CN116392601A - Preparation of composite modified exosome-coated resveratrol and preparation method thereof - Google Patents
Preparation of composite modified exosome-coated resveratrol and preparation method thereof Download PDFInfo
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- CN116392601A CN116392601A CN202310465931.8A CN202310465931A CN116392601A CN 116392601 A CN116392601 A CN 116392601A CN 202310465931 A CN202310465931 A CN 202310465931A CN 116392601 A CN116392601 A CN 116392601A
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- resveratrol
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- cholesterol
- milk
- exosomes
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- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention belongs to the technical field of medical preparations, and relates to a preparation of resveratrol wrapped by a compound modified exosome and a preparation method thereof. The preparation comprises the following components: cholesterol-RGD, milk-derived exosomes and resveratrol. The preparation is prepared by wrapping resveratrol by using milk-derived exosomes and then modifying the exosomes by cholesterol-RGD. The preparation has the advantages of high availability, low cost, low toxicity, high yield and suitability for large-scale production, and can enhance the bioavailability of the encapsulated resveratrol by modifying milk source exosomes with cholesterol-RGD, thereby improving the efficacy of the resveratrol.
Description
Technical Field
The invention belongs to the technical field of medical preparations, and relates to a preparation of resveratrol wrapped by a compound modified exosome and a preparation method thereof.
Background
Resveratrol (resveratrol), also known as polydatin, chemical name: (E) -5- [2- (4-hydroxyphenyl) -vinyl ] -1, 3-benzenediol; 3,5,4 "-trihydroxy stilbene; stilbene triphenols; molecular weight: 228.25, cas number: 501-36-0; physical characteristic parameters: white or off-white needle-like solid powder; melting point: 255-265 ℃; chemical characteristic parameters: is easily dissolved in diethyl ether, chloroform, methanol, ethanol, acetone, etc. It is anthraquinone terpenoid mainly derived from rhizome extract of Polygonum cuspidatum of Polygonaceae, polygonum cuspidatum Sieb. Resveratrol is a natural antioxidant, and has effects in reducing blood viscosity, inhibiting blood platelet coagulation and vasodilation, keeping blood smooth, preventing cancer occurrence and development, and preventing and treating atherosclerosis, coronary heart disease, ischemic heart disease and hyperlipidemia; the tumor inhibiting effect also has estrogen-like effect, and can be used for treating breast cancer. However, resveratrol has very low solubility in water and low bioavailability, which has certain limitations on its use. According to literature report, the oral administration of grape extract containing resveratrol by healthy men shows that the oral absorption rate of resveratrol is 75% and is truly absorbed and utilized by human body but less than 1%, and the main reason is that resveratrol undergoes extensive II-phase metabolic reaction in human body to generate glucuronide and sulfate ester conjugate, so that only trace resveratrol can be detected in blood, and the efficacy of resveratrol is affected.
Most biological (animal, plant and some bacteria) cells in nature are capable of secreting nanoscale vesicle-like structures, collectively referred to as extracellular vesicles, surrounded by phospholipid bilayer membranes. The extracellular vesicles mainly originate from multi-vesicles formed by the invagination of intracellular lysosome particles, are fused with cell membranes through the outer membrane of the multi-vesicles and released into extracellular matrixes, and mainly consist of exosomes (30-200 nm), microvesicles (100-1000 nm) and apoptotic bodies (500-4000 nm), wherein the most researches on the exosomes are carried out. The exosomes can carry various proteins, mRNA, miRNA and the like, participate in intercellular communication and substance exchange, and have good biocompatibility and protective lipid bilayer vesicles, so that the exosomes have the potential of becoming a good natural drug delivery carrier. Recently, research on milk-derived exosomes is gradually enriched, and the milk-derived exosomes, which are one of important components of milk, can carry and transmit signal molecules such as miRNA and the like, become a research hotspot of the current food-derived exosomes.
RGD is a short peptide with the sequence RGD, and specifically consists of arginine, glycine and aspartic acid. RGD exists in a variety of extracellular matrices and can specifically bind to 11 integrins. Cholesterol is an important component of a cell membrane and an exosome phospholipid bilayer, and researches show that cholesterol can prevent disorder of the phospholipid bilayer at a high temperature and interfere ordering of the phospholipid bilayer at a low temperature so as to prevent liquid crystal from forming and keep fluidity of the phospholipid bilayer.
The following methods are generally used for solving the current application problems of resveratrol: (1) Synergistic administration improves bioavailability, e.g., inhibition of metabolic enzyme activity in conjunction with the use of certain ingredients; (2) Designing a prodrug, metabolizing and hydrolyzing the prodrug into resveratrol after entering the body, and delaying the metabolism rate of the resveratrol; (3) The liposome or natural exosome is used for encapsulation by improving the dosage form, so that the performance is improved. At present, research on wrapping resveratrol by using liposome or natural exosome to improve the performance of resveratrol is available, for example, CN110123838A uses embryonic stem cells and exosome derived from induced human pluripotent stem cells as a drug carrier of resveratrol, and CN111436609A uses plant exosome as a drug carrier of resveratrol. However, no preparation which can improve the bioavailability and the solubility of the resveratrol and can enhance the targeting property and the stability of the resveratrol is currently known.
Disclosure of Invention
The invention aims to improve the bioavailability and the solubility of resveratrol and enhance the targeting property and the stability of resveratrol.
Based on the above-mentioned objects, the present application addresses this need in the art by providing cholesterol-RGD modified exosome-encapsulated resveratrol formulations and methods of preparation.
In one aspect, the invention relates to a formulation of cholesterol-RGD modified exosome-encapsulated resveratrol comprising: cholesterol-RGD, milk-derived exosomes and resveratrol. Resveratrol includes cis-resveratrol, trans-resveratrol and polydatin.
Further, in the preparation of the cholesterol-RGD modified exosome coated resveratrol, the ratio of the cholesterol-RGD to the milk-derived exosome to the resveratrol is 0.1-1:10:0.1-1 in terms of mass ratio.
Further, in the preparation of resveratrol wrapped by cholesterol-RGD modified exosomes, the milk-derived exosomes are milk exosomes. Milk-derived exosomes of different raw materials are structurally similar, having the same topological structure and lipid bilayer as the cells, with membrane proteins on the membrane, and large amounts of proteins, nucleic acids and lipids in the contents. Based on the availability, milk exosomes prepared from cow milk are selected as milk source exosomes.
The method for preparing the milk exosomes is not particularly limited, and those skilled in the art can easily know that the methods for separating the milk exosomes are commonly used in ultracentrifugation, size exclusion, immunoaffinity capture, kit methods, microfluidic techniques and the like. Illustratively, the present invention provides a method for preparing a milk exosome comprising: centrifuging fresh milk at 2-8 ℃ for 20min at 2000-3000 g, separating a fat layer from whey, and collecting the whey; adding an equal volume of ultrapure water into whey, adjusting the pH to 4.5-4.6 by using 6N hydrochloric acid, and continuously stirring in the adding process to ensure that the protein precipitation is obvious; putting the mixture into a high-speed refrigerated centrifuge at the temperature of 2-8 ℃, centrifuging for 20min at 5000-6000 g, and collecting supernatant after centrifuging; centrifuging 10000-12000 g for 60-90 min at 2-8deg.C, and collecting supernatant; centrifuging 100000 ～ 150000g for 60-90 min at 2-8 ℃ to obtain precipitate to obtain milk exosomes.
Further, in the preparation of resveratrol wrapped by cholesterol-RGD modified exosome, the preparation method of the cholesterol-RGD comprises the following steps: weighing cholesterol and succinic anhydride, dissolving in pyridine, heating to 45 ℃ for reaction for 72 hours, adding water for dialysis for 3 days, and performing vacuum freeze-drying to obtain cholesterol-succinic anhydride;
dissolving the cholesterol-succinic anhydride in dimethyl sulfoxide, adding DMTMM [4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride ] to activate for 30min, adding RGD, stirring at 45 ℃ for overnight reaction, adding water to dialyze for three days, and performing vacuum freeze-drying to obtain the cholesterol-RGD.
Further, in the preparation of the cholesterol-RGD modified exosome-coated resveratrol, the ratio of the cholesterol to the succinic anhydride is 1:0.2-0.4 in terms of mass ratio; the ratio of the cholesterol-succinic anhydride to the RGD is 1:1-2 based on the mass ratio.
Further, in the preparation of the resveratrol wrapped by the composite modified exosome, the preparation method comprises the following steps: (1) Continuously stirring the solution of the milk-derived exosomes, and then dripping an alcohol solution of resveratrol at room temperature; (2) After ultrasonic treatment, placing at 4 ℃ for incubation for 12-18 h to prepare resveratrol wrapped by milk source exosomes; (3) And adding the cholesterol-RGD into the resveratrol wrapped by the milk-derived exosome at room temperature, performing ultrasonic treatment, incubating at 37 ℃ for 30min, and freeze-drying to obtain the preparation of the resveratrol wrapped by the compound modified exosome.
Further, in the preparation of the resveratrol wrapped by the composite modified exosome, the ultrasonic condition in the step (2) is that the ultrasonic is carried out for 3-5 min under the power of 90-110W; and (3) performing ultrasonic treatment for 3-5 min under the power of 90-110W under the ultrasonic condition.
Further, in the preparation of the resveratrol wrapped by the composite modified exosome, the rotation speed of continuous stirring is 500rpm.
Further, in the preparation of resveratrol wrapped by the composite modified exosome, physiological saline is selected as a solvent for the solution of the milk-derived exosome; the alcohol solution of resveratrol adopts absolute ethyl alcohol as solvent.
Compared with the prior art, the invention has the following beneficial effects or advantages:
(1) The invention provides a preparation of resveratrol wrapped by a compound modified exosome and a preparation method thereof, wherein the resveratrol is wrapped by a milk source exosome, the bioavailability of the resveratrol is improved from 57.82 +/-2.45% to 81.04 +/-2.21% (in-vitro gastrointestinal digestion model simulation data), and the solubility of the resveratrol is improved from 0.3g/L to 5-10 g/L.
(2) The invention provides a preparation of resveratrol wrapped by a compound modified exosome and a preparation method thereof, which selects cholesterol-RGD to further modify resveratrol wrapped by a milk source exosome, and can enhance the targeting property and stability of the preparation. Cholesterol is an important component of cell membranes and exosome membranes, RGD is firstly connected with cholesterol to obtain cholesterol-RGD, and then the cholesterol-RGD is loaded on an exosome phospholipid bilayer, wherein cholesterol can be embedded in the phospholipid bilayer, RGD sequences are exposed on the surface of the exosome, and the exosome is endowed with the capability of targeting integrin receptors. Integrins (integrins) are transmembrane glycoproteins which mediate cell-to-cell adhesion and signal transduction and have regulatory effects on cell proliferation, migration, adhesion, apoptosis and other functions, and are highly expressed on the surfaces of neovascular endothelial cells and various malignant tumors. Integrins are divided into 4 subfamilies according to their binding specificity to extracellular matrix proteins: (1) RGD receptor subfamily containing 8 subtypes; (2) collagen receptor subfamily containing 4 subtypes; (3) a laminin receptor subfamily containing 4 subtypes; (4) leukocyte specific receptor subfamily containing 8 subtypes. Wherein the 8 subtypes of the RGD receptor subfamily are respectively alpha V beta 3, alpha V beta 6, alpha V beta 5, alpha V beta 8, alpha V beta 1, alpha 5 beta 1, alpha 8 beta 1 and alpha IIb beta 3, and each subtype has different functional characteristics. Wherein the alpha V beta 6 receptor subtype is only highly expressed in intestinal tract and respiratory tract epithelial cells, and the expression quantity in the rest epithelial cells is little and is difficult to detect. According to the invention, the resveratrol is wrapped by the cholesterol-RGD composite modified exosome, so that the resveratrol can specifically target intestinal epithelial cells, and on the other hand, the metabolism speed of the resveratrol in the body is reduced due to the protection of the exosome wrapping, so that the bioavailability of the resveratrol is enhanced. Cholesterol is an important component of cell membranes and exosome membranes, and researches show that cholesterol can prevent disorder of phospholipid bilayer when the temperature is high, and can interfere with ordering when the temperature is low, so that liquid crystal is prevented from forming, and fluidity is maintained, so that the stability of exosome can be further enhanced by cholesterol-RGD modification.
(3) The invention provides a preparation of resveratrol coated by composite modified exosomes and a preparation method thereof, and has the advantages of strong availability, low cost, low toxicity and high yield.
Drawings
Fig. 1 shows a form of the milk exosome prepared by the invention under a transmission electron microscope.
Fig. 2 shows the morphology of the preparation of the complex-modified exosome-coated resveratrol prepared in example 1 under a transmission electron microscope.
FIG. 3 is a graph showing targeted uptake of formulations of complex modified exosomes-encapsulated resveratrol by Caco-2 cells.
Detailed Description
In order that those skilled in the art will better understand the technical solution of the present invention, the present invention will be further described with reference to specific examples, but the examples are not intended to limit the present invention.
The experimental methods and the detection methods described in the following examples are all conventional methods unless otherwise specified; the test articles and the raw materials are available on the market unless otherwise specified.
Example 1
The embodiment provides a preparation process of a preparation of cis-resveratrol wrapped by a composite modified exosome.
Fresh milk used in this example was derived from pastures around western-style city, resveratrol (cis-resveratrol, trans-resveratrol and resveratrol glycoside) was derived from Shanghai microphone Biotechnology Co., ltd, cholesterol was derived from Shanghai microphone Biotechnology Co., ltd, and RGD was derived from Shanxi future polypeptide biotechnology Co., ltd.
(1) Separation and extraction of milk exosomes
Centrifuging fresh milk at 2-8 ℃ for 20min at 3000g, separating a fat layer from whey, and collecting the whey; adding an equal volume of ultrapure water into whey, adjusting the pH to 4.5-4.6 by using 6N hydrochloric acid, and continuously stirring in the adding process to ensure that the protein precipitation is obvious; putting the mixture into a high-speed refrigerated centrifuge for centrifugation at 5000g for 20min at a temperature of between 2 and 8 ℃, and collecting supernatant after centrifugation; centrifuging 10000g for 60min at 2-8 ℃ and collecting supernatant; centrifuging at the temperature of 2-8 ℃ for 70min at the speed of 150000g, and taking the precipitate to obtain the milk exosomes.
(2) Preparation of cholesterol-RGD
Weighing cholesterol and succinic anhydride according to a mass ratio of 1:0.388, dissolving in pyridine, heating to 45 ℃ for reaction for 72 hours, adding water for dialysis for 3 days, and performing vacuum freeze-drying to obtain the cholesterol-succinic anhydride. 100mg of cholesterol-succinic anhydride is weighed and dissolved in 20mL of dimethyl sulfoxide, 57mg of DMTMM is added for activation for 30min, 151mg of RGD is finally added, stirring is carried out at 45 ℃ for overnight reaction, water is added for dialysis for three days, and vacuum freeze-drying is carried out to obtain the cholesterol-RGD.
(3) Preparation of complex modified exosome-coated cis-resveratrol
Placing a solution of the milk exosome (solvent is physiological saline with the concentration of 5 mg/mL) on a magnetic stirrer, stirring at 500rpm, and dropwise adding an alcoholic solution of cis-resveratrol (solvent is absolute ethanol with the concentration of 500 mg/mL) into the solution of the milk exosome at room temperature according to the mass ratio of cis-resveratrol to the milk exosome of 1:10 to obtain the solution of the milk exosome coated with cis-resveratrol. Ultrasound for 3min at 90W power, and incubating at 4deg.C overnight; adding cholesterol-RGD into a solution of cis-resveratrol wrapped by milk exosomes at room temperature, wherein the ratio of the cholesterol-RGD, the milk exosomes and the cis-resveratrol is 0.1:10:1 by mass ratio, performing ultrasonic treatment for 3min at 90W power, then placing the solution in 37 ℃ for incubation for 30min, and performing freeze drying to obtain the preparation of the cis-resveratrol wrapped by the composite modified exosomes.
Example 2
The embodiment provides a preparation process of a preparation of trans-resveratrol wrapped by a composite modified exosome.
The material sources were the same as in example 1.
(1) Separation and extraction of milk exosomes
Centrifuging 2000g of fresh milk for 20min at 2-8 ℃, separating a fat layer from whey, and collecting the whey; adding an equal volume of ultrapure water into whey, adjusting the pH to 4.5-4.6 by using 6N hydrochloric acid, and continuously stirring in the adding process to ensure that the protein precipitation is obvious; centrifuging 6000g for 20min at 2-8 ℃ in a high-speed refrigerated centrifuge, and collecting supernatant after centrifuging; centrifuging 12000g for 90min at 2-8 ℃ and collecting supernatant; centrifuging 100000g for 90min at 2-8deg.C, and collecting precipitate to obtain milk exosomes.
(2) Preparation of cholesterol-RGD
Weighing cholesterol and succinic anhydride according to a mass ratio of 1:0.2, dissolving in pyridine, heating to 45 ℃ for reaction for 72 hours, adding water for dialysis for 3 days, and performing vacuum freeze-drying to obtain the cholesterol-succinic anhydride. 100mg of cholesterol-succinic anhydride is weighed and dissolved in 20mL of dimethyl sulfoxide, 57mg of DMTMM is added for activation for 30min, and finally 100mg of RGD is added into the mixture, the mixture is stirred for overnight reaction at 45 ℃, water is added for dialysis for three days, and then the mixture is frozen and freeze-dried in vacuum to obtain the cholesterol-RGD.
(3) Preparation of complex-modified exosome-coated trans-resveratrol
Placing a solution of the milk exosome (solvent is physiological saline with the concentration of 5 mg/mL) on a magnetic stirrer, stirring at 500rpm, and dropwise adding an alcoholic solution of trans-resveratrol (solvent is absolute ethanol with the concentration of 500 mg/mL) into the solution of the milk exosome at room temperature to obtain a solution of the milk exosome coated with trans-resveratrol. Ultrasound at 100W power for 4min, and incubating at 4deg.C overnight; adding cholesterol-RGD into a solution of milk exosome coated trans-resveratrol at room temperature, wherein the ratio of the cholesterol-RGD, the milk exosome and the trans-resveratrol is 0.6:10:0.4 by mass ratio, performing ultrasonic treatment for 4min at 100W power, then placing the solution in 37 ℃ for incubation for 30min, and performing freeze drying to obtain the preparation of the compound modified exosome coated trans-resveratrol.
Example 3
The embodiment provides a preparation process of a preparation of resveratrol glycoside coated by a compound modified exosome.
(1) Separation and extraction of milk exosomes
Centrifuging 2500g fresh milk at 2-8deg.C for 20min, separating fat layer from whey, and collecting whey; adding an equal volume of ultrapure water into whey, adjusting the pH to 4.5-4.6 by using 6N hydrochloric acid, and continuously stirring in the adding process to ensure that the protein precipitation is obvious; centrifuging 5500g for 20min at 2-8deg.C in a high-speed refrigerated centrifuge, and collecting supernatant; centrifuging 11000g for 75min at 2-8 ℃ and collecting supernatant; centrifuging 120000g for 75min at 2-8 ℃ to obtain sediment to obtain milk exosomes. The milk exosomes obtained by separation, as shown in fig. 1, are in the form of disc-shaped vesicles under a transmission electron microscope.
(2) Preparation of cholesterol-RGD
Weighing cholesterol and succinic anhydride according to a mass ratio of 1:0.4, dissolving in pyridine, heating to 45 ℃ for reaction for 72 hours, adding water for dialysis for 3 days, and performing vacuum freeze-drying to obtain the cholesterol-succinic anhydride. 100mg of cholesterol-succinic anhydride is weighed and dissolved in 20mL of dimethyl sulfoxide, 57mg of DMTMM is added for activation for 30min, 200mg of RGD is finally added into the mixture, the mixture is stirred for overnight reaction at 45 ℃, water is added for dialysis for three days, and the mixture is frozen and freeze-dried in vacuum to obtain the cholesterol-RGD.
(3) Preparation of compound modified exosome coated resveratrol glycoside
Placing a solution of milk exosomes (solvent is physiological saline with concentration of 5 mg/mL) on a magnetic stirrer, stirring at 500rpm, and dropwise adding an alcoholic solution of resveratrol glycoside (solvent is absolute ethanol with concentration of 500 mg/mL) into the solution of milk exosomes at room temperature to obtain a solution of the resveratrol glycoside coated by the milk exosomes. Ultrasound at 110W for 5min, and incubating at 4deg.C overnight; adding cholesterol-RGD into a solution of resveratrol glycoside wrapped by milk exosomes at room temperature, wherein the ratio of the cholesterol-RGD, the milk exosomes and the resveratrol glycoside is 1:10:0.1 by mass ratio, performing ultrasonic treatment at 110W for 5min, then placing in 37 ℃ for incubation for 30min, and performing freeze drying to obtain the preparation of the resveratrol glycoside wrapped by the compound modified exosomes. The configuration of the modified exosomes wrapping the resveratrol glycoside and the configuration under a transmission electron microscope are shown in fig. 2, the structure of the modified exosomes is complete, and the exosomes are integrally in a disc-shaped vesicle structure.
Comparative example 1
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.
Comparative example 2
The preparation method of this example is the same as that of example 1, except that cholesterol-RGD modification is not performed.
Example 4
The embodiment provides in vitro bioavailability detection of the complex modified exosome-encapsulated resveratrol.
The preparation (RGD-Res-EVs) of resveratrol coated with the complex modified exosome prepared in the above example and the preparation (Res-EVs) not subjected to cholesterol-RGD modification prepared in the comparative example were freeze-dried, and the two were simulated to digest gastric fluid for 2 hours and intestinal fluid for 4 hours, and then centrifuged and the supernatant was collected by filtration. And (3) weighing resveratrol powder, dissolving in absolute ethyl alcohol, diluting according to concentration to obtain standard liquid, establishing a resveratrol content standard curve, measuring absorbance value at 326nm of a resveratrol characteristic absorption peak, and substituting the absorbance value into the standard curve to calculate the resveratrol content.
The bioavailability of resveratrol was calculated according to the following formula:
wherein c digestive juice refers to the concentration of resveratrol in digestive juice after simulated digestion is completed, v supernatant refers to the volume of supernatant after centrifugal filtration, and m resveratrol is the mass of resveratrol before digestion.
TABLE 1 in vitro bioavailability of complex modified exosome-encapsulated resveratrol
| 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％ |
Example 5
The present example provides cytotoxicity detection of complex modified exosome-encapsulated resveratrol.
The human cloned colon cancer Caco-2 cells are cultured, the cell structure and function are similar to that of differentiated small intestine epithelial cells, the cell sub-microscopic structure research shows that the Caco-2 cells and the human small intestine epithelial cells are in the following conditionsMorphologically similar, with the same cell polarity and tight junctions. After the cells are fused to about 80%, the cells are counted after digestion, 3000 cells/hole are inoculated into a 96-well plate, and the cells are placed into a constant temperature cell incubator with 5% carbon dioxide and 37 ℃ for culturing for 48-72 hours, so that the cells are attached to the wall and grow normally. The preparation (RGD-Res-EVs) of resveratrol coated with the complex modified exosome prepared in the example was diluted with MEM (NEAA) +20% FBS medium to 8 concentration gradients of 10%, 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.1563% and 0.0781% in order by mass fraction, and when the cell plating rate in 96-well plate reached 80%, the culture solution containing RGD-Res-EVs with different concentrations was changed, and after the change, the 96-well plate was placed in an incubator (37 ℃ C., 5% CO) 2 ) Is cultured. After incubation of the cells for 24h, the supernatant was discarded, 100. Mu.L of 10% CCK-8 detection solution was added, incubated at 37℃for 2h in the absence of light, 90. Mu.L was aspirated after incubation, and OD was read at 450 nm. According to the calculation of the formula,
table 2 cytotoxicity assay of complex modified exosome-encapsulated resveratrol
Example 6
The preparation of fluorescence-labeled complex-modified exosome-encapsulated resveratrol (cy 5-RGD-Res-EVs) and the preparation of fluorescence-labeled exosome-encapsulated resveratrol (cy 5-Res-EVs) were prepared by using cy5 fluorescence-labeled cholesterol-RGD and cholesterol alone according to the method of example 1.
Culturing Caco-2 cells, inoculating the digested cells on a 12-pore plate cell climbing sheet after the cells are fused to about 80%, and culturing in a constant-temperature cell incubator at 37 ℃ for 48 hours under the condition of 5% carbon dioxide to enable the cells to adhere to the wall and grow normally. The cy5-RGD-EVs and the cy5-EVs are prepared into 5% solution by using MEM (NEAA) serum-free culture medium, old culture solution in an orifice plate is discarded, the cy5-RGD-EVs and the serum-free culture solution of the cy5-EVs are added, the culture solution is incubated in an incubator for 2 hours, culture solution supernatant is sucked off after 2 hours, PBS buffer solution is used for carefully washing twice, hoechst 33342 is used for staining cell nuclei, and the cell nuclei are observed under a fluorescence microscope after washing.
As shown in fig. 3, under the same incubation time, the fluorescence intensity of the preparation of cholesterol-RGD modified exosome coated resveratrol in the Caco-2 cell is higher than that of the preparation without cholesterol-RGD modification, which indicates that the modified exosome preparation can be targeted and ingested by the Caco-2 cell more quickly.
The present invention may be better implemented as described above, and the above examples are merely illustrative of preferred embodiments of the present invention and not intended to limit the scope of the present invention, and various changes and modifications made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the present invention without departing from the spirit of the design of the present invention.
Claims (9)
1. A preparation of resveratrol wrapped by composite modified exosomes is characterized in that resveratrol is wrapped by milk-derived exosomes to obtain resveratrol wrapped by milk-derived exosomes, and then cholesterol-RGD is used for modifying the resveratrol wrapped by milk-derived exosomes to obtain the preparation.
2. The preparation of the resveratrol wrapped by the composite modified exosomes according to claim 1, wherein the ratio of the cholesterol-RGD, the milk-derived exosomes and the resveratrol is 0.1-1:10:0.1-1 in terms of mass ratio.
3. The preparation of resveratrol encapsulated by composite modified exosomes according to claim 1, wherein the milk-derived exosomes are milk exosomes.
4. The preparation of complex-modified exosome-coated resveratrol according to claim 1, wherein the preparation method of cholesterol-RGD comprises: weighing cholesterol and succinic anhydride, dissolving in pyridine, heating to 45 ℃ for reaction for 72 hours, adding water for dialysis for 3 days, and performing vacuum freeze-drying to obtain cholesterol-succinic anhydride;
dissolving the cholesterol-succinic anhydride in dimethyl sulfoxide, adding DMTMM for activation for 30min, adding RGD, stirring at 45 ℃ for overnight reaction, dialyzing with water for three days, and vacuum freeze-drying to obtain cholesterol-RGD.
5. The preparation of the complex-modified exosome-coated resveratrol according to claim 4, wherein the ratio of the cholesterol to the succinic anhydride is 1:0.2 to 0.4;
the ratio of the cholesterol-succinic anhydride to the RGD is 1:1-2 based on the mass ratio.
6. The preparation of complex-modified exosome-coated resveratrol according to claim 1, wherein the preparation method comprises:
(1) Continuously stirring the solution of the milk-derived exosomes, and then dripping an alcohol solution of resveratrol at room temperature;
(2) After ultrasonic treatment, placing at 4 ℃ for incubation for 12-18 h to prepare resveratrol wrapped by milk source exosomes;
(3) And adding the cholesterol-RGD into the resveratrol wrapped by the milk-derived exosome at room temperature, performing ultrasonic treatment, incubating at 37 ℃ for 30min, and freeze-drying to obtain the preparation of the resveratrol wrapped by the compound modified exosome.
7. The preparation of the resveratrol wrapped by the composite modified exosomes according to claim 6, wherein the ultrasonic condition in the step (2) is ultrasonic for 3-5 min under the power of 90-110W;
and (3) performing ultrasonic treatment for 3-5 min under the power of 90-110W under the ultrasonic condition.
8. The complex-modified-exosome-coated resveratrol formulation according to claim 6, wherein the rotational speed of continuous stirring is 500rpm.
9. The preparation of resveratrol wrapped by the compound modified exosomes according to claim 6, wherein physiological saline is selected as a solvent for the solution of the milk-derived exosomes;
the alcohol solution of resveratrol adopts absolute ethyl alcohol as solvent.
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| CN116785194A (en) * | 2023-08-21 | 2023-09-22 | 天津外泌体科技有限公司 | Milk exosome loaded alkylated cosmetic peptide and application thereof in cosmetics |
| CN118440884A (en) * | 2024-03-27 | 2024-08-06 | 陕西微泌生物科技有限公司 | Preparation method and application of engineering milk exosomes |
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| CN116785194A (en) * | 2023-08-21 | 2023-09-22 | 天津外泌体科技有限公司 | Milk exosome loaded alkylated cosmetic peptide and application thereof in cosmetics |
| CN118440884A (en) * | 2024-03-27 | 2024-08-06 | 陕西微泌生物科技有限公司 | Preparation method and application of engineering milk exosomes |
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