Preservation method and preservation solution for exosomes

Abstract

The invention relates to a preservation method and a preservation solution of exosomes, and belongs to the technical field of biology, the preservation solution comprises trehalose, cane sugar, sodium hyaluronate, polyethylene glycol, dextran sulfate, an HEPES buffer solution and mannitol, the preservation method comprises the following steps: mixing purified exosomes with the exosome protection solution to obtain a mixture, and adding the mixture into the exosome protection solution to obtain the exosome preservation solution. Sub-packaging the mixture, and directly cryopreserving the sub-packaged sample at the temperature of-80 DEG C or lower; in a word, through a rigid network formed by the sodium hyaluronate and the polyethylene glycol, the aggregation and fusion of the exosome in the freezing and thawing process are effectively inhibited. The embodiment shows that after three times of freeze-thaw cycles, the particle size change rate of the exosome is less than 6%, and the particle recovery rate is close to 90%.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method and preservation solution for the preservation of exosomes. Background Technology

[0002] Exosomes, especially engineered ones (e.g., loaded with nucleic acid drugs, proteins, or displaying targeting ligands on their surface), have become a potential breakthrough tool for disease treatment and diagnosis. However, their commercialization faces severe challenges in long-term storage stability. Existing cryopreservation methods (e.g., storage at -80°C in PBS buffer containing trehalose, human serum albumin, or high molecular weight polymers) can partially maintain the physical integrity (particle size, concentration) of exosomes, but after repeated freeze-thaw cycles or long-term storage, the core functional properties of engineered exosomes—the activity of surface-engineered proteins (e.g., targeting ligands, receptors) and the integrity of internal bioactive payloads (e.g., mRNA, siRNA)—can be significantly diminished.

[0003] Existing technologies offer limited protection of the surface functional activity of engineered exosomes that require repeated use (i.e., undergoing multiple freeze-thaw cycles), which becomes a key bottleneck restricting their large-scale application, quality control, and clinical administration.

[0004] The reason for this is that conventional preservatives mainly provide "rigid support" to inhibit physical aggregation and ice crystal damage, but lack a "functional stabilization" mechanism to address the disturbances in the protein microenvironment on the membrane surface during freeze-thaw cycles (such as conformational changes caused by dehydration-rehydration and charge shielding inactivation due to drastic changes in local ionic strength). Therefore, developing a novel preservation solution that can synergistically address the dual degradation of physical integrity and functional activity is an urgent need in this field. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and to propose a method and preservation solution for the preservation of exosomes.

[0006] The technical solution of the present invention is as follows:

[0007] An exosome preservation solution comprising the following components:

[0008] (a) Trehalose and sucrose, with a total concentration of 100-200 mM;

[0009] (b) Sodium hyaluronate with a molecular weight of 1000-1800 kDa and a concentration of 0.05%-0.2% w / v;

[0010] (c) Polyethylene glycol with a molecular weight of 8000-12000 and a concentration of 0.5%-1.5% w / v;

[0011] (d) Dextran sulfate with a molecular weight of 40-100 kDa and a sulfur content of 15%-20%, at a concentration of 0.01%-0.1% w / v;

[0012] (e) HEPES buffer, concentration 10-30 mM;

[0013] (f) Mannitol, at a concentration of 100-200 mM;

[0014] The pH value of the preservation solution is 7.2-7.4.

[0015] Furthermore, the molar ratio of trehalose to sucrose is (1:1) to (3:1).

[0016] Furthermore, the molecular weight of the sodium hyaluronate is 1500±200kDa.

[0017] Furthermore, the molecular weight of the sulfated dextran is 70±20 kDa.

[0018] A method for preserving exosomes, using the above-mentioned exosome preservation solution, includes the following steps:

[0019] S1. The purified exosomes are mixed with the exosome preservation solution to obtain a mixture;

[0020] S2. Dispense the mixture into portions;

[0021] S3. Place the dispensed samples directly at -80°C or lower for freezing.

[0022] Furthermore, the cryopreservation described in step S3 is a one-step cryopreservation method.

[0023] Furthermore, it also includes a resuscitation step: the frozen sample is rapidly shaken in a 37°C water bath to revive, and the revival time does not exceed 2 minutes.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] 1. Excellent physical integrity protection: The rigid network composed of sodium hyaluronate and polyethylene glycol effectively inhibits the aggregation and fusion of exosomes during the freeze-thaw process. Examples show that after three freeze-thaw cycles, the exosome particle size change rate is less than 6%, and the particle recovery rate is close to 90%.

[0026] 2. Breakthrough Functional Activity Retention: Thanks to the introduction of dextran sulfate and its synergy with the rigid network, this invention can significantly maintain the surface functional activity of engineered exosomes. Experiments show that after three freeze-thaw cycles, the surface-exhibited binding activity against HER2 scFv can still be maintained at over 85%, which is significantly better than the control system without dextran sulfate.

[0027] 3. Strong resistance to repeated freeze-thaw cycles: This solution is specifically designed for repeated freeze-thaw scenarios. Its synergistic protection mechanism can effectively cope with the cumulative damage caused by repeated freeze-thaw cycles, providing feasibility for multiple sampling of exosome sample banks and clinical fractionated administration.

[0028] 4. Clear Ingredients and Safety: The formula avoids complex animal-derived components (such as serum) and uses mannitol to replace sodium chloride, which may cause the "salting-out effect." The ingredients are clearer, the biocompatibility is better, and it is more suitable for the development of clinical-grade products. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0030] Example 1

[0031] A cryoprotectant for preserving exosomes, wherein the cryoprotectant comprises the following essential components dissolved in water for injection, with the pH adjusted to 7.2-7.4:

[0032] Basic carbohydrate protectants:

[0033] Trehalose: Concentration 50-100mM.

[0034] Sucrose: Concentration 50-100mM.

[0035] Function: Provides basic water displacement and glass formation capabilities at a low cost with stable results.

[0036] Rigid support network components:

[0037] High molecular weight sodium hyaluronate (HA): molecular weight 1,000-1,800 kDa, concentration 0.05%-0.2% w / v. HA in this molecular weight range is chosen to form a sufficiently long and stable physically entangled network.

[0038] Polyethylene glycol (PEG): molecular weight 8,000-12,000, concentration 0.5%-1.5% w / v. PEG fills the gaps in the HA network, providing additional steric hindrance and inhibiting ice crystal growth.

[0039] Core components of functionally stable networks (key distinguishing features):

[0040] Dextran sulfate (DS): molecular weight 40-100 kDa, sulfur content 15%-20%, concentration 0.01%-0.1% w / v. This is a key innovative additive, and its charge density and molecular weight must be strictly controlled within this range to achieve the best balance between functional stability and biocompatibility.

[0041] Optimize the buffer system:

[0042] Buffer salt: 10-30mM HEPES.

[0043] Osmotic pressure regulator: 100-200mM mannitol.

[0044] Completely avoid using small molecule salts such as NaCl and KCl to eliminate irreversible denaturation of membrane proteins caused by salt crystallization or localized surges in concentration ("salting-out effect") during freeze-thaw cycles.

[0045] Example 2:

[0046] An exosome preservation solution, with the following specific formula:

[0047] Trehalose: 150 mM;

[0048] Sucrose: 50mM;

[0049] Sodium hyaluronate (molecular weight 1500kDa): 0.1% w / v;

[0050] Polyethylene glycol (PEG10,000): 1.0% w / v;

[0051] Dextran sulfate (molecular weight 70 kDa, sulfur content 18%): 0.05% w / v;

[0052] HEPES: 20mM;

[0053] Mannitol: 150mM;

[0054] Dissolve the above components in water for injection, adjust the pH to 7.4 with 1M NaOH, bring the volume to a final volume, filter through a 0.22μm filter membrane for sterilization, and store at 4℃ for later use.

[0055] Example 3

[0056] A method for preserving exosomes, comprising the following:

[0057] Preparation of protective agent: Dissolve each component in water for injection according to the proportions in the above examples, stir until completely dissolved, adjust the pH to 7.2-7.4 with dilute NaOH or HCl, filter with a 0.22μm filter membrane for sterilization, and store at 4℃ for later use.

[0058] Exosome preparation: purified engineered exosomes (e.g., MSC exosomes loaded with siRNA and displaying anti-HER2 scFv on their surface).

[0059] Mixing and freezing: Gently resuspend the exosome precipitate with this cryoprotectant and aliquot into cryovials. Store directly at -80°C using the "one-step" method, avoiding contact with -20°C.

[0060] Resuscitation: Shake rapidly in a 37°C water bath for <2 minutes before use. Use immediately after resuscitation.

[0061] Examples and synergistic effect verification

[0062] Preparation and freeze-thaw stability testing of engineered exosomes

[0063] 1. Preparation of engineered exosomes:

[0064] The mRNA encoding luciferase was introduced into exosomes derived from human mesenchymal stem cells using electroporation, and anti-HER2 single-chain antibody fragments (scFv) were stably expressed on their surface using genetic engineering techniques to obtain targeted engineered exosomes loaded with reporter genes (Exo-scFv-Luc).

[0065] 2. Experimental grouping and treatment:

[0066] Equal amounts of Exo-scFv-Luc were resuspended in the following five liquids:

[0067] Control group A: PBS buffer (pH 7.4).

[0068] Control group B: Basic sugar solution (containing 150mM trehalose, 50mM sucrose, 20mM HEPES, 150mM mannitol, pH 7.4).

[0069] Control group C: Basic sugar solution + 0.1% sodium hyaluronate + 1.0% PEG 10000 (simulating rigid support network).

[0070] Control group D: Basic sugar solution + 0.05% dextran sulfate (simulating functional stable component).

[0071] This invention group: Complete preservation solution prepared in Example 2.

[0072] After aliquoting each resuspended sample, place it directly in an ultra-low temperature freezer at -80℃. Perform three complete cycles of "freezing at -80℃ for 24 hours / rapid recovery in a 37℃ water bath (1.5 minutes)".

[0073] 3. Testing and Results:

[0074] Immediately after the third resuscitation, the samples were subjected to the following tests, and the results are summarized in Table 1.

[0075] Table 1: Comparison of performance indicators of exosomes after 3 freeze-thaw cycles in each experimental group

[0076] detection indicators Control group A (PBS) Control group B (basal solution) Control group C (HA+PEG) Control group D(DS) This invention group Particle size change rate (%) +45.2±6.1 +22.3±4.5 +8.1±2.3 +18.5±3.8 +5.3±1.5 Particle recovery rate (%) 35.7±4.8 68.2±5.1 82.5±3.2 71.4±4.6 89.8±2.7 scFv binding activity retention rate (%) 21.5±5.2 58.7±6.9 65.3±5.5 70.1±4.8 85.6±3.1 Internal luciferase activity retention rate (%) 15.8±4.1 62.3±7.2 70.8±5.0 66.9±5.3 83.4±3.9

[0077] Note: The particle size change rate is based on the initial particle size before freezing; the activity retention rate is calculated with the activity of the fresh sample before freezing as 100%.

[0078] 4. Results Analysis and Proof of Synergistic Effect:

[0079] Physical protection: The control group C (HA+PEG) showed the best performance in terms of particle size stability and particle recovery rate, demonstrating the physical protection advantage of the rigid network. Our invention group further optimized these indicators, showing that the addition of dextran sulfate (DS) positively contributes to the physical network.

[0080] Functional activity protection: Control group D (DS) was superior to control group C (HA+PEG only, 65.3%) in terms of protective surface scFv binding activity (70.1%), highlighting the unique stabilizing effect of DS on membrane protein function.

[0081] Significant synergistic gain: The scFv binding activity retention rate of the group in this invention was as high as 85.6%. This value is significantly higher than the individual effects of control group C (65.3%) and control group D (70.1%). If it were simply a superposition, the expected effect should be between or slightly higher than the two, but the actual result of 85.6% far exceeded this linear expectation, clearly demonstrating a strong synergistic effect between the HA / PEG rigid network and the DS functionally stable network.

[0082] Overall Results: This invention achieves an optimal balance across all core indicators, particularly in maintaining the most critical functional activity of engineered exosomes, demonstrating a breakthrough effect.

[0083] Long-term preservation verification

[0084] The Exo-scFv-Luc was resuspended in the solution of this invention and stored at -80°C. Samples were taken and analyzed after 1 month, 3 months, and 6 months of storage. The results showed that after 6 months of storage, the exosome particle recovery rate was >85%, the scFv binding activity retention rate was >80%, the internal luciferase activity retention rate was >78%, and the particle size distribution remained stable (PDI < 0.2). This demonstrates that the preservation solution of this invention is also effective for long-term storage.

[0085] Comparative example: Verifying the necessity of components

[0086] A preservation solution without dextran sulfate, but with all other components identical to the solution of this invention, was prepared, and the freeze-thaw experiment was repeated. Its scFv binding activity retention rate was only 68.2%, significantly lower than the 85.6% of the solution in this invention, and the particle recovery rate also decreased to approximately 83%. This indirectly confirms that dextran sulfate, as a functionally stable core component, is indispensable for achieving the best synergistic protective effect.

[0087] In summary, the protective solution of this invention utilizes a rigid support network formed by high molecular weight sodium hyaluronate and polyethylene glycol. This network primarily maintains the physical integrity of exosomes (particle size stability and prevention of aggregation) through steric hindrance and inhibition of ice crystal growth. Meanwhile, dextran sulfate, with its high-density negative charge, forms a dynamic hydrated ion cloud-molecular chaperone composite layer on the exosome membrane surface (especially in positively charged protein-modified regions). This layer stabilizes the membrane protein conformation and maintains a local hydration microenvironment, thereby specifically protecting the activity of surface-engineered proteins. The combination of these two components forms a rigid support-functional stability interpenetrating synergistic network, effectively solving the industry-wide problem of functional activity loss in engineered exosomes during freeze-thaw cycles.

[0088] 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 preservation solution for exosomes, characterized in that: It contains the following components: (a) Trehalose and sucrose, with a total concentration of 100-200 mM; (b) Sodium hyaluronate with a molecular weight of 1000-1800 kDa and a concentration of 0.05%-0.2% w / v; (c) Polyethylene glycol with a molecular weight of 8000-12000 and a concentration of 0.5%-1.5% w / v; (d) Dextran sulfate with a molecular weight of 40-100 kDa and a sulfur content of 15%-20%, at a concentration of 0.01%-0.1% w / v; (e) HEPES buffer, concentration 10-30 mM; (f) Mannitol, at a concentration of 100-200 mM; The pH value of the preservation solution is 7.2-7.

4.

2. The exosome preservation solution according to claim 1, characterized in that: The molar ratio of trehalose to sucrose is (1:1) to (3:1).

3. The exosome preservation solution according to claim 1, characterized in that: The molecular weight of the sodium hyaluronate is 1500±200kDa.

4. The exosome preservation solution according to claim 1, characterized in that: The molecular weight of the sulfated dextran is 70±20 kDa.

5. A method for preserving exosomes, characterized in that: Using the exosome protection solution according to any one of claims 1-4, and comprising the following steps: S1. The purified exosomes are mixed with the exosome preservation solution to obtain a mixture; S2. Dispense the mixture into portions; S3. Place the dispensed samples directly at -80°C or lower for freezing.

6. The exosome preservation method according to claim 5, characterized in that, The cryopreservation described in step S3 is a one-step cryopreservation method.

7. The exosome preservation method according to claim 5, characterized in that, It also includes a resuscitation step: rapidly shake the frozen sample in a 37°C water bath to revive it, with the revival time not exceeding 2 minutes.