Functionalized drug-loaded exosomes based on milk exosomes and preparation method and application thereof

Abstract

The present invention belongs to the field of biotechnology and pharmaceutical engineering technology, and discloses a functionalized drug-loaded exosome based on milk exosomes, a preparation method thereof, and an application thereof. The functionalized drug-loaded exosomes include a carrier and a water-insoluble drug, and the water-insoluble drug is loaded inside the carrier; wherein the surface of the carrier is loaded with milk exosomes with functionalized molecules, and the functionalized molecule is any one of phosphatidylserine, a molecule capable of macrophage targeting, and a molecule or polymer that enhances the targeting and stability of milk exosomes; the water-insoluble drug is any one of an antibacterial drug, an antitumor drug, and a gene drug. The functionalized drug-loaded exosomes in the present invention can improve the gastrointestinal stability of oral administration and enhance the efficacy against bacterial pathogens; expand the application scope of milk exosomes in the fields of anti-intestinal bacterial infection and food preservation, and the exosome content in milk is very rich, which is convenient for large-scale preparation and has important application value.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and pharmaceutical engineering technology, and in particular to functionalized drug-loaded exosomes based on milk exosomes, and a preparation method and application thereof. Background Art

[0002] The intestine is an important barrier of the body and is also the place where infection is most likely to occur. Different types of infections can cause intestinal infections, including bacterial infection, fungal infection, or worm infection. Among them, viral intestinal infections caused by MDR bacterial pathogens are becoming more and more serious. Therefore, viral intestinal infections caused by MDR bacterial pathogens are a major problem that needs to be solved urgently.

[0003] Antimicrobial drugs have always been the first choice for controlling and treating microbial infections, and they play a vital role in protecting human and animal health. The widespread and irrational use of antimicrobial drugs has accelerated the emergence, prevalence, and rapid spread of bacterial resistance, causing many clinical antimicrobial drugs to gradually show reduced therapeutic efficiency or even become ineffective during use. Coupled with the frequent discovery of multidrug resistance, such as NDM-1 and MCR-1 in Escherichia coli, Gram-negative bacteria are more seriously resistant to antimicrobial drugs, posing a serious threat to global public health security. Therefore, how to use antimicrobial drugs rationally, improve their therapeutic effects, extend the service life of existing antimicrobial drugs, and overcome the limitations of the efficacy of antimicrobial drugs with poor solubility in oral administration remain to be solved.

[0004] For decades, various synthetic nanoparticle delivery systems have emerged and been used to improve the pharmacokinetic and pharmacodynamic properties of antimicrobial drugs. When carried by a delivery system, the clearance and tissue distribution characteristics of antimicrobial drugs are primarily influenced by the characteristics of the carrier rather than the physicochemical properties of the antimicrobial drug molecules. Therefore, synthetic drug delivery systems have been developed to improve drug efficacy and therapeutic index while minimizing drug toxicity and off-target side effects. Liposomes, micelles, dendrimers, nanocapsules, and nanosponges are the most prominent examples of synthetic drug delivery systems. Although these carriers offer advantages for antimicrobial drug delivery, many obstacles remain in their clinical translation. For example, the safety and stability of the carriers have been important factors restricting their development.

[0005] In recent years, the field of biological or biomimetic drug carriers has developed rapidly. Extracellular vesicles (EVs) are cell-derived membrane structures that can transport various active biomolecules from producer cells to recipient cells, thereby altering the physiological functions of the recipient cells. This ability has attracted considerable attention for EVs for therapeutic applications and as a potential carrier for antimicrobial drugs. However, the low yield and poor stability of EVs hinder the oral delivery of poorly soluble drugs for the treatment of viral enteric infections or other intestinal diseases. Furthermore, existing delivery vehicles are plagued by uncertain safety issues in the food preservation field, which greatly limits their application in this field.

[0006] To this end, the present invention provides a functionalized drug-loaded exosome based on milk exosomes, and a preparation method and application thereof. Summary of the Invention

[0007] In order to address the deficiencies in the above-mentioned prior art, the present invention provides a functionalized drug-loaded exosome based on milk exosomes, and a preparation method and application thereof.

[0008] The present invention provides a functionalized drug-loaded exosome based on milk exosomes and its preparation method and application, which are achieved through the following technical solutions:

[0009] The first object of the present invention is to provide a functionalized drug-loaded exosome based on milk exosomes, comprising a carrier and a water-insoluble drug, wherein the water-insoluble drug is loaded inside the carrier;

[0010] The surface of the carrier is loaded with milk exosomes containing functionalized molecules, wherein the functionalized molecules are any one of phosphatidylserine, molecules capable of macrophage targeting, and molecules or polymers that enhance the targeting and stability of milk exosomes;

[0011] The water-insoluble drug is any one of an antibacterial drug, an antitumor drug and a gene drug.

[0012] Furthermore, the water-insoluble drug is α-mangostin.

[0013] Furthermore, the molecule capable of achieving macrophage targeting is any one of galactose, lactose, mannose and folic acid.

[0014] Furthermore, the molecule or polymer that improves the targeting and stability of milk exosomes is cholesterol or polyethylene glycol PEG.

[0015] Furthermore, when the milk exosomes loaded with functionalized molecules also contain marker proteins;

[0016] The marker protein is any one of TSG101, Flotillin-1, CD63, CD81, Alix, and HSP60.

[0017] Furthermore, the particle size of the carrier is 35 to 1000 nm.

[0018] The second object of the present invention is to provide a method for preparing the functionalized drug-loaded exosomes, comprising the following steps:

[0019] Step 1: Loading water-insoluble drugs into milk exosomes to obtain drug-loaded exosomes;

[0020] Step 2, loading functionalized molecules onto the surface of the drug-loaded exosomes to obtain functionalized drug-loaded exosomes;

[0021] The functionalized molecule is any one of phosphatidylserine, a molecule capable of macrophage targeting, and a molecule or polymer that improves the targeting and stability of milk exosomes.

[0022] Furthermore, in step 1, the loading method is any one of co-incubation, electroporation, ultrasonic treatment, co-extrusion and repeated freezing and thawing.

[0023] Furthermore, the milk exosomes are separated from milk, and the separation method is any one or more of ultracentrifugation technology, size exclusion chromatography technology, density gradient centrifugation technology and immunoseparation technology.

[0024] The third object of the present invention is to provide a use of the above-mentioned functionalized drug-loaded exosomes as a drug delivery system.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention uses milk exosomes, which are abundant in milk, as a carrier. Milk exosomes have a vesicle structure, so that water-insoluble drugs can be loaded into the interior of the milk exosomes to obtain drug-loaded exosomes, thereby significantly improving the solution stability of water-insoluble drugs; the drug-loaded exosomes are then functionalized with functional molecules such as phosphatidylserine, galactose, lactose, mannose and folic acid to obtain targeted functionalized drug-loaded exosomes, thereby making them significantly targeted to macrophages. The drug delivery system formed by the method of the present invention can effectively improve the efficacy of anti-bacterial infection.

[0027] The functionalized drug-loaded exosomes obtained by the present invention have excellent intestinal mucus penetration and are effective in treating necrotizing enterocolitis caused by Clostridium perfringens. Furthermore, modification with cholesterol and polyethylene glycol can enhance stability and intestinal mucus permeability, thereby improving efficacy.

[0028] The functionalized drug-loaded exosomes of the present invention can improve the gastrointestinal stability of oral administration and enhance the efficacy against bacterial pathogens; milk exosomes are derived from milk and are extremely safe, ensuring food safety and antibacterial effectiveness; they expand the application scope of milk exosomes in the fields of anti-intestinal bacterial infection and food preservation. The exosome content in milk is very rich, which is convenient for large-scale preparation and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 To separate the particle size distribution of milk exosomes Exo, curve ULC is the particle size distribution of milk exosomes Exo separated by ultracentrifugation, and curve SEC is the particle size distribution of milk exosomes Exo separated by size exclusion chromatography;

[0030] Figure 2 The curve ULC represents the protein marker identification result of milk exosomes Exo separated by ultracentrifugation, and the curve SEC represents the protein marker identification result of milk exosomes Exo separated by size exclusion chromatography;

[0031] Figure 3 Transmission electron microscopy images of isolated milk exosomes Exo, Figure 3 (a) Transmission electron microscopy image of milk exosomes separated by ultracentrifugation at a scale of 500 μm. Figure 3 (b) is the transmission electron microscope image of the box in Figure (a) at a scale of 50 μm. Figure 3 (c) Transmission electron microscopy image of milk exosomes separated by size exclusion chromatography at a scale of 500 μm. Figure 3 (d) is the transmission electron microscope image of the box in Figure (c) at a scale of 50 μm;

[0032] Figure 4 These are the stability test results of α-mangostin solution and α-mangostin-loaded exosomes solution, where AMG represents the α-mangostin group and AExo represents the α-mangostin-loaded exosomes group;

[0033] Figure 5 These are the test results of the intestinal mucus penetration test of milk exosomes, where FITC represents the fluorescein thiocyanate group and Exo-FITC represents the milk exosome group labeled with fluorescein thiocyanate;

[0034] Figure 6These are the test results of the phosphatidylserine-modified exosomes' ability to eliminate bacteria in macrophages. PBS represents the PBS group, mExo represents the milk exosomes treatment group, AMG represents the α-mangostin treatment group, and PS-AExo represents the phosphatidylserine-modified exosomes loaded with α-mangostin treatment group.

[0035] Figure 7 Drug-loaded exosomes (AExo) for the treatment of VRE fm The test results of mouse intestinal infection caused by CAU369, Figure 7 A is the quantitative distribution of bacterial VRE in the jejunum, ileum, cecum, and colon of mice; Figure 7 B is the number of bacterial VRE in the cecum of mice after treatment, where PBS is the PBS group, LZD is the linezolid treatment group, AMG is the α-mangostin treatment group, and AExo is the exosome treatment group loaded with α-mangostin;

[0036] Figure 8 The results of the test of drug-loaded exosomes AExo in treating necrotic enteritis in chickens caused by Clostridium perfringens are shown in the figure. Figure 8 A is the quantitative distribution of Clostridium perfringens in the jejunum, ileum, cecum and rectum of chickens; Figure 8 B is the number of Clostridium perfringens in the cecum of chickens after treatment, where PBS is the PBS group, Exo is the milk exosome treatment group, AMG is the α-mangostin treatment group, and AExo is the exosome treatment group loaded with α-mangostin. DETAILED DESCRIPTION

[0037] As described in the background art, in order to achieve oral delivery, the present invention delivers poorly soluble drugs for treating viral intestinal infections or other intestinal diseases into the intestine to treat various intestinal infections. The present invention takes into account that milk exosomes are abundant in milk, have high stability, and good safety, making them ideal targets for drug delivery carriers. In addition, milk exosomes contain 43.6 mol% cholesterol in the total lipids, which significantly increases the rigidity of the phospholipid bilayer membrane, thereby having high stability in the gastrointestinal environment. In addition, milk exosomes have good mucus permeability and can penetrate chemical barriers to reach immune barriers. Therefore, the present invention uses milk exosomes as carriers to load water-insoluble drugs into the interior of milk exosomes, which can significantly improve the solution stability of water-insoluble drugs. Then, any one of phosphatidylserine, molecules that can achieve macrophage targeting, and molecules or polymers that enhance the targeting and stability of drug-loaded exosomes is used as a functional molecule. The drug-loaded exosomes are functionalized by the functional molecules to obtain targeted functionalized drug-loaded exosomes, thereby making them significantly targeted to macrophages and improving the efficacy of antibacterial infection. The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0038] The present invention provides functionalized drug-loaded exosomes based on milk exosomes, comprising a carrier and a water-insoluble drug, wherein the water-insoluble drug is loaded inside the carrier; and the functionalized drug-loaded exosomes of the present invention are obtained by the following preparation method:

[0039] Loading water-insoluble drugs into the interior of milk exosomes to obtain drug-loaded exosomes; then loading functionalized molecules onto the surface of the drug-loaded exosomes to obtain functionalized drug-loaded exosomes;

[0040] It should be noted that the present invention does not limit the loading order of water-insoluble drugs and functionalized molecules. The functionalized molecules can be loaded on the surface of the milk exosomes first, and then the water-insoluble drugs can be loaded inside the milk exosomes; or the water-insoluble drugs can be loaded inside the milk exosomes first, and then the functionalized molecules can be loaded on the surface. As long as the interior of the milk exosomes can be loaded with water-insoluble drugs and the surface can be loaded with functionalized molecules, drug-loaded exosomes with targeted functionality can be obtained.

[0041] The present invention does not limit the specific composition of the water-insoluble drug and can be selected according to actual needs, for example, it can be selected from antibacterial drugs, anti-tumor drugs, and gene drugs. Taking into account the low efficacy of BCS II and IV antibacterial drugs against intestinal infections, the present invention uses α-mangostin as a water-insoluble drug and loads it onto targeted functionalized exosomes, thereby improving the solubility of the drug and increasing its solubility, thereby improving the antibacterial efficacy.

[0042] The present invention does not limit the specific method of loading water-insoluble drugs into milk exosomes. Any loading method, such as co-incubation, electroporation, ultrasonic treatment, co-extrusion, and repeated freeze-thaw cycles, can be used as long as the purpose of drug loading is achieved. However, considering the ease and efficiency of loading, the present invention uses an incubation technique to load α-mangostin onto milk exosomes to obtain drug-loaded exosomes.

[0043] The present invention is not limited to the specific type or structure of the functionalized molecule, as long as the drug-loaded exosomes can be targeted and functionalized, for example, any one of phosphatidylserine, molecules that can achieve macrophage targeting (such as galactose, lactose, mannose and folic acid, etc.), and molecules or polymers that enhance the targeting and stability of milk exosomes (such as cholesterol and polyethylene glycol) can be selected to enhance the targeting of the drug-loaded exosomes, thereby not only improving the therapeutic effect but also reducing toxic side effects. The present invention takes into account the reasons for specific receptor recognition-mediated macrophage targeting, and preferably uses phosphatidylserine as the functionalized molecule. The present invention does not limit the specific loading method of the functionalized molecules on the drug-loaded exosomes, as long as the drug-loaded exosomes can be targeted and functionalized. The present invention takes into account the reasons of simplicity and efficiency, and optionally adopts a co-incubation method for loading.

[0044] The present invention takes into account the factors of cost and preparation convenience, uses milk as a source, and isolates and obtains milk exosomes therefrom, and the present invention does not limit the specific separation method of separating milk exosomes from milk, as long as milk exosomes can be isolated and obtained. The milk exosomes of the present invention contain marker proteins (the marker proteins are any one of TSG101, Flotillin-1, CD63, CD81, Alix, and HSP60). The presence of marker proteins can prove that the isolated exosomes are exosomes, and marker proteins are one of the technical means for identifying exosomes. And the present invention can optionally adopt any one or more of ultracentrifugation technology, size exclusion chromatography technology, density gradient centrifugation technology, and immunoseparation technology. Specifically, the following methods can be used to separate and obtain milk exosomes:

[0045] Separation method 1: Centrifuge the milk at 1000-3000g for 5-20 minutes to remove cell spheres and cell debris; collect the supernatant and centrifuge it at 8000-13000g for 10-40 minutes to remove fat globules and casein fragments; collect the supernatant and centrifuge it at 60000-100000g for 30-90 minutes to remove large particles and microvesicles; collect the supernatant and centrifuge it at 120000-300000g for 60-240 minutes to collect exosome particles.

[0046] Separation method 2: Centrifuge the milk at 1000-3000g for 5-20 minutes to remove cell pellets and cell debris; collect the supernatant and centrifuge it again at 8000-13000g for 10-40 minutes to remove fat globules and casein fragments; then use an exosome separation column with a specification of 35-350nm or 70-1000nm, a column volume of 3-100mL, and a sample volume of 0.1-10mL to collect the exosomes.

[0047] Separation method three: Add acetic acid to the pretreated milk at room temperature to pH 4.6, stir and mix for 5 minutes, then centrifuge at 10,000 × g for 10 minutes at 4°C, and filter the supernatant with a 0.22 μm membrane; then use an exosome separation column with a 35-350 nm or 70-1000 nm specification, a column volume of 3-100 mL, and a sample volume of 0.1-10 mL to collect the exosome components.

[0048] Separation method 4: Add 20-30 mM / L EDTA to the pretreated milk at room temperature, stir and mix for 5 minutes, then centrifuge at 10,000 × g for 10 minutes at 4°C, and filter the supernatant with a 0.22 μm membrane; then use an exosome separation column with a 35-350 nm or 70-1000 nm specification, a column volume of 3-100 mL, and a sample volume of 0.1-10 mL to collect the exosome components.

[0049] The particle size of milk exosomes obtained by the above method can reach 35 to 1000 nm.

[0050] Example 1

[0051] The present invention provides a functionalized drug-loaded exosome based on milk exosomes, and the preparation method thereof is as follows:

[0052] S1. Preparation of milk exosomes

[0053] Milk exosomes were isolated and obtained by ultracentrifugation using milk as the source of milk exosomes, and the obtained milk exosomes had a vesicle structure;

[0054] S2. α-mangostin is used as a water-insoluble drug and loaded into the vesicle structure of milk exosomes by co-incubation to obtain drug-loaded exosomes;

[0055] S3. Phosphatidylserine is used as a functional molecule and loaded onto the surface of the vesicle structure of milk exosomes by co-incubation to obtain targeted functionalized exosomes.

[0056] Example 2

[0057] The present invention provides a functionalized drug-loaded exosome based on milk exosomes, and the preparation method thereof differs from that of Example 1 only in that:

[0058] Milk exosomes were isolated and obtained using size exclusion chromatography;

[0059] Galactose was used as the functionalized molecule;

[0060] Using electroporation, α-mangostin was loaded into the interior of the vesicle structure of milk exosomes.

[0061] Example 3

[0062] The present invention provides a functionalized drug-loaded exosome based on milk exosomes, and the preparation method thereof differs from that of Example 1 only in that:

[0063] In this example, milk exosomes were isolated and obtained by density gradient centrifugation;

[0064] Cholesterol was used as the functionalized molecule;

[0065] Using ultrasonic treatment, α-mangostin was loaded into the interior of the vesicle structure of milk exosomes.

[0066] Example 4

[0067] The present invention provides a functionalized drug-loaded exosome based on milk exosomes, and the preparation method thereof differs from that of Example 1 only in that:

[0068] In this example, mannose was used as the functionalized molecule.

[0069] Example 5

[0070] The present invention provides a functionalized drug-loaded exosome based on milk exosomes, and the preparation method thereof differs from that of Example 1 only in that:

[0071] In this embodiment, polyethylene glycol (PEG) is used as the functionalized molecule.

[0072] Experimental part

[0073] The experimental materials used in the present invention were purchased from conventional biochemical reagent manufacturers unless otherwise specified. The quantitative experiments in the following experiments were repeated three times and the results were averaged.

[0074] 1. Isolation of Milk Exosomes

[0075] The present invention adopts ultracentrifugation and size exclusion chromatography to separate exosomes.

[0076] The specific steps for isolating exosomes by ultracentrifugation are as follows:

[0077] First, fresh milk was centrifuged at 2000g and 4°C for 10 minutes to remove dead cells and cell debris; the supernatant was centrifuged at 10000g and 4°C for 30 minutes to remove impurities such as fat globules; the supernatant was centrifuged at 100000g and 4°C for 60 minutes to remove impurities such as extracellular vesicles; the supernatant was centrifuged at 135000g and 4°C for 90 minutes, the supernatant was discarded, and the precipitate was resuspended in PBS (pH 7.4) to obtain the milk exosome suspension.

[0078] The specific steps for isolating exosomes by size exclusion chromatography are:

[0079] First, fresh milk was centrifuged at 2000g at 4°C for 10 minutes to remove dead cells and cell debris. The supernatant was then centrifuged again at 10,000g at 4°C for 30 minutes to remove impurities such as fat globules, and the supernatant was collected. An exosome separation column, qEV (35-350nm, IZON), was rinsed and activated with PBS for 10 minutes. The collected supernatant was then added to the chromatographic column. Once the sample had completely entered the column, it was rinsed with PBS again, and the different fractions were collected to obtain the exosome solution.

[0080] 2. Characterization of Milk Exosomes

[0081] The present invention characterizes the properties of milk exosomes obtained by the above two separation methods as follows:

[0082] 1) Milk exosome particle size and concentration test

[0083] The milk exosomes separated by the two methods were tested for particle size concentration, and the test results were as follows: Figure 1 As shown, the curve ULC is the particle size distribution of milk exosomes Exo obtained by ultracentrifugation, and the curve SEC is the particle size distribution of milk exosomes Exo obtained by size exclusion chromatography. The present invention uses a nanoparticle tracking analyzer to measure the particle size concentration of milk exosomes:

[0084] First, dilute 10 μL of the exosome resuspension to an appropriate amount and mix thoroughly by pipetting. The diluted milk exosome resuspension is then tested on the analyzer at room temperature. Test sample conditions: 0.3 nm - 5 μm, concentration range: 0.1 ppm - 40% w / v.

[0085] Depend on Figure 1 It can be seen that the particle size of milk exosomes separated by size exclusion chromatography is more uniform than that of milk exosomes separated by ultracentrifugation. This may be because size exclusion chromatography avoids the destruction of milk exosomes by centrifugal force, better preserves the integrity of milk exosomes, and selectively elutes them out of the chromatography column according to size.

[0086] 2) Identification of milk exosome protein markers

[0087] First, the milk exosome protein was extracted by lysis, and then the protein concentration (μg / mL) in the extracted sample was determined by BCA method. Figure 2 As shown, the curve ULC is the protein marker identification result of the milk exosome Exo obtained by ultracentrifugation separation, and the curve SEC is the protein marker identification result of the milk exosome Exo obtained by size exclusion chromatography separation.

[0088] And the present invention specifically adopts the following method for identification: prepare separation gel and stacking gel respectively, fill the protein solution with protein denaturation buffer to 25 μL, denature the protein for 3-5min, and quickly put it into low temperature for 1min for standby use. Protein Mreker and sample are added to the loading well. After adding electrophoresis fluid, low voltage 80V is first used to make the sample reach the junction of separation gel and stacking gel, and then the voltage is increased to 120V. After the electrophoresis is completed, cross-flow wet transfer is adopted for transfer, and the PVDF membrane is taken out and placed in the blocking solution, and blocked at room temperature for 1-2h. Take out the PVDF membrane, rinse 3 times in TBST buffer and then incubate with antibodies, and finally expose using ECL chemiluminescent liquid.

[0089] Depend on Figure 2 It can be seen that the exosomes separated by size exclusion chromatography and ultracentrifugation both carry signature proteins: lipid raft marker protein Flotillin 1 and ESCRT complex-associated protein TSG101, indicating that both methods can successfully isolate milk exosomes from milk.

[0090] 3) Transmission electron microscopy of milk exosomes

[0091] In order to observe the structure of the isolated milk exosomes, the present invention conducted transmission electron microscopy tests on the milk exosomes obtained by the above two separation methods. The test results are as follows: Figure 3 The present invention is specifically tested by the following steps:

[0092] a. Pipette 20 μL of milk exosome suspension and dilute it moderately with PBS to obtain a well-dispersed sample for later structural observation;

[0093] b. Use tweezers to gently pick up a 200-mesh copper mesh and place it on clean filter paper;

[0094] c. Pipette 10 μL of the diluted milk exosome suspension onto the copper mesh and let it stand at room temperature for 5 minutes;

[0095] d. Gently remove excess exosome solution with filter paper, then add 10 μL of 1% phosphotungstic acid onto the copper grid for counterstaining for 1 minute.

[0096] e. After the restaining treatment is completed, use filter paper to absorb the excess phosphotungstic acid, and then add 10 μL of PBS buffer on the copper grid.

[0097] Figure 3 middle, Figure 3 (a) Transmission electron microscopy image of milk exosomes separated by ultracentrifugation at a scale of 500 μm. Figure 3 (b) is the transmission electron microscope image of the box in Figure (a) at a scale of 50 μm. Figure 3(c) Transmission electron microscopy image of milk exosomes separated by size exclusion chromatography at a scale of 500 μm. Figure 3 (d) is the transmission electron microscope image of the box in Figure (c) at a scale of 50 μm, and Figure 3 It can be seen that the exosomes separated by size exclusion chromatography and ultracentrifugation are typical vesicle structures with uniform size.

[0098] 3. Evaluation of drug delivery in milk exosomes

[0099] 1) Preparation of functionalized drug-loaded exosomes

[0100] S1. α-mangostin was used as a water-insoluble drug and co-incubated with milk exosomes to produce functionalized drug-loaded exosomes using the following co-incubation method:

[0101] A mixed solution of acetonitrile and ethanol in a volume ratio of 1:1 was used as the solvent, and α-mangostin was placed in it to form an α-mangostin solution with a concentration of 512μg / mL. The α-mangostin solution was then simply mixed with exosomes (protein concentration 2mg / mL) at a ratio of 1:9 (v / v) and co-incubated, keeping the final solvent concentration ≤10% to obtain α-mangostin (AMG)-loaded exosomes. The choice of this solvent concentration has no significant effect on the quality attributes of the exosomes. The specific process is: after mixing the AMG drug solution with the exosomes, incubate at room temperature for 15 minutes, and centrifuge at 10000g for 10 minutes to remove unbound drugs. In order to remove excess solvent, the exosomes loaded with AMG drugs were collected, and nitrogen was continuously passed through to volatilize the organic solvent, thus obtaining drug-loaded exosomes.

[0102] S2. Then, the obtained drug-loaded exosomes were co-incubated with phosphatidylserine (10 mg / mL) at a dosage of 1:20 (v / v) to obtain functionalized drug-loaded exosomes, and the obtained functionalized drug-loaded exosomes were suspended in PBS, aseptically processed through a 0.22 μm syringe, and stored at -80°C.

[0103] 2) Stability evaluation

[0104] a. Standard solution configuration:

[0105] Weigh an appropriate amount of α-mangostin reference substance and dissolve it in dimethyl sulfoxide (DMSO). Mix thoroughly to prepare a 5000 μg / mL standard stock solution. Store in a refrigerator at 4°C until ready for use. Immediately before use, dilute the solution with mobile phase to a desired concentration of standard working solution. The stock solution is usable within one month.

[0106] b. Chromatographic conditions:

[0107] A shim-pack GIST C18 column (4.6×250 mm, 5 μm) was used, the mobile phase was acetonitrile: 0.1% phosphoric acid aqueous solution (90:10, v / v), the detection mode was UV 312 nm, the injection volume was 20 μL, the flow rate was 1.0 mL / min, and the column temperature was 30°C.

[0108] c. Sample processing:

[0109] The functionalized drug-loaded exosomes and α-mangostin aqueous solution were placed at room temperature for 12 hours, and the supernatant was diluted 10 times and filtered through a 0.22 μm filter membrane before being tested for drug concentration. The test results were as follows: Figure 4 As shown, AMG is the α-mangostin group, and AExo is the exosome group loaded with α-mangostin.

[0110] And by Figure 4 It can be seen that the pure α-mangostin aqueous solution is very unstable, the supernatant contains almost no drug, and all the drugs are precipitated to the bottom. After α-mangostin is loaded into milk exosomes, the solution is very stable. After standing for 12 hours, the drug concentration in the supernatant remains almost unchanged, indicating that loading α-mangostin into milk exosomes to form functionalized drug-loaded exosomes can significantly improve the solution stability of α-mangostin.

[0111] 3) Evaluation of exosome intestinal mucus penetration

[0112] The mucus transport properties of milk exosomes were evaluated using an in vitro Transwell model. Native intestinal mucus was obtained from porcine intestine and frozen at -80°C until use.

[0113] Transwell specifications are as follows: 0.4 μm polycarbonate membrane, size 0.33 cm 2 The surface area is used to separate the upper and lower compartments. 600 μL of filtered PBS is placed in the lower chamber, and 40 μL of mucus (a layer about 0.2 mm thick) is added to the upper side of the membrane. After the milk exosomes are fluorescently labeled with FITC, 15 μL of labeled milk exosomes are added to the surface of the mucus layer in each well so that the mucus is not diluted. The well plate is then immediately covered and moved to 37°C and gently shaken. The PBS is removed from the lower chamber within the specified time, and the fluorescence intensity of the solution is measured on a multifunctional microplate reader. The fluorescence intensity of the original exosome solution initially added to the mucus is measured as a blank control. The operation flow chart is shown below. Figure 5 As shown in the middle left picture.

[0114] The test results are as follows Figure 5 As shown in the middle right picture, FITC is the fluorescein thiocyanate group, and Exo-FITC is the milk exosome group labeled with fluorescein thiocyanate. It can be seen that milk exosomes have strong intestinal mucus penetration properties.

[0115] 4) Antibacterial evaluation of functionalized exosomes

[0116] Macrophages were cultured at 10 5 Cells were plated in 24-well plates at 100 cells / well. After the cells adhered to the wall, they were infected with methicillin-resistant Staphylococcus aureus (MRSAT44) at an MOI of 100. After 1 hour of bacterial infection, the supernatant was discarded and the drugs were added for treatment. The cells were divided into 4 groups: PBS treatment group, milk exosome treatment group, AMG treatment group (10 μg / mL), and phosphatidylserine-modified exosome treatment group (equivalent to AMG 10 μg / mL). After 3 hours of treatment, they were treated with 0.1% Triton for 1 minute. After pipetting and lysing the cells, they were serially diluted 10-fold. The dilutions were evenly spread on the culture dishes and moved to a 37°C incubator. The colonies were counted after 18 hours.

[0117] The test results are as follows Figure 6 As shown, PBS is the PBS group, mExo is the milk exosome treatment group, AMG is the α-mangostin treatment group, and PS-AExo is the phosphatidylserine-modified exosomes loaded with α-mangostin treatment group. It can be seen that phosphatidylserine-modified milk exosomes have significantly enhanced antibacterial activity.

[0118] IV. Drug-loaded exosomes treat intestinal infection in mice with vancomycin-resistant Enterococcus faecium

[0119] Bacteria Vancomycin-resistant Enterococcus faecium (Enterococcus faecium CAU369) Preparation: VRE requiring oral administration fm The number of CAU369 is 5×10 8 CFUs, the gavage volume is 400 μL, and the bacterial stock solution needs to be prepared to be 1.2×10 9 CFUs / mL.

[0120] Antibiotic preparation: 0.5 g / L ampicillin (AMP). Give mice free access to drinking water, change it every 3 days, and treat for 5 days to destroy the intestinal flora.

[0121] The specific steps of the experimental operation are as follows:

[0122] (1) ICR mice weighing 18-20 g were treated with 0.5 g / LAMP in drinking water for five days, and then the drug was stopped for one day. 5 × 10 8 CFUs VRE fm CAU369 was gavaged once, with 400 μL of bacterial solution per mouse;

[0123] (2) One hour after infection, mice were treated with PBS, linezolid (LZD 8 mg / kg), AMG (8 mg / kg), or AExo (equivalent to AMG 8 mg / kg). Two days later, small intestinal contents were collected. The contents were weighed, added with 1 mL of PBS buffer, mixed vigorously, and serially diluted tenfold. The diluted contents were plated on selective medium (sodium azide-crystal violet-esculin agar) for VRE counts.

[0124] The test results are as follows Figure 7 As shown, Figure 7 Drug-loaded exosomes (AExo) for the treatment of VRE fm Test results of mouse intestinal infection caused by CAU369, Figure 7 A is the distribution of bacterial VRE in the jejunum, ileum, cecum and colon of mice; Figure 7 B is the number of bacterial VRE in the cecum of mice after treatment, where PBS is the PBS group, LZD is the linezolid treatment group, AMG is the α-mangostin treatment group, and AExo is the exosome treatment group loaded with α-mangostin. Figure 7 It can be seen that VRE fm CAU369 is mainly distributed in the cecum and colon of mice, and the drug-loaded exosomes can significantly reduce the number of bacteria in the cecum and colon of mice.

[0125] 5. Drug-loaded exosomes for the treatment of necrotic enteritis in chickens caused by Clostridium perfringens

[0126] Preparation of Clostridium perfringens bacteria: The number of C. perfringens to be administered orally is 1×10 9 CFUs, the gavage volume is 0.2 mL, and the bacterial stock solution needs to be prepared to 5×10 9 CFUs / mL.

[0127] Antibiotic preparation: 0.5g / L ampicillin and 1g / L streptomycin were given to the chicks for free drinking water for 3 days to destroy the intestinal flora.

[0128] The specific steps of the experimental operation are as follows:

[0129] The experimental chickens were 12 days old and divided into four groups. The chickens had free access to drinking water containing 0.5g / L ampicillin and 1g / L streptomycin for three consecutive days. The chickens were fed fresh sterile water for 1 day and then orally challenged with Clostridium perfringens (1×10 9CFUs, 200μL) infection. The experimental treatment was carried out 1h after infection and was divided into four groups, PBS, milk exosomes, AMG (8mg / kg), AExo (equivalent to AMG 8mg / kg) treatment, and the number of bacteria in the intestine (jejunum, ileum, cecum, rectum) was detected after 2d of treatment. In order to obtain a single-cell suspension, the collected intestinal samples were weighed and homogenized in 1mL of sterile PBS. Serial dilutions of the suspension were inoculated on a Clostridium perfringens selective plate and evenly spread, and the bacterial colony was quantified after culturing at 37°C under anaerobic conditions for 18h. The test results are as follows Figure 8 As shown, Figure 8 The test results of drug-loaded exosomes AExo in treating necrotic enteritis in chickens caused by Clostridium perfringens are shown in Figure 2. Figure 8 A represents the quantitative distribution of Clostridium perfringens in the jejunum, ileum, cecum and rectum of chickens; Figure 8 B is the number of Clostridium perfringens in the cecum of chickens after treatment, where PBS is the PBS group, Exo is the milk exosome treatment group, AMG is the α-mangostin treatment group, and AExo is the exosome treatment group loaded with α-mangostin. Figure 8 It can be seen that Clostridium perfringens C. perfringens is mainly distributed in the cecum and rectum of the chicken intestine, and the functional drug-loaded exosomes obtained by the present invention can significantly reduce the number of bacteria in the chicken cecum.

[0130] Obviously, the above embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A functionalized drug-loaded exosome based on milk exosomes, characterized in that: It comprises a carrier and a water-insoluble drug, wherein the water-insoluble drug is loaded inside the carrier; Wherein, the carrier is a milk exosome with a functional molecule loaded on the surface, and the functional molecule is phosphatidylserine; The water-insoluble drug is α-mangostin; The milk exosomes have a vesicle structure.

2. The functionalized drug-loaded exosomes based on milk exosomes according to claim 1, characterized in that The particle size of the carrier is 35-1000 nm.

3. A method for preparing functionalized drug-loaded exosomes based on milk exosomes according to any one of claims 1-2, characterized in that: The following steps are involved: Step 1: Loading water-insoluble drugs into milk exosomes to obtain drug-loaded exosomes; Step 2, loading functionalized molecules onto the surface of the drug-loaded exosomes to obtain functionalized drug-loaded exosomes; Wherein, the functionalized molecule is phosphatidylserine.

4. The preparation method according to claim 3, wherein In step 1, the loading method is any one of co-incubation, electroporation, ultrasonic treatment, co-extrusion and repeated freezing and thawing.

5. The preparation method according to claim 3, wherein The milk exosomes are separated from milk, and the separation method is any one or more of ultracentrifugation technology, size exclusion chromatography technology, density gradient centrifugation technology and immunoseparation technology.

6. Use of the functionalized drug-loaded exosomes according to any one of claims 1 to 2 in the preparation of a drug for resisting intestinal bacterial infection.

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