Preserving fluid for improving stability of external vesicles as well as preparation method and application of preserving fluid

Abstract

The invention relates to the technical field of protection of external vesicles, in particular to a preserving fluid for improving the stability of the external vesicles and a preparation method and application of the preserving fluid. The invention provides a preserving fluid for improving stability of external vesicles. The preserving fluid comprises an acidic buffer solution and a stabilizer, the acidic buffer solution is a citric acid buffer solution, and the stabilizer is cyclodextrin or a functionalized derivative thereof; and the pH value of the preservation solution is 6.0-7.0. The preserving fluid provided by the invention can stably store the external vesicles under the condition of low temperature (4 DEG C and-80 DEG C), and can effectively inhibit damage, such as membrane rupture and protein inactivation, of the external vesicles in the low-temperature storage process.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of extracellular vesicle protection, and particularly relates to a preservation solution for improving stability of extracellular vesicles, and a preparation method and application thereof. BACKGROUND

[0002] Extracellular vesicles (EVs) are important intercellular signal transmission carriers, and are widely used in the fields of disease diagnosis, drug delivery, immunotherapy, etc.

[0003] Extracellular vesicles are prone to freezing damage or structural changes caused by low temperature during the preservation process. When stored at 4℃ or -80℃, extracellular vesicles will undergo structural changes such as membrane rupture, protein aggregation and nucleic acid degradation. Structural changes will limit the widespread applicability of extracellular vesicles in large-scale research and clinical applications, especially when extracellular vesicles are used for vaccine development or drug delivery systems. Therefore, the stability of extracellular vesicles under extreme temperature conditions is crucial for their clinical applications.

[0004] Existing outer vesicle preservation solutions are mostly based on phosphate buffer solution (PBS), but their stability at lower temperatures is not ideal. To address this issue, existing research has used the addition of special ingredients to improve the stability of the preservative. Zhang et al. (Zhang, T. Y., Tan, P. C., Xie, Y. et al. The combination of trehalose and glycerol: an effective and non-toxic recipe for cryopreservation of human adipose-derived stem cells. Stem Cell Res Ther 11, 460 (2020). https: / / doi.org / 10.1186 / s13287-020-01969-0) and Patel et al. (Patel, M., Park, J. K. & Jeong, B. Rediscovery of poly(ethylene glycol)s as a cryoprotectant for mesenchymal stem cells. Biomater Res 27, 17 (2023). https: / / doi.org / 10.1186 / s40824-023-00356-z) proposed adding certain antifreeze agents or protective molecules, such as glycerol, dimethyl sulfoxide (DMSO), or polyethylene glycol (PEG), to PBS. However, this only improves the preservation effect of the preservative for cell preservation and cannot fully meet the stability required for long-term storage of outer vesicles. Patents WO2019155060A1 and US11337419B2 also propose using specific chemical components, such as sugars, amino acids, or peptides, to improve the stability of outer vesicles. These components can improve the long-term preservation effect of outer vesicles under different temperature conditions by enhancing the antifreeze properties of the outer vesicle membrane, reducing the fluidity of the membrane, and protecting active ingredients in the outer vesicle.

[0005] Existing methods for preserving outer vesicles are often complex and still face certain limitations in practical applications. Therefore, a simple and highly stable preservative is needed to protect outer vesicles. SUMMARY

[0006] The present application provides a preservative for improving the stability of outer vesicles, as well as a preparation method and application thereof. The preservative can significantly improve the stability of outer vesicles at 4°C and -80°C.

[0007] To achieve the above-mentioned purposes, the present application provides the following technical solutions:

[0008] The application provides a storage solution for improving the stability of outer vesicles, comprising an acidic buffer and a stabilizer; the acidic buffer is a citric acid buffer, and the stabilizer is cyclodextrin or a functional derivative thereof; the pH of the storage solution is 6.0-7.0.

[0009] Compared with the traditional PBS storage solution, the storage solution of the application utilizes the special action between the citric acid buffer and the cyclodextrin or the functional derivative thereof, while ensuring that the overall pH is 6.0-7.0, so that the outer vesicles can be stably stored at low temperature (4°C and -80°C).

[0010] Specifically, the cyclodextrin can reduce the aggregation of the outer vesicles, and by wrapping the outer vesicles through the hollow truncated cone structure of the cyclodextrin, the damage of the external environment (such as temperature, light, mechanical stress) to the active ingredients of the outer vesicles can be reduced, and the stability of the outer vesicles at a lower temperature (2-8°C) can be improved. The citric acid buffer effectively reduces the generation of free radicals by chelating metal ions (such as Fe 2+ , Cu 2+ ), and delays the oxidation of biomolecules. Compared with other buffers, the citric acid buffer has a wide range of pH adaptability, and can cover pH 3.0-6.6 by adjusting the proportion, and is suitable for scenes that require acidic to near neutral conditions (such as anticoagulation, antigen repair).

[0011] Finally, the storage solution provided by the application can effectively inhibit the damage of the outer vesicles during low-temperature storage, such as membrane rupture and protein inactivation. At the same time, the storage solution of the application can maintain the significant inhibitory effect of the outer vesicles on Staphylococcus aureus; the activity of Staphylococcus aureus as a biological activity index for evaluating the stability of the outer vesicles, and the significant inhibitory effect can prove that the stability of the outer vesicles has been significantly improved.

[0012] Preferably, the pH of the storage solution is 6.0-6.5.

[0013] Preferably, the pH is adjusted with hydrochloric acid.

[0014] Preferably, the citric acid buffer comprises a citrate.

[0015] Preferably, the final concentration of the citrate in the storage solution is 10-30 mM.

[0016] Further preferably, the final concentration of the citrate in the storage solution is 15-25 mM.

[0017] Further preferably, the final concentration of the citrate in the storage solution is 20 mM.

[0018] Preferably, the citrate is at least one of sodium citrate and trisodium citrate.

[0019] Preferably, the citrate is sodium citrate.

[0020] Preferably, the citrate buffer further comprises citric acid.

[0021] Preferably, the citrate buffer further comprises a soluble salt.

[0022] Preferably, the soluble salt is at least one of a soluble sodium salt, a soluble calcium salt, a soluble magnesium salt, and a soluble potassium salt.

[0023] The soluble sodium salt can be sodium chloride, and the soluble magnesium salt can be magnesium chloride.

[0024] Preferably, the final concentration of the soluble salt in the preservation solution is 60-90 mM.

[0025] Further preferably, the final concentration of the soluble salt in the preservation solution is 80 mM.

[0026] Preferably, the amount of the stabilizer is 3-8 wt% of the preservation solution.

[0027] Further preferably, the amount of the stabilizer is 3-6 wt% of the preservation solution.

[0028] Further preferably, the amount of the stabilizer is 5 wt% of the preservation solution.

[0029] Preferably, the stabilizer is hydroxypropyl-β-cyclodextrin.

[0030] Hydroxypropyl-β-cyclodextrin is connected by 7 glucose units through α-1, 4-glycosidic bonds to form a hollow truncated cone structure, and the hydrophilic outside and the hydrophobic cavity enable it to form a complex with hydrophobic molecules through inclusion, which has various applications in pharmaceutical preparations, including improving solubility, stability, bioavailability, improving drug taste, controlling release rate, targeted delivery, and reducing toxicity, etc., and is an important auxiliary material and tool in the field of pharmaceutical preparations. Hydroxypropyl-β-cyclodextrin can be used as an adjuvant for vaccines, inducing Th2 cell response, promoting specific antibody production, and enhancing long-term immune memory. In addition, hydroxypropyl-β-cyclodextrin can replace traditional surfactants to reduce the particle size dispersion of lipid nanoparticles and improve the stability during freeze-drying.

[0031] The application provides a preparation method of a preservation solution for improving the stability of exovesicles, comprising: preparing a citrate buffer, adding cyclodextrin or a functional derivative thereof, and then adjusting the pH to 6.0-7.0 with hydrochloric acid to obtain the preservation solution for improving the stability of exovesicles.

[0032] The application provides an application of the preservation solution for improving the stability of exovesicles in improving the stability of exovesicles.

[0033] Preferably, the preservation solution can improve the stability of the outer vesicle at-80 to 4 DEG C.

[0034] The outer vesicle can effectively inhibit Staphylococcus aureus at 2 to 8 DEG C, and the particle size of the outer vesicle does not change obviously after being stored at-80 DEG C for 8 weeks. The above effects can prove that the preservation solution has the effect of stabilizing the outer vesicle.

[0035] Therefore, the present application has the following beneficial effects:

[0036] (1) The preservation solution provided by the present application can store the outer vesicle at low temperature (4 DEG C and-80 DEG C) conditions, can effectively inhibit the damage of the outer vesicle during low temperature storage and maintain the function, such as membrane rupture, protein inactivation, etc.

[0037] (2) The preservation solution provided by the present application can effectively inhibit Staphylococcus aureus, and the particle size of the outer vesicle does not change obviously after being stored at-80 DEG C for 8 weeks.

[0038] (3) The idea of preparing the preservation solution provided by the present application can provide more stable outer vesicle resources for the fields of vaccine research and drug delivery, and provide strong support for related research. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The transmission electron micrographs of the outer vesicles in different formulations extracted freshly;

[0040] Figure 2 The Western blot detection results of the marker proteins of the outer vesicles in different formulations extracted freshly;

[0041] Figure 3 The inhibition rate results of Staphylococcus aureus growth by the outer vesicles of formula 3 with different concentrations;

[0042] Figure 4 The inhibition rate results of Staphylococcus aureus growth by the outer vesicles (0.625 mg / mL) in different formulations after being stored at 2 to 8 DEG C for different time;

[0043] Figure 5 The particle size distribution diagram of the outer vesicles in different formulations after being stored at-80 DEG C for 8 weeks. DETAILED DESCRIPTION

[0044] The application will be further described below with reference to specific examples. Those skilled in the art will be able to implement the application based on these descriptions. In addition, the examples of the application described in the following description are generally only examples of a part of the application, rather than all examples. Therefore, all other examples obtained by those skilled in the art based on the examples in the application without creative work shall fall within the scope of protection of the application.

[0045] In this part, the raw material sources are as follows: phosphate buffer solution (PBS), pH 7.4, purchased from labshark; milk is collected from a farm and not pasteurized; rennet is purchased from Clerici; hydroxypropyl-β-cyclodextrin is purchased from Shifeng Biological; sodium citrate is purchased from Shanghai Testing; sodium chloride is purchased from Shanghai Testing; hydrochloric acid, 30% HCl, is purchased from Keda.

[0046]

EXAMPLE

[0047] Extracellular vesicles (mEVs) preparation

[0048] Fresh milk is taken, and casein in the milk is precipitated by using rennet, and then centrifuged at 8000g. The supernatant is taken after centrifugation, and the precipitated protein and fat are removed. After being filtered through a 1.5μm filter and a 0.5μm filter respectively, the supernatant is concentrated by ultrafiltration, and then the protein is removed by size exclusion. The flow-through obtained is the purified extracellular vesicle solution, which is then filtered through a 0.22μm filter to obtain the extracellular vesicle solution.

[0049] Example 1 (Formula 2)

[0050] A preservation solution of a citric acid buffer system is prepared, which is named Formula 2. In 500mL of deionized water, sodium citrate, sodium chloride, and hydroxypropyl-β-cyclodextrin are added, and the pH is adjusted to 6.0 by using 30% HCl to obtain the preservation solution. In the preservation solution, the final concentration of sodium citrate is 20mM, the final concentration of sodium chloride is 80mM, and the amount of hydroxypropyl-β-cyclodextrin is 5wt% of the preservation solution. Bacteria are removed by using a 0.22μm PES membrane, and the solution is stored at room temperature for standby.

[0051] Example 2 (Formula 3)

[0052] A preservation solution of a citric acid buffer system is prepared, which is named Formula 3. In 500mL of deionized water, sodium citrate, sodium chloride, and hydroxypropyl-β-cyclodextrin are added, and the pH is adjusted to 6.5 by using 30% HCl to obtain the preservation solution. In the preservation solution, the final concentration of sodium citrate is 20mM, the final concentration of sodium chloride is 80mM, and the amount of hydroxypropyl-β-cyclodextrin is 5wt% of the preservation solution. Bacteria are removed by using a 0.22μm PES membrane, and the solution is stored at room temperature for standby.

[0053] Comparative Example 1 (Formula 1)

[0054] Prepare the preservation solution for the PBS system. Add hydroxypropyl-β-cyclodextrin to 500 mL of PBS, and adjust the pH to 7.4 with 30% HCl to obtain the preservation solution, named Formula 1. The amount of hydroxypropyl-β-cyclodextrin in the preservation solution is 5 wt%. Sterilize using a 0.22 μm PES membrane and store at room temperature for later use.

[0055] Comparative Example 2 (Formula 4)

[0056] Prepare a preservation solution using a citrate buffer system, designated Formula 2. Add sodium citrate, sodium chloride, and hydroxypropyl-β-cyclodextrin to 500 mL of deionized water. Adjust the pH to 7.4 with 30% HCl to obtain the preservation solution. The final concentration of sodium citrate in the preservation solution is 20 mM, the final concentration of sodium chloride is 80 mM, and the amount of hydroxypropyl-β-cyclodextrin is 5 wt%. Sterilize using a 0.22 μm PES membrane and store at room temperature for later use.

[0057] [Performance Testing]

[0058] 1. Morphological confirmation

[0059] Stability test: The preservation solutions obtained from formulations 1 to 4 and pure PBS preservation solution were added to the ultrafiltration concentration step of "external vesicle preparation" to obtain external vesicles with different preservation solutions, a total of 5 groups corresponding to formulations 1 to 4 and PBS.

[0060] The external vesicles obtained from the "stability test" were identified by transmission electron microscopy and Western blotting, and the results are as follows: Figures 1-2 As shown. Observation Figure 1 It can be seen that the freshly prepared exovesicles have relatively intact structures in all formulations and in the preservation solution of PBS, and the classic saucer-like shape of the exovesicles can be observed. Figure 2 In this study, CD81+ and TSG101+ were used as markers for the determination of external vesicles, which proved that external vesicles were obtained.

[0061] 2. Antibacterial performance evaluation

[0062] The antibacterial effect of external vesicles on Staphylococcus aureus (SA) was evaluated.

[0063] The five groups of external vesicles obtained in “1. Morphological Confirmation-Stability Test” were diluted to 10 mg / mL with the corresponding preservation solutions to obtain five groups of samples containing external vesicles at a concentration of 10 mg / mL.

[0064] Specific antibacterial experiments:

[0065] Staphylococcus aureus culture activated from a single colony was diluted to 5 × 10⁻⁶ using TSB medium. 4The bacteria inhibition experiment was carried out after the CFU / mL was obtained. The bacteria inhibition experiment of the five groups of different storage solutions was carried out according to the following specific experiment.

[0066] The specific experiment of each group of exosome-containing storage solution was as follows: the operation was carried out on a 96-well bacterial culture plate, and the final volume of each well was 100 μL. 200 μL of exosome-containing storage solution (concentration of 10 mg / mL) was added to the second well, and the concentration of the exosome-containing storage solution in the second to sixth wells was 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.313 mg / mL and 0.156 mg / mL respectively by using a two-fold dilution method. 100 μL of diluted S. aureus solution (5 x 10 4 CFU / mL) was added to the experimental wells, mixed well, and incubated at 37°C for 18 hours. Then, the OD600 value was detected, and compared with the OD600 value of the S. aureus solution without exosome addition. The inhibition rate of exosome on S. aureus growth was calculated as follows: inhibition rate = 1- (OD600 of S. aureus solution with exosome addition / OD600 of S. aureus solution without exosome addition).

[0067] During the bacteria inhibition experiment, the exosome-containing storage solution of formula 3 with different concentrations was stored at 2-8°C for 8 weeks, and the samples were taken at 2 weeks, 4 weeks, 6 weeks and 8 weeks to detect the bacteria inhibition effect of the exosome-containing storage solution with different concentrations on the S. aureus solution. The higher the concentration of exosome, the more obvious the bacteria inhibition effect, as shown in Figure 3 .

[0068] The storage results of different storage solutions are shown in Figure 4 . It can be observed that the bacteria inhibition activity of exosome in formula 1 is obviously better than that in PBS at the same concentration of 0.625 mg / mL, which indicates that hydroxypropyl-β-cyclodextrin can improve the thermal stability of exosome at 2-8°C, but the bacteria inhibition effect of exosome in formula 1 decreases significantly after being stored in formula 1 for 8 weeks, and the bacteria inhibition effect of exosome in formula 4 decreases at the 6th week. The exosome in storage solutions 2 and 3 still maintains the bacteria inhibition effect after being stored at 2-8°C for 8 weeks, which indicates that the combination of hydroxypropyl-β-cyclodextrin and certain acidic environment is the best for maintaining the functional stability of exosome.

[0069] 3. Particle size stability evaluation

[0070] The exosome extracted from the exosome in formula 2, formula 4 and PBS in the three groups of different storage solutions after being stored at -80°C for 8 weeks was diluted 40000 times with PBS buffer. The particle size of the exosome was detected by ZetaView, and the results are shown in Figure 5 .

[0071] It can be observed that the particle size of the exosome in formula 2 is smaller than that in formula 4 and PBS, and the particle size of the exosome in formula 4 is smaller than that in PBS. Figure 5It can be seen that the particle size of the vesicles of Formulation 2 did not change significantly after 8 weeks. The vesicles of PBS and Formulation 4 showed peaks at 200-500 nm, indicating the presence of large particles. In particular, the peaks at 200-500 nm increased in number and height after 8 weeks of storage of the vesicles of PBS, indicating that the number of large particles increased over time.

Claims

1. A preservation solution for improving the stability of exovesicles, characterized in that, It includes an acidic buffer solution and a stabilizer; the acidic buffer solution is a citrate buffer solution, and the stabilizer is cyclodextrin or its functionalized derivative; the pH of the preservation solution is 6.0 to 7.

0.

2. The preservation solution as described in claim 1, characterized in that, The pH of the preservation solution is 6.0 to 6.

5.

3. The preservation solution as described in claim 1, characterized in that, The citrate buffer solution comprises citrate; Preferably, the final concentration of the citrate in the preservation solution is 10–30 mM; Preferably, the citrate is at least one of sodium citrate and trisodium citrate; Preferably, the citrate is sodium citrate.

4. The preservation solution as described in claim 3, characterized in that, The citrate buffer also includes citric acid.

5. The preservation solution as described in claim 3, characterized in that, The citrate buffer also includes soluble salts; Preferably, the soluble salt is at least one selected from soluble sodium salt, soluble calcium salt, soluble magnesium salt, and soluble potassium salt; Preferably, the final concentration of the soluble salt in the preservation solution is 60–90 mM.

6. The preservation solution as described in claim 1, characterized in that, The amount of stabilizer used is 3-8 wt% of the preservation solution; Preferably, the stabilizer is hydroxypropyl-β-cyclodextrin.

7. The method for preparing the preservation solution according to any one of claims 1 to 6, characterized in that, include: A citrate buffer solution was prepared by adding cyclodextrin or its functionalized derivatives, and then adjusting the pH to 6.0–7.0 with hydrochloric acid to obtain a preservation solution that enhances the stability of the outer vesicles.

8. The application of the preservation solution according to any one of claims 1 to 6 or the preservation solution prepared by the method according to claim 7 in improving the stability of exovesicles, characterized in that, The preservation solution can improve the stability of the outer vesicles at temperatures ranging from -80°C to 4°C.

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