CN111567798B - Construction method of targeted intestinal slow-release functional factor exosome based on brown algae - Google Patents

Construction method of targeted intestinal slow-release functional factor exosome based on brown algae Download PDF

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CN111567798B
CN111567798B CN202010512675.XA CN202010512675A CN111567798B CN 111567798 B CN111567798 B CN 111567798B CN 202010512675 A CN202010512675 A CN 202010512675A CN 111567798 B CN111567798 B CN 111567798B
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exosome
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active ingredient
brown algae
phosphate buffer
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CN111567798A (en
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蔡路昀
曹爱玲
曹敏杰
刘东红
李秀霞
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Zhejiang University ZJU
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Abstract

The invention discloses a construction method of targeted intestinal slow-release functional factor exosomes based on brown algae, belonging to the field of health-care food. The exosome loaded with the functional factors can target intestinal tracts and has the characteristics of strong penetrating power, high safety, stability in vivo and high targeting property.

Description

Construction method of targeted intestinal slow-release functional factor exosome based on brown algae
Technical Field
The invention belongs to the field of health-care food, and particularly relates to a construction method of a targeted intestinal tract slow-release functional factor exosome based on brown algae.
Background
The exosome is a transport vesicle which is secreted by a living cell and released into an extracellular environment and has the size of 60-100nm, has natural substance transport characteristics, relatively small molecular structure and excellent biocompatibility, is a natural transport carrier and can avoid phagocytosis of an immune system. Active ingredients in food such as carotenoid, polyphenol compounds, organic sulfide and the like have good regulating effect on intestinal flora, intestinal inflammation and intestinal absorption and metabolism, but the active ingredients are easy to degrade and oxidize in heating, acid-base solution and oxidation environments, have the defects of poor targeting property, low bioavailability and the like, and limit the wide application of the active ingredients. Many encapsulation carriers, such as liposomes, microcapsules, emulsions, etc., encapsulate the active ingredients inside them, protecting them from degradation, and improving the stability of the active ingredients, but these carriers have the disadvantages of poor safety, poor targeting, poor penetration, etc.
At present, no related research on extraction of brown algae exosomes exists, and no related report that exosomes load active ingredients to intestinal tracts exists. Therefore, the active ingredients are encapsulated in the exosomes of the brown algae, the exosomes are subjected to surface modification, so that the active ingredients have a stronger intestinal tract targeting effect, the cytotoxicity is reduced, the bioavailability of the active ingredients is improved, a novel exosome extraction raw material is provided, and the application of the exosomes is expanded.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a construction method of a targeted intestinal tract slow-release functional factor exosome based on brown algae. The method can modify exosome to target intestinal tract, improve delivery efficiency of active ingredient, and reduce toxicity.
The purpose of the invention is realized by the following technical scheme: a construction method of targeted intestinal slow-release functional factor hydrophobic exosomes based on brown algae specifically comprises the following steps:
(1) washing fresh brown algae with water for 2-3 times, chopping, homogenizing at 0-4 deg.C for 1-2min to obtain brown algae juice, centrifuging brown algae juice at 2000g for 20min, centrifuging at 10000g for 1 hr, removing residue, filtering, and collecting supernatant. And (3) extracting and purifying the supernatant by using a body fluid exosome extraction kit to obtain brown algae exosome solution, and dissolving the brown algae exosome solution in Phosphate Buffer Solution (PBS) to make the concentration of the brown algae exosome solution be 1-3mg/mL, thereby obtaining an exosome-phosphate buffer solution.
(2) Dissolving 1-3mg of ligand and 1-2mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in 0.1-0.15mL of dimethyl sulfoxide (DMSO), keeping the temperature at 45-55 ℃ for 10-20min, cooling to 20-25 ℃, adding 2-4mL of the exosome-phosphate buffer solution obtained in the step 1, reacting for 2h to obtain a conjugate, purifying by using a sucrose gradient solution, and dissolving the purified exosome in PBS to obtain an exosome dispersion liquid.
(3) Mixing 0.5-1.5mg/mL of hydrophobic active ingredient solution with the exosome dispersion obtained in the step 2, keeping the solute concentration to be more than or equal to 10wt%, standing at room temperature for 12-18min, then centrifuging at 10000g for 10min, then centrifuging at 135000g for 90min at 4 ℃, and collecting precipitates, namely the exosomes loaded with the hydrophobic active ingredient. The solvent of the hydrophobic active ingredient solution is prepared from acetonitrile and ethanol according to a volume ratio of 1: 1. The solute in the hydrophobic active ingredient solution is polyphenol, flavonoid, isoflavonoid, carotene and its derivatives, hydrophobic vitamin, protein or polysaccharide, etc.
The invention also provides a construction method of the targeted intestinal slow-release functional factor hydrophilic exosome based on brown algae, which specifically comprises the following steps:
(1) washing fresh brown algae with water for 2-3 times, chopping, homogenizing at 0-4 deg.C for 1-2min to obtain brown algae juice, centrifuging brown algae juice at 2000g for 20min, centrifuging at 10000g for 1 hr, removing residue, filtering, and collecting supernatant. And (3) extracting and purifying the supernatant by using a body fluid exosome extraction kit to obtain brown algae exosome solution, and dissolving the brown algae exosome solution in Phosphate Buffer Solution (PBS) to make the concentration of the brown algae exosome solution be 1-3mg/mL, thereby obtaining an exosome-phosphate buffer solution.
(2) Dissolving 1-3mg of ligand and 1-2mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in 0.1-0.15mL of dimethyl sulfoxide (DMSO), keeping the temperature at 45-55 ℃ for 10-20min, cooling to 20-25 ℃, adding 2-4mL of the exosome-phosphate buffer solution obtained in the step 1, reacting for 2h to obtain a conjugate, purifying by using a sucrose gradient solution, and dissolving the purified exosome in PBS to obtain an exosome dispersion liquid.
(3) 30-60. mu.L of a solution of a hydrophilic active compound having a concentration of 0.5-1.5mg/mL and 100. mu.L of an exosome dispersion having a concentration of 0.5-1.5mg/mL were mixed in an electroporation cuvette, and the electroporation cuvette was subjected to electroporation using an electroporator under conditions of 350V and 150 ms. And then incubating at 37 ℃ to fuse the membrane of the exosome to obtain the hydrophilic exosome. The solute in the hydrophilic active ingredient solution is hydrophilic polysaccharide, protein and the like which are dissolved in water with the mass fraction of at least 0.001%.
The invention also provides a construction method of the targeted intestinal slow-release functional factor amphoteric exosome based on brown algae, which specifically comprises the following steps:
(1) washing fresh brown algae with water for 2-3 times, chopping, homogenizing at 0-4 deg.C for 1-2min to obtain brown algae juice, centrifuging brown algae juice at 2000g for 20min, centrifuging at 10000g for 1 hr, removing residue, filtering, and collecting supernatant. And (3) extracting and purifying the supernatant by using a body fluid exosome extraction kit to obtain brown algae exosome solution, and dissolving the brown algae exosome solution in Phosphate Buffer Solution (PBS) to make the concentration of the brown algae exosome solution be 1-3mg/mL, thereby obtaining an exosome-phosphate buffer solution.
(2) Dissolving 1-3mg of ligand and 1-2mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in 0.1-0.15mL of dimethyl sulfoxide (DMSO), keeping the temperature at 45-55 ℃ for 10-20min, cooling to 20-25 ℃, adding 2-4mL of the exosome-phosphate buffer solution obtained in the step 1, reacting for 2h to obtain a conjugate, purifying by using a sucrose gradient solution, and dissolving the purified exosome in PBS to obtain an exosome dispersion liquid.
(3) Diluting the exosome with PBS solution to a solution with total protein concentration of 0.1-0.3mg/mL, and then adding an amphiphilic active ingredient PBS solution with the same volume as the solution and the concentration of 0.3-0.6mg/mL to obtain a mixture of the amphiphilic active ingredient and the exosome. The amphiphilic active ingredient is protein, polysaccharide or phospholipid material.
(4) Incubating the mixture of the amphiphilic active ingredient and the exosome for 15-20 hours at room temperature, adding saponin with the concentration of 0.1-0.3wt%, and oscillating for 20 minutes at room temperature to obtain the exosome loaded with the amphiphilic active ingredient.
Further, step 4 may also be: the mixture of amphiphilic active ingredient and exosomes was incubated for 25-35 minutes, then frozen at-80 ℃ for 30-90 minutes, and subsequently thawed at room temperature for 15-30 minutes. Repeating freeze-thaw cycle for 3-4 times to obtain the exosome loaded with the amphiphilic active ingredient.
Further, in the step 2, the ligand is wheat germ agglutinin, lactoferrin or vitamin B12.
Further, the method for purifying by using sucrose gradient solution in step 2 comprises: adding the obtained conjugate into an ultracentrifuge tube, then sequentially adding 30wt%, 45wt% and 60wt% of sucrose solutions, centrifuging for 1.5h at the temperature of 4 ℃ under 100000g, then forming a bright band between the 30wt% and 45wt% of sucrose solutions and between the 45wt% and 60wt% of sucrose solutions, and collecting to obtain the purified exosome.
Further, the pH of the phosphate buffer solutions was 7.4.
Compared with the prior art, the invention has the beneficial effects that: the invention firstly uses brown algae to extract exosome, and compared with more extracting materials such as milk, cells and the like, the brown algae has wider sources and lower cost. The invention adopts the kit to extract the exosome of the plant tissue, is convenient and quick, and related reports are not found. Compared with the non-modified exosome, the exosome can accurately target intestinal cells, the exosome prepared by the invention can load hydrophilic, hydrophobic and amphipathic functional active ingredients, the application of the exosome in the field of health food is expanded, and compared with carriers such as emulsion, microspheres and nanoparticles, the exosome is from plants, has high safety, can widely load various different functional active ingredients, and has the natural advantages of strong penetrating power and high stability. Improves the target slow release effect on intestinal cells, strengthens the local effect of the bioactive components in the intestinal tract, and has better prevention and treatment effect on intestinal tract related diseases.
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FIG. 1 is a transmission electron micrograph of exosomes.
Detailed Description
The pH of the phosphate buffer solution used in the present invention was 7.4.
Example 1
The invention discloses a construction method of a targeted intestinal slow-release functional factor hydrophobic exosome based on brown algae, which specifically comprises the following steps:
(1) washing fresh brown algae with water for 2 times, chopping, homogenizing at 0 deg.C for 1min to obtain brown algae juice, centrifuging at 2000g for 20min, centrifuging at 10000g for 1h, removing residue, filtering, and collecting supernatant. And extracting and purifying the supernatant by using a body fluid exosome extraction kit to obtain a brown algae exosome solution.
The brown algae exosome solution obtained by the method is observed by a transmission electron microscope, and the specific method comprises the following steps: and (3) dropwise adding 10 mu L of exosome on a copper net for precipitating for 1min, sucking surface liquid by using filter paper, dropwise adding 10 mu L of phosphotungstic acid on the copper net for precipitating for 1min, sucking the surface liquid by using the filter paper, drying at normal temperature for 5-10 min, and performing electron microscope detection imaging at 100kV to obtain a transmission electron microscope imaging result. As shown in FIG. 1, the brown algae exosomes have membrane vesicle-like structure, and the outer surface of the vesicle is visible as membranes, and the outer shape is circular or elliptical and the particle size is about 40 nm.
Therefore, the brown algae exosome solution is dissolved in Phosphate Buffered Saline (PBS), and the concentration of the brown algae exosome solution is measured to be 2.68mg/mL, so that an exosome-phosphate buffered solution is obtained.
(2) 1mg of wheat germ agglutinin and 1mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) were dissolved in 0.1mL of dimethyl sulfoxide (DMSO), keeping the temperature at 45 ℃ for 10min, then cooling to 20 ℃, adding 2mL of the exosome-phosphate buffer solution obtained in the step 1, reacting for 2h to obtain a conjugate, adding the obtained conjugate into an ultracentrifuge tube, then sucrose solutions with the concentrations of 30wt%, 45wt% and 60wt% are added in sequence, centrifuging at 100000g at 4 deg.C for 1.5 hr to obtain sucrose solutions with a bright band between 30wt% and 45wt% and between 45wt% and 60wt%, collecting, namely the purified exosome, and then dissolving the purified exosome in PBS to obtain an exosome dispersion liquid.
(3) Mixing 0.5mg/mL kaempferol solution with the exosome dispersion obtained in the step 2, and keeping the concentration of the solvent at 10wt%, wherein the solvent of the kaempferol solution is prepared by mixing acetonitrile and ethanol according to the volume ratio of 1: 1. Standing at room temperature for 12min, centrifuging at 10000g for 10min, centrifuging at 135000g at 4 deg.C for 90min, and collecting precipitate to obtain exosome loaded with hydrophobic active ingredient. And detecting the concentration of the kaempferol remained in the supernatant, calculating the difference value between the initial kaempferol total amount and the kaempferol total amount in the residual solution, and calculating to obtain the exosome loaded with the hydrophobic active ingredients, wherein the exosome is up to 60.8%, so that the brown algae exosome has a good protection effect on the kaempferol and is prevented from being damaged by external conditions.
Carrying out in-vitro digestion experiments on the exosome loaded with the hydrophobic active ingredients obtained by the method, wherein the method comprises the following steps: the exosomes loaded with hydrophobic active ingredient were formulated at a concentration of 2 mg/mL. Loading hydrophobic active ingredient in exosome-PBS solution, adding 1.34 μ L of hydrophobic active ingredient-loaded exosome-PBS solution into 18.5% w/v HCl solution with pH of 2.0 and 2.024 μ L of 80mg/mL pepsin-HCl solution, incubating slowly by rotation at 37 deg.C for 30min, and dissolving 80 μ L of NaHCO solution with concentration of 24mg/mL and pancreatin with concentration of 4mg/mL in 0.1N3In the method, NaHCO with the mass concentration of 0.1N is used3The stability of the hydrophobic active ingredient loaded exosomes was evaluated by adjusting the pH to 6.5, and incubating for 30min under the same conditions, by measuring the particle size and surface charge.
As can be seen from Table 1, the average particle size of the exosomes loaded with the hydrophobic active ingredient before digestion was 103.12. + -. 29.87aThe grain size is uniform and consistent, and no obvious fluctuation exists, which shows that the form of the exosome loaded with the hydrophobic active ingredient can better maintain the original state, and no obvious aggregation and damage exist; the Zeta potential of the compound is-127.91 +/-174.82 mV, the compound has better in vitro stability, and the surface of the compound is charged negatively and accords with the characteristics of exosomes. After in vitro simulated gastrointestinal tract digestion, the grain size and the potential of the exosome are slightly reduced, but no significant difference exists, which indicates that the exosome can still keep better grain size and potential after in vivo gastrointestinal tract digestion, can better protect well-embedded functional factors, and achieves the purpose of stable delivery.
TABLE 1 particle size and potential analysis of exosomes before and after in vitro digestion
Exosomes Average particle diameter/nm Zeta potential/mV
Before digestion 103.12±29.87a -127.91±174.82a
After digestion 75.83±19.48a -101.52±160.99a
In addition, the hydrophobic active ingredient used in the technical scheme of the present invention may be polyphenol, flavonoid, isoflavonoid, carotene and its derivatives, hydrophobic vitamin, protein or polysaccharide, etc., and kaempferol is only one example.
Example 2
The invention also discloses a construction method of the targeted intestinal tract slow-release functional factor hydrophilic exosome based on brown algae, which specifically comprises the following steps:
(1) washing fresh brown algae with water for 3 times, chopping, homogenizing at 4 deg.C for 2min to obtain brown algae juice, centrifuging at 2000g for 20min, centrifuging at 10000g for 1 hr, removing residue, filtering, and collecting supernatant. And (3) extracting and purifying the supernatant by using a body fluid exosome extraction kit to obtain a brown algae exosome solution, and the brown algae exosome is in a membrane vesicle shape, is in a structure of a visible membrane type at the periphery of the vesicle, is circular or elliptical in appearance and has the particle size of about 40nm as confirmed by transmission electron microscope observation. Dissolving the brown algae exosome solution in Phosphate Buffer Solution (PBS) to ensure that the concentration of the brown algae exosome solution is 1-3mg/mL, thereby obtaining exosome-phosphate buffer solution.
(2) Dissolving 3mg of lactoferrin and 2mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in 0.15mL of dimethyl sulfoxide (DMSO), keeping the temperature at 55 ℃ for 20min, then cooling to 25 ℃, adding 4mL of the exosome-phosphate buffer solution obtained in the step 1, reacting for 2h to obtain a conjugate, purifying by using a sucrose gradient solution, and dissolving the purified exosome in PBS to obtain an exosome dispersion liquid.
(3) mu.L of the antimicrobial peptide parasinI solution having a concentration of 1.5mg/mL was mixed with 200. mu.L of the exosome dispersion having a concentration of 1.5mg/mL in an electroporation cuvette, and the electroporation cuvette was electroporated with an electroporator under conditions of 350V and 150 ms. And then incubating at 37 ℃ to fuse the membrane of the exosome to obtain the hydrophilic exosome.
Carrying out in-vitro digestion experiments on the exosomes loaded with the hydrophilic active ingredients obtained by the method, and evaluating the stability of the exosomes loaded with the hydrophilic active ingredients by measuring the particle size and the surface charge. Experimental data show that before and after in vitro digestion, the particle size of the exosome loaded with the hydrophilic active ingredient has no obvious difference, and the Zeta potential has no obvious change, which shows that the exosome loaded with the hydrophilic active ingredient can still keep better particle size and potential after in vivo gastrointestinal tract digestion, can better protect well-embedded functional factors, and achieves the purpose of stable delivery.
In addition, the hydrophilic active ingredient adopted in the technical scheme of the invention can be hydrophilic polysaccharide, protein and the like with the mass fraction of at least 0.001 percent dissolved in water, and the antimicrobial peptide parasin I is only one example.
Example 3
The invention also discloses a construction method of the targeting intestinal slow-release functional factor amphoteric exosome based on brown algae, which specifically comprises the following steps:
(1) washing fresh brown algae with water for 3 times, chopping, homogenizing at 4 deg.C for 2min to obtain brown algae juice, centrifuging at 2000g for 20min, centrifuging at 10000g for 1h, removing residue, filtering, and collecting supernatant. And (3) extracting and purifying the supernatant by using a body fluid exosome extraction kit to obtain brown algae exosome solution, and observing by using a transmission electron microscope to verify that the brown algae exosomes are membrane vesicle-shaped, the outer periphery of the vesicle is in a visible membrane structure, the outer shape is circular or oval, and the particle size is about 40 nm. Dissolving the brown algae exosome solution in Phosphate Buffer Solution (PBS) to ensure that the concentration of the brown algae exosome solution is 1-3mg/mL, thereby obtaining exosome-phosphate buffer solution.
(2) 3mg of vitamin B12 and 2mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) were dissolved in 0.15mL of dimethyl sulfoxide (DMSO) and maintained at 55 ℃ for 20min, followed by cooling to 25 ℃ and 4mL of the exosome-phosphate buffer solution obtained in step 1 was added, and after reacting for 2h, a conjugate was obtained, which was purified using a sucrose gradient solution, and the purified exosome was dissolved in PBS to obtain an exosome dispersion.
(3) And (3) diluting the exosome to a solution with the total protein concentration of 0.1mg/mL by using a PBS solution, and then adding a lecithin PBS solution with the concentration of 0.3mg/mL and the volume equal to that of the solution to obtain a mixture of the amphiphilic active ingredient and the exosome.
(4) And (3) incubating the mixture of the amphiphilic active ingredient and the exosomes for 15 hours at room temperature, adding saponin with the concentration of 0.1 wt%, and oscillating for 20 minutes at room temperature to obtain the exosomes loaded with the amphiphilic active ingredient.
Carrying out in-vitro digestion experiments on the exosomes loaded with the amphiphilic active ingredients obtained by the method, and evaluating the stability of the exosomes loaded with the hydrophilic active ingredients by measuring the particle size and the surface charge. Experimental data show that before and after in vitro digestion, the particle size of the exosome loaded with the amphiphilic active ingredient is not obviously changed, and Zeta potential is not obviously changed, so that the exosome loaded with the amphiphilic active ingredient can still keep better particle size and potential after in vivo gastrointestinal tract digestion, can better protect well-embedded functional factors, and achieves the purpose of stable delivery.
Example 4
Obtaining a mixture of the amphiphilic active ingredient and the exosomes by the method of the steps (1) to (3) in the step 3, incubating the mixture for 20 hours at room temperature, adding saponin with the concentration of 0.3wt%, and oscillating for 20 minutes at room temperature to obtain the exosomes loaded with the amphiphilic active ingredient.
Carrying out in-vitro digestion experiments on the exosomes loaded with the amphiphilic active ingredients obtained by the method, and evaluating the stability of the exosomes loaded with the amphiphilic active ingredients by measuring the particle size and the surface charge. Experimental data show that before and after in vitro digestion, the particle size and Zeta potential of the exosome loaded with the amphiphilic active ingredient are not influenced by receptor in vitro digestion, which indicates that the exosome loaded with the amphiphilic active ingredient can still keep better particle size and potential after being digested by gastrointestinal tracts in vivo, can better protect well-embedded functional factors, and achieves the purpose of stable delivery.
Example 5
The mixture of the amphiphilic active ingredient and exosomes was obtained using the method of steps (1) to (3) in example 3, incubated for 25 minutes, then frozen at-80 ℃ for 30 minutes, and then thawed at room temperature for 15 minutes. Repeating the freeze-thaw cycle for 3 times to obtain the exosome loaded with the amphiphilic active ingredient.
Carrying out in-vitro digestion experiments on the exosomes loaded with the amphiphilic active ingredients obtained by the method, and evaluating the stability of the exosomes loaded with the amphiphilic active ingredients by measuring the particle size and the surface charge. Experimental data show that the particle size of the exosome loaded with the amphiphilic active ingredient has no obvious change before and after in vitro digestion, and the Zeta potential has no obvious change, which shows that the exosome loaded with the amphiphilic active ingredient can still keep better particle size and potential after in vivo gastrointestinal tract digestion, can better protect well-embedded functional factors, and achieves the purpose of stable delivery.
Example 6
The method of steps (1) to (3) of example 3 was used to obtain a mixture of amphiphilic active ingredient and exosomes, which was incubated for 35 minutes, then frozen at-80 ℃ for 90 minutes, followed by thawing at room temperature for 30 minutes. And repeating the freeze-thaw cycle for 4 times to obtain the exosome loaded with the amphiphilic active ingredient.
Carrying out in-vitro digestion experiments on the exosomes loaded with the amphiphilic active ingredients obtained by the method, and evaluating the stability of the exosomes loaded with the amphiphilic active ingredients by measuring the particle size and the surface charge. Experimental data show that the particle size of the exosome loaded with the amphiphilic active ingredient has no significant change and the Zeta potential has no significant change before and after in vitro digestion, which shows that the exosome loaded with the amphiphilic active ingredient can still keep better particle size and potential after in vivo gastrointestinal tract digestion, can better protect well-embedded functional factors and achieves the purpose of stable delivery.
In addition, the amphiphilic active ingredient adopted in the technical scheme of the invention can be protein, polysaccharide or phospholipid substances, and lecithin is only one example.

Claims (5)

1. A construction method of targeted intestinal slow-release functional factor hydrophobic exosomes based on brown algae is characterized by comprising the following steps:
(1) washing fresh brown algae with water for 2-3 times, chopping, homogenizing at 0-4 deg.C for 1-2min to obtain brown algae juice, centrifuging brown algae juice at 2000g for 20min, centrifuging at 10000g for 1h, removing residue, filtering, and collecting supernatant; extracting and purifying the supernatant by using a body fluid exosome extraction kit to obtain brown algae exosome solution, and dissolving the brown algae exosome solution in phosphate buffer solution PBS to make the concentration of the brown algae exosome solution be 1-3mg/mL, so as to obtain exosome-phosphate buffer solution;
(2) dissolving 1-3mg of ligand and 1-2mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC in 0.1-0.15mL of dimethyl sulfoxide DMSO, keeping the temperature at 45-55 ℃ for 10-20min, cooling to 20-25 ℃, adding 2-4mL of the exosome-phosphate buffer solution obtained in the step (1), reacting for 2h to obtain a conjugate, purifying by adopting a sucrose gradient solution, and dissolving the purified exosome in PBS to obtain an exosome dispersion liquid;
the ligand is wheat germ agglutinin, lactoferrin or vitamin B12;
(3) mixing 0.5-1.5mg/mL of hydrophobic active ingredient solution with the exosome dispersion liquid obtained in the step (2), keeping the solute concentration to be more than or equal to 10wt%, standing at room temperature for 12-18min, then centrifuging at 10000g for 10min, then centrifuging at 4 ℃ at 135000g for 90min, and collecting precipitate to obtain the exosome loaded with the hydrophobic active ingredient; the solvent of the hydrophobic active ingredient solution is prepared from acetonitrile and ethanol according to a volume ratio of 1: 1, preparing a composition; the solute in the hydrophobic active ingredient solution is polyphenol, flavonoid, isoflavonoid, carotene, vitamin, protein or polysaccharide.
2. A construction method of targeted intestinal slow-release functional factor hydrophilic exosomes based on brown algae is characterized by comprising the following steps:
(1) washing fresh brown algae with water for 2-3 times, chopping, homogenizing at 0-4 deg.C for 1-2min to obtain brown algae juice, centrifuging at 2000g for 20min, centrifuging at 10000g for 1 hr, removing residue, filtering, and collecting supernatant; extracting and purifying the supernatant by using a body fluid exosome extraction kit to obtain a brown alga exosome solution, and dissolving the brown alga exosome solution in phosphate buffer solution PBS to ensure that the concentration of the brown alga exosome solution is 1-3mg/mL to obtain an exosome-phosphate buffer solution;
(2) dissolving 1-3mg of ligand and 1-2mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC in 0.1-0.15mL of dimethyl sulfoxide DMSO, keeping the temperature at 45-55 ℃ for 10-20min, cooling to 20-25 ℃, adding 2-4mL of the exosome-phosphate buffer solution obtained in the step (1), reacting for 2h to obtain a conjugate, purifying by adopting a sucrose gradient solution, and dissolving the purified exosome in PBS to obtain an exosome dispersion liquid;
the ligand is wheat germ agglutinin, lactoferrin or vitamin B12;
(3) mixing 30-60 mu L of hydrophilic active ingredient solution with the concentration of 0.5-1.5mg/mL and 100-200 mu L of exosome dispersion liquid electroporation test tube with the concentration of 0.5-1.5mg/mL, and performing electroporation on the electroporation test tube by using an electroporator under the conditions of 350V and 150 ms; then incubating at 37 ℃ to fuse the membrane of the exosome to obtain the exosome loaded with the hydrophilic active ingredient; the solute in the hydrophilic active ingredient solution is hydrophilic polysaccharide and protein with the mass fraction of at least 0.001 percent dissolved in water.
3. A construction method of targeted intestinal slow-release functional factor amphoteric exosomes based on brown algae is characterized by comprising the following steps:
(1) washing fresh brown algae with water for 2-3 times, chopping, homogenizing at 0-4 deg.C for 1-2min to obtain brown algae juice, centrifuging at 2000g for 20min, centrifuging at 10000g for 1 hr, removing residue, filtering, and collecting supernatant; extracting and purifying the supernatant by using a body fluid exosome extraction kit to obtain a brown alga exosome solution, and dissolving the brown alga exosome solution in phosphate buffer solution PBS to ensure that the concentration of the brown alga exosome solution is 1-3mg/mL to obtain an exosome-phosphate buffer solution;
(2) dissolving 1-3mg of ligand and 1-2mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC in 0.1-0.15mL of dimethyl sulfoxide DMSO, keeping the temperature at 45-55 ℃ for 10-20min, cooling to 20-25 ℃, adding 2-4mL of the exosome-phosphate buffer solution obtained in the step (1), reacting for 2h to obtain a conjugate, purifying by adopting a sucrose gradient solution, and dissolving the purified exosome in PBS to obtain an exosome dispersion liquid;
the ligand is wheat germ agglutinin, lactoferrin or vitamin B12;
(3) diluting exosome with PBS solution to a solution with total protein concentration of 0.1-0.3mg/mL, and then adding an amphiphilic active ingredient PBS solution with the same volume as the solution and the concentration of 0.3-0.6mg/mL to obtain a mixture of the amphiphilic active ingredient and exosome; the amphiphilic active ingredient is protein, polysaccharide or phospholipid substance;
(4) incubating the mixture of the amphiphilic active ingredient and the exosome for 15-20 hours at room temperature, adding saponin with the concentration of 0.1-0.3wt%, and oscillating for 20 minutes at room temperature to obtain the exosome loaded with the amphiphilic active ingredient; or incubating a mixture of the amphiphilic active ingredient and exosomes for 25-35 minutes, then freezing at-80 ℃ for 30-90 minutes, followed by thawing at room temperature for 15-30 minutes; repeating the freeze-thaw cycle for 3-4 times to obtain the exosome loaded with the amphiphilic active ingredient.
4. The method for constructing according to any one of claims 1 to 3, wherein the purification using a sucrose gradient solution in step (2) is performed by: adding the obtained conjugate into an ultracentrifuge tube, then sequentially adding 30wt%, 45wt% and 60wt% of sucrose solutions, centrifuging for 1.5h at the temperature of 4 ℃ under 100000g, then forming a bright band between the 30wt% and 45wt% of sucrose solutions and between the 45wt% and 60wt% of sucrose solutions, and collecting to obtain the purified exosome.
5. The construction method according to any one of claims 1 to 3, wherein the phosphate buffer solution has a pH of 7.4.
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