Manufacturing method for prolonging in-vivo half-life period of artificial exosome

Abstract

The invention discloses a preparation method for prolonging the in-vivo half-life period of an artificial exosome. The preparation method comprises the following steps: S1, extracting the exosome; s1.1, cell culture: culturing the human liver Kupffer cells for 7 days; s1.2, primary centrifugation: collecting a culture solution, and centrifuging to remove cells and large fragments; s1.3, secondary clarification: transferring the supernatant to a new tube, and centrifuging to further remove smaller cell debris; s1.4, ultracentrifugal precipitation of the exosome: transferring the supernate again, and centrifugally precipitating the exosome; s1.5, re-suspending the exosome: gently re-suspending the precipitate with PBS, and carrying out ultracentrifugation to remove impurities; s2, platelet membrane component extraction; s3, combining the exosome with the platelet membrane component. According to the invention, the in-vivo half-life period of the exosome is greatly prolonged by using a simpler process and cost, and the half-life period can reach 24-48 hours.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome production, and specifically to a method for producing artificial exosomes with an improved in vivo half-life. Background Art

[0002] Exosomes are nanoscale vesicles (with a diameter of about 30 - 150 nanometers) secreted by cells and are widely present in various body fluids such as blood, saliva, and urine. They carry bioactive molecules such as proteins, nucleic acids (such as mRNA, miRNA, DNA), and lipids, and play important roles in cell - cell communication, disease occurrence and development, and medical applications. For example: Exosomes are naturally biocompatible and targetable, can be loaded with chemotherapeutic drugs, siRNA, etc. for precision therapy (such as targeting tumor cells), stem cell - derived exosomes are used to treat tissue injuries such as myocardial infarction and liver fibrosis, and exosomes loaded with tumor antigens are used as personalized vaccines to activate anti - tumor immunity, etc.

[0003] Although exosomes have great application prospects in the medical field, they also have significant drawbacks that hinder their rapid and large - scale application in the medical field. One of the main points is that the half - life of artificially cultured exosomes in vivo is very short. The half - life of exosomes in the human body (i.e., the time required for their concentration in the circulatory system to decrease by half) is affected by various factors, including exosome source, surface modification, administration route, and individual physiological status, etc.

[0004] The half - life of natural exosomes (such as those derived from blood or cell culture) is usually short, about 2 - 6 minutes in mouse models. The main reasons for the rapid clearance are the capture by the mononuclear phagocyte system (MPS) such as the liver and spleen, and kidney filtration (especially for small - sized exosomes). The half - life can be extended through surface modification (such as polyethylene glycolylation, coupling of targeting molecules). For example, the half - life of polyethylene glycol (PEG) - modified exosomes in mice can be extended to 1 hour. Targeting ligands (such as antibodies, peptides) can reduce non - specific clearance and improve the accumulation efficiency in target tissues.

[0005] Therefore, the main strategies and technologies for providing the half - life of exosomes currently are as follows:

[0006] 1. Surface engineering, introducing "self" - labeled proteins such as CD47, CD24, etc. to reduce immune clearance. Or polyethylene glycol (PEG) modification to reduce non - specific binding to plasma proteins.

[0007] 2. Targeted modification, coupling targeting molecules (such as antibodies, aptamers) to improve tissue specificity and reduce systemic exposure.

[0008] 3. Size regulation, screening larger - sized exosomes (>100nm) through purification techniques to reduce kidney filtration.

[0009] Although the above strategies and technologies can improve the half-life of exosomes to a certain extent, their technical processes are numerous, with many uncontrollable factors and high costs, which are not conducive to large-scale industrial production. Moreover, based on current data analysis, the improvement effect is not obvious and still cannot meet the urgent needs of the medical field. Summary of the Invention

[0010] The purpose of the present invention is to provide a method for producing artificial exosomes with an improved in vivo half-life to solve the problems in the prior art.

[0011] To achieve the above purpose, the present invention provides the following technical solution: A method for producing artificial exosomes with an improved in vivo half-life, comprising the following steps:

[0012] S1. Exosome extraction;

[0013] S1.1. Cell culture: Human liver Kupffer cells are cultured for 7 days.

[0014] S1.2. Primary centrifugation: The culture medium is collected and centrifuged to remove cells and large debris.

[0015] S1.3. Secondary clarification: The supernatant is transferred to a new tube and centrifuged to further remove smaller cell debris.

[0016] S1.4. Ultracentrifugation to precipitate exosomes: The supernatant is transferred again and centrifuged to precipitate exosomes.

[0017] S1.5. Exosome resuspension: The precipitate is gently resuspended with PBS and ultracentrifuged to remove impurities.

[0018] S2. Platelet membrane component extraction;

[0019] S2.1. Blood sample treatment: Mouse blood is centrifuged at room temperature to initially separate plasma.

[0020] S2.2. EDTA treatment: 0.8 mM EDTA is added to the plasma and centrifuged at room temperature.

[0021] S2.3. Precipitate resuspension and freeze-thaw cycle: The precipitate is resuspended in PBS containing 1 mM EDTA and a protease inhibitor; the PBS solution containing platelets is placed in liquid nitrogen for freezing, taken out after 20 minutes and thawed in a 37 °C water bath and gently shaken, and this process is repeated a total of 5 times.

[0022] S2.4. Final centrifugation and storage: After the last thawing, the pure platelet membrane component is collected by centrifugation.

[0023] S3. Binding of exosomes and platelet membrane components;

[0024] S3.1, Mixing ratio: Mix exosomes and platelet membrane components with a mass ratio of 1:1 in PBS;

[0025] S3.2, Ultrasonic treatment: Make the mixture uniformly dispersed through ultrasonic treatment;

[0026] S3.3, Gradual extrusion: Use a micro extruder to sequentially pass through polycarbonate membranes with pore sizes of 0.4 μm, 0.2 μm, and 0.1 μm, and repeat each size 10 times;

[0027] S3.4, Preservation: Preserve the homogenized mixture.

[0028] Preferably, the unused exosomes are stored in a -80°C refrigerator; the unused platelet membrane components are stored in a -80°C refrigerator.

[0029] Preferably, the storage conditions in S3.4 are short-term storage at 4°C or long-term storage at -80°C.

[0030] Preferably, in S1.2, centrifugation is carried out at a centrifugal force of 360×g for 10 minutes;

[0031] In S1.3, centrifugation is carried out at a centrifugal force of 10000×g for 30 minutes;

[0032] In S1.4, centrifugation is carried out at a centrifugal force of 10000×g for 100 minutes;

[0033] In S1.5, centrifugation is carried out at a centrifugal force of 10000×g for 100 minutes.

[0034] Preferably, in S2.1, centrifugation is carried out at a centrifugal force of 100×g for 20 minutes;

[0035] In S2.2, centrifugation is carried out at a centrifugal force of 800×g for 20 minutes;

[0036] In S2.4, centrifugation is carried out at a centrifugal force of 10000×g for 10 minutes.

[0037] Preferably, the concentration after mixing in S3.1 does not exceed 2 mg / ml.

[0038] Preferably, the conditions for ultrasonic treatment in S3.2 are: ultrasonic treatment for 3 minutes at 4°C.

[0039] Compared with the prior art, the beneficial effects of the present invention are: With a simpler process and cost, the half-life of exosomes in vivo is greatly improved, reaching 24 - 48 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The drawings are used to provide a further understanding of the present invention and form a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention. In the drawings:

[0041] Figure 1 is a schematic structural diagram of the present invention;

[0042] Figure 2 is a schematic diagram of the size and concentration of the exosomes fused with the present invention;

[0043] Figure 3 is a schematic diagram of the determination of the in vivo half-life of the exosomes after fusion of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0044] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed present invention, but merely represents selected embodiments of the present invention.

[0045] Please refer to Figures 1 - 3 , in an embodiment of the present invention, a method for producing artificial exosomes with an increased in vivo half-life includes the following steps:

[0046] S1. Exosome extraction;

[0047] S1.1. Cell culture: Human liver Kupffer cells are cultured for 7 days (human liver Kupffer cells (Wuhan Shangen Biology, product number SNP-H065), and after culturing the cells for 7 days according to the instructions);

[0048] S1.2. Primary centrifugation: Collect the culture medium and centrifuge to remove cells and large debris;

[0049] S1.3. Secondary clarification: Transfer the supernatant to a new tube and centrifuge to further remove smaller cell debris;

[0050] S1.4. Ultracentrifugation to precipitate exosomes: Transfer the supernatant again and centrifuge to precipitate exosomes;

[0051] S1.5. Exosome resuspension: Gently resuspend the precipitate with PBS and ultracentrifuge to remove impurities;

[0052] S2. Extraction of platelet membrane components;

[0053] S2.1. Blood sample treatment: Mouse blood is centrifuged at room temperature to initially separate plasma;

[0054] S2.2. EDTA treatment: 0.8 mM EDTA (ethylenediaminetetraacetic acid) is added to the plasma and centrifuged at room temperature;

[0055] S2.3. Precipitate resuspension and freeze-thaw cycles: The precipitate is resuspended in PBS containing 1 mM EDTA and protease inhibitor; The PBS solution containing platelets is frozen in liquid nitrogen, taken out after 20 minutes, thawed in a 37 °C water bath and gently shaken, and this process is repeated 5 times in total;

[0056] S2.4. Final centrifugation and preservation: After the last thawing, centrifuge to collect pure platelet membrane components;

[0057] S3. Binding of exosomes to platelet membrane components;

[0058] S3.1. Mixing ratio: Exosomes and platelet membrane components with a mass ratio of 1:1 are mixed in PBS;

[0059] S3.2. Ultrasonic treatment: The mixture is evenly dispersed by ultrasonic treatment;

[0060] S3.3. Gradual extrusion: Using a microextruder (Avanti Polar Lipids, USA), sequentially pass through polycarbonate membranes with pore sizes of 0.4 μm, 0.2 μm, and 0.1 μm, and repeat each size 10 times;

[0061] S3.4. Preservation: The homogenized mixture is preserved.

[0062] Preferably, the unused exosomes are stored in an -80 °C refrigerator; the unused platelet membrane components are stored in an -80 °C refrigerator.

[0063] Preferably, the preservation condition in S3.4 is short-term preservation at 4 °C or long-term preservation at -80 °C.

[0064] Preferably, in S1.2, centrifugation is performed at a centrifugal force of 360×g for 10 minutes;

[0065] In S1.3, centrifugation is performed at a centrifugal force of 10000×g for 30 minutes;

[0066] In S1.4, centrifugation is performed at a centrifugal force of 10000×g for 100 minutes;

[0067] In S1.5, centrifugation is carried out at a centrifugal force of 10,000×g for 100 minutes.

[0068] Preferably, in S2.1, centrifugation is carried out at a centrifugal force of 100×g for 20 minutes;

[0069] In S2.2, centrifugation is carried out at a centrifugal force of 800×g for 20 minutes;

[0070] In S2.4, centrifugation is carried out at a centrifugal force of 10,000×g for 10 minutes.

[0071] Preferably, the concentration after mixing in S3.1 does not exceed 2 mg / ml.

[0072] Preferably, the conditions for ultrasonic treatment in S3.2 are: ultrasonic treatment at 4°C for 3 minutes.

[0073] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for increasing the in vivo half-life of artificial exosomes, characterized in that: The following steps are involved: S1, exosome extraction; S1.

1. Cell culture: Human liver Kupffer cells were cultured for 7 days; S1.2, initial centrifugation: collect the culture medium and centrifuge to remove cells and large debris; S1.3, Secondary clarification: The supernatant is transferred to a new tube and centrifuged to further remove smaller cell debris; S1.4, ultracentrifugation to precipitate exosomes: transfer the supernatant again and centrifuge to precipitate exosomes; S1.

5. Resuspension of exosomes: Gently resuspend the precipitate with PBS and remove impurities by ultracentrifugation; S2, platelet membrane component extraction; S2.

1. Blood sample processing: Mouse blood was centrifuged at room temperature to initially separate plasma; S2.2, EDTA treatment: add 0.8 mM EDTA to the plasma and centrifuge at room temperature; S2.3, pellet resuspension and freeze-thaw cycle: the pellet was resuspended in PBS containing 1 mM EDTA and protease inhibitors; the PBS solution containing platelets was frozen in liquid nitrogen, taken out after 20 minutes, thawed in a 37°C water bath and gently shaken, and this process was repeated 5 times; S2.

4. Final centrifugation and storage: After the final thawing, the pure platelet membrane fraction is collected by centrifugation; S3, exosomes bind to platelet membrane components; S3.1, Mixing ratio: Mix the exosomes and platelet membrane components in PBS at a mass ratio of 1:1; S3.2, ultrasonic treatment: the mixture is evenly dispersed by ultrasonic treatment; S3.3, step-by-step extrusion: using a micro extruder, sequentially pass through polycarbonate membranes with pore sizes of 0.4 μm, 0.2 μm, and 0.1 μm, and repeat 10 times for each size; S3.

4. Storage: Store the homogenized mixture.

2. The method for producing artificial exosomes for increasing the in vivo half-life according to claim 1, characterized in that: Unused exosomes were stored in a -80°C refrigerator; unused platelet membrane components were stored in a -80°C refrigerator.

3. The method for producing artificial exosomes for increasing the in vivo half-life according to claim 1, characterized in that: The storage conditions in S3.4 are 4°C for short-term storage or -80°C for long-term storage.

4. The method for producing artificial exosomes for increasing the in vivo half-life according to claim 1, characterized in that: In S1.2, centrifugation is performed at a centrifugal force of 360×g for 10 minutes; In S1.3, centrifugation is performed at a centrifugal force of 10,000×g for 30 minutes; In S1.4, centrifugation is performed at a centrifugal force of 10,000×g for 100 minutes; In the S1.5, a centrifugal force of 10,000×g is used for centrifugation, and the centrifugation time is 100 minutes.

5. The method for producing artificial exosomes for increasing the in vivo half-life according to claim 1, characterized in that: In S2.1, centrifugation is performed at a centrifugal force of 100×g for 20 minutes; In S2.2, centrifugation is performed at a centrifugal force of 800×g for 20 minutes; In S2.4, a centrifugal force of 10,000×g is used for centrifugation, and the centrifugation time is 10 minutes.

6. The method for producing artificial exosomes for increasing the in vivo half-life according to claim 1, characterized in that: The concentration after mixing in S3.1 does not exceed 2 mg / ml.

7. The method for producing artificial exosomes for increasing the in vivo half-life according to claim 1, characterized in that: The ultrasonic treatment conditions in S3.2 are: ultrasonic treatment at 4° C. for 3 minutes.

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