Application of zwitterion in improvement of cryopreservation efficiency and particle size uniformity of exosome

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to application of zwitterions in improvement of cryopreservation efficiency and particle size uniformity of exosomes. The invention provides the application of zwitterions in any one of the following aspects: 1) the cryopreservation efficiency and the particle size uniformity of the exosome are improved; 2) preparing a reagent for improving cryopreservation efficiency and particle size uniformity of the exosome; wherein the zwitterion is selected from one or a combination of betaine, sulphobetaine and phosphorylcholine. The invention finds that by using the zwitterion, the cryopreservation efficiency of the exosome can be greatly improved, and the protective efficacy of the exosome is effectively enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and specifically relates to the application of zwitterions in improving the cryopreservation efficiency and particle size uniformity of exosomes. Background Art

[0002] Exosomes are small membrane vesicles (30-150nm) containing complex RNA and proteins. All cultured cell types can secrete exosomes, and exosomes exist naturally in body fluids, including blood, saliva, urine, cerebrospinal fluid and breast milk. In recent years, exosomes, as a new type of biological mediator, have shown great potential in biomedical applications. First, as biomarkers, exosomes have potential applications in clinical diagnosis and monitoring. Exosomes carry abundant biomolecules, such as proteins, nucleic acids and metabolites, which can be used as biomarkers for diseases for early diagnosis, prognosis assessment and monitoring of treatment effects. For example, miRNA in exosomes has been widely studied for early diagnosis and prognosis assessment of tumors. Secondly, exosomes have also shown potential application value in drug delivery and gene therapy. Exosomes can be used as a natural nano drug delivery system. By modifying the surface proteins of exosomes or directly loading drugs, targeted delivery of drugs can be achieved, thereby improving the bioavailability of drugs and reducing toxic side effects. In addition, exosomes can also be used as an effective gene delivery tool, entering target cells through the nucleic acids they carry and exerting biological effects, thereby achieving the purpose of gene therapy. Finally, exosomes also have potential applications in tissue repair and regeneration. Studies have shown that exosomes can promote cell proliferation and migration, regulate cell differentiation and repair, and thus play a positive role in the process of tissue repair and regeneration.

[0003] However, a bottleneck in the current exosome technology is that the extracted exosomes are usually stored in PBS or other buffer solutions and then directly frozen. After freezing and thawing, the exosome vesicles will rupture and the effective ingredients in the contents will flow out, thus affecting the use of exosomes in clinical diagnosis and testing.

[0004] CN 112514892A discloses an exosome cryopreservation protection solution and a preparation method thereof. However, the cryopreservation solution has a complex formula and contains human protein components and animal lipid concentrate components, which is not conducive to the next application of exosomes after cryopreservation and resuscitation. Summary of the invention

[0005] The present invention unexpectedly discovered that by using zwitterions such as betaine, sulfobetaine and phosphorylcholine, the cryopreservation efficiency and particle size uniformity of exosomes can be greatly improved.

[0006] Based on the above findings, the present invention first provides the application of zwitterions in any of the following aspects:

[0007] 1) Improve the cryopreservation efficiency and particle size uniformity of exosomes;

[0008] 2) Preparation of reagents for improving exosome cryopreservation efficiency and particle size uniformity;

[0009] Wherein, the zwitterion is selected from one or a combination of betaine, sulfobetaine and phosphorylcholine.

[0010] As a preferred embodiment, each 100 mL of exosome cryopreservation protection solution contains 1 to 10 g of the zwitterion.

[0011] As a further preferred embodiment, the content of the zwitterion is 3 to 10 g / 100 mL, more preferably 5 to 10 g / 100 mL.

[0012] As a preferred embodiment, the exosomes are derived from one or more of stem cells, immune cells, epithelial cells, connective tissue cells, neural cells and virus-infected cells.

[0013] As a preferred embodiment, the exosomes are used to deliver mRNA.

[0014] The present invention also provides an exosome cryopreservation protection solution, which contains zwitterions, and the zwitterions are selected from one or a combination of betaine, sulfobetaine and phosphorylcholine.

[0015] As a preferred embodiment, each 100 mL of exosome cryopreservation protection solution contains 1 to 10 g of the zwitterion.

[0016] As a further preferred embodiment, the content of the zwitterion is 3 to 10 g / 100 mL, more preferably 5 to 10 g / 100 mL.

[0017] As a preferred embodiment, the exosome cryopreservation protection solution also contains 15-25 mM sodium citrate.

[0018] As a further preferred embodiment, the content of sodium citrate is 20±2 mM.

[0019] As a preferred embodiment, the exosome cryopreservation protection solution further contains a Tris-HCl buffer solution with a pH value of 7.0 to 8.0.

[0020] As a further preferred solution, the pH value of the Tris-HCl buffer solution is 7.5±0.3.

[0021] The present invention also provides an exosome cryopreservation method, comprising: mixing the exosome cryopreservation protection solution described in any of the aforementioned schemes with exosomes, and then cryopreserving the mixed solution.

[0022] As a preferred embodiment, the volume ratio of the exosomes to the exosome cryopreservation protection solution is 1:0.5-2.

[0023] As a preferred embodiment, the exosomes are derived from one or more of stem cells, immune cells, epithelial cells, connective tissue cells, neural cells and virus-infected cells.

[0024] As a preferred embodiment, the exosomes are used to deliver mRNA.

[0025] The present invention finds that by using the zwitterions, the cryopreservation efficiency of exosomes can be greatly improved, and the protective effect on exosomes is effectively enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 is the particle size of each group of exosomes in the experimental examples of the present invention.

[0028] Figure 2 is the polydispersity coefficient of the particle size distribution of each group of exosomes in the experimental examples of the present invention. DETAILED DESCRIPTION

[0029] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. Those skilled in the art may make various modifications and changes to the present invention without departing from the scope or spirit of the present invention. For example, a feature described or illustrated as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0030] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0031] As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0032] Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0033] The present invention relates to concentration values, and its meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuations within the range of ±0.1%. For values ​​that are large or do not require too fine control, its meaning is also allowed to include greater fluctuations. For example, 100mM can allow fluctuations within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, its meaning is allowed to include fluctuations of ±10%.

[0034] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0035] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0036] In the present invention, "preferably", "better", "more preferably", and "suitably" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent of each other.

[0037] application

[0038] The present invention relates to the application of zwitterions in any of the following aspects:

[0039] 1) Improve the cryopreservation efficiency and particle size uniformity of exosomes;

[0040] 2) Preparation of reagents for improving exosome cryopreservation efficiency and particle size uniformity;

[0041] Wherein, the zwitterion is selected from one or a combination of betaine, sulfobetaine and phosphorylcholine.

[0042] The present invention unexpectedly discovered that the use of the above-mentioned zwitterions for exosome cryopreservation will be beneficial to significantly improve the cryopreservation efficiency of exosomes, can significantly enhance the protective effect on exosomes, while maintaining the good dispersibility of exosomes in the cryopreservation protection solution, and improve the particle size uniformity of exosomes after cryopreservation recovery.

[0043] In some embodiments, the zwitterions mentioned above can be added to the freezing system when freezing exosomes.

[0044] In some embodiments, the zwitterions can be prepared into reagents or cryopreservation protection solutions in advance and directly mixed with the exosomes when the exosomes are cryopreserved.

[0045] In some embodiments, the zwitterion is betaine, sulfobetaine, or phosphocholine.

[0046] In some embodiments, the zwitterion is a mixture of two of betaine, sulfobetaine, or phosphocholine.

[0047] In some embodiments, the zwitterion is a mixture of betaine, sulfobetaine, and phosphocholine.

[0048] When zwitterions are used in the above manner, the cryopreservation efficiency of exosomes can be greatly improved.

[0049] In some specific embodiments, the CAS number of the betaine is 107-43-7.

[0050] In some specific embodiments, the sulfobetaine is lauramidopropylhydroxysulfobetaine.

[0051] In some specific embodiments, the CAS number of the phosphorylcholine is 107-73-3.

[0052] In some embodiments, 1 to 10 g of the zwitterion is contained in every 100 mL of the exosome cryopreservation protection solution. Such a concentration will be more conducive to improving the particle size uniformity and cryopreservation efficiency of the exosomes.

[0053] In some preferred embodiments, the content of the zwitterion is 3 to 10 g / 100 mL, more preferably 5 to 10 g / 100 mL. By using the zwitterion at the above concentration, the particle size uniformity and cryopreservation efficiency of the exosomes can be further improved.

[0054] As an example, in some specific embodiments, the content of the zwitterion can be 1 g / 100 mL, 2 g / 100 mL, 3 g / 100 mL, 4 g / 100 mL, 5 g / 100 mL, 6 g / 100 mL, 7 g / 100 mL, 8 g / 100 mL, 9 g / 100 mL or 10 g / 100 mL.

[0055] In specific implementation, those skilled in the art can control the content of the zwitterion in the cryopreservation protection solution by adjusting the added amount of the zwitterion or the reagent containing the zwitterion.

[0056] As a preferred embodiment, the exosomes are derived from one or more of stem cells, immune cells, epithelial cells, connective tissue cells, neural cells and virus-infected cells. When the zwitterions of the present invention are used, the cryopreservation efficiency of the exosomes from the above sources can be greatly improved.

[0057] As a preferred embodiment, the exosomes are used to deliver mRNA. After the zwitterionic cryopreservation of cells is applied, the damage to the exosomes themselves is small, which will be beneficial to the subsequent application of the exosomes, such as using them to deliver mRNA corresponding to coronaviruses (SARS-CoV-2, COVID-19, etc.), influenza viruses, Ebola viruses, and various infectious viruses.

[0058] Cryoprotectant

[0059] Based on the above findings, the present invention also provides an exosome cryopreservation protection solution, which contains zwitterions, wherein the zwitterions are selected from one or a combination of betaine, sulfobetaine and phosphorylcholine. The application of the exosome cryopreservation protection solution can greatly improve the cryopreservation efficiency of exosomes.

[0060] In some embodiments, the zwitterion is betaine, sulfobetaine, or phosphocholine.

[0061] In some embodiments, the zwitterion is a mixture of two of betaine, sulfobetaine, or phosphocholine.

[0062] In some embodiments, the zwitterion is a mixture of betaine, sulfobetaine, and phosphocholine.

[0063] In some embodiments, each 100 mL of exosome cryopreservation protection solution contains 1 to 10 g of the zwitterion.

[0064] In some preferred embodiments, the content of the zwitterion is 3 to 10 g / 100 mL, more preferably 5 to 10 g / 100 mL.

[0065] As an example, in some specific embodiments, the content of the zwitterion can be 1 g / 100 mL, 2 g / 100 mL, 3 g / 100 mL, 4 g / 100 mL, 5 g / 100 mL, 6 g / 100 mL, 7 g / 100 mL, 8 g / 100 mL, 9 g / 100 mL or 10 g / 100 mL.

[0066] In some specific embodiments, the CAS number of the betaine is 107-43-7.

[0067] In some specific embodiments, the sulfobetaine is lauramidopropylhydroxysulfobetaine.

[0068] In some specific embodiments, the CAS number of the phosphorylcholine is 107-73-3.

[0069] In some embodiments, the exosome cryopreservation protection solution further contains 15-25 mM sodium citrate, which is more conducive to preventing the aggregation of nanoliposomes, thereby further ensuring the dispersion effect of exosomes.

[0070] As a further preferred embodiment, the content of sodium citrate is 20±2 mM.

[0071] As an example, in some specific embodiments, the content of sodium citrate is 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM or 25mM.

[0072] In some specific embodiments, the CAS number of the sodium citrate is 6132-04-3.

[0073] In some embodiments, the exosome cryopreservation protection solution further contains a Tris-HCl buffer solution with a pH value of 7.0 to 8.0. This is more conducive to ensuring the growth state of the exosomes and also to ensuring the application effect of the exosomes in downstream applications such as mRNA drugs.

[0074] In some more preferred embodiments, the pH value of the Tris-HCl buffer solution is 7.5±0.3.

[0075] As an example, in some specific embodiments, the pH value of the Tris-HCl buffer solution can be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

[0076] In some embodiments, the exosome cryopreservation protection solution contains 1-10 g / 100 mL of the zwitterion, 15-25 mM sodium citrate, and a Tris-HCl buffer solution with a pH value of 7.0-8.0.

[0077] In some more preferred embodiments, the exosome cryopreservation protection solution contains 1-10 g / 100 mL of the zwitterion, 20±2 mM sodium citrate and a Tris-HCl buffer solution with a pH value of 7.5±0.3.

[0078] In some embodiments, those skilled in the art may further add other protective agents and / or antioxidants to the exosome cryopreservation protection solution of the present invention.

[0079] In addition, those skilled in the art can combine the above-mentioned embodiments with common sense to obtain other preferred embodiments of the exosome cryopreservation protection solution of the present invention.

[0080] Exosome cryopreservation method

[0081] The present invention also provides an exosome cryopreservation method, comprising: mixing the exosome cryopreservation protection solution described in any of the aforementioned schemes with exosomes, and then cryopreserving the mixed solution.

[0082] In some embodiments, the volume ratio of the exosomes to the exosome cryopreservation protection solution is 1:0.5 to 2. This will further help improve the cryopreservation effect of the exosomes.

[0083] As an example, in some specific embodiments, the volume ratio of the exosomes to the exosome cryopreservation protection solution is 1:0.5, 1:1, 1:1.5 or 1:2.

[0084] In some embodiments, the exosomes are derived from one or more of stem cells, immune cells, epithelial cells, connective tissue cells, neural cells, and virus-infected cells.

[0085] In some embodiments, the exosomes are used to deliver mRNA.

[0086] In some embodiments, the cryopreservation includes: placing the mixed solution in a -80°C or liquid nitrogen tank for rapid freezing, and then transferring to a suitable long-term storage temperature. The long-term storage temperature is usually selected to be -80°C or the liquid nitrogen gas phase in liquid nitrogen.

[0087] The present invention does not particularly limit other operations involved in the cryopreservation of exosomes. During specific implementation, those skilled in the art can confirm other detailed operations involved in the cryopreservation of exosomes in combination with common knowledge in the art.

[0088] In some specific embodiments, the exosomes are prepared by ultracentrifugation, density gradient centrifugation, etc.

[0089] Example

[0090] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0091] In the following examples, the CAS number of betaine is 107-43-7, purchased from Sigma-Aldrich. The specific category of sulfobetaine is lauramide propyl hydroxysulfobetaine LHSB-35, purchased from Shengci Industrial Materials Co., Ltd. The CAS number of phosphorylcholine is 107-73-3, purchased from Sigma-Aldrich. The CAS number of sodium citrate is 6132-04-3, purchased from Sigma-Aldrich. The CAS number of tris (hydroxymethyl)aminomethane (Tris) in the buffer is 77-86-1, purchased from Sigma-Aldrich.

[0092] In order to facilitate comparison, the exosomes in the following Examples 1 to 4 are all from the same batch of mesenchymal stem cells, which are prepared by ultracentrifugation. The specific steps are:

[0093] 1. Collect exosomes from stem cell cultures. When the cell culture reaches an appropriate stage (usually at the peak of cell growth and exosome secretion), collect the culture supernatant containing the cell culture. This supernatant contains exosomes released by the cells.

[0094] 2. Perform some pretreatment steps in the collected culture supernatant, centrifuge at 300g for 10 minutes to remove cell debris and large particles to purify the supernatant.

[0095] 3. Transfer the pretreated culture supernatant to an ultracentrifuge tube. Ultracentrifugation is to place the sample in an ultracentrifuge and centrifuge at high speed to precipitate and enrich exosomes. Use high-speed centrifugation at 10,000g to separate exosomes, and centrifuge for at least one hour to precipitate exosome particles.

[0096] 4. Washing and resuspension: The precipitated exosome particles were washed with buffer (PBS) to remove potential contaminants and cryopreservation solution. After washing, the exosomes were suspended in PBS.

[0097] However, exosomes obtained from conventional sources and using conventional preparation methods can actually achieve the technical effects of the present invention.

[0098] For experimental methods in the following examples where specific conditions are not specified, reference is made to the instructions given in the present invention first, and may also be made to experimental manuals or conventional conditions in the art, or to other experimental methods known in the art, or to conditions recommended by manufacturers.

[0099] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0100] Example 1

[0101] This embodiment provides a cryopreservation protection solution, which contains: 10g / 100mL betaine, 20mM sodium citrate, and a Tris-HCl buffer solution with a pH value of 7.5.

[0102] The present embodiment further provides a cryopreservation method, which comprises: mixing the exosomes with the above-mentioned cryopreservation protection solution in a volume ratio of 1:1, and placing the mixture at -80°C for rapid freezing and storage.

[0103] Example 2

[0104] This embodiment provides a cryopreservation protection solution, which contains: 1g / 100mL betaine, 20mM sodium citrate, and a Tris-HCl buffer solution with a pH value of 7.5.

[0105] The cryopreservation method of this embodiment is the same as that of embodiment 1, except that the cryopreservation is performed using the cryopreservation protection solution of this embodiment.

[0106] Example 3

[0107] This embodiment provides a cryoprotectant solution, which contains: 10 g / 100 mL sulfobetaine, 20 mM sodium citrate, and a Tris-HCl buffer solution with a pH value of 7.5.

[0108] The cryopreservation method of this embodiment is the same as that of embodiment 1, except that the cryopreservation is performed using the cryopreservation protection solution of this embodiment.

[0109] Example 4

[0110] This embodiment provides a cryoprotectant solution, which contains: 1 g / 100 mL sulfobetaine, 20 mM sodium citrate, and a Tris-HCl buffer solution with a pH value of 7.5.

[0111] The cryopreservation method of this embodiment is the same as that of embodiment 1, except that the cryopreservation is performed using the cryopreservation protection solution of this embodiment.

[0112] Test example

[0113] After being frozen in a -80°C refrigerator for 6 months according to the freezing method in each embodiment, the exosomes were revived and used Nano ZS, Malvern Instruments detected the particle size and polydispersity index (PDI) of the exosomes in each example and compared them with the PBS control group (exosomes were mixed with PBS in a ratio of 1:1 and then frozen). The results are shown in Figure 1-2 .

[0114] From the results, it can be seen that the size of exosomes in each experimental group is stable, and its particle size has increased slightly, but still maintains good dispersibility, and its polydispersity coefficient has no significant change.

[0115] It can be seen that after adding the zwitterions described in the present invention, the freezing efficiency of exosomes is significantly improved, and better protective efficacy is obtained.

[0116] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. Application of zwitterions in any of the following areas: 1) Improve the cryopreservation efficiency of exosomes and the uniformity of particle size after cryopreservation; 2) Preparation of reagents for improving exosome cryopreservation efficiency and particle size uniformity; in, The zwitterion is selected from one or a combination of betaine, sulfobetaine and phosphorylcholine.

2. The use according to claim 1, wherein: Each 100 mL of exosome cryopreservation protection solution contains 1 to 10 g of the zwitterion, preferably the content of the zwitterion is 3 to 10 g / 100 mL, and more preferably 5 to 10 g / 100 mL.

3. The use according to claim 1 or 2, wherein: The exosomes are derived from one or more of stem cells, immune cells, epithelial cells, connective tissue cells, neural cells and virus-infected cells; preferably, the exosomes are used to deliver mRNA.

4. An exosome cryopreservation protection solution, comprising zwitterions, wherein the zwitterions are selected from one or a combination of betaine, sulfobetaine and phosphorylcholine.

5. The exosome cryopreservation protection solution according to claim 4, wherein: Each 100 mL of exosome cryopreservation protection solution contains 1 to 10 g of the zwitterion, preferably the content of the zwitterion is 3 to 10 g / 100 mL, and more preferably 5 to 10 g / 100 mL.

6. The exosome cryopreservation protection solution according to claim 4 or 5, further comprising 15-25 mM sodium citrate; preferably, the content of the sodium citrate is 20±2 mM.

7. The exosome cryopreservation protection solution according to any one of claims 4 to 6, further comprising a Tris-HCl buffer solution with a pH value of 7.0 to 8.0; preferably, the pH value of the Tris-HCl buffer solution is 7.5±0.

3.

8. A method for freezing exosomes, comprising: After mixing the exosome cryoprotectant solution according to any one of claims 4 to 7 with exosomes, the mixed solution is cryopreserved.

9. The method for freezing exosomes according to claim 8, wherein: The volume ratio of the exosomes to the exosome cryopreservation protection solution is 1:0.5-2.

10. The method for freezing exosomes according to claim 8 or 9, wherein: The exosomes are derived from one or more of stem cells, immune cells, epithelial cells, connective tissue cells, neural cells and virus-infected cells; preferably, the exosomes are used to deliver mRNA.

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