A polyethylene glycol hydrogel monolith column and method for batch separation and purification of extracellular vesicles
By combining a polyethylene glycol hydrogel monolithic column with a silica size exclusion separation column, the problems of low yield and low purity in extracellular vesicle separation were solved, achieving efficient and low-cost extracellular vesicle separation and purification.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHAANXI NORMAL UNIV
- Filing Date
- 2024-03-13
- Publication Date
- 2026-06-19
AI Technical Summary
Existing technologies for the isolation of extracellular vesicles suffer from problems such as low yield, low purity, time-consuming process, and the need for expensive equipment. Furthermore, existing methods can damage the integrity of extracellular vesicles.
A polyethylene glycol hydrogel monolithic column and a silica size exclusion chromatography column were used in combination. Biological samples were concentrated using the polyethylene glycol hydrogel monolithic column and then purified using the silica size exclusion chromatography column. This combined the advantages of polymer precipitation, ultrafiltration and size exclusion chromatography to achieve efficient separation and purification.
This method achieves high-yield and high-purity separation of extracellular vesicles, simplifies the operation process, reduces equipment costs, avoids damage to extracellular vesicles, and improves separation efficiency and purity.
Smart Images
Figure CN117942617B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of extracellular vesicle separation technology, specifically relating to a polyethylene glycol hydrogel monolithic column, and a method for separating and purifying extracellular vesicles by using a combination of a polyethylene glycol hydrogel monolithic column and a silica size exclusion separation column. Background Technology
[0002] Extracellular vesicles possess a typical phospholipid bilayer membrane structure and are secreted by all eukaryotic cells and most known archaea and bacteria, with diameters ranging from 50 to 200 nm. The vesicle structure and its nanoparticle size enable them to transport various biological information molecules from the source cell, such as proteins, nucleic acids, and lipids, without being degraded by enzymes in body fluids. They serve multiple functions, including messenger for intercellular signal transduction and carrier for cargo transport.
[0003] The separation principle of extracellular vesicles is mainly based on their physicochemical characteristics. For example, ultracentrifugation, under a certain centrifugal force, separates different components in a sample based on differences in density and size, and is considered the "gold standard" for extracellular vesicle separation. However, this method is time-consuming and requires specialized ultracentrifugation equipment, limiting its clinical application. Furthermore, the structure and biological function of the separated extracellular vesicles may be affected by prolonged ultracentrifugation. Polymer precipitation is the basis of many commercial extraction kits. Highly hydrophilic polymers interact with water molecules surrounding extracellular vesicles, creating a hydrophobic microenvironment that leads to vesicle precipitation. Although the recovery rate may be high, the purity of the precipitated extracellular vesicle sample is low because essentially all soluble particles are precipitated. Moreover, the precipitating reagent cannot be completely removed from the final formulation, potentially affecting downstream characterization or analysis. Compared to separation methods, it is more like a concentration method. Ultrafiltration uses nanofiltration membranes with different molecular weight cutoff values to enrich and separate extracellular vesicles from clinical samples, distinguishing between extracellular and non-extracellular vesicles by size. However, during filtration, shear stress can cause potential degradation and may result in some loss due to membrane clogging. Size exclusion chromatography (SUC) can separate extracellular vesicles with minimal structural damage. Long before its application in the separation and purification of extracellular vesicles, SUC had already been well-developed and widely used for the high-resolution separation of biomacromolecules or large molecular aggregates (such as proteins, polymers, and various liposome particles). Various porous packing materials such as dextran polymers, agarose, and polyacrylamide have been used in SUC separation. However, gel packing materials suffer from problems such as non-uniform particle size, easy collapse, and low reusability.
[0004] The aforementioned separation methods suffer from drawbacks to varying degrees, including insufficient integrity and low purity of extracellular vesicles, as well as being time-consuming and requiring expensive equipment. Therefore, achieving high-yield and high-purity preparation of extracellular vesicles to meet clinical needs is a pressing research direction. Summary of the Invention
[0005] The purpose of this invention is to achieve high-yield and high-purity separation and enrichment of extracellular vesicles, and to solve the problems of time-consuming and labor-intensive purification of extracellular vesicles in large-scale purification, the need for expensive centrifugation equipment, or the impact of precipitation agents on downstream analysis. This invention provides a polyethylene glycol hydrogel monolithic column and a method for batch separation and purification of extracellular vesicles using this monolithic column in conjunction with a silica size exclusion separation column, which is used to separate and enrich extracellular vesicles from large batches of biological samples.
[0006] To achieve the above objectives, the polyethylene glycol hydrogel monolithic column provided by the present invention is prepared by the following steps:
[0007] Step 1: Add polyethylene glycol monomethyl ether and polyethylene glycol diacrylate to a 1× phosphate buffer solution, premix, adjust the pH to 7-8 with a 1 mol / L sodium hydroxide aqueous solution, and then add tetramethylethylenediamine, ammonium sulfate and propylene to obtain a polyethylene glycol polymerization solution.
[0008] Step 2: Take an empty chromatography column, cap it, and add polyethylene glycol polymerization solution. The amount of polyethylene glycol polymerization solution added is 5% to 20% of the column volume. Then place the column at -20℃ for polymerization reaction for 12 to 24 hours. After the reaction is completed, let the column return to room temperature, remove the cap, and wash the gel inside the column with deionized water to obtain a monolithic polyethylene glycol hydrogel column.
[0009] In step 1 above, preferably, the amounts of polyethylene glycol monomethyl ether, polyethylene glycol diacrylate, tetramethylethylenediamine, and propylene added are 8%–12%, 0.1%–0.3%, 0.1%–0.3%, and 0.8%–1.5% of the volume of the polyethylene glycol polymerization solution, respectively, and the mass ratio of the added ammonium sulfate to the volume of the polyethylene glycol polymerization solution is 3–5 mg:100 mL.
[0010] The method for batch isolation and purification of extracellular vesicles provided by this invention includes the following steps:
[0011] Step 1: Add the biological sample to the polyethylene glycol hydrogel monolithic column, with a sample volume to column bed volume ratio of 20:1 to 5:1. After reacting at room temperature for 30 to 120 minutes, compress the polyethylene glycol hydrogel in the column and collect the eluent to obtain the concentrated biological sample.
[0012] Step 2: Add the concentrated biological sample to a silica size exclusion column. The ratio of sample volume to column volume is 1:10 to 1:50. After the concentrated biological sample flows into the column, add 10 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution for elution. The ratio of the elution buffer to sample volume is 20:1 to 40:1. Collect the portion of the elution liquid that reaches 30% to 40% of the buffer volume to obtain extracellular vesicles.
[0013] In step 1 of the above-mentioned batch separation and purification of extracellular vesicles, the preferred ratio of sample loading volume to column bed volume is 10:1 to 15:1.
[0014] In step 1 of the above-mentioned batch separation and purification of extracellular vesicles, the polyethylene glycol hydrogel monolithic column is regenerated after use. The regeneration method is as follows: wash with deionized water and then dry.
[0015] In step 2 of the above-mentioned batch separation and purification of extracellular vesicles, the preparation method of the silica size exclusion separation column is as follows: silica gel with a particle size of 30-90 μm and a pore size of 50-100 nm is added to deionized water and mixed evenly to obtain a silica dispersion; after installing a lower sieve plate with a pore size of 10-20 μm in an empty chromatography column with a bottom cap, the silica dispersion is added, and the column is allowed to settle naturally for more than 24 hours to allow the lower precipitate to reach the required column height. The upper solution is then removed, and an upper sieve plate with a pore size of 10-20 μm is installed to obtain the silica size exclusion separation column.
[0016] In step 2 of the above-mentioned batch separation and purification of extracellular vesicles, the preferred ratio of sample loading volume to column bed volume is 1:20 to 1:30.
[0017] In step 2 of the above-mentioned batch separation and purification of extracellular vesicles, the silica size exclusion column is regenerated after use. The regeneration conditions are as follows: first, wash with 2-4 column volumes of 1% Triton aqueous solution, then wash with 2-4 column volumes of deionized water, then wash with 2-4 column volumes of 70% ethanol aqueous solution, and then wash with 2-4 column volumes of deionized water. If the silica size exclusion column is used continuously, wash with 2-4 column volumes of 10 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution. If the silica size exclusion column is to be stored for a long time, place it in 20% ethanol aqueous solution.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] 1. The polyethylene glycol hydrogel monolithic column of this invention enables simple and efficient concentration of large-volume biological samples. Compared to polymer precipitation, no additional reagents are required; compared to ultrafiltration, no expensive equipment is needed; and it avoids the loss of extracellular vesicles and damage to their integrity caused by ultrafiltration membrane clogging. This saves time and experimental costs while meeting the demand for high yields. Furthermore, its synthesis method is simple and widely applicable, and polyethylene glycol has good biocompatibility, is safe and non-toxic, and will not contaminate biological samples.
[0020] 2. This invention uses a silica-sized size exclusion column to remove impurity proteins from concentrated biological samples, achieving high purity. The silica-sized size exclusion column used has more uniform particle size and pore size, suitable for the separation and purification of extracellular vesicles. Compared with existing commercial agarose and dextran-based bio-based packing materials, it has better rigidity, is less prone to collapse, adapts to high flow rate and high pressure separation environments, has higher resolution, and can be reused more times.
[0021] 3. This invention combines two methods, concentration and purification, to simultaneously meet the requirements for yield and purity during the separation of extracellular vesicles. The method is simple, easy to implement, economical, and environmentally friendly, providing a feasible solution for subsequent batch purification of extracellular vesicles. Attached Figure Description
[0022] Figure 1 This is a comparison of whey concentration before and after concentration in Example 2.
[0023] Figure 2 This is the separation curve of the silica size exclusion separation column in Example 2.
[0024] Figure 3 This is the separation curve of the silica size exclusion separation column in Example 3.
[0025] Figure 4 This is the separation curve of the silica size exclusion separation column in Example 4. Detailed Implementation
[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be clearly and completely described below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.
[0027] Example 1
[0028] Preparation of polyethylene glycol hydrogel monolithic columns
[0029] Step 1: Add 0.2 mL of polyethylene glycol monomethyl ether and 4 μL of polyethylene glycol diacrylate to 1.772 mL of 1× phosphate buffer solution. After premixing, adjust the pH to 7-8 with 1 mol / L sodium hydroxide aqueous solution. Then add 4 μL of tetramethylethylenediamine, 80 μg of ammonium sulfate and 20 μL of propylene to obtain a polyethylene glycol polymerization solution.
[0030] Step 2: Take 25 mL of empty chromatography column, cap it, and add 2 mL of polyethylene glycol polymerization solution. The amount of polyethylene glycol polymerization solution added is 8% of the column volume. Then place the column at -20℃ for polymerization reaction for 12 hours. After the reaction is completed, let the column return to room temperature, remove the cap, and clean the gel inside the column with deionized water to obtain a monolithic polyethylene glycol hydrogel column.
[0031] Example 2
[0032] Isolation and purification of extracellular vesicles from bulk raw milk
[0033] Step 1: Aliquot raw milk into 50 mL centrifuge tubes and centrifuge at 8000×g for 30 minutes at 4°C to remove most of the upper fat and lower cell debris. Then, take the middle layer of milk and centrifuge at 13200×g for 30 minutes at 4°C. After centrifugation, adjust the pH of the milk to approximately 6.0 with a 10% (v / v) acetic acid aqueous solution. Add 0.025 mg of rennet per mL of the centrifuged milk, mix well, and then place in a 37°C water bath until all proteins in the milk sample coagulate and whey precipitates. Collect the supernatant and centrifuge at 5000×g for 10 minutes at 4°C. After centrifugation, filter the supernatant through a 0.45 μm filter membrane to obtain the whey sample to be concentrated. 20 mL of whey sample to be concentrated was added to the polyethylene glycol hydrogel monolithic column of Example 1, with a sample volume to column bed volume ratio of 10:1. After reacting at room temperature for 60 minutes, the polyethylene glycol hydrogel in the column was compressed, and the eluent was collected to obtain the concentrated whey sample. The used polyethylene glycol hydrogel monolithic column can be regenerated by washing with deionized water and drying. The particle concentration of the whey samples before and after concentration was detected by a nanoparticle size tracer (NTA). The results are shown in [Figure 1]. Figure 1 .Depend on Figure 1 As can be seen, the concentration of whey after concentration is approximately 4.5 × 10⁻⁶. 11 particles / ml, compared to the original concentration of 1.5 × 10⁻⁶ 10 The number of particles / ml increased by approximately 30 times.
[0034] Step 2: Add 10 g of silica gel with a particle size of 30 μm and a pore size of 70 nm to 15 mL of deionized water and mix well to obtain a silica gel dispersion. Install a 20 mL empty chromatography column with a capped bottom and a 20 μm sieve plate, then add the silica gel dispersion. Allow it to settle naturally for at least 24 hours to allow the lower precipitate to reach the desired column height. Remove the upper solution, then install a 20 μm sieve plate to obtain a silica size exclusion column. Add 0.4 mL of concentrated whey sample to the silica size exclusion column at a loading volume to column bed volume ratio of 1:25. After the concentrated whey sample flows into the column bed, add 10 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid buffer for elution at a buffer volume to loading volume ratio of 30:1. Collect one fraction for every elution volume equal to the loading volume, for a total of 30 fractions. After use, the silica size exclusion column should be regenerated under the following conditions: first, wash with 2–4 column volumes of 1% Triton aqueous solution, then wash with 2–4 column volumes of deionized water, then wash with 2–4 column volumes of 70% ethanol aqueous solution, and then wash with 2–4 column volumes of deionized water. If the silica size exclusion column is to be used continuously, wash with 2–4 column volumes of 10 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution. If the silica size exclusion column is to be stored for a long period of time, place it in 20% ethanol aqueous solution.
[0035] The protein concentration of each component was determined using a BCA kit. A sample volume of 20 μL was required for the BCA assay. The NTA assay range is 1 × 10⁻⁶. 7 ~1 × 10 8 Between a concentration range of particles / mL, the remaining sample was diluted by the corresponding factor according to this concentration range to determine the particle concentration of each component. The obtained data on protein concentration and particle concentration for each component can reflect its degree of separation. Figure 2 The image shows the elution curves of the concentrated whey sample after loading onto a silica size exclusion column. The horizontal axis represents the elution volume. Curve A represents the extracellular vesicle concentration of each component measured by NTA, and curve B represents the protein concentration of each component measured by BCA. It can be seen that the extracellular vesicle concentration reaches its peak at about 40% of the total elution volume, while the protein concentration reaches its peak at 80% of the total elution volume. This indicates that the extracellular vesicles and proteins are basically separated, achieving the purpose of purification.
[0036] Example 3
[0037] Extracellular vesicles isolated and purified from bulk Cornus officinalis
[0038] Step 1: Take 400g of frozen fresh Cornus officinalis, remove the pits manually (the weight after pitting is 243g), add 300 mL of 10 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid buffer to the pitted Cornus officinalis pulp (including the peel), and juice it. Filter the crude Cornus officinalis juice through gauze to remove the pulp tissue. Centrifuge the filtered liquid at 3000×g for 30 minutes, collect the supernatant, and centrifuge again at 10000×g for 1 hour to obtain the Cornus officinalis clear liquid sample to be concentrated. Add 20 mL of the Cornus officinalis clear liquid sample to the polyethylene glycol hydrogel monolithic column of Example 1, with a sample volume to column bed volume ratio of 10:1. React at room temperature for 30 minutes, then compress the polyethylene glycol hydrogel in the column and collect the eluent to obtain the concentrated Cornus officinalis clear liquid sample. The used polyethylene glycol hydrogel monolithic column can be regenerated by washing with deionized water and drying.
[0039] Step 2: Add 0.4 mL of the concentrated Cornus officinalis extract sample to a silica size exclusion column (prepared using the same method as in Example 2), with a sample volume to column bed volume ratio of 1:25. After the concentrated Cornus officinalis extract sample flows into the column bed, add 10 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution for elution, with an elution buffer to sample volume ratio of 30:1. Collect one fraction for every elution volume equal to the sample volume, collecting a total of 30 fractions. After use, regenerate the silica size exclusion column under the same conditions as in Example 2.
[0040] The protein concentration of each component was determined using a BCA kit. A sample volume of 20 μL was required for the BCA assay. The NTA assay range is 1 × 10⁻⁶. 7 ~1 × 10 8 Between a concentration range of particles / mL, the remaining sample was diluted by the corresponding factor according to this concentration range to determine the particle concentration of each component. The obtained data on protein concentration and particle concentration for each component can reflect its degree of separation. Figure 3 The image shows the elution curves of the concentrated Cornus officinalis extract sample after loading onto a silica size exclusion column. The horizontal axis represents the elution volume. Curve C represents the extracellular vesicle concentration of each component measured by NTA, and curve D represents the protein concentration of each component measured by BCA. It can be seen that the extracellular vesicle concentration reaches its peak at approximately 30% of the total elution volume, while the protein concentration reaches its peak at 70% of the total elution volume. This indicates that the extracellular vesicles and proteins are basically separated, achieving the purpose of purification.
[0041] Example 4
[0042] Extracellular vesicles were isolated and purified from bulk cell supernatants.
[0043] Step 1: Resuspend MCF-7 cells in DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin, and incubate at 37°C in a 5% CO2 incubator. When the cell confluence reaches approximately 90%, discard the old medium and add fresh medium. After 48 hours of incubation, collect the cell culture supernatant. Centrifuge at 500×g for 10 minutes at 4°C to remove suspended cells. Collect the supernatant and centrifuge at 2500×g for 20 minutes at 4°C to remove cell debris, obtaining the cell supernatant sample to be concentrated. Add 20 mL of the cell supernatant sample to the polyethylene glycol hydrogel monolithic column of Example 1, with a sample volume to column bed volume ratio of 10:1. React at room temperature for 30 minutes, then compress the polyethylene glycol hydrogel in the column and collect the eluent to obtain the concentrated cell supernatant sample. The used polyethylene glycol hydrogel monolithic column can be regenerated by washing with deionized water and drying.
[0044] Step 2: Add 0.4 mL of concentrated cell supernatant sample to a silica size exclusion column (prepared in the same way as in Example 2), with a sample volume to column bed volume ratio of 1:25. After the concentrated cell supernatant sample flows into the column bed, add 10 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid buffer for elution, with an elution buffer to sample volume ratio of 30:1. Collect one fraction for every elution volume equal to the sample volume, for a total of 30 fractions. After use, regenerate the silica size exclusion column under the same conditions as in Example 2. Detect the protein concentration in each fraction using a BCA kit; the BCA assay requires a sample volume of 20 μL. The NTA assay range is 1 × 10⁻⁶. 7 ~1 × 10 8 Between a concentration range of particles / mL, the remaining sample was diluted by the corresponding factor according to this concentration range to determine the particle concentration of each component. The obtained data on protein concentration and particle concentration for each component can reflect its degree of separation. Figure 4 The image shows the elution curves of the concentrated cell supernatant sample after loading onto a silica size exclusion column. The horizontal axis represents the elution volume. Curve E represents the extracellular vesicle concentration of each component measured by NTA, and curve F represents the protein concentration of each component measured by BCA. It can be seen that the extracellular vesicle concentration reaches its peak at approximately 37% of the total elution volume, while the protein concentration reaches its peak at approximately 75% of the total elution volume. This indicates that the extracellular vesicles and proteins are basically separated, achieving the purpose of purification.
Claims
1. A method for batch isolation and purification of extracellular vesicles, characterized in that, The method includes the following steps: Step 1: Add the biological sample into the polyethylene glycol hydrogel monolithic column. The ratio of sample volume to column bed volume is 20:1 to 5:
1. After reacting at room temperature for 30 to 120 minutes, compress the polyethylene glycol hydrogel in the column and collect the effluent to obtain the concentrated biological sample. The preparation method of the polyethylene glycol hydrogel monolithic column is as follows: polyethylene glycol monomethyl ether and polyethylene glycol diacrylate are added to a 1× phosphate buffer solution, premixed, and then... The pH was adjusted to 7-8 with a mol / L sodium hydroxide aqueous solution, and then tetramethylethylenediamine, ammonium sulfate, and propylene were added to obtain a polyethylene glycol (PEG) polymerization solution. An empty chromatography column was taken, the bottom cap was closed, and the PEG polymerization solution was added at a volume of 5%-20% of the column volume. The column was then placed at -20°C for polymerization for 12-24 hours. After the reaction, the column was allowed to return to room temperature, the bottom cap was removed, and the gel inside the column was washed with deionized water to obtain a monolithic PEG hydrogel column. The amounts of PEG monomethyl ether, PEG diacrylate, tetramethylethylenediamine, and propylene added were 8%-12%, 0.1%-0.3%, 0.1%-0.3%, and 0.8%-1.5% of the PEG polymerization solution volume, respectively. The mass ratio of ammonium sulfate to the PEG polymerization solution volume was 3-5 mg:100 mL. Step 2: Add the concentrated biological sample to a silica size exclusion column. The ratio of sample volume to column volume is 1:10 to 1:
50. After the concentrated biological sample flows into the column, add 10 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution for elution. The ratio of the elution buffer to sample volume is 20:1 to 40:
1. Collect the portion of the elution liquid that reaches 30% to 40% of the buffer volume to obtain extracellular vesicles.
2. The method for batch isolation and purification of extracellular vesicles according to claim 1, characterized in that, In step 1, the ratio of sample loading volume to column bed volume is 10:1 to 15:
1.
3. The method for batch isolation and purification of extracellular vesicles according to claim 1 or 2, characterized in that, In step 1, the polyethylene glycol hydrogel monolithic column is regenerated after use. The regeneration method is as follows: wash with deionized water and then dry.
4. The method for batch isolation and purification of extracellular vesicles according to claim 1, characterized in that, In step 2, silica gel with a particle size of 30–90 μm and a pore size of 50–100 nm is added to deionized water and mixed thoroughly to obtain a silica gel dispersion. After installing a lower sieve plate with a pore size of 10–20 μm in an empty chromatography column with the bottom cap on, the silica gel dispersion is added and allowed to settle naturally for more than 24 hours to allow the lower precipitate to reach the required column height. The upper solution is then removed, and an upper sieve plate with a pore size of 10–20 μm is installed to obtain the silica size exclusion separation column.
5. The method for batch isolation and purification of extracellular vesicles according to claim 1, characterized in that, In step 2, the ratio of sample loading volume to column bed volume is 1:20 to 1:
30.
6. The method for batch isolation and purification of extracellular vesicles according to any one of claims 1, 4, and 5, characterized in that, In step 2, the silica size exclusion column is regenerated after use. The regeneration conditions are as follows: first, wash with 2 to 4 column volumes of 1% Triton aqueous solution, then wash with 2 to 4 column volumes of deionized water, then wash with 2 to 4 column volumes of 70% ethanol aqueous solution, and then wash with 2 to 4 column volumes of deionized water. If the silica size exclusion column is used continuously, wash with 2 to 4 column volumes of 10 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution. If the silica size exclusion column is to be stored for a long time, place it in 20% ethanol aqueous solution.