CN110699382A - Preparation method of exosome for delivering siRNA - Google Patents
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Abstract
The invention relates to the technical field of exosome delivery siRNA, and provides a preparation method of an exosome for delivering siRNA, wherein in the process of preparing a supernatant, blood plasma stored at-80 ℃ is placed in a water bath at 37 ℃, so that the frozen blood plasma can be thawed and is completely liquid; then separating the exosome from the plasma by a precipitation reagent method, and removing the exosome, dead cells and fragments thereof by a centrifugal method to obtain the exosome; then analyzing the size, yield, purity, form and marker expression characteristics of the exosomes respectively, thereby performing characterization analysis on the characteristics of the separated exosomes and laying a cushion for the subsequent step of encapsulating siRNA into the exosomes; finally, siRNA is encapsulated into the separated exosome through an electroporation method, and free siRNA is removed through a particle size exclusion chromatography method, so that the exosome encapsulated with siRNA with high purity can be obtained, and the efficiency of siRNA delivery by the exosome is improved.
Description
Technical Field
The invention relates to the technical field of siRNA delivery by exosomes, in particular to a preparation method of an exosome for delivering siRNA.
Background
Exosomes are natural vesicle structures, have unique biocompatibility and contain nucleic acid and protein inside, so that exosomes serving as delivery systems are very hot research directions in recent years. Extracellular vesicles, particularly exosomes, have received attention in recent years as novel drug delivery vehicles due to their biological origin, abundance and inherent capacity. The siRNA is small interfering RNA which can cause RNA interference, wherein the RNA interference is used for specifically inhibiting the expression process after gene transcription so as to achieve the purpose of gene silencing; and the exosome is used as a carrier, so that the siRNA can be delivered. siRNA can be successfully loaded into exosomes by electroporation and then exosomes are delivered into cancer cells in vitro, which protocol provides a standard procedure to develop siRNA loaded exosomes for efficient delivery into cancer cells.
Disclosure of Invention
Solves the technical problem
In view of the defects of the prior art, the invention provides a preparation method of an exosome for delivering siRNA, aiming at developing the exosome loaded with siRNA so as to effectively deliver the siRNA into cancer cells.
Technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme:
a method for preparing exosomes for delivering siRNA, comprising the steps of:
s1, preparing a supernatant: firstly, putting plasma stored at minus 80 ℃ in a water bath at 37 ℃, and putting the plasma on ice after the plasma is completely liquid; centrifuging at 2000 Xg for 20-30min to remove cells and debris, sucking the supernatant with a pipette into a new centrifugal tube, and centrifuging at 10000 Xg for 20-30 min; finally, the resulting supernatant was aspirated into a new centrifuge tube and placed on ice until separation began;
s2, separation of exosomes: sucking the supernatant liquid which is separated in the S1 to a proper volume into a new centrifugal tube, adding PBS with the volume of 0.5 times that of the supernatant liquid, and then performing vortex mixing; then 0.2 x (volume of supernatant + volume of PBS) exosome precipitation reagent was added, and after vortex mixing, the mixture was recorded; incubating the mixture at room temperature for 10-15min, centrifuging at room temperature at 10000 Xg for 5-10min, and removing supernatant to obtain particles as exosomes;
s3, characterization and analysis of exosomes: analyzing the exosomes obtained in S2, wherein the analysis comprises the following steps:
a. characterizing exosome size and yield with a nanoparticle tracking analyzer;
b. the exosome purity was characterized by a particle protein ratio assay;
c. detecting the characteristics of the expression of the exosome markers by flow cytometry;
d. characterizing exosome morphology using transmission electron microscopy;
s4, encapsulating siRNA into exosomes: encapsulating siRNA into exosome subjected to S3 characterization analysis through electroporation, and recording the obtained mixture;
s5, removal of free siRNA: and removing free siRNA in the mixture by adopting a particle size exclusion chromatography to obtain the exosome encapsulated with siRNA.
Further, the centrifugation in S1 is performed at room temperature.
Further, the PBS in S2 is preferably 1 XPBS buffer.
Further, in the characterization analysis process of exosomes in S3, the analysis result needs to be recorded.
Further, the specific operation steps for encapsulating the siRNA into the exosome in S4 are as follows:
1) the electroporation rings were pre-cooled on ice for 30min, then mixed as 1: mixing exosome and siRNA in a microcentrifuge tube according to the molar ratio of 60, adding a proper amount of citric acid buffer solution, and mixing to obtain a premix;
2) transferring the premix in the step 1) into an electroporation test tube for electroporation, obtaining a mixture after the electroporation is finished, and taking the mixture out of the test tube by using a pipette for next treatment.
Further, the specific operation steps for removing free siRNA in S5 are as follows:
1) the SEC column was equilibrated by twice filtration of 350 μ LPBS, then 150 μ L of the mixture was dissolved in 350 μ L of filtered PBS and transferred to the SEC column for free siRNA removal, then the first 500 μ L of fractions eluted from the SEC column were collected;
2) add 500. mu.L of filtered PBS to SEC column again, and collect the next 500. mu.L fraction;
3) repeating the step 2) until a total fraction of 10 × 500 μ L is collected, at which time free siRNA in the mixture can be removed to obtain siRNA encapsulated exosomes.
Advantageous effects
The invention provides a preparation method of an exosome for delivering siRNA, and compared with the prior known technology, the preparation method has the following beneficial effects:
in the process of preparing the supernatant, the plasma stored at the temperature of minus 80 ℃ is placed in a water bath at the temperature of 37 ℃, and the frozen plasma can be thawed to be completely liquid; then separating the exosome from the plasma by a precipitation reagent method, and removing the exosome, dead cells and fragments thereof by a centrifugal method to obtain the exosome; then analyzing the size, yield, purity, form and marker expression characteristics of the exosomes respectively, thereby performing characterization analysis on the characteristics of the separated exosomes and laying a cushion for the subsequent step of encapsulating siRNA into the exosomes; finally, siRNA is encapsulated into the separated exosome through an electroporation method, and free siRNA is removed through a particle size exclusion chromatography method, so that the exosome encapsulated with siRNA with high purity can be obtained, and the efficiency of siRNA delivery by the exosome is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of the steps of the present invention;
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
the preparation method of the exosome for delivering the siRNA comprises the following steps:
s1, preparing a supernatant: firstly, putting plasma stored at minus 80 ℃ in a water bath at 37 ℃, and putting the plasma on ice after the plasma is completely liquid; centrifuging at 2000 Xg for 20min to remove cells and debris, sucking the supernatant with a pipette into a new centrifugal tube, and centrifuging at 10000 Xg for 30 min; finally, the resulting supernatant was aspirated into a new centrifuge tube and placed on ice until separation began;
s2, separation of exosomes: sucking the supernatant liquid which is separated in the S1 to a proper volume into a new centrifugal tube, adding PBS with the volume of 0.5 times that of the supernatant liquid, and then performing vortex mixing; then 0.2 x (volume of supernatant + volume of PBS) exosome precipitation reagent was added, and after vortex mixing, the mixture was recorded; incubating the mixture at room temperature for 10min, centrifuging at room temperature at 10000 Xg for 8min, and removing supernatant to obtain particles as exosomes;
s3, characterization and analysis of exosomes: analyzing the exosomes obtained in S2, wherein the analysis comprises the following steps:
a. characterizing exosome size and yield with a nanoparticle tracking analyzer;
b. the exosome purity was characterized by a particle protein ratio assay;
c. detecting the characteristics of the expression of the exosome markers by flow cytometry;
d. characterizing exosome morphology using transmission electron microscopy;
s4, encapsulating siRNA into exosomes: encapsulating siRNA into exosome subjected to S3 characterization analysis through electroporation, and recording the obtained mixture;
s5, removal of free siRNA: and removing free siRNA in the mixture by adopting a particle size exclusion chromatography to obtain the exosome encapsulated with siRNA.
Further, centrifugation in S1 was performed at room temperature.
Further, PBS in S2 is preferably 1 XPBS buffer.
Further, in S3, during the characterization and analysis of exosome, the analysis result needs to be recorded.
Further, the specific operation steps of encapsulating siRNA into exosome in S4 are:
1) the electroporation rings were pre-cooled on ice for 30min, then mixed as 1: mixing exosome and siRNA in a microcentrifuge tube according to the molar ratio of 60, adding a proper amount of citric acid buffer solution, and mixing to obtain a premix;
2) transferring the premix in the step 1) into an electroporation test tube for electroporation, obtaining a mixture after the electroporation is finished, and taking the mixture out of the test tube by using a pipette for next treatment.
Further, the specific operation steps for removing free siRNA in S5 are:
1) the SEC column was equilibrated by twice filtration of 350 μ LPBS, and then 150 μ L of the mixture was dissolved in 350 μ L of filtered PBS and transferred to the SEC column for free siRNA removal, and then the first 500 μ L of fractions eluted from the SEC column were collected;
2) add 500. mu.L of filtered PBS to SEC column again, and collect the next 500. mu.L fraction;
3) repeating the step 2) until a total fraction of 10 × 500 μ L is collected, at which time free siRNA in the mixture can be removed to obtain siRNA encapsulated exosomes.
Example 2:
the preparation method of the exosome for delivering the siRNA comprises the following steps:
s1, preparing a supernatant: firstly, putting plasma stored at minus 80 ℃ in a water bath at 37 ℃, and putting the plasma on ice after the plasma is completely liquid; centrifuging at 2000 Xg for 25min to remove cells and debris, sucking the supernatant with a pipette into a new centrifugal tube, and centrifuging at 10000 Xg for 20 min; finally, the resulting supernatant was aspirated into a new centrifuge tube and placed on ice until separation began;
s2, separation of exosomes: sucking the supernatant liquid which is separated in the S1 to a proper volume into a new centrifugal tube, adding PBS with the volume of 0.5 times that of the supernatant liquid, and then performing vortex mixing; then 0.2 x (volume of supernatant + volume of PBS) exosome precipitation reagent was added, and after vortex mixing, the mixture was recorded; incubating the mixture at room temperature for 15min, centrifuging at room temperature for 5min at 10000 Xg, and removing supernatant to obtain particles as exosomes;
s3, characterization and analysis of exosomes: analyzing the exosomes obtained in S2, wherein the analysis comprises the following steps:
a. characterizing exosome size and yield with a nanoparticle tracking analyzer;
b. the exosome purity was characterized by a particle protein ratio assay;
c. detecting the characteristics of the expression of the exosome markers by flow cytometry;
d. characterizing exosome morphology using transmission electron microscopy;
s4, encapsulating siRNA into exosomes: encapsulating siRNA into exosome subjected to S3 characterization analysis through electroporation, and recording the obtained mixture;
s5, removal of free siRNA: and removing free siRNA in the mixture by adopting a particle size exclusion chromatography to obtain the exosome encapsulated with siRNA.
Further, centrifugation in S1 was performed at room temperature.
Further, PBS in S2 is preferably 1 XPBS buffer.
Further, in S3, during the characterization and analysis of exosome, the analysis result needs to be recorded.
Further, the specific operation steps of encapsulating siRNA into exosome in S4 are:
1) the electroporation rings were pre-cooled on ice for 30min, then mixed as 1: mixing exosome and siRNA in a microcentrifuge tube according to the molar ratio of 60, adding a proper amount of citric acid buffer solution, and mixing to obtain a premix;
2) transferring the premix in the step 1) into an electroporation test tube for electroporation, obtaining a mixture after the electroporation is finished, and taking the mixture out of the test tube by using a pipette for next treatment.
Further, the specific operation steps for removing free siRNA in S5 are:
1) the SEC column was equilibrated by twice filtration of 350 μ LPBS, and then 150 μ L of the mixture was dissolved in 350 μ L of filtered PBS and transferred to the SEC column for free siRNA removal, and then the first 500 μ L of fractions eluted from the SEC column were collected;
2) add 500. mu.L of filtered PBS to SEC column again, and collect the next 500. mu.L fraction;
3) repeating the step 2) until a total fraction of 10 × 500 μ L is collected, at which time free siRNA in the mixture can be removed to obtain siRNA encapsulated exosomes.
Example 3:
the preparation method of the exosome for delivering the siRNA comprises the following steps:
s1, preparing a supernatant: firstly, putting plasma stored at minus 80 ℃ in a water bath at 37 ℃, and putting the plasma on ice after the plasma is completely liquid; centrifuging at 2000 Xg for 30min to remove cells and debris, sucking the supernatant with a pipette into a new centrifugal tube, and centrifuging at 10000 Xg for 25 min; finally, the resulting supernatant was aspirated into a new centrifuge tube and placed on ice until separation began;
s2, separation of exosomes: sucking the supernatant liquid which is separated in the S1 to a proper volume into a new centrifugal tube, adding PBS with the volume of 0.5 times that of the supernatant liquid, and then performing vortex mixing; then 0.2 x (volume of supernatant + volume of PBS) exosome precipitation reagent was added, and after vortex mixing, the mixture was recorded; incubating the mixture at room temperature for 13min, centrifuging at room temperature at 10000 Xg for 10min, and removing supernatant to obtain particles as exosomes;
s3, characterization and analysis of exosomes: analyzing the exosomes obtained in S2, wherein the analysis comprises the following steps:
a. characterizing exosome size and yield with a nanoparticle tracking analyzer;
b. the exosome purity was characterized by a particle protein ratio assay;
c. detecting the characteristics of the expression of the exosome markers by flow cytometry;
d. characterizing exosome morphology using transmission electron microscopy;
s4, encapsulating siRNA into exosomes: encapsulating siRNA into exosome subjected to S3 characterization analysis through electroporation, and recording the obtained mixture;
s5, removal of free siRNA: and removing free siRNA in the mixture by adopting a particle size exclusion chromatography to obtain the exosome encapsulated with siRNA.
Further, centrifugation in S1 was performed at room temperature.
Further, PBS in S2 is preferably 1 XPBS buffer.
Further, in S3, during the characterization and analysis of exosome, the analysis result needs to be recorded.
Further, the specific operation steps of encapsulating siRNA into exosome in S4 are:
1) the electroporation rings were pre-cooled on ice for 30min, then mixed as 1: mixing exosome and siRNA in a microcentrifuge tube according to the molar ratio of 60, adding a proper amount of citric acid buffer solution, and mixing to obtain a premix;
2) transferring the premix in the step 1) into an electroporation test tube for electroporation, obtaining a mixture after the electroporation is finished, and taking the mixture out of the test tube by using a pipette for next treatment.
Further, the specific operation steps for removing free siRNA in S5 are:
1) the SEC column was equilibrated by twice filtration of 350 μ LPBS, and then 150 μ L of the mixture was dissolved in 350 μ L of filtered PBS and transferred to the SEC column for free siRNA removal, and then the first 500 μ L of fractions eluted from the SEC column were collected;
2) add 500. mu.L of filtered PBS to SEC column again, and collect the next 500. mu.L fraction;
3) repeating the step 2) until a total fraction of 10 × 500 μ L is collected, at which time free siRNA in the mixture can be removed to obtain siRNA encapsulated exosomes.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (6)
1. A method for preparing an exosome for delivering siRNA, comprising the following steps:
s1, preparing a supernatant: firstly, putting plasma stored at minus 80 ℃ in a water bath at 37 ℃, and putting the plasma on ice after the plasma is completely liquid; centrifuging at 2000 Xg for 20-30min to remove cells and debris, sucking the supernatant with a pipette into a new centrifugal tube, and centrifuging at 10000 Xg for 20-30 min; finally, the resulting supernatant was aspirated into a new centrifuge tube and placed on ice until separation began;
s2, separation of exosomes: sucking the supernatant liquid which is separated in the S1 to a proper volume into a new centrifugal tube, adding PBS with the volume of 0.5 times that of the supernatant liquid, and then performing vortex mixing; then 0.2 x (volume of supernatant + volume of PBS) exosome precipitation reagent was added, and after vortex mixing, the mixture was recorded; incubating the mixture at room temperature for 10-15min, centrifuging at room temperature at 10000 Xg for 5-10min, and removing supernatant to obtain particles as exosomes;
s3, characterization and analysis of exosomes: analyzing the exosomes obtained in S2, wherein the analysis comprises the following steps:
a. characterizing exosome size and yield with a nanoparticle tracking analyzer;
b. the exosome purity was characterized by a particle protein ratio assay;
c. detecting the characteristics of the expression of the exosome markers by flow cytometry;
d. characterizing exosome morphology using transmission electron microscopy;
s4, encapsulating siRNA into exosomes: encapsulating siRNA into exosome subjected to S3 characterization analysis through electroporation, and recording the obtained mixture;
s5, removal of free siRNA: and removing free siRNA in the mixture by adopting a particle size exclusion chromatography to obtain the exosome encapsulated with siRNA.
2. The method of claim 1, wherein the centrifugation in S1 is performed at room temperature.
3. The method for preparing exosome for delivering siRNA according to claim 1, wherein PBS in S2 is preferably 1 x PBS buffer.
4. The method for preparing exosomes for delivering siRNA according to claim 1, wherein in the step of characterizing and analyzing exosomes in S3, the analysis result needs to be recorded.
5. The method for preparing exosome for delivering siRNA according to claim 1, wherein the specific operation steps for encapsulating siRNA into exosome in S4 are as follows:
1) the electroporation rings were pre-cooled on ice for 30min, then mixed as 1: mixing exosome and siRNA in a microcentrifuge tube according to the molar ratio of 60, adding a proper amount of citric acid buffer solution, and mixing to obtain a premix;
2) transferring the premix in the step 1) into an electroporation test tube for electroporation, obtaining a mixture after the electroporation is finished, and taking the mixture out of the test tube by using a pipette for next treatment.
6. The method for preparing exosome for delivering siRNA according to claim 1, wherein the specific operation steps for removing free siRNA in S5 are as follows:
1) the SEC column was equilibrated by twice filtration of 350 μ LPBS, then 150 μ L of the mixture was dissolved in 350 μ L of filtered PBS and transferred to the SEC column for free siRNA removal, then the first 500 μ L of fractions eluted from the SEC column were collected;
2) add 500. mu.L of filtered PBS to SEC column again, and collect the next 500. mu.L fraction;
3) repeating the step 2) until a total fraction of 10 × 500 μ L is collected, at which time free siRNA in the mixture can be removed to obtain siRNA encapsulated exosomes.
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| WO2023232976A1 (en) | 2022-06-03 | 2023-12-07 | Ags Therapeutics Sas | Extracellular vesicles from genetically-modified microalgae containing endogenously-loaded cargo, their preparation, and uses |
| WO2024088808A1 (en) | 2022-10-24 | 2024-05-02 | Ags Therapeutics Sas | Extracellular vesicles from microalgae, their biodistribution upon intranasal administration, and uses thereof |
| CN118178344A (en) * | 2024-03-21 | 2024-06-14 | 广州市天河诺亚生物工程有限公司 | Preparation method of exosome drug-carrying system, product and application thereof |
| CN118178344B (en) * | 2024-03-21 | 2024-09-06 | 广州市天河诺亚生物工程有限公司 | Preparation method of exosome drug-carrying system, product and application thereof |
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