CN116497018A - Method for promoting cell to ingest exosomes by using low-intensity ultrasound - Google Patents
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Abstract
The invention discloses a method for promoting cells to ingest exosomes by using low-intensity ultrasound, and belongs to the field of biomedical application. The invention firstly proposes that the uptake of the exosome by the target cells or tissues can be promoted by applying certain low-intensity ultrasound to the cells or living animals in vitro. The method promotes the cells in the target area to absorb exosomes through the low-intensity ultrasonic effect, is simple and safe to operate, lays a foundation for clinical combined application of future disease treatment, and has great clinical significance.
Description
Technical Field
The invention relates to the field of biomedical application, in particular to a method for promoting receptor cells to ingest exosomes by low-intensity pulse ultrasonic stimulation.
Background
Exosomes can be divided into microvesicles, apoptotic bodies and exosomes: microbubbles (45-1000 nm) are produced by direct germination of cell membranes; apoptotic bodies (1-2 μm) are formed by random foaming of cell membranes during apoptosis; exosomes (30-200 nm) are vesicles surrounded by a bilayer lipid membrane. Exosomes are secreted by most cells, which contain proteins, lipids and RNAs, which vary depending on the cell from which they are derived and the physiological or pathological conditions, and thus affect the function of the recipient cell. Exosomes, due to their membrane structure, can potentially avoid endo-enzymatic pathways and lysosomal degradation, rendering the "cargo" they carry free from rnases and proteases, preventing them from being phagocytized by monocytes, and can be transported directly into the cytoplasm without unnecessary accumulation in the liver, thus avoiding the first metabolic effect before reaching the target site. Compared with stem cell transplantation, the stem cell-derived exosome has the characteristics of good blood brain barrier permeability, less immune response, less cancerogenic risk, high stability and the like, and has great potential in clinical application including central nervous system. Exosomes studies include basic studies (biological occurrence, secretion, uptake and pharmacokinetic profile of exosomes), biomarker identification (proteins, RNA, DNA carried by exosomes), drug products (natural and engineered extracellular vesicles), biomaterial-based drug delivery (loading proteins, RNA or drugs). In addition, how exosomes are recognized and taken up by specific cells is an important direction to be studied, directly related to the application prospect of targeted therapy.
Current exosome administration techniques include intravenous injection, stereotactic injection, nasal drop methods, etc., with little and lengthy exosomes eventually reaching the target region. Administration of central nervous system disorders has been a challenge, and studies have shown that low intensity ultrasound can temporarily open the blood brain barrier without causing side effects. In recent years, the development of acoustic dynamics and sonogenetics has also led to an increasing emphasis on the use of low intensity ultrasound.
Disclosure of Invention
In view of the above, the present invention provides a method for promoting cellular uptake of exosomes by low intensity ultrasound. The exosome is promoted to be taken up by cells in the target area through the low-intensity ultrasonic effect, the operation is simple and safe, and a foundation is laid for clinical combined application of future disease treatment.
In order to achieve the above purpose, the present invention provides the following technical solutions.
The invention provides a method for promoting cells or tissues to ingest exosomes, which is characterized in that the method is characterized in that low-intensity ultrasound is adopted.
Further, the method specifically comprises the following steps:
step 1: obtaining an exosome;
step 2: the exosomes obtained in the first step are identified, and specifically comprise the steps of adopting concentration measurement, nanoparticle tracking analysis, transmission electron microscope scanning and western blot experiment verification to identify the exosomes;
step 3: adding the exosomes obtained in the step one into a receptor cell culture medium for low-intensity ultrasonic treatment.
Further, the low-intensity ultrasonic treatment conditions in the step 3 are as follows: under the condition of 2MHz, the ultrasonic radiation dose of 90-85200 is adopted.
Further, the method further comprises identifying uptake of the exosome by the recipient cells using immunofluorescent staining and flow cytometry.
Further, the method is applied to the preparation of a central nervous system disease drug delivery system.
The invention also provides application of the low-intensity ultrasonic technology in preparing a central nervous system disease drug delivery system, which is characterized by specifically promoting cells or tissues to take up exosomes.
Further, the application specifically comprises the following steps:
step 1: obtaining an exosome;
step 2: the exosomes obtained in the first step are identified, and specifically comprise the steps of adopting concentration measurement, nanoparticle tracking analysis, transmission electron microscope scanning and western blot experiment verification to identify the exosomes;
step 3: adding the exosomes obtained in the step one into a receptor cell culture medium for low-intensity ultrasonic treatment.
Further, the low-intensity ultrasonic treatment conditions in the step 3 are as follows: under the condition of 2MHz, the ultrasonic radiation dose of 90-85200 is adopted.
Further, the application also comprises the use of immunofluorescent staining and flow cytometry to identify the uptake of exosomes by the recipient cells.
Compared with the prior art, the invention has the beneficial effects.
The exosomes can be used as bioactive nano-drugs or targeted gene therapy vectors, and play a great role in the treatment of various diseases in the future. However, there are organs that may be affected by various factors such as blood flow in the living body, and the uptake effect on the exosomes may be poor. Drug delivery has been a challenge in the central nervous system due to the presence of the blood brain barrier. The invention provides that the low-intensity ultrasound can promote the uptake of cells or tissues into exosomes under the condition of in-vitro cell culture and in living animals for the first time. Low-intensity ultrasound is a very potential therapeutic approach in degenerative diseases of the central nervous system due to its mechanical, cavitation, thermal effects and other effects and safety. The exosomes secreted by the stem cells have a plurality of proteins and nucleic acids with stem cell activity, and the combination of low-intensity ultrasound therapy brings hope for overcoming degenerative diseases of the central nervous system.
The invention promotes the uptake of cells or tissues into exosomes through low-intensity pulse ultrasonic stimulation, solves the problems of small uptake amount reaching a target area, long uptake time and medicines in the exosome administration technology of central nervous system diseases, and has the advantages of simple, convenient, safe and high-efficiency method, low cost and easy popularization.
Drawings
FIG. 1 is a schematic diagram of the structure of a device for promoting the uptake of exosomes by recipient cells by low intensity ultrasound.
Fig. 2 is a graph of analysis results of nano particle sizes of exosomes, wherein a is exosome distribution with different diameters, B is exosome distribution with different volumes, and C is an on-machine measurement picture of NTA.
FIG. 3 is an exosome transmission electron microscope image.
FIG. 4 is a graph showing the results of exosome Western blotting experiments.
FIG. 5 is a graph of fluorescence microscopy results of exosomes taken up by cells, which showed successful uptake of PKH26 stained exosomes by cells.
FIG. 6 is a graph of flow cytometry results of uptake of exosomes by three different recipient cells (from left to right: HT22 cell line, neuro2a cell line, BV2 cell line) at different doses of ultrasound radiation.
Fig. 7 is a graph of results of in vivo imaging of animal models with different ultrasonic radiation doses, which can significantly improve uptake of exosomes by brain tissue.
Detailed Description
The following examples will aid in the understanding of the present invention, but are merely illustrative of the invention and the invention is not limited thereto. The methods of operation in the examples are all conventional in the art.
Example one, different ultrasound radiation doses promote the uptake of exosomes by different recipient cells.
The method specifically comprises the following steps:
step 1: culturing mesenchymal stem cells to 80% density in a cell culture incubator at 37 ℃, collecting cell supernatant, centrifuging at a high speed for 3000Xg 10min,10000Xg 40 minutes, and then ultracentrifugating at 120000Xg for 70 minutes to obtain exosomes;
step 2: performing concentration measurement, nanoparticle tracking analysis, transmission electron microscope scanning and Western blotting experiment verification on the exosomes obtained in the step 1 by adopting a method known in the art (as shown in figures 1-4);
step 3: the exosomes were stained with PKH26 and then ultracentrifuged again to remove excess dye, after which they were added to HT22, neuro2a, BV2 receptor cell culture medium, and after 6-24 hours, different ultrasonic radiation doses were used at 2MHz, with specific experimental groupings as shown in the table below:
；
step 4: after 6-24 hours, the uptake of exosomes by cells and the ultrasound-promoted uptake of exosomes by cells were identified by immunofluorescent staining and flow cytometry.
The results of the experiment are shown in fig. 6, and the experimental groups one, two and three promoted uptake of exosomes by neuroblastoma cell line Neuro2a, astrocyte line NHA and mouse hippocampal neuronal cell line HT22 compared to the control group. The graph shows that when three cells ingest undyed exosomes, a small amount of autofluorescence exists, the number of positive cells is 62, 39 and 150 respectively, and the positive cells respectively account for 0.62%, 0.39% and 1.5% of the total number of cells; the positive cell numbers of exosomes after the cells absorb PKH26 staining without ultrasonic radiation are 7746, 6132 and 1679 respectively, which respectively account for 77.5%, 61.3% and 16.8% of the total cell number; the positive cell number of exosomes after cells uptake PKH26 staining at 90 ultrasonic radiation dose is 81%, 69.1% and 19.3% respectively; the positive cell number of exosomes after cells uptake PKH26 staining is 7870, 6985, 1559 respectively at an ultrasonic radiation dose of 15120, and the percentages are 78.7%, 69.8%, 15.6% respectively; the positive cell numbers of the exosomes after the cells ingest PKH26 staining at the ultrasonic radiation dose of 86200 are 7455, 6991 and 2961 respectively, and the percentages are 74.6%, 69.9% and 29.6% respectively.
Example two, animal experiment procedure:
step 1: the same as in the first embodiment;
step 2: colleague embodiment one;
step 3: the exosomes are dyed by PKH26, then ultracentrifuged to remove redundant dye, dripped into animal bodies by a nasal drop method, and different ultrasonic radiation doses are adopted under the frequency condition of 2MHz after 2-4 hours, and specific experimental groups are shown in the following table:
；
step 4: after 1-6 hours, the conditions of taking up exosomes by cells and promoting the exosomes by cells by ultrasound are identified by animal model living body imaging and immunofluorescence staining.
As shown in fig. 7, the fluorescence intensity of the ultrasonic treatment group is significantly enhanced compared with that of the control group, but the prolongation of the action time or the increase of the ultrasonic radiation dose does not bring more beneficial results, but may cause a problem of safety.
The method for promoting cells to take up exosomes by using low-intensity ultrasound specifically comprises the following steps:
step 1: culturing donor cells to 80% density in a 37 ℃ cell incubator, collecting cell supernatant, centrifuging at a high speed for 3000Xg 10min,10000Xg 40 minutes, and then ultracentrifugating at 120000Xg for 70 minutes to obtain exosomes;
step 2: performing concentration measurement, nanoparticle tracking analysis, transmission electron microscope scanning and Western blotting experiment verification on the exosomes;
step 3: the exosomes are added into a receptor cell culture medium after being stained by PKH26, and the cells are irradiated under different ultrasonic radiation doses after 6-24 hours;
step 4: after 6-24 hours, the uptake of exosomes by cells and the ultrasound-promoted uptake of exosomes by cells were identified by immunofluorescent staining and flow cytometry.
In a fourth embodiment, a method for promoting living tissue of an animal model to take up exosomes by using low-intensity ultrasound specifically comprises the following steps:
step 1: culturing donor cells to 80% density in a 37 ℃ cell incubator, collecting cell supernatant, centrifuging at a high speed for 3000Xg 10min,10000Xg 40 minutes, and then ultracentrifugating at 120000Xg for 70 minutes to obtain exosomes;
step 2: performing concentration measurement, nanoparticle tracking analysis, transmission electron microscope scanning and Western blotting experiment verification on the exosomes;
step 3: the exosomes are stained by PKH26 and enter the brain area of animals by a nasal drop method, and the brain area is irradiated under different ultrasonic radiation doses after 2-4 hours;
step 4: after 1-6 hours, the conditions of uptake of exosomes by brain tissues and promotion of exosomes uptake by brain tissues by ultrasound were observed by live animal imaging.
In summary, the low-intensity ultrasound disclosed by the invention can promote the uptake of exosomes by cells or tissues under in vitro cell culture conditions and in living animals. Low-intensity ultrasound is a very potential therapeutic approach in degenerative diseases of the central nervous system due to its mechanical, cavitation, thermal effects and other effects and safety. The exosomes secreted by the stem cells have a plurality of proteins and nucleic acids with stem cell activity, and the combination of low-intensity ultrasound therapy brings hope for overcoming degenerative diseases of the central nervous system.
Claims (9)
1. A method for promoting the uptake of exosomes by cells or tissues, said method being using low intensity ultrasound.
2. The method according to claim 1, characterized in that it comprises in particular the following steps:
step 1: obtaining an exosome;
step 2: the exosomes obtained in the first step are identified, and specifically comprise the steps of adopting concentration measurement, nanoparticle tracking analysis, transmission electron microscope scanning and western blot experiment verification to identify the exosomes;
step 3: adding the exosomes obtained in the step one into a receptor cell culture medium for low-intensity ultrasonic treatment.
3. The method of claim 2, wherein the low intensity sonication conditions in step 3 are: under the condition of 2MHz, the ultrasonic radiation dose of 90-85200 is adopted.
4. The method of claim 1, further comprising identifying uptake of exosomes by the recipient cells using immunofluorescent staining and flow cytometry.
5. Use of the method of claim 1 for the preparation of a central nervous system disorder drug delivery system.
6. Use of low intensity ultrasound technology for the preparation of a system for administration of a central nervous system disorder, characterized in that it specifically promotes the uptake of exosomes by cells or tissues.
7. The application according to claim 6, characterized in that it comprises in particular the following steps:
step 1: obtaining an exosome;
step 2: the exosomes obtained in the first step are identified, and specifically comprise the steps of adopting concentration measurement, nanoparticle tracking analysis, transmission electron microscope scanning and western blot experiment verification to identify the exosomes;
step 3: adding the exosomes obtained in the step one into a receptor cell culture medium for low-intensity ultrasonic treatment.
8. The use according to claim 6, wherein the low intensity ultrasound treatment conditions in step 3 are: under the condition of 2MHz, the ultrasonic radiation dose of 90-85200 is adopted.
9. The use of claim 6, further comprising identifying uptake of exosomes by the recipient cells using immunofluorescent staining and flow cytometry.
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