Method for improving exosome uptake of cells and application

Abstract

The invention discloses a method for improving exosome uptake of cells and application. The invention firstly discovers that when the trypsin or the sialidase is used for treating the exosome, the uptake function of cells on the exosome can be enhanced, and provides a new application of the trypsin or the sialidase as an enhancer for the uptake of the exosome by the cells, so that the drug delivery efficiency of the exosome is favorably improved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method and application of enhancing cell uptake of exosomes. Background Art

[0002] DC2.4 cells are a mouse bone marrow-derived dendritic cell line that can mimic the functions of dendritic cells, including antigen presentation and the initiation of immune responses. Dendritic cells are key antigen-presenting cells in the immune system, responsible for initiating and regulating immune responses, and can phagocytose and present exogenous antigens such as proteins, pathogens, or tumor antigens. After activation, dendritic cells migrate to lymph nodes, present antigens to T cells, and initiate adaptive immune responses.

[0003] Exosomes are nanoscale extracellular vesicles secreted by cells. They play a key role in intercellular communication and are involved in a variety of biological processes, including immune response, antigen presentation, cell migration, cell differentiation, and tumor invasion. The natural targeting ability of exosomes enables them to effectively deliver drugs to specific tissues and cells. They can enter receptor cells through endocytosis or direct fusion with cell membranes and deliver their contents to the cytoplasm. In addition, exosomes have the characteristics of high bioavailability, biostability, targeting specificity, low toxicity, and low immunogenicity, which makes them show great potential in disease diagnosis, treatment, and drug delivery. However, they still face great challenges in clinical applications, and problems such as low drug delivery efficiency need to be urgently addressed. Summary of the invention

[0004] In view of this, the present invention provides a method and application for enhancing the uptake of exosomes by cells.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for improving the uptake of exosomes by cells comprises treating the exosomes with trypsin or sialidase.

[0007] Preferably, the treatment comprises: incubating the exosomes with trypsin or sialidase at 37° C. with shaking.

[0008] More preferably, the mixture is incubated at 37° C. with shaking at 160-240 rpm for 10 minutes.

[0009] More preferably, the volume ratio of trypsin or sialidase to exosomes is 1:(9.5-11), the mass volume concentration of trypsin is 0.23%-0.27% g / ml, the mass volume concentration of sialidase is 0.1 mg / ml, and the mass volume concentration of exosomes is 4-5 mg / ml.

[0010] Preferably, the cells include but are not limited to DC2.4 cells and γδ T cells.

[0011] Preferably, the exosomes include but are not limited to milk exosomes.

[0012] The present invention also provides the use of trypsin or sialidase as an enhancer for the uptake of exosomes by cells.

[0013] Preferably, the cells include but are not limited to DC2.4 cells and γδ T cells.

[0014] Preferably, the trypsin-treated exosomes can enhance the uptake of exosomes by DC2.4 cells.

[0015] Preferably, the trypsin-treated exosomes can enhance the uptake of exosomes by γδT cells.

[0016] Preferably, the sialidase-treated exosomes can enhance the uptake of exosomes by DC2.4 cells.

[0017] More preferably, the exosomes include but are not limited to milk exosomes.

[0018] The beneficial effects of the present invention are:

[0019] The present invention discovers for the first time that treating exosomes with trypsin or sialidase can enhance the cellular uptake of exosomes, and proposes a new use of trypsin and sialidase as enhancers for cellular uptake of exosomes, thereby helping to increase the drug delivery efficiency of exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 Represents microscopic imaging of milk exosomes;

[0022] Figure 2 Represents the microscopic imaging of milk exosomes treated with trypsin;

[0023] Figure 3 Microscopic imaging showing DC2.4 cells taking up milk exosomes;

[0024] Figure 4 Microscopic imaging showing DC2.4 cells taking up trypsin-treated milk exosomes;

[0025] Figure 5 Microscopic imaging showing DC2.4 cells taking up sialidase-treated milk exosomes;

[0026] Figure 6 It indicates that trypsin-treated milk exosomes can enhance their uptake by DC2.4 cells;

[0027] Figure 7 It indicates that trypsin-treated milk exosomes can enhance their uptake by γδT cells;

[0028] Figure 8 This indicates that sialidase-treated milk exosomes can enhance their uptake by DC2.4 cells. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0031] Example 1 Trypsin-treated milk exosomes can enhance their uptake by DC2.4 cells

[0032] (1) Preparation of milk exosomes: skim pure milk (Yili) was centrifuged at 10000g and 4°C for 30 minutes in a centrifuge to remove the precipitate, remove the top layer of milk skin, transfer the supernatant to a new tube, and repeat at least 3 times until there is no milk skin on the top layer; the supernatant was transferred to a 100KDa ultrafiltration tube and centrifuged at 4000rpm for 10 minutes for concentration, and repeated several times; qEV original Columns 35nm column (IZON) was washed 3 times, and 0.5ml of sample was added. After the flow was completed, 0.5ml of PBS buffer was added. After the flow was completed, 2ml of PBS buffer was added. After the flow was completed, 0.5ml of PBS buffer was added to collect the sample, and repeated 5 times.

[0033] (2) Exosome trypsin treatment and fluorescent labeling: 4.56 mg / ml milk exosomes and 10% volume (i.e., the volume of trypsin added is 10% of the volume of milk exosomes) of trypsin (Solarbio; T1350) were incubated at 37°C with shaking at 200 rpm for 10 minutes, and then immediately separated and purified using qev original columns. Cyanine5 NHS ester was added to milk exosomes and trypsin-treated milk exosomes, respectively, and after incubation at 37°C for one hour, a desalting column was used to remove excess free Cyanine5 NHS ester.

[0034] (3) Prepare DMEM culture medium containing 10% exosome-free serum (SclenCell; 0025) and 1% P / S, take two tubes of culture medium, add Cyanine 5NHS ester-labeled milk exosomes and trypsin-treated milk exosomes, respectively, to ensure the same fluorescence intensity.

[0035] (4) Counting DC2.4 cells. After centrifugation, add 100 μl of DMEM medium containing 10% FBS and 1% P / S for every 20,000 cells to resuspend them and slowly drip them into the 48-well plate along the wall to ensure that the cells are evenly distributed in the wells. DC2.4 cells were cultured at 37°C and 5% CO. 2 , and culture in an incubator with a relative humidity of 90% for 24 hours.

[0036] (5) Discard the supernatant and rinse the cells with PBS.

[0037] (6) Add the culture medium containing milk exosomes and trypsin-treated milk exosomes, slowly drip into a 48-well plate to prevent the cells from being blown away, and incubate in an incubator for 2 h.

[0038] (7) Aspirate and discard the supernatant, blow off the cells with PBS, and centrifuge; resuspend with 200 μL of a mixture containing 0.5 mol / L sodium chloride and 0.2 mol / L acetic acid, centrifuge, and then resuspend with 100 μL of PBS containing 1% FBS.

[0039] (8) Flow cytometry was used to detect the fluorescence intensity of Cyanine 5 in cells, indicating the ability of cells to take up exosomes.

[0040] Microscopic imaging of milk exosomes Figure 1 As shown, the microscopic images of milk exosomes treated with trypsin are shown in Figure 2 As shown in the two figures, it can be seen that the milk exosomes used in this application have a characteristic exosome structure: a distinct membrane, a clear three-dimensional structure, and a saucer-like or concave hemispherical structure. Microscopic imaging of DC2.4 cells taking up milk exosomes is shown in Figure 3As shown, the microscopic imaging of DC2.4 cells taking up trypsin-treated milk exosomes is shown in Figure 4 As shown, Cyanine 5 marked the exosomes, making the exosomes appear red in the image. Untreated milk exosomes were hardly taken up by DC2.4 cells, while milk exosomes treated with trypsin could be taken up by DC2.4 cells in large quantities. Figure 6 The flow cytometry graph shows that the fluorescence level of DC2.4 after taking in trypsin-treated milk exosomes significantly exceeds the fluorescence level of taking in untreated milk exosomes. It can be seen that trypsin-treated milk exosomes can enhance the uptake of DC2.4 cells.

[0041] Example 2 Trypsin-treated milk exosomes can enhance their uptake by γδT cells

[0042] (1) Exosome trypsin treatment and fluorescent labeling: Step Example 1.

[0043] (2) Prepare T cell culture medium containing 10% exosome-free serum. Take two tubes of culture medium and add Cyanine5NHS ester-labeled milk exosomes and trypsin-treated milk exosomes respectively to ensure the same fluorescence intensity.

[0044] (3) Counting: After centrifugation, the γδ T cells were resuspended in PBS, centrifuged again, and then distributed into a 48-well plate. 20,000 cells were added to each well of a 100 μl T cell culture medium containing exosome-free serum.

[0045] (4) The culture medium with milk exosomes was incubated with γδT cells at 37°C and 5% CO 2 , cultured in an incubator with a relative humidity of 90% for 2 hours, taken out, centrifuged to remove the supernatant, resuspended in 200 μL of a mixture of 0.5 mol / L sodium chloride and 0.2 mol / L acetic acid, centrifuged, and then resuspended in 100 μL of PBS containing 1% FBS.

[0046] (5) Flow cytometry was used to detect the fluorescence intensity of Cyanine 5 in cells, indicating the ability of cells to take up exosomes.

[0047] like Figure 7 As shown in the flow cytometry graph, the fluorescence amount of γδT cells after taking up trypsin-treated milk exosomes significantly exceeded the fluorescence amount of untreated milk exosomes. It can be seen that trypsin-treated milk exosomes can enhance their uptake by γδT cells.

[0048] Example 3 Sialidase-treated milk exosomes can enhance their uptake by DC2.4 cells

[0049] (1) Exosome sialidase treatment and fluorescent labeling: 4.56 mg / ml milk exosomes were incubated with 10% volume (i.e., the volume of sialidase added was 10% of the volume of milk exosomes) sialidase (Rhinogen; QPF-005) at 37°C with shaking at 200 rpm for 10 minutes, and then immediately separated and purified using qev original columns. Cyanine5 NHS ester was added to milk exosomes and sialidase-treated milk exosomes, respectively, and after incubation at 37°C for one hour, excess free Cyanine5 NHS ester was removed using a desalting column.

[0050] (2) Prepare DMEM culture medium containing 10% exosome-free serum and 1% P / S, take two tubes of culture medium, add Cyanine 5NHS ester-labeled milk exosomes and sialidase-treated milk exosomes, respectively, and ensure that the fluorescence intensity is the same.

[0051] (3) Counting DC2.4 cells. After centrifugation, add 100 μl of DMEM medium containing 10% FBS and 1% P / S for every 20,000 cells to resuspend them and slowly drip them into the 48-well plate along the wall to ensure that the cells are evenly distributed in the wells. DC2.4 cells were cultured at 37°C and 5% CO. 2 , and culture in an incubator with a relative humidity of 90% for 24 hours.

[0052] (4) Discard the supernatant and rinse the cells with PBS.

[0053] (5) Add culture medium containing milk exosomes and milk exosomes treated with sialidase, slowly drip into a 48-well plate to prevent the cells from being blown away, and incubate in an incubator for 2 h.

[0054] (6) Aspirate and discard the supernatant, blow off the cells with PBS, and centrifuge; resuspend with 200 μL of a mixture containing 0.5 mol / L sodium chloride and 0.2 mol / L acetic acid, centrifuge, and then resuspend with 100 μL of PBS containing 1% FBS.

[0055] (7) Flow cytometry was used to detect the fluorescence intensity of Cyanine 5 in cells, indicating the ability of cells to take up exosomes.

[0056] like Figure 8 As shown in the flow cytometry graph, the fluorescence amount of DC2.4 after taking in sialidase-treated milk exosomes significantly exceeded the fluorescence amount of taking in untreated milk exosomes. It can be seen that sialidase-treated milk exosomes can enhance the uptake of DC2.4 cells.

Claims

1. A method for improving the uptake of exosomes by cells, comprising treating the exosomes with trypsin or sialidase.

2. The method according to claim 1, characterized in that The treatment includes: incubating the exosomes with trypsin or sialidase at 37° C. with shaking.

3. The method according to claim 1, characterized in that The cells include DC2.4 cells and γδ T cells.

4. The method according to any one of claims 1 to 3, characterized in that: The exosomes include milk exosomes.

5. Application of trypsin or sialidase as enhancers of cellular uptake of exosomes.

6. The use according to claim 5, characterized in that: The cells include DC2.4 cells and γδ T cells.

7. The use according to claim 5, characterized in that: The trypsin-treated exosomes can enhance the uptake of exosomes by DC2.4 cells.

8. The use according to claim 5, characterized in that: The trypsin-treated exosomes can enhance the uptake of exosomes by γδT cells.

9. The use according to claim 5, characterized in that: The sialidase-treated exosomes can enhance the uptake of exosomes by DC2.4 cells.

10. The use according to any one of claims 5 to 9, characterized in that: The exosomes include milk exosomes.

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