Precise-delivery astaxanthin vesicle system and preparation method thereof

Abstract

The invention belongs to the technical field of nano-carriers, and particularly relates to a precise-delivery astaxanthin vesicle system and a preparation method thereof, and the precise-delivery astaxanthin vesicle system comprises vesicles, an iRGD peptide ligand and a hyaluronic acid ligand. The carrier disclosed by the invention is high in stability, high in embedding rate for hydrophobic bioactive substances, good in slow release effect, capable of accurately delivering the hydrophobic bioactive substances to a targeted site, high in biological safety, widely applicable to carrying different types of hydrophobic bioactive substances, capable of effectively improving the bioavailability of carried substances and high in application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanocarriers, and particularly relates to a precisely delivered astaxanthin vesicle system and a preparation method thereof. Background Art

[0002] Many bioactive functional factors, such as astaxanthin and lutein, are hydrophobic bioactive substances, which have problems such as low solubility, poor stability and low bioavailability, which limit the development and application of such bioactive functional factors in functional products.

[0003] With the development of nanotechnology, the steady-state of bioactive factors can be effectively achieved through carrier embedding. Relevant research shows that proteins, oligosaccharides, polysaccharides and starches can all be used to prepare nanocarriers for the embedding and delivery of bioactive factors. For example, relevant researchers combined whey protein isolate (WPI) with mannose through Maillard reaction and then modified it with triphenylphosphine bromide (TPP) to prepare a dual-targeted nanoparticle (AXT@TPP-WPI-Man) for the delivery of the bioactive factor astaxanthin. This nanoparticle can target macrophages in the intestinal inflammatory site, significantly improving the bioavailability and anti-inflammatory effect of astaxanthin (CN 117337970 A); amylose can also be used as the matrix material of nanocarriers, and through self-assembly with lipids and active molecules, stable ternary complex nanoparticles are formed for the intestinal targeting delivery of bioactive factors (CN118806728 A); however, there are still certain problems in these carrier preparation methods, mainly including high preparation cost, complex preparation process, insufficient control of sustained-release effect, inability to precisely deliver to the target site and poor biocompatibility. In addition, during the preparation of some carriers, more emulsifiers and highly toxic organic solvents are used, so there are varying degrees of reagent residues, posing certain safety hazards.

[0004] Therefore, developing a carrier with high biosafety, simple preparation method, wide source of raw materials, good sustained-release effect and capable of precisely delivering to the target site is of great significance for improving the bioavailability of such hydrophobic bioactive substances. 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. For this purpose, the present invention provides a precisely delivered astaxanthin vesicle system and a preparation method thereof. The carrier of the present invention has high stability, high encapsulation rate for hydrophobic bioactive substances, good sustained-release effect, can precisely deliver it to the target site, that is, macrophages in the tumor microenvironment, has high biosafety, is widely applicable to transporting different types of hydrophobic bioactive substances, and can effectively improve the bioavailability of the transported substances, with high application value.

[0006] In the first aspect of the present invention, a carrier is proposed. According to an embodiment of the present invention, it includes: vesicles, iRGD peptide ligands, and hyaluronic acid ligands. The carrier according to the embodiment of the present invention has high stability, a high encapsulation rate for hydrophobic bioactive substances, a good sustained-release effect, can accurately deliver them to the target site, namely macrophages in the tumor microenvironment, has high biosafety, is widely applicable to carrying different types of hydrophobic bioactive substances, and can effectively improve the bioavailability of the carried substances, with high application value.

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

[0008] 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.

[0009] According to an embodiment of the present invention, the vesicles are derived from Lactobacillus plantarum.

[0010] According to an embodiment of the present invention, the particle size of the carrier is 20 - 300 nm.

[0011] According to an embodiment of the present invention, the method for preparing the iRGD peptide ligand includes: contacting the iRGD peptide with a carboxyl-functionalized polyethylene glycolylated lipid to form the iRGD peptide ligand.

[0012] According to an embodiment of the present invention, the carboxyl-functionalized polyethylene glycolylated lipid is 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-N-carboxy-polyethylene glycol 2000.

[0013] According to an embodiment of the present invention, the mass ratio of the iRGD peptide to the carboxyl-functionalized polyethylene glycolylated lipid is 1:(0.5 - 10).

[0014] According to an embodiment of the present invention, the method for preparing the hyaluronic acid ligand includes: contacting hyaluronic acid with an amino-functionalized polyethylene glycolylated lipid to form the hyaluronic acid ligand.

[0015] According to an embodiment of the present invention, the amino-functionalized polyethylene glycolylated lipid is N-distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amino.

[0016] According to an embodiment of the present invention, the mass ratio of the hyaluronic acid to the amino-functionalized polyethylene glycolylated lipid is (5 - 20):1.

[0017] According to an embodiment of the present invention, the method for preparing the carrier includes: contacting the iRGD peptide ligand, the hyaluronic acid ligand with a solution containing the vesicles to obtain the carrier.

[0018] According to an embodiment of the present invention, the solution is a phosphate buffer solution.

[0019] According to an embodiment of the present invention, the contacting is carried out in an organic solvent.

[0020] According to an embodiment of the present invention, the organic solvent includes one or more of ethanol, methanol, acetone, and ethyl acetate.

[0021] 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.

[0022] In a second aspect of the present invention, the present invention provides a method for preparing the carrier described in the first aspect. According to an embodiment of the present invention, the method includes: contacting the iRGD peptide ligand, the hyaluronic acid ligand with a solution containing the vesicles to obtain the carrier. According to the preparation method of the embodiment of the present invention, natural and safe excipients are used, and the carrier is prepared by utilizing the charge interaction between amphiphilic substances. The whole preparation process is safe, green and efficient, with the advantages of simple preparation method and high preparation efficiency, and has high application value.

[0023] According to an embodiment of the present invention, the above preparation method may further have the following additional technical features:

[0024] According to an embodiment of the present invention, the solution is a phosphate buffer solution.

[0025] According to an embodiment of the present invention, the contacting is carried out in an organic solvent.

[0026] According to an embodiment of the present invention, the organic solvent includes one or more of ethanol, methanol, acetone, and ethyl acetate.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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).

[0031] According to an embodiment of the present invention, the method for preparing the hyaluronic acid ligand includes: contacting hyaluronic acid with a polyethylene glycolylated lipid containing an amino functional group to form the hyaluronic acid ligand;

[0032] 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.

[0033] 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.

[0034] In a third aspect of the present invention, the present invention provides an embedding body. According to an embodiment of the present invention, it includes: the carrier described in the first aspect; a payload; the payload is embedded in the carrier. The embedding body according to the embodiment of the present invention is suitable for embedding various payloads. While effectively protecting the payload and improving its stability, the accurate delivery and slow release of the payload are achieved through the carrier, further improving the utilization rate of the payload.

[0035] According to an embodiment of the present invention, the above embedding body may further have the following additional technical features:

[0036] According to an embodiment of the present invention, the payload is a hydrophobic bioactive substance.

[0037] According to an embodiment of the present invention, the payload includes one or more of astaxanthin, lutein, and fucoxanthin.

[0038] In a fourth aspect of the present invention, the present invention provides a drug. According to an embodiment of the present invention, it includes the carrier described in the first aspect and / or the embedding body described in the third aspect.

[0039] Those skilled in the art can understand that the features and advantages described above for the carrier or the embedding body also apply to this application and will not be repeated here.

[0040] 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

[0041] 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:

[0042] Figure 1It is the standard curve graph of astaxanthin in Example 4 of the present invention;

[0043] Figure 2 It is the graph of the determination results of the encapsulation rate of different vesicles in Example 4 of the present invention;

[0044] Figure 3 It is the graph of the particle size results of different vesicles in Example 5 of the present invention. Among them, (A) is the particle size graph of Lactobacillus plantarum cell membrane vesicles, (B) is the particle size graph of hyaluronic acid-iRGD blank vesicles, and (C) is the particle size graph of hyaluronic acid-iRGD astaxanthin vesicles;

[0045] Figure 4 It is the transmission electron microscopy result graph of different vesicles in Example 5 of the present invention. Among them, (A) is the transmission electron microscopy result graph of Lactobacillus plantarum cell membrane vesicles, (B) is the transmission electron microscopy result graph of hyaluronic acid-iRGD blank vesicles, and (C) is the transmission electron microscopy result graph of hyaluronic acid-iRGD astaxanthin vesicles;

[0046] Figure 5 It is the Fourier transform infrared spectroscopy result graph of different vesicles in Example 5 of the present invention;

[0047] Figure 6 It is the graph of the in vitro digestion test results of hyaluronic acid-iRGD peptide astaxanthin vesicles in Example 5 of the present invention.

[0048] Figure 7 It is the co-localization result graph of astaxanthin vesicles and macrophages in tumor tissues in vivo in Example 6 of the present invention; among them, (A) in the Control group from left to right are the result graph of DAPI-labeled cell nuclei, the result graph of F4 / 80-labeled macrophages, the Nile red fluorescence result graph, the result graph after the overlap of each label, and the result graph after magnification by 65 times, (B) in the Nile red treatment group from left to right are the result graph of DAPI-labeled cell nuclei, the result graph of F4 / 80-labeled macrophages, the Nile red fluorescence result graph, the result graph after the overlap of each label, and the result graph after magnification by 65 times, (C) in the Nile red-EVs from left to right are the result graph of DAPI-labeled cell nuclei, the result graph of F4 / 80-labeled macrophages, the Nile red fluorescence result graph, the result graph after the overlap of each label, and the result graph after magnification by 65 times; Detailed implementation manners

[0049] The embodiments of the present invention will be described in detail below. The following described embodiments are exemplary and are only used to explain the present invention and should not be construed as a limitation to the present invention.

[0050] It should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, 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 specified, the meaning of "a plurality" is two or more.

[0051] The endpoints and any values in the ranges disclosed herein 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.

[0052] In this text, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0053] In this text, 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 such events or conditions occur, as well as the cases where such events or conditions do not occur.

[0054] Terms and Definitions

[0055] In this text, 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.

[0056] In this text, the "HA ligand" is a hyaluronic acid ligand. Hyaluronic acid is a natural anionic polysaccharide that can interact with the CD44 surface receptor on inflammatory macrophages to increase the uptake of macrophages and help achieve the targeted delivery of the carrier.

[0057] In this text, a "vesicle" refers to a cell membrane structure extracted from Lactobacillus plantarum with the ability of self-assembly, which can embed the payload into its internal hydrophobic domain, improve the stability of the payload, and promote its absorption and utilization.

[0058] In this text, a "dynamic microjet" refers to a high-pressure homogenization technology that uses a high-pressure pump to pass fluid materials through a very narrow microjet channel, generating extremely high shear forces and pressure drops, so that the materials are subjected to strong physical effects when passing through the microjet channel. This technology is mainly used for preparing nano- or sub-micron-sized emulsions, suspensions and solutions, as well as improving the stability and bioavailability of materials.

[0059] carrier

[0060] The present invention provides a carrier. According to an embodiment of the present invention, it includes: vesicles, iRGD peptide ligand, and hyaluronic acid ligand. The carrier according to the embodiment of the present invention has high stability, high encapsulation rate for hydrophobic bioactive substances, good sustained-release effect, can accurately deliver them to the target site, has high biological safety, is widely applicable to different types of hydrophobic bioactive substances, and can effectively improve their bioavailability, with high application value.

[0061] For the carrier according to the present invention, vesicles formed by the principle of microbial self-assembly are combined with iRGD peptide ligand and hyaluronic acid ligand to form the carrier described in the present invention. This carrier can reach target sites such as intestinal epithelial cells in a complete form, and at the same time, with the help of iRGD peptide ligand and hyaluronic acid ligand, it can efficiently target macrophages in the tumor microenvironment and improve the ability of macrophages to uptake the loaded substances, thereby realizing the accurate delivery of loaded substances such as astaxanthin.

[0062] 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 multiple microorganisms that can self-assemble to form extracellular vesicles, are suitable for different application scenarios, and increase the flexibility and adaptability of the carrier.

[0063] According to an 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. It forms extracellular vesicles through cell membrane fragments. The inventors used physical methods such as ultracentrifugation to separate cell membrane fragments from cell organelles to obtain the vesicles required for preparing the carrier of the present invention.

[0064] According to an embodiment of the present invention, the particle size of the carrier is 20 - 300 nm. Thus, the carrier has the advantage of a small molecular weight. When the carrier is actually applied, it can more easily pass through the barriers in the organism, thereby more effectively reaching the target site, and has better sustained-release performance, stability, and targeted delivery ability. Exemplarily, the particle size of the carrier is 20 nm, 40 nm, 60 nm, 80 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 220 nm, 240 nm, 260 nm, 280 nm, 300 nm, preferably 20 - 180 nm, more preferably 120 nm.

[0065] 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 obtaining a stable and amphiphilic iRGD peptide ligand.

[0066] 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 of the 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.

[0067] 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, avoiding waste of resources.

[0068] According to an embodiment of the present invention, the method for preparing the hyaluronic acid ligand includes: contacting hyaluronic acid with a polyethylene glycolylated lipid containing an amino functional group to form the hyaluronic acid ligand. Thus, 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 obtaining a stable hyaluronic acid ligand.

[0069] 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. According to an embodiment of the present invention, the polyethylene glycolylated lipid containing an amino functional group 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.

[0070] According to an embodiment of the present invention, the mass ratio of the hyaluronic acid to the amino-functionalized polyethylene glycolated lipid is (5 to 20):1. Exemplarily, the mass ratio of the hyaluronic acid to the amino-functionalized polyethylene glycolated lipid 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 to 20):1, more preferably 20:1; thus, by adjusting the mass ratio of the two, efficient preparation of the hyaluronic acid ligand is achieved, avoiding waste of resources.

[0071] According to an embodiment of the present invention, the method for preparing the carrier includes: contacting the iRGD peptide ligand, the hyaluronic acid ligand with the solution containing the vesicles to obtain the carrier. Thus, the carrier is prepared by utilizing the charge interaction between amphiphilic substances. The whole preparation process is safe, green and efficient, having the advantages of simple preparation method, high preparation efficiency, etc. The prepared carrier has the integrated properties of the iRGD peptide ligand, the hyaluronic acid ligand and the vesicles, and has high application value.

[0072] According to an embodiment of the present invention, the solution is a phosphate buffer solution. Thus, the vesicles maintain their biological activity in the phosphate buffer solution, which helps the smooth progress of the subsequent operation of preparing the carrier.

[0073] According to an embodiment of the present invention, the contacting is carried out in an organic solvent. Thus, the contacting is carried out in an organic solvent, enabling the iRGD peptide ligand, the hyaluronic acid ligand and the vesicles to be fully contacted, further improving the preparation efficiency of the carrier.

[0074] According to an embodiment of the present invention, the organic solvent includes one or more of ethanol, methanol, acetone and ethyl acetate. Thus, the contacting can be carried out in a variety of organic solvents, suitable for different application scenarios.

[0075] 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. Exemplarily, the working concentration of the iRGD peptide ligand is 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 concentration of the hyaluronic acid ligand is 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 0.1 - 5 g / L, more preferably 1 g / L; the working concentration of the vesicles is 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; thus, by adjusting the working concentrations of the iRGD peptide ligand, the hyaluronic acid ligand, and the vesicles, a green, safe, and stable targeted carrier can be prepared according to specific application requirements, thereby realizing the targeted delivery of the loaded substance.

[0076] Preparation method of the carrier

[0077] The present invention provides a method for preparing the aforementioned carrier. According to an embodiment of the present invention, it includes: contacting the iRGD peptide ligand, the hyaluronic acid ligand with a solution containing the vesicles to obtain the carrier. According to the preparation method of the embodiment of the present invention, natural and safe excipients are used, and the carrier is prepared by utilizing the charge interaction between amphiphilic substances. The entire preparation process is safe, green, and efficient, with advantages such as simple preparation method and high preparation efficiency, and has high application value.

[0078] According to an embodiment of the present invention, the solution is a phosphate buffer solution. Thus, the contact is carried out in an organic solvent, enabling the iRGD peptide ligand, the hyaluronic acid ligand, and the vesicles to come into full contact, further improving the preparation efficiency of the carrier.

[0079] According to an embodiment of the present invention, the contact is carried out in an organic solvent. Thus, the contact is carried out in an organic solvent, enabling the iRGD peptide ligand, the hyaluronic acid ligand, and the vesicles to come into full contact, further improving the preparation efficiency of the carrier.

[0080] 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 can be carried out in a variety of organic solvents, suitable for different application scenarios.

[0081] According to an embodiment of the present invention, the working concentration of the iRGD peptide ligand is 0.1 to 0.5 g / L, the working concentration of the hyaluronic acid ligand is 0.1 to 10 g / L, and the working concentration of the vesicle is 1 to 100 g / L. Exemplarily, the working concentration of the iRGD peptide ligand is 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, preferably 0.2 to 0.5 g / L, more preferably 0.5 g / L; the working concentration of the hyaluronic acid ligand is 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 to 10 g / L, more preferably 10 g / L; the working concentration of the vesicle is 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 to 80 g / L, more preferably 60 g / L; thus, by adjusting the working concentrations of the iRGD peptide ligand, the hyaluronic acid ligand, and the vesicle, a green, safe, stable, and targeted carrier can be efficiently prepared according to specific application requirements, thereby achieving the targeted delivery of the loaded substance.

[0082] 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.

[0083] 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 of the 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.

[0084] According to an embodiment of the present invention, the mass ratio of the iRGD peptide to the carboxyl-functionalized polyethylene glycolated lipid is 1:(0.5 to 10). Exemplarily, the mass ratio of the iRGD peptide to the carboxyl-functionalized polyethylene glycolated lipid 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 to 5), more preferably 1:1; thus, by adjusting the mass ratio of the two, efficient preparation of the iRGD peptide ligand is achieved, avoiding waste of resources.

[0085] According to an embodiment of the present invention, the method for preparing the hyaluronic acid ligand includes: forming the hyaluronic acid ligand by reacting hyaluronic acid with a polyethylene glycolated lipid containing an amino functional group. Thus, carboxyl groups are present in hyaluronic acid, which covalently binds to the polyethylene glycolated lipid containing an amino functional group to form stable amide bonds, thereby obtaining a stable hyaluronic acid ligand.

[0086] According to an embodiment of the present invention, the polyethylene glycolated lipid containing an amino functional group is distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amine. According to an embodiment of the present invention, the polyethylene glycolated lipid containing an amino functional group can be N-distearoyl phosphatidylethanolamine-polyethylene glycol 2000-amino group, which can covalently bind to the carboxyl groups in hyaluronic acid to form stable amide bonds, thereby obtaining a stable hyaluronic acid ligand.

[0087] According to an embodiment of the present invention, the mass ratio of the hyaluronic acid to the polyethylene glycolated lipid containing an amino functional group is (5 to 20):1. Exemplarily, the mass ratio of the hyaluronic acid to the polyethylene glycolated 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 to 20):1, more preferably 20:1; thus, by adjusting the mass ratio of the two, efficient preparation of the hyaluronic acid ligand is achieved, avoiding waste of resources.

[0088] Inclusion body

[0089] The present invention provides an inclusion body. According to an embodiment of the present invention, it includes: the aforementioned carrier; a payload; the payload is embedded in the carrier. The inclusion body according to an embodiment of the present invention is suitable for embedding various payloads, and while effectively protecting the payload and improving its stability, precise delivery and slow release of the payload are achieved through the carrier, further improving the utilization rate of the payload.

[0090] According to an embodiment of the present invention, the payload is a hydrophobic bioactive substance. Thus, the payload being a hydrophobic bioactive substance is suitable for different application scenarios, while improving its stability and further enhancing its bioavailability.

[0091] According to an embodiment of the present invention, the payload includes one or more of astaxanthin, lutein, and fucoxanthin. Thus, the payload can be various hydrophobic bioactive substances such as astaxanthin, which is suitable for different application scenarios.

[0092] Drug

[0093] The present invention provides a drug. According to an embodiment of the present invention, it includes the aforementioned carrier and / or the aforementioned inclusion body.

[0094] Those skilled in the art can understand that the features and advantages described above for the carrier or inclusion body also apply to this application and will not be elaborated here.

[0095] Unless otherwise specified, the formulation of the MRS liquid medium used in the embodiments of the present invention is as follows:

[0096] 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, and 0.25 g of manganese sulfate; adjust the pH value to 6.2 - 6.4, and autoclave (101 Kpa, 121 °C) for 15 min.

[0097] Unless otherwise specified, the formulation of the MRS agar medium used in the embodiments of the present invention is as follows:

[0098] 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, and 15 g of agar powder; adjust the pH value to 6.2 - 6.4, and autoclave (101 Kpa, 121 °C) for 15 min.

[0099] Next, the solutions of the present invention will be explained with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specifically noted in the examples regarding technical or conditions, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0100] Example 1: Cultivation of Lactobacillus plantarum and Preparation of Cell Membrane Vesicles

[0101] 1. Cultivation of Lactobacillus plantarum

[0102] Add the freeze-dried powder of Lactobacillus plantarum (purchased from Suzhou Shienkang Biotechnology Co., Ltd.) into sterile MRS liquid medium and incubate at 37°C for 36 h to obtain the incubated solution of Lactobacillus plantarum.

[0103] After gradient dilution of the incubated solution of Lactobacillus plantarum, spread 0.1 mL of the diluted suspension onto MRS agar medium, culture at 37 ± 1°C for 46 - 50 h, and perform colony counting to confirm the yield of the cell membrane vesicles of Lactobacillus plantarum obtained in subsequent preparations.

[0104] 2. Collection of protoplast precipitate

[0105] Centrifuge the incubated solution of Lactobacillus plantarum 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 some organelles of Lactobacillus plantarum cells), and then centrifuge at 3500 g for 10 min to obtain the protoplast precipitate.

[0106] 3. Preparation of Lactobacillus plantarum cell membrane vesicles

[0107] Wash the protoplast precipitate twice with PBS and resuspend it in PBS. Use ice bath ultrasonic treatment at 24 kHz for 30 min for fragmentation, remove the unbroken protoplasts by centrifugation at 3500 g for 10 min, and remove other organelles by centrifugation at 20000 g for 30 min to obtain the supernatant containing only cell membrane fragments. The supernatant is ultracentrifuged at 100000 g for 1 h to obtain the membrane fragment precipitate; redisperse the membrane fragment precipitate in sterile PBS, treat it under the condition of ultrasonic power of 35 kHz for 30 min, and then circulate it twice under the condition of 100 MPa of dynamic microfluidics to form Lactobacillus plantarum cell membrane vesicles, which are stored at -80°C for standby.

[0108] Example 2: Synthesis of targeting ligand

[0109] 1. DSPE-PEG 2000 -HA targeting ligand synthesis

[0110] Completely dissolve 20 g of hyaluronic acid (HA) in 50 mL of distilled water, add 40 μmol of NHS and 50 μmol of EDC to activate the hydroxyl groups, and stir at 4°C for 4 h to obtain a mixed solution. Add the mixed solution to 50 mL of aqueous solution containing 1 g of DSPE-PEG 2000 -NH2 and stir for 12 h to obtain a viscous solution. Transfer the viscous solution to a dialysis bag and dialyze at room temperature for 48 h, and obtain DSPE-PEG 2000 -HA targeting ligand by freeze-drying.

[0111] 2. Synthesis of DSPE-PEG 2000 -iRGD targeting ligand

[0112] Dissolve 0.05 g of iRGD peptide (iRGD) completely in 50 mL of distilled water, add 40 μmol of NHS and 50 μmol of EDC to activate the amino group, and stir for 4 h at 4 °C to obtain a mixed solution. Mix the mixed solution with 50 mL of aqueous solution containing 0.05 g of DSPE-PEG 2000 -COOH and stir for 12 h to obtain a viscous solution. Transfer the viscous solution to a dialysis bag and dialyze at room temperature for 48 h, and obtain DSPE-PEG 2000 -iRGD targeting ligand by lyophilization.

[0113] Example 3: Preparation of vesicle system

[0114] 1. Preparation of hyaluronic acid vesicles

[0115] Mix the Lactobacillus plantarum cell membrane vesicles prepared in Example 1 and the DSPE-PEG 2000 -HA targeting ligand prepared in Example 2 to obtain a mixed solution. The concentration of Lactobacillus plantarum cell membrane vesicles in the mixed solution is 60 g / L, and the concentration of DSPE-PEG 2000 -HA targeting ligand is 1 g / L. Ultrasonically treat the mixture in an ice bath for 30 min, and then centrifuge at a centrifugal force of 10,000 g for 30 min to remove the unreacted membrane fragments, which is the hyaluronic acid vesicle solution and store it at -80 °C for later use.

[0116] 2. Preparation of iRGD peptide vesicles

[0117] Mix the Lactobacillus plantarum cell membrane vesicles prepared in Example 1 and the DSPE-PEG 2000 -iRGD targeting ligand prepared in Example 2 to obtain a mixed solution. The concentration of Lactobacillus plantarum cell membrane vesicles in the mixed solution is 60 g / L, and the concentration of DSPE-PEG 2000 -iRGD targeting ligand is 0.5 g / L. Ultrasonically treat the mixture in an ice bath for 30 min, and then centrifuge at a centrifugal force of 10,000 g for 30 min to remove the unreacted membrane fragments, which is the iRGD peptide vesicle solution and store it at -80 °C for later use.

[0118] 3. Preparation of hyaluronic acid-iRGD blank vesicles

[0119] Mix the Lactobacillus plantarum cell membrane vesicles prepared in Example 1, the DSPE-PEG 2000 -HA targeting ligand prepared in Example 2, and the DSPE-PEG prepared in Example 22000 Mix with the -iRGD targeting ligand to obtain a mixed solution. The concentration of Lactobacillus plantarum cell membrane vesicles in the mixed solution is 60 g / L, and the concentration of DSPE-PEG 2000 -HA targeting ligand is 1 g / L, and the concentration of DSPE-PEG 2000 -iRGD targeting ligand is 0.5 g / L. Sonicate in an ice bath for 30 min, and then centrifuge at 10,000 g for 30 min to remove un-synthesized membrane fragments, obtaining the hyaluronic acid-iRGD blank vesicle solution, which is stored at -80 °C for later use.

[0120] 4. Preparation of hyaluronic acid-iRGD astaxanthin vesicles

[0121] Taking astaxanthin as an example, mix the hyaluronic acid-iRGD blank vesicles prepared in Example 3(3) with astaxanthin (purchased from Shanghai Macklin Biochemical Co., Ltd.) dissolved in a dichloromethane / absolute ethanol mixture (v / v, 1:3) at a mass ratio of 1:120 (astaxanthin: hyaluronic acid-iRGD blank vesicles). Sonicate in an ice bath for 30 min, centrifuge at 10,000 g for 30 min to remove un-entrapped astaxanthin, and then centrifuge at 100,000 g for 60 min to remove ethanol to obtain a precipitate. Redisperse the precipitate in sterile PBS to obtain the hyaluronic acid-iRGD peptide astaxanthin vesicles, which are stored at -80 °C for later use.

[0122] 5. Preparation of hyaluronic acid astaxanthin vesicles

[0123] Taking astaxanthin as an example, mix the hyaluronic acid vesicles prepared in Example 3(1) with astaxanthin dissolved in absolute ethanol at a mass ratio of 1:120 (astaxanthin: hyaluronic acid astaxanthin vesicles). Sonicate in an ice bath for 30 min, centrifuge at 10,000 g for 30 min to remove un-entrapped astaxanthin, and then centrifuge at 100,000 g for 60 min to remove ethanol to obtain a precipitate. Redisperse the precipitate in sterile PBS to obtain the hyaluronic acid astaxanthin vesicles, which are stored at -80 °C for later use.

[0124] 6. Preparation of iRGD peptide astaxanthin vesicles

[0125] Taking astaxanthin as an example, mix the iRGD peptide vesicles prepared in Example 3(2) with astaxanthin dissolved in absolute ethanol at a mass ratio of 1:120 (astaxanthin: iRGD peptide astaxanthin vesicles). Sonicate in an ice bath for 30 min, centrifuge at 10,000 g for 30 min to remove un-entrapped astaxanthin, and then centrifuge at 100,000 g for 60 min to remove ethanol to obtain a precipitate. Redisperse the precipitate in sterile PBS to obtain the iRGD peptide astaxanthin vesicles, which are stored at -80 °C for later use.

[0126] Example 4: Determination of the encapsulation efficiency of hyaluronic acid-iRGD peptide astaxanthin vesicles

[0127] 1. Extraction of astaxanthin from vesicles

[0128] Mix 1 mL of hyaluronic acid-iRGD peptide astaxanthin vesicles, hyaluronic acid astaxanthin vesicles, and iRGD peptide astaxanthin vesicles prepared in Example 3 with 5 mL of dichloromethane / methanol (2:1, v / v) solution respectively. After vortexing for 10 min, a suspension is obtained. The suspension is centrifuged at 4000 rpm for 15 min, and the dichloromethane layer containing astaxanthin is collected, dried with nitrogen, and redissolved in 5 mL of methanol to obtain astaxanthin solution I (derived from hyaluronic acid-iRGD peptide astaxanthin vesicles), astaxanthin solution II (derived from hyaluronic acid astaxanthin vesicles), and astaxanthin solution III (derived from iRGD peptide astaxanthin vesicles) respectively.

[0129] 2. Plotting of the standard curve and determination and calculation of the astaxanthin content

[0130] (1) Plotting of the astaxanthin standard curve

[0131] Take 50 mg of astaxanthin into a clean 100 mL volumetric flask. Dissolve it with absolute ethanol and make up to 100 mL to obtain a 500 μg / mL astaxanthin stock solution. Accurately weigh 0.5, 1.0, 1.5, 2.0, and 2.5 mL of the standard stock solution into 10 mL volumetric flasks respectively, dilute to the mark with absolute ethanol, shake well, and obtain solutions with mass concentrations of 25, 50, 75, 100, and 125 μg / mL, and filter through a 0.45 μm filter membrane; and use C 18 -HPLC-DAD for determination respectively (the specific condition settings are the same as those in step 2 of Example 4). Plot the astaxanthin standard curve with the mass concentration of the astaxanthin series standard solutions as the abscissa and the corresponding absorption peak area as the ordinate.

[0132] The astaxanthin standard curve is shown in Figure 1 .

[0133] The results show that the regression equation of the astaxanthin standard curve is Y = 0.15273x + 0.06597, and R 2 = 0.9991.

[0134] (2) Calculation of the astaxanthin encapsulation efficiency

[0135] Calculate the encapsulation efficiency of astaxanthin in different vesicle systems according to the astaxanthin standard curve. The specific calculation method is as follows:

[0136]

[0137] The results showed that the encapsulation efficiency of vesicles containing only hyaluronic acid ligand (hyaluronic acid vesicles) and vesicles containing only iRGD peptide ligand (iRGD peptide vesicles) for astaxanthin was in the range of 70 - 75%, and there was no significant difference between the two (p > 0.05); while for the vesicle system containing both hyaluronic acid and iRGD peptide ligand (hyaluronic acid - iRGD peptide blank vesicles), the content of loaded astaxanthin was more than 80%, showing a significant difference compared with the above two vesicles (p < 0.05).

[0138] The results of astaxanthin encapsulation efficiency are shown in Figure 2 。

[0139] Example 5: Determination of Physicochemical Properties of Vesicles

[0140] To deeply understand the physical, chemical and biological properties of the hyaluronic acid vesicles, iRGD peptide vesicles, hyaluronic acid - iRGD peptide vesicles and hyaluronic acid - iRGD peptide astaxanthin vesicles prepared in Example 3, the inventors conducted performance tests on them, including microstructure, particle size potential and release ability. The specific methods are as follows:

[0141] 1. Dynamic Light Scattering

[0142] Using a dynamic light scattering instrument (DLS), the particle size of the vesicles was measured to evaluate the size distribution of the vesicles. The specific parameters were a temperature of 23°C, a scattering angle of 90°, an aqueous solution as the measurement medium, a set viscosity of 0.933 cP, a refractive index of 1.333, an external optical fiber angle of 90°, and a measurement intensity of 300 kHz.

[0143] The size distribution results of different vesicles are shown in Figure 3 。

[0144] The results showed that whether it was Lactobacillus plantarum cell membrane vesicles, hyaluronic acid - iRGD peptide blank vesicles or hyaluronic acid - iRGD peptide astaxanthin vesicles, their particle sizes were all distributed in the range of 100 - 1000 nm. However, compared with the hyaluronic acid - iRGD peptide blank vesicles, the particle size distribution of the hyaluronic acid - iRGD peptide astaxanthin vesicles was more uniform; the potentials of both the hyaluronic acid - iRGD peptide blank vesicles and the hyaluronic acid - iRGD peptide astaxanthin vesicles were negative, indicating that they were both negatively charged. The absolute potential of the hyaluronic acid - iRGD peptide astaxanthin vesicles was higher than that of the Lactobacillus plantarum cell membrane vesicles, and the absolute potential value of the hyaluronic acid - iRGD peptide astaxanthin vesicles was higher than that of both the Lactobacillus plantarum cell membrane vesicles and the hyaluronic acid - iRGD blank vesicles, indicating that the hyaluronic acid - iRGD astaxanthin vesicles were more stable and would not form complexes and precipitates due to charge interaction.

[0145] 2. Scanning Electron Microscopy

[0146] The surface microstructure of the vesicles was further observed using a freeze-scanning electron microscope (FSEM). The specific parameters were as follows: the vesicle solution was placed on the sample stage and rapidly frozen, then the sample stage was moved to the preparation room, and the excess sample material was trimmed using the built-in blade. The sample was sublimated at -90 °C for 20 min, sputter-coated with gold for 1 min, and then placed in the test chamber for analysis. Finally, the vesicle images were recorded using a high-resolution electron microscope at a temperature of -145 °C.

[0147] The scanning electron microscope results of different vesicles are shown in Figure 4 .

[0148] The results showed that the cell membrane vesicles of Lactobacillus plantarum, the hyaluronic acid-iRGD blank vesicles, and the hyaluronic acid-iRGD astaxanthin vesicles all presented uniform spherical shapes, and their diameters were similar to the particle size ranges measured by dynamic light scattering.

[0149] 3. Fourier transform infrared spectroscopy

[0150] The molecular forces and chemical bonds of the vesicles were determined using Fourier transform infrared spectroscopy (FTIR) to analyze the interactions in the vesicles. The specific parameters were as follows: the wavelength range was set to 500 - 4000 cm -1 , the scanning resolution was 4 cm -1 , the number of scans was 32, and the air in the environment was used as the blank background for infrared measurement.

[0151] The Fourier transform infrared spectroscopy results of different vesicles are shown in Figure 5 .

[0152] The results showed that the peaks of astaxanthin monomer at 3033 cm -1 , 2964 cm -1 and 2862 cm -1 increased in the hyaluronic acid-iRGD astaxanthin vesicles, the peak at 3604 cm -1 disappeared, and the peak at 1651 cm -1 weakened. These changes in the peaks indicated that there was a binding interaction between the cell membrane vesicles of Lactobacillus plantarum and astaxanthin, forming inclusion bodies.

[0153] 4. Digestibility test

[0154] Mix 0.4 mL of each vesicle sample with 7.1 mL of simulated oral digestive fluid for 10 min to obtain digestive fluid I. Then, add digestive fluid I to simulated gastric juice (7.5 mL), adjust the pH to 2.0, and digest in a water bath shaker at 37 °C for 120 min to obtain digestive fluid II. Then, adjust the pH of digestive fluid II to 7.0, add it to 10 mL of simulated intestinal fluid, and digest at 37 °C for 120 min. During this period, use 0.01 M NaOH solution and HCl solution to maintain pH = 7.0. During the whole digestion process, take 1 mL of digestive fluid at 10 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min, 210 min, and 240 min respectively, and supplement the same volume of digestive fluid, and measure the astaxanthin content in the digestive fluid.

[0155] Among them, the formula of the simulated oral digestive fluid is as follows: 1.594 mg·mL -1 sodium chloride, 0.328 mg·mL -1 ammonium nitrate, 0.636 mg·mL -1 potassium dihydrogen phosphate, 0.202 mg·mL -1 potassium chloride, 0.308 mg·mL -1 potassium citrate, 0.021 mg·mL -1 sodium dihydrogen urate, 0.198 mg·mL -1 urea, 0.146 mg·mL -1 sodium lactate, 30 mg·mL -1 mucosal protein, and all the components used are prepared with ultrapure water; the formula of the simulated gastric juice is as follows: 2 mg·mL -1 sodium chloride, 7 mL·L -1 hydrochloric acid, 3.2 mg·mL -1 pepsin, and all the components used are prepared with ultrapure water;

[0156] The formula of the simulated intestinal fluid is as follows: salt solution (36.7 mg·mL -1 calcium chloride dihydrate, 218.7 mg·mL -1 sodium chloride), 24 mg·mL -1 lipase, 54 mg·mL -1 bile salts, where the salt solution is prepared with ultrapure water, and lipase and bile salts are prepared with 5 mmol·L -1 phosphate buffer (pH = 7.0).

[0157] The digestion test results of different vesicles are shown in Figure 6 .

[0158] The results showed that due to the temperature of 37 °C and the strong acidic environment of gastric juice, the monomer of astaxanthin degraded rapidly in the whole simulated environment. At the end of the simulated gastric juice digestion, the content of astaxanthin monomer was only 59.56 ± 2.58%, and at the end of the simulated intestinal juice digestion, the content of astaxanthin monomer was less than 45%. This indicates that astaxanthin is relatively sensitive to the environment, prone to degradation, and has a low bioavailability. After being encapsulated by hyaluronic acid-iRGD peptide vesicles, astaxanthin showed a slow-release effect. During the whole process of simulated gastrointestinal juice digestion, the content of astaxanthin was above 80%. This shows that hyaluronic acid-iRGD peptide vesicles can protect astaxanthin from the influence of the environment, enabling it to be absorbed in a relatively large amount and reach the action site to exert a stronger effect.

[0159] Example 6: Co-localization experiment in tumor-bearing mice

[0160] The inventors established a tumor-bearing mouse model by subcutaneously injecting murine colon cancer cells (MC38 cells) into the back of mice. Nile red was used as a hydrophobic probe instead of astaxanthin and encapsulated into hyaluronic acid-iRGD peptide vesicles for the co-localization experiment in tumor-bearing mice. The specific protocol is as follows:

[0161] Eighteen 4- to 5-week-old male C57BL / 6 mice with a body weight of 18-20 g were randomly divided into 3 groups (n = 6), named the control group (Control), the Nile red monomer group (Nile red), and the Nile red vesicle group (Nile red-EVs), with 6 mice in each group. The mice were housed in a specific pathogen-free room with a constant temperature of (22 ± 1) °C, a light / dark cycle of 12 h / 12 h, and a humidity of 40-60%. During the experiment, the mice were allowed to access water and food freely. After 1 week of environmental adaptation, 5×10 5 cells of MC38 cells were subcutaneously injected into the back of the mice. When the volume of the tumor tissue on the back of the mice reached 1000 mm 3 ³, the control group was intragastrically administered with phosphate buffer solution, the Nile red monomer group was intragastrically administered with a Nile red solution dissolved in phosphate buffer solution (the concentration of Nile red was 1.5 g / L), and the Nile red vesicle treatment group was intragastrically administered with a Nile red vesicle solution (the concentration of Nile red was 1.5 g / L, and the concentration of hyaluronic acid-iRGD blank vesicles was 60 g / L). Six hours after oral administration, the tumor tissues of the mice were collected, and the tumor tissues of the mice were cryosectioned. Macrophages in the tumor section tissues were immunofluorescently labeled with F4 / 80 antibody, and fluorescence imaging was performed using a fluorescence inverted microscope.

[0162] The results of the fluorescence intensity of different treatment groups are shown in Figure 7 .

[0163] The results showed that no red fluorescence was shown in the tumor tissues of the mice in the control group, but red fluorescence appeared in the tumor tissues of the mice administered with monomeric nile red and nile red vesicles by gavage. However, the red fluorescence in the tumor tissues of the mice in the monomeric nile red treatment group was weak, while the tumor tissue sections of the mice in the nile red vesicle treatment group had a wider range of nile red fluorescence, and the coincidence rate with the green fluorescence of macrophages was relatively high.

[0164] The above results indicate that under the action of the vesicle system, the hyaluronic acid-iRGD peptide blank vesicles can precisely target the embedded substances to macrophages in the tumor microenvironment.

[0165] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "examples", "specific examples", 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.

[0166] 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. A carrier, characterized in that include: Vesicles, iRGD peptide ligand, and hyaluronic acid ligand.

2. The carrier 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.

3. The carrier according to claim 1, characterized in that The particle size of the carrier is 20 to 300 nm.

4. The carrier 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-carboxyl-polyethylene glycol 2000; Optionally, the mass ratio of the iRGD peptide to the PEGylated lipid containing a carboxyl functional group is 1:(0.5-10).

5. The carrier 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; Optionally, the mass ratio of the hyaluronic acid to the PEGylated lipid containing an amino functional group is (5-20):

1.

6. The carrier according to claim 1, characterized in that The method for preparing the carrier comprises: contacting the iRGD peptide ligand, the hyaluronic acid ligand and a solution containing the vesicle to obtain the carrier; Optionally, the solution is a phosphate buffer solution; Optionally, the contacting is carried out in an organic solvent; Optionally, the organic solvent comprises one or more of ethanol, methanol, acetone and ethyl acetate; Optionally, 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.

7. A method for preparing the carrier according to any one of claims 1 to 6, characterized in that: include: contacting the iRGD peptide ligand, the hyaluronic acid ligand and a solution containing vesicles to obtain the carrier; Optionally, the solution is a phosphate buffer solution; Optionally, the contacting is carried out in an organic solvent; Optionally, the organic solvent comprises one or more of ethanol, methanol, acetone and ethyl acetate; Optionally, 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.

8. The preparation method according to claim 7, 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-carboxyl-polyethylene glycol 2000; Optionally, the mass ratio of the iRGD peptide to the PEGylated lipid containing a carboxyl functional group is 1:(0.5-10).

9. The preparation method according to claim 7, 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; Optionally, the mass ratio of the hyaluronic acid to the PEGylated lipid containing an amino functional group is (5-20):

1.

10. An embedded body, characterized in that: include: The vector according to any one of claims 1 to 6; load; The load is embedded in the carrier.

11. The embedding body according to claim 10, characterized in that: The load is a hydrophobic bioactive substance; Optionally, the loading substance comprises one or more of astaxanthin, lutein and fucoxanthin.

12. A medicine, characterized in that: It comprises the carrier according to any one of claims 1 to 6 and / or the embedding body according to claim 10 or 11.

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