Preparation method and application of exosome vesicles capable of efficiently loading drugs

Abstract

The invention relates to the technical field of exosome loaded drugs, and provides a preparation method and application of exosome vesicles capable of efficiently loading drugs, and the preparation method comprises the following steps: exosome preparation: preparing an exosome sample with enough granularity and purity; pretreatment of exosomes: performing precipitation treatment and collecting the exosomes to prepare exosome precipitates; carrying out loaded medicine pretreatment: treating loaded medicine or a medicine preparation by using a lipid reagent; target drug loading: rapidly treating the exosome precipitate by using a drug lipid component, and then treating by using a buffer system to obtain the exosome load of the drug; and free lipid and drug removal: reducing free lipid and drug residues by re-enriching the exosomes. The problems of high concentration, high purity and low cost in industrial production of the exosome are solved, meanwhile, an efficient and convenient technical method is provided for taking the exosome loaded medicine as a delivery system, and the method can be suitable for the fields of health medical treatment, medical beauty cosmetics and biological medicine products.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome drug loading, and more specifically, it relates to a preparation method and application of exosome vesicles for efficiently loading drugs. Background Art

[0002] Currently, carriers for drug delivery systems include liposomes, microcapsules, microspheres, microemulsions, nanocapsules, nanospheres, and exosomes, etc.; among them, humanized exosomes have received increasing attention and research in the field of drug carriers. They can not only save costs, but also greatly solve various adverse reactions and difficult metabolism problems brought by the carrier system. Exosomes as drug carriers have advantages such as good biocompatibility, biological stability, and tumor targeting.

[0003] There are two ways to load drugs using exosome vesicles as drug delivery carriers: 1. Passive loading method. Based on incubating drugs with exosomes or cells, and then further separating the released exosomes containing drugs. Due to the realization of drug concentration gradients, substances themselves will also migrate into the vesicles during exosome incubation, and this process can be enhanced by applying additional forces, such as shaking or stirring, etc. The loading efficiency of passive drug loading is low, and the output drug concentration is low (about 1%). The method of loading drugs by incubation is usually applied to hydrophobic drugs, etc. 2. Active loading method. It can achieve higher loading efficiency, including sonication (up to 28%), electroporation (up to 5%), extrusion, freeze-thaw, pH gradient (up to 1.7%), and conjugation methods. Another active loading method is to conjugate drugs with exosomes through antibodies or click chemistry. The methods of actively loading drugs are more intense and will cause a large number of exosome vesicles to break, affecting the usable concentration.

[0004] In the prior art, the disadvantages of exosome preparation include: differential ultracentrifugation is complex in operation, time-consuming, with low yield, and difficult to process large batches of samples; density gradient centrifugation: high cost, time-consuming, low throughput, and difficult to achieve large-scale production; in polymer precipitation, the obtained exosomes have low purity, and the mixing of polymer materials may affect downstream operations and analysis, etc.; in size exclusion, particles similar in size to exosomes cannot be effectively separated, and the samples obtained by size exclusion will be severely diluted, and subsequent experiments may require an additional concentration process; in tangential flow ultrafiltration, exosome vesicles cannot be effectively separated, and the extraction purity is low. At the same time, the vesicle structure will be broken during ultrafiltration; microfluidics is suitable for preparing a small amount of exosome samples and is not suitable for large-scale production methods. The method of separating exosomes using kits has led to the emergence of many various kit sets for exosome extraction on the market, which not only increases the laboratory cost, but also makes it difficult to scale up the prepared samples for industrial production, etc. Summary of the Invention

[0005] The present invention provides a method for preparing exosome vesicles with high-efficiency drug loading and its application, which solves the problems of high concentration, high purity and low cost in the industrial production of exosomes. At the same time, it provides an efficient and convenient technical method for loading drugs into exosomes as a delivery system, and can be applied to the fields of health care, medical beauty cosmetics and biological medicine products.

[0006] In the first aspect, the present invention provides a method for preparing exosome vesicles with high-efficiency drug loading, comprising the following steps:

[0007] (1) Exosome preparation: Prepare an exosome sample with sufficient particle size and purity. The liquid sample is removed of large particles or residual debris by centrifugation or filtration, and the tissue sample is obtained a liquid containing exosome vesicles through fragmentation, infiltration with buffer liquid, centrifugation or filtration.

[0008] (2) Exosome pretreatment: Precipitate and collect exosomes to prepare an exosome precipitate. Add pre-cooled 50% drug loading to the solution containing exosome vesicles obtained in step (1) until the final concentration of the drug loading reaches 6%. Place the sample at 2 - 8 °C and let it stand for 20 - 24 h.

[0009] (3) Drug loading pretreatment: Treat the drug loading or drug preparation with a lipid reagent, centrifuge the treatment solution in step (2) to obtain an exosome precipitate; add the buffer liquid described in step (1) to the precipitate for resuspension and washing, and remove the residual solid part by centrifugation or filtration.

[0010] (4) Target drug loading: Rapidly treat the exosome precipitate with the drug lipid component, and then treat it with a buffer system to obtain the exosome loading of the drug. Add it to the exosome precipitate at a volume ratio of 2:1, resuspend and mix well for 30 s, and then immediately add 10 times the volume of the buffer solution of 10 mM PBS of the target drug pretreatment solution for dilution and suspension operation under the condition of pH 7.2 - 7.5.

[0011] (5) Removal of free lipids and drugs: Reduce the residual of free lipids and drugs by re-enriching exosomes.

[0012] Preferably, in step (1), the exosome sample includes exosomes from animal, plant tissues or body fluids.

[0013] Preferably, in step (3), it further includes lipid phase incubation of the drug, and the lipid reagent includes water-soluble reagent or water-insoluble reagent.

[0014] Preferably, in step (4), the buffer system includes NaHCO3, Tris-HCl, PBS, glucose solution or physiological saline.

[0015] Preferably, in step (3), the loaded drug is one of PEG2000, PEG4000, PEG6000, and PEG8000.

[0016] Preferably, a preparation method for efficiently loading drugs into exosome vesicles comprises the following steps:

[0017] (1) Sample pretreatment: For liquid samples, large particles or residual debris can be removed by centrifugation or filtration; for tissue samples, a liquid containing exosome vesicles can be obtained by fragmentation, infiltration with buffer liquid, centrifugation, or filtration. Among them, the buffer liquids used include NaHCO3, Tris-HCl, PBS, glucose solution, physiological saline, etc., and these buffer liquids are all applicable to the present invention;

[0018] (2) Add pre-cooled 50% PEG2000 or PEG4000 or PEG6000 or PEG8000 to the solution containing exosome vesicles obtained in step (1) until the final concentration of PEG reaches 6%. Place the sample at 2 - 8°C and let it stand for 20 - 24 hours;

[0019] (3) Centrifuge the treatment solution in step (2) at the maximum centrifugal force to obtain precipitates such as exosomes; add the buffer liquid described in step (1) to the precipitate for resuspension and washing, and remove the residual solid part by centrifugation or filtration;

[0020] (4) Use a 300KD hollow fiber ultrafiltration system to treat the exosome sample obtained in step (3). When the retained volume reaches 1 / 5 - 1 / 10 of the raw material volume (or weight), wash it 5 - 7 times with 10 mM PBS (pH 7.2 - 7.5) to reduce the residual of miscellaneous proteins or pigments, etc., and finally obtain a concentrated exosome treatment solution;

[0021] (5) Filter and top-filter the concentrated exosome treatment solution in step (4) using a 0.45μm + 0.22μm capsule filter to obtain a concentrated exosome filtrate;

[0022] (6) Fine purification of exosome vesicles: Chromatographically purify the concentrated exosome filtrate obtained in step (5) by molecular exclusion method, where molecular exclusion uses fillers such as Core700 series, Sepharose 4FF, or Sepharose 6FF, etc.; collect the corresponding purified exosome vesicle solution through the UV260 and UV280 peak charts.

[0023] (7) Filter the exosome vesicle sample obtained in step (6) through a 0.22μm filter to obtain a sterile exosome sample.

[0024] Preferably, an exosome high-efficiency drug loading method technology comprises the following steps:

[0025] (1) Drug pretreatment: For the target drug, lipid reagents such as cholesterol mentioned in the present invention are used. Cholesterol is used to dissolve the target drug or mix with the target drug solution. The method of suspension and high-speed centrifugation is used for repeated mixing. Among them, for stable target drugs, violent shaking can be used for suspension, and for unstable samples, repeated pipetting with a pipette or pipette can be selected for suspension. Then, the suspension is centrifuged at 12,000 g and 4 °C for 15 min; the above operations are repeated 3 - 5 times, and finally the target drug pretreatment solution is obtained;

[0026] (2) Exosome vesicle precipitation: 50% PEG6000 is added to the obtained high-purity exosome sample to a final concentration of 6%. Exosome precipitates are obtained by centrifugation;

[0027] (3) The target drug pretreatment solution obtained in step (1) is added to the exosome precipitate in a ratio of 2:1 (volume: mass), and resuspended and mixed evenly for 30 s; then immediately add 10 times the volume of the buffer solution of the target drug pretreatment solution, 10 mM PBS (pH 7.2 - 7.5), for dilution and suspension operation;

[0028] (4) The operation of step (2) can be repeated to remove free cholesterol and the target drug in the solution, which can effectively reduce the influence of free cholesterol and the target drug on experiments or drug use;

[0029] (5) By repeating steps (2), (3), and (4), etc., the exosome loading efficiency of the target drug can be increased.

[0030] In the second aspect, the present invention provides a method for efficiently loading drugs into exosome vesicles for use in the preparation of functional cosmetics.

[0031] In the third aspect, the present invention provides a method for efficiently loading drugs into exosome vesicles for use in the preparation of medical aesthetic cosmetics.

[0032] In the fourth aspect, the present invention provides a method for efficiently loading drugs into exosome vesicles for use in the preparation of medical dressings.

[0033] In the fifth aspect, the present invention provides a method for efficiently loading drugs into exosome vesicles for use in the preparation of health care products.

[0034] In the sixth aspect, the present invention provides a method for efficiently loading drugs into exosome vesicles for use in the preparation of disease prevention or treatment preparation products.

[0035] In summary, the present invention has the following beneficial effects:

[0036] 1. In view of the problems of difficult isolation and purification of current exosomes, low purity of isolation and purification, and difficulty in realizing large-scale production, etc., in combination with the advanced technologies in the current biomedical field, through exploration and innovation, a technical method for preparing high-purity and high-concentration exosomes of the present invention is established. It is applicable to the purification of exosomes from liquid raw materials and solid tissue raw materials, can realize industrial production, and has the advantages of stable technology, high efficiency, and low cost.

[0037] 2. A technical method for efficiently loading drugs into exosomes of the present invention, wherein the loading of large and small molecule chemical drugs and biological drugs can be realized, providing a broader application prospect for scientific research and clinical applications. It provides a new technical method in terms of increasing drug targeting and reducing adverse reactions.

[0038] 3. The exosome vesicles loaded with drugs prepared by the present invention have a wide range of applications. They can be used in the preparation of medical beauty and cosmetic preparations; at the same time, they can be loaded with drugs such as mRNA for efficient application in the vaccine field; at the same time, they can be loaded with tumor-targeted drugs, which can greatly improve the targeted treatment effect while reducing the off-target effect of the targeted drugs.

[0039] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the purification chromatography map of mesenchymal stem cell MSC exosomes in Example 1 of the present invention;

[0041] Figure 2 is the electron microscope detection image of mesenchymal stem cell MSC exosomes in Example 1 of the present invention;

[0042] Figure 3 is the particle size detection result of mesenchymal stem cell MSC exosomes in Example 1 of the present invention;

[0043] Figure 4 is the SDS-PAGE electrophoresis detection image of mesenchymal stem cell MSC exosomes in Example 1 of the present invention;

[0044] Figure 5 is the purification chromatography map of exosomes from animal placenta tissue in Example 2 of the present invention;

[0045] Figure 6 is the electron microscope detection image of exosomes from animal placenta tissue in Example 2 of the present invention;

[0046] Figure 7 is the particle size detection result of exosomes from animal placenta tissue in Example 2 of the present invention;

[0047] Figure 8It is the SDS-PAGE electrophoresis detection diagram of animal placenta tissue exosomes in Example 2 of the present invention;

[0048] Figure 9 It is the exosome particle size measurement diagram in Example 3 of the present invention;

[0049] Figure 10 It is the comparison diagram of drug loading efficiency of exosomes in different ways in Example 3 of the present invention;

[0050] Figure 11 It is the comparison diagram of the effective rate of animal experiments on drug loading of exosomes in different ways in Example 3 of the present invention. Specific implementation mode

[0051] The present invention will be further described in detail below in conjunction with embodiments. It should be particularly noted that: for those not specifying specific conditions in the following embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. Except for special instructions, the raw materials used in the following embodiments can be obtained from ordinary commercial sources.

[0052] A preparation method for efficiently loading drugs into exosome vesicles, comprising the following steps:

[0053] (1) Sample pretreatment: For liquid samples, large particles or residual debris can be removed by centrifugation or filtration; for tissue samples, a liquid containing exosome vesicles can be obtained by crushing, infiltration with buffer liquid, centrifugation or filtration; among them, the buffer liquid used includes NaHCO3, Tris-HCl, PBS, glucose solution, and physiological saline;

[0054] (2) Add pre-cooled 50% PEG2000 or PEG4000 or PEG6000 or PEG8000 to the solution containing exosome vesicles obtained in step (1) until the final concentration of PEG reaches 6%. Place the sample at 2-8°C and let it stand for 20-24 hours;

[0055] (3) Centrifuge the treatment solution in step (2) at the maximum centrifugal force to obtain precipitates such as exosomes; add the buffer liquid described in step (1) to the precipitate for resuspension and washing, and remove the residual solid part by centrifugation or filtration;

[0056] (4) Use a 300KD hollow fiber ultrafiltration system to process the exosome sample obtained in step (3). When the cut-off volume reaches 1 / 5-1 / 10 of the raw material volume (or weight), wash it 5-7 times with 10 mM PBS (pH 7.2-7.5) to reduce the residual of miscellaneous proteins or pigments, etc., and finally obtain the exosome concentrated treatment solution;

[0057] (5) Filter and top filter the exosome concentrated treatment solution in step (4) using a 0.45μm + 0.22μm capsule filter to obtain an exosome concentrated filtrate;

[0058] (6) Fine purification of exosome vesicles. Chromatographically purify the exosome concentrated filtrate obtained in step (5) by molecular exclusion method, where molecular exclusion uses fillers such as Core700 series, Sepharose 4FF, or Sepharose 6FF, etc.; collect the corresponding exosome vesicle purified solution through UV260 and UV280 peak maps.

[0059] (7) Filter the exosome vesicle sample obtained in step (6) through 0.22μm to obtain a sterile exosome sample.

[0060] A high-efficiency drug loading method for exosomes technology, comprising the following steps:

[0061] (1) Drug pretreatment: Use lipid reagents for the target drug, such as cholesterol mentioned in the present invention, to dissolve the target drug or mix the target drug solution with cholesterol; use the methods of suspension and high-speed centrifugation for repeated promotion and mixing. Among them, for stable target drugs, violent shaking can be used for suspension, and for unstable samples, repeated pipetting or blowing with a pipette or pipette can be selected for suspension. Then centrifuge the suspension at 12000g, 4°C for 15 minutes; repeat the above operation 3 - 5 times to finally obtain the target drug pretreatment solution;

[0062] (2) Exosome vesicle precipitation: Add 50% PEG6000 to the obtained high-purity exosome sample to a final concentration of 6%. Obtain exosome precipitates by centrifugation;

[0063] (3) Add the target drug pretreatment solution obtained in step (1) to the exosome precipitate in a ratio of 2:1 (volume: mass), resuspend and mix well for 30s; then immediately add 10 times the volume of the buffer solution of the target drug pretreatment solution, 10mM PBS (pH 7.2 - 7.5), for dilution and suspension operation;

[0064] (4) The operation of step (2) can be repeated to remove free cholesterol and the target drug in the solution, which can effectively reduce the influence of free cholesterol and the target drug on experiments or drug use;

[0065] (5) By repeating steps (2), (3), and (4), etc., the exosome loading efficiency of the target drug can be increased.

[0066] Examples

[0067] Example 1

[0068] Sample information:

[0069] The culture supernatant of mesenchymal stem cells (MSCs) is a frozen stock solution of MSC cell culture supernatant without any treatment, donated by a professional cell company. The whole process of its acquisition is obtained through cold chain transportation, namely dry ice transportation.

[0070] A method for preparing high-concentration and high-purity mesenchymal stem cell (MSC) exosomes, the specific implementation steps include the following aspects:

[0071] (1) Sample pretreatment: For liquid samples, large particles or residual debris can be removed by centrifugation or filtration; among them, dilution treatment is carried out using pre-cooled buffer liquid 10 mM PBS (pH 7.2), the purpose is to increase the sample volume for subsequent operations, and at the same time can effectively improve the subsequent enrichment effect of exosome vesicles;

[0072] (2) Add pre-cooled 50% PEG8000 to the solution containing exosome vesicles obtained in step (1) until the final concentration of PEG8000 reaches 6%. Mix and stir slowly in an ice bath until sufficient, and then place the sample at 8 °C for 24 hours;

[0073] (3) Centrifuge the treatment solution in step (2) at 12000 g, 4 °C for 60 minutes to obtain precipitates such as exosomes; add the buffer liquid described in step (1) to the precipitate for resuspension and washing, and remove the residual solid part by centrifugation or filtration;

[0074] (4) Use a 300KD hollow fiber ultrafiltration system to process the precipitate samples such as exosomes obtained in step (3). When the retention volume reaches 1 / 5 of the raw material volume (or weight), wash 5 times with 10 mM PBS (pH 7.2) to reduce the residual of miscellaneous proteins or pigments, etc., and finally obtain an exosome concentrated treatment solution;

[0075] (5) Filter and top-filter the exosome concentrated treatment solution in step (4) using a 0.45 μm + 0.22 μm capsule filter to obtain an exosome concentrated filtrate;

[0076] (6) Fine purification of exosome vesicles. Chromatographically purify the exosome concentrated filtrate obtained in step (5) by molecular exclusion method. Among them, molecular exclusion uses Core700 series packing materials, and the packed column height > 70 cm. The purpose of using this packing material is to effectively remove the protein components in the sample solution, and at the same time obtain exosome vesicles through the molecular sieve exclusion function, greatly improving the purity of exosome vesicles; collect the corresponding exosome vesicle purification solution through the UV260 and UV280 peak graphs.

[0077] (7) Filter the exosome vesicle sample obtained in step (6) through a 0.22 μm filter to obtain a sterile exosome sample.

[0078] (8) The purified exosome sample is tested to obtain the following information related to sample purity. The test results are as Figures 1-4 shown.

[0079] Figure 1 is the purification chromatography map of mesenchymal stem cell MSC exosomes in Example 1 of the present invention;

[0080] Figure 2 is the electron microscopy detection image of mesenchymal stem cell MSC exosomes in Example 1 of the invention;

[0081] Figure 3 is the particle size detection result of mesenchymal stem cell MSC exosomes in Example 1 of the present invention. The left side is the adjusted parameter, and the right side is the change in particle size;

[0082] Figure 4 is the SDS-PAGE electrophoresis detection image of mesenchymal stem cell MSC exosomes in Example 1 of the present invention, showing the electrophoresis intensity under different conditions.

[0083] Example 2

[0084] Sample information:

[0085] The animal placenta tissue is a frozen sample of untreated placenta tissue donated by the stem cell repository of the cooperative unit. This sample is only used for laboratory scientific research and exploration, and is a non-profit behavior. The acquisition process is obtained by cold chain transportation, that is, dry ice transportation.

[0086] A method for preparing high-concentration and high-purity exosomes from animal placenta tissue, and the specific implementation steps include the following aspects:

[0087] (1) Sample pretreatment: For tissue samples, obtain a liquid containing exosome vesicles through crushing, soaking with buffer liquid, centrifugation or filtration; among them, use pre-cooled buffer liquid 10 mM PBS (pH 7.5) for dilution treatment, the purpose of which is to soak the tissue sample to obtain a large number of exosomes, increase the volume for subsequent operations, and at the same time effectively improve the subsequent enrichment effect of exosome vesicles;

[0088] (2) Add pre-cooled 50% PEG6000 to the solution containing exosome vesicles obtained in step (1) until the final concentration of PEG6000 reaches 8%. Mix and stir slowly under ice bath conditions until sufficient, and then place the sample at 8 °C for 24 hours;

[0089] (3) Centrifuge the treatment solution in step (2) at 12,000 g for 60 minutes at 4 °C to obtain precipitates such as exosomes; add the buffer liquid described in step (1) to the precipitate for resuspension and washing, and remove the residual solid part by centrifugation or filtration;

[0090] (4) Use a 300KD hollow fiber ultrafiltration system to process the precipitate sample such as exosomes obtained in step (3). When the retention volume reaches 1 / 5 of the raw material volume (or weight), wash it 5 - 7 times with 10 mM PBS (pH 7.5) to reduce the residue of impurities such as proteins or pigments, and finally obtain a concentrated exosome treatment solution;

[0091] (5) Filter and top - filter the concentrated exosome treatment solution in step (4) using a 0.45μm + 0.22μm capsule filter to obtain a concentrated exosome filtrate;

[0092] (6) Fine purification of exosome vesicles. Chromatographically purify the concentrated exosome filtrate obtained in step (5) by molecular exclusion method. For molecular exclusion, use Sepharose 6FF series packing material, and the column height filled is > 90 cm. The purpose of using this packing material is to effectively remove the protein components in the sample solution, and at the same time obtain exosome vesicles through the molecular sieve exclusion function, greatly improving the purity of exosome vesicles; collect the corresponding purified exosome vesicle solution through the UV260 and UV280 peak maps.

[0093] (7) Filter the exosome vesicle sample obtained in step (6) through a 0.22μm filter to obtain a sterile exosome sample.

[0094] (8) The purified exosome sample is detected to obtain the following information related to sample purity. The detection results are as Figures 5-8 shown.

[0095] Figure 5 This is the purification chromatography map of exosomes from animal placenta tissue in Example 2 of the present invention;

[0096] Figure 6 This is the electron microscope detection image of exosomes from animal placenta tissue in Example 2 of the present invention;

[0097] Figure 7 This is the particle size detection result of exosomes from animal placenta tissue in Example 2 of the present invention; on the left are the adjusted parameters, and on the right are the changes in particle size;

[0098] Figure 8 This is the SDS - PAGE electrophoresis detection image of exosomes from animal placenta tissue in Example 2 of the present invention.

[0099] Example 3

[0100] Sample information:

[0101] The experimental materials for the exploration and verification of this invention are derived from self-prepared placental tissue exosomes, the drug levodopa for treating Parkinson's disease, and the loading excipient cholesterol.

[0102] A method for efficiently loading drugs into animal placental tissue exosomes, and the specific implementation steps include the following aspects:

[0103] (1) Drug pretreatment: Dissolve levodopa solution with cholesterol; use the methods of suspension and high-speed centrifugation for repeated mixing, and use the methods of repeated pipetting and shaking for suspension. Then centrifuge the suspension at 12,000 g and 4 °C for 15 min; repeat the above operations 5 times, and finally obtain the target drug pretreatment solution;

[0104] (2) Exosome vesicle aggregation: Add 50% PEG6000 to the obtained high-purity exosome sample to a final concentration of 6%. Obtain exosome precipitates by centrifugation;

[0105] (3) Add the target drug pretreatment solution obtained in step (1) to the exosome precipitates in a ratio of 2:1 (volume: mass), and resuspend and mix evenly for 30 s; then immediately add 10 times the volume of the buffer solution of the target drug pretreatment solution, 10 mM PBS (pH 7.5), for dilution and suspension operation;

[0106] (4) The operations in step (2) can be repeated to remove free cholesterol and the target drug in the solution, which can effectively reduce the influence of free cholesterol and the target drug on experiments or drug use;

[0107] (5) By repeating steps (2), (3), and (4), etc., the exosome loading efficiency of the target drug can be increased.

[0108] (6) The analysis of the detection results of the loaded exosomes is as Figures 9-11 shown.

[0109] Figure 9 It is a diagram of the exosome particle size measurement in Example 3 of this invention; where the abscissa is intensity and the ordinate is size.

[0110] Figure 10 It is a comparison diagram of the drug loading efficiency of exosomes in different ways in Example 3 of this invention; they are the freeze-thaw method, the ultrasonic method, and the method of Example 3 of this invention respectively.

[0111] Figure 11 It is a comparison diagram of the effective rate of the animal experiment of drug loading into exosomes in different ways in Example 3 of this invention. They are the freeze-thaw method, the ultrasonic method, and the method of Example 3 of this invention respectively.

[0112] As described above, the above is only an exemplary specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A method for preparing exosome vesicles efficiently loaded with drugs, characterized in that: The following steps are involved: (1) Exosome preparation: Prepare exosome samples with sufficient particle size and purity. Remove large particles or residual debris from liquid samples by centrifugation or filtration. Tissue samples are crushed, infiltrated with buffered liquid, and centrifuged or filtered to obtain liquid containing exosome vesicles. (2) Exosome pretreatment: collect exosomes by precipitation treatment, prepare exosome precipitate, add 50% pre-cooled loading drug to the solution containing exosome vesicles obtained in step (1), until the final loading drug concentration reaches 6%. Place the sample at 2-8°C for 20-24 hours; (3) Pretreatment of loading drugs: treating the loaded drugs or drug preparations with lipid reagents, centrifuging the treatment solution in step (2) to obtain an exosome precipitate; adding the buffer liquid in step (1) to the precipitate for resuspending and washing, and removing the residual solid part by centrifugation or filtration; (4) Target drug loading: The drug lipid component is quickly treated with the exosome precipitate, and then treated with a buffer system to obtain the drug exosome loading, and added to the exosome precipitate at a volume ratio of 2:1, resuspended and mixed for 30 seconds, and then immediately added with 10 times the volume of the target drug pretreatment solution buffer 10mM PBS, and the dilution and suspension operation is performed at a pH of 7.2-7.5; (5) Removal of free lipids and drugs: Reduction of free lipids and drug residues by re-enriching exosomes.

2. The method for preparing exosome vesicles efficiently loaded with drugs according to claim 1, characterized in that: In step (1), the exosome sample includes exosomes from animal or plant tissues or body fluids.

3. The method for preparing exosome vesicles efficiently loaded with drugs according to claim 1, characterized in that: Step (3) also includes lipid phase incubation of the drug, and the lipid reagent includes a water-soluble reagent or a water-insoluble reagent.

4. The method for preparing exosome vesicles efficiently loaded with drugs according to claim 1, characterized in that: In step (4), the buffer system includes NaHCO3, Tris-HCl, PBS, glucose solution or physiological saline.

5. The method for preparing exosome vesicles efficiently loaded with drugs according to claim 1, characterized in that: In step (3), the loaded drug is one of PEG2000, PEG4000, PEG6000, and PEG8000.

6. An exosome vesicle efficiently loaded with drugs prepared as claimed in any one of claims 1 to 5 is used in the preparation of functional cosmetics.

7. An exosome vesicle efficiently loaded with drugs prepared as claimed in any one of claims 1 to 5 is used in the preparation of medical cosmetics.

8. An exosome vesicle efficiently loaded with drugs prepared as claimed in any one of claims 1 to 5 is used in the preparation of medical dressings.

9. An exosome vesicle efficiently loaded with drugs prepared as claimed in any one of claims 1 to 5, wherein the exosome vesicle is used in the preparation of health care products.

10. An exosome vesicle efficiently loaded with drugs prepared as claimed in any one of claims 1 to 5, wherein the exosome vesicle is used in the preparation of disease prevention or treatment preparations.

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