A small molecule compound for promoting exosome production, a composition thereof and application thereof

Abstract

The application discloses a small-molecule compound for promoting exosome production, a composition thereof and application. During culture of mesenchymal stem cells to produce exosomes, the small-molecule compound is added into a culture system at a certain concentration, so that the production of exosomes is further promoted. The small-molecule compound can increase the yield of mesenchymal stem cell exosomes to 1.5 times to more than 10 times of a control group, and does not affect cell activity.

Description

Technical Field

[0001] This invention relates to biotechnology and pharmaceutical technology, and in particular to a small molecule substance that can significantly increase the yield of cell exosomes, as well as the use of this small molecule substance as an additive in cell culture and its application in the large-scale production of exosomes. Background Technology

[0002] Exosomes are nanoscale (30-150 nm) vesicles actively secreted by cells, carrying important biological information such as proteins, nucleic acids, and lipids. They show great potential for applications in intercellular communication, disease diagnosis, drug delivery, and tissue regeneration. However, the natural yield of exosomes is extremely low, making it difficult to meet the large-scale demands of basic research and clinical applications, which has become a major bottleneck restricting their development. Currently, methods to increase exosome yield mainly include: Physical stimulation methods, such as hypoxia, radiation exposure, and shear force stimulation, may cause uncontrollable damage to cells, alter the biological characteristics of exosomes, and have poor reproducibility.

[0003] Genetic engineering: This method involves overexpressing genes related to exosome biogenesis (such as the Rab GTPase family) using gene editing technology. This method is technically complex, costly, carries safety risks, and is not suitable for primary cells or cells used in clinical therapy.

[0004] Biological factor stimulation: This method uses cytokines or growth factors (such as TGF-β) for stimulation. However, biological factors are expensive, have poor stability, and may introduce exogenous pollutants.

[0005] Therefore, there is an urgent need in this field to develop an efficient, stable, safe, and low-cost method to increase exosome production. Small molecule compounds are considered an ideal approach to solving this problem due to their well-defined structures, good stability, ease of synthesis, and standardization.

[0006] Although there are sporadic reports (such as GW4869 potentially increasing production at specific concentrations), the effects are unclear and the applicability is poor. There is a lack of highly efficient small molecule inducers specifically targeting exosome production. Summary of the Invention

[0007] Purpose of the invention: To overcome the limitations of existing technologies, the purpose of this invention is to provide a small molecule compound, its composition, and its application that can efficiently and safely promote the production of various cell exosomes.

[0008] The small molecule substance provided by this invention promotes exosome production. Adding a certain concentration of this substance to the culture system during the exosome production process of mesenchymal stem cells can further promote exosome production. This substance is a β2-adrenoceptor agonist. After binding to the β2-adrenoceptor, it activates the intracellular Gs protein, further activating adenylate cyclase, leading to increased production of cyclic adenosine monophosphate (cAMP). cAMP then transmits signals to protein kinase A (PKA), rapidly promoting cell signaling, increasing cell activity, and enhancing protein expression.

[0009] Technical solution: In a first aspect, the present invention provides a small molecule substance for promoting the production of cell exosomes, wherein the small molecule substance is a small molecule compound represented by general formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof:

[0010] I Preferably, the small molecule compound is 5-[1-hydroxy-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-1,3-benzenediol, with the following chemical structural formula:

[0011] In a second aspect, the present invention provides a cell culture additive comprising an effective dose of the small molecule compound as described in the first aspect and a pharmaceutically or cell culture-acceptable carrier.

[0012] Thirdly, the present invention provides the use of the small molecule compound as described in the first aspect or the cell culture additive as described in the second aspect in the preparation of reagents or kits for promoting cell secretion of exosomes.

[0013] Fourthly, the present invention provides a method for large-scale production of exosomes, characterized by comprising the following steps: (1) Add an effective dose of the small molecule compound as described in the first aspect or the cell culture additive as described in the second aspect to the cell culture system; (2) Continue culturing the cells under suitable conditions, preferably 12-72 hours; (3) Collect the cell culture supernatant and separate and purify the exosomes by at least one of the following methods: differential centrifugation, ultracentrifugation, size exclusion chromatography or polymer precipitation.

[0014] Preferably, the cells are mesenchymal stem cells.

[0015] Preferably, the working concentration of the small molecule is 10 μM - 100 μM, more preferably 50-100 μM.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. High efficiency: The small molecule compounds of the present invention can increase the production of exosomes in various cells by 1.5 to 10 times or more than that in the control group, without affecting cell activity.

[0017] 2. Universality: Experimental results have shown that this small molecule has a significant effect on increasing the production of mesenchymal stem cells.

[0018] 3. Safety: Within the effective concentration range, this small molecule has no significant negative impact on cell morphology and survival rate, and the produced exosomes are morphologically intact and the marker proteins are expressed normally.

[0019] 4. Simple operation and low cost: It only needs to be added during routine cell culture, without the need for complex equipment or genetic manipulation, making it easy to standardize and mass-produce. It can generate convection, making it suitable for culturing cells that require eddy currents. Attached Figure Description

[0020] Figure 1 The chemical structural formula of the small molecule 5-[1-hydroxy-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-1,3-benzenediol described in Example 1 of this invention is shown below. Figure 2 Morphological images of human umbilical cord mesenchymal stem cell-derived exosomes after treatment with 5-[1-hydroxy-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-1,3-benzenediol, observed under an electron microscope after ultracentrifugation; Figure 3 For cell morphology and survival rate, control group A and experimental group B were used. Figure 4 The concentration of exosomes in cell supernatant after treatment with 5-[1-hydroxy-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-1,3-benzenediol was analyzed using nanoparticle tracking technology (A) and compared with the control group (B). Figure 5 A statistical graph showing the concentration of exosomes in cell supernatants after treatment with different concentrations of 5-[1-hydroxy-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-1,3-benzenediol for analysis using nanoparticle tracking technology; Figure 6 To detect the expression of marker proteins CD9(a), CD63(b), and CD81(c) in exosomes obtained after treatment with 5-[1-hydroxy-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-1,3-benzenediol by flow cytometry. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be further described below.

[0022] The compound 5-[1-hydroxy-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-1,3-benzenediol used in the following examples was purchased from Yuanye Biotechnology (Catalog No.: T92925-1ml).

[0023] Example 1: Verification of the effect of the compound in promoting exosome production Human umbilical cord mesenchymal stem cells were cultured. In the culture system, the experimental group was given the small molecule substance 5-[1-hydroxy-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-1,3-benzenediol of this invention at a working concentration of 50 μM; the control group was given an equal volume of solvent. After culturing for a certain period, the cell culture supernatant was collected.

[0024] Exosomes were isolated and purified by ultracentrifugation. Transmission electron microscopy was used to observe the morphology of the exosomes, and the results showed that the exosomes obtained in the experimental group exhibited typical cup-shaped or spherical vesicle structures (see [link to study]). Figure 2 ).

[0025] Nanoparticle tracking analysis was used to quantify exosomes in both groups. The results showed that the exosome concentration in the experimental group was significantly higher than that in the control group (see...). Figure 4 ).

[0026] Example 2: Identification of exosome marker protein expression The expression of surface marker proteins in the exosome samples obtained after treatment with the small molecule substance described in Example 1 was detected by nanoflow cytometry. The results showed that the obtained exosomes highly expressed the characteristic markers CD9, CD63, and CD81 of mesenchymal stem cell-derived exosomes (see [link to original text]). Figure 6 This indicates that the exosomes are intact and of a clear origin.

[0027] Example 3: Observation of cell morphology and viability Cell morphology in the experimental and control groups of Example 1 was observed under an optical microscope. The results showed that, within the effective concentration range, the addition of the small molecule substance had an effect on the morphology of mesenchymal stem cells (see...). Figure 3 The fact that the substance showed no significant negative impact on survival rate indicates that it has good biological safety.

[0028]

[0029] Example 4: Validation of the effects of different concentrations In a mesenchymal stem cell culture system, different concentrations (10 μM, 50 μM, and 100 μM) of the aforementioned small molecule substance were added, and exosomes were collected and quantitatively analyzed after culture. The results showed that within the concentration range of 10 μM to 100 μM, exosome production increased with increasing concentration, with the most significant increase observed at concentrations of 50-100 μM (see [link to relevant documentation]). Figure 5 ).

[0030] The above are merely preferred embodiments of the present invention and do not constitute any limitation on the present invention. Any equivalent substitutions or modifications made by those skilled in the art to the technical solutions and content disclosed in the present invention without departing from the scope of the present invention shall be deemed to have remained within the protection scope of the present invention.

Claims

1. Use of small molecule compounds of general formula I, their stereoisomers, or pharmaceutically acceptable salts thereof in promoting exosome production, the structure of general formula I is as follows: 。 2. The use according to claim 1, characterized in that, The compound is 5-[1-hydroxy-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-1,3-benzenediol, and its chemical structural formula is as follows: 。 3. The use according to claim 1 or 2, characterized in that, The intended use is in the preparation of reagents or kits for promoting the secretion of exosomes by cells.

4. The use according to claim 1 or 2, characterized in that, The cells are mesenchymal stem cells; the concentration of the compound used is 10 μM to 100 μM.

5. A cell culture additive for promoting the production of exosomes, characterized in that, The compound of general formula (I) of claim 1 or 2, its stereoisomer, or a pharmaceutically acceptable salt thereof, comprising an effective dose.

6. The cell culture additive according to claim 5, characterized in that, The compound is 5-[1-hydroxy-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl]-1,3-benzenediol; the effective dose is 10 μM to 100 μM; and it also contains a pharmaceutically or cell culture-acceptable carrier.

7. The cell culture additive according to claim 6, characterized in that, The carrier is selected from phosphate buffer, cell culture medium, dimethyl sulfoxide, and combinations thereof.

8. A method for producing exosomes, characterized in that, In a cell culture system, an effective dose of the compound, stereoisomer, or pharmaceutically acceptable salt thereof of any one of claims 1-4, or the cell culture additive of any one of claims 5-7, is added, the cells are cultured, the cell culture supernatant is collected, and the exosomes are separated and purified.

9. The method according to claim 8, characterized in that, After adding the compound or cell culture additive, continue culturing the cells for 12 to 72 hours.

10. The method according to claim 8, characterized in that, After the culture is completed, the cell culture supernatant is collected and the exosomes are separated and purified by at least one of the following methods: differential centrifugation, ultracentrifugation, size exclusion chromatography or polymer precipitation.

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