CN116036298B

CN116036298B - Application of milk exosomes in preparation of drug carrier

Info

Publication number: CN116036298B
Application number: CN202111263036.5A
Authority: CN
Inventors: 王伊; 宋海峰; 凌焱; 徐铭枝; 董亚南
Original assignee: Guangwu Huiwen Biotechnology Beijing Co ltd
Current assignee: Guangwu Huiwen Biotechnology Beijing Co ltd
Priority date: 2021-10-28
Filing date: 2021-10-28
Publication date: 2024-06-04
Anticipated expiration: 2041-10-28
Also published as: CN116036298A

Abstract

The invention relates to the field of pharmaceutical preparations, in particular to application of milk exosomes in preparation of a pharmaceutical carrier. The invention adopts the milk exosome as the drug carrier, the exosome is derived from milk, is safe and reliable, has the functional property of the milk exosome, and can realize the oral administration of protein drugs by taking the milk exosome as the drug carrier. Oral administration of liraglutide is achieved by loading liraglutide with milk exosomes, treating type two diabetes.

Description

Application of milk exosomes in preparation of drug carrier

Technical Field

The invention relates to the field of pharmaceutical preparations, in particular to application of milk exosomes in preparation of a pharmaceutical carrier.

Background

Exosomes refer to small vesicles (30-150 nm) containing complex RNAs and proteins, which nowadays are specifically disc-shaped vesicles with diameters of 40-100 nm. Exosomes were first found in sheep reticulocytes in 1983, and Johnstone named "exosomes" in 1987. A variety of cells secrete exosomes under both normal and pathological conditions. Mainly derived from the multivesicular body formed by the invagination of the lysosome particles in cells, and released into extracellular matrix after being fused with cell membranes through the outer membrane of the multivesicular body.

The existing exosome purification technology at present mainly comprises the following methods

1. Ultracentrifugation (differential centrifugation)

The super-separation method is the most commonly used exosome purification means, is a gold standard for exosome extraction, but has smaller scale, time-consuming process, unstable recovery rate (possibly related to rotor type) and questioned purity; in addition, repeated centrifugation may damage the vesicles, thereby reducing their quality.

2. Density gradient centrifugation

Under the action of overspeed centrifugal force, the sucrose solution is formed into a density layer which is continuously distributed from low to high, and the method is a zonal separation method. Density gradient centrifugation has the problem of cumbersome and time-consuming steps.

3. Ultrafiltration centrifugation

Ultrafiltration centrifugation may block the filter pores, resulting in a shortened membrane life and a lower separation efficiency.

4. Magnetic bead immunization method

The magnetic bead method has low exosome extraction efficiency, the exosome biological activity is easily influenced by pH and salt concentration, the downstream experiment is not facilitated, and the exosome biological activity is difficult to widely popularize.

5. PEG-base precipitation method

Polyethylene glycol (PEG) precipitation exosomes present a number of problems: such as low purity and recovery, high impurity protein content (false positive), non-uniform particle size, and the generation of difficult-to-remove polymers, and mechanical forces or chemical additives such as tween-20, which can damage exosomes, etc.

6. Kit extraction

Commercial exosome extraction kit, extract exosome scale is little, and can produce many hybrid protein, exosome purity and rate of recovery are low.

Therefore, the invention aims to solve the problem of a method for efficiently extracting milk exosomes in a large scale without depending on centrifugation.

More importantly, the drug loading of the exosomes is a difficult point, and the invention realizes and improves the drug loading of the exosomes to protein drugs.

The common administration mode of protein medicine at present is injection administration, the injection administration is complex, and safety risks exist.

Disclosure of Invention

In view of the above, the invention adopts the milk exosome as the drug carrier, the exosome is derived from milk, is safe and reliable, has the functional property of the milk exosome, and can realize the oral administration of protein drugs by using the milk exosome as the drug carrier. Oral administration of liraglutide is achieved by loading liraglutide with milk exosomes, treating type two diabetes.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides application of milk exosomes in preparation of drug carriers. In some specific embodiments of the invention, the drug comprises a protein drug. In some embodiments of the invention, the drug comprises liraglutide.

The invention also provides application of the milk exosome as a drug carrier in preparing drugs for preventing and/or treating diseases. In some embodiments of the invention, the drug comprises a protein drug. In some embodiments of the invention, the drug comprises liraglutide. In some embodiments of the invention, the route of administration of the drug is oral. In some embodiments of the invention, the disease comprises type II diabetes.

More importantly, the invention also provides an oral medicament which comprises an active ingredient and milk exosomes serving as medicament carriers.

In the invention, the preparation method of the milk exosome in the application or the oral medicine comprises the following steps:

Step 1, pretreatment;

step2, preliminary purification and concentration;

Step 3, fine purification;

The pretreatment includes fat removal and/or protein removal;

The primary purification and concentration adopts tangential flow ultrafiltration;

the purification comprises one or more of CIMmultus QA column purification, captocore 700 column purification and S400 molecular sieve chromatography purification.

In some embodiments of the invention, removing fat in step 1 comprises:

(1) Removing fat by adopting a method of standing natural sedimentation and filtration; and/or

(2) Removing fat by adopting a centrifugal method;

The filtration includes depth filtration and/or capsule filtration.

In some embodiments of the invention, the protein removal in step 1 is performed by a protein solubilization and/or protein precipitation process;

The protein dissolving method comprises a sodium citrate dissolving method;

the precipitated protein method comprises one or a combination of EDTA precipitated protein method, sodium phosphate precipitated protein method and ammonium sulfate precipitated protein method.

In some embodiments of the invention, the sodium citrate dissolution method comprises the steps of: mixing equal volume of milk with sodium citrate solution with mass concentration of 2%, shaking in ice bath, and filtering with filter membrane.

The sodium citrate dissolution method specifically comprises the following steps: pouring an equal volume of milk into a sodium citrate solution with the mass concentration of 2%, shaking the milk sample by a shaking table for 60 minutes in an ice bath, clarifying the milk sample, and filtering the clarified milk sample by a 0.2 mu m filter membrane.

In some embodiments of the invention, the EDTA-precipitated protein method comprises the steps of: mixing equal volume of milk with EDTA solution, standing at room temperature, centrifuging, and filtering with filter membrane; the concentration of the EDTA solution was 0.35M and the pH was 7.0.

In some embodiments of the present invention, the sodium citrate dissolution method is specifically: pouring the obtained milk into EDTA solution, precipitating at room temperature, standing for 15min, centrifuging at 12000rpm for 40min, and filtering the supernatant with 0.45 μm and 0.2 μm filter membrane.

In some embodiments of the invention, the sodium phosphate solution has a concentration of 1m and a ph of 6.3.

In some embodiments of the invention, the sodium phosphate precipitation protein method comprises the steps of:

the specific steps of the sodium phosphate precipitation protein method are different for different milk types:

(1) Whole milk

Centrifuging milk at 16000rpm for 30min to remove fat, mixing equal volume of milk with sodium phosphate solution, stirring, precipitating, and freezing at-80deg.C;

(2) Skimmed milk

Centrifuging milk at 16000rpm for 30min to remove fat, mixing equal volume of milk with sodium phosphate solution, stirring, precipitating, standing at room temperature, centrifuging at 3500rpm for 10min, collecting supernatant, and filtering with a filter membrane.

In some embodiments of the invention, the sodium phosphate precipitation protein method specifically comprises the steps of: taking milk 16000rpm, centrifuging for 30min to remove fat, pouring an equal volume of milk into sodium phosphate solution, stirring for 15min with a magnetic stirrer at 350rpm, centrifuging for 10min with 3500rpm of the precipitated milk sample, and filtering the supernatant with 0.8 μm,0.45 μm and 0.2 μm filter membranes in sequence.

In some embodiments of the invention, the ammonium sulfate precipitation protein process comprises the steps of: mixing the milk with the ammonium sulfate solution in equal volume, standing at room temperature, centrifuging, and filtering with a filter membrane; the concentration of the ammonium sulfate solution was 3.0M.

In some embodiments of the invention, the ammonium sulfate precipitation protein process specifically comprises the steps of: pouring an equal volume of milk into an ammonium sulfate solution, stirring with a glass rod while pouring, precipitating at room temperature, standing for 1.5h, centrifuging at 6000rpm for 20min, and filtering the supernatant with 0.8 μm,0.45 μm and 0.2 μm filter membranes in sequence.

The invention also provides a method for treating diseases, which takes milk exosomes as drug carriers and carries drugs to be applied to animals.

In some embodiments of the invention, the drug comprises a protein drug.

In some embodiments of the invention, the drug comprises liraglutide.

In some embodiments of the invention, the route of administration of the drug is oral.

In some embodiments of the invention, the disease comprises type II diabetes.

The invention adopts the milk exosome as the drug carrier, the exosome is derived from milk, is safe and reliable, has the functional property of the milk exosome, and can realize the oral administration of protein drugs by taking the milk exosome as the drug carrier. The oral administration of the liraglutide is realized by loading the liraglutide by using milk exosomes, so that the type II diabetes is treated.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a process route diagram for extracting milk exosomes by TFF+BE-SEC and UC;

FIG. 2 shows morphological manifestations of exosomes extracted by 3 methods under TEM (Bar=500 nm) (A: TFF; B: TFF+BE-SEC; C: UC);

FIG. 3 shows the particle size distribution of exosomes extracted by 3 methods (A: TFF; B: TFF+BE-SEC; C: UC.);

FIG. 4 shows a SEC-HPLC chromatogram of the extraction of milk exosomes by 3 methods (A: TFF; B: TFF+BE-SEC; C: UC.);

FIG. 5 shows the liraglutide solution after sonication (left, more precipitate separated out after sonication, solution is colloidal) and milk exosome-liraglutide mixture (right);

FIG. 6 shows Core700 column purified milk exosomes sonicated loaded with liraglutide samples;

FIG. 7 shows Core700 column purification of sonicated liraglutide samples;

FIG. 8 shows samples after QA column purification of Milk exosomes and liraglutide sonications;

FIG. 9 shows QA column purification of sonicated liraglutide samples;

FIG. 10 shows the effect of the Liraglutide solution and exosome-Liraglutide mixture on luciferase expression after sonication and Core700 (positive control was 0.375ug/ml of non-sonicated Liraglutide stock drug treatment group);

FIG. 11 shows conditions for exploring the use of electrotransport methods to achieve exosome-loaded PKH 67; wherein, FIG. 11A shows the electrotransport PKH 67-exosome mixture (100. Mu.F capacitance) under different voltage conditions, sample FITC positive rate and particle concentration; FIG. 11B shows FITC positive rate and particle concentration of samples for electrotransport PKH 67-exosome cocktail at 500V voltage at different capacitances;

FIG. 12 shows Core700 column purified milk exosomes electroporated loaded with liraglutide samples;

FIG. 13 shows Core700 column purification electroporation treatment of liraglutide samples;

FIG. 14 shows the effect of liraglutide solution and exosome-liraglutide mixture after electrotransformation and core700 on luciferase expression (positive control 0.375ug/ml of liraglutide stock drug treatment group without electrotransformation);

FIG. 15 shows the conditions explored to achieve exosome transloading with PKH67 using the incubation method; wherein, FIG. 15 is a left graph showing the positive rate of FITC and the concentration of particles in a sample obtained by incubating PKH 67-exosome mixture (0.5 h) under different temperature conditions; FIG. 15 right graphically depicts FITC positive rate and particle concentration of samples incubated at 50℃for different times;

FIG. 16 shows Core700 column purification electroporation treatment of liraglutide samples;

FIG. 17 shows the effect of liraglutide solution and exosome-liraglutide mixture after co-incubation and Core700 on luciferase expression (positive control 0.375ug/ml of liraglutide drug substance treatment group without co-incubation);

FIG. 18 shows the cellular effects of exosomes loaded with different drugs;

FIG. 19 shows the nano-flow detection of the number of positive exosome particles in mice serum at various time points of gavage administration;

FIG. 20 shows nanoflow detection of positive exosome particle counts in mice serum at various time points of sublingual administration;

FIG. 21 shows the sublingual administration of different drug-loaded exosomes to mice, after 1 hour of gastric glucose solution, with time-dependent detection of blood glucose changes in mice;

FIG. 22 shows the sublingual administration of different concentrations of exosomes-liraglutide to mice, and the random blood glucose changes in mice with time;

figure 23 shows the change in fasting blood glucose of mice over time after 6 hours of fasting of mice with different concentrations of exosome-liraglutide administered sublingually.

Detailed Description

The invention discloses application of milk exosomes in preparation of drug carriers, and a person skilled in the art can properly improve process parameters by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

In the application of the milk exosome provided by the invention in the preparation of the drug carrier, the raw materials and the reagents used can be purchased from the market.

The invention is further illustrated by the following examples:

EXAMPLE 1 Industrial scalable purification Process of bovine exosomes

1) Pretreatment of milk samples with different protein removal methods

EDTA precipitation protein method: mixing skimmed milk with EDTA with the same volume, different concentration and different pH, incubating at room temperature for 15min, and precipitating casein. The concentration of EDTA is 0.15-0.35 mol.L ^－1, and the pH value is 6.0-8.0. 12 000 r.min ^－1, centrifuged for 40min, and the supernatant filtered sequentially through 0.80, 0.45 and 0.20 μm filters.

B. Sodium phosphate precipitation protein method: an equal amount of skim milk was added to the sodium phosphate solution. Several preliminary experiments were performed on the concentration and pH range of sodium phosphate, such as using 0.5 mol.L ^－1 (pH 5.3, pH 6.5, and pH 7.6), 1.0 mol.L ^－1 (pH 5.2, pH6.3, and pH 7.6), and 2.0 mol.L ^－1 (pH 6.0, pH 7.0, and pH 8.0) sodium phosphate. The protein was precipitated overnight at 4℃and the supernatant after precipitation was filtered through 0.80, 0.45 and 0.20 μm filters in this order.

C. Sodium citrate solubilization method: skim milk was mixed with equal amounts of 2%, 4%, 6%, 8% and 16% sodium citrate, incubated on ice for 2h to dissolve casein, and filtered through a 0.20 μm filter.

2) TFF method for primary purification and concentration of milk exosomes

The pretreated milk supernatant is treated by a hollow fiber column with the relative molecular mass of 750 000. Setting the maximum pressure of the system parameter TMP to be 2 atmospheres, and setting the alarm pressure to be 2 atmospheres; volume: the minimum volume is 100mL, and the alarm volume is 100mL. The sample was poured into a circulating cup and replaced with 1 XPBS until the concentration of protein penetrated was 0.00 g.L ^－1, and the liquid in the cup was concentrated to 1/20 of the stock solution to collect the sample.

3) BE-SEC and UC pure milk exosomes BE-SEC:

The Pure 25 purification system adopts ultrapure water to wash, the HiScreen Capto Core column is connected with the ultrapure water to wash 3 column volumes continuously, buffer solution 1 XPBS is adopted to balance 3 column volumes after the washing is finished, the initially Pure sample is purified by adopting the HiScreen CaptoCore column with the flow rate of 2mL min ^－1, and the sample at the peak position of the flow passing through is collected, namely the milk exosome.

Experimental results:

As shown in FIG. 1, a pretreatment sample was obtained by a protein precipitation method, and a purification method of TFF combined with BE-SEC was used to obtain milk exosomes. Comparing with UC, which is the traditional exosome extraction method, the method proves that the obtained sample has higher yield and purity.

As shown in FIG. 2, membrane structures were observed under TEM for the exosomes extracted by TFF, BE-SEC and UC, and were in the form of a saucer or disc. However, the UC extracted exosome under TEM has a more heterogeneous shooting background and more protein aggregates. Protein precipitation pre-treated samples and purification methods using TFF in combination with BE-SEC were relatively clean in background.

The results of detecting the particle size, concentration and purity of exosomes using NanoFCM are shown in fig. 3: the average grain diameter of the exosome obtained by TFF is (66.92 +/-16.39) nm, the yield is 3.6X10 ¹³ grains L ^-1, and the purity is 58%; the diameter of milk exosomes prepared by TFF+BE-SEC is concentrated and is 30-150 nm, the average grain diameter of exosomes is (66.41 +/-15.81) nm, the yield is 2.5X10. 10 ¹³ grains L ^-1, and the purity is 90%; the average particle size of the exosomes obtained by UC is (68.87 +/-18.04) nm, the yield is 2.4X10- ¹¹ particles L ^-1, and the purity is 25%. The method shows that the exosome prepared by TFF+BE-SEC has higher yield and higher purity.

As shown in fig. 4: HPLC was used to detect the content of free protein (particle size smaller than exosome size) in exosome samples. The retention time of the milk exosomes extracted by the 3 methods on the TSKgel G6000 PWXL column is 8min. However, the ratio of TFF to exosome samples extracted by BE-SEC is smaller than that of impurity peaks (RT >8 min) of samples obtained by UC, and the final product obtained by TFF+BE-SEC is more represented as a single peak.

By adopting a purification method of preprocessing milk by protein precipitation and combining TFF with BE-SEC, an exosome sample with higher yield and purity can BE obtained, more importantly, TFF can realize the initial purification and concentration of the sample from a few liters to hundreds of liters, and the TFF and BE-SEC are combined, so that the large-scale preparation of milk exosome can BE finally realized.

EXAMPLE 2 milk exosome drug-loading Process-ultrasonic Loading method

Principle of: the ultrasonic waves can disturb the arrangement of lipid chains in the membrane to form temporary small holes, so that the exosomes are subjected to low-frequency continuous ultrasonic waves, the encapsulated air is inflated by the sound waves, the inner pressure of the vesicles is increased to reach the elastic limit of the bilayer, the phospholipid layer is provided with pores, and the drug is diffused into the liposome through the pores due to the high concentration of the drug outside the membrane and the difference of osmotic pressure.

The operation steps are as follows:

Ultrasonic loading optimal conditions were explored using PKH 67: after PKH67 and milk exosomes are incubated for 30min at room temperature, the cell disruptors fumbly the ultrasonic power respectively

150W/300w/450w/600w/800w/1000w/1200w, and explore ultrasound time

0S/50s/250s/500s/750s/20min/30min/1h/2h/4h/6h. The number of particles in the sample after sonication was measured using NanoFCM and the PKH67 positive rate. And according to the optimized ultrasonic loading conditions, exploring the ultrasonic volume, and detecting the particle number, the particle size and the PKH67 positive rate of the sample by utilizing NanoFCM after ultrasonic.

Experimental results:

TABLE 1 influence of ultrasound Power and ultrasound time on exosome load positivity

TABLE 2 influence of ultrasound Power and ultrasound time on exosome particle count (10 ¹⁰ particals/mL)

TABLE 3 influence of different ultrasound volumes on exosome loading (600 w ultrasound 500 s)

The loading efficiency of exosomes after the ultrasonic waves with different powers is greatly different, and the positive rate after the ultrasonic waves is between 5% and 90%; the power of 450W-1200W, the ultrasonic time exceeds 20min, the loading efficiency gap is not obvious, and the loading efficiency is basically stabilized at about 80%. As the ultrasound time is prolonged, the particle count decreases. And can realize the loading scale of a 3ml-500ml system, and the particle number and the loading positive rate after loading are relatively stable.

Ultrasonic loading of liraglutide:

Mixing 1×10 ¹¹ exosome particles with 5mg liraglutide, performing ultrasound, purifying with Core700 or QA column, and significantly changing solution state before and after ultrasound, as shown in fig. 5: the liraglutide solution after ultrasonic treatment (left, more precipitate is separated out after ultrasonic treatment, the solution is colloid) and the milk exosome-liraglutide mixed solution (right).

Taking 25mg of liraglutide, carrying out ultrasonic treatment on a mixed solution of 25mg of liraglutide and 5×10 ¹¹ exosome particles, and collecting flow-through after ultrasonic treatment by the Core 700. As shown in FIGS. 6-7, a 5mL Core700 column was used to collect 2-4mL of the flow-through sample after the beginning of the sample injection, which was the exosome particles loaded with liraglutide, and the free liraglutide was adsorbed on the column and eluted.

250Mg of liraglutide and 250mg of liraglutide are mixed with 5X 10 ¹² exosome particles, and after ultrasonic treatment, the mixture is purified by a QA column, and flow through and elution of each component sample are collected. As shown in fig. 8 to 9, the liraglutide-loaded exosomes and the free liraglutide were eluted at different salt concentrations, respectively, according to the difference in the charged amounts.

GLP-1R-CHO cells were inoculated into 96-well plates, and after cell culture for 24 hours, the medium was changed to a solution containing either blank milk exosomes, or 10 ⁵/mL,10⁶/mL,10⁷/mL,10⁸/mL,10⁹/mL,10¹⁰/mL mill-exosomes-liraglutide, and liraglutide at the corresponding particle concentration for 5 hours. And (3) collecting samples according to the specification of the luciferase kit, adding 100ul of cell lysate without holes to lyse cells for 5min, collecting the cell lysate, centrifuging at 1200rpm for 5min, adding a substrate (incubating for 5 min), and detecting fluorescence by an enzyme-labeled instrument. The results are shown in FIG. 10 and Table 4.

TABLE 4 Table 4

Conclusion of experiment: from the comparison of the liquid shape of the raw material medicine and the milk exosome-liraglutide mixed liquid after ultrasonic treatment, the liraglutide is not separated out but loaded on the exosome when the exosome is contained. After ultrasonic treatment of the milk exosome-liraglutide mixed solution, QA purification is carried out, and a chromatogram shows that the absorption ratio of the sample after ultrasonic loading in high-salt elution (conductivity 70.44 Ms/cm) A260/A280 is increased to 1.88, the exosome is broken through a double-layer membrane after ultrasonic treatment, the exosome is an absorption peak of the released nucleic acid content, and the exosome particle number is reduced after ultrasonic treatment time is prolonged. After purification by core700 or QA, the cells were treated by proportional dilution and the measured luciferase expression was seen to be dose dependent. The 1 multiplied by 10 ¹⁰ particle number milk exosome-liraglutide treated cells remarkably improve the expression quantity of cell luciferase, which shows that the loading of the milk exosome on the liraglutide is realized and the biological activity is still realized.

EXAMPLE 3 milk exosome drug-carrying Process-electroporation method

Principle of: electroporation is a microbiological technique in which an electric field is applied to cells to increase the permeability of the cell membrane, allowing chemicals, drugs or DNA to be introduced into the cells.

The operation steps are as follows:

The optimal conditions for electroporation drug loading were explored using PKH 67: after incubation of PKH67 with milk exosomes for 30min at room temperature, the electroporation apparatus was performed at a voltage of 100-500v and a capacitance of 100-500 uF. The number of particles, the particle size and the PKH67 positive rate of the samples after the electric transformation are detected by NanoFCM.

And (3) carrying out electrotransformation on 25mg of liraglutide and a mixed solution of 25mg of liraglutide and 5×10 ¹¹ exosome particles, removing free liraglutide by core700 after electrotransformation, and collecting flow through.

GLP-1R-CHO cells were inoculated into 96-well plates, and after cell culture for 24 hours, the medium was changed to a solution containing either blank milk exosomes, or 10 ⁵/mL,10⁶/mL,10⁷/mL,10⁸/mL,10⁹/mL,10¹⁰/mL mill-exosomes-liraglutide, and liraglutide at the corresponding pellet concentration for 5 hours. Collecting sample according to the specification of luciferase kit, adding 100ul of cell lysate without holes to lyse cells for 5min, collecting cell lysate, centrifuging at 1200rpm for 5min, adding substrate (incubating for 5 min), and detecting fluorescence with enzyme-labeled instrument.

The experimental results are shown in FIGS. 11 to 14 and tables 5 to 7:

TABLE 5 influence of voltage on exosome loading

Voltage V	Positive rate%	Concentration particle count X10 ¹⁰/mL
			0	7.12	8.55
100	8.2	2.8
			200	28.5	2.52
300	39	2.26
			400	62.9	1.56
500	59.5	2.14

TABLE 6 influence of capacitance on exosome loading

Capacitor uF	Positive rate%	Concentration particle count X10 ¹⁰/mL
			0	6.35	8.55
100	46.8	3.2
			200	51.8	3.78
300	71.2	2.78
			400	69.8	2.96
500	57.1	2.11

TABLE 7

EXAMPLE 4 milk exosome drug-carrying Process-Co-incubation method

Principle of: the medicine is adsorbed on the medicine carrier by utilizing the interaction force between substances.

The operation steps are as follows:

Optimal conditions for co-incubation drug loading were explored using PKH 67: after incubation of PKH67 with milk exosomes for 30min at room temperature, the samples were incubated at 4℃at 37℃for 30min,1h,2h,500rpm shaking. The number of particles, the particle size and the PKH67 positive rate of the samples after the co-incubation are detected by NanoFCM.

Taking 25mg of liraglutide, incubating and incubating 25mg of liraglutide with 5×10 ¹¹ exosome particle mixed solution, removing free liraglutide by Core700, and collecting flow-through.

The results of the experiments are shown in FIGS. 14 to 17 and tables 8 to 10.

TABLE 8 influence of incubation temperature on exosome loading

Temperature (temperature)	Positive rate%	Concentration particle count X10 ¹⁰/mL
			4℃	7.12	8.55
37℃	8.2	3.18
			42℃	28.5	3.32
50℃	49	3.26
			60℃	22.9	1.56

Table 9 influence of incubation period on exosome loading

Duration of time	Positive rate%	Concentration particle count X10 ¹⁰/mL
			0.5h	32.1	3.6
1h	46.8	3.78
			2h	29.8	3.78
4h	21.2	2.78
			8h	9.8	1.96

TABLE 10 cell efficacy by Co-incubation

Conclusion of experiment: after purification by core700, the treated cells were diluted in proportion and examined for luciferase expression in a dose-dependent manner. The 1 multiplied by 10 ¹⁰ particle number milk exosome-liraglutide treated cells remarkably improve the expression quantity of cell luciferase, which shows that the loading of the milk exosome on the liraglutide is realized and the biological activity is still realized.

EXAMPLE 5 milk exosomes loaded with different T2DM drugs

Currently, there are a variety of drugs on the market for the treatment of type two diabetes mellitus (type 2diabetes mellitus,T2DM), such as insulin de-glu, liraglutide, cable Ma Lutai, and Tirzepatide which are already marketed abroad. The invention realizes that milk exosomes are loaded with different T2DM medicaments, so that the administration modes of the medicaments can be changed from injection into oral administration, and the dilemma of no oral administration of diabetes medicaments in the market is solved.

The operation is as follows:

1) According to the drug loading mode of the milk exosome introduced in 4.2, an electroporation method is selected, and the milk exosome is used for respectively loading insulin deluge, insulin deltoid, liraglutide, and cord Ma Lutai and Tirzepatide and purifying by a core700 column;

2) GLP-1R-CHO cells are inoculated to a 96-well plate, after cell culture is carried out for 24 hours, the culture medium is replaced by a blank milk exosome or 10 ⁵/mL,10⁶/mL,10⁷/mL,10⁸/mL,10⁹/mL,10¹⁰/mL milk exosome loaded with different drugs, and the culture is carried out for 5 hours. And (3) collecting samples according to the specification of the luciferase kit, adding 100ul of cell lysate without holes to lyse cells for 5min, collecting the cell lysate, centrifuging at 1200rpm for 5min, adding a substrate (incubating for 5 min), and detecting fluorescence by an enzyme-labeled instrument.

The experimental results are shown in fig. 18 and table 11:

TABLE 11 cell efficacy of exosomes loaded with different drugs

Conclusion of experiment:

From the results, milk exosomes loaded with different drugs can release GLP from cells and are dose dependent. The invention can realize loading of different types of protein polypeptide medicines.

EXAMPLE 6 sublingual administration of milk exosomes

Principle of: according to the invention, by utilizing the characteristics of non-keratinization of sublingual mucosa, rich capillaries and high blood flow speed, aiming at the problems that the milk exosome is easily damaged by gastric acid after passing through gastrointestinal tract, so that the bioavailability of the loaded medicine is low, the liraglutide can be degraded after being orally taken, and the like, the milk exosome loaded with the liraglutide is prepared into a sublingual tablet, so that the oral administration of the liraglutide is realized, and the liraglutide can be absorbed through the sublingual mucosa, directly enter into blood circulation through jugular vein and superior vena cava, and has rapid effect; and the medicine is taken without water, is placed under the tongue for dissolving, and has convenient administration and good taste.

Experimental operation:

A. PKH67 marked milk exosomes are respectively subjected to gastric lavage, sublingual administration and enteric capsule preparation;

B. after administration, blood of mice at different time points is collected, and the number of cationic particles in the blood is detected by utilizing nano flow to determine the blood-in absorption effect of exosomes.

The results are shown in FIGS. 19 to 20 and tables 12 to 14:

Table 12 intragastric administration data

TABLE 13 sublingual administration data

Table 14 enteric capsule data

	mean	SD
			0min	4.58×10⁷	2.67×10⁷
30min	6.10×10⁷	1.89×10⁷
			1h	1.53×10⁷	9.87×10⁶
2h	1.53×10⁷	8.22×10⁶
			3h	3.05×10⁷	1.24×10⁷
4h	6.86×10⁷	2.78×10⁷
			5h	1.30×10⁸	9.18×10⁶
8h	1.53×10⁷	7.35×10⁶
			22h	9.92×10⁷	3.22×10⁷

The mice were given PKH67 labelled milk exosomes by gavage, blood was collected at different time points to detect positive particles, each mouse was gavaged with 4X 10 ¹¹ particles, the highest particles were detected at 5min, and 2X 10 ⁹ positive particles were detected in the blood of the mice, thus, equivalent to 0.5% exosomes entering the blood. When PKH 67-labeled milk exosomes were sublingually given, the number of positive particles detected for 5min was the greatest, approximately 2X 10 ¹⁰ positive particles, corresponding to 5% of exosomes entering the blood. While the enteric capsule administration was attempted, the number of positive particles in the blood of mice was not significantly increased in the administered group. Thus, the bioavailability of sublingual administration is significantly higher than that of intragastric administration.

EXAMPLE 7 milk exosome sublingual tablet

Experimental operation: the liraglutide-loaded lyophilized powder of milk exosome is mixed with disintegrating agent, filler, binder, lubricant and correctant to prepare sublingual tablet. In each 1000 pieces of liraglutide milk exosome sublingual tablets, the content of main medicine is 10 ⁵-10¹⁷ particles, the content of disintegrating agent is 1-50g, and the content of filling agent is 20-400g; 0-50g of flavoring agent, 5-100g of adhesive and 1-10 g of lubricant;

The disintegrating agent is selected as follows: microcrystalline cellulose, crosslinked sodium carboxymethyl cellulose, crosslinked polyvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, starch, tween, and sodium dodecyl sulfate;

The filler selected is: microcrystalline cellulose, microcrystalline cellulose-mannitol, microcrystalline cellulose-micro powder silica gel, lactose, starch, modified starch, mannitol, sorbitol, xylitol, erythrose, trehalose, pregelatinized starch, powdered sugar, glucose, dextrin, calcium sulfate, or a mixture of two or more thereof.

The flavoring agent is selected as follows: stevioside, sugar powder, glycyrrhizin, aspartame, sucralose, cyclamate, thaumatin, saccharin and the like.

The adhesive is selected as follows: purified water, starch slurry, hydroxypropyl methylcellulose, polyvinylpyrrolidone, carbomer, dextrin, gelatin slurry, acacia slurry, sodium alginate and syrup.

The lubricant used was: any one or more than two of stearic acid, magnesium stearate, calcium stearate, micro powder silica gel, talcum powder, hydrogenated vegetable oil, polyethylene glycol 4000, polyethylene glycol 6000, sodium dodecyl sulfate, magnesium sulfate of the order of twelve ten thousand and sodium fumarate.

The optimal formula is as follows: 1X 10 ¹⁵ particle exosome-liraglutide, 4g croscarmellose sodium, 80g mannitol, 25g purified water, 3.5g magnesium stearate, prepared according to the following preparation steps

The preparation method comprises the following steps:

the first step: freeze-drying the main medicine, and sieving with 80-120 mesh sieve;

And a second step of: drying filler, disintegrating agent, correctant, lubricant, pulverizing, sieving with 80-120 mesh sieve, and pre-treating;

And a third step of: uniformly mixing the pretreated main medicine, the filling agent, the flavoring agent and the disintegrating agent;

fourth step: adding binder into the above boredom, making into soft material, granulating, and drying;

fifth step: adding lubricant into the dry particles, mixing uniformly, and tabletting to obtain the finished product.

Example 8

Adding 0.6g mannitol into 1X 10 ¹³ grain number main medicine liraglutide milk exosome, freeze drying, mixing with 55g microcrystalline cellulose, 1g disintegrant sodium carboxymethyl cellulose and 1g lubricant magnesium stearate, oven drying, pulverizing, sieving, mixing, granulating with 20 mesh sieve, air drying until water content is less than or equal to 3%, adding lubricant, mixing, and tabletting to obtain liraglutide milk exosome sublingual tablet.

Example 9

Experimental operation:

Loading insulin deluge, insulin deltoid, liraglutide, and cord Ma Lutai, tirzepatide into milk exosomes by electroporation method, purifying to obtain drug-loaded exosomes, and preparing sublingual tablet according to the above formulation;

Sublingual tablets with a particle count of 1X 10 ¹¹ were administered sublingually to each mouse, and after glucose injection, the effect of the mice on glucose tolerance was examined.

The results are shown in FIG. 21 and Table 15:

TABLE 15

Analysis of results:

By utilizing milk exosomes, loading of various hypoglycemic agents can be achieved. The drug-loaded exosome can realize obvious blood sugar reducing effect within 15min through oral administration. And can maintain the stability of blood sugar and improve the tolerance reaction of mice to sugar.

Example 10

The method of electroporation is used to load and purify liraglutide (including, but not limited to, insulin deluge, cord Ma Lutai, tirzepatide) into milk exosomes to obtain drug-loaded exosomes, and to prepare sublingual tablets according to the optimal formulation of sublingual tablets, the results of loading liraglutide into milk exosomes are described herein as an example.

Different concentrations of liraglutide-loaded milk exosomes were administered sublingually to diabetic mice and the random blood glucose of the mice was measured.

The results are shown in FIG. 22 and Table 16:

Table 16

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Conclusion: from the results of random blood glucose, liraglutide-loaded exosomes can significantly reduce blood glucose.

Example 11

Charging liraglutide (including, but not limited to, insulin deluge, cord Ma Lutai, tirzepatide) into milk exosomes by electroporation, purifying to obtain drug-loaded exosomes, and preparing sublingual tablets, wherein the results of charging liraglutide into milk exosomes are described as an example;

After 6h of fasted diabetic mice, different concentrations of liraglutide-loaded milk exosomes were orally administered to test the fasting blood glucose of the mice.

The results are shown in FIG. 23 and Table 17:

TABLE 17

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Conclusion: from the results of fasting blood glucose, liraglutide-loaded exosomes can significantly lower blood glucose.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and variations could be made by those skilled in the art without departing from the principles of the present invention, and such modifications and variations should also be considered as being within the scope of the present invention.

Claims

1. Use of milk exosomes in the preparation of a pharmaceutical carrier;

the administration route of the medicine is oral administration;

The preparation method of the milk exosome comprises the following steps:

Step 1, pretreatment;

step2, preliminary purification and concentration;

Step 3, fine purification;

The pretreatment includes fat removal and/or protein removal;

The primary purification and concentration adopts tangential flow ultrafiltration; the purification comprises captocore 700 column purification;

The fat removal in step1 comprises:

(2) Removing fat by adopting a centrifugal method;

the filtering includes depth filtration and/or capsule filter filtration;

The protein removal in the step 1 adopts a protein dissolving method and/or a protein precipitation method;

The protein dissolving method comprises a sodium citrate dissolving method;

the precipitated protein method comprises one or the combination of EDTA precipitated protein method, sodium phosphate precipitated protein method and ammonium sulfate precipitated protein method;

The sodium citrate dissolution method specifically comprises the following steps: pouring an equal volume of milk into a sodium citrate solution with the mass concentration of 2%, shaking the milk in a shaking table for 60min in an ice bath, clarifying the milk sample, and filtering the clarified milk sample with a 0.2 mu m filter membrane;

The EDTA precipitated protein method specifically comprises the following steps: pouring an equal volume of milk into EDTA solution, precipitating at room temperature, standing for 15min, centrifuging at 12000rpm for 40min, and filtering the supernatant with 0.45 μm and 0.2 μm filter membrane; the concentration of the EDTA solution is 0.35M, and the pH value is 7.0;

the sodium phosphate precipitation protein method comprises the following steps:

(1) Whole milk

(2) Skimmed milk

Centrifuging milk at 16000rpm for 30min to remove fat, mixing equal volume of milk with sodium phosphate solution, stirring, precipitating, standing at room temperature, centrifuging at 3500rpm for 10min, collecting supernatant, and filtering with a filter membrane;

The concentration of the sodium phosphate solution is 1M, and the pH value is 6.3;

The sodium phosphate precipitation protein method specifically comprises the following steps: taking 16000rpm of milk, centrifuging for 30min to remove fat, pouring an equal volume of milk into a sodium phosphate solution, stirring for 15min by a magnetic stirrer at 350rpm, centrifuging for 10min at 3500rpm of a precipitated milk sample, and sequentially filtering the supernatant with 0.8 μm,0.45 μm and 0.2 μm filter membranes;

The ammonium sulfate precipitated protein method specifically comprises the following steps: pouring an equal volume of milk into an ammonium sulfate solution, stirring with a glass rod while pouring, precipitating at room temperature, standing for 1.5h, centrifuging at 6000rpm for 20min, and sequentially filtering the supernatant with 0.8 μm,0.45 μm and 0.2 μm filter membranes; the concentration of the ammonium sulfate solution is 3.0M;

the drug includes one or more of insulin deluge, insulin detention, liraglutide, cord Ma Lutai or Tirzepatide.

2. The use of claim 1, wherein the medicament comprises liraglutide.

3. The application of milk exosomes as drug carriers in preparing drugs;

the administration route of the medicine is oral administration;

The preparation method of the milk exosome comprises the following steps:

Step 1, pretreatment;

step2, preliminary purification and concentration;

Step 3, fine purification;

The pretreatment includes fat removal and/or protein removal;

The purification comprises captocore 700 column purification;

The fat removal in step1 comprises:

(2) Removing fat by adopting a centrifugal method;

the filtering includes depth filtration and/or capsule filter filtration;

The protein dissolving method comprises a sodium citrate dissolving method;

(1) Whole milk

(2) Skimmed milk

4. The use of claim 3, wherein the medicament comprises liraglutide.

5. The use according to claim 3 or 4, wherein the disease treated with the medicament comprises type II diabetes.