A method for stable preservation of exosomes at room temperature

Abstract

This invention relates to the field of biomedical technology, specifically to a method for stable preservation of exosomes at room temperature. The method includes: extracting exosomes; mixing the exosomes with cholesterol-modified single-stranded DNA, followed by ultrafiltration to obtain DNA-surface-engineered exosomes; resuspending the DNA-surface-engineered exosomes in PBS buffer and storing them at 20-40°C. The method for preserving exosomes provided by this invention allows for stable preservation at room temperature, is simple to operate, has good biocompatibility, and facilitates exosome storage.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a method for stable preservation of exosomes at room temperature. Background Technology

[0002] Exosomes are tiny extracellular vesicles (30-150 nm) secreted by cells, possessing a phospholipid bilayer structure. They can carry various biological information substances such as proteins, lipids, and nucleic acids. Exosomes act as a medium for intercellular communication, mediating information exchange and substance transfer between cells. Furthermore, their nanoscale size, high biocompatibility, low immunogenicity, and ability to cross biological barriers make them ideal drug delivery carriers. Multiple studies have shown that exosomes can achieve the efficient delivery of small molecule drugs, proteins, nucleic acids, and nanoparticles, treating a variety of diseases.

[0003] The vesicle structure of exosomes, with its lipid bilayer, protects internal biomolecules from various enzymes in body fluids, thus maintaining their integrity and biological activity. However, after isolation, storage conditions such as temperature, pressure, or solvent exposure can lead to membrane rupture and denaturation / shedding of surface proteins, affecting the structure and function of exosomes. Therefore, the intact preservation of exosomes is a crucial prerequisite for studying their biological roles and functions in vivo. Improving the in vitro stability of exosomes is a key factor limiting the development of exosome-based clinical detection and therapeutic vector technologies.

[0004] Exosomes are unstable and easily inactivated. To maintain their structural integrity, these formulations must be maintained and transported at relatively low temperatures to preserve their biological activity. However, the loss of exosome biological activity often occurs during storage. They are typically formulated as liquids and stored at ultra-low temperatures (e.g., not higher than -60°C), transported under frozen conditions, and require thawing before use. Furthermore, prolonged ultra-low temperature storage, repeated freeze-thaw cycles, or improper use can cause a rapid and significant decline in the biological activity of exosomes, thus limiting their normal use. Summary of the Invention

[0005] The technical problem solved by this invention is the preservation of exosomes under low-temperature freezing conditions. Exosomes need to be thawed before use. If the freezing time is too long and repeated freeze-thaw cycles occur during use or if the use is improper, the biological activity of exosomes will rapidly and significantly decrease.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for stable preservation of exosomes at room temperature includes the following steps:

[0008] Step S1: Extract exosomes;

[0009] Step S2: Mix the exosomes with cholesterol-modified single-stranded DNA evenly, and then perform ultrafiltration to obtain DNA-engineered exosomes.

[0010] Step S3: Resuspend the DNA-engineered exosomes in PBS buffer and store them at 20-40°C.

[0011] Preferably, in step S1, the extraction of exosomes includes:

[0012] Step S11: Take the exosome extract, centrifuge the exosome extract, take the supernatant, filter it, and obtain the cell supernatant;

[0013] Step S12: Centrifuge the cell supernatant at high speed to remove the supernatant, and resuspend the resulting precipitate in PBS buffer to obtain the exosomes.

[0014] Preferably, in step S11, the centrifugation process includes: first centrifuging the exosome extract at a speed of 2800-3200g for 15-25min, and then centrifuging the resulting supernatant at a speed of 9000-11000g for 25-35min.

[0015] Preferably, in step S11, the centrifugation process is carried out at 4-6°C.

[0016] Preferably, in step S12, the PBS buffer is a sterile PBS buffer.

[0017] Preferably, in step S12, the ultracentrifugation speed is 95000g-105000g and the time is 1-2h.

[0018] Preferably, in step S1, the exosome extract includes one of cell culture medium, body fluid, milk, and plant juice.

[0019] Preferably, in step S2, the step of mixing the exosomes with the cholesterol-modified single-stranded DNA includes: mixing the exosomes with the cholesterol-modified single-stranded DNA and shaking at room temperature, wherein the shaking speed is 300-700 rpm and the time is 3-7 min.

[0020] Preferably, in step S2, the single-stranded DNA used to prepare the cholesterol-modified single-stranded DNA is selected from straight-stranded DNA containing 7-28 bases.

[0021] Preferably, in step S2, the cholesterol-modified single-stranded DNA is a straight-stranded DNA with cholesterol modified at the 5' or 3' end.

[0022] Compared with existing technologies, this invention modifies exosomes using cholesterol-modified single-stranded DNA. Leveraging the hydrophilic and hydrophobic properties of cholesterol, the single-stranded DNA inserts into the phospholipid bilayer of the exosome, constructing a dense nucleic acid shell on the vesicle surface. Because the DNA is negatively charged, there is a certain Coulombic repulsion between adjacent exosomes, reducing direct contact and fusion between them, stabilizing the colloidal structure, and thus allowing exosomes to be stably preserved at room temperature. The method for room-temperature stable preservation of exosomes provided by this invention is simple to operate, has good biocompatibility, and facilitates exosome storage. Attached Figure Description

[0023] Figure 1 This is a flowchart illustrating the process of preserving exosomes at room temperature in an embodiment of the present invention;

[0024] Figure 2 The images show a comparison of transmission electron microscopy (TEM) images of exosomes stored at 37°C for 0 h and 72 h in Example 1 and Comparative Example 1, respectively.

[0025] Figure 3 This is a comparison diagram of the DLS particle size distribution of exosomes during the preservation process in Example 1 and Comparative Example 1;

[0026] Figure 4 This is a comparison of the average particle size of exosomes during the preservation process in Example 1 and Comparative Example 1;

[0027] Figure 5 This is a comparison chart of the polydispersity coefficients of exosomes during preservation in Example 1 and Comparative Example 1. Detailed Implementation

[0028] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0029] It should be noted that, unless otherwise specified, the features in the embodiments of this invention can be combined with each other. The terms "comprising," "including," "containing," and "having" are non-limiting, meaning that other steps and other components that do not affect the results can be added. The above terms cover the terms "composed of" and "substantially composed of." Unless otherwise specified, the materials, equipment, and reagents are commercially available.

[0030] like Figure 1 As shown in the figure, an embodiment of the present invention provides a method for stable preservation of exosomes at room temperature, comprising the following steps:

[0031] Step S1: Extract exosomes;

[0032] Step S2: Mix the exosomes with cholesterol-modified single-stranded DNA evenly, and then perform ultrafiltration to obtain DNA-engineered exosomes.

[0033] Step S3: Resuspend the DNA-engineered exosomes in PBS buffer and store them at 20-40°C.

[0034] Compared with existing technologies, the embodiments of this invention modify exosomes with cholesterol-modified single-stranded DNA. Leveraging the hydrophilic and hydrophobic properties of cholesterol, the single-stranded DNA inserts into the phospholipid bilayer of the exosome, constructing a dense nucleic acid shell on the vesicle surface. Because the DNA is negatively charged, there is a certain Coulombic repulsion between adjacent exosomes, reducing direct contact and fusion between them, stabilizing the colloidal structure, and thus allowing exosomes to be stably preserved at room temperature. The method for room-temperature stable preservation of exosomes provided by this invention is simple to operate, has good biocompatibility, and facilitates exosome storage.

[0035] Preferably, in step S1, the extraction of exosomes includes:

[0036] Step S11: Take the exosome extract, centrifuge the exosome extract, take the supernatant, filter it, and obtain the cell supernatant;

[0037] Step S12: Centrifuge the cell supernatant at high speed to remove the supernatant, and resuspend the resulting precipitate in PBS buffer to obtain exosomes.

[0038] It should be noted that the PBS buffer used in this invention is one of the most widely used buffers in biochemical research. Its main components are Na2HPO4, KH2PO4, NaCl and KCl. It is generally used as a solvent to dissolve and protect the reagents.

[0039] Specifically, in step S11, the centrifugation process includes: first centrifuging the exosome extract at 2800-3200g for 15-25 minutes, and then centrifuging the resulting supernatant at 9000-11000g for 25-35 minutes. Preferably, in step S12, the ultracentrifugation speed is 95000g-105000g, and the time is 1-2 hours.

[0040] In an embodiment of the present invention, in step S11, the centrifugation process is performed at 4-6°C. Centrifugation at 4-6°C results in more stable extracted exosomes.

[0041] In an embodiment of the present invention, in step S12, the PBS buffer is a sterile PBS buffer.

[0042] In an embodiment of the present invention, in step S1, the exosome extract includes one of cell culture medium, body fluid, milk, and plant juice.

[0043] In an embodiment of the present invention, step S2, which involves uniformly mixing the exosomes with the cholesterol-modified single-stranded DNA, includes: mixing the exosomes with the cholesterol-modified single-stranded DNA and shaking at room temperature, wherein the shaking speed is 300-700 rpm and the time is 3-7 min. Mixing the exosomes with the cholesterol-modified single-stranded DNA at room temperature with shaking is more conducive to uniform mixing.

[0044] In an embodiment of the present invention, in step S2, the single-stranded DNA used to prepare the cholesterol-modified single-stranded DNA is selected from linear DNA containing 7-28 bases.

[0045] In an embodiment of the present invention, in step S2, the cholesterol-modified single-stranded DNA is a straight-stranded DNA with cholesterol modified at the 5' or 3' end.

[0046] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0047] It should be noted that the cholesterol-modified single-stranded DNA used in this embodiment of the invention was purchased from Sangon Biotech (Shanghai) Co., Ltd., with the sequence: 5'-AAAATACGAGCTGAGAATCCT-3', and purified by HPLC. The 5' end of this single-stranded DNA is modified with a cholesterol group. This cholesterol-modified single-stranded DNA can be synthesized using a laboratory DNA synthesizer or customized when purchasing the single-stranded DNA; in this embodiment, it was customized by Sangon Biotech (Shanghai) Co., Ltd.

[0048] Example 1

[0049] 1.1 Extraction of exosomes

[0050] 1.1.1 Take 50 mL of human breast cancer cell MDA-MB-231 culture medium in a centrifuge tube and centrifuge at 3000 g for 20 minutes. Transfer the supernatant after centrifugation to a new centrifuge tube and centrifuge at 10000 g for 30 minutes. Take the supernatant and filter it through a 0.22 μm filter to obtain cell supernatant.

[0051] 1.1.2 Centrifuge the cell supernatant at 100,000g for 90 minutes, remove the supernatant, and resuspend the precipitate obtained at the bottom of the centrifuge tube in 500μL PBS buffer to obtain exosomes.

[0052] 1.2 Preparation of DNA-engineered exosomes

[0053] Exosomes were mixed with cholesterol-modified DNA, shaken at 500 rpm for 5 minutes, and ultrafiltration was used to remove free nucleic acids to obtain DNA-engineered exosomes.

[0054] 1.3 The DNA-engineered exosomes were resuspended in 200 μL of PBS buffer solution and placed in a constant temperature incubator for static storage at 37°C.

[0055] Example 2

[0056] 2.1 Extraction of exosomes

[0057] 2.1.1 Take 50 mL of human breast cancer cell MDA-MB-231 culture medium in a centrifuge tube and centrifuge at 3000 g for 20 minutes. Transfer the supernatant after centrifugation to a new centrifuge tube and centrifuge at 10000 g for 30 minutes. Take the supernatant and filter it through a 0.22 μm filter to obtain cell supernatant.

[0058] 2.1.2 Centrifuge the cell supernatant at 100,000g for 90 minutes, remove the supernatant, and resuspend the precipitate obtained at the bottom of the centrifuge tube in 500μL PBS buffer to obtain exosomes.

[0059] 2.2 Preparation of DNA-engineered exosomes

[0060] Exosomes were mixed with cholesterol-modified DNA, shaken at 500 rpm for 5 minutes, and ultrafiltration was used to remove free nucleic acids to obtain DNA-engineered exosomes.

[0061] 2.3. The DNA-engineered exosomes were resuspended in 200 μL of PBS buffer solution and placed in a constant temperature incubator for static storage at 20°C.

[0062] Example 3

[0063] 3.1 Extraction of exosomes

[0064] 3.1.1 Take 50 mL of human breast cancer cell MDA-MB-231 culture medium in a centrifuge tube and centrifuge at 3000 g for 20 minutes. Transfer the supernatant after centrifugation to a new centrifuge tube and centrifuge at 10000 g for 30 minutes. Take the supernatant and filter it through a 0.22 μm filter to obtain cell supernatant.

[0065] 3.1.2 Centrifuge the cell supernatant at 100,000g for 90 minutes, remove the supernatant, and resuspend the precipitate obtained at the bottom of the centrifuge tube in 500μL PBS buffer to obtain exosomes.

[0066] 3.2 Preparation of DNA-engineered exosomes

[0067] Exosomes were mixed with cholesterol-modified DNA, shaken at 500 rpm for 5 minutes, and ultrafiltration was used to remove free nucleic acids to obtain DNA-engineered exosomes.

[0068] 3.3 The DNA-engineered exosomes were resuspended in 200 μL of PBS buffer solution and placed in a constant temperature incubator for static storage at 40°C.

[0069] Comparative Example 1

[0070] Take 50 mL of human breast cancer cell culture medium MDA-MB-231 in a centrifuge tube and centrifuge at 3000g for 20 minutes. Transfer the supernatant after centrifugation to a new centrifuge tube and centrifuge at 10000g for 30 minutes. Take the supernatant and filter it through a 0.22 μm filter to obtain the cell supernatant.

[0071] The cell supernatant was centrifuged at 100,000g for 90 minutes, the supernatant was removed, and the precipitate obtained at the bottom of the centrifuge tube was resuspended in 500μL PBS buffer to obtain exosomes.

[0072] The exocrine cells were resuspended in 200 μL of PBS buffer solution and placed in a constant temperature incubator for static storage at 37°C.

[0073] Experimental Example 1

[0074] The diameter data of exosomes preserved in Example 1 and Comparative Example 1 were collected every 24 hours using an Anton Paar Litesizer 500 nanometer particle size analyzer. The results are as follows: Figure 2-4 As shown, Figure 2 Image a in Figure 1 is a transmission electron microscope (TEM) image of the exosomes prepared in Comparative Example 1 after 0 hours of preservation. Figure 2 Image b is a transmission electron microscope (TEM) image of DNA-surface-engineered exosomes prepared in Example 1 after 0 hours of storage. Figure 2 Image c is a transmission electron microscope image of the exosomes prepared in Comparative Example 1 after 72 hours of preservation. Figure 2 Image d in the image is a transmission electron microscope image of DNA-engineered exosomes prepared in Example 1 after 72 hours of storage. Figures 2-4 As can be seen, the exosomes preserved in Comparative Example 1 underwent vesicle rupture and fusion over time, with some exosomes forming large vesicle structures with diameters of 1 to 2 micrometers. In contrast, the diameter of the DNA-engineered exosomes in Example 1 remained stable at around 100 nm. This demonstrates that the method provided in this embodiment of the invention can stably preserve exosomes at room temperature.

[0075] The polydispersity index data of exosomes preserved in Example 1 and Comparative Example 1 were collected every 24 hours using an Anton Paar Litesizer 500 nanometer particle size analyzer. The results are as follows: Figure 5 As shown, from Figure 5 It can be seen that the polydispersity index of the exosomes preserved in Comparative Example 1 continuously increases over time, indicating a decrease in the uniformity of exosome diameter. In contrast, the DNA surface-engineered exosomes preserved in Example 1 maintain essentially unchanged diameter uniformity. This further verifies that the method provided in this embodiment of the invention can stably preserve exosomes at room temperature.

[0076] It should be noted that, Figure 2 The vertical axis "percent" represents the percentage of content. Figures 3-5 In this example, natural exosomes correspond to Example 1, and exosome-DNA corresponds to Example 1.

[0077] Furthermore, it should be noted that although the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A method for stable preservation of exosomes at room temperature, characterized in that, Includes the following steps: Step S1: Extract exosomes; Step S2: Mix the exosomes with cholesterol-modified single-stranded DNA until homogeneous, and then perform ultrafiltration to obtain DNA-surface-engineered exosomes; wherein, mixing the exosomes with cholesterol-modified single-stranded DNA until homogeneous includes: mixing the exosomes with the cholesterol-modified single-stranded DNA and shaking at room temperature, wherein the shaking speed is 300-700 rpm and the time is 3-7 min; the single-stranded DNA used to prepare the cholesterol-modified single-stranded DNA is selected from straight-stranded DNA containing 7-28 bases; the cholesterol-modified single-stranded DNA is straight-stranded DNA with cholesterol modified at the 5' end or 3' end; Step S3: Resuspend the DNA-engineered exosomes in PBS buffer and store them at 20-40°C.

2. The method for stable preservation of exosomes at room temperature according to claim 1, characterized in that, In step S1, the extraction of exosomes includes: Step S11: Take the exosome extract, centrifuge the exosome extract, take the supernatant, filter it, and obtain the cell supernatant; Step S12: Centrifuge the cell supernatant at high speed to remove the supernatant, and resuspend the resulting precipitate in PBS buffer to obtain the exosomes.

3. The method for stable preservation of exosomes at room temperature according to claim 2, characterized in that, In step S11, the centrifugation process includes: first centrifuging the exosome extract at a speed of 2800-3200g for 15-25min, and then centrifuging the supernatant at a speed of 9000-11000g for 25-35min.

4. The method for stable preservation of exosomes at room temperature according to claim 3, characterized in that, In step S11, the centrifugation process is carried out at 4-6°C.

5. The method for stable preservation of exosomes at room temperature according to claim 2, characterized in that, In step S12, the PBS buffer is a sterile PBS buffer.

6. The method for stable preservation of exosomes at room temperature according to claim 2, characterized in that, In step S12, the ultracentrifugation speed is 95000g-105000g, and the time is 1-2h.

7. The method for stable preservation of exosomes at room temperature according to claim 1, characterized in that, In step S1, the exosome extract includes one of cell culture medium, body fluid, milk, and plant juice.

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