A method for preparing a protective agent composition for exosome freeze-drying

Abstract

This invention belongs to the field of medical technology, specifically relating to a method for preparing a protective agent composition for exosome freeze-drying, comprising the following steps: Step 1, Raw material preparation: Prepare sufficient exosome suspension, mannitol, sucrose, trehalose, glycine, and a buffer system. The buffer system can be any one of physiological saline, PBS, Tris-HCl, or pure water. Step 2, Protective agent composition solution preparation: Combine mannitol, sucrose, trehalose, and glycine from Step 1 according to the following three ratios. This invention, through the combination of mannitol, sucrose, trehalose, and glycine, achieves synergistic protection through physical support, vitrification fixation, and chemical buffering, with significantly better effects than any single component. This formulation can greatly reduce frostbite damage, ensuring that the particle recovery rate after exosome freeze-drying and rehydration remains stable at over 85%, and the particle size distribution shows no significant change compared to before freeze-drying, effectively preventing aggregation.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a method for preparing a protective agent composition for exosome freeze-drying. Background Technology

[0002] Exosomes are nanoscale, lipid bilayer-enclosed structures carrying proteins, lipids, RNA metabolites, growth factors, and cytokines, and have attracted widespread attention due to their crucial role in mediating intercellular communication both locally and systemically. Exosomes originate from the endocytic pathway in most cells and are released from vesicle bodies (MVBs) into almost all biological fluids, such as blood, saliva, and urine. Exosome formation can be divided into four processes: invagination, endosome formation, fusion, and secretion. Initially, early endosomes are formed by invagination of the plasma membrane, subsequently transforming into late endosomes containing MVBs. After MVBs fuse with the plasma membrane, exosomes are continuously released from the cell. The abundance, composition, and functional properties of exosomes depend on their cell / tissue origin, physiological / pathological state, and even the microenvironment of the parent cell. The main function of exosomes is their ability to transmit information to neighboring cells and even distant cells from which the exosome originates, thereby influencing their function. More importantly, exosomes have been developed as therapeutic carriers for treating diseases. An advantage of using exosomes as delivery carriers is their ability to load hydrophilic substances within or between lipid bilayers. The substances are shielded by a double membrane, effectively protecting them from enzymatic degradation. Furthermore, the long circulation characteristics of mesenchymal stem cell exosomes prolong their residence time in the blood, reducing the clearance rate of the substances they carry. Exosomes generated from mesenchymal stem cells can target specific cell types via their surface receptors. Exosomes have also been shown to cross biological barriers and enter deep tissues. Exosomes exhibit excellent biocompatibility, biodegradability, stability, and low immunogenicity. Small molecules, nucleic acid drugs, or bioactive molecules can be functionally incorporated into exosome-based nanocarriers and then transported to the target site to achieve therapeutic effects.

[0003] The applications of exosomes can be expanded through appropriate preservation methods. Exosomes are typically stored at -80°C; however, some reports indicate that long-term storage under these conditions is not stable. Previous reports have shown that exosomes are smaller in size when stored at 4°C or 37°C compared to freshly isolated exosomes, suggesting structural changes or degradation. Furthermore, storage at -80°C for 4 days alters the morphology of exosomes, biological activity may decrease over 28 days, and storage for 2 years leads to exosomal RNA degradation. These findings indicate that exosome storage is an important issue. Lyophilization is a technique used to preserve various types of biological materials, such as proteins, plasma, and live cells. Furthermore, lyophilization has been used to improve the long-term stability of nanoscale drug delivery carriers. Therefore, lyophilization can improve the preservation stability of exosomes. Summary of the Invention

[0004] The purpose of this invention is to provide an exosome freeze-drying protectant composition with clearly defined components and optimized proportions, and to propose a process method for exosome freeze-drying and its application, so that exosomes can be more fully preserved and transported, and still have good biocompatibility and good biological activity after long-term storage, which has broad application prospects in the field of disease treatment.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a protective agent composition for exosome freeze-drying, comprising the following steps: Step 1: Raw material preparation: Prepare sufficient exosome suspension, mannitol, sucrose, trehalose, glycine and buffer system. The buffer system can be any one of physiological saline, PBS, Tris-HCl or pure water. The protective agent composition consists of mannitol, sucrose, trehalose, and glycine; Step 2, Preparation of the Protectant Composition Solution: Combine the mannitol, sucrose, trehalose, and glycine from Step 1 in the following three proportions: The first type contains 4% mannitol, 3% sucrose, 2% trehalose, and 1% glycine. The second type contains 7% mannitol, 3% sucrose, 2% trehalose, and 1% glycine. The third type contains 10% mannitol, 3% sucrose, 2% trehalose, and 1% glycine. Step 3: Preparation of the protective agent solution: Dissolve mannitol, sucrose, trehalose, and glycine in a buffer system, stir until completely dissolved, bring to a final volume, and filter to remove bacteria; Step 4: Mixing exosomes with the protective agent: Mix the prepared protective agent solution with the exosome suspension at a ratio of 9:1, gently blow to mix evenly, and filter using a 0.22μm filter; Step 5, Packaging: The exosome suspension is mixed with the protective agent composition solution from Step 1 and Step 2, and then sterilized, freeze-dried, and sealed to obtain exosome lyophilized powder.

[0006] As a method for preparing a protective agent composition for exosome freeze-drying according to the present invention, preferably, the sterilization, freeze-drying, and filling and sealing steps in the fifth step are as follows: First stage: target temperature -45.0℃, heating time 0min, target vacuum 0kPa, holding time 3h, holding time 0min; Second stage: target temperature -28.0℃, heating time 60min, target vacuum 0kPa, holding time 1h, holding time 0min; Third stage: target temperature -45.0℃, heating time 0min, target vacuum 0kPa, holding time 2h, holding time 0min; Fourth stage: target temperature -40.0℃, heating time 30min, target vacuum 10kPa, holding time 1h, holding time 0min; Fifth stage: target temperature -30.0℃, heating time 60min, target vacuum 10kPa, holding time 2h, holding time 0min; Stage 6: Target temperature -28.0℃, heating time 30min, target vacuum 10kPa, holding time 8h, holding time 0min; Stage 7: Target temperature -25.0℃, heating time 30min, target vacuum 10kPa, holding time 4h, holding time 0min; Eighth stage: target temperature -20.0℃, heating time 30min, target vacuum 10kPa, holding time 2h, holding time 0min; Ninth stage: target temperature -15.0℃, heating time 30min, target vacuum 10kPa, holding time 1h, holding time 0min; Stage 10: Target temperature -10.0℃, heating time 30min, target vacuum 10kPa, holding time 1h, holding time 0min; Stage 11: Target temperature -5.0℃, heating time 30min, target vacuum 10kPa, holding time 0h, holding time 0min; Stage 12: Target temperature 0.0℃, heating time 30min, target vacuum 10kPa, holding time 0h, holding time 0min; Thirteenth stage: target temperature 10.0℃, heating time 60min, target vacuum 10kPa, holding time 1h, holding time 0min; Fourteenth stage: target temperature 20.0℃, heating time 60min, target vacuum 0kPa, holding time 6h, holding time 0min; Stage 15: Target temperature 0.0℃, heating time 0min, target vacuum 0, constant temperature time 0h, constant temperature time 0min.

[0007] In a preferred embodiment of the present invention, a protective agent composition for cryo-drying of exosomes is prepared by maintaining a vacuum of 10 kPa during the cryo-drying process.

[0008] Compared with the prior art, the beneficial effects of the present invention are: 1. By combining mannitol, sucrose, trehalose and glycine, a synergistic protection of physical support, vitrification fixation and chemical buffering is achieved, with a significantly better effect than any single component.

[0009] 2. This formula can greatly reduce frostbite damage, keep the particle recovery rate of exosomes after freeze-drying and rehydration stable at over 85%, and the particle size distribution is not significantly different from that before freeze-drying, effectively preventing aggregation.

[0010] 3. The exosomes after lyophilization and reconstitution maintained a high positive rate of marker proteins (CD9, CD63, CD81, TSG101), and retained more than 95% of key biological activities such as cellular uptake and targeting.

[0011] 4. The product has an excellent appearance and shape. The resulting freeze-dried cake has a firm, full structure that does not shrink. It remelts quickly, usually within 30 seconds, and has no flocculent insoluble matter.

[0012] 5. Excellent stability; compared to liquid preservation, the shelf life of freeze-dried exosomes far exceeds that of liquid preservation.

[0013] 6. The formula is well-defined and easy to industrialize. All components are pharmaceutical excipients, which are safe, inexpensive, readily available, and have fixed proportions. The process is stable and very suitable for large-scale industrial production. Attached Figure Description

[0014] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a technical roadmap for the freeze-drying of exosomes according to the present invention.

[0015] Figure 2 These are images showing the morphology of exosomes before and after freeze-drying according to the present invention.

[0016] Figure 3 These are transmission electron microscopy (TEM) images of exosomes before and after freeze-drying according to the present invention.

[0017] Figure 4 A schematic diagram of the movement trajectory of nanoparticles before and after exosome freeze-drying.

[0018] Figure 5 Exosome particle size distribution diagrams: (A) Liquid exosomes before lyophilization; (B) Lyophilized exosomes of Formula 1; (C) Lyophilized exosomes of Formula 2; (D) Exosome particle size distribution diagram of Formula 4.

[0019] Figure 6 Statistical charts of exosome particle diameter and particle concentration: (A) Statistical chart of exosome particle diameter; (B) Statistical chart of exosome particle concentration. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Please see Figure 1-6 The present invention provides the following technical solution: a method for preparing a protective agent composition for exosome freeze-drying, comprising the following steps: Step 1: Raw material preparation: Prepare sufficient exosome suspension, mannitol, sucrose, trehalose, glycine and buffer system. The buffer system can be any one of physiological saline, PBS, Tris-HCl or pure water. The protective agent composition consists of mannitol, sucrose, trehalose, and glycine; Step 2, Preparation of the Protectant Composition Solution: Combine the mannitol, sucrose, trehalose, and glycine from Step 1 in the following three proportions: The first type contains 4% mannitol, 3% sucrose, 2% trehalose, and 1% glycine. The second type contains 7% mannitol, 3% sucrose, 2% trehalose, and 1% glycine. The third type contains 10% mannitol, 3% sucrose, 2% trehalose, and 1% glycine. Step 3: Preparation of the protective agent solution: Dissolve mannitol, sucrose, trehalose, and glycine in a buffer system, stir until completely dissolved, bring to a final volume, and filter to remove bacteria; Step 4: Mixing exosomes with the protective agent: Mix the prepared protective agent solution with the exosome suspension at a ratio of 9:1, gently blow to mix evenly, and filter using a 0.22μm filter; Step 5, Packaging: The exosome suspension is mixed with the protective agent composition solution from Step 1 and Step 2, and then sterilized, freeze-dried, and sealed to obtain exosome lyophilized powder.

[0022] After the freeze-drying process is completed, the freeze-dried cake is sealed under vacuum or nitrogen purging to obtain a white or off-white, loose, porous freeze-dried cake. Store in the dark at 2-8°C or room temperature.

[0023] Preferably, the sterilization, freeze-drying, and canning / sealing steps in the fifth step are as follows: First stage: target temperature -45.0℃, heating time 0min, target vacuum 0kPa, holding time 3h, holding time 0min; Second stage: target temperature -28.0℃, heating time 60min, target vacuum 0kPa, holding time 1h, holding time 0min; Third stage: target temperature -45.0℃, heating time 0min, target vacuum 0kPa, holding time 2h, holding time 0min; Fourth stage: target temperature -40.0℃, heating time 30min, target vacuum 10kPa, holding time 1h, holding time 0min; Fifth stage: target temperature -30.0℃, heating time 60min, target vacuum 10kPa, holding time 2h, holding time 0min; Stage 6: Target temperature -28.0℃, heating time 30min, target vacuum 10kPa, holding time 8h, holding time 0min; Stage 7: Target temperature -25.0℃, heating time 30min, target vacuum 10kPa, holding time 4h, holding time 0min; Eighth stage: target temperature -20.0℃, heating time 30min, target vacuum 10kPa, holding time 2h, holding time 0min; Ninth stage: target temperature -15.0℃, heating time 30min, target vacuum 10kPa, holding time 1h, holding time 0min; Stage 10: Target temperature -10.0℃, heating time 30min, target vacuum 10kPa, holding time 1h, holding time 0min; Stage 11: Target temperature -5.0℃, heating time 30min, target vacuum 10kPa, holding time 0h, holding time 0min; Stage 12: Target temperature 0.0℃, heating time 30min, target vacuum 10kPa, holding time 0h, holding time 0min; Thirteenth stage: target temperature 10.0℃, heating time 60min, target vacuum 10kPa, holding time 1h, holding time 0min; Fourteenth stage: target temperature 20.0℃, heating time 60min, target vacuum 0kPa, holding time 6h, holding time 0min; Stage 15: Target temperature 0.0℃, heating time 0min, target vacuum 0, constant temperature time 0h, constant temperature time 0min.

[0024] Preferably, the vacuum during the freeze-drying process is maintained at 10 kPa.

[0025] Example 1: Preparation and freeze-drying of exosomes: Materials and Methods: Reagents and consumables: Mannitol (Aladdin), sucrose (Macgene), seaweed salt (Aladdin), glycine (Zhongke Maichen), physiological saline (Chenxin), 0.22μm (Jet), 10mL syringe (Shuguang Doctor), 50mL centrifuge tube (Jet), Pasteur pipette (Jet), centrifuge (Xiangyi), lyophilizer (Sihuan Lyophilizer), 10mL pipette (Jet).

[0026] Experimental steps: 1. Transfer the collected cell culture medium supernatant into 50ml centrifuge tubes, 20ml per tube, and centrifuge at 3000rpm for 20min at room temperature to remove dead cells and cell debris.

[0027] 2. Collect the supernatant after centrifugation, remove large particles and vesicles using a 0.22μm filter membrane, add 5ml of 5XPEG6000 reagent, and separate the layers. Mix well and let stand overnight at 4℃.

[0028] 3. Centrifugation: Centrifuge at 4℃, 10000g for 60 minutes. White flocculent precipitate will be visible on the bottom and walls of the centrifuge tube. Discard the supernatant in the centrifuge tube. 4. Depending on the amount of precipitate, add an appropriate amount of sterile water for injection to resuspend, wash once, and centrifuge again at 4°C, 15000g, for 60 minutes. 5. After centrifugation, discard the supernatant and resuspend the exosomes in an appropriate amount of physiological saline according to the amount of exosomes obtained to obtain the exosome solution; 6. Add 9 mL of physiological saline and 1 mL of exosome solution to obtain exosome fluid before lyophilization.

[0029] 7. (1) Dissolve 0.4g mannitol, 0.3g sucrose, 0.2g trehalose and 0.1g glycine in 9mL of physiological saline according to formula 1, add 1mL of exosome solution, and obtain the sample before freeze-drying.

[0030] (2) Dissolve 0.7g mannitol, 0.3g sucrose, 0.2g trehalose and 0.1g glycine in 9mL of physiological saline according to formula 2, add 1mL of exosome solution, and obtain the sample before freeze-drying.

[0031] (3) Dissolve 1.0g mannitol, 0.3g sucrose, 0.2g trehalose and 0.1g glycine in 9mL of physiological saline according to formula 2, add 1mL of exosome solution, and obtain the sample before freeze-drying.

[0032] 8. Perform freeze-drying using the freeze-drying procedure described above.

[0033] In this embodiment, exosome solution was obtained through extraction and purification. The exosome preservative composition was mixed with the exosomes and freeze-dried using a lyophilizer. The freeze-dried exosome sample was obtained, and its macroscopic morphology was a fluffy white "cake-like" solid (e.g., ...). Figure 2 (As shown).

[0034] Example 2: Microstructure of exosomes before and after freeze-drying: To investigate whether lyophilization of exosomes damages their internal structure, this embodiment further uses transmission electron microscopy (TEM) to observe the microscopic internal structure of exosomes before and after lyophilization. TEM results clearly reveal the microscopic morphology of exosomes; the vast majority of vesicles in the sample are round or near-round, showing clear bilayer membrane boundaries, indicating that their membrane structure is intact and that lyophilization did not damage the internal structure of the exosomes (e.g., Figure 3 (As shown).

[0035] Example 3: Nanoparticle tracking analysis before and after exosome lyophilization: To assess the impact of freeze-drying on the physical characteristics of exosomes, nanoparticle tracking analysis (NTA) was used to characterize the exosomes. The exosome particles extracted from all experimental groups exhibited typical exosome size characteristics, primarily ranging from 80 to 200 nm. Specifically, the exosome particle size before freeze-drying was 105 nm, with a particle concentration of (2.3 ± 0.1) × 10^7. Compared to before freeze-drying, the exosome particle size after freeze-drying in formulation 1 was 106.1 nm, with a particle concentration of (1.6 ± 0.05) × 10^7 (p < 0.001), while the exosome particle size after freeze-drying in formulation 2 was 105.8 nm, with a particle concentration of (2.22 ± 0.06) × 10^7. The exosome particle size after lyophilization according to Formula 3 was 102.9 nm, and the particle concentration was (2.23±0.06)×10^7. There was no significant difference in the average particle size of exosomes among all experimental groups (p>0.05). Figure 6 A) Regarding exosome particle concentration, the exosome particle concentration was significantly reduced after lyophilization using formulation 1, but there was no significant difference between the exosome particle concentration and that before lyophilization using formulations 2 and 3. This result clearly indicates that the exosome lyophilization protectant compositions of formulations 2 and 3 do not damage the structure or particle concentration of exosomes during lyophilization.

[0036] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a cryoprotectant composition for exosome freeze-drying, characterized in that, Includes the following steps: Step 1: Raw material preparation: Prepare sufficient exosome suspension, mannitol, sucrose, trehalose, glycine and buffer system. The buffer system can be any one of physiological saline, PBS, Tris-HCl or pure water. The protective agent composition consists of mannitol, sucrose, trehalose, and glycine; Step 2, Preparation of the Protectant Composition Solution: Combine the mannitol, sucrose, trehalose, and glycine from Step 1 in the following three proportions: The first type contains 4% mannitol, 3% sucrose, 2% trehalose, and 1% glycine. The second type contains 7% mannitol, 3% sucrose, 2% trehalose, and 1% glycine. The third type contains 10% mannitol, 3% sucrose, 2% trehalose, and 1% glycine. Step 3: Preparation of the protective agent solution: Dissolve mannitol, sucrose, trehalose, and glycine in a buffer system, stir until completely dissolved, bring to a final volume, and filter to remove bacteria; Step 4: Mixing exosomes with the protective agent: Mix the prepared protective agent solution with the exosome suspension at a ratio of 9:1, gently blow to mix evenly, and filter using a 0.22μm filter; Step 5, Packaging: The exosome suspension is mixed with the protective agent composition solution from Step 1 and Step 2, and then sterilized, freeze-dried, and sealed to obtain exosome lyophilized powder.

2. The method for preparing a cryoprotectant composition for exosome freeze-drying according to claim 1, characterized in that: The sterilization, freeze-drying, and canning / sealing steps in the fifth step are as follows: First stage: target temperature -45.0℃, heating time 0min, target vacuum 0kPa, holding time 3h, holding time 0min; Second stage: target temperature -28.0℃, heating time 60min, target vacuum 0kPa, holding time 1h, holding time 0min; Third stage: target temperature -45.0℃, heating time 0min, target vacuum 0kPa, holding time 2h, holding time 0min; Fourth stage: target temperature -40.0℃, heating time 30min, target vacuum 10kPa, holding time 1h, holding time 0min; Fifth stage: target temperature -30.0℃, heating time 60min, target vacuum 10kPa, holding time 2h, holding time 0min; Stage 6: Target temperature -28.0℃, heating time 30min, target vacuum 10kPa, holding time 8h, holding time 0min; Stage 7: Target temperature -25.0℃, heating time 30min, target vacuum 10kPa, holding time 4h, holding time 0min; Eighth stage: target temperature -20.0℃, heating time 30min, target vacuum 10kPa, holding time 2h, holding time 0min; Ninth stage: target temperature -15.0℃, heating time 30min, target vacuum 10kPa, holding time 1h, holding time 0min; Stage 10: Target temperature -10.0℃, heating time 30min, target vacuum 10kPa, holding time 1h, holding time 0min; Stage 11: Target temperature -5.0℃, heating time 30min, target vacuum 10kPa, holding time 0h, holding time 0min; Stage 12: Target temperature 0.0℃, heating time 30min, target vacuum 10kPa, holding time 0h, holding time 0min; Thirteenth stage: target temperature 10.0℃, heating time 60min, target vacuum 10kPa, holding time 1h, holding time 0min; Fourteenth stage: target temperature 20.0℃, heating time 60min, target vacuum 0kPa, holding time 6h, holding time 0min; Stage 15: Target temperature 0.0℃, heating time 0min, target vacuum 0, constant temperature time 0h, constant temperature time 0min.

3. The method for preparing a cryoprotectant composition for exosome freeze-drying according to claim 1, characterized in that: The vacuum during the freeze-drying process is maintained at 10 kPa.

Images