CN115806937A - Application of unbiased separation and enrichment of extracellular vesicles by using lipid affinity polypeptide pHLIP - Google Patents
Application of unbiased separation and enrichment of extracellular vesicles by using lipid affinity polypeptide pHLIP Download PDFInfo
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Abstract
The invention relates to an application of unbiased separation and enrichment of extracellular vesicles by using a lipid affinity polypeptide pHLIP, belonging to the technical field of biology. The invention firstly provides a method for unbiased separation and enrichment of sEVs from a biological sample by utilizing the interaction of pHLIP and sEVs lipid bilayers. Compared with the classical ultracentrifugation method, the method is simple, rapid, efficient and low in cost, large instruments are not required to be used in the whole separation process, and the method does not need modification of other antibodies and aptamers, so that the method has a good clinical application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of unbiased separation and enrichment of extracellular vesicles by using a lipid affinity polypeptide pHLIP.
Background
Extracellular vesicles (sves), a type of vesicular material secreted by cells, contain a phospholipid bilayer structure and are widely distributed in various body fluids. Among them, extracellular vesicles having a size of 30-200nm are defined as exosomes. Due to the high content of sEVs, which persist in all physiological and disease stages, while their inherent stability ensures the integrity of the biomolecules they carry, various techniques have been developed for the isolation of sEVs, including ultracentrifugation, polymer precipitation, etc., and although traditional sEVs isolation methods are widely used in scientific research, they cannot handle micro-samples, have long processing times, have low purity of the resulting sEVs, and risk of breakage of the sEVs. In addition, when antibodies or aptamers are used for immunocapturing sEVs, due to the high heterogeneity of sEVs molecular composition, the traditional immunoseparation method can lose relevant sEVs with low expression or no relevant antigen expression, and lose important information related to tumors. Therefore, the establishment of a rapid, efficient and deletion-free method for separating and enriching sEVs is urgently needed clinically.
pH (Low) insertion peptide (pHLIP) (NH) containing 36 amino acids 2 -CAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT-COOH) is derived from the C helix of bacteriorhodopsin. pHLIP binds strongly to the surface of the lipid bilayer when the pH is higher than 7. Under acidic conditions, pHLIP can cross lipid bilayers to form transmembrane helices. In addition, upon binding or insertion of pHLIP into the lipid bilayer, it has less interference with the lipid bilayer and they do not induce membrane permeation or pore formation, and thus pHLIP can interact with the lipid bilayer of sEVs without bias in separating and enriching sEVs.
Disclosure of Invention
The invention solves the technical difficulties of unbiased separation and enrichment of lower sEVs in the prior art from a sample, and provides a simple method for unbiased separation and enrichment of sEVs by using the binding capacity of the lipid affinity polypeptide pHLIP and the lipid bilayer of an extracellular vesicle. The method is simple, rapid, efficient, low in cost, free of biased enrichment, and free of large-scale instruments in the whole separation process.
According to the purpose of the invention, the application of unbiased separation and enrichment of extracellular vesicles by using lipid affinity polypeptide pHLIP is provided, wherein the lipid affinity polypeptide pHLIP modified on the surface of a micro-nano material is incubated with a sample containing the extracellular vesicles, the sequence of the lipid affinity polypeptide pHLIP is shown as SEQ ID NO:1, and the lipid affinity polypeptide pHLIP is combined with a lipid bilayer of the extracellular vesicles; and after incubation, centrifuging, and precipitating to obtain the extracellular vesicles.
Preferably, the micro-nano material is a magnetic microsphere, a polystyrene microsphere, a ZnO nano material or TiO 2 And (3) nano materials.
Preferably, the specific preparation method of the lipid affinity polypeptide pHLIP modified on the surface of the micro-nano material comprises the following steps: firstly modifying polydopamine on the surface of a micro-nano material, and then modifying streptavidin to be connected with the polydopamine; modifying biotin on the lipid affinity polypeptide pHLIP, and modifying the lipid affinity polypeptide pHLIP on the surface of the micro-nano material through the action of the streptavidin and the biotin.
Preferably, the biotin is modified with a disulfide bond.
Preferably, a dithiothreitol solution or tris (2-carboxyethyl) phosphine is added to the obtained precipitate, and the dithiothreitol solution or the tris (2-carboxyethyl) phosphine breaks the disulfide bond on the biotin, so that extracellular vesicles are released from the micro-nano material.
Preferably, the precipitate is washed with a phosphate buffer.
Preferably, the co-incubation time is 20min to 60min, and the temperature is 18 ℃ to 30 ℃.
Generally, compared with the prior art, the technical scheme conceived by the invention mainly has the following technical advantages:
(1) The method can modify pHLIP on the surface of the micro-nano material only through simple biological coupling, the modification process and the method are simple, the cost is low, the interaction of pHLIP and a lipid bilayer is utilized, the unbiased separation and enrichment of sEVs can be easily realized, and the method does not need modification of other antibodies and aptamers and does not need large-scale instruments.
(2) According to the invention, the micro-nano material has a larger specific surface area, so that more binding sites can be used for pHLIP modification.
(3) In the invention, preferably, pHLIP modified by biotin with disulfide bonds is used for capturing sEVs, and the captured sEVs can be released without loss for downstream analysis through the action of dithiothreitol or tri (2-carboxyethyl) phosphine and the disulfide bonds.
Drawings
FIG. 1 shows the successful verification of streptavidin modification.
FIG. 2 shows the confirmation of pHLIP acting on cell membrane.
FIG. 3 shows the optimization of pHLIP with cell membrane action time.
FIG. 4 is an SEM image of pHLIP-modified polystyrene microspheres capturing sEVs.
FIG. 5 is a graph showing the effect of incubation time of pHLIP-modified polystyrene microspheres with sEVs on capture efficiency.
FIG. 6 shows the capture efficiency of pHLIP-modified polystyrene microspheres on different cell-derived sEVs.
FIG. 7 is an immunological characterization of sEVs released from pHLIP modified polystyrene microsphere surface: (a) prior to release; and (b) after release.
FIG. 8 is a qPCR of the deletion mutation of the downstream EGFR19 exon in a material capture mock sample sEVs (1. Mu.L PC9 cell-derived sEVs + 99. Mu.L healthy human serum).
FIG. 9 shows sanger sequencing fragments from material capture to mock samples of sEVs (1. Mu.L PC9 cell-derived sEVs + 99. Mu.L healthy human serum) with EGFR19 exon deletion mutations.
Figure 10 is qPCR with deletion mutations in downstream EGFR19 exons capturing patient serum svs from material.
FIG. 11 is a diagram showing the modification of pHLIP and binding to extracellular vesicles according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The invention relates to a method for unbiased separation and enrichment of sEVs, which comprises the steps of co-incubating pHLIP and serum sEVs, and obtaining sEVs after washing and releasing; the method also comprises the step of modifying pHLIP on the surface of the micro-nano material before co-incubation.
In some embodiments, the micro-nano material may comprise a polyphenylEthylene microball, znO nano material and TiO 2 Nano materials, etc.
In some embodiments, one of the modification methods is to modify polydopamine on the surface of the micro-nano material, then modify streptavidin, and in addition, modify biotin on the pHLIP short peptide, and modify pHLIP on the surface of the micro-nano material through the action of the streptavidin and the biotin.
In some embodiments, the co-incubation time is 20min to 60min and the temperature is 18 ℃ to 30 ℃.
In some embodiments, the washing comprises washing 2 times with PBS solution; the release was 100 μ M dithiothreitol solution or tris (2-carboxyethyl) phosphine.
The invention provides a method for modifying pHLIP on the surface of a micro-nano material, which comprises the following steps:
(1) In micro-nano material (magnetic microsphere, polystyrene, znO, tiO) 2 Nanospheres, etc.) surface-modified polydopamine;
(2) Modifying streptavidin on the surface of the material obtained in the step (1);
(3) Modifying biotin on the pHLIP short peptide;
(4) Modifying the material surface obtained in step (2) with the biotin-modified pHLIP obtained in step (3).
Preferably, the surface of the micro-nano material is modified with polydopamine by the following steps:
(1) Preparing a 1mg/mL dopamine solution by using a pH =8.5 10mM Tris-HCl buffer solution;
(2) The micro-nano material is rapidly and uniformly dispersed in the dopamine solution and reacts for 15min.
Preferably, streptavidin is modified by the following steps:
(1) Prepare 20 μ g/mL streptavidin solution with pH =8.5 10mM Tris-HCl buffer;
(2) And uniformly dispersing the polydopamine modified micro-nano material in a streptavidin solution, and reacting for 30min.
Preferably, biotin is modified on the pHLIP short peptide by the following steps: 0.2mg of pHLIP and 0.27mg of biotin were dissolved in 200. Mu.L of a mixed solution of PBS and DMSO, reacted at 37 ℃ for 2 hours, and dialyzed against a dialysis bag of 1000Da for 24 hours to remove excess biotin.
Preferably, biotinylated pHLIP is modified on streptavidin-modified micro-nano material by the following steps: the streptavidin modified micro-nano material is uniformly dispersed in 20ug/mL biotinylation pHLIP solution and reacts for 30min.
The invention utilizes a biological coupling technology to modify pHLIP on a micro-nano material for unbiased separation and enrichment of sEVs, and takes polystyrene as an example and adopts the following steps:
(1) A dopamine solution was prepared by dissolving 1mg of dopamine in 1mL of Tris-HCl buffer (10 mM, pH 8.5);
(2) Modifying polydopamine with polystyrene, uniformly dispersing 1mg of polystyrene in dopamine solution, reacting for 15min, centrifuging, and washing with PBS for 3 times;
(3) Preparing a streptavidin solution, and diluting 1mg/mL of streptavidin mother liquor to 20 mu g/mL by using a Tris-HCl buffer solution (10 mM, pH 8.5);
(4) Reacting polydopamine modified polystyrene with streptavidin, reacting the material obtained in the step (2) with 2mL of the streptavidin solution obtained in the step (3) for 30min, centrifuging, and washing with PBS for 3 times;
(5) pHLIP modified biotin, dissolving 0.2mg of pHLIP and 0.27mg of biotin in a mixed solution of PBS and DMSO, reacting at room temperature overnight, and dialyzing in a 1000Da dialysis bag for 24h;
(6) Reacting streptavidin-modified polystyrene with biotinylated pHLIP, reacting the material obtained in step (4) with 1mL of 20. Mu.g/mL biotinylated pHLIP (10mM, pH 7.4, PBS) for 30min, centrifuging, and washing with PBS for 3 times;
(7) And (4) reacting the material obtained in the step (6) with 100 mu L of extracellular vesicle standard sample solution or patient serum for 20min, centrifuging, and washing with PBS (phosphate buffer solution) for 3 times.
In some embodiments, the micro-nano material is magnetic microsphere, polystyrene, znO, tiO 2 And the like.
The following examples are provided to illustrate the method and application of pHLIP for unbiased separation and enrichment of extracellular vesicles.
Example 1
The method for unbiased separation and enrichment of extracellular vesicles by pHLIP comprises the following steps:
(1) A dopamine solution was prepared by dissolving 1mg of dopamine in 1mL of Tris-HCl buffer (10 mM, pH 8.5);
(2) Polystyrene modified polydopamine, 1mg of polystyrene is uniformly dispersed in a freshly prepared dopamine solution, shaking table reaction is carried out for 15min, centrifugation is carried out at 10000rpm for 5min, and PBS washing is carried out for 3 times;
(3) Preparing a streptavidin solution, and diluting 1mg/mL of streptavidin mother liquor to 20 mu g/mL by using a Tris-HCl buffer solution (10 mM, pH 8.5);
(4) Reacting polydopamine modified polystyrene with streptavidin, reacting the material obtained in the step (2) with 2mL of the streptavidin solution obtained in the step (3) for 30min, and centrifuging at 10000rpm for 5min; FIG. 1 shows the successful verification of streptavidin modification. The appearance of a distinct red fluorescence on the polystyrene spheres indicates successful modification of streptavidin. (the specific method is: 1mg streptavidin modified polystyrene reacts with 100 μ L10 ug/mL biotinylated mouse anti-human IgG 30min,10000rpm centrifugates for 5min, PBS washes 3 times, 5% bovine serum albumin solution seals for 30min,10000rpm centrifugates for 5min, PBS washes 3 times, 100 μ L10 ug/mL Cy3 modified goat anti-mouse IgG reacts for 30min,10000rpm centrifugates for 5min, PBS washes 3 times, and the fluorescence is observed).
(5) pHLIP-modified biotin, 0.2mg of pHLIP and 0.27mg of biotin were dissolved in a mixed solution of 200. Mu.L PBS and DMSO (3, 1, v), subjected to shaking overnight at room temperature, dialyzed in a 1000Da dialysis bag for 24 hours, and the dialyzate was changed with PBS at 2h,4h and 10h, respectively; FIG. 2 shows the effect of pHLIP on cell membranes. The red fluorescence on the cell membrane indicates that pHLIP can interact with the cell membrane. (specific method: first, MCF-7 cells were cultured in a 96-well plate, after the cells grew to an appropriate density, the medium was removed, the cells were washed with PBS 3 times, the cells were reacted with 10. Mu.g/mL biotinylated pHLIP for 20min, washed with PBS 3 times, blocked with 5% bovine serum albumin for 30min, the cells were reacted with 10. Mu.g/mL Cy 3-modified streptavidin for 1 min, and the cells were washed with PBS 3 times and then observed for fluorescence.) FIG. 3 is an optimization of the action time of pHLIP and cell membranes. The balance can be achieved by allowing pHLIP to act on cell membrane for 20 min. (the specific method is that MCF-7 cells are cultured in a 96-well plate firstly, when the cells grow to a proper density, the culture medium is removed, the cells are washed by PBS 3 times, the cells are respectively reacted with 10 mu g/mL biotinylation pHLIP for 10min, 20min, 30min, 40min and 60min, the cells are washed by PBS 3 times, 5 percent bovine serum albumin is used for sealing for 30min, the cells are reacted with 10 mu g/mL Cy3 modified streptavidin for 15min, and the change of the fluorescence intensity is observed after the cells are washed by PBS 3 times).
(6) Reacting streptavidin-modified polystyrene with biotinylated pHLIP, shaking-reacting the material obtained in step (4) with 1mL of 20. Mu.g/mL biotinylated pHLIP (10mM, pH 7.4, PBS) for 30min, centrifuging at 10000rpm for 5min, and washing with PBS for 3 times;
(7) Reacting the material obtained in step (6) with 100 μ L sEVs standard solution or patient serum for 20min, centrifuging at 10000rpm for 5min, and washing with PBS for 3 times. FIG. 4 is an SEM image of pHLIP-modified polystyrene microspheres capturing sEVs. In the figure, a micron-level sphere is a pHLIP modified polystyrene microsphere, a nanometer-level sphere is sEVs, and the chart shows that the pHLIP modified polystyrene can successfully capture the sEVs (the specific method is that after a sEVs standard sample is reacted with a material, 2.5% (V/V) glutaraldehyde is used for fixing overnight, and then ethanol solutions with the concentration gradients of 30%, 50%, 70%, 90%, 95% and 100% are used for dewatering for 15min step by step, after the fixing is finished, the extra salt is removed by washing with deionized water, and the drying is carried out at room temperature, so that SEM imaging can be carried out).
FIG. 5 is a graph showing the effect of incubation time of pHLIP-modified polystyrene microspheres with sEVs on capture efficiency. The pHLIP modified polystyrene microspheres and sEVs act for 20min to reach balance. (the specific method is that 1mg of pHLIP modified polystyrene reacts with 100 mu L of sEVs standard sample solution for 10min, 20min, 30min and 40min respectively, and the change of different incubation times on the sEVs capture efficiency is inspected).
FIG. 6 shows the capture efficiency of pHLIP-modified polystyrene microspheres on different cell-derived sEVs. Due to the unbiased property of pHLIP, the pHLIP modified polystyrene microspheres have basically consistent capture efficiency on extracellular vesicles derived from MCF-7 cells, PC9 cells and A549 cells.
FIG. 7 is an immunological characterization of sEVs released from pHLIP modified polystyrene microsphere surfaces. Before release (a) and after release (b). As can be seen from the figure, the fluorescence on the pHLIP-modified polystyrene disappeared after the release of the fluorescently labeled extracellular vesicles. Indicating successful release of extracellular vesicles. (the specific method is that the biotin with disulfide bonds is used for replacing common biotin during material synthesis, after the material is reacted with sEVs, 5% of bovine serum albumin is used for blocking, then the biotin-modified anti-CD9 antibody is reacted with sEVs, then 5% of bovine serum albumin is used for blocking, finally SA-Cy3 is injected, fluorescence is observed by an inverted fluorescence microscope to obtain (a), then 100 mu M dithiothreitol is used for reacting with the material for 30min, as the dithiothreitol can reduce the disulfide bonds, exosome is released, and the fluorescence on the material disappears to obtain (b)).
FIG. 8 is a qPCR of the deletion mutation of the downstream EGFR19 exon in a material capture mock sample sEVs (1. Mu.L PC9 cell-derived sEVs + 99. Mu.L healthy human serum). As can be seen, EGFR19 exon deletion mutations were successfully detected in mock samples. ( The specific method comprises the following steps: adding 1 mu L of sEVs standard sample derived from PC9 cells into 99 mu L of serum of a healthy person, simulating a clinical sample, acting with a material, extracting DNA in situ by using a QIAamp DNAmicro Kit according to the instruction, and detecting the mutation of the EGFR gene by using a human EGFR gene mutation detection Kit. Wherein QC is the quality control group )
FIG. 9 shows sanger sequencing fragments from material capture to mock samples of sEVs (1. Mu.L PC9 cell-derived sEVs + 99. Mu.L healthy human serum) with EGFR19 exon deletion mutations. As can be seen, EGFR19 exon deletion mutations were successfully detected. ( mu.L of PC9 cell-derived sEVs standards were added to 99. Mu.L of serum from healthy persons, clinical specimens were simulated, DNA was extracted in situ with the QIAamp DNA micro Kit according to the instructions after interaction with the material, and PCR amplification was performed. The amplification primers are shown in Table 1. The amplification system is shown in Table 2. The amplification reactions are shown in Table 3. The PCR product was purified with a purification kit and subjected to sanger sequencing. )
TABLE 1 EGFR-specific primer sequences
TABLE 2 PCR amplification reaction System for coding region of EGFR Gene
TABLE 3 PCR amplification reaction procedure for coding regions of the EGFR gene
Figure 10 is qPCR with deletion mutations in downstream EGFR19 exons capturing patient serum svs from material. As can be seen, the EGFR19 exon deletion mutation was successfully detected in the mock sample. ( The specific method comprises the following steps: after the material acted with sEVs in patient serum, DNA was extracted in situ with QIAamp DNA Micro Kit according to the instructions, and then mutation of EGFR gene was detected with a human EGFR gene mutation detection Kit. Where QC is the quality control group. )
FIG. 11 is a diagram showing the modification of pHLIP and binding to extracellular vesicles according to the present invention. Firstly, polydopamine is modified on polystyrene microspheres, then streptavidin is modified on the polydopamine, and pHLIP is successfully modified on the polystyrene microspheres through the action between the streptavidin and biotin so as to achieve the purpose of separating and enriching sEV.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (7)
1. The application of utilizing lipid affinity polypeptide pHLIP to unbiased separation and enrichment of extracellular vesicles is characterized in that the lipid affinity polypeptide pHLIP modified on the surface of a micro-nano material and a sample containing the extracellular vesicles are incubated together, the sequence of the lipid affinity polypeptide pHLIP is shown as SEQ ID NO:1, and the lipid affinity polypeptide pHLIP is combined with a lipid bilayer of the extracellular vesicles; and after incubation, centrifuging, and precipitating to obtain the extracellular vesicles.
2. The application of the non-biased separation and enrichment of extracellular vesicles using the lipid affinity polypeptide pHLIP according to claim 1, wherein the micro-nano material is a magnetic microsphere, a polystyrene microsphere, a ZnO nano material or TiO 2 And (3) nano materials.
3. The application of the lipid affinity polypeptide pHLIP to unbiased separation and enrichment of extracellular vesicles as claimed in claim 1 or 2, wherein the specific preparation method of the lipid affinity polypeptide pHLIP modified on the surface of the micro-nano material comprises: firstly modifying polydopamine on the surface of a micro-nano material, and then modifying streptavidin to be connected with the polydopamine; modifying biotin on the lipid affinity polypeptide pHLIP, and modifying the lipid affinity polypeptide pHLIP on the surface of the micro-nano material through the action of the streptavidin and the biotin.
4. The use of the lipid affinity polypeptide pHLIP for unbiased separation and enrichment of extracellular vesicles as claimed in claim 3, wherein the biotin is modified with disulfide bonds.
5. The use of the lipo affinity polypeptide pHLIP for unbiased separation and enrichment of extracellular vesicles as claimed in claim 4, wherein dithiothreitol solution or tris (2-carboxyethyl) phosphine is added to the resulting precipitate, and the dithiothreitol solution or tris (2-carboxyethyl) phosphine disrupts the disulfide bonds in biotin, thereby releasing extracellular vesicles from the micro-nano material.
6. The use of the lipid affinity polypeptide pHLIP for unbiased separation and enrichment of extracellular vesicles as claimed in claim 1, wherein the pellet is washed with phosphate buffer.
7. The use of the lipid affinity polypeptide pHLIP for unbiased separation and enrichment of extracellular vesicles as claimed in claim 1, wherein the co-incubation time is 20-60 min and the temperature is 18-30 ℃.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20120039990A1 (en) * | 2010-08-13 | 2012-02-16 | Reshetnyak Yana K | Liposome Compositions and Methods of Use Thereof |
| CN109689031A (en) * | 2016-07-11 | 2019-04-26 | 医福斯治疗有限公司 | The metabolic drug of EV loads |
| WO2021226589A1 (en) * | 2020-05-08 | 2021-11-11 | The University Of Kansas | Immunomagnetic compositions for the ph-specific capture of extracellular vesicles |
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|---|---|---|---|---|
| US20120039990A1 (en) * | 2010-08-13 | 2012-02-16 | Reshetnyak Yana K | Liposome Compositions and Methods of Use Thereof |
| CN109689031A (en) * | 2016-07-11 | 2019-04-26 | 医福斯治疗有限公司 | The metabolic drug of EV loads |
| WO2021226589A1 (en) * | 2020-05-08 | 2021-11-11 | The University Of Kansas | Immunomagnetic compositions for the ph-specific capture of extracellular vesicles |
Non-Patent Citations (1)
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| 张建奇: ""肿瘤靶向的pHLIP-SPION 磁共振对比剂的合成及其在肝癌MRI 成像应用的研究"", 《中国优秀硕士学位论文全文数据库（电子期刊） 医药卫生科技辑》, 31 March 2020 (2020-03-31), pages 060 - 130 * |
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