Breast-cancer-targeted milk-derived exosome drug delivery system, and preparation method therefor and use thereof

Abstract

Disclosed are a breast-cancer-targeted milk-derived exosome drug delivery system, and a preparation method therefor and the use thereof, which belong to the technical field of pharmaceuticals. The method for preparing the breast-cancer-targeted milk-derived exosome drug delivery system involves: adding sodium citrate to animal milk to remove casein, performing differential centrifugation under a low-temperature condition by using a centrifuge, separating and enriching milk-derived exosomes, performing ultrasonic drug loading on the drug solution and the milk-derived exosome suspension at 37°C, and performing freeze drying to obtain drug-loaded exosomes. In the preparation method, the exosomes are obtained by taking animal milk as a source. The source of animal milk is abundant and the operation of the method is simple, which reduces the production cost. Moreover, natural ligands on the milk-derived exosomes can specifically target and bind to breast-cancer cells, so that uptake by cancer cells is enhanced, the cytotoxicity of the drug to normal cells is reduced, and the effect of specifically treating breast cancer is achieved. The present application is widely used in the field of targeted delivery of drugs for breast cancer treatment.

Description

A breast cancer-targeted mammary exosome drug delivery system and its preparation method and application Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a breast cancer-targeting mammary exosome drug delivery system, a preparation method, and an application thereof. Background Art

[0002] Breast cancer (BC) accounts for 25-30% of all new cancer cases in women and is the leading cause of death among women worldwide. Commonly used clinical anti-cancer drugs have significant shortcomings, often exhibiting low water solubility, non-specific tumor targeting, rapid elimination from the body, and severe side effects. Traditional drug-loaded targeted formulations easily interact with other biological molecules in the body, leading to three different problems: toxicity, immunogenicity, and rapid clearance by the mononuclear phagocyte system. Therefore, there is an urgent need to develop new drug delivery systems with stability, high targeting specificity, and low tumor toxicity to address the unmet medical needs faced by breast cancer patients.

[0003] Exosomes are natural vesicles of nanometer size (30-200 nm in diameter) secreted by cells in response to environmental stimulation or self-activation, and their membranes have a lipid bilayer structure. Studies have found that exosomes are considered to be an important medium for intercellular communication, and the processes of cell absorption and secretion of exosomes are interrelated. As "messengers" for cell communication in organisms, exosomes are absorbed and secreted by a variety of different mechanisms and have specific effects on different cells. In addition, the membrane structure of exosomes can protect cargo (such as nucleic acids, proteins, and drugs) from environmental influences, thereby extending the half-life and improving the in vivo stability of the delivery material. At the same time, due to good safety, biocompatibility, and low immunogenicity, exosomes have become a very promising drug delivery carrier that can deliver drugs to the cytoplasm with minimal toxicity.

[0004] The isolation and purification of exosomes has long been a concern for researchers, and obtaining high-purity exosomes is crucial for subsequent research. Traditional exosomes are mostly extracted from cells and plants and often undergo engineering modifications, resulting in low yields and high costs, making large-scale production difficult. Furthermore, the quality uniformity of exosomes is highly dependent on their source and extraction method. Currently, there is a lack of stable and reliable sources and standardized extraction methods for exosomes, necessitating the development of more cost-effective, simpler, and more efficient sources and extraction methods.

[0005] Summary of the Invention

[0006] To address the poor targeting, high cytotoxicity, and severe side effects of existing breast cancer-targeted formulations, the present invention provides a breast cancer-targeted milk-derived exosome drug delivery system, its preparation method, and its application. This invention utilizes exosomes as drug delivery vehicles. Compared to existing breast cancer-targeted formulations, exosomes can deliver drugs to breast tumor tissue more efficiently and stably, while also offering greater biosafety. To overcome the low yield, unstable source, and difficulty in large-scale production of traditional exosomes, this invention utilizes milk as the source of exosomes, leveraging the natural components of milk-derived exosomes to target breast cancer.

[0007] The present invention provides the following technical solutions:

[0008] A method for preparing a breast cancer-targeting exosome drug delivery system comprises the following steps:

[0009] (1) Centrifuge fresh animal milk at 2500-5000 g for 5-30 min at 1-5°C to obtain whey;

[0010] (2) Take equal volumes of whey and sodium citrate solution, mix them, shake them in an ice bath for 1-10 hours, centrifuge them at 9000-12000g for 30-60 minutes at 1-5°C, and collect the whey;

[0011] (3) The whey obtained in step (2) is ultracentrifuged at 40,000 to 70,000 g for 40 to 90 minutes at 1 to 5° C., and the resulting precipitate is the milk-derived exosome drug delivery system.

[0012] Furthermore, the mass concentration of the sodium citrate solution in step (2) is 2% to 10%, and the mixture is shaken in an ice bath for 2 to 6 hours.

[0013] The present invention provides a milk-derived exosome drug delivery system prepared by the above preparation method.

[0014] Furthermore, the milk-derived exosome drug delivery system has natural targeting properties for breast cancer cells.

[0015] Furthermore, the milk-derived exosome drug delivery system is unmodified, and the targeting ligands are natural components on the milk-derived exosome membrane, including proteins, fats, and polysaccharides.

[0016] The present invention provides a drug-loaded milk-derived exosome drug delivery system, which is obtained by loading drugs or drug-containing particles into the milk-derived exosome drug delivery system using an ultrasonic method.

[0017] Furthermore, the specific process of the ultrasound method is: mixing the milk-derived exosome drug delivery system with the drug or drug-containing particles in PBS buffer, ultrasound-treated under ice-water bath conditions with an ultrasound power of 70 to 90 W, ultrasound-treated for 30 seconds, and then placed in an ice-water bath for 30 to 60 seconds. After repeating the ultrasound operation for 5 to 7 cycles, the drug-loaded milk-derived exosome drug delivery system is obtained.

[0018] Furthermore, the drugs include small molecule compound drugs, peptide drugs, protein drugs and gene drugs.

[0019] Furthermore, the small molecule compound drugs include paclitaxel and doxorubicin.

[0020] The present invention provides a method for preparing a drug-loaded emulsion-derived exosome freeze-dried preparation, comprising the following steps:

[0021] 1) Evenly mixing the lyoprotectant and the drug-loaded emulsion-derived exosome drug delivery system, wherein the mass ratio of the lyoprotectant to the drug-loaded emulsion-derived exosome drug delivery system is 0.5 to 30:1;

[0022] 2) After freeze-drying, the drug-loaded milk-derived exosome freeze-dried preparation is obtained.

[0023] Furthermore, the lyophilization protective agent in step 1) is composed of 2-4% trehalose, 2-4% mannitol and pH 7.4 PBS buffer.

[0024] Furthermore, the freeze-drying procedure of step 2) is:

[0025] Pre-freezing: 1h, end temperature -80℃, 1h, end temperature -40℃;

[0026] Sublimation: 1h, end temperature -30℃, 1h, end temperature -20℃, 1h, end temperature -10℃;

[0027] Desorption drying: 6h, end temperature 0℃.

[0028] The present invention also provides a drug-loaded milk-derived exosome freeze-dried preparation prepared by the above preparation method.

[0029] The present invention also provides the use of the above-mentioned milk-derived exosome drug delivery system in the preparation of breast cancer targeted drugs.

[0030] Furthermore, the drug specifically targets breast cancer cells, including Luminal A, Ki67 low-expressing, Luminal B, HER-2+ and triple-negative breast cancer cells.

[0031] The present invention has the following beneficial effects compared to the prior art:

[0032] The present invention provides the preparation and application of a novel breast cancer-targeted drug delivery system. This system utilizes milk-derived exosomes as drug delivery vehicles. Milk-derived exosomes can efficiently load and stably deliver drugs such as small molecules, nucleic acids, and proteins. Milk-derived exosomes possess a robust bimolecular phospholipid structure. The internal cavity facilitates the loading of large quantities of water-soluble substances, while the hydrophobic microphase between the bimolecular phospholipids encapsulates hydrophobic substances. This unique structure protects the contents from degradation and dilution in the harsh extracellular environment, preventing capture and clearance by the reticuloendothelial system, thereby achieving a targeted effect. Furthermore, the natural, unique lipid structure and protein on the surface of milk-derived exosomes facilitate targeted fusion with breast cancer cells, releasing the contents or drugs into target cells. This allows for specific targeted intervention, thereby enhancing therapeutic efficacy and reducing toxicity. This approach successfully overcomes the shortcomings of traditional targeted agents, such as easy clearance, poor targeting efficacy, and severe side effects. Furthermore, using milk-derived exosomes as drug carriers exhibits excellent biocompatibility and non-immunogenicity, which can improve effective utilization and bioavailability while reducing drug clearance. Compared to traditional exosomes, milk-derived exosomes are widely available, have a stable structure, and are readily available. This invention has successfully developed an exosome-targeted drug delivery system that is inexpensive and can be industrially produced on a large scale. This system can effectively enhance the therapeutic efficacy of drugs against breast cancer while reducing their side effects, demonstrating promising economic and market prospects and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0034] Figure 1 shows a transmission electron micrograph of milk-derived exosomes (A) and a picture of milk-derived exosomes and drug-loaded milk-derived exosome freeze-dried powder (B);

[0035] FIG2 is a characterization of the particle size distribution and surface potential of milk-derived exosomes by dynamic light scattering;

[0036] Figure 3 shows the characterization of the particle size distribution and surface potential of drug-loaded emulsion-derived exosomes by dynamic light scattering;

[0037] FIG4 is a graph showing the in vitro release curves of drug-loaded emulsion-derived exosomes in normal body fluid environment and tumor microenvironment;

[0038] Figure 5 shows the stability of the lyophilized formulation of drug-loaded emulsion-derived exosomes at 4°C and 25°C after hydration;

[0039] Figure 6 is a comparison of the targeting effects of the milk-derived exosome drug delivery system and conventional drug-loaded liposomes at the cellular level, wherein A is a quantitative analysis of the binding ability of conventional drug-loaded liposomes and the milk-derived exosome drug delivery system to breast cancer cells by flow cytometry, and B is a qualitative analysis of the binding ability of conventional drug-loaded liposomes and the milk-derived exosome drug delivery system to breast cancer cells by confocal fluorescence uptake of cells;

[0040] Figure 7 is a comparison of the tumor targeting effects of the milk-derived exosome drug delivery system and conventional drug-loaded liposomes in tumor-bearing nude mice evaluated by fluorescence imaging quantitative method;

[0041] Figure 8 shows the therapeutic effect of the milk-derived exosome drug delivery system on breast cancer;

[0042] Figure 9 shows the safety evaluation results of the milk-derived exosome drug delivery system. DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with specific examples, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or replacements made to the inventive method, steps, and conditions are intended to fall within the scope of the present invention. Unless otherwise specified, the experiments and techniques, reagents, and materials used in the examples are all commercially available.

[0044] Example 1 Preparation and characterization of milk-derived exosomes (MEVs)

[0045] (1) Preparation of milk-derived exosomes (MEVs)

[0046] Fresh bovine milk was centrifuged at 3000g for 20 minutes at 4°C. The upper fat layer was carefully discarded, and the intermediate whey was collected. Sodium citrate solutions of varying concentrations (2%, 4%, 6%, 8%, and 10%) were prepared and equal volumes of the intermediate whey were added to the sodium citrate solutions. The samples were shaken on an ice-bath for 120 minutes until the samples became clear. The milky whey fraction was centrifuged at 10,000g for 40 minutes at 4°C. The upper fat layer was carefully discarded, and the intermediate whey was collected. The intermediate whey fraction was centrifuged at 50,000g for 60 minutes at 4°C. The milky white, translucent precipitate was collected, which was the milky exosome (MEV). The resulting milky exosomes were resuspended in PBS buffer (pH 7.4) and stored at -20°C until ready for use.

[0047] (2) Characterization of milk-derived exosomes (MEVs)

[0048] Transmission electron microscopy and dynamic light scattering were used to observe the structure, morphology, size, and potential of the milk-derived exosomes. The results are shown in Figures 1 and 2. The results show that the milk-derived exosomes prepared in this example had an average particle size of 100-200 nm, a typical cup-shaped structure, a hydrated particle size of 121.8 ± 1.5 nm, and a PDI of 0.16 ± 0.02, which is less than 0.2, indicating good monodispersity. The zeta potential was -14.7 mV ± 0.1.

[0049] Example 2 Preparation, Characterization and In Vitro Drug Release Evaluation of Paclitaxel-Loaded Emulsion-Derived Exosomes (MEV-PTX)

[0050] (1) Preparation and characterization of paclitaxel-loaded emulsion-derived exosomes (MEV-PTX)

[0051] A 0.1 mg / ml paclitaxel solution was prepared in methanol, and emulsified exosomes (2.4 mg / ml) and paclitaxel solution were loaded at a mass ratio of 10:1. The drug loading method was ultrasonic power 80 W, and after each ultrasonication for 30 seconds, it was placed in an ice water bath for 40 seconds. After repeating the ultrasonic operation for 6 cycles, paclitaxel-loaded emulsified exosomes (MEV-PTX) were obtained.

[0052] The paclitaxel-loaded emulsion-derived exosome solution was centrifuged at 130,000 g for 30 min at 4°C using an ultracentrifuge. The resulting precipitate was washed three times with pH 6.8 PBS buffer and then resuspended in an appropriate amount of PBS.

[0053] Dynamic light scattering technology was used to analyze the size and potential of paclitaxel-loaded emulsion-derived exosomes.

[0054] As shown in Figure 3, the hydrated particle size of the paclitaxel-loaded emulsion-derived exosomes was 127.8 ± 1.5 nm, and the PDI was 0.18 ± 0.02, which was less than 0.2, indicating good monodispersity. Furthermore, the zeta potential was -12.6 mV ± 0.1.

[0055] (2) Drug loading and in vitro release of paclitaxel-loaded emulsion-derived exosomes (MEV-PTX)

[0056] Ultrafiltration was used to determine the encapsulation efficiency and drug loading of paclitaxel-loaded emulsion-derived exosomes. A precise amount of 0.5 ml of paclitaxel-loaded emulsion-derived exosomes was placed in an ultrafiltration tube and centrifuged at 10,000 g for 10 minutes at 4°C. The supernatant was aspirated into a 1 ml volumetric flask, diluted to the mark with methanol, and shaken thoroughly. After filtration through a 0.22 μm microporous membrane, the filtrate was injected into a liquid chromatograph to determine the free paclitaxel content. Another 0.5 ml of paclitaxel-loaded emulsion-derived exosomes was placed in a 1 ml volumetric flask, broken by ultrasonication, and diluted to the mark with methanol. The total paclitaxel content in the paclitaxel-loaded emulsion-derived exosomes was determined using the same method.

[0057] Calculation showed that the encapsulation efficiency of paclitaxel-loaded emulsion-derived exosomes was 75.4% and the drug loading capacity was 11.7%.

[0058] The in vitro release of paclitaxel-loaded emulsified exosomes was investigated using dynamic dialysis. Two milliliters of paclitaxel-loaded emulsified exosomes were placed in a 3000 kDa dialysis bag. The bag was tied tightly at both ends to prevent leakage and then placed in 20 ml of PBS (pH 7.4) containing 0.2% Tween 80. The bag was shaken at 800 rpm for 24 hours at 37°C. At 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours, 2 ml of the release medium was aspirated. The same volume of release medium was then added and filtered through a 0.22 μm microporous membrane. The paclitaxel content in the release medium was determined by HPLC. Another 2 ml of paclitaxel-loaded emulsified exosomes were placed in 20 ml of PBS (pH 5.4) containing 0.2% Tween 80 and the same procedure was performed at 37°C. Cumulative release was calculated. The release curve is shown in FIG4 . After 24 hours, the cumulative drug release of paclitaxel-loaded emulsion-derived exosomes in the normal body fluid environment was 72%, and the cumulative drug release in the tumor environment was 78%.

[0059] Example 3 Preparation and Stability of Freeze-dried Formulation

[0060] (1) Preparation of paclitaxel-loaded emulsified exosome freeze-dried preparation

[0061] Dissolve 2% trehalose and 4% mannitol in 1 ml of pH 7.4 PBS buffer to prepare a lyoprotectant. Mix paclitaxel-loaded emulsified exosomes (2.4 mg / ml) with the lyoprotectant in a 1:1 volume ratio. Place the mixed solution in a vial and lyophilize according to the following procedure:

[0062] Pre-freezing: 1h, end temperature -80℃, 1h, end temperature -40℃;

[0063] Sublimation: 1h, end temperature -30℃, 1h, end temperature -20℃, 1h, end temperature -10℃;

[0064] Desorption drying: 6h, end temperature 0℃;

[0065] The paclitaxel-loaded emulsion-derived exosome freeze-dried preparation was obtained, the morphology of which is shown in FIG1B . The freeze-dried preparation needs to be stored in a dry environment at 4°C.

[0066] (2) Stability of paclitaxel-loaded emulsified exosome freeze-dried preparation

[0067] The lyophilized paclitaxel-loaded emulsion-derived exosomes were rehydrated with the same volume of water as before lyophilization. The hydrated solutions were placed at 4°C and room temperature (25°C) for 24 hours. The particle size of the hydrated lyophilized preparation was measured by dynamic light scattering at 0, 0.5, 1, 2, 4, and 6 hours. The test results are shown in Figure 5. The hydrated paclitaxel-loaded emulsion-derived exosome lyophilized preparation showed good stability within 6 hours.

[0068] Example 4 Targeting effect of paclitaxel-loaded emulsion exosomes (MEV-PTX)

[0069] (1) Quantitative analysis of the targeting effect of paclitaxel-loaded emulsion-derived exosomes (MEV-PTX) at the cellular level

[0070] MBA-MD-231 cells were digested and counted, and 200,000 cells were seeded into 6-well plates per well and cultured overnight in a cell culture incubator. After all the cells adhered, the culture medium was replaced with fresh serum-free culture medium, and 1 ml of the same number of particles of paclitaxel-loaded emulsified exosomes and paclitaxel-loaded liposomes were added, respectively, and incubated at 37 ° C for 3 hours. After incubation, the culture medium was discarded, centrifuged at 3000 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in 300 μl of pre-cooled PBS and detected by flow cytometry. The results are shown in Figure 6A. The uptake effect of MBA-MD-231 cells on paclitaxel-loaded emulsified exosomes (MEV-PTX) was significantly enhanced compared to paclitaxel-loaded liposomes (liposomes), proving that the milk-derived exosome nanoformulation can specifically target breast cancer cells.

[0071] (2) Qualitative analysis of the targeting effect of paclitaxel-loaded emulsion-derived exosomes (MEV-PTX) at the cellular level

[0072] MBA-MD-231 cells were digested and counted, then seeded at 50,000 cells per well in a 24-well plate and cultured overnight at 37°C in a 5% CO2 incubator to allow adherent growth. DID-paclitaxel-loaded emulsion exosomes and DID-paclitaxel-loaded liposomes were prepared in PBS buffer (pH 7.4) to a suspension of 800 μg / mL and added to the corresponding wells to a final concentration of 200 μg / mL. After 4 hours of culture, the old medium was discarded, and 200 μL of 4% paraformaldehyde was added to each well and incubated at 37°C for 20 minutes to fix the cells. The paraformaldehyde was aspirated, and 300 μL of Hoechst stain was added to each well. The plates were stained at 37°C for 20 minutes. After washing three times with PBS buffer (pH 7.4), an appropriate concentration of mounting solution was added to a glass slide. The cell slides were placed on the slide, with the cell surface in contact with the mounting solution. The fixed cells were observed using a confocal microscope. The results are shown in Figure 6B. The uptake of paclitaxel-loaded emulsion-derived exosomes (MEV-PTX) by MBA-MD-231 cells was significantly enhanced compared with that of paclitaxel-loaded liposomes (liposomes), demonstrating the specific targeting effect of the emulsion-derived exosome nanoformulation on breast cancer cells.

[0073] (3) Targeting effect of paclitaxel-loaded emulsion-derived exosomes (MEV-PTX) at the animal level

[0074] MBA-MD-231 cells were cultured to the logarithmic growth phase, collected, and diluted to 1×10 7 Cell suspension of 1000 cells / ml was inoculated under the fourth mammary fat pad on the left side of nude mice at a dose of 0.2 ml per mouse. Mice with good tumor growth were selected as breast cancer models. 3 At about 14:00 p.m., nude mice were weighed and randomly divided into a DIR-paclitaxel-loaded emulsion-derived exosome group and a DIR-paclitaxel-loaded liposome group for tail vein injection. Each nude mouse was injected with a preparation of the same fluorescence intensity. The nude mice were sacrificed 1, 2, 4, 8, and 12 hours after administration, and the heart, liver, spleen, lung, and kidney, as well as the tumor, were removed. The fluorescence intensity of the isolated organs was observed and quantified under a small animal imaging device. The ratio of the fluorescence intensity in the tumor to the fluorescence intensity in the liver, spleen, and lung of the paclitaxel-loaded emulsion-derived exosome and paclitaxel-loaded liposome groups was compared. The results are shown in Figure 7. The targeting effect of paclitaxel-loaded emulsion-derived exosomes (MEV-PTX) on tumor tissue was significantly stronger than that of paclitaxel-loaded liposomes (liposomes), demonstrating that the emulsion-derived exosome nanoformulation has a specific targeting effect on breast cancer tumor tissue in vivo.

[0075] Example 5 Antitumor Effect of Paclitaxel-Loaded Emulsion Exosomes (MEV-PTX)

[0076] MBA-MD-231 cells were cultured to the logarithmic growth phase, collected, and diluted to 1×107 Cell suspension of 1000 cells / ml was inoculated under the fourth mammary fat pad on the left side of nude mice at a dose of 0.2 ml per mouse. Mice with good tumor growth were selected as breast cancer models. 3 At about 14 days, the nude mice were weighed and randomly divided into PBS group, paclitaxel-loaded emulsion-derived exosomes (MEV-PTX) group, and paclitaxel-loaded liposomes (liposome) group. The dosage was calculated based on paclitaxel (15 mg / kg). Tumor-bearing nude mice were injected with 0.1 ml of PBS, paclitaxel-loaded emulsion-derived exosomes, and paclitaxel-loaded liposomes through the tail vein on days 3, 6, 9, 12, and 15. Before each administration, the short diameter (a) and long diameter (b) of the tumor of each group of nude mice were measured, and the tumor volume (V) was calculated. V = (a 2 b) / 82, the tumor was dissected and weighed after 15 days. Figure 8 shows the anti-tumor effect of paclitaxel-loaded emulsion-derived exosomes in nude mice. It can be seen that paclitaxel-loaded emulsion-derived exosomes can significantly inhibit the growth of breast cancer tumors and have a better anti-tumor effect.

[0077] Example 6 Safety Evaluation of Paclitaxel-Loaded Emulsion-Derived Exosomes (MEV-PTX)

[0078] MBA-MD-231 cells were cultured to the logarithmic growth phase, collected, and diluted to 1×10 7 Cell suspension of 1000 cells / ml was inoculated under the fourth mammary fat pad on the left side of nude mice at a dose of 0.2 ml per mouse. Mice with good tumor growth were selected as breast cancer models. 3 At about 14 days, the nude mice were weighed and randomly divided into PBS group, paclitaxel-loaded emulsion exosomes (MEV-PTX) group, and paclitaxel-loaded liposomes (liposome) group. The dosage was calculated based on paclitaxel (15 mg / kg). Tumor-bearing nude mice were injected with 0.1 ml of PBS, paclitaxel-loaded emulsion exosomes, and paclitaxel-loaded liposomes through the tail vein on days 3, 6, 9, 12, and 15. The weight of the tumor-bearing nude mice was weighed and recorded every day. Figure 9 shows the results of the safety evaluation experiment of paclitaxel-loaded emulsion exosomes. There was no significant decrease in the weight of nude mice in the PBS and paclitaxel-loaded emulsion exosomes groups, and the paclitaxel-loaded emulsion exosomes were safe in animals.

[0079] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The technical solutions described in the above embodiments may be modified, or some or all of the technical features therein may be replaced by equivalents; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the present invention.

Claims

1. A method for preparing a breast cancer-targeting exosome drug delivery system, characterized in that: The steps include: (1) fresh animal milk is centrifuged at 2500-5000 g for 5-30 min at 1-5° C. to obtain whey; (2) taking an equal volume of whey and sodium citrate solution, mixing them, shaking them in an ice bath for 1 to 10 hours, centrifuging them at 9000 to 12000 g for 30 to 60 minutes at 1 to 5°C, and collecting the whey; (3) The whey obtained in step (2) is ultracentrifuged at 40,000 to 70,000 g for 40 to 90 minutes at 1 to 5° C., and the resulting precipitate is the milk-derived exosome drug delivery system.

2. The preparation method according to claim 1, characterized in that: The mass concentration of the sodium citrate solution in step (2) is 2% to 10%, and the solution is shaken in an ice bath for 2 to 6 hours.

3. A milk-derived exosome drug delivery system prepared by the preparation method according to claim 1 or 2.

4. A drug-loaded milk-derived exosome drug delivery system, obtained by loading drugs or drug-containing particles into the milk-derived exosome drug delivery system according to claim 3 using an ultrasonic method.

5. The drug-loaded emulsion-derived exosome drug delivery system according to claim 4, characterized in that: The specific process of the ultrasound method is: mix the milk-derived exosome drug delivery system with the drug or drug-containing particles in PBS buffer, ultrasound under ice-water bath conditions, the ultrasound power is 70-90W, ultrasound for 30s and then placed in an ice-water bath for 30-60s, repeat the ultrasound operation for 5-7 cycles to obtain the drug-loaded milk-derived exosome drug delivery system.

6. The drug-loaded emulsion-derived exosome drug delivery system according to claim 4, characterized in that: The drugs include small molecule compound drugs, peptide drugs, protein drugs and gene drugs; the small molecule compound drugs include paclitaxel and doxorubicin.

7. A method for preparing a drug-loaded milk-derived exosome freeze-dried preparation, characterized in that: The steps include: 1) mixing the lyophilized protective agent and the drug-loaded emulsion-derived exosome drug delivery system according to any one of claims 4 to 6 evenly, wherein the mass ratio of the lyophilized protective agent to the drug-loaded emulsion-derived exosome drug delivery system is 0.5 to 30:1; 2) After freeze-drying, the drug-loaded milk-derived exosome freeze-dried preparation is obtained.

8. The preparation method according to claim 7, characterized in that: Step 1) the lyophilization protective agent is composed of 2-4% trehalose, 2-4% mannitol and pH 7.4 PBS buffer; The freeze drying procedure of step 2) is: Pre-freezing: 1h, end temperature -80℃, 1h, end temperature -40℃; Sublimation: 1h, end temperature -30℃, 1h, end temperature -20℃, 1h, end temperature -10℃; Desorption drying: 6h, end temperature 0℃.

9. The drug-loaded milk-derived exosome freeze-dried preparation prepared by the preparation method according to claim 7 or 8.

10. Use of the milk-derived exosome drug delivery system according to claim 1 or 2 in the preparation of breast cancer targeted drugs.

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