Method for significantly enhancing efficiency of drug loading into extracellular vesicles and medicinal preparation prepared by the method

Abstract

To provide a preparation method capable of significantly improving the efficiency of drug loading into extracellular vesicles.SOLUTION: A preparation method for significantly improving the efficiency of drug loading into extracellular vesicles, where extracellular vesicles having an ammonium sulfate concentration gradient are blended in a balanced crystalloid solution, thereby allowing a target drug prepared in the balanced crystalloid solution to be loaded into the extracellular vesicles.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to extracellular vesicle bioengineering technology, and in particular to a method for significantly improving the drug loading efficiency of extracellular vesicles. [Background technology]

[0002] Exosomes, or extracellular vesicles (EVs), are microvesicles with an average diameter of 30-200 nanometers (nm) and are composed of a lipid bilayer. They are formed by invagination of the cell membrane through a host cell, loading biomolecules into the intraluminal vesicles, which are then released from the host cell. The exosomes loaded with biomolecules then enter recipient cells and release the biomolecules into the recipient cells, thereby achieving the purpose of intercellular communication.

[0003] Due to their biocompatibility and special functions, exosomes have gradually become a mainstream tool for disease treatment or cell diagnosis in the biological and medical industries. Through bioengineering, targeted drugs can be loaded into the exosome cavity, allowing the exosomes to carry targeted drugs in the human body, deliver them to specific sites and / or specific cells, and then release the targeted drugs within the cavity.

[0004] However, the efficiency of loading exosomes with targeted drugs is extremely low, and exosomes often lose their activity or even burst after drug loading, preventing exosomes from achieving the effect of loading large amounts of drugs and making it difficult to increase the concentration of drugs loaded in exosomes. Furthermore, the choice of solution used during drug loading is also extremely important to achieve the goal of loading targeted drugs into exosomes. Traditionally, buffer solutions (e.g., PBS) tend to precipitate drugs, which affects the efficiency of drug encapsulation in exosomes. Therefore, the development of high-efficiency, large-volume drug-loading technologies for extracellular vesicles that do not affect activity is becoming increasingly important. Summary of the Invention [Problem to be solved by the invention]

[0005] In order to develop a technology for loading drugs into extracellular vesicles with high efficiency without affecting activity, the present invention provides a preparation method that can significantly improve the drug loading efficiency of extracellular vesicles. [Means for solving the problem]

[0006] The preparation method of the present invention, which can significantly improve the drug loading efficiency of extracellular vesicles, includes the following steps: Preparing an extracellular vesicle concentrate containing the extracellular vesicles; The extracellular vesicle concentrate is uniformly mixed with an ammonium sulfate solution of an appropriate concentration, and after at least a portion of the ammonium sulfate solution enters the cavities of the extracellular vesicles, at least a portion of the ammonium sulfate solution remaining outside the lipid bilayer of the extracellular vesicles is removed and replaced with an equilibrium crystallization solution to form an extracellular vesicle reaction solution, thereby generating an ammonium sulfate concentration gradient between the outside of the lipid bilayer of the extracellular vesicles and the inside of the cavities; A step of compounding a target drug into an equilibrium crystal solution to form a drug solution, mixing the drug solution with the extracellular vesicle reaction solution and then incubating the drug solution, allowing the target drug to enter the cavity through an ammonium sulfate concentration gradient, and then removing the drug solution that has not entered the cavity to complete loading of the target drug into the extracellular vesicles; Includes: [Effects of the Invention]

[0007] In addition, in the process of forming the ammonium sulfate concentration gradient and loading a drug, the present invention uses the equilibrium crystallization solution as the main additive solution (e.g., the extracellular vesicle reaction solution, the drug solution). Furthermore, the drug solution that does not enter the cavities of the extracellular vesicles is also removed by dialysis through the equilibrium crystallization solution. This eliminates the need for repeated exchange of different reaction solutions during the process from the establishment of the ammonium sulfate concentration gradient to the completion of loading of the target drug. This maintains the stability of the reaction environment of the extracellular vesicles during the reaction, improving the quality and efficiency of drug loading of the extracellular vesicles. The inventive step of the present invention is that the equilibrium crystallization solution can mix substances from two different media without causing precipitation or destroying the original properties of the substances. The present invention mixes a target drug and a biological product without damaging the integrity and activity of biological or lipid membranes, and the product produced by this method has biological activity, safety, and therapeutic efficacy in animal and cell experiments. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is an explanatory diagram of the drug loading process provided by the present invention. [Figure 2] 1 is a block diagram of the preparation method provided by the present invention. [Figure 3] FIG. 1 shows the first experimental results provided by the present invention. [Figure 4] FIG. 10 shows the second experimental result provided by the present invention. [Figure 5] FIG. 10 shows the third experimental result provided by the present invention. [Figure 6] FIG. 10 shows the fourth experimental result provided by the present invention. [Figure 7] FIG. 5 shows the fifth experimental result provided by the present invention. [Figure 8A] FIG. 6 shows the sixth experimental result provided by the present invention. [Figure 8B] FIG. 6 shows the sixth experimental result provided by the present invention. [Figure 9A]FIG. 7 shows the seventh experimental result provided by the present invention. [Figure 9B] FIG. 7 shows the seventh experimental result provided by the present invention. [Figure 9C] FIG. 7 shows the seventh experimental result provided by the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0009] Referring to Figures 1 and 2, the present invention provides a method for preparing drug-loaded extracellular vesicles, which are used to load (or fill) a target drug into a cavity 12 formed by extracellular vesicles surrounded by a lipid bilayer 11. Extracellular vesicles include, but are not limited to, exosomes 10, microvesicles (microparticles), apoptosis bodies, and oncosomes. In the present invention, the exosomes 10, which are small and fragile, are selected for the following explanation. The preparation method includes the following steps.

[0010] In step S1, an exosome concentrate 101 is prepared. The method and source of the exosome concentrate 101 are not limited, and any known technique for obtaining the exosomes 10 can be included within the scope of the present invention.

[0011] In this example, the exosomes 10 are collected from a human embryonic kidney cell line (HEK293T) by cell culture. The culture medium in which the human embryonic kidney cell line (HEK293T) is cultured is collected, concentrated, and replaced with phosphate buffered saline (PBS) to form a transition solution. The transition solution is then concentrated to form an exosome concentrate 101.

[0012] Here, the culture medium can be concentrated using a tangential flow filtration (TFF) system.

[0013] Here, after the transition solution was detected by nanoparticle tracking analysis (NTA), the particle concentration of the exosomes 10 was 10 9 ~10 13 / mL.

[0014] The volume of the culture medium is 1 liter (L), and the volume of the culture medium after concentration is concentrated 10 to 100 times. The volume of the transition solution is 3 to 4 milliliters (mL), and the transition solution is concentrated to approximately 1 mL of the exosome concentrate 101 using an Amicon 3K concentration centrifuge tube.

[0015] Furthermore, the exosomes 10 may be modified exosomes that have been modified by bioengineering. For example, the exosomes 10 may be modified to carry a target protein on their surface after modification. Modified exosomes carrying a target protein can be formed by fusing a commonly found transmembrane protein (tetraspanin), such as CD63, CD81, CD82, or CD9, in the lipid bilayer 11 of the exosomes 10 with the target protein using fusion protein technology, thereby allowing the exosomes 10 to carry the target protein and form the modified exosomes.

[0016] Step S2, ammonium sulfate concentration gradient: The exosome concentrate 101 and an ammonium sulfate solution 102 of an appropriate concentration are uniformly mixed to form an exosome mixed solution. After the ammonium sulfate solution 102 enters the cavities 12 of the exosomes 10, the ammonium sulfate solution 102 outside the lipid bilayer 11 of the exosomes 10 is removed and replaced with an equilibrium crystallization solution to form an exosome reaction solution 103. At this time, the exosome reaction solution 103 generates an ammonium sulfate concentration gradient outside the lipid bilayer 11 and within the cavities 12 of each exosome 10.

[0017] Here, the volume ratio of the exosome concentrate 101 to the ammonium sulfate solution 102 is 1:50 to 1:200.

[0018] Here, the appropriate concentration of the ammonium sulfate solution 102 is preferably 0.1 to 5M.

[0019] Here, the ammonium sulfate solution 102 is introduced into the exosomes 10 by ultrasonic vibration (sonication), addition of a surfactant (surfactant), or extrusion (extrusion).

[0020] Here, the ultrasonic vibration method (sonication) may be to treat the exosome mixture with ultrasonic waves at a power of 20 to 80 watts for 4 minutes.

[0021] After treatment with a surfactant, the membrane permeability of the exosomes 10 can be increased, allowing the ammonium sulfate solution 102 to enter the exosomes 10. The surfactant contains saponin or triton, and in this embodiment, the exosome mixture reacts with saponin at a concentration of 0.001 to 0.05%.

[0022] Here, the extrusion method involves extruding the exosome mixture through an airtight syringe by repeatedly extruding a polycarbonate membrane with a pore size of 0.1 to 0.3 μm into the membrane. This produces uniformly sized exosomes 10 by physical force, while simultaneously pumping the ammonium sulfate solution 102 into the cavities 12 of the exosomes 10 to form the ammonium sulfate concentration gradient. In another embodiment, an auto-extrusion method can be used in place of the airtight syringe, where a high-pressure liposome / exosome extruder applies stable pressure to the polycarbonate membrane. Preferably, the stable pressure is 50 to 500 psi.

[0023] Here, the ammonium sulfate solution 102 remaining outside the lipid bilayer 11 of the exosomes 10 is removed by tangential flow filtration (TFF) or size-exclusion chromatography, and replaced with the equilibrium crystallization solution to form the exosome reaction solution 103.

[0024] Here, the equilibrium crystalloid solution may be a lactated Ringer's buffer or a Plasma Lyte A (PLA) solution. In this example, the following description will be given assuming that the equilibrium crystalloid solution is lactated Ringer's buffer.

[0025] Furthermore, the exosome reaction solution 103 is filtered through a syringe filter with a pore size of 0.22 μm.

[0026] Furthermore, the particle concentration of the exosomes 10 in the exosome reaction solution 103 detected by an NTA nanoparticle analyzer is preferably 109 ~10 13 particles / mL.

[0027] Step S3, loading the targeted drug: The targeted drug is mixed with an equilibrium crystallization solution (lactated Ringer's buffer) to form a drug solution 104, which is then mixed with the exosome reaction solution 103 and incubated. The drug solution 104 is then introduced into the cavity 12 of the exosomes 10 via the ammonium sulfate concentration gradient. Any remaining drug solution 104 that has not entered the cavity 12 of the exosomes 10 is then removed, completing the loading of the targeted drug into the cavity 12 of the exosomes 10. During the loading process, the targeted drug is placed in the equilibrium crystallization solution, which prevents the precipitation of the targeted drug and ensures stable construction of the exosomes 10 in the ammonium sulfate concentration gradient.

[0028] The reaction amount or reaction concentration of the targeting drug varies depending on the selection of the targeting drug (such as the chemical structure, molecular weight, or pharmacokinetics of the targeting drug). In this embodiment, the effective concentration of the targeting drug in the drug solution 104 is 0.5 to 5 mg / mL, and the reaction amount of the targeting drug reacting with the exosome reaction solution 103 is 500 to 1000 μg.

[0029] Here, the number of the exosomes 10 in the exosome reaction solution 103 that reacts with the drug solution 104 is 10 11 There are individuals.

[0030] The reaction time for incubating the drug solution 104 and the exosome reaction solution 103 is 10 to 60 minutes.

[0031] Here, the drug solution 104 that has not entered the cavity 12 of the exosome 10 is removed by dialysis or size-exclusion chromatography.

[0032] In this example, dialysis is used to remove the drug solution 104 that has not entered the cavity 12 of the exosomes 10. The exosome reaction solution 103 after reaction with the drug solution 104 is placed in a dialysis cassette with a pore size, and the dialysis cassette is placed in a beaker containing the lactated Ringer's solution for dialysis. Based on the principle that the volume of the target drug after dissolving in the drug solution 104 is smaller than the pore size and the volume of the exosomes 10 is larger than the pore size, the drug solution 104 that has not entered the exosomes 10 can be precipitated through the dialysis cassette.

[0033] The dialysis time for the dialysis method is 16 to 24 hours.

[0034] The present invention further analyzes the loading effect (or filling effect) of the exosomes 10 after loading the targeting drug, compares the exosomes 10 loaded with the targeting drug with a standard curve of the targeting drug, and uses the standard curve to estimate the amount of the targeting drug loaded in the exosomes 10, thereby evaluating the efficiency and quality of the preparation method for exosomes 10 loaded with the drug provided by the present invention.

[0035] The targeting agent is not limited, but preferably contains an amine group. The amine group and sulfate group form crystals, allowing the targeting agent to differentially enter the cavity 12 of the exosome 10 through the ammonium sulfate concentration gradient. The crystallization reaction with ammonium sulfate also increases the efficiency of the targeting agent entering the exosome 10. In this example, the targeting agents are doxorubicin hydrochloride (Dox-HCl) and temozolomide (TMZ). A 2 mg / mL Dox-HCl standard curve was first prepared by serially diluting the Dox-HCl solution and readings at 480 nm. A 1 mg / mL TMZ standard curve was then prepared by serially diluting the TMZ solution and readings at 330 nm.

[0036] Furthermore, additives can be added simultaneously to the exosome mixture or the ammonium sulfate solution in step S2, and the additives are not limited. The purpose of adding the additive is to assist in the establishment of the ammonium sulfate concentration gradient and promote the drug loading effect in the subsequent step S3. For example, the additive may be a pH adjuster, catalyst, coenzyme, surfactant, or other chemical or drug. Here, the additive may be the saponin provided above.

[0037] See Table 1 and Figure 3. First, to verify the effectiveness of the lactated Ringer's solution as the main solution in the above-mentioned exosome-containing drug preparation method, the present invention prepares the exosomes 10 and the target drug in a preservation solution currently commonly used for preserving and reacting the exosomes 10, respectively, as a comparative example. Examples 1 to 3 differ from Comparative Examples 1 to 3 in that the particle concentrations of the exosomes 10 in the exosome reaction solution are different, but the reaction amounts of the exosomes 10 and the target drug are the same in each of the Examples and Comparative Examples.

[0038] Here, the preservative solution is prepared by mixing sucrose, polysorbate 80, and sodium acetate, each of which is a solution commonly used for preserving and stabilizing pharmaceutical and protein materials.

[0039] [Table 1]

[0040] In Figure 3, a paired sample t-test was performed for each of the Examples and Comparative Examples. It can be seen that Examples 1 to 3 have a better drug loading effect than Comparative Examples 1 to 3, with the drug loading efficiency being approximately four times higher than that of the Comparative Examples. Therefore, it can be seen that the exosome 10 can improve the loading efficiency (or loading efficiency) of the target drug in lactated Ringer's solution.

[0041] Referring to Table 2, we next determined whether the establishment of the ammonium sulfate concentration gradient affected the drug loading of the exosomes 10 and compared the drug loading effects after establishing the ammonium sulfate concentration gradient using incubation and ultrasonic vibration. The ammonium sulfate solution 102 in Experiment 1 and Experiment 1A was prepared from lactated Ringer's solution.

[0042] In Experiment 1, the exosome concentrate 101 and the ammonium sulfate solution 102 were incubated with the targeting drug after establishing an ammonium sulfate concentration gradient using ultrasonic vibration. In Experiment 1A, the exosome concentrate 101 and the ammonium sulfate solution 102 were uniformly mixed to form an exosome mixture, which was then incubated to passively load the exosomes 10 with the ammonium sulfate solution 102. A control group was also used, in which the exosome concentrate 101 and the PBS were uniformly mixed and then incubated. Compared to the control group, Experiment 1A demonstrated that the exosomes 10 could indeed load the targeting drug after establishing an ammonium sulfate concentration gradient. Comparing the results of Experiment 1 and the three control groups, establishing an ammonium sulfate concentration gradient using ultrasonic vibration significantly improved the loading effect of the targeting drug on the exosomes, optimizing the overall drug loading capacity.

[0043] [Table 2]

[0044] Next, referring to Table 3 and Figure 4, the optimal reaction concentration of the targeting drug doxorubicin hydrochloride (Dox-HCl) was determined, and the present invention performs a process in which the exosomes 10 are loaded with 500 μg and 1000 μg of doxorubicin hydrochloride (Dox-HCl) in reaction amounts, respectively, and the targeting drug in step S3. Here, in Experiments 2 to 6, the ammonium sulfate concentration gradient was established using ultrasonic vibration (sonication).

[0045] [Table 3]

[0046] Figure 4 compares Experiments 2, 3, and 5 with Experiments 4 and 6 using an unpaired t-test. It can be seen that the administration of 1000 μg of Dox-HCl in Experiments 4 and 6, which reacted with exosomes 10, resulted in a superior drug loading efficiency compared to the administration of 500 μg of Dox-HCl in Experiments 2, 3, and 5, and increased the drug loading amount by nearly three times.

[0047] Furthermore, Experiments 2, 3, and 5 were compared with Experiment 1 to confirm whether the ultrasonic vibration method and extrusion method after establishing the ammonium sulfate concentration gradient affected the loading efficiency of the targeted drug entering the exosomes 10. As shown in Figure 5, when the ammonium sulfate concentration gradient was established using ultrasonic vibration and 500 μg of Dox-HCl was administered and reacted with exosomes 10, a better drug loading efficiency was obtained than when the ammonium sulfate concentration gradient was established by incubation, and the drug loading amount reached approximately 70 times (see Figure 5).

[0048] Further, see Table 4 and Figures 6 and 7. To test whether the establishment of the ammonium sulfate concentration gradient by the extrusion method and the auto-extrusion method affects the efficiency of loading the exosomes 10 with the targeting drug, the present invention performs the exosomes 10 of Experimental Groups 7 to 12 by the extrusion method, after which the ammonium sulfate concentration gradient is established, and then performs the step of loading each experimental group with 500 μg and 1000 μg of doxorubicin hydrochloride (Dox-HCl) and the targeting drug in step S3. Experimental Groups 13 to 15 are exosomes 10 in which the ammonium sulfate concentration gradient was established by the auto-extrusion method, and then performs the step of loading each experimental group with 500 μg and 1000 μg of doxorubicin hydrochloride (Dox-HCl) and the targeting drug in step S3. Experimental groups 9 to 12 and 13 to 15 were then compared with the aforementioned Experiments 4 to 5. In these experiments, the pressure provided by the high-pressure liposome / exosome extruder in the automated extrusion method was 50 to 500 psi.

[0049] [Table 4]

[0050] In Figure 6, a one-way ANOVA was used to compare the results, and it was found that there was no significant difference in the effect of the ammonium sulfate concentration gradient generated by the ultrasonic vibration method, extrusion method, and auto-extrusion method on the loading effect of the target drug into the exosomes 10, but the ammonium sulfate concentration gradient generated by the extrusion method and auto-extrusion method slightly increased the overall drug loading into the exosomes 10.

[0051] Next, referring to Figure 7, TMZ was used as the targeting drug, and after the ammonium sulfate concentration gradient was established, the loading efficiency of the exosomes 10 was confirmed. In accordance with the results described above, after the ammonium sulfate concentration gradient was established, the exosomes 10 were able to load the targeting drug. By combining this with the automated extrusion method, the drug loading amount of the exosomes 10 could be increased by approximately three times (loading concentration).

[0052] In the present invention, the equilibrium crystal solution is used as the main additive solution (e.g., the exosome reaction solution 103 and the drug solution 104) in the process of forming the ammonium sulfate concentration gradient in step 2 and loading the drug in step 3. Furthermore, when removing the drug solution 104 that has not entered the cavity 12 of the exosomes 10, it is dialyzed through the equilibrium crystal solution. This eliminates the need to repeatedly exchange different reaction solutions for the exosomes 10 from the establishment of the ammonium sulfate concentration gradient to the completion of loading the target drug, thereby maintaining the stability of the reaction environment during the reaction of the exosomes 10 and improving the quality and efficiency of drug loading of the exosomes 10.

[0053] To further verify that the drug-loaded exosomes 10 are internalized by cells and have the ability to release the drug and kill the cells, see Figures 8A and 8B. In a cytotoxicity experiment, breast cancer cells (MDA-MB-231) were mixed with the exosomes 10 loaded with Dox-HCl as the targeting drug and cultured for 48 hours (Figure 8A). The exosomes 10 in this example were capable of targeting human leukocyte antigen G (HLA-G). The control group was a group directly administered with the targeting drug (Dox-HCl) to treat the breast cancer cells (Figure 8B).

[0054] As can be seen from the results, in the group in which the targeted drug was directly administered to treat breast cancer cells (Figure 8B), the drug concentration required for the targeted drug to kill 20% of the cancer cells (EC20) was 0.76 μg / mL. As shown in Figure 8A, the drug concentration required for the exosomes 10 loaded with the targeted drug to kill 20% of the cancer cells (EC20) was 0.062 μg / mL. The experimental results demonstrate that the exosomes 10 loaded with the targeted drug can effectively deliver the drug to target cells and reduce the effective drug concentration. According to the reference, Schaller TH, et al. (Schaller TH, Snyder DJ, Spasojevic I, et al. First in human dose calculation of a single-chain bispecific antibody targeting glioma using the MABEL approach. Journal for ImmunoTherapy of Cancer 2020;8:e000213. doi:10.1136 / jitc-2019-000213), the drug dose in the cytotoxicity experiment was officially converted to the drug dose when used in the human body. The minimum effective dose (MABEL) of the targeted drug for directly treating breast cancer cells was 32.56 μg / kg, but the minimum effective dose (MABEL) of the Dox-HCl-loaded exosome 10 was 2.66 μg / kg, which was enough to kill 20% of the cancer cells, a significant improvement of more than 10 times.

[0055] Furthermore, Figures 9A-9C show the cytotoxicity of the exosomes 10 loaded with the targeting drug Dox-HCl compared with that of liposome injections (Dox@Lipo) currently used in clinical settings. In the cellular uptake experiment of breast cancer cells shown in Figure 9A, the targeting drug Dox-HCl was loaded at loading concentrations of 0.25 μM, 1 μM, and 2 μM into the exosomes 10 (Dox@EOX) and liposome injections (Dox@Lipo), respectively, and then co-cultured with breast cancer cells for 3 hours. Flow cytometry was used to detect the percentage of the targeting drug signal internalized by the breast cancer cells. The results demonstrate that the exosomes 10 loaded with the targeting drug Dox-HCl (Dox@EOX) significantly outperformed the liposome injections (Dox@Lipo) in both the rate and percentage of delivery of the targeting drug into breast cancer cells.

[0056] Figure 9B shows the results of co-culture of breast cancer cells with the exosome 10 loaded with the targeting drug Dox-HCl (Dox@EOX) and the liposome injection loaded with the targeting drug Dox-HCl (Dox@Lipo) for 5 hours, calculating the cell viability of the breast cancer cells, and comparing the toxic effects on breast cancer cells after treatment with the exosome 10 loaded with the targeting drug Dox-HCl (Dox@EOX) and the liposome injection loaded with the targeting drug Dox-HCl (Dox@Lipo). After performing statistics using an unpaired sample t-test, it was shown that the exosome 10 loaded with the target drug Dox-HCl had superior cytotoxicity to the liposome injection (Dox@Lipo) loaded with the target drug Dox-HCl at loading concentrations of 4 μM, 2 μM, and 1 μM. It was shown that the exosome 10 loaded with the drug had a higher probability of killing cancer cells in a short period of time, with a cell killing rate increased by approximately 15% compared to the liposome injection (Dox@Lipo) loaded with the target drug Dox-HCl.

[0057] Referring to Figure 9C, the efficacy and safety of the exosomes 10 loaded with the targeted drug were confirmed using a breast cancer animal model. In this example, breast cancer cells (MDA-MB-231) were orthotopically injected into the mammary fat of mice, and tumors were cultured to establish the breast cancer animal model. Mice were divided into drug treatment groups 1 to 3 and control group 1. Drug treatment groups 1 to 3 were treated with the targeted drug Dox-HCl directly (Dox-HCl), with liposome injections loaded with the targeted drug Dox-HCl (Dox@Lipo), and with exosomes 10 loaded with the targeted drug Dox-HCl (Dox@EXO), respectively. Control group 1 was administered a placebo (PBS) (Ctrl).

[0058] The breast cancer animal models of drug-treated groups 1-3 and control group 1 were injected with the breast cancer cells (MDA-MB-231) via the tail vein for six consecutive weeks. Tumor progression was measured and imaged based on tumor luminescence signals using an in vivo real-time imaging system (IVIS Imaging System). Results showed that two weeks after the final administration (day 49), the tumor luminescence signal intensity in the Dox@EXO group was significantly lower than that of the other drug-treated and control groups. The tumor luminescence signal intensity in control group 1 (Ctl) was approximately 21.8-fold higher than that of the Dox@EXO group. The tumor inhibition rates, calculated in terms of tumor volume, for drug-treated groups 1-3 were 73.8% for drug-treated group 1 (Dox-HCl), 76.9% for drug-treated group 2 (Dox@Lipo), and 97.8% for drug-treated group 3 (Dox@EXO), respectively. It is clear that the exosomes 10 prepared by the method provided by the present invention can exert better drug effects after loading the target drug.

[0059] Furthermore, to demonstrate that the exosome 10 loaded with the target drug is biocompatible and safe for in vivo use, see Table 5. In animal model safety experiments, immunocompetent mice were divided into a control group, a carrier control group, a drug-treated group (Dox), and carrier-treated groups 1-3 (Dox@EXO 1-3). They were injected weekly with lactated Ringer's solution, the exosome 10, the target drug (doxorubicin, Dox-HCl), and the exosome 10 loaded with the target drug Dox-HCl at loading concentrations of 2 mg / kg, 6 mg / kg, and 12 mg / kg (Dox@EXO). After each injection, blood was collected from the mice, serum biochemical values ​​were analyzed, and organ function was monitored. One-way analysis of variance (One-Way ANOVA) showed significant differences in serum liver function-related values, such as albumin and alkaline phosphatase, between the group directly injected with the target drug and the control group administered lactated Ringer's solution. However, no significant differences were observed in serum biochemical values ​​between the exosome 10 (Dox@EXO1-3) loaded with the target drug Dox-HCl at concentrations of 2 mg / kg, 6 mg / kg, and 12 mg / kg and the control group. During the experiment, the physical activity of each mouse in the Dox@EXO1-3 group was normal (not shown), demonstrating that the exosome 10 provided by the present invention is safe for biological use.

[0060] [Table 5] Abbreviation: ALB=Albumin; ALP=Alkaline Phosphatase; AMY=Amylase; CK=Creatine kinase; LDH=Lactate Dehydrogenase *: p≦0.05

[0061] Comprehensive analysis of the above results demonstrates that the method for drug loading of exosomes 10 provided herein significantly improves the loading efficiency and loading concentration of one or more small molecule drugs (e.g., doxorubicin and TMZ) into exosomes 10, effectively delivering the drugs to target cells and reducing drug diffusion loss without causing toxic side effects. The above examples are intended to illustrate the technical concepts and features of the present application, and are intended to enable those skilled in the art to understand and practice the content of the present application. They do not limit the scope of protection of the present application. For example, the aforementioned doxorubicin can be replaced with other drugs. Any equivalent changes or modifications made in accordance with the spirit and content of the present application are also intended to be within the scope of protection of the present application. [Explanation of symbols]

[0062] 10 Exosomes 101 Exosome concentrate 102 Ammonium sulfate solution 103 Exosome reaction solution 104 Drug Solution 11 Lipid bilayer 12 Cavity S1 Step S2 Step S3 Step

Claims

1. A preparation method for significantly improving the drug loading efficiency of extracellular vesicles, which is used to load a target drug into a cavity formed by covering an extracellular vesicle with a lipid bilayer, Preparing an extracellular vesicle concentrate containing the extracellular vesicles; The extracellular vesicle concentrate is uniformly mixed with an ammonium sulfate solution of an appropriate concentration, and after at least a portion of the ammonium sulfate solution enters the cavities of the extracellular vesicles, at least a portion of the ammonium sulfate solution remaining outside the lipid bilayer of the extracellular vesicles is removed and replaced with an equilibrium crystallization solution to form an extracellular vesicle reaction solution, thereby generating an ammonium sulfate concentration gradient between the outside of the lipid bilayer of the extracellular vesicles and the inside of the cavities; A step of mixing a target drug into an equilibrium crystal solution to form a drug solution, mixing the drug solution with the extracellular vesicle reaction solution and then standing to react, allowing the target drug to enter the cavity through an ammonium sulfate concentration gradient, and then removing the drug solution that has not entered the cavity to complete loading of the target drug into the extracellular vesicles; A preparation method for significantly improving the drug loading efficiency of extracellular vesicles comprising:

2. 2. The method of claim 1, wherein the equilibrium crystallization solution is lactated Ringer's buffer or plasma Wright A solution.

3. 2. The method according to claim 1, wherein the concentration of the ammonium sulfate solution is 0.1 to 5M.

4. 4. The method of claim 3, wherein the ammonium sulfate solution contains an additive that assists in establishing the ammonium sulfate concentration gradient or promotes entry of the target drug into the cavity.

5. 2. The method according to claim 1, wherein the volume ratio of the extracellular vesicle concentrate to the ammonium sulfate solution is 1:50 to 1:

200.

6. The method according to claim 1, wherein the ammonium sulfate solution is forced into the extracellular vesicles by ultrasonic vibration, saponin reaction, or extrusion.

7. 7. The method of claim 6, wherein the extrusion method is a manual or automatic extrusion method.

8. 2. The method of claim 1, wherein the effective concentration of the target drug in the drug solution is 0.5 to 5 mg / mL.

9. The extracellular vesicles are exosomes, and the particle concentration of the exosomes in the extracellular vesicle reaction solution is 10 ９ ~10 １３ The method of claim 1, wherein the concentration of the granules is 10 ...

10. A preparation method for significantly improving the drug loading efficiency of extracellular vesicles, the method being used to load a targeting drug into a cavity formed by the lipid bilayer of the extracellular vesicles, the targeting drug comprising an amine group; Preparing an extracellular vesicle concentrate containing the extracellular vesicles; The extracellular vesicle concentrate is uniformly mixed with an ammonium sulfate solution of an appropriate concentration, and after at least a portion of the ammonium sulfate solution enters the cavities of the extracellular vesicles, at least a portion of the ammonium sulfate solution remaining outside the lipid bilayer of the extracellular vesicles is removed and added to an equilibrium crystallization solution to form an extracellular vesicle reaction solution, thereby generating an ammonium sulfate concentration gradient between the outside of the lipid bilayer of the extracellular vesicles and the inside of the cavities; A step of mixing a target drug into an equilibrium crystal solution to form a drug solution, mixing the drug solution with the extracellular vesicle reaction solution and then standing to react, allowing the target drug to enter the cavity through the ammonium sulfate concentration gradient, and then removing the drug solution that has not entered the cavity to complete loading of the target drug into the extracellular vesicles; A preparation method for significantly improving the drug loading efficiency of extracellular vesicles comprising:

11. A preparation method for significantly improving the drug loading efficiency of extracellular vesicles as described in claim 10, wherein the ammonium sulfate solution contains an additive that assists in the creation of an ammonium sulfate concentration gradient or promotes the entry of the target drug into the cavity.

12. The reaction amount of the target drug in the drug solution is 500 to 1000 μg, the extracellular vesicles are exosomes, and the particle concentration of the exosomes in the extracellular vesicle reaction solution is 10 ９ ~10 １３ A preparation method for significantly improving the drug loading efficiency of extracellular vesicles described in claim 10, wherein the drug loading efficiency is 1000particles / mL.

13. 11. The preparation method according to claim 10, wherein the equilibrium crystallization solution is lactated Ringer's buffer or plasma Wright A solution.

14. The preparation method according to claim 10, wherein the concentration of the ammonium sulfate solution is 0.1 to 5M.

15. The preparation method according to claim 10, wherein the volume ratio of the extracellular vesicle concentrate to the ammonium sulfate solution is 1:50 to 1:

200.

16. The preparation method according to claim 10, wherein the targeting drug is doxorubicin hydrochloride or temozolomide.

17. A pharmaceutical formulation prepared by the method of claim 1.

18. A pharmaceutical formulation prepared by the method of claim 10.

19. 19. The pharmaceutical preparation according to claim 18, which is used to kill cancer cells or inhibit tumor growth and contains a targeted agent that is doxorubicin hydrochloride or temozolomide.

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