Application of pHLIP, a lipid affinity peptide, for unbiased separation and enrichment of extracellular vesicles

Abstract

The present invention relates to the application of unbiased separation and enrichment of extracellular vesicles using a lipophilic polypeptide pHLIP, and belongs to the field of biotechnology. The present invention proposes for the first time a method for unbiased separation and enrichment of sEVs from biological samples using the interaction between pHLIP and the lipid bilayer of sEVs. Compared with the classical ultracentrifugation method, this method is simple, rapid, efficient, and low-cost. No large instrument is required in the entire separation process, and the present invention does not require modification of other antibodies or aptamers, and has good clinical application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and more specifically, relates to the application of utilizing lipophilic polypeptide pHLIP to separate and enrich extracellular vesicles without bias. Background Art

[0002] Extracellular vesicles (sEVs) are a type of vesicle secreted by cells. They contain a phospholipid bilayer structure and are widely distributed in various body fluids. Among them, people define extracellular vesicles with a size between 30-200nm as exosomes. Due to the high content of sEVs and their continued presence in all physiological and disease stages, and the inherent stability of sEVs that ensures the integrity of the biological molecules they carry, a variety of technologies have been developed for the separation of sEVs, including ultracentrifugation and polymer precipitation. Although traditional sEVs separation methods are widely used in scientific research, they cannot handle trace samples, have a long processing time, and the purity of the obtained sEVs is low, and sEVs are at risk of damage. In addition, when using antibodies or nucleic acid aptamers to capture sEVs, due to the high heterogeneity of sEVs molecular composition, traditional immunoseparation methods often lose relevant sEVs with low or no relevant antigen expression and lose important information related to tumors. Therefore, there is an urgent need to establish a rapid, efficient, and loss-free sEVs separation and enrichment method in clinical practice.

[0003] The pH(low) insertion peptide (pHLIP) containing 36 amino acids (NH2-CAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT-COOH) is derived from the C helix of bacteriorhodopsin. When the pH is greater than 7, pHLIP can strongly bind to the surface of the lipid bilayer. Under acidic conditions, pHLIP can pass through the lipid bilayer and form a transmembrane helix. In addition, once pHLIP binds or inserts into the lipid bilayer, it has little interference with the lipid bilayer. They do not induce membrane permeation or form pores. Therefore, pHLIP can interact with the lipid bilayer of sEVs and separate and enrich sEVs without bias. Summary of the invention

[0004] The present invention solves the technical difficulties of unbiased separation and enrichment of low-content sEVs from specimens in the prior art. The present invention utilizes the ability of the lipophilic polypeptide pHLIP to bind to the lipid bilayer of extracellular vesicles to provide a simple method for unbiased separation and enrichment of sEVs by pHLIP. The method is simple, rapid, efficient, low-cost, unbiased enrichment, and no large-scale instruments are required during the entire separation process.

[0005] According to the purpose of the present invention, there is provided an application of unbiased separation and enrichment of extracellular vesicles using a lipophilic polypeptide pHLIP, wherein a lipophilic polypeptide pHLIP modified on the surface of a micro-nano material is co-incubated with a sample containing extracellular vesicles, wherein the sequence of the lipophilic polypeptide pHLIP is shown in SEQ ID NO: 1, and the lipophilic polypeptide pHLIP is bound to the lipid bilayer of the extracellular vesicle; after incubation, centrifugation is performed, and the precipitate is the separated extracellular vesicle.

[0006] Preferably, the micro-nano material is magnetic microspheres, polystyrene microspheres, ZnO nanomaterials or TiO2 nanomaterials.

[0007] Preferably, the specific preparation method of the lipophilic polypeptide pHLIP modified on the surface of the micro-nano material is: first modifying polydopamine on the surface of the micro-nano material, and then modifying streptavidin to connect with polydopamine; modifying biotin on the lipophilic polypeptide pHLIP, and modifying the lipophilic polypeptide pHLIP on the surface of the micro-nano material through the action of streptavidin and biotin.

[0008] Preferably, the biotin is modified with a disulfide bond.

[0009] Preferably, dithiothreitol solution or tris(2-carboxyethyl)phosphine is added to the obtained precipitate, and the dithiothreitol solution or tris(2-carboxyethyl)phosphine destroys the disulfide bond on biotin, so that the extracellular vesicles are released from the micro-nano material.

[0010] Preferably, the precipitate is washed with phosphate buffer.

[0011] Preferably, the co-incubation time is 20 min to 60 min, and the temperature is 18° C. to 30° C.

[0012] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[0013] (1) The present invention can modify pHLIP on the surface of micro-nano materials only through simple bioconjugation. The modification process and method are simple and low-cost. By utilizing the interaction between pHLIP and lipid bilayer, unbiased separation and enrichment of sEVs can be easily achieved. In addition, the present invention does not require modification of other antibodies or aptamers, and does not require large-scale instruments.

[0014] (2) Since the micro-nano material of the present invention has a larger specific surface area, more binding sites can be used for pHLIP modification.

[0015] (3) Preferably, in the present invention, sEVs are captured by modifying pHLIP with disulfide bonds, and the captured sEVs can be released intact through the action of dithiothreitol or tris(2-carboxyethyl)phosphine on the disulfide bonds for downstream analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Verification of successful streptavidin modification.

[0017] Figure 2 To verify that pHLIP acts on the cell membrane.

[0018] Figure 3 To optimize the interaction time between pHLIP and cell membrane.

[0019] Figure 4 SEM image of pHLIP-modified polystyrene microspheres capturing sEVs.

[0020] Figure 5 The effect of incubation time of pHLIP-modified polystyrene microspheres with sEVs on the capture efficiency.

[0021] Figure 6 The capture efficiency of sEVs from different cell sources by pHLIP-modified polystyrene microspheres.

[0022] Figure 7 Immunocharacterization of sEVs released from the surface of pHLIP-modified polystyrene microspheres: (a) before release; (b) after release.

[0023] Figure 8 The material captured simulated sample sEVs (1μL PC9 cell-derived sEVs + 99μL healthy human serum), and its downstream EGFR exon 19 deletion mutation was detected by qPCR.

[0024] Fig. 9 The material captured the simulated sample sEVs (1μL PC9 cell-derived sEVs + 99μL healthy human serum), and its Sanger sequencing fragment of EGFR exon 19 deletion mutation.

[0025] Fig.10 The material was captured from patient serum sEVs and the downstream EGFR exon 19 deletion mutation was detected by qPCR.

[0026] Fig.11 Schematic diagram of pHLIP modification and binding to extracellular vesicles of the present invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0028] The method for unbiased separation and enrichment of sEVs of the present invention comprises co-incubating pHLIP with serum sEVs, and obtaining sEVs after washing and releasing; and before co-incubation, pHLIP is modified on the surface of micro-nano material.

[0029] In some embodiments, the micro-nano material may include polystyrene microspheres, ZnO nanomaterials, TiO2 nanomaterials, etc.

[0030] In some embodiments, one of the modification methods is to modify the surface of the micro-nano material with polydopamine and then modify streptavidin. In addition, biotin is modified on the pHLIP short peptide, and pHLIP is modified on the surface of the micro-nano material through the action of streptavidin and biotin.

[0031] In some embodiments, the co-incubation time is 20 min to 60 min, and the temperature is 18° C. to 30° C.

[0032] In some embodiments, the washing comprises washing twice with a PBS solution; and the releasing agent is a 100 μM dithiothreitol solution or tris(2-carboxyethyl)phosphine.

[0033] The present invention provides a method for modifying pHLIP on the surface of a micro-nano material, comprising the following steps:

[0034] (1) Modify polydopamine on the surface of micro-nano materials (magnetic microspheres, polystyrene, ZnO, TiO2 nanospheres, etc.);

[0035] (2) modifying the surface of the material obtained in step (1) with streptavidin;

[0036] (3) Modification of biotin on pHLIP short peptide;

[0037] (4) The biotin-modified pHLIP obtained in step (3) is used to modify the surface of the material obtained in step (2).

[0038] Preferably, polydopamine is modified on the surface of the micro-nano material by the following steps:

[0039] (1) Prepare a 1 mg / mL dopamine solution using 10 mM Tris-HCl buffer (pH 8.5);

[0040] (2) The micro-nano material was quickly and evenly dispersed in the dopamine solution and reacted for 15 minutes.

[0041] Preferably, streptavidin is modified by the following steps:

[0042] (1) Prepare a 20 μg / mL streptavidin solution using 10 mM Tris-HCl buffer (pH 8.5);

[0043] (2) The polydopamine-modified micro-nanomaterials were evenly dispersed in the streptavidin solution and reacted for 30 minutes.

[0044] Preferably, biotin is modified on the pHLIP short peptide by the following steps: 0.2 mg pHLIP and 0.27 mg biotin are dissolved in 200 μL of a mixed solution of PBS and DMSO, reacted at 37° C. for 2 h, and dialyzed with a 1000 Da dialysis bag for 24 h to remove excess biotin.

[0045] Preferably, biotinylated pHLIP is modified on the streptavidin-modified micro-nano material by the following steps: the streptavidin-modified micro-nano material is uniformly dispersed in a 20 ug / mL biotinylated pHLIP solution and reacted for 30 min.

[0046] The present invention utilizes bioconjugation technology to modify pHLIP on micro-nano materials for unbiased separation and enrichment of sEVs. Taking polystyrene as an example, the following steps are adopted:

[0047] (1) Prepare dopamine solution by dissolving 1 mg dopamine in 1 mL Tris-HCl buffer (10 mM, pH 8.5);

[0048] (2) Polystyrene modified polydopamine: 1 mg of polystyrene was evenly dispersed in the dopamine solution, reacted for 15 min, centrifuged, and washed 3 times with PBS;

[0049] (3) Prepare streptavidin solution by diluting 1 mg / mL streptavidin stock solution to 20 μg / mL with Tris-HCl buffer (10 mM, pH 8.5);

[0050] (4) Reaction of polydopamine-modified polystyrene with streptavidin: react the material obtained in step (2) with 2 mL of the streptavidin solution obtained in step (3) for 30 min, centrifuge, and wash with PBS three times;

[0051] (5) pHLIP-modified biotin: 0.2 mg pHLIP and 0.27 mg biotin were dissolved in a mixed solution of PBS and DMSO, reacted at room temperature overnight, and dialyzed in a 1000 Da dialysis bag for 24 h;

[0052] (6) Reaction of streptavidin-modified polystyrene with biotinylated pHLIP: the material obtained in step (4) was reacted with 1 mL 20 μg / mL biotinylated pHLIP (10 mM, pH 7.4, PBS) for 30 min, centrifuged, and washed 3 times with PBS;

[0053] (7) The material obtained in step (6) was reacted with 100 μL of extracellular vesicle standard solution or patient serum for 20 min, centrifuged, and washed 3 times with PBS.

[0054] In some embodiments, the micro-nano material is magnetic microspheres, polystyrene, ZnO, TiO2, etc.

[0055] The following examples provide a detailed description of the method and application of pHLIP for unbiased separation and enrichment of extracellular vesicles.

[0056] Example 1

[0057] The method for pHLIP unbiased separation and enrichment of extracellular vesicles of the present invention comprises the following steps:

[0058] (1) Prepare dopamine solution by dissolving 1 mg dopamine in 1 mL Tris-HCl buffer (10 mM, pH 8.5);

[0059] (2) Polystyrene modified polydopamine: 1 mg of polystyrene was evenly dispersed in the freshly prepared dopamine solution, shaken for 15 min, centrifuged at 10,000 rpm for 5 min, and washed with PBS three times;

[0060] (3) Prepare streptavidin solution by diluting 1 mg / mL streptavidin stock solution to 20 μg / mL with Tris-HCl buffer (10 mM, pH 8.5);

[0061] (4) reacting polydopamine-modified polystyrene with streptavidin, reacting the material obtained in step (2) with 2 mL of the streptavidin solution obtained in step (3) for 30 min, and centrifuging at 10,000 rpm for 5 min; Figure 1 Verification of successful streptavidin modification. Obvious red fluorescence on the polystyrene sphere indicates successful streptavidin modification. (Specific method: 1mg streptavidin-modified polystyrene reacts with 100μL10 ug / mL biotinylated mouse anti-human IgG for 30min, centrifuges at 10000rpm for 5min, washes 3 times with PBS, blocks with 5% bovine serum albumin solution for 30min, centrifuges at 10000rpm for 5min, washes 3 times with PBS, reacts with 100μL10ug / mL Cy3-modified goat anti-mouse IgG for 30min, centrifuges at 10000rpm for 5min, washes 3 times with PBS, and observes fluorescence).

[0062] (5) pHLIP-modified biotin: 0.2 mg pHLIP and 0.27 mg biotin were dissolved in 200 μL of a mixed solution of PBS and DMSO (3:1, v:v). The reaction was allowed to react overnight on a shaker at room temperature. The cells were dialyzed in a 1000 Da dialysis bag for 24 h. The dialysate was PBS, and the dialysate was changed at 2 h, 4 h, and 10 h of dialysis. Figure 2 This is the verification that pHLIP acts on the cell membrane. The red fluorescence on the cell membrane indicates that pHLIP can act on the cell membrane. (The specific method is: first culture MCF-7 cells in a 96-well plate, and when the cells grow to a suitable density, remove the culture medium, wash the cells 3 times with PBS, react the cells with 10μg / mL biotinylated pHLIP for 20 minutes, wash 3 times with PBS, block with 5% bovine serum albumin for 30 minutes, react the cells with 10μg / mL Cy3-modified streptavidin for 15 minutes, and observe the fluorescence after washing 3 times with PBS.) Figure 3 To optimize the interaction time between pHLIP and cell membrane. pHLIP can reach equilibrium after 20 minutes of interaction with cell membrane. (The specific method is: first culture MCF-7 cells in a 96-well plate, and when the cells grow to a suitable density, remove the culture medium, wash the cells 3 times with PBS, react the cells with 10μg / mL biotinylated pHLIP for 10min, 20min, 30min, 40min, and 60min, wash 3 times with PBS, block with 5% bovine serum albumin for 30min, react the cells with 10μg / mL Cy3-modified streptavidin for 15min, and observe the change in fluorescence intensity after washing 3 times with PBS).

[0063] (6) The streptavidin-modified polystyrene was reacted with the biotinylated pHLIP. The material obtained in step (4) was reacted with 1 mL of 20 μg / mL biotinylated pHLIP (10 mM, pH 7.4, PBS) on a shaker for 30 min, centrifuged at 10,000 rpm for 5 min, and washed with PBS three times;

[0064] (7) The material obtained in step (6) was reacted with 100 μL sEVs standard solution or patient serum for 20 min, centrifuged at 10,000 rpm for 5 min, and washed three times with PBS. Figure 4 This is a SEM image of pHLIP-modified polystyrene microspheres capturing sEVs. The micrometer-level balls in the image are pHLIP-modified polystyrene microspheres, and the nanometer-level balls are sEVs. It can be seen from the image that pHLIP-modified polystyrene can successfully capture sEVs (the specific method is: after the sEVs standard sample reacts with the material, it is fixed with 2.5% (V / V) glutaraldehyde overnight, and then dehydrated step by step for 15 minutes with a concentration gradient of 30%, 50%, 70%, 90%, 95%, and 100% ethanol solution. After fixation, wash with deionized water to remove excess salt, dry at room temperature and take SEM).

[0065] Figure 5 The effect of incubation time of pHLIP-modified polystyrene microspheres with sEVs on the capture efficiency. pHLIP-modified polystyrene microspheres and sEVs can reach equilibrium after 20 minutes of reaction. (The specific method is: 1 mg of pHLIP-modified polystyrene reacts with 100 μL of sEVs standard solution for 10 minutes, 20 minutes, 30 minutes, and 40 minutes, respectively, to investigate the changes in sEVs capture efficiency at different incubation times).

[0066] Figure 6 The capture efficiency of pHLIP-modified polystyrene microspheres for sEVs from different cell sources. Due to the non-biased nature of pHLIP, the capture efficiency of pHLIP-modified polystyrene microspheres for extracellular vesicles from MCF-7 cells, PC9 cells, and A549 cells is basically the same.

[0067] Figure 7 This is the immunological characterization of sEVs released from the surface of pHLIP-modified polystyrene microspheres. (a) is before release, and (b) is after release. As can be seen from the figure, after the fluorescently labeled extracellular vesicles are released, the fluorescence on the pHLIP-modified polystyrene disappears. This indicates that the extracellular vesicles are successfully released. (The specific method is: when the material is synthesized, ordinary biotin is replaced with biotin with a disulfide bond. After the material reacts with sEVs, it is blocked with 5% bovine serum albumin. Then the biotin-modified anti-CD9 antibody reacts with sEVs, followed by blocking with 5% bovine serum albumin, and finally SA-Cy3 is injected, and the fluorescence is observed with an inverted fluorescence microscope, which is (a). Then 100μM dithiothreitol is used to react with the above material for 30 minutes. Since dithiothreitol can reduce disulfide bonds, the exosomes will be released and the fluorescence on the material will disappear, which is (b)).

[0068] Figure 8 The material captured the simulated sample sEVs (1μL PC9 cell-derived sEVs + 99μL healthy human serum), and its downstream EGFR exon 19 deletion mutation qPCR. As can be seen from the figure, the EGFR exon 19 deletion mutation can be successfully detected in the simulated sample. (The specific method is: add 1μL PC9 cell-derived sEVs standard to 99μL healthy human serum to simulate clinical samples. After reacting with the material, use the QIAamp DNA Micro Kit to extract DNA in situ according to the instructions, and then use the human EGFR gene mutation detection kit to detect EGFR gene mutations. QC is the quality control group)

[0069] Fig. 9The material captured the simulated sample sEVs (1μL PC9 cell-derived sEVs + 99μL healthy human serum), and its EGFR exon 19 deletion mutation sanger sequencing fragment. As can be seen from the figure, the EGFR exon 19 deletion mutation can be successfully detected. (1μL PC9 cell-derived sEVs standard was added to 99μL healthy human serum to simulate clinical samples. After reacting with the material, DNA was extracted in situ using the QIAamp DNA Micro Kit according to the instructions, and then PCR amplification was performed. The amplification primers are shown in Table 1. The amplification system is shown in Table 2. The amplification reaction is shown in Table 3. The PCR product was purified with a purification kit and then sanger sequencing was performed.)

[0070] Table 1 EGFR specific primer sequences

[0071]

[0072] Table 2 PCR amplification reaction system of EGFR gene coding region

[0073]

[0074]

[0075] Table 3 PCR amplification reaction procedure of EGFR gene coding region

[0076]

[0077] Fig.10 The material captured the patient's serum sEVs, and the downstream EGFR exon 19 deletion mutation was detected by qPCR. As shown in the figure, the EGFR exon 19 deletion mutation can be successfully detected in the simulated sample. (The specific method is: after the material reacts with the sEVs in the patient's serum, the DNA is extracted in situ using the QIAamp DNA Micro Kit according to the instructions, and then the EGFR gene mutation detection kit is used to detect the EGFR gene mutation. QC is the quality control group.)

[0078] Fig.11 The figure is a schematic diagram of pHLIP modification and binding to extracellular vesicles of the present invention. First, polydopamine is modified on the polystyrene microspheres, and then streptavidin is modified on the polydopamine. Through the interaction between streptavidin and biotin, pHLIP is successfully modified on the polystyrene microspheres to achieve the purpose of separating and enriching sEVs.

[0079] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of lipophilic polypeptide pHLIP for unbiased separation and enrichment of extracellular vesicles in serum, characterized in that: The lipophilic polypeptide pHLIP modified on the surface of the micro-nano material is co-incubated with a sample containing extracellular vesicles, wherein the sequence of the lipophilic polypeptide pHLIP is shown in SEQ ID NO: 1, and the lipophilic polypeptide pHLIP is modified with biotin; the lipophilic polypeptide pHLIP is bound to the lipid bilayer of the extracellular vesicles; after incubation, centrifugation is performed, and the precipitate is the separated extracellular vesicles.

2. The use of the lipophilic polypeptide pHLIP for unbiased separation and enrichment of extracellular vesicles in serum as claimed in claim 1, characterized in that: The micro-nano material is magnetic microspheres, polystyrene microspheres, ZnO nanomaterials or TiO2 nanomaterials.

3. The use of the lipophilic polypeptide pHLIP for unbiased separation and enrichment of extracellular vesicles in serum as claimed in claim 1 or 2, characterized in that: The specific preparation method of the lipophilic polypeptide pHLIP modified on the surface of the micro-nano material is: first modifying polydopamine on the surface of the micro-nano material, and then modifying streptavidin to connect with polydopamine; modifying the lipophilic polypeptide pHLIP on the surface of the micro-nano material through the action of streptavidin and biotin.

4. The use of the lipophilic polypeptide pHLIP for unbiased separation and enrichment of extracellular vesicles in serum as claimed in claim 3, characterized in that: The biotin is modified with a disulfide bond.

5. The use of the lipophilic polypeptide pHLIP for unbiased separation and enrichment of extracellular vesicles in serum as claimed in claim 4, characterized in that: A dithiothreitol solution or tris(2-carboxyethyl)phosphine is added to the obtained precipitate, and the dithiothreitol solution or tris(2-carboxyethyl)phosphine destroys the disulfide bond on biotin, so that the extracellular vesicles are released from the micro-nano material.

6. The use of the lipophilic polypeptide pHLIP for unbiased separation and enrichment of extracellular vesicles in serum as claimed in claim 1, characterized in that: The precipitate was washed with phosphate buffer.

7. The method of claim 1 for unbiased separation and enrichment of serum using the lipophilic polypeptide pHLIP The use of extracellular vesicles in the present invention is characterized in that The co-incubation time is 20 min to 60 min. The temperature is 18℃~30℃.

Images