Active drug loading method for extracellular vesicles

Abstract

The invention relates to the technical field of extracellular vesicles, in particular to an extracellular vesicle active drug loading method which comprises the following preparation steps: S1, collecting cell supernatant, collecting the cell supernatant under the condition of 4 DEG C, and performing multi-stage centrifugation to remove impurities to obtain purified extracellular vesicles; s2, dissolving a target drug in a corresponding solvent to prepare a drug solution with the concentration of 0.1-5 mg / mL; s3, mixing the purified extracellular vesicles obtained in S1 with the medicine solution obtained in S2 according to the volume ratio of 1: 1, and performing low-frequency ultrasonic treatment in a pulse mode under the ice bath condition of 4 DEG C; s4, incubating the mixed system subjected to ultrasonic treatment at 37 DEG C for 25-35 minutes, and meanwhile, adding a cholesterol solution with the final concentration of 0.005-0.01% to assist membrane repair; the problems of contradiction between the drug loading efficiency and the integrity of the extracellular vesicles, poor drug universality, low purification efficiency and insufficient application stability in the existing active drug loading technology of the extracellular vesicles are specifically solved through the whole process optimization of precise purification, efficient drug loading, directional repair and deep refining.

Description

Technical Field

[0001] This invention relates to the field of extracellular vesicle technology, and more specifically to an active drug delivery method for extracellular vesicles. Background Technology

[0002] Current drug delivery systems, such as nanoparticle drug delivery systems, typically use various stabilizers to prepare stable nanosystems. After loading drugs, they can actively or passively aggregate to target organs, cells, or organelles, enhancing drug targeting. Using nanoparticles to load drugs can also improve drug stability and solubility, promote transmembrane drug transport, prolong drug circulation time to improve drug efficacy, and reduce the toxicity of free drugs to non-target organs / cells.

[0003] Nanomedicine can facilitate the clinical translation of drugs and improve patient treatment side effects, but currently only a few polymeric nanomedicines have been approved by the FDA and used clinically. Of the currently approved nanomedicines, few are recommended as first-line treatments, and some only show better efficacy in a small subset of patients. Although existing engineered nanotechnology can overcome the limitations of traditional drug delivery technologies and shows the potential to enhance drug efficacy, some shortcomings remain. For example, liposome-loaded drugs are prone to leakage and have poor stability; inorganic nanoparticles often use heavy metal formulations, which have low solubility and high risk of toxic side effects. Therefore, developing safe and highly targeted nanosystems as delivery carriers remains a major scientific challenge in the biomedical field.

[0004] Extracellular vesicles are cell-derived nanoscale vesicles, mainly including exosomes and microvesicles. Under different types of endogenous or exogenous stimuli, the cell membrane can detach to produce vesicles of different sizes, or endosomes can secrete them. Vesicles of 100-1000 nm are called microvesicles, while those of 30-150 nm are called exosomes. Extracellular vesicles are distributed in biological fluids, carrying biomolecules such as nucleic acids, proteins, and lipids through circulation. They can also cross the blood-brain barrier, playing an important role in intercellular communication. Furthermore, the typical lipid bilayer structure of extracellular vesicles protects nucleic acids from degradation, making them ideal carriers for nucleic acids in gene therapy, thus possessing the potential to serve as biomolecule delivery carriers.

[0005] Furthermore, after being released into the extracellular environment, extracellular vesicles can be taken up by different types of cells, primarily engaging in cell signal transduction and communication through two pathways: binding to specific cell membrane molecules and endocytosis into target cells. Once inside the cell, extracellular vesicles can release their cargo into the target cell and perform its biological functions. Because extracellular vesicles carry the phospholipid bilayer structure of the source cell during cell detachment, they can fuse more effectively with recipient cells. Moreover, modifying the surface of extracellular vesicles can achieve more efficient targeting, enabling them to deliver drugs, proteins, and other biomolecules to target tissues.

[0006] In conclusion, extracellular vesicles are ideal delivery carriers for drugs and biomolecules. Compared with synthetic carriers such as liposomes or other nanoparticles, extracellular vesicles, as products of cells, exhibit better biocompatibility, lower immunogenicity, and the ability to cross biological barriers such as the blood-brain barrier. Extracellular vesicles hold great promise as nanocarriers for the treatment of various diseases, potentially improving clinical efficacy and safety.

[0007] To improve drug delivery efficiency, traditional methods often use continuous ultrasound. However, the local high temperature generated by ultrasound can cause disorder of the phospholipid bilayer of the EV membrane and denaturation of membrane proteins, ultimately causing more than 30% of EVs to rupture or aggregate. Even if they do not rupture completely, the damage to the membrane structure will lead to a significant increase in the subsequent drug leakage rate. The drug leakage rate often exceeds 40% within 48 hours, which is far from meeting the clinical demand for "long-term delivery" of drug carriers.

[0008] Electroporation for drug loading: High-voltage pulses are used to create transient channels on the EV membrane. Although this method has a certain drug loading effect on nucleic acid drugs, the voltage parameters are difficult to control precisely: if the voltage is too low, there will be insufficient channels and the drug loading rate will be less than 25%; if the voltage is too high, the EV membrane will be irreversibly damaged, with a damage rate of more than 28%. In addition, the strong electric field generated by electroporation will cause the nucleic acid drug to degrade, further reducing the drug efficacy.

[0009] Repeated freeze-thaw / compression drug loading: Drug loading is achieved by repeatedly freezing and thawing at -80℃ and 37℃ or by forcibly compressing the membrane. The former can easily lead to the EV membrane fusing to form large aggregates, losing the permeation advantage of the nanocarrier. The latter, due to excessive mechanical force, causes the leakage rate of EV contents to exceed 30%, destroying its natural biological activity.

[0010] In the EV drug delivery process, the conflict between "impurity removal" and "EV recovery" has remained unresolved, making it difficult to balance the purity and yield of drug-loaded EVs.

[0011] EV purification stage: Existing technologies mostly rely on a single purification method: Although ultracentrifugation can precipitate EVs, it cannot remove soluble proteins and apoptotic bodies with densities close to those of EVs, resulting in a final EV purity of only about 80%. The residual protein impurities will compete with drugs for binding sites in subsequent drug loading, reducing drug loading efficiency. Although size exclusion chromatography can separate EVs from small molecule impurities, EVs are easily adsorbed by the gel matrix, with a recovery rate of less than 60%, and it cannot remove protein aggregates with particle sizes close to those of EVs, affecting the stability of subsequent drug loading.

[0012] Post-drug loading purification stage: After drug loading is completed, it is necessary to remove free drugs that have not entered EVs to avoid non-targeted toxicity in vivo. However, existing purification methods are inefficient: single sucrose density gradient centrifugation can only remove less than 70% of free drugs, and the remaining free drugs will be toxic to normal tissues; single ultrafiltration centrifugation results in EV loss of more than 40% due to membrane retention effect, and is prone to EV aggregation, which cannot meet the needs of large-scale production. Summary of the Invention

[0013] The purpose of this invention is to address the problems of contradiction between drug loading efficiency and extracellular vesicle integrity, poor drug universality, low purification efficiency, and insufficient application stability in existing extracellular vesicle active drug loading technologies by optimizing the entire process of precise purification, efficient drug loading, targeted repair, and deep purification.

[0014] To achieve the above objectives, the present invention provides the following technical solution:

[0015] A method for active drug delivery to extracellular vesicles includes the following preparation steps:

[0016] S1. Collect cell supernatant. After collecting the cell supernatant at 4°C, remove impurities by multi-stage centrifugation to obtain purified extracellular vesicles.

[0017] S2. Dissolve the target drug in the corresponding solvent to prepare a drug solution with a concentration of 0.1-5 mg / mL;

[0018] S3. The purified extracellular vesicles of S1 and the drug solution of S2 are mixed at a volume ratio of 1:1 and subjected to low-frequency ultrasound treatment in pulse mode under 4°C ice bath conditions.

[0019] S4. Place the ultrasonically treated mixture at 37°C for 25-35 minutes, and add a cholesterol solution with a final concentration of 0.005%-0.01% to assist in membrane repair.

[0020] S5. First, perform initial purification by size exclusion chromatography to collect the characteristic peak components of extracellular vesicles. Then, purify by centrifugation with a 10%-40% sucrose density gradient to collect drug-loaded extracellular vesicles in the 20%-30% density range.

[0021] As a preferred embodiment of the present invention, the multi-stage centrifugation in S1 includes removing cell debris by centrifugation at 300 rpm for 10-15 min at 4°C, removing apoptotic bodies by centrifugation at 2000 rpm for 20-30 min, filtering the cell supernatant through a 0.22 μm filter membrane, precipitating the cell supernatant by ultracentrifugation at 100000 rpm for 80-100 min, and resuspending the cell supernatant in a buffer solution and repeating the ultracentrifugation once.

[0022] As a preferred embodiment of the present invention, the target drug in S2 includes one of small molecule chemotherapy drugs, nucleic acid drugs, or protein drugs.

[0023] As a preferred embodiment of the present invention, the final concentration of the extracellular vesicles after mixing with the drug solution in step S3 is 0.6-0.8 mg / mL, and the total volume of the mixing system is 200-500 μL.

[0024] As a preferred embodiment of the present invention, the size exclusion chromatography in S5 uses a Sephacryl S-500HR column, the mobile phase is buffer, the flow rate is 0.5 mL / min, and the characteristic peaks of extracellular vesicles with a retention time of 18-22 min are collected. The sucrose density gradient centrifugation conditions are 100,000 centrifugation force for 110-130 min.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] This invention addresses the problems of existing active drug delivery technologies for extracellular vesicles, such as the contradiction between drug delivery efficiency and extracellular vesicle integrity, poor drug universality, low purification efficiency, and insufficient application stability, through a comprehensive optimization of the entire process including precise purification, efficient drug loading, targeted repair, and deep purification. Pulsed ultrasound and a 4°C ice bath temperature control reduce the extracellular vesicle breakage rate, improving the loading rates of doxorubicin, siRNA, and insulin compared to passive incubation. Subsequent cholesterol repair reduces drug leakage after 48 hours. No special modifications are required, making it compatible with small molecule chemotherapy drugs and nucleic acid-based drugs. For pharmaceutical and protein drugs, this technology overcomes the limitations of single-drug compatibility in existing technologies. Multi-stage centrifugation improves the purity and recovery rate of extracellular vesicles, while two-stage purification increases the removal rate of free drugs and the overall recovery rate of extracellular vesicles. It resolves the contradiction that high purity leads to low recovery rate. The parameters for each step are clearly defined and can be achieved with conventional equipment. It is reproducible from small-scale to pilot-scale. The coefficient of variation of extracellular vesicle particle size and drug loading between batches is reduced, resulting in less free drug residue, reduced toxicity to normal tissues, preservation of targeting markers on the surface of extracellular vesicles, increased uptake by tumor cells, and reduced immunogenicity risk. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the raw materials used in the examples and comparative examples are all disclosed in the prior art, such as those that can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0029] Example:

[0030] This invention provides a technical solution:

[0031] A method for active drug delivery to extracellular vesicles includes the following preparation steps:

[0032] Step 1: Collect cell supernatant. Under 4°C conditions, collect cell supernatant and remove impurities through multi-stage centrifugation to obtain purified extracellular vesicles.

[0033] Human umbilical cord mesenchymal stem cells were cultured to 80% confluence, then replaced with exosome-free FBS-free DMEM / F12 medium. After 48 hours of further culture, the cell supernatant was collected and stored at 4°C. The supernatant was transferred to a 50 mL centrifuge tube and centrifuged at 300 rpm for 12 min at 4°C. The cell debris at the bottom was discarded, and the supernatant was collected. The supernatant was transferred to a new centrifuge tube and centrifuged at 2000 rpm for 25 min at 4°C to remove apoptotic bodies. The supernatant was then vacuum filtered through a 0.22 μm polyethersulfone membrane to remove cells with a diameter >220 nm. Impurities and particles were filtered, and the supernatant was transferred to an ultracentrifuge tube. The tube was centrifuged at 100,000 rpm for 90 minutes at 4°C. The supernatant was discarded, and the bottom precipitate was gently resuspended in 1 mL of pre-cooled buffer, avoiding vigorous resuscitation to prevent rupture of extracellular vesicles. The resuspended precipitate was then brought to 10 mL with buffer, and centrifuged again at 100,000 rpm for 90 minutes. Finally, the precipitate was resuspended in 2 mL of buffer to purify the extracellular vesicles. The purity of the extracellular vesicles was detected using nanoflow cytometry, and the concentration of extracellular vesicles was determined using the BCA protein quantification method and adjusted to 1.2-1.6 mg / mL in preparation for subsequent mixing.

[0034] Step 2: Dissolve the target drug in the corresponding solvent to prepare a drug solution with a concentration of 0.1-5 mg / mL;

[0035] Take 2 mg of doxorubicin powder, add 1 mL of buffer solution, vortex for 10 s until completely dissolved, sterilize through a 0.22 μm filter membrane, and store at 4 °C protected from light. The concentration is 2 mg / mL. Take 0.5 mg of cholesterol lyophilized powder, add 1 mL of enzyme-free water, gently pipette 5 times until dissolved, aliquot into 100 μL / tube, store at -80 °C, thaw before use, and the concentration is 0.5 mg / mL. Take 0.1 mL of insulin injection solution, add 0.9 mL of 0.01 M hydrochloric acid solution, mix well, and measure the concentration as 1 mg / mL. Insulin is stable under acidic conditions; avoid aggregation.

[0036] Step 3: Mix the purified extracellular vesicles from Step 1 with the drug solution from Step 2 at a volume ratio of 1:1, and perform low-frequency sonication in pulse mode under 4°C ice bath conditions. The target drug in Step 2 includes one of small molecule chemotherapy drugs, nucleic acid drugs, or protein drugs. The final concentration of extracellular vesicles after mixing with the drug solution in Step 3 is 0.6-0.8 mg / mL, and the total volume of the mixing system is 200-500 μL.

[0037] Take purified extracellular vesicles and mix them with any of the above-mentioned drug solutions at a volume ratio of 1:1. For example, take 200 μL of extracellular vesicles and 200 μL of doxorubicin solution, and after mixing, the final concentration of extracellular vesicles is 0.7 mg / mL, with a total volume of 400 μL. Prepare cholesterol and insulin groups respectively, and transfer them to 1.5 mL enzyme-free mini centrifuge tubes. Place the mini centrifuge tubes in an ice bath at 4°C, and insert the ultrasonic probe 1 cm below the liquid surface, avoiding contact with the tube wall. Set the parameters as follows: power 20W, pulse mode, 30s on / 30s off, total processing time 120s, for a total of 4 cycles. During the ultrasonic process, gently shake the mini centrifuge tube once every 30s to ensure uniform mixing, and monitor the system temperature with a thermometer. The temperature should always be <10°C to avoid thermal damage to the extracellular vesicle membrane.

[0038] Step 4: Incubate the ultrasonically treated mixture at 37°C for 25-35 minutes, while adding a cholesterol solution with a final concentration of 0.005%-0.01% to assist in membrane repair.

[0039] After sonication, cholesterol stock solution was added to the mixing system. The amount added was calculated based on a final concentration of 0.008%. The small centrifuge tube was gently inverted three times to mix. The small centrifuge tube was then placed in a 37°C incubator and incubated for 30 minutes. The doxorubicin group required incubation in the dark. The small centrifuge tube was wrapped with aluminum foil to prevent drug photolysis. During incubation, the tube was gently shaken every 10 minutes to promote cholesterol embedding into the extracellular vesicle membrane and repair the transient pores caused by sonication.

[0040] Step 5: First, perform initial purification by size exclusion chromatography to collect the characteristic peak components of extracellular vesicles. Then, purify by centrifugation with a 10%-40% sucrose density gradient to collect drug-loaded extracellular vesicles in the 20%-30% density range.

[0041] Connect the Sephacryl S-500HR column to a high-performance liquid chromatography (HPLC) system. Use buffer as the mobile phase at a flow rate of 0.5 mL / min. Equilibrate three column volumes until the baseline stabilizes. Load the incubated mixture onto the HPLC system. Collect 0.5 mL of eluent per tube. Analyze the extracellular vesicle particle concentration in each tube using nanoflow cytometry. Collect the fractions retained for 18–22 min, discarding any peaks containing free drug or protein impurities. Use a gradient mixer to separate 10%, 20%, 30%, and 40% sucrose solutions. Add the 13.2 mL ultracentrifuge tubes in the order of high concentration at the bottom and low concentration at the top. Let them stand at 4℃ for 30 min to form a continuous density gradient. Slowly add the extracellular vesicle components collected by SEC to the top of the sucrose gradient. Centrifuge at 4℃ and 100,000 centrifugation force for 120 min. After centrifugation, use a pipette to collect 0.5 mL from the top of the centrifuge tube, for a total of 24 tubes. Use a refractometer to measure the sucrose concentration in each tube. Collect tubes 8-12 corresponding to a density of 20%-30%, which are the high-purity drug-loaded extracellular vesicles.

[0042] Furthermore, in step one, the multi-stage centrifugation includes centrifugation at 300 rpm for 10-15 min at 4°C to remove cell debris, centrifugation at 2000 rpm for 20-30 min to remove apoptotic bodies, filtration of the cell supernatant through a 0.22 μm filter, ultracentrifugation at 100,000 rpm for 80-100 min to precipitate the cell supernatant, resuspending the cell supernatant in PB buffer and repeating the ultracentrifugation once. The entire process is performed at 4°C using multi-stage centrifugation, which significantly reduces the fluidity of extracellular vesicle membranes and the rate of protein denaturation, avoiding the loss of the phospholipid bilayer of extracellular vesicle membranes due to room temperature or high temperature environments. The process involves sequencing and denaturation of surface-targeting proteins, which are crucial for the natural targeted delivery of extracellular vesicles. The preservation of these proteins directly determines the in vivo targeting efficiency of subsequent drug-loaded extracellular vesicles. This reduces the risk of extracellular vesicle rupture or leakage due to thermal stress. Combined with transmission electron microscopy observations in the documented examples, it is demonstrated that this low-temperature condition can control the extracellular vesicle breakage rate to below 5%, far superior to the breakage rate of over 30% in existing continuous ultrasonic purification methods. The protocol employs a gradient setting of low, medium, and high centrifugal forces to achieve precise separation based on the density / particle size differences of different impurities, avoiding co-precipitation or excessive loss of extracellular vesicles and impurities.

[0043] Centrifuge at 300 for 10-15 min: Only cell debris with higher density is precipitated, while extracellular vesicles with lower density remain in the supernatant. This can reduce the early loss of extracellular vesicles due to excessive centrifugation, and the initial impurity removal efficiency is over 90%.

[0044] Centrifuge at 2000 for 20-30 min: Targeted removal of apoptotic bodies. If these impurities remain, they will compete for drug binding sites during subsequent ultrasonic drug loading, reducing drug loading efficiency. This step can increase the removal rate of apoptotic bodies to 95%, clearing the way for efficient drug loading in the future.

[0045] 100,000 RPM ultracentrifugation for 80-100 min: This centrifugal force is the optimal parameter for extracellular vesicle precipitation. It ensures sufficient precipitation of extracellular vesicles while avoiding excessive compression and rupture of the extracellular vesicle membrane due to excessive centrifugal force, or insufficient precipitation and reduced recovery rate due to insufficient centrifugation time. According to the document's examples, this step can achieve an extracellular vesicle recovery rate of over 85%, significantly better than existing single ultracentrifugation. Inserting a 0.22 μm filter membrane filtration step between multi-stage centrifugation can specifically remove residual impurities with a diameter >220 nm. This pore size will not trap the target extracellular vesicles and can eliminate interference from oversized impurities in subsequent experiments. If such impurities remain, they will occupy the characteristic peak positions of extracellular vesicles in subsequent size exclusion chromatography purification, resulting in incomplete separation of extracellular vesicles from free drug after drug loading. After filtration, the coefficient of variation of extracellular vesicle particle size distribution can be controlled within 20%, significantly improving uniformity. This process ensures that the ultrasonic energy is evenly applied to each extracellular vesicle during subsequent ultrasonic drug loading, preventing localized over-ultrasound rupture or under-ultrasound rupture due to extracellular vesicle aggregation, which would result in low drug loading efficiency. After the first ultracentrifugation, soluble proteins, small molecule metabolites, and other impurities may remain in the extracellular vesicle precipitate. Resuspension with buffer can dissolve these impurities, and a second ultracentrifugation further removes them. The final purity of the extracellular vesicles can reach over 95%, far exceeding the 80% purity achieved by existing single ultracentrifugation. High-purity extracellular vesicles can prevent impurities from competing with drugs for binding sites, directly improving the active drug loading efficiency in the subsequent step three. The osmotic pressure and pH of the buffer are close to the physiological environment, preventing other buffers from causing extracellular vesicle membrane rupture or leakage of contents. Combined with the gentle resuspension operation during repeated centrifugation, the final integrity rate of extracellular vesicles can be maintained at over 90%, laying the foundation for membrane repair in the subsequent step four and drug loading and purification in the fifth step.

[0046] The extracellular vesicles purified by this multi-stage centrifugation process can be directly adapted to subsequent ultrasonic drug loading, cholesterol repair, and two-step purification procedures. The low-impurity soluble proteins, apoptotic bodies, and large-sized particles reduce the loss caused by non-specific binding of drugs to impurities during ultrasonic drug loading, ensuring that drugs preferentially enter the extracellular vesicles. The highly intact and active extracellular vesicle membrane structure can rapidly respond to the cholesterol repair in step four after ultrasonic perforation, improving the membrane micropore closure efficiency by 30%, reducing subsequent drug leakage, and ultimately ensuring the in vivo delivery effect of drug-loaded extracellular vesicles.

[0047] Furthermore, in step five, size exclusion chromatography uses an ephacryl-500HR column with a PB mobile phase and a flow rate of 0.5 mL / min. It collects the characteristic peaks of extracellular vesicles with a retention time of 18-22 min. Sucrose density gradient centrifugation is performed at 100,000 rpm for 110-130 min, efficiently removing free drug, preserving the structural integrity of extracellular vesicles, and improving the purity and stability of drug-loaded extracellular vesicles. The purification scheme in step five, through the synergistic design of precise chromatographic separation and density gradient purification, solves the pain points of existing extracellular vesicle purification technologies, such as incomplete removal of free drug, high extracellular vesicle loss rate, and interference with drug loading by impurities. SEC initial purification can quickly remove more than 80% of the free drug, reducing the drug load of subsequent sucrose gradient centrifugation. If sucrose gradient centrifugation is performed directly, a large amount of free drug will dissolve in the sucrose solution, and some residue will remain even after centrifugation. With the two steps combined, the total free drug removal rate is ≥96.2±1.5%, far higher than SEC alone or single SEC alone. Sucrose centrifugation prevents premature leakage of free drugs during subsequent storage or in vivo delivery, ensuring the efficacy of drug-loaded extracellular vesicles. This includes reducing the toxicity of free drugs to normal tissues and minimizing extracellular vesicle loss, balancing purity and recovery rate. SEC (Sequencing and Degradation) minimizes extracellular vesicle adsorption loss through gentle elution, while sucrose gradient centrifugation ensures precise density stratification, preventing the co-discarding of extracellular vesicles and impurities. These two steps work synergistically to achieve a total extracellular vesicle recovery rate ≥78.3±3.2%, resolving the contradiction of decreased recovery rate with increased purity in existing purification techniques. This approach is well-suited to the application requirements of drug-loaded extracellular vesicles. The purified extracellular vesicles possess high purity, high integrity, and low drug residue, directly meeting the needs of subsequent in vivo targeted delivery or in vitro activity testing. High purity avoids immune responses caused by impurities such as protein aggregates, high integrity ensures the membrane barrier function of drug-loaded extracellular vesicles, and low drug residue reduces interference from free drugs in efficacy assessment, such as preventing misidentification of free drugs as drug-loaded extracellular vesicles in in vitro cell experiments.

[0048] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for active drug delivery to extracellular vesicles, characterized in that, The preparation steps include the following: S1. Collect cell supernatant. After collecting the cell supernatant at 4°C, remove impurities by multi-stage centrifugation to obtain purified extracellular vesicles. S2. Dissolve the target drug in the corresponding solvent to prepare a drug solution with a concentration of 0.1-5 mg / mL; S3. The purified extracellular vesicles of S1 and the drug solution of S2 are mixed at a volume ratio of 1:1 and subjected to low-frequency ultrasound treatment in pulse mode under 4°C ice bath conditions. S4. Place the ultrasonically treated mixture at 37°C for 25-35 minutes, and add a cholesterol solution with a final concentration of 0.005%-0.01% to assist in membrane repair. S5. First, perform initial purification by size exclusion chromatography to collect the characteristic peak components of extracellular vesicles. Then, purify by centrifugation with a 10%-40% sucrose density gradient to collect drug-loaded extracellular vesicles in the 20%-30% density range.

2. The method for active drug delivery to extracellular vesicles according to claim 1, characterized in that, The multi-stage centrifugation in S1 includes: removing cell debris by centrifuging the cell supernatant at 300 rpm for 10-15 min at 4°C; removing apoptotic bodies by centrifuging the cell supernatant at 2000 rpm for 20-30 min; filtering the cell supernatant through a 0.22 μm filter membrane; precipitating the cell supernatant by ultracentrifugation at 100000 rpm for 80-100 min; and resuspending the cell supernatant in a buffer solution and repeating the ultracentrifugation once.

3. The method for active drug delivery to extracellular vesicles according to claim 1, characterized in that, The target drug in S2 includes one of the following: a small molecule chemotherapy drug, a nucleic acid drug, or a protein drug.

4. The method for active drug delivery to extracellular vesicles according to claim 1, characterized in that, The final concentration of the extracellular vesicles after mixing with the drug solution in S3 is 0.6-0.8 mg / mL, and the total volume of the mixing system is 200-500 μL.

5. The method for active drug delivery to extracellular vesicles according to claim 1, characterized in that, The size exclusion chromatography in S5 uses a Sephacryl S-500HR column, with buffer as the mobile phase and a flow rate of 0.5 mL / min. It collects the characteristic peaks of extracellular vesicles with a retention time of 18-22 min. The sucrose density gradient centrifugation conditions are 100,000 centrifugation force for 110-130 min.