CN115161260A - 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 application of Mal-PEG6-NHS-ester in exosome tracing modification and an exosome modification method. The invention uses Mal-PEG6-NHS-ester as connecting material, which can connect single-chain DNA to the exosome membrane; the method utilizes Mal-PEG6-NHS-ester to modify the exosome, not only has simple and convenient operation steps and easy realization, but also further shortens the time required by the reaction, and can simply realize the process of connecting the single-stranded DNA with the tracer group to the exosome.
Description
Technical Field
The invention relates to the technical field of exosome modification, in particular to application of Mal-PEG6-NHS-ester in exosome tracing modification and an exosome modification method.
Background
The exosome is a tiny extracellular vesicle which is secreted by cells and is positioned between 30nm and 150nm, is widely present in various cell culture supernatants and various body fluids, and can transfer various bioactive components which are wrapped in a double-layer membrane structure. The exosome can be used as an excellent carrier for drug delivery due to the advantages of biological source and low antigenicity, however, the exosome membrane needs to be subjected to surface modification to obtain tracer property in order to observe the distribution of the exosome entering the body particularly among each organ and each tissue. The current membrane modification method for obtaining tracer for exosome mainly comprises: 1. inducing and expressing fusion protein of endogenous exosome membrane protein and fluorescent protein (such as expression Lamp2b-GFP fusion protein to trace exosome) to express on an exosome membrane; 2. the polypeptide and protein with fluorescence are directly connected to an exosome membrane by means of mediation of organic compounds, namely a chemical modification method. In the chemical modification method of the exosome membrane, an organic solution DSPE-PEG or NHS-PEG is commonly used, in the scheme, DSPE and NHS are responsible for connecting with hydrophobic exosome lipid membrane, and PEG can be combined with substances such as protein, polypeptide and the like due to the hydrophilicity. When chemical ligation is carried out, firstly, DSPE-PEG or NHS-PEG is 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-ligation and an exosome are incubated for 3 hours to obtain the exosome connected with the target protein, so that the exosome can obtain tracer. Although the above method can achieve the purpose of modification, it has disadvantages in that: 1. the reaction needs longer time; 2. the reaction system is complex and the experimental conditions are harsh.
Disclosure of Invention
In order to solve the problems, the invention provides application of Mal-PEG6-NHS-ester in exosome tracing modification and an exosome modification method. The method utilizes Mal-PEG6-NHS-ester to modify the exosome, not only has simple and convenient operation steps and easy realization, but also further shortens the time required by the reaction and can simply realize the process of connecting the single-stranded DNA with the tracer group to the exosome.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides application of Mal-PEG6-NHS-ester in exosome modification.
The invention also provides a modifier for modifying the substance containing the lysine residue, wherein the modifier comprises single-stranded DNA modified by sulfydryl and Mal-PEG6-NHS-ester connected with the sulfydryl; the sulfhydryl and Mal-on Mal-PEG6-NHS-ester are connected through covalent bond.
Preferably, the lysine residue-containing substance comprises exosomes.
The invention also provides a method for modifying exosomes, which comprises the following steps:
carrying out first mixed incubation on sulfhydryl-modified single-stranded DNA and Mal-PEG6-NHS-ester to obtain a single-stranded DNA-Mal-PEG6-NHS-ester connector;
secondly, mixing and incubating the single-chain DNA-Mal-PEG6-NHS-ester connector and the exosome to obtain a modified exosome;
or
Thirdly, mixing and incubating Mal-PEG6-NHS-ester and exosome to obtain exosome connected with Mal-PEG6-NHS-ester;
and fourthly, mixing and incubating the exosome connected with the Mal-PEG6-NHS-ester with the sulfhydryl modified single-stranded DNA to obtain the modified exosome.
Preferably, the dosage proportion of the sulfhydryl modified single-stranded DNA, mal-PEG6-NHS-ester and the exosome is 11.7nmol:100 μ g:20 to 60 mu g.
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 to 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 to 25 ℃.
Preferably, the mixed incubation time of the sulfhydryl-modified single-stranded DNA and Mal-PEG6-NHS-ester is 10min;
the single-chain DNA-Mal-PEG6-NHS-ester connector and the exosome are mixed and incubated for 30min;
the incubation time of the mixture of the Mal-PEG6-NHS-ester and the exosome is 30min;
the exosome connected with Mal-PEG6-NHS-ester and the sulfhydryl modified single-stranded DNA are mixed and incubated for 10min.
Preferably, after the modified exosome is obtained, ultracentrifugation is adopted for purification treatment; the centrifugal force of the ultracentrifugation was 100,000 Xg, and the time of the ultracentrifugation was 70min.
Preferably, the modified exosome further comprises: and (3) complementarily combining the complementary single-stranded DNA modified by the fluorescent group at the 5' end with the modified exosome to obtain the exosome with the tracing effect.
Has the advantages that:
the invention provides application of Mal-PEG6-NHS-ester in exosome modification. Mal-PEG6-NHS-ester (abbreviation: SM (PEG) 6) is used as an organic medium for connecting an exosome and single-stranded DNA, wherein Mal-full name Maleimide-, namely Maleimide, can react with sulfhydryl to form a covalent bond; NHS-ester is N-hydroxysuccinimide ester, and has lysine residue (primary amine, -NH) with amino acid in structure of cell membrane, free protein, polypeptide, etc 2 ) The capacity of combination, namely, the Mal-PEG6-NHS-ester is used as a connecting substance, so that the single-stranded DNA can be connected to an exosome membrane; the method utilizes Mal-PEG6-NHS-ester to modify the exosome, not only has simple and convenient operation steps and easy realization, but also further shortens the time required by the reaction, and can simply realize the process of connecting the single-stranded DNA with the tracer group to the exosome.
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 embodiments will be briefly described below.
FIG. 1 is a schematic diagram of Mal-PEG6-NHS-ester connecting single-stranded DNA and exosome;
FIG. 2 is a schematic diagram of the modified exosome ligated complementary DNA;
FIG. 3 shows the result of flow analysis of FITC channel fluorescence intensity;
FIG. 4 shows the results of cell flow analysis of death after co-incubation of unmodified or modified exosomes, wherein NC denotes incubation of unmodified U251 exosome set, U251exo-NHS-Alexa488 denotes incubation of Alexa 488-complementary single stranded DNA-NHS-U251 exosomes, i.e. modified exosome set;
FIG. 5 shows confocal fluorescence microscopy results, where blue signals are nuclear Hoechst staining and green punctate signals are tracer exosome signals.
Detailed Description
The invention provides application of Mal-PEG6-NHS-ester in exosome modification.
The invention also provides a modifier for modifying the substance containing the lysine residue, wherein the modifier comprises single-stranded DNA modified by sulfydryl and Mal-PEG6-NHS-ester connected with the sulfydryl; the sulfhydryl and Mal-on Mal-PEG6-NHS-ester are connected through covalent bond.
In the present invention, the lysine residue-containing substance preferably includes an exosome.
The invention also provides a method for modifying exosomes, which comprises the following steps:
mixing and incubating sulfydryl modified single-stranded DNA and Mal-PEG6-NHS-ester to obtain a single-stranded DNA-Mal-PEG6-NHS-ester connector;
and mixing and incubating the single-chain DNA-Mal-PEG6-NHS-ester connector and the exosome to obtain the modified exosome.
The single-stranded DNA modified by sulfydryl and Mal-PEG6-NHS-ester are mixed and incubated to obtain the single-stranded DNA-Mal-PEG6-NHS-ester connector. In the invention, the molar mass ratio of the sulfhydryl modified single-stranded DNA to Mal-PEG6-NHS-ester is preferably 11.7nmol:100 μ g. The method for modifying the thiol-modified single-stranded DNA of the present invention is not particularly limited, and may be a modification method known to those skilled in the art. The present invention preferably modifies the thiol group at the 5' end of the single-stranded DNA.
In the present invention, the mixed incubation of the thiol-modified single-stranded DNA and Mal-PEG6-NHS-ester preferably comprises: purifying the sulfhydryl modified single-stranded DNA; the purification treatment preferably comprises: the thiol-modified single-stranded DNA was made to a concentration of 0.39mM with sterile water, and then 30. Mu.L of the DNA was purified by passing through a NAP-5 column, and the DNA remaining 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) at pH7.4 in advance.
The Phosphate Buffered Saline (PBS) of the present invention preferably comprises the following components in the following concentrations: 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 buffered saline is preferably 7.2 to 7.4.
In the invention, the pH value of the mixed incubation of the sulfhydryl-modified single-stranded DNA and Mal-PEG6-NHS-ester is preferably 7.2-7.4, and more preferably 7.2; the mixed incubation temperature of the sulfhydryl-modified single-stranded DNA and Mal-PEG6-NHS-ester is preferably 15-25 ℃, and more preferably 18-22 ℃; the time for mixing and incubating the sulfhydryl modified single-stranded DNA and Mal-PEG6-NHS-ester is preferably 10min. The invention forms covalent bond by the reaction of sulfydryl on single-chain DNA and Mal-group on Mal-PEG6-NHS-ester under proper incubation condition, thus connecting the single-chain DNA and Mal-PEG6-NHS-ester and obtaining the single-chain DNA-Mal-PEG6-NHS-ester connector.
In the present invention, after the thiol-modified single-stranded DNA is mixed and incubated with Mal-PEG6-NHS-ester, the method preferably further comprises: and (3) purifying the mixed incubated mixture by using a NAP-5 column and eluting by using phosphate buffered saline to obtain a purified single-stranded DNA-Mal-PEG6-NHS-ester connector. The method can remove redundant Mal-PEG6-NHS-ester through elution treatment.
After the single-chain DNA-Mal-PEG6-NHS-ester connector is obtained, the single-chain DNA-Mal-PEG6-NHS-ester connector and the exosome are mixed and incubated to obtain the modified exosome.
In the invention, the molar mass ratio of the single-stranded DNA-Mal-PEG6-NHS-ester connector to the exosome is preferably 11.7nmol:20 to 60. Mu.g, more preferably 11.7nmol:40 μ g.
In the present invention, the exosomes preferably include one or more of the human glioblastoma cell line U251 exosome, the glioblastoma cell line T98G exosome and the mouse brain microvascular endothelial cell line b end.3 exosome.
In the present invention, the method for isolating exosomes preferably comprises: and ultracentrifuging culture medium supernatant to obtain precipitate as the exosome. In the present invention, the centrifugal force of the ultracentrifugation treatment is preferably 100,000 Xg, and the time of the ultracentrifugation is preferably 70min.
In the invention, the pH value of the mixed incubation of the single-stranded DNA-Mal-PEG6-NHS-ester connector and the exosome is preferably 7.2-7.4, and more preferably 7.2; the temperature of mixed incubation of the single-chain DNA-Mal-PEG6-NHS-ester connector and the exosome is preferably 15-25 ℃, and more preferably 18-22 ℃; the time for mixing and incubating the single-stranded DNA-Mal-PEG6-NHS-ester connector and the exosome is preferably 30min. The invention relates to a method for preparing single-stranded DNA-Mal-PEG6-NHS-ester by incubating NHS-ester (N-hydroxysuccinimide ester) on a single-stranded DNA-Mal-PEG6-NHS-ester and lysine residues (primary amine, -NH) on an exosome under proper incubation conditions 2 ) And (3) combining to obtain the single-stranded DNA-Mal-PEG 6-NHS-ester-exosome, namely connecting the single-stranded DNA to the exosome through Mal-PEG6-NHS-ester so as to modify the exosome.
In the invention, after the single-stranded DNA-Mal-PEG6-NHS-ester connector is mixed and incubated with the exosome, the preferred method further comprises ultracentrifugation treatment, and the precipitate is obtained and is the modified exosome; the rotational speed of the ultracentrifugation treatment is preferably 100,000 Xg, and the ultracentrifugation time is preferably 70min.
After obtaining the modified exosome, the method preferably further comprises: and (3) complementarily combining the complementary single-stranded DNA modified by the fluorescent group at the 5' end with the modified exosome to obtain the exosome with the 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 invention can connect the fluorescent group to the exosome membrane through the complementary reaction of single-stranded DNA, so as to obtain the exosome with the tracing function, thereby having good development prospect.
The invention provides an exosome modification method, which comprises the following steps:
mixing and incubating Mal-PEG6-NHS-ester and an exosome to obtain an exosome connected with Mal-PEG6-NHS-ester;
and mixing and incubating the exosome connected with Mal-PEG6-NHS-ester with the sulfhydryl modified single-stranded DNA to obtain the modified exosome.
The invention mixes Mal-PEG6-NHS-ester with exosomeAnd (4) incubating to obtain an exosome connected with Mal-PEG6-NHS-ester. In the invention, the mass ratio of the Mal-PEG6-NHS-ester to the exosome is 5:1. the invention uses proper incubation condition to make NHS-ester on Mal-PEG6-NHS-ester and lysine residue (primary amine, -NH) on exosome 2 ) And combining to obtain an exosome connected with Mal-PEG6-NHS-ester.
In the present invention, the exosomes preferably include one or more of the human glioblastoma cell line U251 exosome, the glioblastoma cell line T98G exosome and the mouse brain microvascular endothelial cell line b end.3 exosome.
In the present invention, the method for isolating exosomes preferably comprises: and ultracentrifuging culture medium supernatant to obtain precipitate as the exosome. In the present invention, the centrifugal force of the ultracentrifugation treatment is preferably 100,000 Xg, 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, and more preferably 7.2; the temperature of mixed incubation of the Mal-PEG6-NHS-ester and the exosome is preferably 15-25 ℃, and more preferably 18-22 ℃; the period of incubation of the Mal-PEG6-NHS-ester and the exosome in a mixing mode is preferably 30min.
In the invention, after the Mal-PEG6-NHS-ester is mixed and incubated with exosome, the method further comprises the following steps: and (3) purifying the mixed incubated mixture by using a NAP-5 column and eluting by using phosphate buffered saline to obtain the purified exosome connected with Mal-PEG6-NHS-ester. The method can remove redundant Mal-PEG6-NHS-ester through elution treatment.
After the exosome connected with the Mal-PEG6-NHS-ester is obtained, the exosome connected with the Mal-PEG6-NHS-ester and the sulfhydryl modified single-stranded DNA are mixed and incubated to obtain the modified exosome. In the invention, the molar mass ratio of the sulfhydryl-modified single-stranded DNA to the exosome connected with Mal-PEG6-NHS-ester is preferably 11.7nmol:20 to 60. Mu.g, more preferably 11.7nmol:40 μ g. The modification method of the sulfhydryl modified single-stranded DNA has no special requirements, and the modification method known to the skilled person can be adopted. The present invention preferably modifies the thiol group at the 5' end of the single-stranded DNA.
In the invention, the pH value of the mixed incubation of the exosome connected with Mal-PEG6-NHS-ester and the sulfhydryl modified single-stranded DNA is preferably 7.2-7.4, and more preferably 7.2; the temperature of mixed incubation of the exosome connected with Mal-PEG6-NHS-ester and the sulfhydryl modified single-stranded DNA is preferably 15-25 ℃, and more preferably 18-22 ℃; the time for mixing and incubating the exosome connected with Mal-PEG6-NHS-ester and the sulfhydryl modified single-stranded DNA is preferably 10min. According to the invention, through a proper incubation condition, a Mal-group on an exosome connected with Mal-PEG6-NHS-ester reacts with a sulfhydryl on single-stranded DNA to form a covalent bond, so that the single-stranded DNA-Mal-PEG 6-NHS-ester-exosome is obtained, namely the single-stranded DNA is connected on the exosome through the Mal-PEG6-NHS-ester, thereby modifying the exosome.
In the present invention, the exosome connected with Mal-PEG6-NHS-ester preferably further comprises, after mixed incubation with thiol-modified single-stranded DNA: ultracentrifugation treatment to obtain precipitate as modified exosome; the rotational speed of the ultracentrifugation treatment was 100,000 Xg, and the ultracentrifugation time was 70min.
After obtaining the modified exosome, the method preferably further comprises: and (3) complementarily combining the complementary single-stranded DNA modified by the fluorescent group at the 5' end with the modified exosome to obtain the exosome with the 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 invention can connect the fluorescent group to the exosome membrane through the complementary reaction of single-stranded DNA, so as to obtain the exosome with the tracing function, thereby having good development prospect.
In order to further illustrate the present invention, the application of Mal-PEG6-NHS-ester in the tracing modification of exosome and the modification method of exosome provided by the present invention are described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A method for modifying exosome comprises the following steps (the schematic diagram is shown in figure 1):
1) Separating natural exosomes in the culture supernatant of the U251 cells by an ultracentrifugation method (100,000 Xg, 70 min) and resuspending the exosomes by PBS so that the final concentration of the exosomes is 1 mu g/mu L;
2) 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 weight: 6077 ng/nL), followed by taking 30. Mu.L (i.e., 11.7nmol of single-stranded DNA) of the DNA purified by passing through a NAP-5 column (the NAP-5 column was previously equilibrated with PBS of pH 7.4), and the DNA remaining in the column after passing through the column was eluted with 0.5mLPBS to obtain a DNA eluate;
3) Mixing the DNA eluent with 20 mu LMal-PEG6-NHS-ester (the molar mass ratio of the 5' -SH single-stranded DNA1 to the Mal-PEG6-NHS-ester in the mixed solution is 11.7nmol:100 mu g) and incubating for 10min at room temperature (15-25 ℃) to obtain a mixed solution;
4) Purifying the mixed solution by using a NAP-5 column and eluting by using 1mL of LPBS to finally obtain 1mL of eluent containing NHS-single-stranded DNA1 from which redundant Mal-PEG6-NHS-ester is removed, wherein the DNA concentration is 43.5 ng/mu L, namely 7.16nmol/mL (7.16 nM);
5) Mixing 40 μ L of the U251 exosomes prepared in step 1) with 1mL of an eluent containing NHS-single-stranded DNA1 (when the molar mass ratio of NHS-single-stranded DNA1 to U251 exosomes in the mixture was detected to be 7.16nmol:40 mu g) and incubating for 30min at room temperature to obtain 1mL of exosome-NHS-single-stranded DNA1;
6) The exosome-NHS-single stranded DNA1 was then re-centrifuged using ultracentrifugation (100,000 Xg, 70 min) to obtain a precipitate as purified chemically modified exosomes.
Example 2
A method of exosome modification consisting of the steps of:
1) Obtaining an exosome-NHS-single-stranded DNA1 mixed solution according to steps 1) to 5) in example 1;
2) Incubating the exosome-NHS-single-stranded DNA1 and 10 mu L of 0.39 mu M complementary single-stranded DNA2 carrying FITC fluorescent group at 0 ℃ for 30min to obtain exosome-NHS-complementary double-stranded DNA-FITC (the schematic diagram is shown in figure 2);
3) The exosome-NHS-complementary double stranded DNA-FITC was re-centrifuged using ultracentrifugation (100,000 Xg, 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 an exosome-NHS-single stranded DNA1 mixed solution according to steps 1) to 5) in 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 fluorescent groups at 0 ℃ for 30min to obtain an exosome-NHS-complementary double-stranded DNA-Alexa488 (the schematic diagram is shown in figure 2);
3) The exosome-NHS-complementary double stranded DNA-Alexa488 was re-centrifuged using ultracentrifugation (100,000 Xg, 70 min) to obtain a pellet as purified chemically modified exosomes.
Example 4
A method similar to example 1, the only difference being that the volume of U251 exosomes in step 5) is 20 μ Ι _.
Example 5
A method similar to example 1, with the only difference that the volume of U251 exosomes in step 5) was 30 μ Ι _.
Example 6
A method similar to example 1, the only difference being that in step 5) the exosomes are T98G exosomes.
Example 7
A method similar to example 1, the only difference being that in step 5) the exosomes are b end.3 exosomes.
Example 8
A method similar to example 3, except that the exosome-NHS-single-stranded DNA1 obtained in example 6 was replaced with the exosome-NHS-single-stranded DNA1 obtained in example 2) in step 2).
Example 9
A method similar to example 3, except that the exosome-NHS-single-stranded DNA1 obtained in example 7 was replaced with the exosome-NHS-single-stranded DNA1 obtained in example 2) in step 2).
Application example 1
Endothelial cell complete medium (1 mL per well) was added to 6-well plates, and the formulation of the endothelial cell complete medium was: ECM +5% FBS (fetal bovine serum) +1% ECGS (endothelial cell growth factor) +1% penicillin, brands are: sciencell;
the 6-well plates to which the medium was added were inoculated with endothelial cells at 5 x 10 per well 5 (ii) individual cells; culturing for 24h after inoculation;
after 24h of culture, replacing the culture medium with a complete culture medium containing the chemically modified exosome prepared in example 2 (the concentration of the chemically modified exosome in the culture medium is 40 mug/muL), culturing for 12h after replacement, and performing FITC channel fluorescence intensity flow analysis after the culture is finished;
a control group (NC-HUVEC) was also set: the control treatment procedure was as above, the only difference being the replacement of chemically modified exosomes by equimolar unmodified exosomes and equimolar FITC-double stranded DNA.
The results of the experiment are shown in FIG. 3.
As can be seen from FIG. 3, the average fluorescence intensity of umbilical vein endothelial cells (HUVECs) which take in the FITC-labeled exosomes is significantly higher than that of the control group, i.e., the exosomes-NHS-single stranded DNA 1-single stranded DNA2-FITC is successfully constructed.
Application example 2
Endothelial cell complete medium (1 mL per well) was added to 6-well plates, and the formulation of the endothelial cell complete medium was: ECM +5% FBS (fetal bovine serum) +1% ECGS (endothelial cell growth factor) +1% penicillin, brands are: sciencell;
the 6-well plates with added medium were seeded with endothelial cells in 5 × 10 cells per well 5 (ii) individual cells; culturing for 24h after inoculation;
after 24h 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 was 30 μ g/μ L), and after 12h of culture, after the culture was finished, the dead cells in each group were stained and labeled with 7-AAD, and the fluorescence intensity of the PC5.5 channel of the cells was analyzed by flow analysis, and the results are shown in fig. 4;
wherein the control group (NC) represents incubation of the unmodified U251 exosome group, the U251exo-NHS-Alexa488 group represents incubation of the Alexa 488-complementary single stranded DNA-NHS-U251 exosomes, i.e., the modified exosome group: the control treatment process was the same as above, the only difference being that the chemically modified exosomes were replaced by unmodified natural exosomes of equal mass.
As can be seen from fig. 4, the proportion of 7AAD positive cells in the group of incubated modified exosomes, i.e. the proportion of cells undergoing death, was not different from that in the control group, indicating that the chemical modification tracing exosomes at this dose had no obvious toxic effect on the cells.
Application example 3
HT-22 cell complete medium (1 mL per well) was added to 6-well plates, the complete medium formulation being: DMEM +10% FBS (fetal bovine serum) +1% penicillin, brand: gibco;
the 6-well plates to which the medium was added were seeded with HT-22 endothelial cells in an amount of 5 × 10 cells per well 5 (ii) individual cells; culturing for 24h after inoculation;
after 24h of culture, the culture medium was replaced with a complete culture medium containing the chemically modified exosomes prepared in example 9 (the concentration of the chemically modified exosomes in the culture medium was 30 μ g/μ L), and after 12h of culture, each group of cells was subjected to Hoechst dye-lined nuclei and then observed under a confocal inverted fluorescence microscope after the culture was completed;
the results are shown in FIG. 5, where blue signals are nuclear Hoechst staining and green punctual signals are tracer exosome signals. This result indicates that the tracer chemical modification of exosomes was successful.
In conclusion, the exosome modification method provided by the invention not only has simple and convenient operation steps and is easy to realize, but also further shortens the time required by reaction, and can simply realize the process of connecting the single-stranded DNA with the tracer group to the exosome.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
- Application of Mal-PEG6-NHS-ester in exosome modification.
- 2. A modifier for modifying substances containing lysine residues is characterized by comprising sulfhydryl-modified single-stranded DNA and Mal-PEG6-NHS-ester connected with the sulfhydryl; the sulfhydryl and Mal-on Mal-PEG6-NHS-ester are connected through covalent bond.
- 3. The modified body of claim 2, wherein the lysine residue-containing substance comprises an exosome.
- 4. A method of exosome modification comprising the steps of:carrying out first mixed incubation on sulfhydryl-modified single-stranded DNA and Mal-PEG6-NHS-ester to obtain a single-stranded DNA-Mal-PEG6-NHS-ester connector;secondly, mixing and incubating the single-chain DNA-Mal-PEG6-NHS-ester connector and the exosome to obtain a modified exosome;orThirdly, mixing and incubating Mal-PEG6-NHS-ester and exosome to obtain exosome connected with Mal-PEG6-NHS-ester;and fourthly, mixing and incubating the exosome connected with the Mal-PEG6-NHS-ester with the sulfhydryl modified single-stranded DNA to obtain the modified exosome.
- 5. The method according to claim 4, wherein the thiol-modified single-stranded DNA, mal-PEG6-NHS-ester and exosome are used in an amount ratio of 11.7nmol:100 μ g:20 to 60 mu g.
- 6. The method according to claim 4 or 5, wherein the pH value of the first mixed incubation, the pH value of the second mixed incubation, the pH value of the third mixed incubation and the pH value of the fourth mixed incubation are respectively 7.2-7.4, and the temperature of the first mixed incubation, the second mixed incubation, the third mixed incubation and the fourth mixed incubation are respectively 15-25 ℃.
- 7. The method according to claim 4 or 5, wherein the thiol-modified single-stranded DNA is incubated with Mal-PEG6-NHS-ester for 10min;the single-chain DNA-Mal-PEG6-NHS-ester connector and the exosome are mixed and incubated for 30min;the incubation time of the mixture of the Mal-PEG6-NHS-ester and the exosome is 30min;the exosome connected with Mal-PEG6-NHS-ester and the sulfhydryl modified single-stranded DNA are mixed and incubated for 10min.
- 8. The method according to claim 4 or 5, wherein after obtaining the modified exosome, further comprising performing a purification treatment by ultracentrifugation; the centrifugal force of the ultracentrifugation was 100,000 Xg, and the time of the ultracentrifugation was 70min.
- 9. The method according to claim 4 or 5, wherein obtaining the modified exosome further comprises: and (3) complementarily combining the complementary single-stranded DNA modified by the fluorescent group at the 5' end with the modified exosome to obtain the exosome with the tracing effect.
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