CN115340978A

CN115340978A - Preparation method of milk-derived exosome

Info

Publication number: CN115340978A
Application number: CN202211282350.2A
Authority: CN
Inventors: 王文丽; 牛水芹; 翟艳华; 何新军; 许可
Original assignee: Suzhou Weisilkang Technology Co ltd
Current assignee: Suzhou Weisilkang Technology Co ltd
Priority date: 2022-10-19
Filing date: 2022-10-19
Publication date: 2022-11-15
Anticipated expiration: 2042-10-19
Also published as: CN115340978B

Abstract

The invention relates to a preparation method of milk-derived exosomes. The preparation method comprises the following steps: removing fat and milk protein in the milk to obtain acidic whey with pH value of 4~6; (II) concentrating the acidic whey to obtain acidic concentrated whey; thirdly, adjusting the pH value of the acidic concentrated whey to 6.5 to 7.5 by using an alkaline substance to obtain neutral whey; and (IV) separating and purifying the neutral whey to obtain the milk-derived exosome. The preparation method can obtain the milk-derived exosome with higher purity and higher yield at lower cost, can provide an idea for the industrial preparation of the milk-derived exosome, and provides a support for the mining of the medicinal value of the natural milk-derived exosome and the development of a drug delivery technology based on the milk-derived exosome.

Description

Preparation method of milk-derived exosome

Technical Field

The invention relates to a preparation method of exosome, in particular to a method for separating exosome from emulsion.

Background

Extracellular Vesicles (EVs) are tiny membrane vesicles having a membrane structure secreted by most cells including eukaryotes and prokaryotes, and are mainly classified into exosomes, microvesicles (MVs) or Microparticles (MPs) and apoptotic bodies according to size and origin. The exosome is a vesicle formed by fusing multivesicular bodies (MVBs) and a plasma membrane, wherein the MVs are formed by directly budding outwards from the plasma membrane, and apoptotic bodies are released after apoptosis. The biological role of EVs has been widely reported over the past decades. EVs are nanoscale vesicles that can transfer a carried bioactive molecule from a donor cell to a recipient cell through various mechanisms such as membrane fusion, receptor-ligand interaction, endocytosis or phagocytosis, and thus can serve as a carrier for intercellular information communication, for the transfer of proteins, lipids, nucleic acids, and metabolic molecules, and for effector stimulation and regulation of downstream recipient cells. Compared with the traditional nano material, EVs has the advantages of biocompatibility, biodegradability, low toxicity, non-immunogenicity and the like, and is one of the most promising candidates in nano medicine.

In nanomedicine, most of the research is focused on exosomes and MVs/MPs, with less research on apoptotic bodies. With the development of research on extracellular vesicles, the functions of natural exosomes separated from various body fluids such as plasma, saliva, urine, cerebrospinal fluid and emulsion are gradually discovered, and a material basis is laid for the exploitation of the value of the natural exosomes in disease diagnosis and clinical application. However, the industrial extraction and purification of exosomes often require expensive large-scale cell culture to obtain larger amounts of upstream raw materials, and reducing the upstream cost is the biggest problem facing the pharmaceutical industry of exosomes. Compared with exosomes from other sources such as plasma, exosomes from milk sources (milk-derived exosomes for short) such as milk exosomes have the advantages of lower immunogenicity, higher biocompatibility, natural targeting capability, good biological barrier permeability and the like, and meanwhile, the milk-derived exosomes also have the advantages of wide sources and low raw material cost, and can be used as a preferred carrier of natural drugs and nano-drugs. There are also literature reports that milk-derived exosomes can survive in the strongly acidic environment in the stomach, and milk exosomes would be one of the most promising oral delivery vehicle systems.

The extraction technology of the exosome mainly comprises an ultracentrifugation method, a density gradient centrifugation method, a chemical precipitation method, a size exclusion method, an immunocapture method and the like. Ultracentrifugation separates the supernatant of the exosome through different centrifugal forces and centrifugal times according to the difference of sedimentation speeds of the exosome, the protein, cell debris, cells, organelles and other substances in a sample; the density gradient centrifugation method realizes separation by using density difference of exosome and other solutes; the chemical precipitation method changes the solubility and the dispersibility of exosome through polyethylene glycol (PEG) and the like, so that components with lower solubility are separated out from the solution; the size exclusion method is a method for separating and extracting by using a chromatographic column according to the size of the exosome, and can obtain relatively complete exosomes.

The emulsion is rich in a large amount of free protein and fat, and is very easy to interfere the extraction and purification of the exosome, and the method is a general method, and the quality and the yield of the milk-derived exosome cannot be considered simultaneously when the emulsion is used for extracting and purifying the milk-derived exosome. The purity is ensured, the yield cannot be improved, and accordingly, the production cost and the use cost of the exosome are high, and the production efficiency is low; the quality of the exosome such as purity, integrity of a membrane structure and the like is reduced when the yield is ensured, and accordingly, the downstream test on the natural biological activity of the milk-derived exosome is interfered, and the development of a drug delivery system based on the milk-derived exosome is influenced.

Patent CN 111012924A discloses a preparation method of a milk exosome, wherein skim milk is obtained through centrifugation, acid adjustment is performed to remove casein, and centrifugation and microfiltration are combined to obtain the milk exosome.

Patent CN 109468265B discloses a method for extracting cow's milk exosomes, which removes milk lipid layer by centrifugation, adjusts acid of supernatant, removes casein by using rennin, collects cow's milk exosomes by microfiltration, and has poor yield and purity, and also has cow's milk exosome damage.

Patent CN113061571 a discloses a separation and identification method of holstein cow milk exosomes, which performs cow milk exosome purification by a combination of means such as centrifugal milk protein removal, density gradient centrifugation and the like.

Despite the prior art, a preparation method of the milk-derived exosome which can give consideration to both the quality and the yield of the exosome is always lacked. The method which can give consideration to both the quality and the yield of the exosome is provided, and the problems of mining the medicinal value of the milk-derived exosome and drug delivery development based on the milk-derived exosome are mainly solved.

Disclosure of Invention

The technical problem solved by the invention is to overcome the defects of the prior art and provide an improved preparation method of milk-derived exosomes.

In order to solve the technical problems, the invention adopts the following technical scheme:

a preparation method of milk-derived exosomes takes milk as a raw material, and comprises the following working procedures:

(1) Removing fat and milk protein in the milk to obtain acidic whey with pH of 4~6;

(2) Concentrating said acidic whey to obtain an acidic concentrated whey;

(3) Adjusting the pH value of the acidic concentrated whey to 6.5-7.5 by using an alkaline substance to obtain neutral whey;

(4) And separating and purifying the neutral whey to obtain the milk-derived exosome.

According to a further preferred embodiment of the present invention, in the step (3), the pH of the acidic concentrated whey is adjusted to 6.8 to 7.2 by using an alkaline substance. In some particularly preferred and specific embodiments, the adjusted pH is controlled to 7.0.

Further, in the step (3), the selection of the basic substance is not limited for the purpose of adjusting pH, but as a preferred embodiment of the present invention, the basic substance is preferably one or a combination of plural kinds selected from sodium hydroxide, disodium hydrogen phosphate, and sodium hydrogen phosphate. In some particularly preferred embodiments according to the present invention, the alkaline substance is an aqueous solution of NaOH with a concentration of 4mol/L to 6 mol/L. For example, a 4mol/L NaOH aqueous solution, a 4.5mol/L NaOH aqueous solution, a 5mol/L NaOH aqueous solution, a 5.5mol/L NaOH aqueous solution, or a 6mol/L NaOH aqueous solution.

According to a preferred embodiment of the present invention, in step (2), concentration is carried out by tangential flow ultrafiltration. Further preferably, the tangential flow ultrafiltration adopts a hollow fiber column with the molecular weight cut-off of 100kDa to 500kDa. According to a further preferred embodiment of the present invention, the molecular weight cut-off of the hollow fiber column is from 100kDa to 200kDa, such as a hollow fiber column with a molecular weight cut-off of 100kDa, a hollow fiber column with a molecular weight cut-off of 120kDa, a hollow fiber column with a molecular weight cut-off of 150kDa, a hollow fiber column with a molecular weight cut-off of 180kDa, a hollow fiber column with a molecular weight cut-off of 200kDa, and the like.

Preferably, in the step (2), the concentration is 5 to 10 times, for example, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, or the like.

According to a further preferred embodiment of the invention, in step (2) the acidic concentrated whey is subjected to microfiltration using a microfiltration membrane before the tangential flow ultrafiltration. Further preferably, the microfiltration comprises multistage microfiltration of the acid whey, wherein the pore size of the membranes used in the multistage microfiltration is sequentially reduced.

Taking three-stage microfiltration as an example for illustration, first-stage microfiltration is performed on acidic whey to obtain primary trapped fluid, then second-stage microfiltration is performed on the primary trapped fluid to obtain secondary trapped fluid, and finally third-stage microfiltration is performed on the secondary trapped fluid to obtain filtrate for concentration, wherein the pore diameter of a filter membrane adopted by the first-stage microfiltration is larger than the pore diameter of a filter membrane adopted by the second-stage microfiltration is larger than the pore diameter of a filter membrane adopted by the third-stage microfiltration.

In some specific and preferred embodiments according to the present invention, the multi-stage microfiltration is a three-stage microfiltration, wherein the first stage microfiltration uses a membrane having a pore size of 0.5 μm to 0.8 μm, the second stage microfiltration uses a membrane having a pore size of 0.3 μm to 0.5 μm, and the third stage microfiltration uses a membrane having a pore size of 0.2 μm to 0.25 μm.

According to a further preferred embodiment of the present invention, in the step (1), the pH of the acidic whey is 4.3 to 4.8, for example, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8.

Preferably, in the step (1), the fat in the milk is removed to obtain whey, then the pH value of the whey is adjusted by using an acidic substance to precipitate milk protein, and finally the precipitated milk protein is removed to obtain acidic whey, wherein:

the acidic substance used is not particularly limited, and may be, for example, one or a combination of more of hydrochloric acid, glacial acetic acid, citric acid, and phosphoric acid. In some specific and preferred embodiments, in the step (1), the acidic substance is a hydrochloric acid aqueous solution with a concentration of 5mol/L to 7 mol/L. For example, a 5mol/L hydrochloric acid aqueous solution, a 5.5mol/L hydrochloric acid aqueous solution, a 6mol/L hydrochloric acid aqueous solution, a 6.5mol/L hydrochloric acid aqueous solution, a 7mol/L hydrochloric acid aqueous solution, and the like.

Preferably, the fat and the precipitated milk protein are removed from the emulsion by centrifugation, respectively. Still more preferably, the centrifugal force used in the centrifugation is 1500g to 6500g. It is still further preferred that the emulsion is subjected to a first stage of centrifugation at a first centrifugal force, the whey from the first stage of centrifugation is subjected to a second stage of centrifugation at a second centrifugal force, the pH of the whey from the second stage of centrifugation is adjusted using an acidic substance, and then a third stage of centrifugation is performed at a third centrifugal force to obtain said acidic whey, said first centrifugal force and said third centrifugal force being greater than said second centrifugal force. Still more preferably, the first centrifugal force is 5000g to 7000g, the second centrifugal force is 1500g to 2500g, and the third centrifugal force is 4500g to 6500g. Still more preferably, the time of the first-stage centrifugation and/or the second-stage centrifugation is 5-30 min, and the time of the third-stage centrifugation is 20-40 min.

Preferably, in the step (4), the neutral whey is subjected to size exclusion chromatography and density gradient centrifugation sequentially to obtain an exosome gradient layer, and then the exosome gradient layer is centrifuged to obtain a precipitate, wherein the precipitate is the milk-derived exosome.

Further preferably, the size exclusion chromatography uses a size exclusion chromatography column with exclusion molecular weight of 650kDa to 750kDa. Such as a molecular exclusion chromatography column with an exclusion molecular weight of 650kDa, a molecular exclusion chromatography column with an exclusion molecular weight of 680kDa, a molecular exclusion chromatography column with an exclusion molecular weight of 700kDa, a molecular exclusion chromatography column with an exclusion molecular weight of 720kDa, a molecular exclusion chromatography column with an exclusion molecular weight of 740 kDa.

Further preferably, the density gradient centrifugation employs a discontinuous density gradient layering fluid, wherein:

the stratified liquid can be iodixanol solution or glucose solution. Preferably, the mass fraction of the layering liquid ranges from 5% to 50%. Still more preferably, the mass fraction of the layering liquid decreases from bottom to top according to a gradient of 5% -15%.

The centrifugal force of the density gradient centrifugation is preferably 100000g to 200000g. Still more preferably, the centrifugation time of the density gradient centrifugation is 5h to 20h, such as 5h, 8h, 10h, 12h, 14h, 16h, 18h, 20h and the like.

The surface of the layering liquid is preferably sealed by using PBS buffer, tris buffer or HEPES buffer.

In the step (4), the precipitate is preferably obtained by centrifuging the exosome gradient layer under the centrifugal force of 100000g to 120000g. More preferably, the time for centrifuging the exosome gradient layer is 1h to 2h. Further preferably, the pellet is resuspended using PBS buffer, tris buffer, HEPES buffer or sterile, enzyme-free water and then stored.

The invention also provides a preparation method of the milk-derived exosome, which takes the milk as the raw material and comprises the following steps:

(1) Removing fat in the emulsion by centrifugation to obtain whey;

(2) Adjusting the pH value of the whey to 4~6 by adopting an acidic substance to separate out lactoprotein;

(3) Removing milk protein by centrifugation to obtain acidic whey;

(4) Microfiltering the acidic whey, and concentrating the filtrate by tangential flow to obtain acidic concentrated whey;

(5) Adjusting the pH value of the acidic concentrated whey to 6.5 to 7.5 by using an alkaline substance to obtain neutral whey;

(6) Separating and purifying the neutral whey by adopting a molecular exclusion chromatographic column to obtain an exosome crude extract;

(7) Separating and purifying the crude exosome extracting solution by adopting density gradient centrifugation, and collecting an exosome gradient layer;

(8) And centrifuging the exosome gradient layer, and collecting precipitates to obtain the milk-derived exosomes.

Preferably, in the step (2), the pH value of the whey is adjusted to 4.3-4.8 by using a hydrochloric acid aqueous solution with the concentration of 5-7 mol/L. Preferably, in the step (5), the pH value of the acidic concentrated whey is adjusted to 6.8-7.2 by adopting a NaOH aqueous solution with the concentration of 4-6 mol/L.

Preferably, in the step (1), firstly, the emulsion is subjected to first-stage centrifugation under a first centrifugal force, then, whey obtained by the first-stage centrifugation is subjected to second-stage centrifugation under a second centrifugal force, and the whey obtained by the second-stage centrifugation is collected, wherein the first centrifugal force is preferably 5000g to 7000g, the second centrifugal force is preferably 1500g to 2500g, and the time of the first-stage centrifugation and the time of the second-stage centrifugation are 5min to 30min respectively.

Preferably, the centrifugal force used in the step (3) is 4500g to 6500g, and the centrifugal time is 20min to 40min.

Preferably, the centrifugal force of the density gradient centrifugation in the step (7) is 100000g to 200000g, and the centrifugation time is 5h to 20h.

Preferably, the centrifugal force adopted in the step (8) is 100000g to 120000g, and the centrifugal time is 1h to 2h.

Preferably, all the centrifugings in the preparation method of the milk-derived exosome are carried out at 0-8 ℃.

Preferably, in the step (4), the microfiltration includes performing a first stage microfiltration on the acidic whey to obtain a primary retentate, performing a second stage microfiltration on the primary retentate to obtain a secondary retentate, and performing a third stage microfiltration on the secondary retentate to obtain a filtrate for concentration, wherein: the aperture of the filter membrane adopted by the first-stage microfiltration is 0.5-0.8 μm, the aperture of the filter membrane adopted by the second-stage microfiltration is 0.3-0.5 μm, and the aperture of the filter membrane adopted by the third-stage microfiltration is 0.2-0.25 μm.

Preferably, in the step (4), the tangential flow concentration adopts a hollow fiber column with the molecular weight cut-off of 100kDa to 500kDa. The concentration times are 5 to 10 times.

Preferably, in the step (6), the size exclusion chromatographic column has an exclusion molecular weight of 650 kDa-750 kDa.

Preferably, in the step (7), the layering solution adopted in the density gradient centrifugation is iodixanol solution or glucose solution, the mass fraction of the layering solution is in the range of 5% -50%, and the mass fraction of the layering solution decreases gradually from bottom to top according to the gradient of 5% -15%. Preferably, the surface of the layering solution is sealed by using a PBS buffer solution.

Preferably, the precipitate collected in step (8) is stored after being resuspended using PBS buffer, tris buffer, HEPES buffer or sterile enzyme-free water.

Preferably, one or more of goat milk, mare milk, camel milk or other species derived emulsions.

Preferably, the emulsion is 0.001% -5% of fat by mass.

There is no particular limitation on the obtainment of the emulsion, and pasteurized milk sold at normal temperature storage, and pasteurized milk sold at 4 ℃ storage can be used, and the types include skim milk, semi-skim milk, and whole milk.

According to preferred embodiments of the present invention, they contribute relatively better effects in at least one of improving the quality (purity, concentration, uniformity, membrane structural integrity, etc.) of exosomes, and the yield of exosomes.

The invention also provides the milk-derived exosome prepared by the preparation method of the milk-derived exosome and application of the milk-derived exosome in a drug delivery system.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the following advantages:

the exosome obtained by the method has higher purity and particle concentration, and meanwhile, the membrane structure is complete and does not deform, so that the exosome prepared by the method has better quality compared with the exosome prepared by the existing method; in addition, the method has high yield and low cost.

Drawings

FIG. 1 is a transmission electron micrograph of the exosome heavy suspension prepared in example 1;

FIG. 2 is a transmission electron micrograph of the exosome heavy suspension prepared in comparative example 1;

FIG. 3 is a transmission electron micrograph of the exosome heavy suspension prepared in comparative example 2;

FIG. 4 is a transmission electron micrograph of the exosome-purified liquid prepared in example 3;

FIG. 5 is an exosome particle size distribution map of the exosome heavy suspension prepared in example 1;

FIG. 6 is an exosome particle size distribution diagram of the exosome heavy suspension prepared in example 2;

FIG. 7 is an exosome particle size distribution diagram of the exosome purification solution prepared in example 3;

FIG. 8 is an exosome particle size distribution diagram of the exosome heavy suspension prepared in comparative example 1;

fig. 9 is an exosome particle size distribution diagram of the exosome heavy suspension prepared in comparative example 2.

Detailed Description

The milk-derived exosome has natural advantages as a drug in-vivo delivery system, but compared with the exosomes derived from other sources such as blood and the like, the difficulty in extracting and purifying the milk-derived exosome is obviously improved. At present, few reports are provided for preparation methods of milk-derived exosomes. Milk sources contain large amounts of milk proteins and fats, making many purification methods that work well in cell culture supernatants unusable. Although some methods for extracting milk-derived milk protein are known at present, when the method is used for preparing milk-derived exosome, more or less damage to the milk-derived exosome cannot be avoided, or the later-stage biological activity of the milk-derived exosome extract is influenced, and in the subsequent preparation process, the purity and yield of the milk-derived exosome are difficult to be considered. The present invention aims to solve the aforementioned problems.

The invention has an unexpected positive effect on ensuring the structural integrity and non-deformation of the subsequently prepared exosome vesicle membrane by integrally designing the process, in particular adjusting the pH of the acidic whey to be neutral after the acidic whey is concentrated and before the acidic whey is subjected to subsequent separation and purification. The invention further organically combines the isoelectric precipitation, tangential flow concentration, size exclusion, density gradient centrifugation and ultracentrifugation of milk protein, and not only obtains high-quality exosomes with complete structure, no deformation and high purity, but also has high yield. The method provided by the invention is beneficial to realizing the industrial preparation of the milk-derived exosome and provides powerful support for the mining of the medicinal value of the natural milk-derived exosome and the development of a drug delivery technology based on the milk-derived exosome.

Definition of terms

The term "milk", without specific definition, refers to milk or colostrum derived from cow, human, buffalo, goat, sheep, camel, donkey, horse, reindeer, moose or yak.

The term "milk-derived exosome" refers to exosome obtained by separating milk serving as a raw material. The term "milk source" is used interchangeably with the term "isolated from milk".

The term "room temperature" means an ambient temperature of 25. + -. 5 ℃.

The term "skim milk", also known as whole skim milk, is normal milk that has had a portion of its fat removed to reduce the fat content to 0.5% and below, and that has had almost all of the water removed from the milk and does not contain any artificial additives.

The term "whey" relates to the liquid phase remaining after the fat in milk has been removed and the proteins have been precipitated and removed.

The term "drug delivery system" refers to a formulation that delivers a pharmaceutically active ingredient to a desired body site and/or provides for the timely release of a therapeutic agent. In the present application, the pharmaceutically active ingredient deliverable by a milk-derived exosome comprises a small molecule drug or a biotherapeutic agent, which is not naturally present in milk-derived exosomes, said biotherapeutic agent being selected from a peptide, protein, polysaccharide or nucleic acid selected from single-or double-stranded DNA, iRNA, siRNA, shRNA, mRNA, non-coding RNA (ncRNA), antisense RNA, LNA, morpholino oligonucleotide or analogues or conjugates thereof.

The term "isolated and purified" means that the test sample containing the analyte of interest and the interfering substance are physically isolated or separated from each other.

The term "centrifugal force" refers to the apparent outward force that pulls the rotating body away from the center of rotation. The method is preferably a mechanical method, more preferably by applying centrifugal force in a rotating device such as a centrifuge.

The term "membrane" means a semi-permeable material that can be used to separate components in a feed fluid into a permeate that passes through the material and a retentate that is retained by the material.

The term "density gradient centrifugation" refers to the formation of a continuous or discontinuous density gradient in a centrifuge tube with a certain medium, mixing a test sample containing an analyte of interest with a bottom layer, and layering and separating the analyte of interest by the action of a centrifugal force field.

The term "wash liquid" refers to a fluid released from a chromatography column or chromatography membrane.

The technical solution of the present invention is further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. The implementation conditions used in the examples can be further adjusted according to specific requirements, and the implementation conditions not noted are generally those in routine experiments. The following examples and comparative examples are given only by way of example of cow's milk, other milk sources, such as goat's milk, etc., being equally applicable to the above preparation process.

In the present invention, the apparatus, raw materials and reagents used are commercially available unless otherwise specified. In the present invention, the centrifuge used is a low temperature centrifuge. The milk used in the following examples and comparative examples was specifically deyoyo skim pure milk, and was stored at room temperature.

Example 1

The embodiment provides a preparation method of milk exosomes, which comprises the following specific steps:

s1, taking 50mL of milk, centrifuging at 4 ℃ for 10min at 6000g, and collecting middle layer whey A.

S2, centrifuging the whey A at the temperature of 4 ℃ and 2000g for 20min, and collecting the middle layer whey B.

And S3, regulating the pH value of the whey B to 4.60 by using 6mol/L HCl aqueous solution, standing at room temperature for 10min, and separating out the milk protein.

S4, centrifuging at 4 ℃ and 5000g for 30min, removing milk protein, and collecting the middle layer to obtain whey C (acid whey).

S5, filtering the whey C by a filter membrane of 0.8 mu m, and collecting filtrate to obtain whey D.

S6, filtering the whey D through a 0.45-micron filter membrane, and collecting filtrate to obtain whey E.

S7, filtering the whey E through a 0.22-micron filter membrane, and collecting filtrate to obtain whey F.

S8, performing tangential flow filtration and concentration on the whey F through a 100kDa hollow fiber column by a concentration multiple of 6.73 times, and collecting concentrated whey G (acidic concentrated whey).

And S9, adjusting the pH value of the whey G to 7.0 by using 4mol/L NaOH aqueous solution to obtain neutral whey H.

S10, passing 5mL of neutral whey H through a molecular exclusion chromatographic column (Cytiva Capto Core 700 BE-SEC column) with the exclusion molecular weight of 700kDa, wherein the volume of a column packing is 10mL, after the neutral whey H passes through the column, performing column passing washing by using 1mL of 1 XPBS, and combining a washing liquid with concentrated whey after the column passing to obtain the milk exosome crude extract.

S11, mixing 5.2mL of milk exosome crude extract with 10.6mL of 60% iodixanol solution to obtain 40% iodixanol bottom solution, and transferring the 40% iodixanol bottom solution into an upright centrifuge tube, wherein the content of the 40% iodixanol bottom solution is about 40% of that of a centrifuge system. 5.85mL of 30% iodixanol diluent, 5.85mL of 20% iodixanol diluent and 5.85mL of 10% iodixanol diluent are sequentially and gradually added above the 40% iodixanol bottom liquid, and finally 5.85mL of 1 XPBS buffer is sealed above the liquid surface of the uppermost layer to complete the preparation of a density gradient centrifugation system.

S12, centrifuging for 18 hours at 4 ℃ and 186000g to separate the milk exosomes from other components in the milk exosome crude extract.

S13. The milk exosome gradient layer migrating to the interface of 10% iodixanol diluent and 20% iodixanol diluent was carefully extracted in a volume of about 6mL.

S14, diluting the extracted milk exosome gradient liquid by using 1 XPBS buffer solution, then centrifuging for 90min at 4 ℃ and 110000g, after the centrifugation is finished, resuspending the exosome sediment attached to the bottom of the tube wall by using 200 mu L of 1 XPBS buffer solution, and storing at 4 ℃.

Example 2

And S3, adjusting the pH value of the whey B to 4.60 by using 6mol/L HCl aqueous solution, standing for 10min at room temperature, and separating out the milk protein.

S4, centrifuging at 4 ℃ and 5000g for 30min, and collecting middle layer whey C (acid whey).

S5, filtering the whey C by using a 0.8-micron filter membrane, and collecting filtrate to obtain whey D.

S6, filtering the whey D by using a 0.45-micron filter membrane, and collecting filtrate to obtain whey E.

S7, filtering the whey E by using a 0.22-micron filter membrane, and collecting filtrate to obtain whey F.

S8, performing tangential flow filtration and concentration on the whey F through a 100kDa hollow fiber column by a concentration multiple of 6.73 times, and collecting the whey G (acid concentrated whey).

S10, mixing 5.2mL of neutral whey H with 10.6mL of 60% iodixanol solution to form 40% iodixanol bottom liquid, and transferring the 40% iodixanol bottom liquid into an upright centrifuge tube, wherein the concentration of the bottom liquid is about 40% of that of a centrifuge system. 5.85mL of 30% iodixanol diluent, 5.85mL of 20% iodixanol diluent and 5.85mL of 10% iodixanol diluent are gradually added above the 40% iodixanol bottom liquid in sequence, and finally 5.85mL of 1 XPBS buffer is sealed above the liquid surface of the uppermost layer to complete the preparation of the density gradient centrifugation system.

S11, centrifuging at 4 ℃ for 18H at 186000g to separate milk exosomes from other components in neutral whey H.

S12. The milk exosome gradient layer migrating to the interface of 10% iodixanol diluent and 20% iodixanol diluent was carefully extracted.

S13, purifying the milk exosome gradient layer solution by a molecular exclusion chromatographic column (Cytiva Capto Core 700 BE-SEC column) with the exclusion molecular weight of 700kDa, wherein the column packing volume is 10mL, after neutral concentrated whey H passes through the column, carrying out column passing washing by using 1mL of 1 XPBS buffer solution, and combining a washing solution with the concentrated whey after passing through the column to obtain the milk exosome purified solution.

S14, centrifuging the purified solution for 90min at the temperature of 4 ℃ at the speed of 110000g, after centrifugation is finished, resuspending the exosome precipitate attached to the bottom of the tube wall by 200 mu L of 1 XPBS, and storing at the temperature of 4 ℃.

Example 3

S10, 5.2mL of neutral whey H and 10.6mL of 60% iodixanol solution are mixed to form 40% iodixanol bottom solution, and the 40% iodixanol bottom solution is transferred to an upright centrifuge tube, which accounts for about 40% of the centrifuge system. 5.85mL of 30% iodixanol diluent, 5.85mL of 20% iodixanol diluent and 5.85mL of 10% iodixanol diluent are gradually added above the 40% iodixanol bottom layer liquid in sequence, and finally 5.85mL of 1 XPBS is sealed above the liquid surface of the uppermost layer to complete the preparation of the density gradient centrifugation system.

S11, centrifuging at 4 ℃ and 186000g for 18H to separate exosomes from other components in neutral whey H.

S12. The milk exosome gradient layer migrating to the interface of 10% iodixanol diluent and 20% iodixanol diluent was carefully extracted in a volume of about 6mL.

S13, diluting the milk exosome gradient layer solution by using 1 XPBS buffer solution, then centrifuging for 90min at the temperature of 4 ℃ at 110000g, and after the centrifugation is finished, resuspending the exosome sediment attached to the bottom of the tube wall by using 200 mu L of 1 XPBS buffer solution.

S14, purifying the exosome by using a molecular exclusion chromatographic column (Cytiva Capto Core 700 BE-SEC column) with the exclusion molecular weight of 700kDa, wherein the volume of a column packing is 2.5mL, after the gradient layer solution passes through the column, washing the column by using 0.9mL of 1 XPBS buffer, combining the column washing solution and the gradient layer column passing purification solution into SEC purification solution of the exosome, and storing the SEC purification solution at 4 ℃ with the final volume of 1.0 mL.

Example 4

This example provides a method for preparing milk exosomes, which is substantially the same as example 1, except that in step S9, pH is adjusted to 6.5.

Example 5

This example provides a method for preparing milk exosomes, which is substantially the same as example 1, except that in step S9, pH is adjusted to 7.5.

Example 6

This example provides a method for preparing milk exosomes, which is substantially the same as example 1, except that only primary filtration membrane filtration is performed, and the pore size of the filtration membrane is 0.45 μm.

Comparative example 1

The comparative example provides a preparation method of a milk exosome, which comprises the following specific steps:

And S3, adjusting the pH value of the whey B to 4.60 by using 6mol/L HCl aqueous solution, and standing at room temperature for 10min to obtain acidic whey.

S4, centrifuging the acidic whey at the temperature of 4 ℃ for 30min at the speed of 5000g, and collecting the middle layer whey C.

S5, filtering the whey C by using a 0.8-micron filter membrane, and collecting the primary filtered whey D.

S6, filtering the primary filtered whey D by using a 0.45-micron filter membrane, and collecting secondary filtered whey E.

S7, filtering the whey E by using a 0.22 mu m filter membrane, and collecting final filtered whey F.

S8, performing tangential flow filtration and concentration on the final filtered whey F through a 100kDa hollow fiber column, wherein the concentration multiple is 6.73 times, and collecting concentrated whey G.

S9, mixing 5.2mL of concentrated whey G with 10.6mL of 60% iodixanol solution to obtain 40% iodixanol bottom solution, and transferring the bottom solution to an upright centrifuge tube, wherein the bottom solution accounts for about 40% of the centrifuge system. 5.85mL of 30% iodixanol diluent, 5.85mL of 20% iodixanol diluent and 5.85mL of 10% iodixanol diluent are sequentially and gradually added above 40% iodixanol bottom liquid, and finally 5.85mL of 1 XPBS is sealed above the liquid surface of the uppermost layer to complete the preparation of a density gradient centrifugation system.

S10, centrifuging at 4 ℃ and 186000G for 18h to separate exosomes from other components in concentrated whey G.

S11, carefully extracting the milk exosome gradient layer transferred between 10% iodixanol diluent and 20% iodixanol diluent, wherein the volume is about 6mL, diluting the extracted milk exosome gradient liquid with 1 x PBS, and centrifuging for 90min at the temperature of 4 ℃ and at the temperature of 110000 g.

S12, after the centrifugation is finished, the exosome precipitate attached to the bottom of the tube wall is resuspended by 200 mu L of 1 XPBS and is stored at 4 ℃.

Comparative example 2

This comparative example provides a method of preparing milk exosomes, which is substantially the same as example 1, except that the S9 step is omitted.

Exosome identification experiment

Identifying the extracted exosomes through different detection indexes, wherein whether the structure of the extracted exosome membrane is complete and the size of the extracted exosome membrane accords with expectation is observed through a Transmission Electron Microscope (TEM); analyzing the particle size and the particle number concentration of exosomes by a nanoparticle tracking technology (NTA), and evaluating the exosome extraction efficiency according to the number of exosome particles capable of being extracted per milliliter of emulsion; protein concentration was measured by BCA method, and the purity of exosomes was evaluated according to the number of exosome particles per unit protein (ratio of exosome particle concentration to protein concentration), with a larger ratio indicating higher purity.

And (3) observing the exosome condition in the exosome suspension or the purified liquid finally obtained in the example and the comparative example by a Transmission Electron Microscope (TEM), wherein the structure of the exosome prepared in the example is obviously more complete and has no deformation compared with the comparative example. As can be seen from comparison of fig. 1 to fig. 4, under the same magnification, the exosome membrane structure observed in the exosome suspension prepared in example 1 is complete and has no deformation (a typical vesicle in a "cup-and-dish" structure), the size of the exosome membrane structure is more expected, the number of exosomes is more, and meanwhile, the background impurities are significantly less; the number of undeformed exosomes observed in the exosome weight suspension prepared in comparative example 2 was significantly reduced; the amount of undeformed exosomes observed in the exosome suspension prepared in comparative example 1 was very small, with more background impurities. In three examples, namely example 1, example 2 and example 3, the typical vesicles observed in the exosome suspension prepared in example 1 are the largest in number, the most complete in structure and the least in impurities, which is obviously better than those in example 2 and example 3.

The particle size and particle number concentration of the exosome heavy suspension or the purification solution prepared in each example above were analyzed by nanoparticle tracking technology (NTA), and the results are shown in fig. 5 to 9, which indicate that: the particle size distribution of the exosome prepared in example 1 is narrow, the peak particle size is 140.9nm, the average particle size is 159.1nm, the uniformity is good, and the particle number concentration of the exosome is 4.64E +11 (particle number/mL), which is obviously superior to that of other examples and comparative examples.

The concentration of the protein remaining in each prepared exosome heavy suspension or purified solution was measured according to the BCA protein quantification method, and the number of exosome particles per unit protein was calculated as exosome particle number concentration/protein concentration, and the results are shown in table 1.

In addition, the amount of exosome obtained per ml of milk was calculated from the number concentration of exosome particles in the exosome weight suspension or the purified liquid prepared according to the preparation methods of each example described above, and the results are shown in table 1.

Table 1 shows that the exosome concentration and purity are best for example 1. Although table 1 shows that the exosome particle concentration and the obtained amount of exosomes per ml of milk were also higher in the exosome suspension of comparative example 1 and comparative example 2, it can be seen from the results of transmission electron microscopy that the exosomes prepared in comparative example 1 and comparative example 2 have a large number of deformed exosomes, and the number concentration of undeformed exosome particles is actually much smaller than the data in table 1.

The present invention is described in detail for the purpose of understanding the content of the present invention and implementing the same by those skilled in the art, and the present invention is not limited to the embodiments described above, and all equivalent changes or modifications made according to the spirit of the present invention should be covered by the scope of the present invention.

Claims

1. A preparation method of milk-derived exosomes takes milk as a raw material, and is characterized in that: the preparation method comprises the following steps:

(2) Concentrating said acidic whey to obtain an acidic concentrated whey;

2. The method for producing milk-derived exosomes according to claim 1, characterized in that: adjusting the pH value of the acidic concentrated whey to 6.8-7.2 by using an alkaline substance.

3. The method for producing milk-derived exosomes according to claim 2, characterized in that: adjusting the pH of said acidic concentrated whey to 7.0 with an alkaline substance.

4. The method for producing milk-derived exosomes according to claim 1, characterized in that: in the step (3), the alkaline substance includes one or more of sodium hydroxide, disodium hydrogen phosphate, and sodium hydrogen phosphate.

5. The method for producing milk-derived exosomes according to claim 4, characterized in that: the alkaline substance is NaOH aqueous solution with the concentration of 4-6 mol/L.

6. The method for producing milk-derived exosomes according to claim 1, characterized in that: in step (2), the acidic whey is concentrated by tangential flow ultrafiltration.

7. The method for producing milk-derived exosomes according to claim 6, characterized in that: the tangential flow ultrafiltration adopts a hollow fiber column with the molecular weight cutoff of 100kDa to 500kDa.

8. The method for producing milk-derived exosomes according to claim 1, characterized in that: in the step (2), the concentration is 5 to 10 times.

9. The method for producing milk-derived exosomes according to claim 6, characterized in that: in the step (2), microfiltration is performed on the acidic whey by using a microfiltration membrane before tangential flow ultrafiltration.

10. The method for producing milk-derived exosomes according to claim 9, characterized in that: the microfiltration comprises the step of carrying out multistage microfiltration on the acidic whey, wherein the pore diameters of filter membranes adopted in the multistage microfiltration are sequentially reduced.

11. The method for producing milk-derived exosomes according to claim 10, characterized in that: the multistage microfiltration comprises three-stage microfiltration, wherein the aperture of a filter membrane adopted by the first-stage microfiltration is 0.5-0.8 mu m, the aperture of a filter membrane adopted by the second-stage microfiltration is 0.3-0.5 mu m, and the aperture of a filter membrane adopted by the third-stage microfiltration is 0.2-0.25 mu m.

12. The method for producing milk-derived exosomes according to claim 1, characterized in that: in the step (1), the pH value of the acidic whey is 4.3 to 4.8.

13. The method for producing milk-derived exosomes according to claim 1, characterized in that: in the step (1), the acidic whey is obtained by removing fat in the milk to obtain whey, then adjusting the pH of the whey with an acidic substance to precipitate milk protein, and finally removing the precipitated milk protein.

14. The method for producing milk-derived exosomes according to claim 13, characterized in that: the acidic substance is one or more of hydrochloric acid, glacial acetic acid, citric acid and phosphoric acid.

15. The method for producing milk-derived exosomes according to claim 14, characterized in that: the acidic substance is a hydrochloric acid aqueous solution with the concentration of 5-7 mol/L.

16. The method for producing milk-derived exosomes according to claim 13, characterized in that: in the step (1), the fat and the precipitated milk protein in the emulsion are removed by centrifugation.

17. The method for producing milk-derived exosomes according to claim 16, characterized in that: the centrifugal force adopted by the centrifugal method is 1500g to 6500g.

18. The method for producing milk-derived exosomes according to claim 13, characterized in that: subjecting the emulsion to a first centrifugation under a first centrifugal force, subjecting the whey obtained from the first centrifugation to a second centrifugation under a second centrifugal force, adjusting the pH of the whey obtained from the second centrifugation using an acidic substance, and then subjecting the whey to a third centrifugation under a third centrifugal force, wherein the first centrifugal force and the third centrifugal force are greater than the second centrifugal force, to obtain the acidic whey.

19. The method for producing milk-derived exosomes according to claim 18, characterized in that: the first centrifugal force is 5000 g-7000 g, the second centrifugal force is 1500 g-2500 g, and the third centrifugal force is 4500 g-6500g.

20. The method for producing milk-derived exosomes according to claim 18, characterized in that: the time of the first-stage centrifugation and/or the second-stage centrifugation is 5min to 30min, and the time of the third-stage centrifugation is 20min to 40min.

21. The method for producing milk-derived exosomes according to claim 1, characterized in that: in the step (4), the neutral whey is subjected to size exclusion chromatography and density gradient centrifugation sequentially to obtain an exosome gradient layer, and then the exosome gradient layer is centrifuged to obtain a precipitate, wherein the precipitate is the milk-derived exosome.

22. The method of producing milk-derived exosomes according to claim 21, characterized in that: the molecular exclusion chromatography adopts a molecular exclusion chromatographic column with exclusion molecular weight of 650kDa to 750kDa.

23. The method of producing milk-derived exosomes according to claim 21, characterized in that: the density gradient centrifugation adopts a layering solution with discontinuous density gradient, and the layering solution is iodixanol solution or glucose solution.

24. The method of producing milk-derived exosomes according to claim 23, characterized in that: the mass fraction range of the layering liquid is 5-50%.

25. A method of producing milk-derived exosomes according to claim 23 or 24, characterized in that: the mass fraction of the layering liquid is gradually reduced from bottom to top according to the gradient of 5-15%.

26. The method of producing milk-derived exosomes according to claim 23, characterized in that: the surface of the layering solution is sealed by using PBS buffer solution, tris buffer solution or HEPES buffer solution.

27. The method of producing milk-derived exosomes according to claim 21, characterized in that: the centrifugal force of the density gradient centrifugation is 100000g to 200000g.

28. A method of producing milk-derived exosomes according to claim 21 or 27, characterized in that: the centrifugation time of the density gradient centrifugation is 5h to 20h.

29. The method of producing milk-derived exosomes according to claim 21, characterized in that: and centrifuging the exosome gradient layer under the condition that the centrifugal force is 100000g to 200000g to obtain the precipitate.

30. A method of producing milk-derived exosomes according to claim 21 or 29, characterized in that: and the time for centrifuging the exosome gradient layer is 1h to 2h.

31. The method of producing milk-derived exosomes according to claim 21, characterized in that: and the precipitate is stored after being re-suspended by using PBS buffer solution, tris buffer solution, HEPES buffer solution or sterile enzyme-free water.

32. A preparation method of milk-derived exosomes takes milk as a raw material, and is characterized in that: the preparation method comprises the following steps:

(1) Removing fat from the emulsion by centrifugation to obtain whey;

(3) Removing milk protein by centrifugation to obtain acidic whey;

(7) Separating and purifying the crude exosome extract by adopting density gradient centrifugation, and collecting an exosome gradient layer;

33. The method of producing milk-derived exosomes according to claim 32, characterized in that: in the step (2), the pH value of the whey is adjusted to 4.3-4.8 by adopting a hydrochloric acid aqueous solution with the concentration of 5-7 mol/L.

34. The method of producing milk-derived exosomes according to claim 32, characterized in that: in the step (5), naOH water solution with the concentration of 4-6 mol/L is adopted to adjust the pH value of the acidic concentrated whey to 6.8-7.2.

35. The method of producing milk-derived exosomes according to claim 32, characterized in that: in the step (1), firstly, carrying out first-stage centrifugation on the emulsion under a first centrifugal force, then carrying out second-stage centrifugation on whey obtained by the first-stage centrifugation under a second centrifugal force, and collecting the whey obtained by the second-stage centrifugation, wherein the first centrifugal force is 5000-7000 g, the second centrifugal force is 1500 g-2500 g, and the time of the first-stage centrifugation and the time of the second-stage centrifugation are respectively 5 min-30min;

and/or the centrifugal force adopted in the step (3) is 4500 g-6500g, and the centrifugal time is 20 min-40min;

and/or the centrifugal force of density gradient centrifugation in the step (7) is 100000g to 200000g, and the centrifugation time is 5h to 20h;

and/or the centrifugal force adopted in the step (8) is 100000g to 200000g, and the centrifugal time is 1h to 2h;

and/or all centrifugings in the preparation method of the milk-derived exosome are carried out at 0-8 ℃.

36. The method of producing milk-derived exosomes according to claim 32, characterized in that: in the step (4), the microfiltration comprises performing primary microfiltration on the acidic whey to obtain primary trapped fluid, performing secondary microfiltration on the primary trapped fluid to obtain secondary trapped fluid, and performing tertiary microfiltration on the secondary trapped fluid to obtain filtrate for concentration, wherein the aperture of a filter membrane adopted by the primary microfiltration is 0.5-0.8 μm, the aperture of a filter membrane adopted by the secondary microfiltration is 0.3-0.5 μm, and the aperture of a filter membrane adopted by the tertiary microfiltration is 0.2-0.25 μm;

and/or, in the step (4), the tangential flow concentration adopts a hollow fiber column with the molecular weight cut-off of 100kDa to 500kDa;

and/or in the step (4), the concentration multiple is 5 to 10 times;

and/or, in the step (6), the exclusion molecular weight of the molecular exclusion chromatographic column is 650 kDa-750 kDa;

and/or in the step (7), a layering liquid adopted by the density gradient centrifugation is iodixanol solution or glucose solution, the mass fraction range of the layering liquid is 5% -50%, the mass fraction of the layering liquid decreases progressively from bottom to top according to a gradient of 5% -15%, and the surface of the layering liquid is sealed by PBS buffer solution;

and/or, the precipitate collected in the step (8) is stored after being resuspended by using PBS buffer, tris buffer, HEPES buffer or sterile and enzyme-free water.

37. A method of producing milk-derived exosomes according to claim 1 or 32, characterized in that: the emulsion is one or more of cow milk, goat milk, mare milk, camel milk or other species-derived emulsions; and/or the mass percentage of fat in the emulsion is 0.001-5%.

38. A milk-derived exosome prepared by the method for preparing a milk-derived exosome according to any one of claims 1 to 37.

39. Use of the milk-derived exosome of claim 38 in a drug delivery system.