous health benefits, prebiotics face the following major challenges when administered orally: Prebiotics are easily degraded by gastric acid and digestive enzymes in the stomach, resulting in a decrease in their effective concentration in the small intestine and colon, thus affecting their subsequent physiological effects; prebiotics have a large molecular weight and are difficult to directly absorb in the small intestine, requiring fermentation by beneficial bacteria in the colon to exert their role in regulating intestinal flora; and oral prebiotics are relatively dispersed in the intestine, making it difficult for them to efficiently reach their target sites (such as the colon), thus affecting their efficacy.

[0003] Exosomes are transport vesicles secreted by living cells and released into the extracellular environment. They typically range in diameter from 30 to 150 nm and possess natural transport properties, a relatively small molecular structure, and excellent biocompatibility. Exosomes can shuttle and carry bioactive molecules such as proteins, lipids, RNA, and DNA. Due to their natural cellular origin, they have low immunogenicity and can avoid phagocytosis by the immune system. Furthermore, exosomes play a vital role in intercellular communication, delivering their cargo to recipient cells through direct fusion with cell membranes or endocytosis.

[0004] In recent years, research on gut-targeted exosomes has gained increasing attention. For example, studies in mouse models have shown that gut-targeted exosomes can significantly increase drug concentrations in the colon and prolong drug action. Furthermore, some studies have explored the use of gut-targeted exosomes in the treatment of inflammatory bowel disease, finding that they can effectively reduce intestinal inflammation and promote intestinal mucosal repair. Despite their significant advantages in drug delivery, exosomes currently face challenges in loading prebiotics for gut-targeted delivery, including low loading efficiency, insufficient targeting, and poor stability. Summary of the Invention

[0005] In order to solve the problems in the prior art, the present invention provides a prebiotic-loaded intestinal-targeted exosome, a preparation method and application thereof. The preparation method can significantly improve the loading efficiency and intestinal targeting of prebiotics by optimizing the extraction of exosomes, the loading of prebiotics and the intestinal-targeted modification process, enhance the stability of exosomes in the gastrointestinal tract, and thus improve the delivery efficiency and therapeutic effect of prebiotics in the intestine.

[0006] In order to achieve the above objectives, the present invention provides a method for preparing prebiotic-loaded intestinal-targeted exosomes, comprising the following steps:

[0007] Step 1: Gene editing technology is used to construct a plasmid containing an intestinal cell targeting peptide sequence, which is then transfected into cells. The transfected cells are screened with antibiotics to obtain a cell line that stably expresses the intestinal cell targeting peptide.

[0008] Step 2: When the obtained cell line stably expressing the intestinal cell targeting peptide is cultured to 70% to 80% confluence, prebiotics are added and the culture is continued for 24 to 48 hours to allow the cells to take up the prebiotics and release the prebiotic-loaded exosomes;

[0009] Step 3: Collect the cell culture supernatant, centrifuge and resuspend multiple times under low temperature conditions to obtain intestinal-targeted exosomes loaded with prebiotics.

[0010] Preferably, the gene editing technology in step 1 adopts the CRISPR / Cas9 system or TALEN technology.

[0011] Preferably, the transfection in step 1 adopts liposome transfection or electroporation, and the transfection conditions of the liposome transfection method are: the ratio of liposome to plasmid is (2-5):1 (μL:μg), the transfection medium is serum-free DMEM or DMEM with a serum concentration of 1%-5%, and the transfection time is 4-8 hours; the transfection conditions of the electroporation method are: the electric field strength is 1000-1500 V / cm, the pulse time is 10-30 ms, and the number of pulses is 3-7 times.

[0012] Preferably, the cells into which the plasmid is transfected in step 1 include HEK293 cells, COS-7 cells, Hela cells or SF9 cells.

[0013] Preferably, the transfected cells are screened using a culture medium containing 400-1200 μg / mL G418, a culture medium containing 0.5-10 μg / mL Promycin, a culture medium containing 50-300 μg / mL Hygromycin B, or a culture medium containing 2-20 μg / mL Blasticidin S, and the screening time is 2-6 weeks.

[0014] Preferably, in step 1, flow cytometry and / or immunofluorescence are used to detect the expression level of the targeting peptide, and cell lines with a targeting peptide expression level greater than or equal to 1000 MFI, a positive cell ratio greater than or equal to 80%, and a fluorescent signal evenly distributed on the cell surface are screened out.

[0015] Preferably, the prebiotics in step 2 include fructooligosaccharides, inulin or xylo-oligosaccharides, the concentration of fructooligosaccharides is 100-300 μg / mL, the concentration of inulin is 200-400 μg / mL, and the concentration of xylo-oligosaccharides is 300-600 μg / mL.

[0016] Preferably, the step 3 includes:

[0017] Initial centrifugation: Collect the cell culture supernatant and centrifuge at 200-400 rpm for 3-15 minutes;

[0018] Differential centrifugation: Take the supernatant and centrifuge at 8000-12000 rpm at 2-6°C for 20-40 minutes;

[0019] Ultracentrifugation: Take the supernatant and centrifuge at 100,000-150,000 rpm at 2-6°C for 45-75 minutes to collect the exosome precipitate;

[0020] Resuspend and re-centrifuge: Resuspend the pellet in 0.5-10 mL of sterile PBS and centrifuge again at 100,000-150,000 rpm at 2-6°C for 45-75 minutes to collect the pellet.

[0021] Final resuspension: Resuspend the pellet with 0.5-10 mL of sterile PBS to obtain intestinal-targeted exosomes loaded with prebiotics.

[0022] The present invention also provides a prebiotic-loaded intestinal-targeted exosome, which is prepared using the above-mentioned method for preparing prebiotic-loaded intestinal-targeted exosomes. The diameter of the prebiotic-loaded intestinal-targeted exosome is 30 to 150 nm.

[0023] The present invention also provides a use of the above-mentioned prebiotic-loaded intestinal-targeted exosomes in the preparation of medicines or health products for regulating intestinal microecology, preventing and treating intestinal diseases.

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

[0025] 1. Enhanced Targeting: The CEP plasmid, constructed through gene editing technology, enables cells to stably express an intestinal cell-targeting peptide. This targeting peptide specifically binds to receptors on the surface of intestinal cells, thereby improving the enrichment and uptake efficiency of exosomes in the intestine. Compared to existing non-targeted exosomes, the intestinal-targeted exosomes of this invention can more precisely reach the intestine, reduce distribution in other tissues or organs, improve therapeutic efficacy, and reduce potential side effects.

[0026] 2. Effective loading and protection of prebiotics: Prebiotics (such as oligofructose) are added during the cell culture process. Through cellular uptake and release of exosomes, the prebiotics are encapsulated within the exosomes. The double-layer membrane structure of exosomes can protect prebiotics from damage by the digestive tract environment (such as gastric acid and bile). Compared with direct oral administration of prebiotics, exosomes loaded with prebiotics can better protect prebiotics, allowing them to remain active after reaching the intestine and improving their bioavailability.

[0027] 3. Improve the yield and quality of exosomes: Through gene editing and screening of stable cell lines, ensure that the cells can efficiently express intestinal cell-targeting peptides. This optimized cell line can stably release exosomes during culture, and the loaded prebiotics can further promote the formation of exosomes. Compared with traditional exosome preparation methods, the present invention can increase the yield of exosomes, and through optimized separation steps, namely multiple centrifugation and resuspension, purer exosomes can be obtained, reducing the interference of impurities.

[0028] 4. Enhanced exosome stability and bioactivity: Prebiotic-loaded exosomes undergo multiple centrifugation and resuspension processes during preparation to remove impurities such as cell debris and organelles. Low-temperature operation also reduces exosome degradation. The exosomes produced by this method exhibit enhanced stability and bioactivity, enabling them to better perform their functions in the intestine, such as regulating intestinal flora and improving intestinal barrier function.

[0029] 5. Potentially Enhanced Therapeutic Effects: Intestinal-targeted exosomes loaded with prebiotics can precisely deliver prebiotics to the intestine, while the exosomes themselves also regulate cellular function. This dual mechanism of action can better exert the physiological functions of prebiotics. When treating intestinal-related diseases (such as inflammatory bowel disease and irritable bowel syndrome), the exosomes of the present invention can provide more effective treatment options and enhance therapeutic effects. DETAILED DESCRIPTION

[0030] The present invention will be further explained below in conjunction with specific embodiment. Obviously, the embodiment described is a part of the application's embodiment, rather than all embodiments. Based on the embodiment in the application, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present application. In addition, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods of the following examples that do not specify specific experimental conditions are usually according to conventional experimental conditions. Unless otherwise specified, reagents and raw materials used in the present invention all can be obtained commercially.

[0031] The present invention first provides a method for preparing intestinal-targeted exosomes loaded with prebiotics, comprising the following steps:

[0032] Step 1: Use gene editing technology to construct a plasmid containing the intestinal cell targeting peptide sequence, named plasmid CEP (intestinal cell targeting peptide expression plasmid). Transfect the plasmid CEP into a suitable cell line, such as HEK293 cells, COS-7 cells, Hela cells or SF9 cells. Use conventional transfection methods such as liposome transfection or electroporation to introduce the plasmid into the cells. The transfected cells are screened with antibiotics (such as G418) to screen out cell lines that can stably express intestinal cell targeting peptides. After multiple subcultures, its stability and the expression level of the targeting peptide are verified, and finally a cell line that can stably express the intestinal cell targeting peptide is obtained.

[0033] Preferably, the gene editing technology uses the CRISPR / Cas9 system or TALEN technology.

[0034] Preferably, the transfection conditions of the liposome transfection method are: the ratio of liposome to plasmid is (2-5):1 (μL:μg), the transfection medium is serum-free DMEM or low-serum (serum concentration is 1%-5%) DMEM, and the transfection time is 4-8 hours; the transfection conditions of the electroporation method are: the electric field strength is 1000-1500 V / cm, the pulse time is 10-30 ms, and the number of pulses is 3-7 times.

[0035] Preferably, the transfected cells are screened using a culture medium containing 400-1200 μg / mL G418, a culture medium containing 0.5-10 μg / mL Promycin, a culture medium containing 50-300 μg / mL Hygromycin B, or a culture medium containing 2-20 μg / mL Blasticidin S, and the screening time is 2-6 weeks.

[0036] Preferably, in step 1, flow cytometry and / or immunofluorescence are used to detect the expression level of the targeting peptide, and cell lines with a targeting peptide expression level higher than 1000 MFI, a positive cell ratio exceeding 80%, and a fluorescent signal evenly distributed on the cell surface are screened out.

[0037] Step 2: Inoculate the cell line stably expressing the enterocyte-targeting peptide obtained in Step 1 into a culture flask. When the cells have grown to 70% to 80% confluence, add a prebiotic at a concentration determined by the type of prebiotic. Continue culturing the cells for 24 to 48 hours to allow the cells to absorb the prebiotic and release the prebiotic-loaded exosomes.

[0038] Preferably, the prebiotics include fructooligosaccharides, inulin or xylo-oligosaccharides, the concentration of fructooligosaccharides is 100-300 μg / mL, the concentration of inulin is 200-400 μg / mL, and the concentration of xylo-oligosaccharides is 300-600 μg / mL.

[0039] Step 3: Collect the cell culture supernatant and isolate exosomes according to the following steps:

[0040] Initial centrifugation: Take the supernatant and centrifuge at 200-400 rpm for 3-15 minutes to remove cell debris and large particles.

[0041] Differential centrifugation: Take the supernatant and centrifuge at 8000-12000 rpm at 2-6°C for 20-40 minutes to remove larger particles such as organelles.

[0042] Ultracentrifugation: Take the supernatant and centrifuge at 100,000-150,000 rpm at 2-6°C for 45-75 min to collect the exosome precipitate.

[0043] Resuspension and re-centrifugation: Take the precipitate, resuspend it with 0.5-10 mL of sterile PBS, centrifuge at 100,000-150,000 rpm at 2-6°C for 45-75 minutes, and collect the precipitate again.

[0044] Final resuspension: Take the precipitate and resuspend it in 0.5-10 mL of sterile PBS to obtain intestinal-targeted exosomes loaded with prebiotics.

[0045] The present invention also provides a prebiotic-loaded intestinal-targeted exosome prepared by the above-mentioned preparation method, with a diameter of 30 to 150 nm, natural material transport properties, relatively small molecular structure and excellent biocompatibility. The above-mentioned prebiotic-loaded intestinal-targeted exosomes are used in the preparation of drugs or health products for regulating intestinal microecology, preventing and treating intestinal diseases. The intestinal cell-targeted peptide sequence can specifically bind to receptors on the surface of intestinal epithelial cells. By loading prebiotics (such as FOS / GOS) into the exosomes and modifying the intestinal targeting molecules (such as CSKSSDYQC peptide), the precise delivery of prebiotics is achieved, and the treatment includes inflammatory bowel disease, irritable bowel syndrome, etc.

[0046] By optimizing exosome extraction, prebiotic loading, and intestinal-targeted modification processes, the present invention significantly improves prebiotic loading efficiency and intestinal targeting, enhances exosome stability in the gastrointestinal tract, and thus improves prebiotic delivery efficiency and therapeutic efficacy in the intestine. The intestinal-targeted exosomes of the present invention have enhanced targeting, higher prebiotic protection efficiency, more stable structure, and better bioactivity. They can be widely used in the development of drugs or health products for regulating intestinal microecology and preventing and treating intestinal diseases, and have important clinical application value and market prospects.

[0047] The present invention will be explained in more detail below with reference to specific embodiments.

[0048] Example 1: Small-scale preparation of intestinal-targeted exosomes loaded with fructooligosaccharides

[0049] Step 1: Construct a plasmid CEP containing an intestinal cell targeting peptide sequence using gene editing technology. The targeting peptide sequence is designed to specifically bind to receptors on the surface of intestinal epithelial cells. The plasmid CEP is transfected into HEK293 cells using liposome transfection. The transfection conditions are: the ratio of liposomes to plasmids is 3:1 (μL:μg), the transfection medium is serum-free DMEM, and the transfection time is 6 hours. The transfected cells are screened with a culture medium containing 600μg / mL G418 for 2 weeks. The expression level of the targeting peptide is detected by flow cytometry, and cell lines that stably express the intestinal cell targeting peptide are screened.

[0050] Step 2: Inoculate the screened cell lines stably expressing the enterocyte-targeting peptide into T25 culture flasks, adding 10 mL of complete medium to each flask. When the cells reach 70% to 80% confluence, add fructo-oligosaccharides (200 μg / mL). Continue culturing the cells for 24 hours to allow for cellular uptake of the fructo-oligosaccharides and the release of fructo-oligosaccharide-loaded exosomes.

[0051] Step 3: Initial Centrifugation: Collect the cell culture supernatant and centrifuge at 300 rpm for 5 minutes to remove cell debris and large particles. Differential Centrifugation: Collect the supernatant and centrifuge at 10,000 rpm at 4°C for 30 minutes to remove larger particles such as organelles. Ultracentrifugation: Collect the supernatant and centrifuge at 120,000 rpm at 4°C for 60 minutes to collect the exosome pellet. Resuspension and Recentrifugation: Resuspend the pellet in 2 mL of sterile PBS and centrifuge again at 120,000 rpm at 4°C for 60 minutes to collect the pellet. Final Resuspension: Resuspend the pellet in 2 mL of sterile PBS to obtain the intestinal-targeted exosomes loaded with fructo-oligosaccharide.

[0052] Example 2: Medium-scale preparation of inulin-loaded intestinal-targeted exosomes

[0053] Step 1: Construct the CEP plasmid as in Example 1. Transfect the CEP plasmid into HEK293 cells using electroporation. Transfection conditions are: electric field strength of 1200 V / cm, pulse duration of 20 ms, and 5 pulses. The transfected cells are screened using culture medium containing 800 μg / mL G418 for 3 weeks. Immunofluorescence analysis of the expression level of the targeting peptide is used to identify cell lines that stably express the enterocyte-targeting peptide.

[0054] Step 2: Inoculate the screened cell lines stably expressing the enterocyte-targeting peptide into T75 culture flasks, adding 30 mL of complete medium to each flask. When the cells reach 70% to 80% confluence, add inulin at a concentration of 300 μg / mL. Continue culturing the cells for 48 hours to allow the cells to uptake the inulin and release the inulin-loaded exosomes.

[0055] Step 3: Initial Centrifugation: Collect the cell culture supernatant and centrifuge at 300 rpm for 10 minutes to remove cell debris and large particles. Differential Centrifugation: Collect the supernatant and centrifuge at 10,000 rpm at 4°C for 30 minutes to remove larger particles such as organelles. Ultracentrifugation: Collect the supernatant and centrifuge at 120,000 rpm at 4°C for 60 minutes to collect the exosome pellet. Resuspension and Recentrifugation: Resuspend the pellet in 3 mL of sterile PBS and centrifuge again at 120,000 rpm at 4°C for 60 minutes to collect the pellet. Final Resuspension: Resuspend the pellet in 3 mL of sterile PBS to obtain the inulin-loaded, gut-targeted exosomes.

[0056] Example 3: Large-scale preparation of intestinal-targeted exosomes loaded with xylooligosaccharides

[0057] Step 1: Construct plasmid CEP as in Example 1. Transfect plasmid CEP into HEK293 cells using liposome transfection. The transfection conditions are as follows: the ratio of liposome to plasmid is 4:1 (μL:μg), the transfection medium is serum-free DMEM, and the transfection time is 8 hours. The transfected cells are screened with a medium containing 1000 μg / mL G418 for 4 weeks. The expression level of the targeting peptide is detected by dual flow cytometry and immunofluorescence to screen out cell lines that stably express the intestinal cell targeting peptide.

[0058] Step 2: The screened cell lines stably expressing the enterocyte-targeting peptide were seeded into a 10-layer cell culture flask, with 100 mL of complete medium added to each layer. When the cells reached 70% to 80% confluence, xylooligosaccharides were added at a concentration of 500 μg / mL. The cells were cultured for 48 hours to allow for the cells to uptake the xylooligosaccharides and release the xylooligosaccharide-loaded exosomes.

[0059] Step 3: Initial Centrifugation: Collect the cell culture supernatant and centrifuge at 300 rpm for 10 minutes to remove cell debris and large particles. Differential Centrifugation: Take the supernatant and centrifuge at 10,000 rpm at 4°C for 30 minutes to remove larger particles such as organelles. Ultracentrifugation: Take the supernatant and centrifuge at 120,000 rpm at 4°C for 60 minutes to collect the exosome pellet. Resuspension and Recentrifugation: Resuspend the pellet in 5 mL of sterile PBS and centrifuge again at 120,000 rpm at 4°C for 60 minutes to collect the pellet. Final Resuspension: Resuspend the pellet in 5 mL of sterile PBS to obtain the xylo-oligosaccharide-loaded intestinal-targeted exosomes.

[0060] 1. Experimental Methods

[0061] Cell culture: Caco-2 cells (human colon adenocarcinoma cells, used to simulate intestinal epithelial cells) were seeded into 24-well plates, 2 mL of complete culture medium was added to each well, and cultured to 70% to 80% confluence.

[0062] Exosome treatment: Prebiotic-loaded intestinal-targeted exosomes were added to the Caco-2 cell culture medium at different concentration gradients (e.g., 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL), and a blank control group without exosomes was set up.

[0063] Detection of cell metabolites: After 24 hours of culture, the cell supernatant was collected and the short-chain fatty acids and lactic acid content were detected.

[0064] Cell barrier function detection: The effect of exosomes on the barrier function of Caco-2 cells was evaluated by detecting the transmembrane electrical resistance (TEER) value and the transmittance of fluorescently labeled dextran.

[0065] 2. Experimental Results

[0066] 1. Detection of Cellular Metabolites

[0067] Table 1: Short-chain fatty acids (SCFA) content in Caco-2 cell supernatant

[0068]

[0069] Table 2: Lactic acid content in Caco-2 cell supernatant

[0070]

[0071]

[0072] 2. Cell Barrier Function Detection

[0073] Table 3: Caco-2 cell transmembrane electrical resistance (TEER) values

[0074]

[0075] Table 4: Transmission rate of fluorescently labeled dextran in Caco-2 cells

[0076]

[0077] The experimental results of Table 1, Table 2, Table 3 and Table 4 show that the intestinal-targeted exosomes loaded with prebiotics of the present invention can significantly promote the metabolic activity of Caco-2 cells, increase the synthesis and secretion of short-chain fatty acids (acetic acid, propionic acid, butyric acid) and lactic acid, which may provide more energy and metabolic intermediates for the cells, thereby enhancing the metabolic capacity of the cells. In addition, the transmembrane resistance of the Caco-2 cells in the exosome-treated group was significantly higher than that in the blank control group, indicating that exosomes can enhance the transmembrane resistance of the cells and reduce the transmittance of fluorescently labeled dextran, indicating that exosomes can regulate tight junctions between cells, enhance the barrier function of cells, and reduce intestinal permeability. These results further confirm that the intestinal-targeted exosomes loaded with prebiotics have significant biological activity in regulating intestinal microecology and improving intestinal barrier function, and are expected to become a new type of intestinal health regulator for the prevention and treatment of intestinal-related diseases.

[0078] Example 4:

[0079] The preparation method comprises the following steps:

[0080] Step 1: Gene editing was performed using the CRISPR / Cas9 system to construct a plasmid CEP containing an intestinal cell targeting peptide sequence. The plasmid CEP was transfected into COS-7 cells. The plasmid was introduced into the cells using the liposome transfection method. The transfection conditions were as follows: the ratio of liposomes to plasmids was 2:1 (μL:μg), the transfection medium was DMEM with a serum concentration of 1%, and the transfection time was 4 hours. The transfected cells were screened with a culture medium containing 0.5μg / mL Promycin for 2 weeks to screen out cell lines that could stably express intestinal cell targeting peptides. After multiple subcultures, the expression level of the targeting peptide was detected by flow cytometry. Cell lines with a targeting peptide expression level higher than 1000 MFI, a positive cell ratio of more than 80%, and a fluorescent signal evenly distributed on the cell surface were screened. Finally, a cell line that could stably express intestinal cell targeting peptides was obtained.

[0081] Step 2: Inoculate the cell line stably expressing the enterocyte-targeting peptide obtained in Step 1 into a culture flask. When the cells reach 70% growth, add 100 μg / mL of oligofructose. Continue culturing the cells for 24 hours to allow the cells to absorb the prebiotics and release the prebiotic-loaded exosomes.

[0082] Step 3: Collect the cell culture supernatant and isolate exosomes according to the following steps:

[0083] Initial centrifugation: Take the supernatant, centrifuge at 200 rpm for 3 minutes to remove cell debris and large particles.

[0084] Differential centrifugation: Take the supernatant and centrifuge at 8000 rpm at 2°C for 20 minutes to remove larger particles such as organelles.

[0085] Ultracentrifugation: Take the supernatant, centrifuge at 100,000 rpm at 2°C for 45 min, and collect the exosome precipitate.

[0086] Resuspension and re-centrifugation: Take the precipitate, resuspend it with 0.5 mL of sterile PBS, centrifuge at 100,000 rpm at 2°C for 45 minutes, and collect the precipitate again.

[0087] Final resuspension: Take the precipitate and resuspend it in 0.5 mL of sterile PBS to obtain intestinal-targeted exosomes loaded with prebiotics.

[0088] Example 5:

[0089] The preparation method comprises the following steps:

[0090] Step 1: TALEN gene editing technology was used to construct a plasmid CEP containing the intestinal cell targeting peptide sequence, and the plasmid CEP was transfected into Hela cells. The plasmid was introduced into the cells by electroporation. The transfection conditions of the electroporation method were: electric field strength of 1000 V / cm, pulse time of 10 ms, and number of pulses 3 times. The transfected cells were screened with a culture medium containing 50 μg / mL Hygromycin B for 2 weeks to screen out cell lines that can stably express the intestinal cell targeting peptide. After multiple subcultures, its stability and the expression level of the targeting peptide were verified. The expression level of the targeting peptide was detected by flow cytometry or immunofluorescence. Cell lines with a targeting peptide expression level higher than 1000 MFI, a positive cell ratio of more than 80%, and a fluorescent signal evenly distributed on the cell surface were screened. Finally, a cell line that can stably express the intestinal cell targeting peptide was obtained.

[0091] Step 2: Inoculate the cell line stably expressing the enterocyte-targeting peptide obtained in Step 1 into a culture flask. When the cells reach 70% growth, add 200 μg / mL of inulin. Continue culturing the cells for 24 hours to allow the cells to absorb the prebiotic and release the prebiotic-loaded exosomes.

[0092] Step 3: Collect the cell culture supernatant and isolate exosomes according to the following steps:

[0093] Initial centrifugation: Take the supernatant and centrifuge at 250 rpm for 5 minutes to remove cell debris and large particles.

[0094] Differential centrifugation: Take the supernatant and centrifuge at 10,000 rpm at 3°C ​​for 25 minutes to remove larger particles such as organelles.

[0095] Ultracentrifugation: Take the supernatant, centrifuge at 120,000 rpm at 3°C ​​for 60 min, and collect the exosome precipitate.

[0096] Resuspension and re-centrifugation: Take the precipitate, resuspend it in 1 mL of sterile PBS, centrifuge at 120,000 rpm at 3°C ​​for 50 min, and collect the precipitate again.

[0097] Final resuspension: Take the precipitate and resuspend it in 1 mL of sterile PBS to obtain intestinal-targeted exosomes loaded with prebiotics.

[0098] Example 6:

[0099] The preparation method comprises the following steps:

[0100] Step 1: CRISPR / Cas9 gene editing was used to construct a plasmid containing an enterocyte-targeting peptide sequence, CEP, which was then transfected into SF9 cells. The plasmid was introduced into the cells using lipofectamine transfection. Transfection conditions were as follows: a liposome-to-plasmid ratio of ~5:1 (μL:μg), DMEM with 5% serum, and an 8-hour transfection time. Transfected cells were screened for 6 weeks in a medium supplemented with 1200 μg / mL G418 to identify cell lines that stably expressed the enterocyte-targeting peptide. After multiple subcultures, the stability and expression levels of the targeting peptide were verified. Flow cytometry or immunofluorescence was used to detect targeting peptide expression. Cell lines expressing targeting peptides with an MFI greater than 1000, a positive cell ratio exceeding 80%, and a uniform distribution of fluorescent signals on the cell surface were identified. Finally, cell lines stably expressing the enterocyte-targeting peptide were identified.

[0101] Step 2: Inoculate the cell line stably expressing the enterocyte-targeting peptide obtained in Step 1 into a culture flask. When the cells reach 80% growth, add 300 μg / mL of oligofructose. Continue culturing the cells for 48 hours to allow the cells to absorb the prebiotics and release the prebiotic-loaded exosomes.

[0102] Step 3: Collect the cell culture supernatant and isolate exosomes according to the following steps:

[0103] Initial centrifugation: Take the supernatant and centrifuge at 400 rpm for 15 minutes to remove cell debris and large particles.

[0104] Differential centrifugation: Take the supernatant and centrifuge at 12,000 rpm at 6°C for 40 minutes to remove larger particles such as organelles.

[0105] Ultracentrifugation: Take the supernatant, centrifuge at 150,000 rpm at 6°C for 75 min, and collect the exosome precipitate.

[0106] Resuspension and re-centrifugation: Take the precipitate, resuspend it in 10 mL of sterile PBS, centrifuge at 150,000 rpm at 6°C for 75 minutes, and collect the precipitate again.

[0107] Final resuspension: Take the precipitate and resuspend it in 10 mL of sterile PBS to obtain intestinal-targeted exosomes loaded with prebiotics.

[0108] Example 7:

[0109] The preparation method comprises the following steps:

[0110] Step 1: TALEN gene editing technology was used to construct a plasmid CEP containing an intestinal cell targeting peptide sequence, and the plasmid CEP was transfected into COS-7 cells. The plasmid was introduced into the cells by electroporation. The transfection conditions of the electroporation method were as follows: electric field strength of 1500 V / cm, pulse time of 30 ms, and number of pulses of 7 times. The transfected cells were screened with a culture medium containing 10 μg / mL Promycin for 5 weeks to screen out cell lines that could stably express intestinal cell targeting peptides. After multiple subcultures, the stability and expression level of the targeting peptide were verified. The expression level of the targeting peptide was detected by flow cytometry or immunofluorescence. Cell lines with a targeting peptide expression level higher than 1000 MFI, a positive cell ratio of more than 80%, and a fluorescent signal evenly distributed on the cell surface were screened out, and finally a cell line that could stably express intestinal cell targeting peptides was obtained.

[0111] Step 2: The cell line stably expressing the intestinal cell targeting peptide obtained in step 1 was inoculated into a culture flask. When the cells grew to 75%, inulin at a concentration of 400 μg / mL was added and the cells were cultured for another 30 hours to allow the cells to take up prebiotics and release prebiotic-loaded exosomes.

[0112] Step 3: Collect the cell culture supernatant and isolate exosomes according to the following steps:

[0113] Initial centrifugation: Take the supernatant and centrifuge at 300 rpm for 9 minutes to remove cell debris and large particles.

[0114] Differential centrifugation: Take the supernatant and centrifuge at 10,000 rpm at 5°C for 30 minutes to remove larger particles such as organelles.

[0115] Ultracentrifugation: Take the supernatant, centrifuge at 130,000 rpm at 5°C for 50 min, and collect the exosome precipitate.

[0116] Resuspension and re-centrifugation: Take the precipitate, resuspend it in 5 mL of sterile PBS, centrifuge at 120,000 rpm at 5°C for 60 min, and collect the precipitate again.

[0117] Final resuspension: Take the precipitate and resuspend it in 5 mL of sterile PBS to obtain intestinal-targeted exosomes loaded with prebiotics.

[0118] Example 8:

[0119] The preparation method comprises the following steps:

[0120] Step 1: Gene editing technology using the CRISPR / Cas9 system was used to construct a plasmid CEP containing an enterocyte-targeting peptide sequence. The plasmid CEP was transfected into Hela cells. The plasmid was introduced into the cells using liposome transfection. The transfection conditions were as follows: a liposome-to-plasmid ratio of 4:1 (μL:μg), a transfection medium of 3% serum in DMEM, and a transfection time of 6 hours. The transfected cells were screened in a medium containing 50 μg / mL Hygromycin B for 3 weeks to identify cell lines that stably expressed the enterocyte-targeting peptide. After multiple subcultures, the stability and expression level of the targeting peptide were verified. The expression level of the targeting peptide was detected by flow cytometry or immunofluorescence. Cell lines with a targeting peptide expression level greater than 1000 MFI, a positive cell ratio exceeding 80%, and a fluorescent signal evenly distributed on the cell surface were selected, ultimately obtaining a cell line that stably expressed the enterocyte-targeting peptide.

[0121] Step 2: The cell line stably expressing the intestinal cell targeting peptide obtained in step 1 was inoculated into a culture flask. When the cells grew to 75%, 300 μg / mL of oligoxylose was added and the cells were cultured for another 40 hours to allow the cells to absorb prebiotics and release prebiotic-loaded exosomes.

[0122] Step 3: Collect the cell culture supernatant and isolate exosomes according to the following steps:

[0123] Initial centrifugation: Take the supernatant and centrifuge at 350 rpm for 10 min to remove cell debris and large particles.

[0124] Differential centrifugation: Take the supernatant and centrifuge at 9000 rpm at 2°C for 25 minutes to remove larger particles such as organelles.

[0125] Ultracentrifugation: Take the supernatant, centrifuge at 140,000 rpm at 6°C for 50 min, and collect the exosome precipitate.

[0126] Resuspension and re-centrifugation: Take the precipitate, resuspend it in 8 mL of sterile PBS, centrifuge at 120,000 rpm at 3°C ​​for 60 min, and collect the precipitate again.

[0127] Final resuspension: Take the precipitate and resuspend it in 7 mL of sterile PBS to obtain intestinal-targeted exosomes loaded with prebiotics.

[0128] Example 9:

[0129] The preparation method comprises the following steps:

[0130] Step 1: TALEN gene editing technology was used to construct a plasmid CEP containing the intestinal cell targeting peptide sequence, and the plasmid CEP was transfected into SF9 cells. The plasmid was introduced into the cells by electroporation. The transfection conditions of the electroporation method were: electric field strength of 1400 V / cm, pulse time of 25 ms, and number of pulses of 5 times. The transfected cells were screened with a culture medium containing 20 μg / mL Blasticidin S for 5 weeks to screen out cell lines that can stably express the intestinal cell targeting peptide. After multiple subcultures, its stability and the expression level of the targeting peptide were verified. The expression level of the targeting peptide was detected by flow cytometry or immunofluorescence. Cell lines with a targeting peptide expression level higher than 1000 MFI, a positive cell ratio of more than 80%, and a fluorescent signal evenly distributed on the cell surface were screened. Finally, a cell line that can stably express the intestinal cell targeting peptide was obtained.

[0131] Step 2: The cell line stably expressing the intestinal cell targeting peptide obtained in step 1 was inoculated into a culture flask. When the cells grew to 77%, 600 μg / mL of xylooligosaccharide was added and the cells were cultured for another 30 hours to allow the cells to absorb the prebiotics and release the prebiotic-loaded exosomes.

[0132] Step 3: Collect the cell culture supernatant and isolate exosomes according to the following steps:

[0133] Initial centrifugation: Take the supernatant and centrifuge at 250 rpm for 5 minutes to remove cell debris and large particles.

[0134] Differential centrifugation: Take the supernatant and centrifuge at 11,000 rpm at 5°C for 40 minutes to remove larger particles such as organelles.

[0135] Ultracentrifugation: Take the supernatant, centrifuge at 130,000 rpm at 2°C for 70 min, and collect the exosome precipitate.

[0136] Resuspension and re-centrifugation: Take the precipitate, resuspend it in 4 mL of sterile PBS, centrifuge at 110,000 rpm at 6°C for 50 min, and collect the precipitate again.

[0137] Final resuspension: Take the precipitate and resuspend it in 9 mL of sterile PBS to obtain intestinal-targeted exosomes loaded with prebiotics.

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing intestinal-targeted exosomes loaded with prebiotics, characterized in that: The following steps are involved: Step 1: Gene editing technology is used to construct a plasmid containing an intestinal cell targeting peptide sequence, which is then transfected into cells. The transfected cells are screened with antibiotics to obtain a cell line that stably expresses the intestinal cell targeting peptide. Step 2: When the obtained cell line stably expressing the intestinal cell targeting peptide is cultured to 70% to 80% confluence, prebiotics are added and the culture is continued for 24 to 48 hours to allow the cells to take up the prebiotics and release the prebiotic-loaded exosomes; Step 3: Collect the cell culture supernatant, centrifuge and resuspend multiple times under low temperature conditions to obtain intestinal-targeted exosomes loaded with prebiotics.

2. The method for preparing intestinal-targeted exosomes loaded with prebiotics according to claim 1, characterized in that: The gene editing technology in step 1 adopts CRISPR / Cas9 system or TALEN technology.

3. The method for preparing a prebiotic-loaded intestinal-targeted exosome according to claim 1, characterized in that: The transfection in step 1 adopts liposome transfection method or electroporation method. The transfection conditions of the liposome transfection method are: the ratio of liposome to plasmid is (2-5):1 (μL:μg), the transfection medium is serum-free DMEM or DMEM with a serum concentration of 1%-5%, and the transfection time is 4-8 hours; the transfection conditions of the electroporation method are: the electric field strength is 1000-1500 V / cm, the pulse time is 10-30 ms, and the number of pulses is 3-7 times.

4. The method for preparing prebiotic-loaded intestinal-targeted exosomes according to claim 3, characterized in that: The cells into which the plasmid is transfected in step 1 include HEK293 cells, COS-7 cells, Hela cells or SF9 cells.

5. The method for preparing prebiotic-loaded intestinal-targeted exosomes according to any one of claims 1 to 4, characterized in that: The transfected cells are screened using a culture medium containing 400-1200 μg / mL G418, a culture medium containing 0.5-10 μg / mL Promycin, a culture medium containing 50-300 μg / mL Hygromycin B or a culture medium containing 2-20 μg / mL Blasticidin S. The screening time is 2-6 weeks.

6. The method for preparing prebiotic-loaded intestinal-targeted exosomes according to claim 5, characterized in that: In step 1, flow cytometry or immunofluorescence is used to detect the expression level of the targeting peptide, and cell lines with a targeting peptide expression level greater than or equal to 1000 MFI, a positive cell ratio greater than or equal to 80%, and a fluorescent signal evenly distributed on the cell surface are screened out.

7. The method for preparing prebiotic-loaded intestinal-targeted exosomes according to claim 1, characterized in that: In step 2, the prebiotics include oligofructose, inulin or oligoxylose, the concentration of oligofructose is 100-300 μg / mL, the concentration of inulin is 200-400 μg / mL, and the concentration of oligoxylose is 300-600 μg / mL.

8. The method for preparing prebiotic-loaded intestinal-targeted exosomes according to claim 1, characterized in that: The step 3 includes: Initial centrifugation: Collect the cell culture supernatant and centrifuge at 200-400 rpm for 3-15 minutes; Differential centrifugation: Take the supernatant and centrifuge at 8000-12000 rpm at 2-6°C for 20-40 minutes; Ultracentrifugation: Take the supernatant and centrifuge at 100,000-150,000 rpm at 2-6°C for 45-75 minutes to collect the exosome precipitate; Resuspend and re-centrifuge: Resuspend the pellet in 0.5-10 mL of sterile PBS and centrifuge again at 100,000-150,000 rpm at 2-6°C for 45-75 minutes to collect the pellet. Final resuspension: Resuspend the pellet with 0.5-10 mL of sterile PBS to obtain intestinal-targeted exosomes loaded with prebiotics.

9. A prebiotic-loaded intestinal-targeted exosome, characterized in that: The prebiotic-loaded intestinal-targeted exosomes are prepared by the preparation method of any one of claims 1 to 8, wherein the diameter of the prebiotic-loaded intestinal-targeted exosomes is 30 to 150 nm.

10. Use of the prebiotic-loaded intestinal-targeted exosomes according to claim 9 in the preparation of medicines or health products for regulating intestinal microecology, preventing and treating intestinal diseases.

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