Application of astaxanthin vesicles in preventing and relieving colon cancer

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of astaxanthin vesicles in preventing and relieving colon cancer, and the astaxanthin vesicles have at least one of the following applications: oxidation resistance; anti-inflammation; the antibacterial effect is achieved; the phagocytic ability of macrophages is promoted; the intestinal health is improved; tumor cell proliferation is resisted; preventing and / or treating tumors; the preparation method of the embedding body comprises the following steps: S1, mixing an iRGD peptide ligand, a hyaluronic acid ligand and a solution containing vesicles to obtain a carrier; s2, enabling the carrier to be in contact with astaxanthin, so as to obtain the embedding body. According to the application, the astaxanthin is embedded into the targeting carrier, so that the astaxanthin is effectively protected from being influenced by an in-vivo environment, and meanwhile, the astaxanthin is accurately targeted to target parts such as macrophages in a tumor microenvironment, so that the concentration of a medicine at a diseased region is improved, the side effect on normal tissues is reduced, and the curative effect of the medicine is improved. The astaxanthin can safely and efficiently reach a target site and exert multiple functions of oxidation, anti-inflammation, antibiosis and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the use of astaxanthin vesicles in the prevention and alleviation of colon cancer. Background Art

[0002] As a potent antioxidant, astaxanthin (AXT) resists oxidative damage of diseased cells and regulates the release of inflammatory mediators through its antioxidant properties, thus showing an important inhibitory effect on tumor cells in the treatment of various tumors such as colon cancer, breast cancer, and cervical cancer. However, due to its instability in vivo, easy degradation, and poor water solubility, it greatly affects its ability to reach the tumor site and exert its therapeutic effect.

[0003] Currently, various delivery systems such as emulsions, liposomes, complexes, and nanoparticles have been proven to improve the stability and bioavailability of astaxanthin. These carriers mainly include solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), microcapsules, etc. Although such delivery systems have made certain progress in improving the stability and bioavailability of astaxanthin, there are still certain problems. For example, the existing astaxanthin delivery systems lack sufficient targeting, resulting in inaccurate drug distribution in the body, thus affecting the therapeutic effect of astaxanthin drugs; in addition, the preparation processes of some astaxanthin delivery systems are complex and difficult to meet the requirements of industrial production. For example, a dual-targeted astaxanthin nanoparticle (Application No.: CN117337970A) uses a polymer formed by the Maillard reaction between separated protein and mannose to modify triphenylphosphonium bromide, enabling astaxanthin to target and accumulate at macrophages in the intestinal inflammation site. However, it has problems such as a complex preparation process; the preparation method of a novel anti-inflammatory astaxanthin derivative (Application No.: CN114213438A) uses free astaxanthin as the main backbone, esterifies it with succinic anhydride and then grafts ROS-responsive functional groups to obtain an astaxanthin derivative, effectively improving the water dispersibility of astaxanthin and having ROS responsiveness, making it have a better anti-inflammatory effect in vivo. However, its stability and biocompatibility still need to be further investigated.

[0004] Therefore, it is necessary to develop an embedding body with biosafety, a simple preparation method, and the ability to accurately deliver astaxanthin to the target site, so as to achieve a safer, more stable, efficient, and targeted drug delivery of astaxanthin. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent. To this end, the present invention provides the use of astaxanthin vesicles in the prevention and alleviation of colon cancer. The use of the present invention, by encapsulating astaxanthin into a targeting carrier, while effectively protecting astaxanthin from the influence of the in vivo environment, precisely targets astaxanthin to target sites such as macrophages in the tumor microenvironment. Thus, the concentration of the drug at the lesion site is increased, and the side effects on normal tissues are reduced, enabling astaxanthin to safely and efficiently reach the target site and exert various functions such as oxidation, anti-inflammation, and anti-bacterial.

[0006] The present invention proposes the use of an inclusion body in the preparation of a drug. According to an embodiment of the present invention, the drug has at least one of the following uses: antioxidant; anti-inflammatory; anti-bacterial; promoting the phagocytic ability of macrophages; improving intestinal health; anti-tumor cell proliferation; preventing and / or treating colitis. Among them, the method for preparing the inclusion body includes: S1: Mixing an iRGD peptide ligand, a hyaluronic acid ligand with a solution containing vesicles to obtain a carrier; S2: Contacting the carrier with astaxanthin to obtain the inclusion body. According to the use of the embodiment of the present invention, by encapsulating astaxanthin into a targeting carrier, while enabling it to reach target sites such as intestinal epithelial cells in a complete form, effectively targeting it to macrophages in the tumor microenvironment with the help of the iRGD peptide ligand and the hyaluronic acid ligand, and improving the ability of macrophages to uptake astaxanthin. Thus, the concentration of the drug at the lesion site is increased, and the side effects on normal tissues are reduced, enabling astaxanthin to safely and efficiently reach the target site and exert various functions such as oxidation, anti-inflammation, and anti-bacterial.

[0007] According to an embodiment of the present invention, the above uses may further have the following additional technical features:

[0008] According to an embodiment of the present invention, the mixing is carried out in an organic solvent.

[0009] According to an embodiment of the present invention, the organic solvent includes one or more of ethanol, methanol, acetone, and ethyl acetate.

[0010] According to an embodiment of the present invention, the working concentration of the iRGD peptide ligand is 0.1 - 0.5 g / L, the working concentration of the hyaluronic acid ligand is 0.1 - 10 g / L, and the working concentration of the vesicles is 1 - 100 g / L.

[0011] According to an embodiment of the present invention, the working concentration of the carrier is 40 - 160 g / L, and the working concentration of astaxanthin is 0.5 - 10 g / L.

[0012] According to an embodiment of the present invention, the vesicles are derived from one or more of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, and Streptococcus mutans.

[0013] According to a preferred embodiment of the present invention, the vesicles are derived from Lactobacillus plantarum.

[0014] According to an embodiment of the present invention, the method for preparing the iRGD peptide ligand comprises: contacting the iRGD peptide with a polyethylene glycolylated lipid containing a carboxyl functional group to form the iRGD peptide ligand.

[0015] According to an embodiment of the present invention, the polyethylene glycolylated lipid containing a carboxyl functional group is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol 2000.

[0016] According to an embodiment of the present invention, the mass ratio of the iRGD peptide to the polyethylene glycolylated lipid containing a carboxyl functional group is 1:(0.5 - 10).

[0017] According to an embodiment of the present invention, the method for preparing the hyaluronic acid ligand comprises: contacting hyaluronic acid with a polyethylene glycolylated lipid containing an amino functional group to form the hyaluronic acid ligand.

[0018] According to an embodiment of the present invention, the polyethylene glycolylated lipid containing an amino functional group is N-distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amino.

[0019] According to an embodiment of the present invention, the mass ratio of the hyaluronic acid to the polyethylene glycolylated lipid containing an amino functional group is (5 - 20):1.

[0020] According to an embodiment of the present invention, the tumor includes one or more of colon cancer, bladder cancer, liver cancer, and oral cancer.

[0021] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0023] Figure 1 It is a result graph of the body weight change and health status change of each group of mice in Example 2 of the present invention. Among them, (A) is the result graph of the disease activity index of each group of mice when drinking 2% DSS water in the first round, (B) is the result graph of the disease activity index of each group of mice when drinking DSS water in the second round, and (C) is the result graph of the body weight change of each group of mice;

[0024] Figure 2This is the result graph of the number and volume of colon tumors in each group of mice in Example 2 of the present invention. Among them, (A) is the result graph of the volume of colon tumors in each group of mice, (B) is the result graph of the number of colon tumors in each group of mice, * represents p < 0.05, ** represents p < 0.01, and ns represents no significance;

[0025] Figure 3 This is the result graph of H&E staining and Alcian blue staining of colon tissues of each group of mice in Example 2 of the present invention. Among them, (A) is the H&E staining result of colon tissues of mice in the Control group, (B) is the H&E staining result of colon tissues of mice in the Model group, (C) is the H&E staining result of colon tissues of mice in the ATX group, (D) is the H&E staining result of colon tissues of mice in the ATX-EVs group, (E) is the H&E staining result of colon tissues of mice in the Hi-EVs group, (F) is the Alcian blue staining result of colon tissues of mice in the Control group, (G) is the Alcian blue staining result of colon tissues of mice in the Model group, (H) is the Alcian blue staining result of colon tissues of mice in the ATX group, (I) is the Alcian blue staining result of colon tissues of mice in the ATX-EVs group, (J) is the Alcian blue staining result of colon tissues of mice in the Hi-EVs group;

[0026] Figure 4 This is the result graph of histopathological scores and goblet cell counts of colon tissues of each group of mice in Example 2 of the present invention. Among them, (A) is the result graph of histopathological scores of colon tissues of each group of mice, (B) is the result graph of goblet cell counts of colon tissues of each group of mice, * represents p < 0.05, ** represents p < 0.01, and **** represents p < 0.0001;

[0027] Figure 5 This is the result graph of the expression levels of inflammatory factors in colon tissues of each group of mice in Example 2 of the present invention. Among them, (A) is the result graph of the expression level of TNF-α in colon tissues of each group of mice, (B) is the result graph of the expression level of IL-1β in colon tissues of each group of mice, (C) is the result graph of the expression level of IL-6 in colon tissues of each group of mice, (D) is the result graph of the expression level of Cox-2 in colon tissues of each group of mice, * represents p < 0.05, ** represents p < 0.01;

[0028] Figure 6This is the result graph of the Nile red fluorescence intensity measured for macrophage uptake in Example 3 of the present invention. (A) In the Control group, from left to right are the result graph of DAPI-labeled macrophage cell nuclei, the result graph of DIO-labeled macrophage cell membranes, the Nile red fluorescence result graph, the result graph after the overlap of each label, and the enlarged schematic diagram of a single macrophage. (B) In the Hi-EVs group, from left to right are the result graph of DAPI-labeled macrophage cell nuclei, the result graph of DIO-labeled macrophage cell membranes, the Nile red fluorescence result graph, the result graph after the overlap of each label, and the enlarged schematic diagram of a single macrophage. (C) In the Nile red treatment group, from left to right are the result graph of DAPI-labeled macrophage cell nuclei, the result graph of DIO-labeled macrophage cell membranes, the Nile red fluorescence result graph, the result graph after the overlap of each label, and the enlarged schematic diagram of a single macrophage. (D) In the Nile red-EVs treatment group, from left to right are the result graph of DAPI-labeled macrophage cell nuclei, the result graph of DIO-labeled macrophage cell membranes, the Nile red fluorescence result graph, the result graph after the overlap of each label, and the enlarged schematic diagram of a single macrophage. (E) is the result graph of the statistical result of the red fluorescence intensity in the Nile red treatment group and the Nile red-EVs treatment group, **** indicates p < 0.0001;

[0029] Figure 7 This is the result graph of the fluorescence intensity measured for astaxanthin promoting macrophage phagocytosis of cell debris in Example 5 of the present invention, ** indicates p < 0.01, **** indicates p < 0.0001;

[0030] Figure 8 This is the result graph of the laser confocal observation of macrophages in each group in Example 6 of the present invention; among them, (A) for macrophages, from left to right are the result graph of DAPI-labeled macrophage cell nuclei, the result graph of DIO-labeled macrophage cell membranes, the fluorescence result graph of Nile red-labeled debris, and the result graph after the overlap of each label. (B) For macrophages + astaxanthin, from left to right are the result graph of DAPI-labeled macrophage cell nuclei, the result graph of DIO-labeled macrophage cell membranes, the fluorescence result graph of Nile red-labeled debris, and the result graph after the overlap of each label. (C) For macrophages + cell debris, from left to right are the result graph of DAPI-labeled macrophage cell nuclei, the result graph of DIO-labeled macrophage cell membranes, the fluorescence result graph of Nile red-labeled debris, and the result graph after the overlap of each label. (D) For macrophages + astaxanthin + cell debris, from left to right are the result graph of DAPI-labeled macrophage cell nuclei, the result graph of DIO-labeled macrophage cell membranes, the fluorescence result graph of Nile red-labeled debris, and the result graph after the overlap of each label;

[0031] Figure 9 This is the result graph of the effect of astaxanthin on the fluorescence intensity of cell debris in Example 6 of the present invention, ** indicates p < 0.01;

[0032] Figure 10 It is a graph showing the changes in tumor volume of each group of mice in Example 7 of the present invention;

[0033] Figure 11 It is a graph showing the changes in tumor volume and weight of each group of mice sacrificed in Example 7 of the present invention. Among them, (A) is the graph of the changes in tumor volume of each group of sacrificed mice, and (B) is the graph of the changes in tumor weight of each group of sacrificed mice. * represents p < 0.05, and ** represents p < 0.01. Detailed implementation manners

[0034] The embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0035] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise stated, the meaning of "a plurality" is two or more.

[0036] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0037] In this article, the term "comprising" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0038] In this article, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where the events or conditions occur and the cases where the events or conditions do not occur.

[0039] Terms and definitions

[0040] In this article, the "iRGD peptide ligand" is a class of polypeptides that can specifically recognize the tumor microenvironment. By recognizing the tumor microenvironment, it helps to achieve the targeted delivery and penetration of the carrier into the tumor.

[0041] In this text, "HA ligand" refers to a hyaluronic acid ligand. Hyaluronic acid is a natural anionic polysaccharide that can interact with the CD44 surface receptor on inflammatory macrophages to increase macrophage uptake, facilitating the targeted delivery of the carrier.

[0042] In this text, "vesicle" refers to a cell membrane structure formed by self-assembly of the cell membrane extracted from Lactobacillus plantarum, which can embed the payload into its internal hydrophobic domain, improving the stability of the payload and promoting its absorption and utilization.

[0043] In this text, "AOM" is azoxymethane, a chemical carcinogen commonly used to induce the development of colon cancer in experimental animals to establish an animal model of colon cancer, assisting in the study of the pathogenesis, preventive measures, and treatment methods of colon cancer.

[0044] Use

[0045] The present invention provides a use of an inclusion body in the preparation of a drug. According to an embodiment of the present invention, the drug has at least one of the following uses: antioxidant; anti-inflammatory; antibacterial; promoting the phagocytic ability of macrophages; improving intestinal health; anti-tumor cell proliferation; preventing and / or treating colitis; wherein, the method for preparing the inclusion body includes: S1: mixing an iRGD peptide ligand, a hyaluronic acid ligand with a solution containing vesicles to obtain a carrier; S2: contacting the carrier with astaxanthin to obtain the inclusion body. According to the use of the embodiment of the present invention, by embedding astaxanthin into a targeted carrier, enabling it to reach target sites such as intestinal epithelial cells in a complete form, while effectively targeting macrophages in the tumor microenvironment with the help of the iRGD peptide ligand and the hyaluronic acid ligand, and improving the ability of macrophages to uptake astaxanthin. Thus, the concentration of the drug at the lesion site is increased, and the side effects on normal tissues are reduced, enabling astaxanthin to safely and efficiently reach the target site and exert various functions such as oxidation, anti-inflammation, and antibacterial.

[0046] Exemplarily, astaxanthin, as a potent natural antioxidant, can effectively scavenge free radicals in the body, reduce the damage of oxidative stress response to cells. By delivering astaxanthin drugs through the embedding body of the present invention, while protecting astaxanthin from oxidation in the body environment, it can ensure that it still maintains high antioxidant activity when reaching the target site, thereby exerting the antioxidant ability of astaxanthin; secondly, relevant research shows that astaxanthin has significant anti-inflammatory properties, can regulate the release of inflammatory mediators, and inhibit inflammatory reactions. By delivering astaxanthin drugs through the embedding body of the present invention, astaxanthin can be accurately delivered to the inflammatory site, increase the concentration of the drug at the lesion site, enhance the anti-inflammatory effect, and at the same time reduce the impact on normal tissues, thereby exerting the anti-inflammatory ability of astaxanthin; in addition, relevant research shows that astaxanthin has significant anti-tumor properties, can inhibit the growth and metastasis of tumor cells through various mechanisms such as inducing apoptosis, inhibiting the cell cycle process, and blocking the signal transduction pathway of tumor cells. By delivering astaxanthin drugs through the embedding body of the present invention, while enabling it to reach target sites such as intestinal epithelial cells in a complete form, it can effectively target macrophages in the tumor microenvironment with the help of iRGD peptide ligand and hyaluronic acid ligand, improve the concentration of the drug at the tumor site and the ability of macrophages to uptake astaxanthin, and thereby efficiently achieve the effects of astaxanthin drugs in anti-tumor cell proliferation and preventing / treating tumors, etc.

[0047] According to an embodiment of the present invention, the mixing is carried out in an organic solvent. Thus, carrying out the mixing in an organic solvent enables astaxanthin, the iRGD peptide ligand, the hyaluronic acid ligand and the vesicles to fully contact, and efficiently prepares a targeting carrier, thereby enhancing the embedding efficiency of astaxanthin.

[0048] According to an embodiment of the present invention, the organic solvent includes one or more of ethanol, methanol, acetone and ethyl acetate. Thus, the contact between the iRGD peptide ligand, the hyaluronic acid ligand and the vesicles can be carried out in a variety of organic solvents, which is suitable for different application scenarios.

[0049] According to an embodiment of the present invention, the working concentration of the iRGD peptide ligand is 0.1 - 0.5 g / L, the working concentration of the hyaluronic acid ligand is 0.1 - 10 g / L, and the working concentration of the vesicles is 1 - 100 g / L. Thus, by adjusting the working concentrations of the iRGD peptide ligand, hyaluronic acid ligand, and vesicles, a green, safe, stable, and uniform targeted carrier can be prepared according to specific application requirements. Exemplarily, the working concentrations of the iRGD peptide ligand are 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, preferably 0.2 - 0.5 g / L, more preferably 0.5 g / L; the working concentrations of the hyaluronic acid ligand are 0.1 g / L, 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, preferably 5 - 10 g / L, more preferably 10 g / L; the working concentrations of the vesicles are 1 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, preferably 20 - 80 g / L, more preferably 60 g / L.

[0050] According to an embodiment of the present invention, the working concentration of the carrier is 40 - 160 g / L, and the working concentration of astaxanthin is 0.5 - 10 g / L. Thus, by adjusting the working concentrations of the carrier and astaxanthin, the embedding efficiency of astaxanthin is further improved, and an astaxanthin embedding body with better efficacy is prepared, thereby achieving efficient targeted delivery of astaxanthin. Exemplarily, the working concentrations of the carrier are 40 g / L, 60 g / L, 80 g / L, 100 g / L, 120 g / L, 140 g / L, 160 g / L, preferably 80 - 140 g / L, more preferably 120 g / L; the working concentrations of astaxanthin are 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, preferably 0.5 - 5 g / L, more preferably 1 g / L.

[0051] According to an embodiment of the present invention, the vesicles are derived from one or more of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, and Streptococcus mutans. Thus, the vesicles are derived from a variety of microorganisms that can produce extracellular vesicles with self-assembly ability, are suitable for different application scenarios, and increase the flexibility and adaptability of the preparation of the astaxanthin embedding body of the present invention.

[0052] According to a preferred embodiment of the present invention, the vesicles are derived from Lactobacillus plantarum. According to a preferred embodiment of the present invention, the vesicles are derived from Lactobacillus plantarum, which is a microorganism listed by the European Food Safety Authority as having a qualified safety status. The extracellular vesicles are formed by cell membrane fragments. The inventors used physical methods such as ultracentrifugation to separate the cell membrane fragments from the cell organelles, and obtained the vesicles required for preparing the inclusion bodies of the present invention.

[0053] According to an embodiment of the present invention, the method for preparing the iRGD peptide ligand includes: contacting the iRGD peptide with a polyethylene glycolylated lipid containing a carboxyl functional group to form the iRGD peptide ligand. Thus, the iRGD peptide contains an amino group, which covalently binds to the polyethylene glycolylated lipid containing a carboxyl functional group to form a stable amide bond, thereby preparing a stable and amphiphilic iRGD peptide ligand, and further laying a structural foundation for preparing the inclusion body.

[0054] According to an embodiment of the present invention, the polyethylene glycolylated lipid containing a carboxyl functional group is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol 2000. According to an embodiment of the present invention, the polyethylene glycolylated lipid containing a carboxyl functional group may be 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol 2000, which can covalently bind to the amino group in the iRGD peptide to form a stable amide bond, thereby obtaining a stable and amphiphilic iRGD peptide ligand.

[0055] According to an embodiment of the present invention, the mass ratio of the iRGD peptide to the polyethylene glycolylated lipid containing a carboxyl functional group is 1:(0.5 - 10). Exemplarily, the mass ratio of the iRGD peptide to the polyethylene glycolylated lipid containing a carboxyl functional group is 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, preferably 1:(0.5 - 5), more preferably 1:1; thus, by adjusting the mass ratio of the two, the efficient preparation of the iRGD peptide ligand is achieved.

[0056] According to an embodiment of the present invention, the method for preparing the hyaluronic acid ligand includes: contacting the hyaluronic acid with a polyethylene glycolylated lipid containing an amino functional group to form the hyaluronic acid ligand. Thus, the hyaluronic acid contains a carboxyl group, which covalently binds to the polyethylene glycolylated lipid containing an amino functional group to form a stable amide bond, thereby preparing a stable hyaluronic acid ligand.

[0057] According to an embodiment of the present invention, the amino-functionalized polyethylene glycolylated lipid is N-distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amino. According to an embodiment of the present invention, the amino-functionalized polyethylene glycolylated lipid may be N-distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amino, which can covalently bind to the carboxyl group in hyaluronic acid to form a stable amide bond, thereby obtaining a stable hyaluronic acid ligand.

[0058] According to an embodiment of the present invention, the mass ratio of the hyaluronic acid to the amino-functionalized PEGylated phospholipid (the polyethylene glycolylated lipid containing an amino functional group) is (5-20):1. Exemplarily, the mass ratio of the hyaluronic acid to the amino-functionalized PEGylated phospholipid (the polyethylene glycolylated lipid containing an amino functional group) is 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, preferably (15-20):1, more preferably 20:1; thus, by adjusting the mass ratio of the two, efficient preparation of the hyaluronic acid ligand is achieved.

[0059] According to an embodiment of the present invention, the tumor includes one or more of colon cancer, bladder cancer, liver cancer, and oral cancer. Thus, through astaxanthin in the inclusion body, the anti-tumor cell proliferation and anti-tumor effects of astaxanthin drugs against different tumor cells are achieved. Exemplarily, during the occurrence and development of colon cancer, oxidative stress is a key factor. As a potent antioxidant, astaxanthin can effectively scavenge free radicals in the body and reduce the damage of oxidative stress to cells; secondly, chronic inflammation is one of the important inducing factors of colon cancer. Astaxanthin can reduce the inflammatory response of the colonic mucosa by inhibiting the expression of inflammatory factors, thereby reducing the risk of inflammation transforming into cancer; in addition, astaxanthin can significantly enhance the phagocytic ability of macrophages, enabling them to more effectively scavenge cancer cells and cell debris; thus, by delivering astaxanthin drugs through the inclusion body of the present invention, while protecting the biological activity of astaxanthin by encapsulating it in vesicles, it can be effectively targeted to macrophages in the tumor microenvironment with the help of iRGD peptide ligand and hyaluronic acid ligand, and improve the ability of macrophages to uptake astaxanthin, thereby achieving precise delivery of astaxanthin drugs, and further exerting the functions of astaxanthin such as antioxidant, anti-inflammatory, and promoting macrophage phagocytic ability to inhibit the growth of colon cancer cells.

[0060] Unless otherwise specified, the formula of the MRS liquid medium used in the embodiments of the present invention is:

[0061] Dissolve the following components in 1 L of distilled water: 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 2 g of dipotassium hydrogen phosphate, 2 g of diammonium citrate, 5 g of sodium acetate, 20 g of glucose, 1 mL of Tween 80, 0.5 g of magnesium sulfate, 0.25 g of manganese sulfate; adjust the pH value to 6.2 - 6.4, and autoclave (101 Kpa, 121 °C) for 15 min.

[0062] Unless otherwise specified, the formula of the MRS agar medium used in the examples of the present invention is as follows:

[0063] Dissolve the following components in 1 L of distilled water: 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 2 g of dipotassium hydrogen phosphate, 2 g of diammonium citrate, 5 g of sodium acetate, 20 g of glucose, 1 mL of Tween 80, 0.5 g of magnesium sulfate, 0.25 g of manganese sulfate, 15 g of agar powder; adjust the pH value to 6.2 - 6.4, and autoclave (101 Kpa, 121 °C) for 15 min.

[0064] The solution of the present invention will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specifically noted in the examples, the techniques or conditions described in the literature in this field or according to the product instructions are followed. For reagents or instruments not indicated by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0065] Example 1: Preparation of inclusion bodies

[0066] 1. Cultivation of Lactobacillus plantarum

[0067] Add the freeze-dried powder of Lactobacillus plantarum (purchased from Suzhou Shienkang Biotechnology Co., Ltd.) into the sterile MRS liquid medium, and incubate at 37 °C for 36 h to obtain the Lactobacillus plantarum incubation solution.

[0068] After gradient dilution of the Lactobacillus plantarum incubation solution, spread 0.1 mL of the diluted suspension onto the MRS agar medium, and culture at 37 ± 1 °C for 46 - 50 h, and perform colony counting to confirm the yield of Lactobacillus plantarum cell membrane vesicles obtained in the subsequent preparation.

[0069] 2. Collection of protoplast precipitation

[0070] Centrifuge the Lactobacillus plantarum incubation solution at 8000 g for 15 min to collect Lactobacillus plantarum cells, then treat the Lactobacillus plantarum cells with lysozyme at 37 °C for 24 h (to remove the cell wall and part of the organelles of Lactobacillus plantarum cells), and then centrifuge at 3500 g for 10 min to obtain the protoplast precipitation.

[0071] 3. Preparation of Lactobacillus plantarum cell membrane vesicles

[0072] The protoplast precipitate was washed twice with PBS and resuspended in PBS. It was disrupted by ultrasonic treatment in an ice bath at 24 kHz for 30 min. Unbroken protoplasts were removed by centrifugation at 3500 g for 10 min. After centrifugation at 20000 g for 30 min (to remove other organelles), the supernatant containing only cell membrane fragments was obtained. The supernatant was ultracentrifuged at 100000 g for 1 h to obtain a membrane fragment precipitate. The membrane fragment precipitate was redispersed in sterile PBS, treated at an ultrasonic power of 35 kHz for 30 min, and then circulated twice under the condition of 100 MPa by dynamic microfluidics to obtain Lactobacillus plantarum cell membrane vesicles, which were stored at -80 °C for later use.

[0073] 4. DSPE-PEG 2000 Synthesis of -HA targeting ligand

[0074] 20 g of hyaluronic acid (HA) was completely dissolved in 50 mL of distilled water. 40 μmol of NHS and 50 μmol of EDC were added to activate the hydroxyl groups, and the mixture was stirred at 4 °C for 4 h to obtain a mixed solution. The mixed solution was added to 50 mL of an aqueous solution containing 1 g of DSPE-PEG 2000 -NH 2 and stirred for 12 h to obtain a viscous solution. The viscous solution was transferred to a dialysis bag and dialyzed at room temperature for 48 h, and then obtained DSPE-PEG 2000 -HA targeting ligand by freeze-drying.

[0075] 5. DSPE-PEG 2000 Synthesis of -iRGD peptide targeting ligand

[0076] 0.05 g of iRGD peptide (iRGD) was completely dissolved in 50 mL of distilled water. 40 μmol of NHS and 50 μmol of EDC were added to activate the amino groups, and the mixture was stirred at 4 °C for 4 h to obtain a mixed solution. The mixed solution was mixed with 50 mL of an aqueous solution containing 0.05 g of DSPE-PEG 2000 -COOH and stirred for 12 h to obtain a viscous solution. The viscous solution was transferred to a dialysis bag and dialyzed at room temperature for 48 h, and then obtained DSPE-PEG 2000 -iRGD peptide targeting ligand by freeze-drying.

[0077] 6. Preparation of hyaluronic acid-iRGD blank vesicles

[0078] The Lactobacillus plantarum cell membrane vesicles prepared in step 3, the DSPE-PEG 2000 -HA targeting ligand prepared in step 4, and the DSPE-PEG 2000-iRGD targeting ligand was mixed, and the mixing conditions of the three were as follows: DSPE-PEG 2000 -HA targeting ligand, DSPE-PEG 2000 -The mass concentration ratio of iRGD targeting ligand and vesicles was 1:0.5:60; after mixing, it was ultrasonically treated in an ice bath for 30 min, and then centrifuged at a centrifugal force of 10000 g for 30 min to remove the unreacted membrane fragments, obtaining the hyaluronic acid-iRGD blank vesicle solution, that is, the hyaluronic acid-iRGD blank vesicle, which was stored at -80 °C for later use.

[0079] 7. Preparation of inclusion bodies

[0080] The hyaluronic acid-iRGD blank vesicles prepared in step 6 and astaxanthin dissolved in absolute ethanol (purchased from Shanghai Macklin Biochemical Co., Ltd.) were mixed at a mass ratio of 1:120 (astaxanthin: hyaluronic acid-iRGD blank vesicles), ultrasonically treated in an ice bath for 30 min, centrifuged at 10000 g for 30 min to remove the unentrapped astaxanthin, and then centrifuged at 100000 g for 60 min to remove ethanol to obtain a precipitate. The precipitate was redispersed in sterile PBS to obtain hyaluronic acid-iRGD peptide astaxanthin vesicles, that is, the astaxanthin vesicle system, which was stored at -80 °C for later use.

[0081] Example 2: Establishment of a mouse model of colon cancer and detection of related indicators

[0082] 1. Establishment of a C57BL / 6 mouse model of colon cancer induced by AOM / DSS

[0083] The inventors established a C57BL / 6 mouse model of colon cancer induced by AOM / DSS, and the specific protocol was as follows:

[0084] Thirty 6-week-old C57BL / 6 male mice with a body weight of 20 - 25 g were randomly divided into 5 groups (n = 6), and were respectively named the control group (Control), the model group (Model), the astaxanthin group (AXT), the astaxanthin vesicle system group (AXT-EVs), and the blank vesicle group (Hi-EVs), with 6 mice in each group. The feeding environment of each mouse was a specific pathogen-free room with a constant temperature (22 ± 1 °C), light / dark cycle (12 h / 12 h), and humidity (40 - 60%); during the experiment, the mice were allowed to obtain water and food freely; 2 weeks before the official start of the experiment, each mouse was pre-intervened with an astaxanthin / astaxanthin vesicle delivery system (astaxanthin was purchased from Shanghai Macklin Biochemical Co., Ltd.) (the start time of pre-intervention was recorded as the 1st week), and the experiment was carried out for 10 weeks. The feeding conditions of the mice in each group during the experiment were:

[0085] (1) Control group: Normal drinking water;

[0086] (2) Model group: Inject AOM (intraperitoneal injection at 10 mg / kg·bw) in the 3rd week, followed by 1 week of 2% DSS water and 2 weeks of normal drinking water. This process is repeated 2 rounds;

[0087] (3) AXT group: Inject AOM (intraperitoneal injection at 10 mg / kg·bw) in the 3rd week, followed by 1 week of 2% DSS water and 2 weeks of normal drinking water. This process is repeated 2 rounds. During the experiment, all mice in this group are intervened by intragastric administration of astaxanthin (40 μg astaxanthin / day);

[0088] (4) AXT-EVs group: Inject AOM (intraperitoneal injection at 10 mg / kg·bw) in the 3rd week, followed by 1 week of 2% DSS water and 2 weeks of normal drinking water. This process is repeated 2 rounds. During the experiment, all mice in this group are intervened by intragastric administration of astaxanthin vesicle system (40 μg astaxanthin / day);

[0089] (5) Hi-EVs group: Inject AOM (intraperitoneal injection at 10 mg / kg·bw) in the 3rd week, followed by 1 week of 2% DSS water and 2 weeks of normal drinking water. This process is repeated 2 rounds. During the experiment, all mice in this group are intervened by intragastric administration of hyaluronic acid-iRGD blank vesicles (40 μg astaxanthin / day).

[0090] 2. Detection of physiological indexes of colon cancer mice

[0091] (1) Detection of changes in mouse body weight

[0092] During the experiment, observe and record the body weight and disease activities of mice every day until the 10th week.

[0093] The results of changes in body weight and health status of mice in each group are shown in Figure 1 .

[0094] The results show that: The body weight of mice in the Model group decreased significantly during the intervention with 2% DSS water; after intervention with astaxanthin and astaxanthin vesicles, the body weight of mice in the AXT group and the AXT-EVs group decreased more slowly, especially in the AXT-EVs group.

[0095] The above results indicate that: The astaxanthin vesicle system can effectively alleviate the decrease in mouse body weight.

[0096] (2) Detection of the number and volume of colon tumors in mice

[0097] In the 10th week of the experiment, sacrifice each mouse and take out the colon, observe and record the number and volume of colon tumors.

[0098] The results of the number and volume of colon tumors in mice in each group are shown in Figure 2 .

[0099] The results showed that: compared with the Model group, the AXT group had a tendency to inhibit tumorigenesis, but there was no significant difference compared with the Model group (p > 0.05); while in the mice of the AXT-EVs group, no generation of colon tumors was found in the colon.

[0100] The above results indicate that: the astaxanthin vesicle system can accurately target the colon and effectively inhibit the growth of colon tumors.

[0101] (3) Histopathological staining

[0102] At the 10th week of the experiment, each mouse was sacrificed and the colon tissue was taken out, fixed, embedded, sectioned, and the sections were stained with H&E (hematoxylin-eosin) and Alcian blue, and observed and photographed using a microscope; and histopathological scoring was performed according to the staining results, and the scoring criteria are shown in Table 1.

[0103] Table 1 Histopathological scoring criteria

[0104]

[0105] The H&E staining and Alcian blue staining results of the colon tissues of mice in each group are shown in Figure 3 .

[0106] The histopathological scoring and the statistical results of the number of goblet cells in the colon tissues of mice in each group are shown in Figure 4 .

[0107] The results showed that: the colon tissue of the Control group mice had intact colon mucosa and goblet cells, and there was no infiltration of inflammatory cells into the tissue; obvious inflammatory lesions appeared in the colon tissues of the Model group and Hi-EVs group mice, specifically manifested as damage and disappearance of mucosal epithelial cells, damage to goblet cells, enlarged cell gaps, infiltration of inflammatory cells in the goblet cell gaps, and a large number of inflammatory cell infiltrations in the submucosa; compared with the Model group and Hi-EVs group, the astaxanthin vesicle system in the AXT-EVs group improved the colon inflammation of the mice after intervention, with less damage to goblet cells, reduced cell gaps, relatively intact mucosal epithelium, and less inflammatory cell infiltration; the goblet cells of the AXT group mice were distorted, and the symptoms were alleviated compared with the Model group and Hi-EVs group, but the effect was not as excellent as that of the astaxanthin vesicle system intervention in the AXT-EVs group; in addition, there was no significant difference in histopathological scoring between the AXT group and the Model group.

[0108] The above results indicate that: the astaxanthin vesicle system can cause astaxanthin to accumulate at the intestinal lesion site, effectively relieve intestinal inflammation and intestinal barrier damage, and thus alleviate the occurrence of intestinal cancer.

[0109] (4) Detection of inflammatory factor expression levels

[0110] At the 10th week of the experiment, each mouse was sacrificed and the colon tissue was removed. RNA was extracted, and the expression levels of inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and cyclooxygenase-2 (Cox-2) were analyzed at the transcriptional level using quantitative real-time PCR (qPCR).

[0111] The results of the expression levels of inflammatory factors in the colon tissues of each group of mice are shown in Figure 5 .

[0112] The results showed that the expression levels of the inflammatory factors TNF-α, IL-1β, IL-6, and Cox-2 were relatively high in the Model group and the Hi-EVs group; after intervention with astaxanthin, although the expression levels of the inflammatory factors TNF-α, IL-1β, IL-6, and Cox-2 in the AXT group decreased, there was no significant difference in the expression of inflammatory factors compared with the Model group (p < 0.05); compared with the Model group, the expression levels of the inflammatory factors TNF-α, IL-1β, IL-6, and Cox-2 in the AXT-EVs group were significantly decreased (p < 0.05).

[0113] The above results indicate that the astaxanthin vesicle system can effectively reduce the levels of inflammatory factors in the colon tissue.

[0114] Example 3: In vitro cell uptake experiment

[0115] RAW264.7 macrophages (from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China)) were seeded at 2 × 10 4The cells were inoculated at a density into a 20-mm-diameter confocal dish and cultured in complete medium DMEM for 24 h. Nile red, which also has hydrophobic properties, was used instead of astaxanthin as a fluorescent probe to explore the targeting and absorption-promoting characteristics of the vesicles. This experiment was divided into four groups: (A) control group (Control), (B) blank vector group (hyaluronic acid-iRGD blank vesicles, Hi-EVs), (C) Nile red monomer group (Nile red), and (D) Nile red vesicle group (hyaluronic acid-iRGD blank vesicles + Nile red, Nile red-EVs); phosphate buffer (control group, 2 mL), hyaluronic acid-iRGD blank vesicles (blank vector group, 60 g / L), Nile red monomer (Nile red monomer group, concentration of 0.75 g / L), and Nile red vesicle solution (Nile red vesicle group, Nile red concentration of 0.75 g / L, vector concentration of 60 g / L) were added to the confocal dish containing RAW264.7 macrophages and treated for 6 h. Then, it was washed twice with PBS to remove the unphagocytosed vesicle system. The cells were fixed on the confocal dish, the nucleus was labeled with DAPI, and the cell membrane was labeled with DIO. The uptake ability of macrophages was observed under a confocal microscope.

[0116] The confocal results of macrophage uptake ability are shown in Figure 6 .

[0117] The results showed that no red fluorescence was shown in both (A) the control group and (B) the blank vector group; however, when Nile red or the vesicle system encapsulating Nile red was added to the macrophages, after the phagocytosis of macrophages, red fluorescence was observed in the cytoplasm of macrophages in (C) the Nile red monomer group; and after Nile red was encapsulated in vesicles, stronger fluorescence intensity was observed in the cytoplasm of macrophages in (D) the Nile red vesicle group. After fluorescence intensity statistics showed that in (D) the Nile red vesicle group, due to the HA targeting ligand that can target the CD44 receptor of macrophages and the iRGD peptide targeting ligand that promotes absorption in the vesicles, there was a significant difference in the red fluorescence intensity between the macrophage treatment group containing Nile red vesicles and the macrophage fluorescence intensity in (C) the Nile red monomer group with only Nile red monomer (p < 0.05).

[0118] The above results indicate that under the action of the vesicle system, macrophages can accurately and rapidly uptake the substances encapsulated in the vesicles, such as astaxanthin.

[0119] Example 4: Cell Phagocytosis Experiment

[0120] RAW264.7 macrophages (from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China)) were seeded at a density of 5×10 4The cells were seeded into a 96-well plate at a cell density of cells / well and cultured in complete DMEM medium for 18 - 24 h; then the medium was removed and PBS was added, and the cells were cultured at 37 °C for 1 - 2 h; then macrophages were treated with different concentrations of astaxanthin (0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM, 20 μM) (experimental group, n = 3) at 37 °C for 2 h respectively; the control group was macrophages untreated with astaxanthin (control group, n = 3); then CFDA-labeled cell debris was added respectively, and the ratio of macrophages to cell debris was 1:4, and the cells were incubated at 37 °C for 1 h; then Trypan blue was used for quenching, and the fluorescence intensity was measured using a fluorescence microplate reader; the change level of macrophage phagocytosis was measured by the percentage increase in the fluorescence intensity of macrophages in the experimental groups with different concentrations of astaxanthin compared to the fluorescence intensity of macrophages in the control group.

[0121] The results of the fluorescence intensity measurement of astaxanthin promoting macrophage phagocytosis of cell debris are shown in Figure 7 。

[0122] The results showed that in the concentration range of 0.01 μM - 10 μM of astaxanthin, compared with the wells untreated with astaxanthin, the fluorescence intensity increased by 70 - 90%.

[0123] The above results indicate that: the cell debris has been phagocytosed into RAW264.7 macrophages, and the presence of astaxanthin in the concentration range of 0.01 μM - 10 μM significantly promoted the phagocytosis of macrophages.

[0124] Example 5: Laser confocal experiment

[0125] RAW264.7 macrophages were seeded into a confocal dish at a cell density of 2×10 4 cells / well and cultured in complete DMEM medium for 24 h. This experiment was divided into four groups, namely: (A) macrophage group, (B) macrophage + astaxanthin, (C) macrophage + cell debris, (D) macrophage + astaxanthin + cell debris group. In group C, astaxanthin was added for intervention for 6 h, and then Nile red-labeled cell debris was added. Among them, the addition amount of astaxanthin in each group was 0.1 mM, and the addition amount of Nile red-labeled cell debris was 8×10 4 cells debris, and the cells were co-cultured at 37 °C for 2 h. Then, the cells were washed twice with PBS, the cell membrane was labeled with DIO, and the nucleus was labeled with DAPI, and the phagocytosis of cell debris by macrophages in each group was observed under laser confocal microscopy.

[0126] The results of laser confocal observation of macrophages in each group are shown in Figure 8 。

[0127] The results of the effect of astaxanthin on the fluorescence intensity of cell debris are shown in Figure 9 。

[0128] The results showed that no red fluorescence was observed in either (A) the macrophage group or (B) the macrophage + astaxanthin group; however, when cell debris labeled with Nile red was added to the macrophages, after phagocytosis by the macrophages, red fluorescence was observed in the cytoplasm of the macrophages in (C) the macrophage + cell debris group; and after intervention with astaxanthin, in (D) the macrophage + astaxanthin + cell debris group, stronger fluorescence intensity was observed in the cytoplasm of the macrophages. After fluorescence intensity statistics, it was shown that there was a significant difference in the red fluorescence intensity in the macrophages after astaxanthin intervention in (D) the macrophage + astaxanthin + cell debris group and the fluorescence intensity in the macrophages without astaxanthin intervention in (C) the macrophage + cell debris group (p < 0.05).

[0129] The above results indicate that under the action of astaxanthin, the ability of macrophages to phagocytose cell debris is increased.

[0130] Example 6: Inhibition of tumor tissue growth in tumor-bearing mice by astaxanthin vesicles

[0131] 1. Establishment of tumor-bearing mouse model

[0132] The inventors induced the establishment of a C57BL / 6 tumor-bearing mouse model using murine colon cancer cells (MC38) and MC38 cell debris. The specific protocol is as follows:

[0133] Twenty-four 4- to 5-week-old C57BL / 6 male mice weighing 18-20 g were randomly divided into 4 groups (n = 6), named the control group (Control), the model group (Model), the astaxanthin group (AXT), and the astaxanthin vesicle system group (AXT-EVs), with 6 mice in each group. The feeding environment for each mouse was a specific pathogen-free room with a constant temperature (22 ± 1°C), light / dark cycle (12 h / 12 h), and humidity (40-60%). During the experiment, the mice were allowed free access to water and food; 2 weeks before the official start of the experiment, each mouse was pre-intervened with an astaxanthin / astaxanthin vesicle delivery system (astaxanthin was purchased from Shanghai Macklin Biochemical Co., Ltd.) (the start time of pre-intervention was recorded as week 1). The experiment was conducted for 6 weeks. The conditions for injecting MC38 cells in each group of mice during the experiment were as follows:

[0134] (1) Control group: Inject MC38 live cells at week 3 (1 × 10 4 Living cells) by subcutaneous injection in the back;

[0135] (2) Model group: Inject MC38 live cells and MC38 cell debris at week 3 (1 × 10 4 Living cells + 9 × 10 5 Debris) by subcutaneous injection in the back;

[0136] (3) AXT group: Inject live MC38 cells and MC38 cell debris at week 3 (subcutaneous injection in the back, 1×10 4 Living cells + 9×10 5 Debris), and intervene with intragastric administration of astaxanthin throughout the experiment (40 μg astaxanthin / day);

[0137] (4) AXT-EVs group: Inject live MC38 cells and MC38 cell debris at week 3 (subcutaneous injection in the back, 1×10 4 Living cells + 9×10 5 Debris), and intervene with intragastric administration of astaxanthin vesicle system throughout the experiment (40 μg astaxanthin / day);

[0138] 2. Detection of physiological indexes of tumor-bearing mice

[0139] (1) Detection of tumor tissue volume in mice

[0140] After the tumor tissue volume of the mice reaches 100 mm 3 or more, measure and record the tumor volume every other day.

[0141] The changes in tumor volume of mice in each group are shown in Figure 10 .

[0142] The results showed that: compared with the Model group, the AXT group had a tendency to inhibit tumor formation, but there was no significant difference compared with the Model group (p > 0.05); while the tumor volume on the back of the mice in the AXT-EVs group was similar to that of the control group mice, and there were significant differences compared with the Model group and the AXT group (p < 0.05).

[0143] The above results indicate that: astaxanthin vesicles can accurately target the tumor microenvironment and effectively inhibit the growth of tumor tissue.

[0144] (2) Detection of tumor volume and weight in mice

[0145] At the 6th week of the experiment, sacrifice each mouse and take out the tumor, and observe and record the volume and weight of the tumor.

[0146] The changes in tumor volume and weight of mice in each group are shown in Figure 11 .

[0147] The results showed that the tumor tissue volume and weight of the AXT group mice were lower than those of the Model group, but there was no significant difference compared with the Model group (p > 0.05); the tumor volume and weight of the AXT-EVs group were significantly smaller than those of the Model group and the AXT group, and there were significant differences in volume and weight compared with both groups (p < 0.05).

[0148] The above results indicate that astaxanthin vesicles can precisely target the tumor microenvironment and effectively inhibit the growth of tumor tissue.

[0149] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0150] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of an embedded body in preparing a drug, characterized in that: The drug has at least one of the following uses: Antioxidant; Anti-inflammatory; Antibacterial; Promote the phagocytic ability of macrophages; Improves intestinal health; Anti-tumor cell proliferation; Prevention and / or treatment of colitis; Prevention and / or treatment of tumors; Wherein, the method for preparing the embedded body comprises: S1: mixing the iRGD peptide ligand, the hyaluronic acid ligand and the solution containing the vesicles to obtain a carrier; S2: contacting the carrier with astaxanthin to obtain the embedded body.

2. The use according to claim 1, characterized in that The mixing is carried out in an organic solvent; Optionally, the organic solvent comprises one or more of ethanol, methanol, acetone and ethyl acetate.

3. The use according to claim 1, characterized in that The working concentration of the iRGD peptide ligand is 0.1-0.5 g / L, the working concentration of the hyaluronic acid ligand is 0.1-10 g / L, and the working concentration of the vesicle is 1-100 g / L; Optionally, the working concentration of the carrier is 40 to 160 g / L, and the working concentration of astaxanthin is 0.5 to 10 g / L.

4. The use according to claim 1, characterized in that The vesicles are derived from one or more of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei and Streptococcus mutans; Preferably, the vesicles are derived from Lactobacillus plantarum.

5. The use according to claim 1, characterized in that: The method for preparing the iRGD peptide ligand comprises: contacting the iRGD peptide with a PEGylated lipid containing a carboxyl functional group to form the iRGD peptide ligand; Optionally, the PEGylated lipid containing a carboxyl functional group is 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol 2000.

6. The use according to claim 1, characterized in that: The mass ratio of the iRGD peptide to the PEGylated lipid containing a carboxyl functional group is 1:(0.5-10).

7. The use according to claim 1, characterized in that The method for preparing the hyaluronic acid ligand comprises: contacting hyaluronic acid with a PEGylated lipid containing an amino functional group to form the hyaluronic acid ligand; Optionally, the PEGylated lipid containing an amino functional group is N-distearoylphosphatidylacetamide-polyethylene glycol 2000-amino.

8. The use according to claim 1, characterized in that The mass ratio of the hyaluronic acid to the PEGylated lipid containing an amino functional group is (5-20):

1.

9. The use according to claim 1, characterized in that The tumor includes one or more of colon cancer, bladder cancer, liver cancer and oral cancer.

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