Stabilizer composition for exosome refrigeration and use thereof

Abstract

This invention belongs to the field of exosome preservation technology and discloses a stabilizer composition for exosome cryopreservation and its application. The stabilizer composition for exosome cryopreservation provided by this invention consists of the following components and their amounts: phytosphingosine: 0.1%-0.2%; cerebroside sulfate: 0.05%-0.1%; trehalose: 2%-5%; lipid-soluble antioxidant: 0.01%-0.05%; HEPES buffer: 10-20 mM; metal ion chelating agent: 0.01%-0.05%. The composition of this invention forms a "gradient protection" through the membrane insertion of phytosphingosine and the shallow negative charge shielding of cerebroside sulfate, enabling stable cryopreservation of exosomes for more than one week with a bioactivity retention of >85%. This preservative is animal-free, easy to use, and effective, making it suitable for the clinical translation and practical application of exosomes.

Description

Technical Field

[0001] This invention belongs to the field of exosome preservation technology, and relates to a stabilizer composition for exosome cold storage and its application. Background Technology

[0002] Exosomes are extracellular vesicles with a diameter of 30-150 nanometers, released after the fusion of multiple vesicles with the plasma membrane. They carry bioactive molecules such as proteins, nucleic acids (e.g., mRNA, miRNA), and lipids, participating in intercellular communication and physiological and pathological regulation. Exosomes from different sources have different functions. Mesenchymal stem cell exosomes carry proteins, nucleic acids, and lipids derived from the mother cell and regulate tissue repair and immune regulation through paracrine mechanisms, exhibiting advantages of low immunogenicity and high biocompatibility. As a representative of "cell-free therapy," they hold promise for overcoming the limitations of stem cell therapy and have great potential in disease treatment and engineered modification. With the continuous expansion of exosome applications, their safe and effective preservation has become crucial. Liquid-preserved exosomes not only have a short shelf life (typically only 48 hours at 4°C), but PBS buffer or solution systems with mismatched densities with the exosomes may also lead to uneven distribution or aggregation of exosomes. Therefore, developing an exosome protective solution that can maintain the biological activity and stability of exosomes and has a density similar to that of exosomes is of great significance, whether for maintaining the integrity of exosomes, ensuring the stability of the activity of the nucleic acids and proteins they carry, or reducing exosome aggregation.

[0003] The current cryopreservation method for exosome preservation mainly relies on -80°C. This method has high equipment and transportation costs, and the unavoidable freeze-thaw cycles will cause uncontrollable physical damage and loss of activity to exosomes, which seriously affects the reproducibility of experiments and batch consistency of products.

[0004] Secondly, there are issues with the addition of protective agents: existing protective agents often have significant shortcomings. For example, most of those using animal-derived proteins (such as bovine serum albumin, BSA) pose risks of pathogens and immunogenicity; human serum albumin (HSA) is limited in supply and also carries the risk of bloodborne pathogens; simple sugars (such as trehalose and mannitol) lack membrane stabilizing capabilities, resulting in limited protective effects. Furthermore, common buffer systems (such as PBS) have a density mismatch with exosomes, easily leading to uneven distribution, sedimentation, and aggregation of exosomes, exacerbating physical damage.

[0005] Although existing studies have attempted to achieve long-term preservation through freeze-drying (lyophilization) technology, this process is cumbersome and time-consuming, making it more suitable for long-term storage and unable to meet the short-term, convenient, and ready-to-use refrigerated preservation needs of research or clinical practice. While some existing patents (such as CN117281109A) attempt to address the low-temperature protection problem, their protective agents are singular in composition and lack complex functional components that can effectively inhibit exosome aggregation, provide steric hindrance, and stabilize membrane structures, resulting in insufficient protective effects. Furthermore, existing technologies have failed to systematically solve the critical physical problem of matching the density of the preservation solution with that of the exosomes. Density matching is an important prerequisite for preventing aggregation due to sedimentation / floating and maintaining the uniform distribution and integrity of exosomes.

[0006] As industry standards rise, exosome production is increasingly adopting animal-free culture media to eliminate batch variations, exogenous contamination, and pathogen risks. The preservation solutions, from production to storage, also aim to replace the existing methods that rely on animal-derived proteins, placing higher demands on storage conditions. Therefore, there is an urgent need to develop a compatible animal-free preservation technology to create a complete solution from production to storage.

[0007] In summary, existing exosome preservation technologies suffer from several problems, including severe freeze-thaw damage, reliance on ultra-low temperature equipment, limited effectiveness or safety risks of existing cryoprotectants, aggregation caused by mismatched preservation solution densities, and an inability to meet the urgent need for convenient refrigerated preservation under the new trend of animal-free production. Summary of the Invention

[0008] The purpose of this invention is to overcome the above-mentioned deficiencies and provide an exosome cryopreservation solution with no animal origin, clearly defined components, and matched density, as well as its application in exosome preservation. This preservation solution can maintain the physical integrity, high dispersibility, biological activity, and functional stability of exosomes for extended periods under conventional refrigeration conditions of 2-8°C, thereby providing key technical support for the standardized storage, convenient transportation, and immediate clinical application of exosomes.

[0009] To achieve the above objectives, the present invention adopts the following technical solution;

[0010] A stabilizer composition for exosome cold storage, comprising the following components and amounts:

[0011] Phytosphingosine: 0.1%-0.2% w / v;

[0012] Cerebroside sulfate: 0.05%-0.1% w / v;

[0013] Trehalose: 2%-5% w / v;

[0014] Fat-soluble antioxidants: 0.01%-0.05% w / v;

[0015] Buffer solution: 10-20 mM;

[0016] Metal ion chelating agent: 0.01%-0.05% w / v;

[0017] Preferably, the ratio of phytosphingosine to cerebroside sulfate is 2:1 to 4:1.

[0018] Preferably, the fat-soluble antioxidant is selected from vitamin E-TPGS, vitamin E succinate, or coenzyme Q10.

[0019] Preferably, the metal ion chelating agent is selected from disodium EDTA, EGTA, or sodium citrate.

[0020] Preferably, the buffer solution is a HEPES buffer solution with a pH of 7.0-7.4.

[0021] This invention also protects a method for cryopreservation of exosomes, comprising the following steps:

[0022] (1) Mix the exosomes with the stabilizer composition for refrigerated exosomes;

[0023] (2) Store at 2~8℃ away from light.

[0024] Furthermore, the exosomes are mesenchymal stem cell exosomes. Even further, the concentration of the exosomes is 10. 8 ~10 11 particles / mL.

[0025] Sulfatides are characterized by the sulfation of the 3-hydroxyl group at the galactose position. The sulfate group is hydrophilic, giving the surface a significant negative charge. This negative charge effectively prevents the aggregation of exosomes through electrostatic repulsion; at the same time, the sulfate group forms a thick hydration layer, generating a strong steric hindrance effect, further maintaining overall stability.

[0026] Phytosphingosine has a C18 long-chain hydrocarbon group that partially inserts into the hydrophobic core of the membrane, while its characteristic trihydroxy structure (3 -OH groups) forms a wide network of intermolecular hydrogen bonds in the interfacial region, which plays a structural anchoring role, effectively preventing membrane deformation and maintaining membrane curvature stability.

[0027] This invention combines cerebroside sulfate and phytosphingosine. The addition of phytosphingosine achieves a good balance between membrane stability and fluidity; the negative surface charge of cerebroside sulfate synergistically provides anti-aggregation protection with the hydration layer. Importantly, neither of these lipids disrupts the internal hydrophobic structure of the membrane, thus fully preserving the biological activity of exosomes.

[0028] Compared with the prior art, the advantages of the present invention are:

[0029] This invention primarily utilizes a "gradient protection" mechanism formed by the membrane insertion of phytosphingosine and the superficial negative charge shielding of cerebroside sulfate to achieve stable cold storage preservation of exosomes for more than one week, maintaining >85% of their biological activity. This preservative is animal-free, easy to use, and highly effective, making it suitable for the clinical translation and practical application of exosomes. Attached Figure Description

[0030] Figure 1 This is a particle size distribution diagram for Example 1.

[0031] Figure 2 This is the particle size distribution diagram for the blank control.

[0032] Figure 3 This is a comparison graph showing the changes in the particle protein ratio between Example 1 and the control.

[0033] Figure 4 This is a comparison graph showing the changes in MDA content between Example 1 and the control. Detailed Implementation

[0034] The following specific embodiments of the present invention will provide a detailed and comprehensive description of the technical solutions of the present invention. It should be noted that the provided embodiments represent only a part of the present invention, and not all of it. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available reagents and materials. Commercially available raw materials are filtered to remove insoluble matter and other impurities using common filtration methods. The exosomes used in the following examples are all mesenchymal stem cell exosomes (conventionally extracted or commercially available), and the concentration of exosomes tested is 10. 8 ~10 11 particles / mL.

[0036] Example 1

[0037] A stabilizer composition for exosome cold storage, comprising the following components and amounts:

[0038] Phytosphingosine: 0.2 g

[0039] Cerebroside sulfate: 0.1 g

[0040] Trehalose: 3 g

[0041] Vitamin E-TPGS: 0.03 g

[0042] HEPES: 15 mM

[0043] Disodium EDTA: 0.02 g

[0044] Inject physiological saline: Add to 100 mL

[0045] Preparation steps:

[0046] 1. Dissolve phytosphingosine and cerebroside sulfate in 5 mL of ethanol (to aid dissolution);

[0047] 2. Add vitamin E-TPGS and sonicate (40 kHz, 5 min) to form a uniform dispersion;

[0048] 3. Dissolve trehalose, HEPES, and disodium EDTA in injectable saline;

[0049] 4. Slowly add the dispersion from step 2 to the solution from step 3, and stir (600 rpm, 30 min).

[0050] 5. Add water to a final volume of 100 mL;

[0051] 6. Sterilize by filtration through a 0.22 μm filter membrane, dispense, and store at 4°C protected from light.

[0052] Physicochemical parameter determination:

[0053] - pH: 7.2±0.1

[0054] - Osmotic pressure: 294±10 mOsm / kg

[0055] - Density: 1.14±0.01 g / cm³

[0056] Effect verification:

[0057] I. Changes in the particulate protein ratio:

[0058] (1) To investigate the stability of exosomes in PBS solution as a blank control group at 4℃: the particle protein ratio and particle size distribution were detected for 7 consecutive days.

[0059] (2) To investigate the stability of the particle-to-protein ratio of the protective solution in Example 1 at 4°C: The exosome sample was diluted 10 times with the protective solution as the solvent and stored in a 4°C refrigerator. The particle-to-protein ratio and particle size distribution were detected daily for 7 consecutive days.

[0060] Each sample was stored at 4°C, and the particle size distribution and particle-to-protein ratio of each sample were measured daily. The exosome particle size distribution in Example 1 is as follows: Figure 1 As shown, the protective agent provided in the examples can maintain the particle size distribution area, effectively prevent exosome aggregation, and maintain the stability of the particle-to-protein ratio. The exosome particle size distribution range increased in the blank control, such as... Figure 2 As shown, its particle-to-protein ratio decreases significantly at 4°C. Figure 3 As shown, PBS alone cannot effectively prevent exosome aggregation at 4°C, thus failing to ensure the stability of exosome particle number and protein content.

[0061] two, Zeta potential

[0062] (1) Investigating the Zeta potential of exosomes in PBS solution at 4℃:

[0063] Exosomal zeta potentials were measured and recorded for 7 consecutive days.

[0064] (2) Investigate the Zeta potential of the protective solution containing exosomes at 4℃:

[0065] The exosome samples were diluted 10 times with a protective solution and stored in a 4°C refrigerator. The exosome zeta potential was measured daily for 7 consecutive days, and the data were recorded.

[0066] The exosomes in Example 1 can maintain the stability of the zeta potential, thereby ensuring the stability of the exosome particles and preventing them from agglomerating. It can be seen that the protective agent provided in the example can ensure that the zeta potential of exosomes remains stable at 4°C, while the absolute value of the zeta potential of exosomes in the blank control group gradually decreases at 4°C, indicating that the repulsive effect between particles weakens and the particles are more prone to aggregation. As shown in Table 1, this demonstrates that PBS alone cannot effectively ensure the stability of the zeta potential of exosomes.

[0067] Table 1. Changes in Zeta potential in Example 1 and the control group

[0068] III. Membrane Flowability

[0069] (1) To investigate the membrane fluidity of exosomes in PBS solution at 4℃

[0070] Exosomes were labeled with the fluorescent probe DPH, and the membrane fluidity of the exosomes was detected by a fluorescence polarimeter. The data were recorded continuously for 7 days.

[0071] (2) Investigate the membrane fluidity of the protective solution containing exosomes at 4℃.

[0072] The exosome samples were diluted 10 times with a protective solution as the solvent and stored in a 4°C refrigerator. The exosomes were labeled daily with a fluorescent probe DPH, and the membrane fluidity of the exosomes was detected by a fluorescence polarimeter. The data were recorded after 7 consecutive days of detection.

[0073] Tests revealed that the exosomes in Example 1 maintained good membrane fluidity, ensuring sufficient mobility and facilitating fusion with the cell membrane. In the control group, the membrane fluidity of exosomes gradually decreased at 4°C, with reduced movement of lipid and protein molecules on the membrane, as shown in Table 2. Table A represents exosomes with good membrane fluidity; Table B represents exosomes with moderate membrane fluidity; and Table C represents exosomes with poor membrane fluidity. This indicates that PBS alone cannot effectively guarantee stable exosome membrane fluidity.

[0074] Table 2. Changes in membrane fluidity between Example 1 and the control group.

[0075] IV. MDA Content

[0076] (1) Investigate the MDA content of exosomes in PBS solution at 4℃.

[0077] The MDA content in exosomes was detected using the TBA method (thiobarbituric acid method) for 7 consecutive days, and the data were recorded.

[0078] (2) Investigate the cell uptake efficiency of exosome-containing protective solution at 4℃.

[0079] The exosome samples were diluted 10 times with a protective solution as the solvent and stored in a 4°C refrigerator. The MDA content in the exosomes was detected daily using the TBA method for 7 consecutive days, and the data were recorded.

[0080] The exosomes in Example 1 can slow down the growth of MDA in exosomes, thereby alleviating oxidative damage to exosomes. The curve showing the change in MDA content in exosomes is shown below. Figure 4 As shown in the figure, the protective agent provided in the example can slow down lipid peroxidation and reduce the growth of MDA. However, the MDA content of exosomes in the control group did not increase at 4°C, and the oxidative stress level in exosomes increased. This indicates that PBS alone cannot effectively ensure the normal level of exosome MDA and reduce the oxidative damage to exosomes.

[0081] V. TEM Morphology

[0082] (1) To investigate the morphology of exosomes in PBS solution at 4℃

[0083] On day 7, the morphology of exosomes was observed using TEM to evaluate the overall structural integrity, dispersion, and presence of aggregation or clustering of exosomes.

[0084] (2) Investigate the morphology of the protective fluid containing exosomes at 4℃.

[0085] On day 7, the morphology of exosomes was observed using TEM to evaluate the overall structural integrity, dispersion, and presence of aggregation or clustering of exosomes.

[0086] In Example 1, the exosomes maintained their standard goblet vesicle structure after 7 days, exhibiting intact morphology, good dispersibility, and virtually no aggregation or clumping. In the control group, the exosomes showed morphological and structural damage after 7 days. Only a small number of goblet vesicle structures were observed, with poor dispersibility and significant particle aggregation. This indicates that at 4°C, PBS alone cannot effectively maintain the integrity and dispersibility of exosomes.

[0087] To investigate the effects of each component, stabilizer composition preservation solutions for each set of examples and comparative examples were prepared, and their compositions are shown in Table 3:

[0088] Table 3. Compositions and dosages of compositions in Examples 1 to Comparative Examples 6

[0089] *The antioxidant in the table is Vitamin E-TPGS; the buffer is HEPES; and the metal ion chelating agent is disodium EDTA.

[0090] On day 7, the morphology of exosomes was observed using TEM to evaluate the overall structural integrity, dispersion, and presence of aggregation or clustering. The results of the exosome morphology observations are shown in Table 4.

[0091] Table 4. Preservation morphology effects of stabilizer compositions in preservation solutions of each group of examples and comparative examples.

[0092] From the perspective of morphology maintenance, using only PBS buffer as a blank control revealed a significant aggregation and degradation effect, resulting in complete loss of activity. Comparative Examples 1 and 2, using either cerebroside sulfate or phytosphingosine alone, still could not meet the requirements for long-term preservation of exosome morphology integrity. Combining Comparative Examples 1 and 2, the composition requires a combination of cerebroside sulfate and phytosphingosine to provide good support for the morphological integrity of exosomes under refrigeration. The addition of both has a significant synergistic effect on the protective effect of the protective solution in this application. Comparative Example 3, which only added trehalose, and Comparative Example 4, which had an excessively high proportion of phytosphingosine, both failed to maintain the exosome morphology well and provide adequate protection. Furthermore, Comparative Examples 5 and 6, even after replacing part of the protective component, could not avoid the aggregation effect. This indicates that specific protective components and their combination are necessary to achieve good preservation under refrigeration conditions.

[0093] The morphology of exosomes was observed after two weeks of refrigeration using the protective solution provided in Example 1. The results showed that the exosomes retained their cup-shaped vesicle structure, maintained their intact morphology, and exhibited virtually no aggregation or clustering. Therefore, the exosome refrigeration composition preservation solution of this invention, without animal-derived components, can maintain the physical integrity, high dispersibility, biological activity, and functional stability of exosomes for an extended period under conventional refrigeration conditions of 2-8°C.

[0094] Obviously, the above embodiments of the present invention are merely examples to clearly illustrate the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A stabilizer composition for exosome cold storage, characterized in that, Substances consisting of the following components and their contents composition: Phytosphingosine: 0.1%-0.2%; Cerebroside sulfate: 0.05%-0.1%; Trehalose: 2%-5%; Fat-soluble antioxidants: 0.01%-0.05%; HEPES buffer: 10-20 mM; Metal ion chelating agent: 0.01%-0.05%.

2. The stabilizer composition according to claim 1, characterized in that, The ratio of phytosphingosine to cerebroside sulfate is 2:1 to 4:

1.

3. The stabilizer composition according to claim 1, characterized in that, The fat-soluble antioxidant is selected from vitamin E-TPGS, vitamin E succinate, or coenzyme Q10.

4. The stabilizer composition according to claim 1, characterized in that, The metal ion chelating agent is selected from disodium EDTA, EGTA, or sodium citrate.

5. The stabilizer composition according to claim 1, characterized in that, The pH of the HEPES buffer solution is 7.0-7.

4.

6. A method for cryopreservation of exosomes, characterized in that, Includes the following steps: (1) Mixing exosomes with the stabilizer composition according to any one of claims 1 to 5; (2) Store at 2~8℃ away from light.

7. The method according to claim 6, characterized in that, The exosomes are mesenchymal stem cell exosomes.

8. The method according to claim 6, characterized in that, The concentration of the exosomes was 10. 8 ~10 11 particles / mL.

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