A composite modified exosome-encapsulated resveratrol preparation and its preparation method

Abstract

The present invention belongs to the technical field of medical preparations and relates to a preparation comprising composite modified exosomes encapsulating resveratrol and a method for preparing the preparation. The preparation comprises cholesterol-RGD, milk-derived exosomes, and resveratrol. The preparation is prepared by encapsulating resveratrol with milk-derived exosomes and then modifying the exosomes with cholesterol-RGD. This preparation has the advantages of high availability, low cost, low toxicity, high yield, and suitability for large-scale production. Cholesterol-RGD modification of milk-derived exosomes enhances the bioavailability of the encapsulated resveratrol and improves its efficacy.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical preparations and relates to a preparation of composite modified exosomes encapsulating resveratrol and a preparation method thereof. Background Art

[0002] Resveratrol, also known as Polygonum cuspidatum aglycone, chemical name: (E)-5-[2-(4-hydroxyphenyl)-vinyl]-1,3-benzenediol; 3,5,4"-trihydroxystilbene; stilbene triphenol; molecular weight: 228.25, CAS number: 501-36-0; physical characteristic parameters: white or off-white needle-shaped solid powder; melting point: 255-265℃; chemical characteristic parameters: easily soluble in ether, chloroform, methanol, ethanol, acetone, etc. It is an anthraquinone terpenoid compound, mainly derived from the rhizome extract of Polygonum cuspidatum Sieb.et Zucc. of the Polygonaceae family. It can also be obtained by synthetic methods. Resveratrol is a natural antioxidant that can reduce blood viscosity and inhibit blood Platelet coagulation and vasodilation maintain blood flow, preventing the occurrence and progression of cancer. It also has preventive and therapeutic effects against atherosclerosis, coronary heart disease, ischemic heart disease, and hyperlipidemia. Its tumor-suppressing effects and estrogen-like effects make it useful in treating diseases such as breast cancer. However, resveratrol's extremely low solubility in water and low bioavailability limit its application. Pharmacokinetic studies of oral administration of grape extract containing resveratrol in healthy men have reported a 75% oral absorption rate, but less than 1% is actually absorbed and utilized by the body. This is primarily due to extensive Phase II metabolism of resveratrol in the body, producing glucuronides and sulfate esters. This results in only trace amounts of resveratrol being detected in the blood, limiting its efficacy.

[0003] Most biological cells in nature (animals, plants, and some bacteria) secrete nanoscale vesicles enclosed by phospholipid bilayers, collectively known as extracellular vesicles (EVs). EVs primarily originate from multivesicular bodies formed by the invagination of intracellular lysosomal microsomes. These structures fuse with the cell membrane and are released into the extracellular matrix. They are primarily composed of exosomes (30–200 nm), microvesicles (100–1000 nm), and apoptotic bodies (500–4000 nm), with exosomes being the most studied. Exosomes can carry a variety of proteins, mRNAs, and miRNAs, participating in intercellular communication and material exchange. Their excellent biocompatibility and protective lipid bilayer structure make them promising natural drug delivery vehicles. In recent years, research on milk-derived exosomes has grown exponentially. As a key component of milk, milk-derived exosomes are capable of carrying and transmitting signaling molecules such as miRNAs, making them a hot topic in food-derived exosome research.

[0004] RGD is a short peptide with the sequence RGD, specifically composed of arginine, glycine, and aspartic acid. RGD is present in various extracellular matrices and can specifically bind to 11 integrins. Cholesterol is a key component of the phospholipid bilayer of cell membranes and exosomes. Studies have shown that cholesterol can prevent disordering of the phospholipid bilayer at high temperatures, while disrupting its ordering at low temperatures, preventing the formation of liquid crystals and maintaining their fluidity.

[0005] There are generally several ways to address the current application problems of resveratrol: (1) synergistic administration to improve bioavailability, such as synergistic use with certain ingredients to inhibit the activity of metabolic enzymes; (2) designing prodrugs that are metabolized and hydrolyzed into resveratrol after entering the body, slowing its metabolic rate; (3) improving the dosage form by using liposomes or natural exosomes for encapsulation to improve performance. Currently, there are studies on encapsulating resveratrol with liposomes or natural exosomes to improve its performance. For example, CN110123838A uses exosomes derived from embryonic stem cells and induced human pluripotent stem cells as drug carriers for resveratrol, and CN111436609A uses plant exosomes as drug carriers for resveratrol. However, there are currently no formulations that can simultaneously improve the bioavailability and solubility of resveratrol and enhance the targeting and stability of resveratrol. Summary of the Invention

[0006] The present invention aims to improve the bioavailability and solubility of resveratrol and, at the same time, enhance the targeting and stability of resveratrol.

[0007] Based on the above objectives, the present application addresses this need in the field by providing a cholesterol-RGD modified exosome-encapsulated resveratrol preparation and a preparation method.

[0008] In one aspect, the present invention relates to a cholesterol-RGD modified exosome-encapsulated resveratrol preparation, comprising cholesterol-RGD, milk-derived exosomes, and resveratrol. Resveratrol includes cis-resveratrol, trans-resveratrol, and resveratrol glycosides.

[0009] Furthermore, in the cholesterol-RGD modified exosomes encapsulating resveratrol preparation provided by the present invention, the ratio of cholesterol-RGD, milk-derived exosomes and resveratrol is 0.1-1:10:0.1-1 by mass ratio.

[0010] Furthermore, in the cholesterol-RGD-modified exosome-encapsulated resveratrol formulation provided herein, the milk-derived exosomes are cow's milk exosomes. Milk-derived exosomes from different raw materials are structurally similar, possessing the same topological structure and lipid bilayer as cells, with membrane proteins on their membranes and abundant proteins, nucleic acids, and lipids within their contents. Based on availability, cow's milk exosomes were selected as the milk-derived exosomes.

[0011] The present invention does not specifically limit the preparation method of milk exosomes. Those skilled in the art will readily appreciate that commonly used methods for isolating milk exosomes include ultracentrifugation, size exclusion, immunoaffinity capture, kit method, and microfluidics. For example, the present invention provides a method for preparing milk exosomes, comprising: centrifuging fresh milk at 2000-3000 g for 20 minutes at 2-8°C to separate the fat layer from the whey, and collecting the whey; adding an equal volume of ultrapure water to the whey, adjusting the pH to 4.5-4.6 with 6N hydrochloric acid, and continuously stirring during the addition process until a significant protein precipitation is observed; centrifuging in a high-speed refrigerated centrifuge at 2-8°C at 5000-6000 g for 20 minutes, and collecting the supernatant after centrifugation; centrifuging at 10000-12000 g for 60-90 minutes at 2-8°C to collect the supernatant; and centrifuging at 100000-150000 g for 60-90 minutes at 2-8°C to obtain the precipitate of milk exosomes.

[0012] Furthermore, in the cholesterol-RGD modified exosomes encapsulating resveratrol preparation provided by the present invention, the preparation method of the cholesterol-RGD comprises: weighing cholesterol and succinic anhydride, dissolving them in pyridine, heating to 45° C. to react for 72 hours, adding water for dialysis for 3 days, and then vacuum freezing and lyophilizing to obtain cholesterol-succinic anhydride;

[0013] The cholesterol-succinic anhydride was dissolved in dimethyl sulfoxide, and DMTMM [4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride] was added for activation for 30 minutes. Finally, RGD was added thereto, and the mixture was stirred at 45° C. for overnight reaction. After dialyzing with water for three days, the mixture was vacuum-frozen and freeze-dried to obtain the cholesterol-RGD.

[0014] Furthermore, in the cholesterol-RGD modified exosome-encapsulated resveratrol preparation provided by the present invention, the ratio of the cholesterol to the succinic anhydride is 1:0.2-0.4 by mass ratio; the ratio of the cholesterol-succinic anhydride to the RGD is 1:1-2 by mass ratio.

[0015] Furthermore, the preparation of the composite modified exosome-encapsulated resveratrol provided by the present invention comprises the following preparation methods: (1) continuously stirring the solution of the milk-derived exosomes, and then dropping an alcohol solution of resveratrol at room temperature; (2) incubating at 4°C for 12 to 18 hours after ultrasonication to obtain milk-derived exosome-encapsulated resveratrol; (3) adding the cholesterol-RGD to the milk-derived exosome-encapsulated resveratrol at room temperature, incubating at 37°C for 30 minutes after ultrasonication, and freeze-drying to obtain the composite modified exosome-encapsulated resveratrol preparation.

[0016] Furthermore, in the preparation of resveratrol encapsulated by the composite modified exosomes provided by the present invention, the ultrasonic conditions in step (2) are ultrasonication at a power of 90 to 110 W for 3 to 5 minutes; and the ultrasonic conditions in step (3) are ultrasonication at a power of 90 to 110 W for 3 to 5 minutes.

[0017] Furthermore, in the preparation of resveratrol encapsulated by composite modified exosomes provided by the present invention, the rotation speed of the continuous stirring is 500 rpm.

[0018] Furthermore, in the composite modified exosome-encapsulated resveratrol preparation provided by the present invention, the solution of milk-derived exosomes uses physiological saline as a solvent; and the alcohol solution of resveratrol uses anhydrous ethanol as a solvent.

[0019] Compared with the prior art, the present invention has the following beneficial effects or advantages:

[0020] (1) The present invention provides a preparation of resveratrol encapsulated by composite modified exosomes and a preparation method thereof, wherein milk-derived exosomes are used to encapsulate resveratrol, thereby increasing the bioavailability of resveratrol from 57.82±2.45% to 81.04±2.21% (in vitro gastrointestinal digestion model simulation data) and the solubility of resveratrol from 0.3 g / L to 5-10 g / L.

[0021] (2) The present invention provides a preparation of resveratrol encapsulated by composite modified exosomes and a preparation method thereof. Cholesterol-RGD is selected to further modify the resveratrol encapsulated by milk-derived exosomes, which can enhance the targeting and stability of the preparation. Cholesterol is an important component of cell membranes and exosome membranes. The present invention first connects RGD with cholesterol to obtain cholesterol-RGD, and then loads cholesterol-RGD on the phospholipid bilayer of the exosomes, wherein cholesterol can be embedded in the phospholipid bilayer, exposing the RGD sequence on the surface of the exosomes, giving the exosomes the ability to target integrin receptors. Integrin is a type of transmembrane glycoprotein that can mediate adhesion and signal transduction between cells and cells and between cells and extracellular matrix, and has a regulatory effect on functions such as cell proliferation, migration, adhesion and apoptosis. It is highly expressed on the surface of neovascular endothelial cells and various malignant tumor cells. According to the specificity of binding to extracellular matrix proteins, integrins are divided into four subfamilies: (1) RGD receptor subfamily, containing 8 subtypes; (2) collagen receptor subfamily, containing 4 subtypes; (3) laminin receptor subfamily, containing 4 subtypes; (4) leukocyte-specific receptor subfamily, containing 8 subtypes. The 8 subtypes of the RGD receptor subfamily are αVβ3, αVβ6, αVβ5, αVβ8, αVβ1, α5β1, α8β1 and αⅡbβ3, and each subtype has different functional characteristics. Among them, the αVβ6 receptor subtype is highly expressed only in the intestinal and respiratory epithelial cells, and its expression level in other epithelial cells is very low and difficult to detect. The present invention uses cholesterol-RGD composite modified exosomes to encapsulate resveratrol, so that it can specifically target intestinal epithelial cells. On the other hand, due to the protective nature of the exosome encapsulation, the metabolic rate of resveratrol in the body is reduced, thereby enhancing the bioavailability of resveratrol. Cholesterol is an important component of cell membranes and exosome membranes. Studies have shown that when the temperature is high, cholesterol can prevent the disorder of the phospholipid bilayer, and when the temperature is low, it can interfere with its order, prevent the formation of liquid crystals, and maintain its fluidity. Therefore, the modification of cholesterol-RGD can further enhance the stability of the exosomes themselves.

[0022] (3) The present invention provides a composite modified exosome-encapsulated resveratrol preparation and a preparation method thereof, which has the advantages of high availability, low cost, low toxicity and high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the morphology of the milk exosomes prepared in the present invention under transmission electron microscopy.

[0024] Figure 2 The morphology of the composite modified exosome-encapsulated resveratrol preparation prepared in Example 1 under transmission electron microscopy.

[0025] Figure 3Targeted uptake of resveratrol-encapsulated composite modified exosomes by Caco-2 cells. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention.

[0027] The experimental methods and detection methods described in the following examples are conventional methods unless otherwise specified; the test supplies and raw materials are all commercially available unless otherwise specified.

[0028] Example 1

[0029] This embodiment provides a process for preparing a preparation of a composite modified exosome-encapsulated cis-resveratrol preparation.

[0030] The fresh milk used in this example was sourced from pastures around Xi'an, resveratrol (cis-resveratrol, trans-resveratrol, and resveratrol glycosides) was sourced from Shanghai MacLean Biochemical Technology Co., Ltd., cholesterol was sourced from Shanghai MacLean Biochemical Technology Co., Ltd., and RGD was sourced from Shaanxi Future Polypeptide Biotechnology Co., Ltd.

[0031] (1) Isolation and extraction of milk exosomes

[0032] Fresh milk was centrifuged at 3000g for 20 minutes at 2-8°C to separate the fat layer from the whey, and the whey was collected; an equal volume of ultrapure water was added to the whey, and the pH was adjusted to 4.5-4.6 with 6N hydrochloric acid. Stirring was continued during the addition process until obvious protein precipitation was visible; the milk was placed in a high-speed refrigerated centrifuge at 2-8°C and centrifuged at 5000g for 20 minutes, and the supernatant was collected after centrifugation; the milk was centrifuged at 10000g for 60 minutes at 2-8°C and the supernatant was collected; the milk was centrifuged at 150000g for 70 minutes at 2-8°C to obtain the precipitate to obtain milk exosomes.

[0033] (2) Preparation of cholesterol-RGD

[0034] Cholesterol and succinic anhydride were weighed in a mass ratio of 1:0.388 and dissolved in pyridine. The mixture was heated to 45°C for 72 hours, dialyzed with water for 3 days, and then lyophilized to obtain cholesterol-succinic anhydride. 100 mg of cholesterol-succinic anhydride was dissolved in 20 mL of dimethyl sulfoxide and activated with 57 mg of DMTMM for 30 minutes. Finally, 151 mg of RGD was added, stirred at 45°C overnight, dialyzed with water for three days, and then lyophilized to obtain cholesterol-RGD.

[0035] (3) Preparation of composite modified exosomes encapsulating cis-resveratrol

[0036] A milk exosome solution (solvent: physiological saline, concentration: 5 mg / mL) was placed on a magnetic stirrer and stirred at 500 rpm. At room temperature, a cis-resveratrol alcohol solution (solvent: anhydrous ethanol, concentration: 500 mg / mL) was added dropwise to the milk exosome solution at a cis-resveratrol: milk exosome mass ratio of 1:10 to obtain a milk exosome-encapsulated cis-resveratrol solution. The solution was then sonicated at 90 W for 3 minutes and incubated at 4°C overnight. Cholesterol-RGD was then added to the milk exosome-encapsulated cis-resveratrol solution at room temperature. The mass ratio of cholesterol-RGD, milk exosomes, and cis-resveratrol was 0.1:10:1. The solution was sonicated at 90 W for 3 minutes, incubated at 37°C for 30 minutes, and freeze-dried to obtain a composite modified exosome-encapsulated cis-resveratrol preparation.

[0037] Example 2

[0038] This embodiment provides a process for preparing a preparation of a composite modified exosome-encapsulated trans-resveratrol.

[0039] The sources of materials are the same as in Example 1.

[0040] (1) Isolation and extraction of milk exosomes

[0041] Fresh milk was centrifuged at 2000g for 20 minutes at 2-8°C to separate the fat layer from the whey, and the whey was collected; an equal volume of ultrapure water was added to the whey, and the pH was adjusted to 4.5-4.6 with 6N hydrochloric acid. Stirring was continued during the addition process until obvious protein precipitation was visible; the milk was placed in a high-speed refrigerated centrifuge at 2-8°C and centrifuged at 6000g for 20 minutes, and the supernatant was collected after centrifugation; the milk was centrifuged at 12000g for 90 minutes at 2-8°C and the supernatant was collected; the milk was centrifuged at 100000g for 90 minutes at 2-8°C to obtain the precipitate to obtain milk exosomes.

[0042] (2) Preparation of cholesterol-RGD

[0043] Cholesterol and succinic anhydride were weighed in a mass ratio of 1:0.2 and dissolved in pyridine. The mixture was heated to 45°C for 72 hours, dialyzed with water for 3 days, and then lyophilized to obtain cholesterol-succinic anhydride. 100 mg of cholesterol-succinic anhydride was dissolved in 20 mL of dimethyl sulfoxide and activated with 57 mg of DMTMM for 30 minutes. Finally, 100 mg of RGD was added, stirred at 45°C overnight, dialyzed with water for three days, and then lyophilized to obtain cholesterol-RGD.

[0044] (3) Preparation of composite modified exosomes encapsulating trans-resveratrol

[0045] A milk exosome solution (solvent: physiological saline, concentration: 5 mg / mL) was placed on a magnetic stirrer and stirred at 500 rpm. An alcoholic solution of trans-resveratrol (solvent: anhydrous ethanol, concentration: 500 mg / mL) was added dropwise to the milk exosome solution at room temperature to obtain a milk exosome-encapsulated trans-resveratrol solution. The solution was ultrasonicated at 100 W for 4 minutes and incubated at 4°C overnight. Cholesterol-RGD was added to the milk exosome-encapsulated trans-resveratrol solution at room temperature. The mass ratio of cholesterol-RGD, milk exosomes, and trans-resveratrol was 0.6:10:0.4. The solution was ultrasonicated at 100 W for 4 minutes, incubated at 37°C for 30 minutes, and freeze-dried to obtain a composite modified exosome-encapsulated trans-resveratrol preparation.

[0046] Example 3

[0047] This embodiment provides a preparation process of a composite modified exosome-encapsulated resveratrol preparation.

[0048] (1) Isolation and extraction of milk exosomes

[0049] Fresh milk was centrifuged at 2500g for 20 minutes at 2-8°C to separate the fat layer from the whey, and the whey was collected; an equal volume of ultrapure water was added to the whey, and the pH was adjusted to 4.5-4.6 with 6N hydrochloric acid. Stirring was continued during the addition process until obvious protein precipitation was visible; the milk was placed in a high-speed refrigerated centrifuge at 2-8°C and centrifuged at 5500g for 20 minutes, and the supernatant was collected after centrifugation; the milk was centrifuged at 11000g for 75 minutes at 2-8°C and the supernatant was collected; the milk was centrifuged at 120000g for 75 minutes at 2-8°C to obtain the precipitate to obtain milk exosomes. Figure 1 The above shows the morphology of the isolated milk exosomes under a transmission electron microscope, which appears as a disc-shaped vesicle structure.

[0050] (2) Preparation of cholesterol-RGD

[0051] Cholesterol and succinic anhydride were weighed in a mass ratio of 1:0.4 and dissolved in pyridine. The mixture was heated to 45°C for 72 hours, dialyzed with water for 3 days, and then lyophilized to obtain cholesterol-succinic anhydride. 100 mg of cholesterol-succinic anhydride was dissolved in 20 mL of dimethyl sulfoxide and activated with 57 mg of DMTMM for 30 minutes. Finally, 200 mg of RGD was added and the mixture was stirred at 45°C overnight. The mixture was dialyzed with water for three days, and then lyophilized to obtain cholesterol-RGD.

[0052] (3) Preparation of composite modified exosomes encapsulating resveratrol glycosides

[0053] The milk exosome solution (solvent is normal saline, concentration is 5 mg / mL) is placed on a magnetic stirrer and stirred at 500 rpm. The alcohol solution of resveratrol glycoside (solvent is anhydrous ethanol, concentration is 500 mg / mL) is added dropwise to the milk exosome solution at room temperature to obtain a milk exosome-encapsulated resveratrol glycoside solution. Ultrasonicate at a power of 110 W for 5 minutes and incubate at 4°C overnight. Cholesterol-RGD is added to the milk exosome-encapsulated resveratrol glycoside solution at room temperature. The mass ratio of cholesterol-RGD, milk exosomes and resveratrol glycoside is 1:10:0.1. Ultrasonicate at a power of 110 W for 5 minutes and then incubate at 37°C for 30 minutes. After freeze-drying, the composite modified exosome-encapsulated resveratrol glycoside preparation is obtained. Figure 2 Shown are the composite-modified exosomes encapsulating resveratrol glycosides and their morphology under transmission electron microscopy. After modification, the exosome structure is complete and the overall structure appears as a disc-shaped vesicle.

[0054] Comparative Example 1

[0055] The preparation method of this example is the same as that of Example 1, except that cholesterol-RGD modification and exosome encapsulation are not performed.

[0056] Comparative Example 2

[0057] The preparation method of this example is the same as that of Example 1, except that cholesterol-RGD modification is not performed.

[0058] Example 4

[0059] This example provides an in vitro bioavailability assay of resveratrol encapsulated by composite-modified exosomes.

[0060] The composite modified exosomes encapsulating resveratrol (RGD-Res-EVs) prepared in the above example and the cholesterol-RGD-free exosomes (Res-EVs) prepared in the comparative example were freeze-dried and digested in simulated gastric fluid for 2 hours and intestinal fluid for 4 hours, respectively. The supernatants were then centrifuged and filtered to collect the supernatants. Resveratrol powder was weighed and dissolved in anhydrous ethanol. The standard solution was diluted to the desired concentration to create a standard curve for resveratrol content. The absorbance at 326 nm, the characteristic absorption peak of resveratrol, was measured and the resveratrol content was calculated using the standard curve.

[0061] The bioavailability of resveratrol was calculated according to the following formula:

[0062]

[0063] Where cdigestate refers to the concentration of resveratrol in the digestate after simulated digestion, vsupernatant refers to the volume of the supernatant after centrifugation, and mresveratrol is the mass of resveratrol before digestion.

[0064] Table 1 In vitro bioavailability of resveratrol encapsulated by composite modified exosomes

[0065] Grouping In vitro bioavailability (%) Example 1 78.01±1.25％ Example 2 81.04±2.21％ Example 3 75.64±0.12％ Comparative Example 1 57.82±2.45％ Comparative Example 2 64.71±3.12％

[0066] Example 5

[0067] This example provides a cytotoxicity assay for resveratrol encapsulated by composite-modified exosomes.

[0068] Culture human colon cancer Caco-2 cells. These cells have similar structure and function to differentiated small intestinal epithelial cells, possessing structures such as microvilli. Submicroscopic studies have shown that Caco-2 cells are morphologically similar to human small intestinal epithelial cells, sharing the same cell polarity and tight junctions. Once the cells reach approximately 80% confluency, digest them, count them, and seed 3,000 cells per well in a 96-well plate. Incubate in a 5% CO2, 37°C incubator for 48-72 hours to allow them to adhere and grow normally. In terms of mass fraction, the composite modified exosome-encapsulated resveratrol preparation (RGD-Res-EVs) prepared in the embodiment was diluted with MEM (NEAA) + 20% FBS culture medium to 8 concentration gradients of 10%, 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.1563% and 0.0781%. When the cell plating rate in the 96-well plate reached 80%, the medium was replaced with a culture medium containing different concentrations of RGD-Res-EVs. After the liquid change was completed, the 96-well plate was placed in an incubator (37°C, 5% CO2) for culture. After the cells were incubated for 24 hours, the supernatant was discarded, 100 μL of 10% CCK-8 detection solution was added, and the cells were incubated in the dark at 37°C for 2 hours. After the incubation was completed, 90 μL was aspirated and the OD value was read at 450nm. According to the formula,

[0069] Table 2 Cytotoxicity detection of resveratrol encapsulated by composite modified exosomes

[0070]

[0071] Example 6

[0072] Cholesterol-RGD and simple cholesterol were fluorescently labeled using cy5, and the fluorescently labeled composite modified exosomes encapsulating resveratrol preparations (cy5-RGD-Res-EVs) and fluorescently labeled exosomes encapsulating resveratrol preparations (cy5-Res-EVs) were prepared according to the method of Example 1.

[0073] Caco-2 cells were cultured until the cells reached approximately 80% fusion. The cells were digested and seeded onto a 12-well plate cell slide and cultured in a constant temperature cell incubator at 37°C with 5% carbon dioxide for 48 hours to allow them to adhere to the wall and grow normally. Cy5-RGD-EVs and cy5-EVs were prepared into a 5% solution using MEM (NEAA) serum-free medium. The old culture medium in the well plate was discarded, and the serum-free culture medium of cy5-RGD-EVs and cy5-EVs was added and incubated in the incubator for 2 hours. After 2 hours, the culture supernatant was aspirated and the cells were carefully rinsed twice with PBS buffer. The cell nuclei were stained with Hoechst 33342 and observed under a fluorescence microscope after washing.

[0074] like Figure 3 As shown, under the same incubation time, the fluorescence intensity of the cholesterol-RGD modified exosomes encapsulating resveratrol preparation inside the Caco-2 cells was higher than that of the preparation without cholesterol-RGD modification, indicating that the modified exosome preparation can be targeted and taken up by Caco-2 cells faster.

[0075] As described above, the present invention can be better implemented. The above embodiments only describe the preferred implementation methods of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the present invention.

Claims

1. A composite modified exosome-encapsulated resveratrol preparation, characterized in that: First, resveratrol is encapsulated by milk-derived exosomes to obtain milk-derived exosome-encapsulated resveratrol, and then cholesterol-RGD is used to modify the milk-derived exosome-encapsulated resveratrol to obtain the preparation; The preparation method of cholesterol-RGD comprises: weighing cholesterol and succinic anhydride, dissolving them in pyridine, heating them to 45° C. for reaction for 72 hours, adding water for dialysis for 3 days, and then vacuum freezing and lyophilizing to obtain cholesterol-succinic anhydride; The cholesterol-succinic anhydride was dissolved in dimethyl sulfoxide, and DMTMM was added for activation for 30 minutes. Finally, RGD was added thereto, stirred at 45° C. for overnight reaction, dialyzed with water for three days, and then freeze-dried in a vacuum freezer to obtain cholesterol-RGD.

2. The composite modified exosome-encapsulated resveratrol preparation according to claim 1, characterized in that: In terms of mass ratio, the ratio of cholesterol-RGD, milk-derived exosomes and resveratrol is 0.1~1:10:0.1~1.

3. The composite modified exosome-encapsulated resveratrol preparation according to claim 1, characterized in that: The milk-derived exosomes are milk exosomes.

4. The composite modified exosome-encapsulated resveratrol preparation according to claim 1, characterized in that: The ratio of the cholesterol to the succinic anhydride is 1:0.2-0.4 by mass; In terms of mass ratio, the ratio of the cholesterol-succinic anhydride to the RGD is 1:1-2.

5. The composite modified exosome-encapsulated resveratrol preparation according to claim 1, characterized in that: The preparation method comprises: (1) The milk-derived exosome solution is continuously stirred, and then an alcohol solution of resveratrol is added dropwise at room temperature; (2) After ultrasound, the mixture was incubated at 4°C for 12-18 hours to obtain resveratrol encapsulated in milk-derived exosomes; (3) Adding the cholesterol-RGD to the milk-derived exosome-encapsulated resveratrol at room temperature, incubating at 37°C for 30 minutes after ultrasonication, and freeze-drying to obtain the composite modified exosome-encapsulated resveratrol preparation.

6. The composite modified exosome-encapsulated resveratrol preparation according to claim 5, characterized in that: The ultrasonic condition in step (2) is 90-110 W of power for 3-5 minutes; The ultrasonic condition described in step (3) is ultrasonication at a power of 90 to 110 W for 3 to 5 minutes.

7. The composite modified exosome-encapsulated resveratrol preparation according to claim 5, characterized in that: The rotation speed of the continuous stirring is 500 rpm.

8. The composite modified exosome-encapsulated resveratrol preparation according to claim 5, characterized in that: The solution of milk-derived exosomes uses physiological saline as solvent; The resveratrol alcohol solution uses anhydrous ethanol as solvent.

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