CN115161260B - Application of Mal-PEG6-NHS-ester in exosome tracing modification and exosome modification method - Google Patents
Application of Mal-PEG6-NHS-ester in exosome tracing modification and exosome modification method Download PDFInfo
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Abstract
The invention relates to the technical field of exosome modification, in particular to an application of Mal-PEG6-NHS-ester in exosome tracing modification and a method for exosome modification. According to the invention, mal-PEG6-NHS-ester is used as a connecting substance, so that single-stranded DNA can be connected to an exosome membrane; the Mal-PEG6-NHS-ester is used for modifying the exosome, so that the operation steps are simple and easy to realize, the reaction time is further shortened, and the process of connecting the single-stranded DNA with the tracer group to the exosome can be simply realized.
Description
Technical Field
The invention relates to the technical field of exosome modification, in particular to an application of Mal-PEG6-NHS-ester in exosome tracing modification and a method for exosome modification.
Background
The exosomes are tiny extracellular vesicles which are secreted by cells and are positioned between 30nm and 150nm, widely exist in various cell culture supernatants and various body fluids, and can transfer various bioactive components wrapped in a double-layer membrane structure. Because of the advantages of biological origin and low antigenicity, exosomes can be used as excellent carriers for drug delivery, however, to observe the distribution of exosomes entering the body, particularly between organs and tissues, it is also necessary to modify the exosome membrane surface in order to obtain tracery properties. The current membrane modification method for obtaining traceability for exosomes mainly comprises the following steps: 1. inducing the expression of fusion proteins of endogenous exosome membrane proteins and fluorescent proteins (such as the expression of Lamp2b-GFP fusion proteins to trace exosomes) on exosome membranes; 2. the polypeptide and protein with fluorescence are directly connected to exosome membrane by means of organic compound mediation, namely chemical modification method. In the chemical modification method of exosome membrane, organic solution DSPE-PEG or NHS-PEG is used, wherein DSPE and NHS are responsible for connecting with hydrophobic exosome lipid membrane, and PEG can be combined with protein, polypeptide and other substances due to hydrophilicity. When chemical connection is carried out, DSPE-PEG or NHS-PEG is firstly incubated with target protein and polypeptide overnight in advance to obtain DSPE-PEG/NHS-PEG connected with the target protein, namely DSPE/NHS-PEG-protein/peptide, and then the pre-connector is incubated with the exosome for 3 hours to obtain the exosome connected with the target protein, so that the exosome obtains tracer. Although the above method can achieve the modification purpose, it has the following disadvantages: 1. the reaction time is long; 2. the reaction system is complex, and the experimental conditions are more severe.
Disclosure of Invention
In order to solve the problems, the invention provides an application of Mal-PEG6-NHS-ester in exosome trace modification and a method for exosome modification. The Mal-PEG6-NHS-ester is used for modifying the exosome, so that the operation steps are simple and easy to realize, the reaction time is further shortened, and the process of connecting the single-stranded DNA with the tracer group to the exosome can be simply realized.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an application of Mal-PEG6-NHS-ester in exosome modification.
The invention also provides a modifier for modifying a substance containing lysine residues, the modifier comprising a thiol-modified single-stranded DNA and Mal-PEG6-NHS-ester linked to the thiol group; the thiol group and Mal-on Mal-PEG6-NHS-ester are linked by a covalent bond.
Preferably, the lysine residue-containing substance includes exosomes.
The invention also provides a method for exosome modification, which comprises the following steps:
incubating the sulfhydryl modified single-stranded DNA with Mal-PEG6-NHS-ester for the first time to obtain a single-stranded DNA-Mal-PEG6-NHS-ester connector;
incubating the single-stranded DNA-Mal-PEG6-NHS-ester connector with exosomes in a second mixing mode to obtain modified exosomes;
or alternatively
Thirdly mixing Mal-PEG6-NHS-ester with exosome for incubation to obtain exosome connected with Mal-PEG6-NHS-ester;
and fourthly, mixing and incubating the exosomes connected with Mal-PEG6-NHS-ester with the sulfhydryl modified single-stranded DNA to obtain modified exosomes.
Preferably, the dosage ratio of the sulfhydryl modified single-stranded DNA, mal-PEG6-NHS-ester and exosome is 11.7nmol:100 μg: 20-60 mug.
Preferably, the pH values of the first mixed incubation, the second mixed incubation, the third mixed incubation and the fourth mixed incubation are respectively 7.2-7.4, and the temperatures of the first mixed incubation, the second mixed incubation, the third mixed incubation and the fourth mixed incubation are respectively 15-25 ℃.
Preferably, the thiol-modified single-stranded DNA is mixed with Mal-PEG6-NHS-ester for incubation for 10min;
the time for mixing and incubating the single-stranded DNA-Mal-PEG6-NHS-ester connector and the exosome is 30min;
the mixing incubation time of the Mal-PEG6-NHS-ester and the exosome is 30min;
the exosomes with Mal-PEG6-NHS-ester attached were incubated with thiol-modified single stranded DNA for 10min.
Preferably, after the modified exosomes are obtained, the purification treatment is further carried out by adopting ultracentrifugation; the centrifugal force of the ultracentrifugation was 100,000Xg, and the time of the ultracentrifugation was 70min.
Preferably, the modified exosomes are obtained further comprising: complementary single-stranded DNA with fluorescent group at 5' end is complementarily combined with the modified exosome to obtain exosome with tracing function.
The beneficial effects are that:
the invention provides an application of Mal-PEG6-NHS-ester in exosome modification. The inventionMal-PEG6-NHS-ester (abbreviated as SM (PEG) 6) is used as an organic medium for connecting exosomes and single-stranded DNA, wherein Mal-full name Maleimide-, namely Maleimide, can react with sulfhydryl groups to form covalent bonds; NHS-ester is N-hydroxysuccinimide ester, and has lysine residue (primary amine, -NH) with amino acid in cell membrane or free protein, polypeptide and other structure 2 ) The ability to bind, using Mal-PEG6-NHS-ester as the binding substance, can link single-stranded DNA to exosome membrane; the Mal-PEG6-NHS-ester is used for modifying the exosome, so that the operation steps are simple and easy to realize, the reaction time is further shortened, and the process of connecting the single-stranded DNA with the tracer group to the exosome can be simply realized.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a schematic diagram of Mal-PEG6-NHS-ester ligation of single-stranded DNA to exosomes;
FIG. 2 is a schematic diagram of modified exosome ligation complementary DNA;
FIG. 3 shows the results of FITC channel fluorescence intensity flow assay;
FIG. 4 is a flow-through analysis of cells that die after co-incubation of unmodified or modified exosomes, wherein NC represents incubation of the unmodified U251 exosome group, U251exo-NHS-Alexa488 represents incubation of Alexa 488-complementary single-stranded DNA-NHS-U251 exosomes, i.e., modified exosome group;
FIG. 5 shows confocal fluorescence microscopy observations, wherein blue signal is nuclear Hoechst staining and green spot signal is tracer exosome signal.
Detailed Description
The invention provides an application of Mal-PEG6-NHS-ester in exosome modification.
The invention also provides a modifier for modifying a substance containing lysine residues, the modifier comprising a thiol-modified single-stranded DNA and Mal-PEG6-NHS-ester linked to the thiol group; the thiol group and Mal-on Mal-PEG6-NHS-ester are linked by a covalent bond.
In the present invention, the lysine residue-containing substance preferably includes exosomes.
The invention also provides a method for exosome modification, which comprises the following steps:
mixing and incubating the sulfhydryl modified single-stranded DNA with Mal-PEG6-NHS-ester to obtain a single-stranded DNA-Mal-PEG6-NHS-ester connector;
and mixing and incubating the single-stranded DNA-Mal-PEG6-NHS-ester connector with the exosome to obtain the modified exosome.
The invention incubates the sulfhydryl modified single-stranded DNA with Mal-PEG6-NHS-ester to obtain the single-stranded DNA-Mal-PEG6-NHS-ester connector. In the present invention, the molar mass ratio of the thiol-modified single-stranded DNA to Mal-PEG6-NHS-ester is preferably 11.7nmol:100 μg. The method for modifying the thiol-modified single-stranded DNA according to the present invention is not particularly limited, and modification methods known to those skilled in the art may be employed. In the present invention, thiol modification is preferably performed at the 5' -end of single-stranded DNA.
In the present invention, the thiol-modified single-stranded DNA preferably comprises, before incubation with Mal-PEG 6-NHS-ester: purifying the sulfhydryl modified single-stranded DNA; the purification treatment preferably comprises: the thiol-modified single-stranded DNA was prepared to a concentration of 0.39mM with sterile water, then 30. Mu.L of the DNA was purified by passing through NAP-5 column, and the DNA left in the column after passing through the column was eluted with 0.5mL of PBS; the NAP-5 column was equilibrated with Phosphate Buffered Saline (PBS) pH7.4 in advance.
The Phosphate Buffered Saline (PBS) of the present invention preferably comprises the following concentrations of components: 210mg/L of potassium dihydrogen phosphate, 9000mg/L of sodium chloride and 726mg/L of disodium hydrogen phosphate heptahydrate; the pH value of the phosphate buffer physiological saline is preferably 7.2-7.4.
In the present invention, the pH value of the mixed incubation of the thiol-modified single-stranded DNA and Mal-PEG6-NHS-ester is preferably 7.2-7.4, more preferably 7.2; the temperature of mixed incubation of the sulfhydryl-modified single-stranded DNA and Mal-PEG6-NHS-ester is preferably 15-25 ℃, more preferably 18-22 ℃; the time for incubation of the thiol-modified single-stranded DNA mixed with Mal-PEG6-NHS-ester is preferably 10min. According to the invention, through proper incubation conditions, sulfydryl on the single-stranded DNA reacts with Mal-groups on Mal-PEG6-NHS-ester to form covalent bonds, so that the single-stranded DNA is connected with Mal-PEG6-NHS-ester to obtain the single-stranded DNA-Mal-PEG6-NHS-ester connector.
In the present invention, the thiol-modified single-stranded DNA preferably further comprises, after incubation with Mal-PEG 6-NHS-ester: the mixed incubation mixture was purified by NAP-5 column and eluted with phosphate buffered saline to give purified single stranded DNA-Mal-PEG6-NHS-ester linker. The invention can remove the excessive Mal-PEG6-NHS-ester by elution treatment.
After obtaining a single-stranded DNA-Mal-PEG6-NHS-ester connector, the invention mixes and incubates the single-stranded DNA-Mal-PEG6-NHS-ester connector with an exosome to obtain a modified exosome.
In the present invention, the molar mass ratio of the single-stranded DNA-Mal-PEG6-NHS-ester linker to the exosome is preferably 11.7nmol:20 to 60. Mu.g, more preferably 11.7nmol:40 μg.
In the present invention, the exosomes preferably include one or more of human glioblastoma cell line U251 exosomes, glioblastoma cell line T98G exosomes, and mouse brain microvascular endothelial cell line bend.3 exosomes.
In the present invention, the method for isolating exosomes preferably comprises: ultracentrifugation of the culture supernatant to obtain a precipitate as the exosome. In the present invention, the centrifugal force of the ultracentrifugation treatment is preferably 100,000×g, and the ultracentrifugation time is preferably 70min.
In the present invention, the pH value of the mixed incubation of the single-stranded DNA-Mal-PEG6-NHS-ester linker and the exosome is preferably 7.2-7.4, more preferably 7.2; the temperature for mixed incubation of the single-stranded DNA-Mal-PEG6-NHS-ester connector and the exosome is preferably 15-25 ℃, more preferably 18-22 ℃; the time for incubation of the single-stranded DNA-Mal-PEG6-NHS-ester linker mixed with exosomes is preferably 30min. The invention combines NHS-ester (N-hydroxysuccinimide ester) on single-stranded DNA-Mal-PEG6-NHS-ester with lysine residue (primary amine, -NH) on exosome by proper incubation conditions 2 ) Combined with, fromAnd obtaining a single-stranded DNA-Mal-PEG 6-NHS-ester-exosome, namely connecting the single-stranded DNA to the exosome through Mal-PEG6-NHS-ester, thereby modifying the exosome.
In the invention, after the single-stranded DNA-Mal-PEG6-NHS-ester connector is mixed and incubated with an exosome, the method preferably further comprises ultracentrifugation treatment to obtain precipitate as the modified exosome; the rotational speed of the ultracentrifugation treatment is preferably 100,000×g, and the ultracentrifugation time is preferably 70min.
The modified exosomes are preferably further comprising: complementary single-stranded DNA with fluorescent group at 5' end is complementarily combined with the modified exosome to obtain exosome with tracing function.
In the present invention, the temperature of the complementary binding is preferably 0 ℃; the time for the complementary binding is preferably 30min. The fluorescent group can be connected to the exosome membrane through the complementary reaction of the single-stranded DNA, so that the exosome with the tracing effect is obtained, and the method has good development prospect.
The invention provides a method for exosome modification, which comprises the following steps:
mixing Mal-PEG6-NHS-ester with exosome for incubation to obtain exosome connected with Mal-PEG6-NHS-ester;
mixing and incubating the exosomes connected with Mal-PEG6-NHS-ester with sulfhydryl modified single-stranded DNA to obtain modified exosomes.
Mal-PEG6-NHS-ester is mixed with exosome for incubation, so that exosome connected with Mal-PEG6-NHS-ester is obtained. In the invention, the mass ratio of Mal-PEG6-NHS-ester to exosome is 5:1. the invention combines NHS-ester (N-hydroxysuccinimide ester) on Mal-PEG6-NHS-ester with lysine residue (primary amine, -NH) on exosome by proper incubation conditions 2 ) And binding to obtain Mal-PEG 6-NHS-ester-linked exosomes.
In the present invention, the exosomes preferably include one or more of human glioblastoma cell line U251 exosomes, glioblastoma cell line T98G exosomes, and mouse brain microvascular endothelial cell line bend.3 exosomes.
In the present invention, the method for isolating exosomes preferably comprises: ultracentrifugation of the culture supernatant to obtain a precipitate as the exosome. In the present invention, the centrifugal force of the ultracentrifugation treatment is preferably 100,000×g, and the ultracentrifugation time is preferably 70min.
In the invention, the pH value of the mixed incubation of Mal-PEG6-NHS-ester and exosome is preferably 7.2-7.4, more preferably 7.2; the temperature of mixing and incubating Mal-PEG6-NHS-ester and exosome is preferably 15-25 ℃, more preferably 18-22 ℃; the time for incubation of Mal-PEG6-NHS-ester mixed with exosomes is preferably 30min.
In the present invention, after the Mal-PEG6-NHS-ester is mixed with exosomes for incubation, the method further comprises: the mixed incubation mixture was purified by NAP-5 column and eluted with phosphate buffered saline to give purified Mal-PEG 6-NHS-ester-linked exosomes. The invention can remove the excessive Mal-PEG6-NHS-ester by elution treatment.
After obtaining the exosomes connected with Mal-PEG6-NHS-ester, the exosomes connected with Mal-PEG6-NHS-ester are mixed and incubated with sulfhydryl modified single-stranded DNA to obtain modified exosomes. In the present invention, the molar mass ratio of the thiol-modified single-stranded DNA to the exosome to which Mal-PEG6-NHS-ester is attached is preferably 11.7nmol:20 to 60. Mu.g, more preferably 11.7nmol:40 μg. The method for modifying the thiol-modified single-stranded DNA according to the present invention is not particularly limited, and modification methods known to those skilled in the art may be employed. In the present invention, thiol modification is preferably performed at the 5' -end of single-stranded DNA.
In the present invention, the pH value of the mixed incubation of the Mal-PEG 6-NHS-ester-linked exosomes and the thiol-modified single-stranded DNA is preferably 7.2-7.4, more preferably 7.2; the mixed incubation temperature of the exosomes connected with Mal-PEG6-NHS-ester and the sulfhydryl modified single-stranded DNA is preferably 15-25 ℃, more preferably 18-22 ℃; the time for incubation of the Mal-PEG 6-NHS-ester-linked exosomes mixed with thiol-modified single-stranded DNA is preferably 10min. According to the preparation method, a Mal-group on an exosome connected with Mal-PEG6-NHS-ester reacts with a sulfhydryl group on single-stranded DNA to form a covalent bond under proper incubation conditions, so that the single-stranded DNA-Mal-PEG 6-NHS-ester-exosome is obtained, namely, the single-stranded DNA is connected to the exosome through Mal-PEG6-NHS-ester, so that the exosome is modified.
In the present invention, it is preferable that the mixed incubation of Mal-PEG 6-NHS-ester-linked exosomes with thiol-modified single-stranded DNA further comprises: ultracentrifugation to obtain a precipitate as a modified exosome; the rotational speed of the ultracentrifugation treatment was 100,000Xg, and the ultracentrifugation time was 70min.
The modified exosomes are preferably further comprising: complementary single-stranded DNA with fluorescent group at 5' end is complementarily combined with the modified exosome to obtain exosome with tracing function.
In the present invention, the temperature of the complementary binding is preferably 0 ℃; the time for the complementary binding is preferably 30min. The fluorescent group can be connected to the exosome membrane through the complementary reaction of the single-stranded DNA, so that the exosome with the tracing effect is obtained, and the method has good development prospect.
For further explanation of the present invention, the use of Mal-PEG6-NHS-ester in exosome tracer modification and exosome modification method provided herein will be described in detail with reference to the examples, but they should not be construed as limiting the scope of the invention.
Example 1
A method for exosome modification, which comprises the following steps (schematic diagram is shown in figure 1):
1) Ultracentrifugation (100,000Xg, 70 min) separates the native exosomes in the U251 cell culture supernatant and resuspension with PBS to a final exosome concentration of 1 μg/μl;
2) The 5'-SH single-stranded DNA1 (5' -end thiol-modified single-stranded DNA) was prepared with sterile water to a molar concentration of 0.39mM (single-stranded DNA1 molecular mass: 6077 ng/nL), followed by purifying the DNA by NAP-5 column (NAP-5 column was equilibrated with PBS pH7.4 in advance), taking 30. Mu.L (i.e., 11.7nmol of single-stranded DNA), eluting the DNA left in the column after passing the column with 0.5mLPBS to obtain a DNA eluent;
3) The DNA eluate was mixed with 20. Mu. LMal-PEG6-NHS-ester (molar mass ratio of 5' -SH single-stranded DNA1 to Mal-PEG6-NHS-ester in the mixture: 11.7nmol:100 μg) and incubating at room temperature (15-25 ℃) for 10min to obtain a mixed solution;
4) The mixture was purified by NAP-5 column and eluted with 1mLPBS, resulting in 1mL of NHS-containing single-stranded DNA1 with the excess Mal-PEG6-NHS-ester removed, at a DNA concentration of 43.5 ng/. Mu.L, i.e., 7.16nmol/mL (7.16 nM);
5) 40. Mu.L of the U251 exosome prepared in step 1) was mixed with 1mL of an eluent containing NHS-single-stranded DNA1 (the molar mass ratio of NHS-single-stranded DNA1 to U251 exosome in the mixed solution at this time was examined to be 7.16nmol:40 μg) and incubated at room temperature for 30min to give 1mL of exosome-NHS-single-stranded DNA1;
6) The exosome-NHS-single stranded DNA1 was then re-centrifuged by ultracentrifugation (100,000Xg, 70 min) to give a precipitate as purified chemically modified exosomes.
Example 2
A method of exosome modification, consisting of the steps of:
1) Obtaining exosome-NHS-single-stranded DNA1 mixed solution according to the steps 1) to 5) in the example 1;
2) Incubating the exosome-NHS-single-stranded DNA1 with 10 mu L of 0.39 mu M complementary single-stranded DNA2 carrying FITC fluorescent groups at 0 ℃ for 30min to obtain exosome-NHS-complementary double-stranded DNA-FITC (schematic diagram is shown in figure 2);
3) The exosomes-NHS-complementary double-stranded DNA-FITC were re-centrifuged using ultracentrifugation (100,000xg, 70 min) to give a precipitate as purified chemically modified exosomes.
Example 3
A method of exosome modification, consisting of the steps of:
1) Obtaining exosome-NHS-single-stranded DNA1 mixed solution according to the steps 1) to 5) in the example 1;
2) Incubating the exosome-NHS-single-stranded DNA1 with 10 mu L of 0.39 mu M complementary single-stranded DNA2 carrying Alexa488 fluorescence groups at 0 ℃ for 30min to obtain exosome-NHS-complementary double-stranded DNa-Alexa488 (schematic diagram is shown in figure 2);
3) The exosome-NHS-complementary double-stranded DNA-Alexa488 was re-centrifuged using ultracentrifugation (100,000Xg, 70 min) to give a chemically modified exosome that precipitated as purified.
Example 4
A similar procedure to example 1 is followed, the only difference being that in step 5) the volume of the U251 exosomes is 20 μl.
Example 5
A similar procedure to example 1 is followed, the only difference being that in step 5) the volume of the U251 exosomes is 30 μl.
Example 6
A process similar to that of example 1, the only difference being that in step 5) the exosomes are T98G exosomes.
Example 7
A process similar to that of example 1, the only difference being that in step 5) the exosome is the bEnd.3 exosome.
Example 8
A method similar to example 3, the only difference being that the exosome-NHS-single stranded DNA1 described in step 2) is replaced with the exosome-NHS-single stranded DNA1 obtained in example 6.
Example 9
A method similar to example 3, the only difference being that the exosome-NHS-single stranded DNA1 described in step 2) is replaced with the exosome-NHS-single stranded DNA1 obtained in example 7.
Application example 1
Endothelial cell complete medium (1 mL per well) was added to the 6-well plate, and the formulation of the endothelial cell complete medium was: ecm+5% fbs (fetal bovine serum) +1% ecgs (endothelial cell growth factor) +1% penicillin, all brands are: scientific cell;
inoculating endothelial cells into a 6-well plate added with a culture medium, wherein the inoculum size is 5 x 10 per well 5 A cell; culturing for 24h after inoculation;
after 24 hours of culture, the culture medium was replaced with a complete culture medium containing the chemically modified exosomes prepared in example 2 (the concentration of the chemically modified exosomes in the culture medium is 40 μg/μl), after replacement, the culture was performed for 12 hours, and after the completion of culture, FITC channel fluorescence intensity flow analysis was performed;
control (NC-HUVEC) was also set: the control group treatment procedure was identical with the only difference that the chemically modified exosomes were replaced with equimolar unmodified exosomes and equimolar FITC-double stranded DNA.
The experimental results are shown in FIG. 3.
As can be seen from FIG. 3, the average fluorescence intensity of umbilical vein endothelial cells (HUVECs) that ingest FITC-labeled exosomes was significantly higher than that of the control group, i.e., exosome-NHS-single-stranded DNA 1-single-stranded DNA2-FITC construction was successful.
Application example 2
Endothelial cell complete medium (1 mL per well) was added to the 6-well plate, and the formulation of the endothelial cell complete medium was: ecm+5% fbs (fetal bovine serum) +1% ecgs (endothelial cell growth factor) +1% penicillin, all brands are: scientific cell;
inoculating endothelial cells into a 6-well plate added with a culture medium, wherein the inoculum size is 5 x 10 per well 5 A cell; culturing for 24h after inoculation;
after 24 hours of culture, the culture medium was replaced with a complete culture medium containing the chemically modified exosomes prepared in example 3 (the concentration of the chemically modified exosomes in the culture medium is 30 μg/μl), after 12 hours of culture after replacement, after the end of culture, each group of dead cells was labeled with 7-AAD, and after staining, flow analysis was performed on the cell PC5.5 channel fluorescence intensity, and the results are shown in fig. 4;
wherein control (NC) represents the incubated unmodified U251 exosome group, U251exo-NHS-Alexa488 group represents the incubated Alexa 488-complementary single-stranded DNA-NHS-U251 exosome, i.e. modified exosome group: the control group treatment procedure was identical to that described above, with the only difference that the chemically modified exosomes were replaced with unmodified natural exosomes of equal quality.
As can be seen from fig. 4, the proportion of 7AAD positive cells in the incubated modified exosome group, i.e. the proportion of cells that die, was not different from the control group, indicating that the chemically modified tracer exosome at this dose had no significant toxic effect on the cells.
Application example 3
HT-22 cell complete medium (1 mL per well) was added to a 6-well plate, the complete medium formulation being: dmem+10% fbs (fetal bovine serum) +1% penicillin, all brands are: gibco;
6-well plates with medium added are inoculated with HT-22 endothelial cells at an amount of 5X 10 per well 5 A cell; culturing for 24h after inoculation;
after 24h of culture, the culture medium is replaced by a complete culture medium containing the chemical modification exosomes prepared in example 9 (the concentration of the chemical modification exosomes in the culture medium is 30 mug/mul), after replacement, the culture is carried out for 12h, after the culture is finished, hoechst dye lining is carried out on each group of cells, and then the cells are observed under a confocal inverted fluorescence microscope;
the results are shown in FIG. 5, where blue signal is nuclear Hoechst staining and green spot signal is tracer exosome signal. This result indicates successful tracer chemical modification of the exosomes.
In summary, the exosome modification method provided by the invention is simple and easy to implement in operation steps, further shortens the reaction time, and can simply implement the process of connecting the single-stranded DNA with the tracer group to the exosome.
While the invention has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present invention, which is therefore to be limited only by the appended claims.
Claims (4)
1. A method of exosome modification comprising the steps of:
incubating the sulfhydryl modified single-stranded DNA with Mal-PEG6-NHS-ester for 10min to obtain a single-stranded DNA-Mal-PEG6-NHS-ester connector;
incubating the single-stranded DNA-Mal-PEG6-NHS-ester connector with the exosome for 30min in a second mixing mode to obtain a modified exosome;
or alternatively
Thirdly mixing Mal-PEG6-NHS-ester with exosome, and incubating for 30min to obtain exosome connected with Mal-PEG6-NHS-ester;
mixing and incubating the exosomes connected with Mal-PEG6-NHS-ester with thiol-modified single-stranded DNA for 10min to obtain modified exosomes;
the modified exosomes are obtained and then comprise: complementary single-stranded DNA with fluorescent group modification at the 5' end is complementarily combined with the modified exosome to obtain the exosome with tracing function;
mal-is known as Maleimide-, i.e., maleimide group; NHS-ester is known by its full name as N-hydroxysuccinimide ester.
2. The method according to claim 1, wherein the ratio of the amount of thiol-modified single-stranded DNA, mal-PEG 6-NHS-ster and exosome is 11.7nmol:100 μg: 20-60 mug.
3. The method according to claim 1 or 2, wherein the pH values of the first mixed incubation, the second mixed incubation, the third mixed incubation and the fourth mixed incubation are respectively 7.2-7.4, and the temperatures of the first mixed incubation, the second mixed incubation, the third mixed incubation and the fourth mixed incubation are respectively 15-25 ℃.
4. The method according to claim 1 or 2, further comprising purifying by ultracentrifugation after obtaining the modified exosomes; the centrifugal force of the ultracentrifugation was 100,000Xg, and the time of the ultracentrifugation was 70min.
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