A method for preparing drug-loaded exosomes and their application

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a method for preparing and applying drug-loaded exosomes. The drug loading method includes the following steps: loading a drug into a porous nanomaterial to obtain a drug-loaded porous nanomaterial; loading the drug-loaded porous nanomaterial into an exosome to obtain a drug-loaded porous nanomaterial coated with exosomes; and then degrading the porous nanomaterial to obtain drug-loaded exosomes. This invention combines the advantages of porous nanomaterials and exosomes, improving the drug loading rate of exosomes and increasing the amount of drug that exosomes can carry. Furthermore, by degrading the porous nanomaterials within the exosomes in vitro before use, the drug-loaded exosomes prepared by this method have the advantages of high drug loading rate and good biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing drug-loaded exosomes and their application. Background Technology

[0002] Exosomes are small vesicles enclosed in a lipid bilayer, ranging in size from approximately 30 to 200 nm. They are generated by the invagination of endosomes and possess the same topological structure as cells. Their components include nucleic acids, amino acids, proteins, lipids, and metabolites, which can reflect their cellular origin. In particular, exosomes derived from cancer cells can therefore serve as ideal biomarkers for early cancer screening and diagnosis. Furthermore, due to their unique lipid bilayer-enclosed structure, exosomes can also be used as biocamouflage materials, showing great potential in drug delivery with minimal impact on cytotoxicity and immunogenicity. This pathway of intercellular exosome transport plays an important role in many aspects of human health and disease, including development, immunity, tissue homeostasis, cancer, and neurodegenerative diseases. Based on these and other properties, exosomes are being developed as therapeutic agents in various disease models. Nevertheless, current exosome drug delivery methods are relatively limited, and the drug loading rate is generally low, necessitating the development of novel and efficient methods for preparing drug-loaded exosomes. Summary of the Invention

[0003] Therefore, the present invention aims to overcome the shortcomings of the existing technology in terms of single drug loading method and low drug loading rate, thereby providing a new method for preparing drug-loaded exosomes and its application.

[0004] Therefore, the present invention provides a method for drug delivery via exosomes, comprising the following steps:

[0005] Step S1: Loading drugs into porous nanomaterials and then into exosomes to obtain drug-loaded porous nanomaterials;

[0006] Step S2: Load the drug-loaded porous nanomaterial into the exosome to obtain drug-loaded porous nanomaterials coated with exosomes;

[0007] Step S3: Degrade porous nanomaterials to obtain drug-loaded exosomes.

[0008] Furthermore, the porous nanomaterial satisfies one or more of the following A:

[0009] A. The porous nanomaterials include inorganic porous nanomaterials, organic porous nanomaterials, and inorganic-organic porous hybrid nanomaterials;

[0010] B. The size of the porous nanomaterial is 20-120 nm;

[0011] C. The size of the internal pores of the porous nanomaterial is less than 20 nanometers;

[0012] D. The size of the exosomes is 80-150 nm;

[0013] E. The particle size of the porous nanomaterial is smaller than that of the exosomes.

[0014] Optionally, the porous nanomaterial is at least one of metal-organic framework materials, porous calcium carbonate materials, and disulfide-bonded polymer nanomaterials;

[0015] Optionally, the metal-organic framework material is ZIF-8 material; the disulfide-linked polymer nanomaterial is a disulfide-linked poly(ethylene glycol)-b-poly(ε-caprolactone) block polymer.

[0016] Furthermore, step S1 includes the following steps: immersing the porous nanomaterial in a drug solution for 20-120 minutes to load the porous nanomaterial onto its surface and interior.

[0017] Furthermore, the drug is a chemical drug or a biological drug.

[0018] Furthermore, the concentration of the drug in the drug solution is 1 μM-100 mM.

[0019] Furthermore, the step of loading the drug-loaded porous nanomaterial into exosomes to obtain exosome-coated porous nanomaterials includes the following steps: mixing the drug-loaded porous nanomaterial with an exosome solution, and introducing the drug-loaded porous nanomaterial into the exosome using a physical-mechanical method or a chemical reagent method; optionally, the physical-mechanical method is at least one of the following: freeze-thaw method, extrusion method, ultrasonic method, electroporation method, and extrusion method, and the chemical reagent used in the chemical reagent method is a saponin (e.g., steroidal saponin, triterpenoid saponin).

[0020] Furthermore, the method for degrading porous nanomaterials is a chemical degradation method; preferably, the chemical degradation method includes at least one of acid treatment, alkali treatment, or thiol compound treatment.

[0021] Furthermore, the acid treatment involves placing the drug-loaded porous nanomaterials coated with exosomes in an acidic buffer solution with a pH of 3.0-5.0 to degrade the porous nanomaterials; the alkaline treatment involves placing the drug-loaded porous nanomaterials coated with exosomes in an alkaline buffer solution with a pH of 8.0-11.0 to degrade the porous nanomaterials; the thiol compound treatment involves placing the drug-loaded porous nanomaterials coated with exosomes in a 10μM-50mM water-soluble thiol compound solution to degrade the porous nanomaterials; preferably, the acidic buffer solution is an acetate buffer solution, the alkaline buffer solution is a disodium hydrogen phosphate buffer solution, and the thiol compound solution is a glutathione solution.

[0022] Furthermore, in step S3, the degradation of porous nanomaterials is carried out in vitro.

[0023] Furthermore, after obtaining the drug-loaded exosomes in step S3, the procedure also includes a step of further functionalizing or engineering the drug-loaded exosomes; the functionalization includes targeted functionalization.

[0024] Furthermore, the functionalization involves fusing drug-loaded exosomes with antibodies, aptamers, or targeting peptides to obtain drug-loaded exosomes that can target specific lesion sites; the engineering modification involves transfecting donor cells with a plasmid encoding fusion, causing the donor cells to secrete on the surface of the exosomes, and then using the engineered exosomes for subsequent drug loading to obtain engineered drug-loaded exosomes.

[0025] The present invention also provides a single-ingredient or compound preparation, wherein the single-ingredient or compound preparation includes the above-mentioned drug-loaded exosomes and pharmaceutically acceptable excipients; optionally, the dosage form of the single-ingredient or compound preparation includes powder, pills, granules, tablets, capsules, suspensions, gels, suppositories, creams, emulsions, sprays, injections, and injections.

[0026] The technical solution of this invention has the following advantages:

[0027] This invention provides a method for drug delivery via exosomes, comprising the following steps: loading a drug into a porous nanomaterial to obtain a drug-loaded porous nanomaterial; loading the drug-loaded porous nanomaterial into an exosome to obtain a drug-loaded porous nanomaterial coated with exosomes; and degrading the porous nanomaterial to obtain drug-loaded exosomes. This invention combines the advantages of porous nanomaterials and exosomes, improving the drug loading rate of exosomes and increasing the amount of drug that exosomes can carry. Furthermore, by degrading the porous nanomaterial within the exosomes in vitro before use, the drug-loaded exosomes prepared by this method possess the advantages of high drug loading rate and good biocompatibility. Attached Figure Description

[0028] Figure 1 The morphology of ZIF-8 / PTX, EXO / ZIF-8 / PTX and EXO / Zn / PTX under transmission electron microscopy;

[0029] Figure 2The fluorescence quantitative statistics of MCF-7 cells on different materials are shown in (a) and (b); where (Ⅰ), (Ⅱ), (Ⅲ), (Ⅳ), (Ⅴ) and (Ⅵ) represent the blank control group, free PTX group, EXO / PTX group, ZIF-8 / PTX group, EXO / ZIF-8 / PTX group and EXO / Zn / PTX group, respectively; the vertical axis in figure a is the fluorescence intensity value, and the vertical axis in figure b is the uptake rate of tumor cells on different materials; in the figures, ** indicates P value <0.01, and * indicates P value <0.05;

[0030] Figure 3 The effects of different treatment groups on the survival rate (a) and cell cycle of MCF-7 cells (b) are shown in Figure a. In Figure a, numbers 1-8 represent the blank control group, EXO group, ZIF-8 group, free PTX group, EXO / PTX group, ZIF-8 / PTX group, EXO / ZIF-8 / PTX group, and EXO / Zn / PTX group, respectively. The vertical axis of Figure a represents tumor cell survival rate, and Figure b shows the percentage of tumor cells in each cell cycle relative to the total number of tumor cells. ** indicates a P-value < 0.01, and * indicates a P-value < 0.05.

[0031] Figure 4 The graph shows the quantitative statistical analysis of fluorescence imaging at the tumor sites in tumor-bearing mice in the free PTX group, ZIF-8 / PTX group, and EXO / Zn / PTX group. The vertical axis represents the fluorescence intensity value at the tumor site in the tumor-bearing mice, and the horizontal axis represents the fluorescence imaging time at the tumor site in the mice after injection of different materials. ** indicates P value < 0.01.

[0032] Figure 5 The therapeutic effects of different treatment groups on tumor-bearing mice are shown in (a) and the tumor images of each group are taken in vitro (b); (Ⅰ), (Ⅱ), (Ⅲ), (Ⅳ), (Ⅴ) and (Ⅵ) represent the blank control group, free PTX group, EXO / PTX group, ZIF-8 / PTX group, EXO / ZIF-8 / PTX group and EXO / Zn / PTX group, respectively. Detailed Implementation

[0033] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0034] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0035] Example 1

[0036] This embodiment provides a method for drug delivery via exosomes, including the following steps:

[0037] (1) Exosome isolation: MCF-7 (breast cancer cells) cells were cultured at T175 cm⁻¹. 2 In cell culture flasks, the culture medium was DMEM high-glucose medium (containing 10% exosome-free FBS and 1% streptomycin-penicillin). After the cells reached confluence, the cell supernatant was collected. The cell supernatant was centrifuged sequentially at 200 and 2000 Rcf for 10 min to remove cells and cell debris. Cells were isolated from the supernatant using a commercial exosome extraction kit (Exosome Extraction Kit, EXORG24B, Liaoning Runji Biotechnology Co., Ltd.) based on the PEG precipitation method, yielding 5 mL of exosomes (5 × 10⁶ cells / mL). 11 The exosomes (number per mL) are denoted as Exo, and their particle size is 100 nm.

[0038] (2) Drug loading of porous nanomaterials: 0.1 mL of metal-organic framework material (particle size of 50 nm) ZIF-8 (0.4 mg / mL, solvent of ethanol) and 1 mL of paclitaxel (PTX, 20 μm / mL, solvent of ethanol) were incubated together at 20 °C for 60 min. The precipitate was then collected by centrifugation at 10000 rpm for 10 min to obtain ZIF-8 loaded with PTX (i.e. drug-loaded porous nanomaterial), denoted as ZIF-8 / PTX.

[0039] (3) Drug loading onto exosomes: 0.1 mg of PTX / ZIF-8 was loaded with 0.2 mL of exosome solution (5 × 10⁻⁶). 9 The mixture was prepared by mixing exosomes (each per mL) and then coating the ZIF-8 surface with exosomes using a liposome extruder.

[0040] (4) Preparation of drug-loaded exosomes: Add 5 mL of pH 5.0 acetate buffer, acidify for 30 min, and replace the buffer with an ultrafiltration tube to obtain drug-loaded exosomes, denoted as Exo / Zn / PTX.

[0041] Example 2

[0042] This embodiment provides a method for drug delivery via exosomes, including the following steps:

[0043] (1) Exosome isolation: 5 mL of peripheral blood cells were collected and centrifuged sequentially at 150 and 1500 Rcf for 10 min to remove cells and cell debris. 2 mL of exosomes (5 × 10⁻⁶ cells / mL) were obtained by separating cells using a commercial exosome extraction kit based on PEG precipitation (Exosome Extraction Kit, EXORG24B, Liaoning Runji Biotechnology Co., Ltd.). 12 The exosomes (number per mL) were denoted as Exo-2, and the particle size of the exosomes was 100 nm.

[0044] (2) Drug loading of porous nanomaterials: 0.1 mL of mesoporous calcium carbonate material (CaCO3, particle size 40 nm) (0.4 mg / mL, solvent is deionized water) was incubated with 1 mL of oxaliplatin (Pt, 20 μM, solvent is deionized water) at 20 °C for 60 min. The precipitate was then collected by centrifugation at 10000 rpm for 10 min to obtain mesoporous calcium carbonate material loaded with oxaliplatin (i.e. drug-loaded porous nanomaterial), denoted as Pt / CaCO3.

[0045] (3) Drug loading onto exosomes: 0.1 mg of Pt / CaCO3 was loaded with 0.2 mL of exosome solution (5 × 10⁻⁶). 9 The mixture was prepared by mixing (exosomes / mL) and then coating the CaCO3 surface with exosomes using a liposome extruder.

[0046] (4) Preparation of drug-loaded exosomes: Add 5 mL of pH 5.0 acetate buffer, acidify for 30 min, and replace the buffer with an ultrafiltration tube to obtain drug-loaded exosomes, denoted as EXO / Ca / Pt.

[0047] Example 3

[0048] This embodiment provides a method for drug delivery via exosomes, including the following steps:

[0049] (1) Exosome isolation: Mesenchymal stem cells were cultured at T175 cm⁻¹. 2 In cell culture flasks, the culture medium was DMEM high-glucose medium (containing 10% exosome-free FBS and 1% streptomycin-penicillin). After the cells reached confluence, the cell supernatant was collected. The cell supernatant was centrifuged sequentially at 200 and 2000 Rcf for 10 min to remove cells and cell debris. Cells were isolated from the supernatant using a commercial exosome extraction kit (Exosome Extraction Kit, EXORG24B, Liaoning Runji Biotechnology Co., Ltd.) based on the PEG precipitation method, yielding 5 mL of exosomes (5 × 10⁶ cells / mL). 11 The exosomes (number per mL) are denoted as Exo, and their particle size is 100 nm.

[0050] (2) Drug loading of porous polymer nanomaterials: 0.1 mL of disulfide-linked poly(ethyleneglycol)-b-poly(epsilon-caprolactone) block polymer (PEG-SS-PCL, particle size 60 nm) polymer nanomaterials (0.4 mg / mL, deionized water) were co-incubated with 1 mL of oxaliplatin (Pt, 20 μM, deionized water) at 20 °C for 80 min. The precipitate was then collected by centrifugation at 10000 rpm for 10 min to obtain PEG-SS-PCL loaded with oxaliplatin (i.e. drug-loaded porous polymer nanomaterials), denoted as PEG-SS-PCL / Pt.

[0051] (3) Drug loading of exosomes: 0.1 mg of PEG-SS-PCL / Pt was loaded with 0.2 mL of exosome solution (5 × 10⁻⁶). 9 The mixture was prepared by mixing exosomes (each per mL) and then coating the surface of the PEG-SS-PCL with exosomes using a liposome extruder.

[0052] (4) Preparation of drug-loaded exosomes: Add 5 mL of 10 mM GSH solution, treat for 30 min, and replace the buffer with an ultrafiltration tube to obtain drug-loaded exosomes, denoted as EXO / PEG-SS-PCL / Pt.

[0053] Comparative Example 1

[0054] This comparative example provides a method for drug delivery via exosomes, the preparation method of which is basically the same as that in Example 1, except that the step of "degrading ZIF-8 by acid treatment and replacing the buffer solution with an ultrafiltration tube" in step (3) is omitted. Instead, 0.1 mg PTX / ZIF-8 is delivered to 0.2 mL of exosome solution (5 × 10⁻⁶ mL). 9 After mixing (exosomes / mL), the exosomes are coated onto the surface of ZIF-8 using a liposome extruder to obtain drug-loaded exosomes, denoted as EXO / ZIF-8 / PTX.

[0055] Comparative Example 2

[0056] This comparative example provides a method for drug delivery via exosomes, the preparation method of which is basically the same as that in Example 1, except that this comparative example does not include step (2) in Example 1, but instead directly delivers 1 ml of PTX to 0.2 mL of exosome solution (5 × 10⁻⁶). 9 The exosomes were mixed (each per mL) and coated onto the surface of ZIF-8 using a liposome extruder. Then, the exosomes were acid-treated according to step (4) in Example 1 to obtain drug-loaded exosomes, denoted as EXO / PTX.

[0057] Experimental Example 1

[0058] The morphology of the drug-loaded porous nanomaterial ZIF-8 / PTX in Example 1, the drug-loaded exosome EXO / Zn / PTX, and the drug-loaded exosome in Comparative Example 1 were observed using transmission electron microscopy. The results are as follows: Figure 1 As shown in Figures a, b, and c.

[0059] The drug loading rates (n=3) of the drug-loaded porous nanomaterial ZIF-8 / PTX, drug-loaded exosome EXO / Zn / PTX in Example 1, and exosomes in Comparative Examples 1 and 2 were tested according to the following method:

[0060] The content of PTX in the solution before and after drug loading was determined by high performance liquid chromatography (HPLC). The drug solution and drug-loaded exosomes were separated by ultrafiltration, and the remaining drug solution after drug loading on the exosomes was collected. A Thermo Hypersil GOLDC18 column (100 mm × 2.1 mm, 1.9 μm) was used with water (A) and acetonitrile (B) as the mobile phase, using gradient elution (0–5 min, B 40%; 5–10 min, B 40%–80%; 10–11 min, B 80%–40%; 11–15 min, B 40%); detection wavelength 227 nm; column temperature 40 °C; flow rate 0.6 mL / min; detection wavelength 227 nm.

[0061] Preparation of standard solution: Accurately measure 0.1 ml (200 μm / ml) of the test drug solution and place it in a 1.25 ml volumetric flask. Dilute to the mark with ethanol to prepare a control solution with a concentration of 16 μm / ml. The formula for calculating the drug loading rate is as follows:

[0062]

[0063] The amount of drug added is the amount of drug used in step (2) of Example 1 before drug loading, and is calculated by multiplying the initial drug concentration by the drug volume. The amount of drug remaining is the amount of free drug remaining after drug loading is completed in step (2) of Example 1, and is calculated by multiplying the drug concentration by the drug volume after loading.

[0064] The experimental results are shown in Table 1. When using exosomes alone to load PTX, the drug loading rate was only 13.73±1.51%, significantly (P<0.05) lower than that of the ZIF-8 / PTX group (39.02±3.78%), indicating that using ZIF-8 can significantly improve the PTX loading capacity. There was no significant difference in drug loading rates between the ZIF-8 / PTX and EXO / ZIF-8 / PTX groups, indicating that subsequent exosome coating operations do not affect the drug loading rate. Finally, there was no significant difference in drug loading rates between the EXO / ZIF-8 / PTX and EXO / Zn / PTX groups, indicating that ZIF-8 degradation does not affect the drug loading rate and can avoid the potential adverse effects of nanomaterials on organisms. These results indicate that this exosome-delivered drug carrier can improve drug loading efficiency.

[0065] Table 1. Drug loading rates of EXO / Zn / PTX exosomes in Comparative Examples 1 and 2.

[0066] Drug loading rate ZIF-8 / PTX 37.02±3.78％ EXO / Zn / PTX 36.37±3.53％ EXO / ZIF-8 / PTX 36.57±2.78％ EXO / PTX 13.73±1.51％

[0067] Experiment Example 2

[0068] The expression levels of drug-loaded porous nanomaterial ZIF-8 / PTX, drug-loaded exosome EXO / Zn / PTX in Example 1, drug-loaded exosomes in Comparative Examples 1 and 2, and free PTX in tumor cells in the PBS group were detected. In order to study the uptake capacity of MCF-7 cells by the modified drug loading system, FITC-labeled PTX was used instead of PTX in the experiment, while the other experimental conditions remained unchanged.

[0069] The specific experimental method is as follows:

[0070] PBS was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0071] Free PTX solution: Dissolve PTX in ethanol to prepare a PTX solution with a concentration of 20 μg / mL;

[0072] EXO / PTX solution: Disperse EXO / PTX in PBS to prepare a 1 μg / mL EXO / PTX solution;

[0073] ZIF-8 / PTX solution: Disperse ZIF-8 / PTX in PBS to prepare a 1 μg / mL ZIF-8 / PTX solution;

[0074] EXO / ZIF-8 / PTX solution: Disperse EXO / ZIF-8 / PTX in PBS to prepare a 1 μg / mL EXO / ZIF-8 / PTX solution;

[0075] EXO / Zn / PTX solution: Disperse EXO / Zn / PTX in PBS to prepare a 1 μg / mL EXO / Zn / PTX solution;

[0076] MCF-7 cells were seeded in confocal culture dishes (30,000 cells per dish). After 24 hours of growth, 0.1 ml of free PTX, EXO / PTX, ZIF-8 / PTX, EXO / ZIF-8 / PTX, and EXO / Zn / PTX (1 μg / mL) were added respectively, and the cells were incubated for 4 hours, followed by washing with PBS. Then, DAPI staining solution (purchased from Beyotime Biotechnology Co., Ltd.) was added to stain the cell nuclei for 10 minutes. Next, the cells were imaged using a confocal laser scanning microscope (CLSM, Leica). The obtained fluorescence images were quantitatively analyzed using ImageJ software.

[0077] To further quantify the uptake capacity of MCF-7 cells for this exosome-loaded drug system, MCF-7 cells were seeded in 6-well plates (30,000 cells per well). After 24 hours of growth, free PTX, EXO / PTX, ZIF-8 / PTX, EXO / ZIF-8 / PTX, and EXO / Zn / PTX were added and incubated for 2 and 6 hours, respectively. Finally, the cells were collected and washed with PBS. The fluorescence intensity of the cell suspensions in each group was detected by flow cytometry (Agilent, BD, USA). The uptake rates of tumor cells for different materials were calculated to be 0%, 9.41±0.55%, 29.49±0.92%, 45.25±1.48%, 59.01±1.25%, and 74.35±0.99%, respectively. The results are shown in the table below. Figure 2 As shown in Figures a and b, Figure a represents the fluorescence intensity of different nanomaterials in MCF-7 cells, and Figure b represents the uptake rate of different nanomaterials by MCF-7 cells. The results in Figures a and b show that MCF-7 cells have difficulty uptakeing free PTX, but loading with ZIF-8 (ZIF-8 / PTX) can improve their uptake capacity. Comparing ZIF-8 / PTX and EXO / ZIF-8 / PTX, under the same PTX content, the fluorescence intensity of the EXO / ZIF-8 / PTX group was significantly higher (P<0.05) than that of the ZIF-8 / PTX group, indicating that exosomes can improve the uptake of nanomaterials by cells. Finally, comparing the EXO / ZIF-8 / PTX and EXO / Zn / PTX groups, the fluorescence intensity of EXO / Zn / PTX was significantly higher (P<0.05) than that of the EXO / ZIF-8 / PTX group, indicating that degrading ZIF-8 can further improve the cells' uptake capacity of this exosome-delivered drug carrier.

[0078] Flow cytometry analysis revealed that MCF-7 cells exhibited the highest drug uptake rate of EXO / Zn / PTX at 6 hours, which was significantly higher (P<0.05) than the ZIF-8 / PTX and EXO / ZIF-8 / PTX groups. This indicates that this exosome-delivered drug carrier can enhance the uptake rate by tumor cells.

[0079] Experimental Example 3

[0080] The toxicity of exosomes (EXO), porous nanomaterials (ZIF-8), drug-loaded porous nanomaterials (ZIF-8 / PTX), drug-loaded exosomes (EXO / Zn / PTX), drug-loaded exosomes (EXO / Zn / PTX), drug-loaded exosomes (Comparative Examples 1-2), Free PTX, and the blank control group to tumor cells, their effects on tumor cell cycle, and their killing effects on tumor cells were tested using the following methods:

[0081] EXO solution: Disperse EXO in PBS to prepare a 1 μg / mL EXO / Zn / PTX solution;

[0082] ZIF-8 solution: Disperse ZIF-8 in PBS to prepare a 1 μg / mL ZIF-8 solution;

[0083] The preparation methods for ZIF-8 / PTX, EXO / Zn / PTX, and the drug-loaded exosomes and free PTX solutions in Comparative Examples 1-2 are the same as those in Experimental Example 2.

[0084] MCF-7 cells were seeded in 96-well plates (5000 cells per well). After 24 hours, 0.1 ml of EXO, ZIF-8, ZIF-8 / PTX, EXO / Zn / PTX, and drug-loaded exosomes and free PTX from Comparative Examples 1-2 were added and incubated for 24 hours. Then, 10 μL of CCK-8 solution (purchased from Beyotime Biotechnology Co., Ltd.) was added and incubated for another 2 hours. Finally, the OD value of each well at 450 nm was measured using a microplate reader (Infinite M200 PRO, TECAN, Austria).

[0085] MCF-7 cells were seeded in 12-well plates (20,000 cells per well). After 24 hours, different materials (1 μg / mL) were added and incubated for another 24 hours. After incubation, the supernatant was collected, and the cells were washed twice with PBS. Cells were then collected after trypsin digestion and centrifuged at 1000 rpm for 5 minutes. Staining solution was added to each group of cell samples according to the kit instructions (Cell Cycle and Apoptosis Detection Kit, purchased from Beyotime Biotechnology Co., Ltd., Shanghai). Subsequently, cell cycle analysis was performed on each group of cell samples using flow cytometry (Agilent, BD, USA).

[0086] The tumor cell viability rates of the blank control group, EXO group, ZIF-8 group, free PTX group, EXO / PTX group, ZIF-8 / PTX group, EXO / ZIF-8 / PTX group, and EXO / Zn / PTX group were measured to be 100.0±3.04%, 100.1±3.14%, 95.1±2.26%, 86.89±3.71%, 63.04±3.98%, 40.83±3.26%, 33.71±1.48%, and 18.49±2.61%, respectively. The experimental results are as follows: Figure 3 As shown in figure a, it can be seen from the figure that the cancer cells treated with EXO / Zn / PTX had the lowest survival rate, meaning that EXO / Zn / PTX exhibited the strongest cytotoxicity against cancer cells; Figure 3 As shown in b, most cells in the blank control group and the free PTX group were in the G0 / G1 phase of the cell cycle. On the other hand, cells treated with EXO / PTX, ZIF-8 / PTX, EXO / ZIF-8 / PT, and EXO / Zn / PTX were mostly in the G2 / M phase. Compared with ZIF-8 / PTX and EXO / ZIF-8 / PTX treatment, the EXO / Zn / PTX group showed an increase in the percentage of cells in the G2 / M phase and a decrease in the percentage of cells in the G0 / G1 phase. These results demonstrate that EXO / Zn / PTX can effectively block the G2 / M phase of the cell cycle, thereby inhibiting the proliferation of tumor cells. Therefore, this exosome drug delivery system can enhance the killing effect of PTX on tumor cells.

[0087] Experiment Example 4

[0088] The drug-loaded porous nanomaterial ZIF-8 / PTX, drug-loaded exosomes EXO / Zn / PTX, and free PTX in Example 1 were tested for their targeted drug delivery capabilities to subcutaneous tumors. The experimental results are as follows: Figure 4 As shown, to investigate the uptake capacity of MCF-7 cells by the modified drug delivery system, FITC-labeled PTX was used instead of PTX in the experiment, while all other experimental conditions remained unchanged. The specific experimental method was as follows:

[0089] (1) Preparation of the test drug solution

[0090] PBS, Free-PTX, EXO / PTX, ZIF-8 / PTX, EXO / ZIF-8 / PTX, and EXO / Zn / PTX were the same as in Experiment 2.

[0091] (2) Processing method

[0092] MCF-7 tumor cells were subcutaneously injected into female Balb / c nude mice (4 weeks old) (3 × 10⁻⁶ cells per mouse). 6A mouse model of tumor bearing was established using 100 cells. Approximately 7 days after inoculation, the tumor grew to 100 mm. 3 After screening, further experiments were conducted. Mice were randomly divided into 6 groups (PBS, Free-PTX, EXO / PTX, ZIF-8 / PTX, EXO / ZIF-8 / PTX, and EXO / Zn / PTX), with 5 mice in each group. The drugs were administered via tail vein injection every three days, at a dose of 0.1 ml (1 μg / ml). In vivo fluorescence imaging was performed on day 10.

[0093] Twelve hours after injection of free PTX, ZIF-8 / PTX, and EXO / Zn / PTX, the fluorescence intensity at the tumor sites in tumor-bearing mice reached its maximum in all groups. The fluorescence intensity at the tumor sites in the free PTX group, ZIF-8 / PTX group, and EXO / Zn / PTX group was measured in tumor-bearing mice. The experimental results are as follows: Figure 4 As shown, free PTX cannot actively accumulate at the tumor site. However, PTX can be delivered to the tumor site after being loaded with ZIF-8. Furthermore, after exosome encapsulation and acid treatment to degrade ZIF-8, the fluorescence intensity of the EXO / Zn / PTX group was significantly higher than that of the ZIF-8 group (P<0.05), indicating that EXO / Zn / PTX can target and deliver the drug into tumor cells, improving the drug's biodistribution at the tumor site. Therefore, this exosome drug delivery method can target subcutaneous tumor-bearing cells in vivo and improve the drug's biodistribution at the tumor site.

[0094] Experimental Example 5

[0095] The effects of the drug-loaded porous nanomaterial ZIF-8 / PTX, the drug-loaded exosome EXO / Zn / PTX in Example 1, and the drug-loaded exosomes in Comparative Examples 1 and 2 on tumor growth in tumor-bearing mice were determined. The experimental results are as follows: Figure 5 As shown, the specific experimental method is as follows:

[0096] MCF-7 tumor cells were subcutaneously injected into female Balb / c nude mice (4 weeks old) (3 × 10⁻⁶ cells per mouse). 6 A mouse model of tumor bearing was established using 100 cells. Seven days after inoculation, the tumor grew to 100 mm. 3 Following screening, further experiments were conducted. Mice were randomly divided into six groups (PBS, Free-PTX, EXO / PTX, ZIF-8 / PTX, EXO / ZIF-8 / PT, and EXO / Zn / PTX), with five mice in each group. Various nanomaterials were injected via tail vein every three days, and the drug was injected via tail vein every three days, with each injection being 0.1 ml (1 μg / ml). Two weeks later, all mice were sacrificed, and tumor tissue was dissected and photographed.

[0097] Seven days after inoculation, the mean tumor volume in mice in the PBS, Free-PTX, EXO / PTX, ZIF-8 / PTX, EXO / ZIF-8 / PT, and EXO / Zn / PTX groups was 1527.57 ± 91.51 mm. 3 1173.01±65.94mm 3 815.98±74.89mm 3 517.09±37.61mm 3 351.72±24.31mm 3 and 66.35±10.14mm 3 The experimental results are as follows Figure 5 As shown. From Figure 5 Figures a and b, along with the experimental results above, show that the tumor volume in mice delivered via EXO / Zn / PTX was significantly smaller than in other groups (P<0.05), and superior to the ZIF-8 / PTX and EXO / ZIF-8 / PTX groups. This indicates that this exosome drug delivery carrier can enhance the in vivo antitumor effect in subcutaneous tumor-bearing mouse models.

[0098] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method of preparing drug-loaded exosomes, characterized by, The method comprises the following steps: S1 step: loading drugs into porous nanomaterials to obtain drug-loaded porous nanomaterials; S2 step: loading the drug-loaded porous nanomaterials into exosomes to obtain exosome-coated drug-loaded porous nanomaterials; S3 step: degrading the porous nanomaterials to obtain drug-loaded exosomes; The porous nanomaterials are at least one of metal organic framework materials, porous calcium carbonate materials, and disulfide-linked polymer nanomaterials. In the S3 step, the step of degrading the porous nanomaterials is performed in vitro.

2. The method of claim 1, wherein the drug-loaded exosome is prepared by the method comprising the steps of: The particle size of the porous nanomaterials is smaller than the particle size of the exosomes.

3. The method of claim 2, wherein the drug-loaded exosome is prepared by the method comprising the steps of: The porous nanomaterials or exosomes meet one or more of the following A-C: A. The particle size of the porous nanomaterials is 20-120 nm; B. The particle size of the internal pores of the porous nanomaterials is less than 20 nm; C. The particle size of the exosomes is 80-150 nm.

4. The method of claim 1 or 2, wherein the drug-loaded exosome is prepared by the method comprising the steps of: In the S1 step, the following steps are included: co-incubating the porous nanomaterials with a drug solution for 20-120 min to load the drugs onto the surface and inside of the porous nanomaterials.

5. The method of claim 4, wherein the drug-loaded exosome is prepared by the method comprising the steps of: The concentration of the drugs in the drug solution is 1 μM-100 mM.

6. The method of claim 1 or 2, wherein the drug-loaded exosome is prepared by the method comprising the steps of: In the S2 step, the following steps are included: mixing the drug-loaded porous nanomaterials with an exosome solution, and using a physical and mechanical method or a chemical reagent method to introduce the drug-loaded porous nanomaterials into the inside of the exosomes.

7. The method of claim 6, wherein the drug-loaded exosome is prepared by the method comprising the steps of: The physical and mechanical method is at least one of freeze-thaw method, extrusion method, ultrasonic method, and electroporation method, and the chemical reagent used in the chemical reagent method is saponin.

8. The method for preparing drug-loaded exosomes according to claim 1, characterized in that, The method of degrading the porous nanomaterials is a chemical degradation method.

9. The method for preparing drug-loaded exosomes according to claim 8, characterized in that, The chemical degradation method includes at least one of acid treatment, alkali treatment, and mercapto compound treatment.

10. The method for preparing drug-loaded exosomes according to claim 9, characterized in that, The acid treatment is degrading the porous nanomaterials by placing the exosome-coated drug-loaded porous nanomaterials in an acidic buffer with a pH value of 3.0-5.0; the alkali treatment is degrading the porous nanomaterials by placing the exosome-coated drug-loaded porous nanomaterials in an alkaline buffer with a pH value of 8.0-11.0; and the mercapto compound treatment is degrading the porous nanomaterials by placing the exosome-coated drug-loaded porous nanomaterials in a 10 μM-50 mM water-soluble mercapto compound solution.

11. The method of claim 10, wherein the drug-loaded exosome is prepared by the method comprising the steps of: The acidic buffer is an acetic acid buffer, the alkaline buffer is a disodium hydrogen phosphate buffer, and the mercapto compound solution is a glutathione solution.

12. The method for preparing drug-loaded exosomes according to claim 1 or 2, characterized in that, After obtaining the drug-loaded exosomes in the S3 step, a further functionalization or engineering modification step is included.

13. The method of claim 12, wherein the drug-loaded exosome is prepared by the method comprising the steps of: The functionalization includes targeted functionalization.

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