Exosome co-delivery composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an exosome co-delivery composition as well as a preparation method and application thereof. According to the exosome co-delivery composition provided by the invention, a system A utilizes mannitol-chitosan oligosaccharide to embed an active component exosome and probiotics to construct a chitosan oligosaccharide-mannitol nano-coating system, so that the survival rate and the structural stability of the active component can be improved; chitosan oligosaccharide in the system B forms a three-dimensional nanogel network through ionic cross-linking, butyrate and glutathione are embedded, a stable nanogel matrix is formed, a chitosan oligosaccharide-glutathione-butyrate nanogel system is constructed, multi-path protection, targeted release and synergistic interaction of probiotics, exosome, butyrate and glutathione are achieved through synergism of the three-dimensional nanogel network and the butyrate and the glutathione, and the chitosan oligosaccharide-glutathione-butyrate nanogel system has the advantages of being good in biocompatibility, good in biocompatibility and good in biocompatibility. And therefore, a remarkable comprehensive promotion effect is generated in the aspects of intestinal barrier repair, flora reconstruction, anti-inflammation, anti-oxidation and energy supply.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an exosome co-delivery composition, its preparation method, and its application. Background Technology

[0002] Intestinal inflammation is a common digestive system disease, including various types such as chronic gastroenteritis and leaky gut. Its pathogenesis is closely related to intestinal flora imbalance, intestinal barrier damage, and abnormal inflammatory responses. Currently, bioactive substances such as probiotics and exosomes show good application potential in the treatment of intestinal inflammation. However, these active substances suffer from poor stability and are easily destroyed in the harsh environment of the gastrointestinal tract, such as gastric acid and pancreatic enzymes, resulting in low oral bioavailability and difficulty in fully exerting their therapeutic effects. For example, Chinese patents CN120078157A and CN118592600A are both single delivery systems that simply mix chitosan with probiotics and exosomes, and their gastrointestinal repair effects are not good. Therefore, there is an urgent need for a composition that can improve the stability of active substances and achieve targeted release. Summary of the Invention

[0003] The purpose of this invention is to provide an exosome co-delivery composition, its preparation method, and its application. The exosome co-delivery composition can achieve targeted release and synergistic effect of multiple active ingredients, providing a new solution for the treatment of intestinal inflammation.

[0004] This invention provides an exosome co-delivery composition comprising system A and system B; By weight, system A comprises 1-30 parts of exosome preparation, 1-60 parts of chitosan oligosaccharide-mannitol, and 1-30 parts of probiotics; wherein the chitosan oligosaccharide-mannitol encapsulates the exosomes and probiotics. By mass, system B comprises 1-60 parts of chitosan oligosaccharide, 1-20 parts of glutathione, and 1-20 parts of butyrate; the chitosan oligosaccharide encapsulates glutathione and butyrate.

[0005] As a preferred embodiment, the mass ratio of system A to system B is 1:1 to 10:1.

[0006] As a preferred embodiment, the mass ratio of chitosan oligosaccharide to mannitol in the chitosan oligosaccharide-mannitol mixture is 10:1 to 20:1.

[0007] The present invention also provides a method for preparing the exosome co-delivery composition described above, comprising the following steps: Exosome preparations, probiotics, and water were mixed to obtain a mixture. The mixture was then added dropwise to a chitosan oligosaccharide-mannitol solution for self-assembly to obtain system A. Butyrate solution and glutathione solution were added dropwise to a chitosan oligosaccharide solution for self-assembly to obtain system B. The exosome co-delivery composition is obtained by mixing system A and system B.

[0008] As a preferred embodiment, the concentration of chitosan oligosaccharide in the chitosan oligosaccharide-mannitol solution is 10-20 mg / mL, and the concentration of mannitol is 1-2 mg / mL.

[0009] As a preferred embodiment, after adding the mixture dropwise to the chitosan oligosaccharide-mannitol solution for self-assembly, the method further includes adjusting the chitosan oligosaccharide concentration to 1~5 mg / mL.

[0010] As a preferred embodiment, the concentration of chitosan oligosaccharide in the chitosan oligosaccharide solution is 10~20 mg / mL.

[0011] As a preferred embodiment, after adding butyrate solution and glutathione solution dropwise to chitosan oligosaccharide solution for self-assembly, the method further includes adjusting the chitosan oligosaccharide concentration to 1~5 mg / mL.

[0012] As a preferred embodiment, the self-assembly method includes stirring; the stirring speed is 250~1000 rpm; and the stirring time is 2~4 h.

[0013] The present invention also provides the application of the exosome co-delivery composition described above or the exosome co-delivery composition prepared by the above preparation method in the preparation of a medicament for treating intestinal inflammation.

[0014] Beneficial effects: This invention provides an exosome co-delivery composition comprising system A and system B. By weight, system A comprises 1-30 parts of exosome preparation, 1-60 parts of chitosan oligosaccharide-mannitol, and 1-30 parts of probiotics; the chitosan oligosaccharide-mannitol encapsulates the exosomes and probiotics. By weight, system B comprises 1-60 parts of chitosan oligosaccharide, 1-20 parts of glutathione, and 1-20 parts of butyrate; the chitosan oligosaccharide encapsulates the glutathione and butyrate. The present invention describes a system A that utilizes mannitol-chitosan oligosaccharide to encapsulate active ingredient exosomes and probiotics, constructing a "chitosan oligosaccharide-mannitol nano-encapsulation system," which can improve the survival rate and structural stability of active ingredients. Mannitol, as a bioprotective agent, can inhibit membrane protein denaturation, reduce osmotic stress, and act as a low-temperature or lyophilization stabilizer to protect the structure of active substances. Chitosan provides acid resistance protection, sustained-release control, and an antibacterial barrier. The two work synergistically to make sensitive active substances more stable, more resistant to gastric acid, and faster rehydration, while enhancing shell density, toughness, and membrane stability. In System B, chitosan oligosaccharides form a three-dimensional nanogel network through ionic cross-linking. The amino groups of chitosan oligosaccharides can form hydrogen bonds-electrostatic networks / physical cross-links with the carboxyl groups of butyrate and the carboxyl and thiol groups of glutathione (GSH), forming a stable nanogel matrix and constructing a "chitosan oligosaccharide-glutathione-butyrate nanogel system". The resulting System B is stable in gastric acid and slowly disintegrates at neutral pH, protecting GSH from oxidation and achieving sustained release of butyrate, reducing gastric irritation, and targeting the intestinal tract to release butyrate and glutathione. This invention constructs two synergistic delivery structures—a chitosan oligosaccharide-mannitol nanoencapsulation system and a chitosan oligosaccharide-glutathione-butyrate nanogel system—to achieve multi-pathway protection, targeted release, and synergistic effects of probiotics, exosomes, butyrate, and glutathione. This results in a significant comprehensive promoting effect on intestinal barrier repair, flora reconstruction, anti-inflammatory and antioxidant effects, and energy supply, achieving gastrointestinal repair effects far exceeding those of single components or simple mixtures.

[0015] This invention also provides a method for preparing an exosome co-delivery composition, comprising the following steps: mixing an exosome preparation, probiotics, and water to obtain a mixture; adding the mixture dropwise to a chitosan oligosaccharide-mannitol solution for self-assembly to obtain system A; adding butyrate solution and glutathione solution dropwise to a chitosan oligosaccharide solution for self-assembly to obtain system B; and mixing system A and system B to obtain the exosome co-delivery composition. The preparation method of this invention is simple and can encapsulate active ingredients, improving stability.

[0016] The present invention also provides the application of the exosome co-delivery composition in the preparation of a drug for treating intestinal inflammation. The exosome co-delivery composition can achieve multi-pathway protection, targeted release and synergistic effect of probiotics, exosomes, butyrate and glutathione, thereby producing a significant comprehensive promoting effect in intestinal barrier repair, flora reconstruction, anti-inflammatory and antioxidant effects and energy supply. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 A flowchart for constructing a cell line enteritis model and detecting drug efficacy; Figure 2 The graph shows the IL-6 detection results for each treatment group in Application Example 1; Figure 3 The graph shows the IL-1β detection results for each treatment group in Example 1. Figure 4 Figure 1 shows the TNF-α detection results for each treatment group in Example 1. Figure 5 The graph shows the COX-2 detection results for each treatment group in Application Example 1; Figure 6 The graph shows the DAO content detection results for each treatment group in Application Example 1; Figure 7 The graph shows the results of D-LA content detection in each treatment group of Application Example 1; Figure 8 The graph shows the MTT test results for each treatment group in Example 1. Figure 9 The graph shows the detection results for each treatment group in Example 2, where A represents the IL-6 detection result; B represents the IL-1β detection result; C represents the TNF-α detection result; and D represents the COX-2 detection result. Figure 10 The graph shows the DAO content detection results for each treatment group in Example 2. Figure 11 The graph shows the results of D-LA content detection in each treatment group in Example 2. Figure 12 The graph shows the MTT test results for each treatment group in Example 2. Detailed Implementation

[0019] This invention provides an exosome co-delivery composition comprising system A and system B; By weight, system A comprises 1-30 parts of exosome preparation, 1-60 parts of chitosan oligosaccharide-mannitol, and 1-30 parts of probiotics; wherein the chitosan oligosaccharide-mannitol encapsulates the exosomes and probiotics. By mass, system B comprises 1-60 parts of chitosan oligosaccharide, 1-20 parts of glutathione, and 1-20 parts of butyrate; the chitosan oligosaccharide encapsulates glutathione and butyrate.

[0020] Unless otherwise specified, all raw materials involved in this invention are obtained through conventional commercial methods.

[0021] To facilitate the specification of the relative amounts of each component, the exosome co-delivery composition of this invention is defined in the form of System A and System B. In System A of this invention, the chitosan oligosaccharide-mannitol encapsulates exosomes and probiotics. Mannitol, as a bioprotective agent, can inhibit membrane protein denaturation, reduce osmotic stress, and also act as a low-temperature stabilizer and lyophilization stabilizer to protect the active components; chitosan can form a dense antibacterial barrier, resisting gastric acid erosion and achieving a sustained-release effect of the active ingredients. When chitosan oligosaccharide is used alone to form a film, the resulting product (microcapsule particles) is prone to collapse during drying and has an uneven structure, leading to easy breakage of the microcapsule particles. However, the addition of mannitol can inhibit excessive entanglement between chitosan chains, improve its flexibility, and reduce the risk of drying cracking. The synergistic effect of chitosan oligosaccharide and mannitol can enhance the density and toughness of the shell, improve membrane stability, make it more resistant to gastric acid, and allow for faster rehydration, thereby improving the survival rate and structural stability of the active ingredients (probiotics and exosomes). Exosomes are natural nanocarriers, but their entry into specific cells remains limited. Chitosan, being positively charged, strongly adsorbs onto negatively charged cell surface molecules (mainly glycoproteins), thereby enhancing exosome adhesion to the cell membrane, improving endocytosis efficiency, and significantly increasing the delivery of miRNAs and proteins. Furthermore, exosomes and probiotics are easily damaged by gastric acid, pancreatic enzymes, plasma protein lysis, high temperatures, and strong shearing environments. Chitosan oligosaccharide-mannitol can form a protective shell, allowing exosomes and probiotics to remain intact in harsh environments. They maintain structural stability even in the acidic environment of gastric acid at pH 2-3, isolating proteases and enhancing the stability of exosomes and probiotics. This prevents the degradation of exosome contents (such as miRNAs, proteins, and lipids) and reduces membrane lipid damage caused by oxidative stress. In System A of this invention, chitosan oligosaccharide-mannitol encapsulates probiotics and exosomes, forming a three-in-one intestinal microecological co-delivery and regulation system. Specifically, chitosan oligosaccharide is preferentially utilized by probiotics, promoting an increase in acid-producing bacteria, which in turn increases the content of short-chain fatty acids (SCFAs), improving the survival and colonization rate of probiotics and competitively inhibiting the proliferation of pathogenic bacteria. As a cationic polysaccharide, chitosan oligosaccharide can encapsulate exosomes, forming a protective shell in the acidic environment of the stomach. Upon dissociation in the small intestine and colon, it releases exosomes and probiotics, promoting cellular uptake. Furthermore, chitosan oligosaccharide enhances exosome absorption, significantly improving the oral absorption efficiency of exosomes. Exosomes can reduce intestinal inflammation, providing a more favorable environment for probiotics, repairing the intestinal barrier, inhibiting the penetration and occupation of microecological niches by pathogenic bacteria, inhibiting inflammatory pathways (NF-κB / NLRP3), and further improving the colonization ability of probiotics. System A of this invention achieves multiple benefits, including targeted intestinal delivery, protection of active ingredient activity, and prebiotic promotion.

[0022] By weight, System A of the present invention comprises 1 to 30 parts of exosome preparation. In specific embodiments of the present invention, the weight of the exosome preparation can be any value from 1 to 30 parts, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 30 parts. As one embodiment, the exosome preparation includes milk-derived exosome preparations and / or plant-derived exosome preparations. The exosomes of the present invention can serve as effective therapeutic carriers for genes and drugs, maintaining their intact particle form during gastrointestinal transport, thereby achieving effective and stable absorption of the encapsulated contents, thus exhibiting excellent improvement effects on intestinal inflammatory symptoms.

[0023] As one implementation method, the milk-derived exosome preparation includes lyophilized milk-derived exosome powder; the lyophilized milk-derived exosome powder is obtained by freeze-drying milk-derived exosomes. This invention does not impose any special restrictions on the source of the milk-derived exosomes; commercially available sources are acceptable. In a specific embodiment of this invention, the method for preparing the milk-derived exosomes includes the following steps: (1) filtering milk and collecting the clarified liquid; (2) concentrating the clarified liquid by ultrafiltration to obtain a fresh milk clarified filtrate; (3) performing aqueous two-phase extraction on the fresh milk clarified filtrate to obtain a preliminary purified product; (4) removing impurities from the preliminary purified product using a gel column and collecting the eluted components; (5) centrifuging the eluted components again to collect the precipitate, thereby obtaining the milk-derived exosomes. In one implementation, the filtration in step (1) uses a hydrophilic and lipophilic nano-interface membrane; the ultrafiltration concentration in step (2) uses a tangential flow ultrafiltration device with a molecular weight cutoff of 100 kDa to 300 kDa, and the small molecule target product is obtained by timely filtration through the ultrafiltration membrane; the aqueous two-phase extraction in step (3) uses an aqueous two-phase solution composed of polyethylene glycol and dextran, and the number of aqueous two-phase extractions is 2 to 3; the packing material of the gel column in step (4) is highly cross-linked agarose; the centrifugation conditions in step (5) are 120,000 × g, centrifugation at 4°C for 90 min. In one implementation, the milk-derivedexosomes include milk-derivedexosomes. In this invention, the milk-derivedexosomes should be accurately described as "milk-derivedexosome-like nano-vesicles", and the particle size range may vary depending on the separation and purification methods. In this invention, the particle size of the milk-derived exosomes is 80-120 nm; as one embodiment, the number of particles of the milk-derived exosomes reaches 10. 12The exosomes, measured in particles / g, are observed as complete and clear exosomes under an electron microscope and are then freeze-dried for preservation. The milk-derived exosomes of this invention possess a natural lipid bilayer and a protective structure, exhibiting strong resistance to gastric acid and digestive enzymes. Furthermore, these milk-derived exosomes can cross the intestinal epithelium and be absorbed systemically or enter intestinal mucosal cells.

[0024] In one embodiment, the plant exosome preparation includes a plant-derived exosome lyophilized powder preparation. In another embodiment, the preparation method of the plant-derived exosome lyophilized powder preparation preferably includes the following steps: homogenizing a mixture of plant and water to obtain a homogenate; centrifuging the homogenate at low speed to obtain a first supernatant; centrifuging the first supernatant at medium speed to obtain a second supernatant; centrifuging the second supernatant at ultracentrifugation, collecting the precipitate, resuspending it, and washing it to obtain the plant-derived exosomes; and freeze-drying the plant-derived exosomes to obtain the plant-derived exosome lyophilized powder preparation. In one embodiment, the mass-to-volume ratio of plant and water is 1 g:5 mL. In one embodiment, the low-speed centrifugation speed is 5000 × g. In one embodiment, the low-speed centrifugation time is 10 min. In one embodiment, the medium-speed centrifugation speed is 10000 × g. In one embodiment, the medium-speed centrifugation time is 20 min. In one embodiment, the ultracentrifugation speed is 100,000 × g, and the ultracentrifugation time is 20 min. In another embodiment, the freeze-drying process includes freezing at -50℃ to -40℃ for 1 to 2 h, followed by vacuum removal of moisture and maintenance at -80℃ for 24 to 48 h. In another embodiment, the invention further includes purification of the freeze-dried plant-derived exosomes. In another embodiment, purification is performed after the freeze-drying process. In another embodiment, the purification uses a 0.22 μm filter membrane. In another embodiment, the plant includes one or more of ginger, turmeric, and yam; in yet another embodiment, the plant includes ginger. The plant-derived exosomes of this invention are derived from edible plants, are easily accepted by the market, and can be used in the preparation of vegetarian and clean-label products. Furthermore, the plant-derived exosomes can be taken up by intestinal macrophages or epithelial cells.

[0025] Based on the mass fraction of the exosome preparation, system A comprises 1 to 60 parts of chitosan oligosaccharide-mannose. In specific embodiments of the present invention, the mass fraction of chitosan oligosaccharide-mannose can be any value from 1 to 60 parts, for example, 1, 2, 3, 5, 8, 10, 12, 15, 18, 20, 22, 25, 27, 29, 30, 31, 33, 35, 37, 39, 40, 42, 45, 48, 50, 52, 54, 55, 58, or 60 parts. In the present invention, the chitosan oligosaccharide-mannitol is a mixture of chitosan oligosaccharide and mannitol, and the mass ratio of chitosan oligosaccharide to mannitol is 10:1 to 20:1. In a specific embodiment of the present invention, the mass ratio of chitosan oligosaccharide to mannitol can be any value from 10:1 to 20:1, such as 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1. A suitable mass ratio of chitosan oligosaccharide to mannitol can better encapsulate the contents and improve stability, while also resulting in smaller particle size after encapsulation. As one embodiment, the degree of polymerization of the chitosan oligosaccharide is 2 to 20. In a specific embodiment of the present invention, the degree of polymerization of the chitosan oligosaccharide can be any value from 2 to 20, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Chitosan oligosaccharide with a degree of polymerization of 2 to 20 has good encapsulation properties and stability, while maintaining a sufficiently small particle size after encapsulation. In this invention, the degree of polymerization of chitosan oligosaccharides in system A is preferably higher than that in system B; since the exosomes and probiotics in system A have large molecular weights, selecting chitosan oligosaccharides with a high degree of polymerization for encapsulation can achieve more effective encapsulation and stability.

[0026] Based on the mass fraction of the exosomes, system A includes 1 to 30 parts of probiotics. In a specific embodiment of the present invention, the mass fraction of the probiotics can be any value from 1 to 30 parts, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 30 parts. As one embodiment, the probiotics include Bifidobacterium (…). Bifidobacterium ), Lactobacillus ( Lactobacillus Lactobacillus () Lacticaseibacillus ), Lactobacillus mucinus ( Limosilactobacillus Lactobacillus () LactiplantibacillusOne or more of the following: Bifidobacterium, as a core colonizing bacterium of the intestine, can first adhere to the intestinal mucosa to form a stable flora structure, occupy an ecological niche to inhibit the colonization of harmful bacteria, and provide a suitable environment for the proliferation and function of other probiotics; Lactobacillus can inhibit the growth and reproduction of pathogenic bacteria in the intestine by producing lactic acid, antimicrobial peptides and other substances, and stimulate the activation of intestinal mucosal immune cells, regulate the body's innate and adaptive immune responses, and improve the overall anti-infection ability and immune balance; Lactobacillus can regulate intestinal smooth muscle contraction, improve intestinal peristalsis, alleviate symptoms related to intestinal dysfunction, and can also regulate through gut-brain axis signaling. It regulates central functions such as mood and sleep, achieving bidirectional gut-brain regulation; *Lactobacillus mucinus* targets the intestinal mucosal barrier, adhering to the intestinal mucus layer, promoting the secretion of mucin by intestinal epithelial cells, thickening the intestinal mucus barrier, and repairing the tight junctions between damaged intestinal epithelial cells, reducing intestinal leakage caused by increased intestinal permeability; *Lactobacillus plantarum* scavenges reactive oxygen free radicals in the body by synthesizing antioxidants such as superoxide dismutase and glutathione, reducing oxidative stress damage, while regulating intestinal mucosal immune tolerance, reducing the probability of abnormal immune responses, and improving the body's tolerance to external stimuli such as environment and food.

[0027] In one implementation, the effective viable count of the probiotics in system A is 10. 4 100 million / g.

[0028] In one implementation, the bifidobacteria include Bifidobacterium adolescentis (Bifidobacterium adolescentis). Bifidobacterium adolescents is Bifidobacterium animalis ( Bifidobacteriumanimalis Bifidobacterium bifidum ( Bifidobacterium bifidum ), Bifidobacterium breve Bifidobacterium breve ) and Bifidobacterium longum ( Bifidobacterium longum One or more of the following are included. As one embodiment, the lactobacillus includes Lactobacillus acidophilus (…). Lactobacillus acidophilus Lactobacillus curvature () Lactobacillus crispatus Lactobacillus delbrueckii ( ), Lactobacillus bulgaricus Lactobacillus gasseri ( Lactobacillus gasseri Lactobacillus helveticus ( Lactobacillus helveticus Lactobacillus johnsonii ( Lactobacillus johnsonii ) and Lactobacillus hummophys ( Lactobacillus kefiranofaciens One or more of the following. As one embodiment, the lactobacillus includes *Lactobacillus casei* (…). Lacticaseibacillus casei Lactobacillus paracasei ( Lacticaseibacillus paracasei Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus One or more of the following. As one embodiment, the *Lactobacillus mucinus* includes *Lactobacillus fermentum* (…). Limosilactobacillus fermentum ) and / or Lactobacillus reuteri ( Limosilactobacillus reuteriAs one embodiment, the lactobacillus includes *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum ).

[0029] In System B of this invention, chitosan oligosaccharide encapsulates glutathione and butyrate. The chitosan oligosaccharide forms a three-dimensional nanogel network through ionic cross-linking. The amino groups of the chitosan oligosaccharide can form hydrogen bonds, electrostatic networks, and physical cross-links with the carboxyl groups of butyrate and the carboxyl and thiol groups of glutathione (GSH), forming a stable nanogel matrix. Butyrate is a major energy substrate for colonic epithelial cells, providing 70% of ATP, and can restore the mucosal barrier, enhance tight junction proteins, inhibit inflammatory factors (IL-6, TNF-α), and promote endogenous GSH synthesis. Glutathione can scavenge ROS, protect mucosal cells, and alleviate chronic inflammation. However, glutathione is extremely unstable in gastric acid and is easily oxidized to ineffective GSSG (oxidized glutathione). In System B of this invention, chitosan oligosaccharides can form nanogels. These nanogels are stable in gastric acid and slowly disintegrate in a neutral pH environment. Encapsulating butyrate and glutathione with chitosan oligosaccharides effectively protects the targeted release of GSH in the intestine, maintains glutathione activity, and enhances mucosal sustained release and absorption. Simultaneously, the three-dimensional nanogel network of System B enables sustained release of butyrate, reducing gastric irritation and targeting colonic release.

[0030] Based on the mass fraction of the exosome preparation, system B comprises 1 to 60 parts of chitosan oligosaccharide. In specific embodiments of the present invention, the mass fraction of chitosan oligosaccharide can be any value from 1 to 60 parts, for example, 1, 2, 3, 5, 8, 10, 12, 15, 18, 20, 22, 25, 27, 29, 30, 31, 33, 35, 37, 39, 40, 42, 45, 48, 50, 52, 54, 55, 58, or 60 parts. The degree of polymerization of the chitosan oligosaccharide has been discussed above and will not be repeated here.

[0031] Based on the mass fraction of chitosan oligosaccharides in system B, system B includes 1 to 20 parts of glutathione. In a specific embodiment of the present invention, the mass fraction of glutathione can be any value from 1 to 20 parts, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 parts. As one embodiment, the glutathione is reduced glutathione. Glutathione can scavenge ROS, protect mucosal cells, reduce chronic inflammation, and protect tight junction structures.

[0032] Based on the mass fraction of chitosan oligosaccharides in system B, system B includes 1-20 parts of butyrate. In specific embodiments of the present invention, the mass fraction of butyrate can be any value from 1 to 20 parts, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 parts. As one embodiment, the butyrate includes sodium butyrate. Butyrate is the source of 70% of the ATP in the colonic epithelium, and can restore the mucosal barrier, enhance tight junction proteins, inhibit inflammatory factors (IL-6, TNF-α), and promote the endogenous synthesis of GSH. The present invention selects sodium butyrate, which is beneficial for nanogel formation, achieving sustained release, and sodium butyrate is low in cost and has a mature supply chain.

[0033] In one implementation, the mass ratio of system A to system B is 1:1 to 10:1. In specific embodiments of the present invention, the mass ratio of system A to system B can be any ratio from 1:1 to 10:1, such as 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. System A and system B of the present invention have a synergistic effect. In system A, probiotics can increase the content of SCFAs (short-chain fatty acids) in the intestine, producing more butyrate, which, together with butyrate in system B, further promotes the proliferation of probiotics and enhances gut microbiota homeostasis. Exosomes in system A can inhibit NLRP3 inflammasomes, promote epithelial cell migration, and promote wound closure; glutathione in system B can reduce oxidative stress, making the exosome loading more efficient. The two work synergistically to double the promotion of intestinal epithelial healing and regeneration, inhibiting inflammatory aging, making it more suitable for chronic gastroenteritis, leaky gut individuals, and those with impaired gastric barrier. Furthermore, both System A and System B contain chitosan oligosaccharides. In System A, chitosan oligosaccharides work together with mannitol to encapsulate probiotics and exosomes, thereby improving the stability of active ingredients. In System B, chitosan oligosaccharides form nanogels, which improve the sustained-release effect and can also act as prebiotics to promote the proliferation of probiotics.

[0034] This invention constructs two synergistic delivery structures, System A (chitosan oligosaccharide-mannitol nanoencapsulation system) and System B (chitosan oligosaccharide-glutathione-butyrate nanogel system), to achieve multi-pathway protection, targeted release, and synergistic effects of probiotics, exosomes, butyrate, and glutathione. This results in a significant comprehensive promoting effect on intestinal barrier repair, flora reconstruction, anti-inflammatory and antioxidant effects, and energy supply, achieving gastrointestinal repair effects far exceeding those of single ingredients or simple mixtures.

[0035] The present invention also provides a method for preparing the exosome co-delivery composition described above, comprising the following steps: mixing exosome preparation, probiotics and water to obtain a mixture, and adding the mixture dropwise to a chitosan oligosaccharide-mannitol solution for self-assembly to obtain system A; Butyrate solution and glutathione solution were added dropwise to chitosan oligosaccharide solution to induce self-assembly, thus obtaining system B. The exosome co-delivery composition is obtained by mixing system A and system B.

[0036] This invention mixes exosome preparations, probiotics, and water to obtain a mixture. In one embodiment, the mass content of the exosome preparation in the mixture is 2%. In another embodiment, the mass content of the probiotics in the mixture is 10%. If the probiotic concentration in the mixture of this invention is too high, it will lead to instability of the encapsulation system and a reduction in the efficacy of the resulting encapsulated product. Therefore, limiting the probiotic concentration to 10% stabilizes the system.

[0037] In one embodiment, chitosan oligosaccharide, mannitol, and water are dissolved to obtain a chitosan oligosaccharide-mannitol solution. In another embodiment, the concentration of chitosan oligosaccharide in the chitosan oligosaccharide-mannitol solution is 10-20 mg / mL. In specific embodiments of the present invention, the concentration of chitosan oligosaccharide in the chitosan oligosaccharide-mannitol solution can be any value within the range of 10-20 mg / mL, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / mL. In yet another embodiment, the concentration of mannitol in the chitosan oligosaccharide-mannitol solution is 1-2 mg / mL. Limiting the concentrations of chitosan oligosaccharide and mannitol in this invention makes the preparation system more stable.

[0038] After obtaining the chitosan oligosaccharide-mannitol solution, the present invention adds the mixture dropwise to the chitosan oligosaccharide-mannitol solution for self-assembly, obtaining system A. As one embodiment, the self-assembly method includes stirring; the stirring speed is 250-1000 rpm; in a specific embodiment of the present invention, the stirring speed can be any value within 250-1000 rpm, for example, 250, 300, 350, 400, 450, 500, 550, 600, 750, 800, 850, 900, 950, or 1000 rpm. As one embodiment, the stirring time is 2-4 h. In a specific embodiment of the present invention, the stirring time can be any value within 2-4 h, for example, 2, 2.5, 3, 3.5, or 4 h. As one embodiment, the dropwise addition rate is 2.6 mL / min. The present invention's dropwise addition of the mixture to the chitosan oligosaccharide-mannitol solution is beneficial for improving encapsulation efficiency and stability. The present invention involves adding the mixture dropwise to a chitosan oligosaccharide-mannitol solution, which allows for a sufficiently small volume ratio of exosomes and probiotics, resulting in smaller encapsulated particle sizes. In a specific embodiment of the present invention, the self-assembly process of adding the mixture dropwise to the chitosan oligosaccharide-mannitol solution includes: adding the mixture dropwise to the chitosan oligosaccharide-mannitol solution at a rate of 2.6 mL / min using a peristaltic pump at 500 rpm, and stirring at 500 rpm for 2 h. The self-assembly process of the present invention begins with the adsorption of the protonated amine groups of chitosan oligosaccharides onto the negatively charged surface of the target particles (exosomes, probiotics), and mannitol enhances the shell density through hydrogen bonding intercalation and segment plasticization, ultimately forming stable nanoparticles.

[0039] As one embodiment, after adding the mixture dropwise to the chitosan oligosaccharide-mannitol solution for self-assembly, the method further includes adjusting the chitosan oligosaccharide concentration to 1-5 mg / mL. In specific embodiments of the present invention, the chitosan oligosaccharide concentration can be adjusted to any value within the range of 1-5 mg / mL, for example, 1, 2, 3, 4, or 5 mg / mL. In one specific embodiment of the present invention, adjusting the chitosan oligosaccharide concentration to 1 mg / mL is beneficial for making the particle size distribution of the encapsulated material (system A) more uniform. As one embodiment, after obtaining system A, the method further includes: lyophilizing system A. The present invention does not particularly limit the lyophilization process; conventional lyophilization methods in the art can be used.

[0040] This invention involves the dropwise addition of butyrate solution and glutathione solution to a chitosan oligosaccharide solution for self-assembly, resulting in system B. As one embodiment, the concentration of chitosan oligosaccharide in the chitosan oligosaccharide solution is 10-20 mg / mL. In specific embodiments of this invention, the concentration of chitosan oligosaccharide in the chitosan oligosaccharide solution can be any value within the range of 10-20 mg / mL, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / mL. As one embodiment, the concentration of the butyrate solution is 1 mg / mL; the concentration of the glutathione solution is 1 mg / mL. As one embodiment, the self-assembly method includes stirring; the stirring speed is 250~1000 rpm; in a specific embodiment of the present invention, the stirring speed can be any value among 250~1000 rpm, for example 250, 300, 350, 400, 450, 500, 550, 600, 750, 800, 850, 900, 950 or 1000 rpm. As one embodiment, the stirring time is 2~4 h. In a specific embodiment of the present invention, the stirring time can be any value among 2~4 h, for example 2, 2.5, 3, 3.5 or 4 h. As one embodiment, the dropwise addition rate is 2.6 mL / min. The present invention's method of dropwise addition of the butyrate solution and glutathione solution to the chitosan oligosaccharide solution can make the butyrate solution and glutathione solution sufficiently small, resulting in a smaller particle size of the encapsulated material. In one specific embodiment of the present invention, the self-assembly of butyrate solution and glutathione solution by dropwise addition to chitosan oligosaccharide solution includes: adding the butyrate solution and glutathione solution dropwise to the chitosan oligosaccharide solution at a rate of 2.6 mL / min using a peristaltic pump at 500 rpm, and stirring at 500 rpm for 2 h. As one embodiment, after mixing the butyrate solution, glutathione solution, and chitosan oligosaccharide solution, the method further includes: adjusting the chitosan oligosaccharide concentration to 1 mg / mL. As another embodiment, after obtaining system B, the method further includes: lyophilization. The present invention adjusts the chitosan oligosaccharide concentration to 1 mg / mL, which is beneficial for forming a stable small-particle-size encapsulation system.

[0041] This invention does not specify the preparation order of system A and system B. After obtaining system A and system B, they are mixed to obtain the exosome co-delivery composition. The mass ratio of system A and system B has been discussed above and will not be repeated here. The preparation method of this invention produces system A, which consists of chitosan oligosaccharide-mannitol-encapsulated exosomes and probiotics in nanoparticles, and system B, which consists of chitosan oligosaccharide-glutathione-butyrate nanogel particles. After mixing, each maintains its original nanostructure and stability.

[0042] This invention also provides the application of the exosome co-delivery composition described above or the exosome co-delivery composition prepared by the above preparation method in the preparation of drugs for treating intestinal inflammation. The exosome co-delivery composition of this invention, by constructing two synergistic delivery structures—a "chitosan oligosaccharide-mannitol nanoencapsulation system" and a "chitosan oligosaccharide-glutathione-butyrate nanogel system"—achieves multi-pathway protection, targeted release, and synergistic effect of probiotics, exosomes, butyrate, and glutathione. This results in a significant comprehensive promoting effect on intestinal barrier repair, flora reconstruction, anti-inflammatory and antioxidant effects, and energy supply, and can be used to prepare drugs for treating intestinal inflammation.

[0043] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0044] In a specific embodiment of the present invention, the raw materials used are sourced from the following sources: Chitosan oligosaccharide was purchased from EXO Longevity Biotech Limited, catalog number EXO20250101; mannitol was purchased from EXO Longevity Biotech Limited, catalog number EXO202506012; glutathione was purchased from EXO Longevity Biotech Limited, catalog number EXO202401112; butyrate (sodium butyrate) was purchased from EXO Longevity Biotech Limited, catalog number EXO202401019. Example 1 Preparation of exosome co-delivery composition based on plant-derived exosomes 1. Preparation of plant-derived exosomes. The preparation method is described in patent 202510242011.9. The specific steps are as follows: (1) Take 100 g of fresh ginger, add 500 mL of water, and homogenize to obtain a homogenate; (2) Centrifuge at low speed of 5000×g for 10 min and take the first supernatant; (3) Centrifuge at medium speed of 10000×g for 20 min and take the second supernatant; (4) Centrifuge at high speed of 100000×g, collect the precipitate, resuspend it with physiological saline and wash it 3 times to obtain ginger-derived exosomes; (5) Freeze at -45℃ for 1.5 h, remove water under vacuum and freeze dry at -80℃ for 36 h, and purify it through a 0.22 μm filter membrane to obtain plant-derived exosome freeze-dried powder.

[0045] 2. Preparation of System A: (1) Weigh chitosan oligosaccharide (degree of polymerization 10) and mannitol, dissolve them in water and bring the volume to 1000 mL to prepare a mixed solution of 10 mg / mL chitosan oligosaccharide + 1 mg / mL mannitol; (2) Weigh 200 mg (10 mg / mL) of the above plant-derived exosome lyophilized powder.6 1000 mg of lyophilized Bifidobacterium infantis powder (purchased from EXO LONGEVITY BIOTECH LIMITED, strain BI15) was dissolved in water to obtain a mixed solution of exosomes and probiotics; the concentration of exosomes in the mixed solution was 2%, the concentration of Bifidobacterium infantis was 10%, and the viable count of Bifidobacterium infantis was 10. 4 100 million / g; (3) Under 500 rpm stirring, the exosome + probiotic mixed solution was added dropwise to the chitosan oligosaccharide-mannitol mixed solution at a speed of 2.6 mL / min using a peristaltic pump, and stirring was continued for 2 h; (4) Water was added to adjust the concentration of chitosan oligosaccharide and mannitol in the system to 1 mg / mL, and the system was stored at 4℃ in the dark, and then freeze-dried at -80℃ to obtain system A nanoparticles.

[0046] 3. Preparation of System B: (1) Weigh 10 g of chitosan oligosaccharide (degree of polymerization 8), dissolve it in water and make up to 1 L to prepare a 10 mg / mL chitosan oligosaccharide solution; (2) Weigh 1 g of butyrate and 1 g of reduced glutathione, dissolve them in water and make up to 1 L to obtain a 1 mg / mL butyrate + 1 mg / mL reduced glutathione composite solution; (3) Under stirring at 500 rpm, use a peristaltic pump to add the composite solution dropwise to the chitosan oligosaccharide solution at a rate of 2.6 mL / min, and continue stirring for 2 h; (4) Add water to adjust the concentration of chitosan oligosaccharide in the system to 1 mg / mL, store at 4℃ in the dark, and then freeze-dry at -80℃ to obtain the composite nanogel particles of System B.

[0047] 4. Assembly of the composition: The nanoparticles of system A and the composite nanogel particles of system B are mixed evenly at a mass ratio of 1:1 to obtain the exosome co-delivery composition.

[0048] Comparative Example 1 A composition is prepared in the same way as in Example 1, except that: after obtaining the plant-derived exosome freeze-dried powder, the chitosan oligosaccharide, mannitol, and Bifidobacterium infantis freeze-dried powder from step 2, and the chitosan oligosaccharide, butyrate, and reduced glutathione from step 3 are directly mixed to obtain the composition, which is referred to as the mixture.

[0049] Comparative Example 2 A composition is prepared in the same way as in Example 1, except that in step 2, chitosan oligosaccharide, mannitol, plant-derived exosome lyophilized powder, and Bifidobacterium infantis lyophilized powder are directly mixed to obtain mixture A; the final composition is denoted as mixture A + system B.

[0050] Comparative Example 3 A composition is prepared in the same way as in Example 1, except that in step 3, chitosan oligosaccharide, butyrate and reduced glutathione are directly mixed to obtain mixture B; the resulting composition is referred to as system A + mixture B.

[0051] Application Example 1 1. Culture and passage of HT-29 cell line: (1) Passage of HT-29 cell line when the density in cell culture plate reaches more than 80%. (2) Digestion: Discard the old culture medium, add 1-2 mL of 0.25% (w / v) trypsin to the culture plate, place it in a 37℃ incubator for 2-4 min to digest, observe the cell digestion under a microscope, if most of the cells become round and detach, quickly return to the operating table, tap the culture flask a few times and add 1-2 mL of complete culture medium to stop digestion. Gently pipette the cells, after they are completely detached, aspirate them into a centrifuge tube and centrifuge (1000 rpm, 5 min). (3) Seeding: Discard the supernatant and resuspend the cells in 1-3 mL of complete culture medium. Transfer the resuspended cell suspension to two new cell culture plates.

[0052] 2. Establishment of cell line inflammation models and drug regimen: according to Figure 1 The following are the specific steps for constructing a cell line enteritis model and detecting drug efficacy: Pass the HT-29 cell line into well plates for the experiment. Once the HT-29 cell line reaches approximately 70% confluence, the experiment can begin. The HT-29 cell line is randomly divided into 7 groups, as follows: Control group (CTRL): Cultured under normal conditions at 37℃.

[0053] Inflammation Treatment Group (IMF): Cells were pre-induced for 9 h with complete medium containing 20 ng / mL IFN-γ, and then induced for 15 h with complete medium containing 1 μg / mL LPS. After the induction culture was completed, the cells were cultured normally for 24 h.

[0054] Positive drug treatment group (IMF+5-ASA): The procedure was the same as that of the inflammation treatment group, except that the HT-29 cell line was treated with 500 μg / mL 5-aminosalicylic acid (5-ASA) for 24 h after the induction culture was completed.

[0055] IMF+ mixture group: The procedure was the same as the inflammation treatment group, except that the HT-29 cell line was treated with the mixture prepared in Comparative Example 1 at 300 μg / mL for 24 h after the induction culture.

[0056] IMF + Mixture A + System B: The procedure was the same as the inflammation treatment group, except that the HT-29 cell line was treated with Mixture A + System B prepared in Comparative Example 2 for 24 h after the induction culture was completed.

[0057] IMF+ System A + Mixture B: The procedure was the same as the inflammation treatment group, except that the HT-29 cell line was treated with System A + Mixture B prepared in Comparative Example 3 for 24 h after the induction culture was completed.

[0058] IMF+ loading co-delivery group: The operation was the same as the inflammation treatment group, except that: after the induction culture, the HT-29 cell line was treated with the exosome co-delivery composition prepared in Example 1 at a concentration of 300 μg / mL for 24 h.

[0059] 3. Result Detection After step 2 is completed, cells are collected to obtain the sample to be tested, and the following tests are performed: (1) RT-qPCR test: RNA was extracted from cells in each of the above treatment groups using an RNA kit and then reverse transcribed into cDNA. RT-qPCR was then performed to detect IL-16, IL-1β, TNF-α, and COX-2 (cyclooxygenase-2), with GAPDH as an internal reference gene. The primer sequences for the RT-qPCR are shown in Table 1. The reaction system was 20 μL: 10 μL TB Green Premix Ex Taq II (2×), 0.8 μL upstream primer (10 μmol / L), 0.8 μL downstream primer (10 μmol / L), 2 μL cDNA template, and 6.4 μL sterile deionized water. The reaction program was: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; 60℃ annealing and extension for 30 s, for a total of 40 cycles. The detection results are as follows: Figures 2-5 As shown in Table 2, Figure 2 IL-1B is the same as IL-1β. Figure 3 TNFA is the same as TNF-α.

[0060] Table 1 Primer sequence information

[0061] Table 2 Detection results of different treatment groups

[0062] IL-16, IL-1β, and TNF-α are all cellular inflammatory genes. qPCR results showed that the expression of these three inflammatory genes increased after induced inflammation, and the expression of COX-2, a gene representing intestinal inflammation, was also significantly upregulated, indicating a clear cellular inflammatory response. After induced inflammation, both the mixture (Comparative Example 1) and the exosome co-delivery composition (Example 1) showed a significant decrease in the expression of cellular inflammatory genes. Specifically, treatment with the exosome co-delivery composition described in this invention reduced the expression of IL-6, IL-1β, TNF-α, and COX-2 by 62%, 57%, 32%, and 50%, respectively, compared to the inflammatory treatment group.

[0063] (2) Immunofluorescence (IF) detection of target protein expression and localization COX-2 plays an important role in apoptosis and tumorigenesis. In ulcerative colitis, COX-2 expression is increased. Immunofluorescence was used to detect COX-2 expression, as follows: ① Cell fixation: Discard the culture medium from the cells of each treatment group above, gently wash the cells twice with pre-cooled PBS for 5 min each time; add 4% PFA fixative to each well and fix at room temperature for 15-20 min; wash with PBS 3 times for 5 min each time to remove residual fixative.

[0064] ② Blocking and primary antibody incubation: Discard PBS, add 5% BSA blocking solution to each well, and block at room temperature for 1 h to block non-specific binding; discard the blocking solution, add primary antibody diluted with 5% BSA according to the instructions, and incubate at 4℃ overnight; the next day, take out the 6-well plate, warm it to room temperature for 30 min, and wash it with PBS 3 times for 5 min each time to remove unbound primary antibody.

[0065] ③ Secondary antibody incubation and DAPI staining: Add fluorescent secondary antibody (1:500 diluted in 5% BSA), incubate at room temperature in the dark for 1 hour; rinse 3 times with PBS in the dark for 5 minutes each time to completely remove unbound secondary antibody; add DAPI staining solution, stain at room temperature in the dark for 5 minutes to stain cell nuclei; rinse 3 times with PBS in the dark for 5 minutes each time to remove excess DAPI.

[0066] ④ Mounting and Microscopic Examination: Carefully remove the slide with the cell side down using tweezers and gently attach it to a glass slide containing an anti-fluorescence quencher, avoiding air bubbles. Observe under a fluorescence microscope, setting the excitation wavelength (488 nm for Alexa Fluor 488, 594 nm for Alexa Fluor 594, and 359 nm for DAPI), acquire images, and save them. Five fields of view are randomly selected from each sample, and the fluorescence intensity of the target protein is analyzed using Image-Pro Plus software to quantitatively assess the relative protein expression level.

[0067] The experimental results showed that COX-2 fluorescence in cells increased compared to the control group after the inflammation model was constructed. Fluorescence expression decreased after using the mixture (Comparative Example 1) and the exosome co-delivery composition (Example 1), demonstrating that the expression of intestinal inflammatory response proteins decreased after use.

[0068] (3) DAO (diamine oxidase) test The samples from each group were processed according to the DAO reagent kit (purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., catalog number AC10279) instructions, including steps such as fragmentation and color development. The samples were then added to blank 96-well plates, and the absorbance was measured using a microplate reader. The data were processed, and the results are as follows: Figure 6 As shown in Table 3.

[0069] Table 3 Enzyme detection results of different groups

[0070] DAO is widely present in various organs of animals and can reflect the integrity and degree of damage to the intestinal mechanical barrier. Results showed that DAO expression was increased in the inflammation model, and the DAO content decreased by 31% compared to IMF after using the exosome co-delivery composition (Example 1).

[0071] (4) D-LA (D-lactic acid) test Cell samples collected from each group were processed, fragmented, and developed according to the instructions of the D-LA kit (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number BC5350). Samples were then added to blank 96-well plates, and absorbance was measured using a microplate reader. The data were processed, and the results are as follows: Figure 7 And as shown in Table 3 D-LA is an important indicator for assessing glucose and aerobic metabolism. In cases of intestinal inflammation, intestinal cells release more lactic acid, and lactic acid levels are commonly used to determine the extent of intestinal inflammation. In our experiments, we observed an increase in D-LA levels when the intestinal inflammation model was established. However, after using a mixture (Comparative Example 1) and an exosome co-delivery composition (Example 1), the D-LA levels decreased to a level similar to the control group.

[0072] (5) MTT (trade name: thiazolyl blue) test.

[0073] The test samples from each group were plated into 96-well flat-bottomed plates, with 5000 cells per well. After the experiment, MTT dissolution reagent was added according to the instructions of the MTT kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd., C0009S). After dissolution, the absorbance was measured using a microplate reader, and the data were processed. The results are as follows: Figure 8 As shown in Table 3.

[0074] MTT can be reduced by dehydrogenases in the body to form a crystalline, dark purple product called formazan. The stronger the cell proliferation activity, the more purple product is produced, the darker the color, and the higher the reading on the microplate reader. Experimental results showed that when cells were in an inflammatory state, the MTT readings of the groups were significantly reduced, and after using the mixture (Comparative Example 1) and the exosome co-delivery composition (Example 1), the readings recovered to levels similar to the control group.

[0075] Example 2 The exosome co-delivery composition based on milk exosomes was prepared using the same method as in Example 1. The preparation method of the milk exosomes was performed according to patent 202410629303.3, with the following steps: (1) Fresh milk (source: Chaocui Biotechnology (Xianning) Co., Ltd., batch number: 20231031-1) was filtered through a hydrophilic-lipophilic nano-interface membrane, and the clarified liquid was collected to remove fat particles from the milk; (2) The milk was then filtered through a membrane with a pore size of 100 kDa~300 kDa. Ultrafiltration was performed using a tangential flow ultrafiltration device with a kDa ultrafiltration membrane. The first concentration was carried out to remove some small molecules of protein and water. The clarified fresh milk was concentrated by 10 to 20 times. (3) The clarified filtrate of the concentrated fresh milk was centrifuged using a two-phase solution prepared by polyethylene glycol and dextran. The second concentration and preliminary purification were carried out. The exosomes were compressed into the dextran phase and the upper phase was removed. This process was repeated 1 to 2 times. At this time, the clarified exosomes were concentrated by 60 to 150 times. (4) Finally, other impurities and apoptotic bodies were removed by separating the gel column with highly cross-linked agarose as the filler. Exosomes of different particle sizes were purified. (5) The precipitate was collected by centrifugation again. The centrifugation conditions were 120,000 × g and 4°C for 90 min to obtain the milk-derived exosomes. (6) The freeze-drying operation was the same as step 1 in implementation 1 to obtain the freeze-dried milk exosome powder.

[0076] Comparative Example 4 A composition is prepared in the same way as in Example 2, except that: after obtaining the plant-derived exosome freeze-dried powder, the chitosan oligosaccharide, mannitol, and Bifidobacterium infantis freeze-dried powder from step 2, and the chitosan oligosaccharide, butyrate, and reduced glutathione from step 3 are directly mixed to obtain the composition, which is referred to as a mixture.

[0077] Comparative Example 5 A composition is prepared in the same way as in Example 2, except that in step 2, chitosan oligosaccharide, mannitol, plant-derived exosome lyophilized powder, and Bifidobacterium infantis lyophilized powder are directly mixed to obtain mixture A; the final composition is denoted as mixture A + system B.

[0078] Comparative Example 6 A composition is prepared in the same way as in Example 2, except that in step 3, chitosan oligosaccharide, butyrate and reduced glutathione are directly mixed to obtain mixture B; the resulting composition is referred to as system A + mixture B.

[0079] Application Example 2 1. Culture and passage of HT-29 cell line, the specific operation is the same as in application example 1.

[0080] 2. The establishment of the cell line inflammation model and the drug regimen are the same as in Application Example 1, with the following differences: IMF+ mixture group: After induction culture, HT-29 cell lines were treated with the mixture prepared in Comparative Example 4 at 300 μg / mL for 24 h.

[0081] IMF+ mixture A+ system B group: After the induction culture was completed, HT-29 cell line was treated with mixture A+ system B prepared by comparison 5 at 300 μg / mL for 24 h.

[0082] IMF+ System A + Mixture B: After induction culture, HT-29 cell lines were treated with IMF+ System A + Mixture B prepared in Comparative Example 6 at a concentration of 300 μg / mL for 24 h.

[0083] IMF+ loading co-delivery group: After induction culture, HT-29 cell lines were treated for 24 h with the exosome co-delivery composition prepared in Example 2 at a concentration of 300 μg / mL.

[0084] 3. Result detection: The detection method is the same as in application example 1, except that (2) immunofluorescence detection is not performed. The detection results are as follows: Figures 9-12 As shown in Tables 4 and 5, Figure 9 The B-cell label records that IL-1B is IL-1β.

[0085] Table 4 Detection results of different treatment groups

[0086] Table 5 Enzyme detection results of different groups

[0087] The results showed that after inducing an inflammatory response, both the mixture (Comparative Example 4) and the exosome co-delivery composition (Example 2) showed a significant reduction in the expression of inflammatory genes in cells. Specifically, after treatment with the exosome co-delivery composition of the present invention, the expression of IL-6, IL-1β, TNF-α and COX-2 was reduced by 62%, 51%, 34% and 55%, respectively, compared with the inflammation treatment group.

[0088] The results showed that the DAO and D-LA contents increased in the inflammation model. After using the mixture (Comparative Example 4) and the exosome co-delivery composition (Example 2), the DAO contents decreased and the D-LA contents decreased to a level similar to that of the control group.

[0089] The MTT assay results showed that when cells were in an inflammatory state, the MTT readings of the groups were significantly reduced, but recovered to levels similar to the control group after using the mixture (Comparative Example 4) and the exosome co-delivery composition (Example 2).

[0090] As discussed above, system A of the exosome co-delivery composition prepared using the method of this invention utilizes mannitol-chitosan oligosaccharide to encapsulate the active ingredient exosomes and probiotics, constructing a chitosan oligosaccharide-mannitol nano-encapsulation system to improve the survival rate and structural stability of the active ingredient. In system B, chitosan oligosaccharide forms a three-dimensional nanogel network through ionic cross-linking, encapsulating butyrate and glutathione to form a stable nanogel matrix, constructing a chitosan oligosaccharide-glutathione-butyrate nanogel system. These two systems synergistically achieve multi-pathway protection, targeted release, and synergistic effects of probiotics, exosomes, butyrate, and glutathione, thereby producing significant comprehensive promoting effects in intestinal barrier repair, flora reconstruction, anti-inflammatory and antioxidant effects, and energy supply.

[0091] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An exosome co-delivery composition, characterized in that, The exosome co-delivery composition comprises system A and system B; By weight, system A comprises 1-30 parts of exosome preparation, 1-60 parts of chitosan oligosaccharide-mannitol, and 1-30 parts of probiotics; The chitosan oligosaccharide-mannitol encapsulates exosomes and probiotics; By mass, system B comprises 1-60 parts of chitosan oligosaccharide, 1-20 parts of glutathione, and 1-20 parts of butyrate; the chitosan oligosaccharide encapsulates glutathione and butyrate.

2. The exosome co-delivery composition according to claim 1, characterized in that, The mass ratio of system A to system B is 1:1 to 10:

1.

3. The exosome co-delivery composition according to claim 1, characterized in that, The mass ratio of chitosan oligosaccharide to mannitol in the chitosan oligosaccharide-mannitol mixture is 10:1 to 20:

1.

4. A method for preparing the exosome co-delivery composition according to any one of claims 1 to 3, characterized in that, Includes the following steps: Exosome preparations, probiotics and water are mixed to obtain a mixture. The mixture is then added dropwise to a chitosan oligosaccharide-mannitol solution for self-assembly to obtain system A. Butyrate solution and glutathione solution were added dropwise to chitosan oligosaccharide solution to induce self-assembly, thus obtaining system B. The exosome co-delivery composition is obtained by mixing system A and system B.

5. The preparation method according to claim 4, characterized in that, The concentration of chitosan oligosaccharide in the chitosan oligosaccharide-mannitol solution is 10-20 mg / mL, and the concentration of mannitol is 1-2 mg / mL.

6. The preparation method according to claim 5, characterized in that, After adding the mixture dropwise to the chitosan oligosaccharide-mannitol solution for self-assembly, the process further includes adjusting the chitosan oligosaccharide concentration to 1~5 mg / mL.

7. The preparation method according to claim 4, characterized in that, The concentration of chitosan oligosaccharide in the chitosan oligosaccharide solution is 10~20 mg / mL.

8. The preparation method according to claim 7, characterized in that, After adding butyrate solution and glutathione solution dropwise to chitosan oligosaccharide solution for self-assembly, the process also includes adjusting the chitosan oligosaccharide concentration to 1~5 mg / mL.

9. The preparation method according to claim 4, characterized in that, The method for self-assembly includes stirring; the stirring speed is 250~1000 rpm; the stirring time is 2~4 h.

10. The use of the exosome co-delivery composition according to any one of claims 1 to 3 or the exosome co-delivery composition prepared by the preparation method according to any one of claims 4 to 9 in the preparation of a medicament for treating intestinal inflammation.

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