A method for distinguishing and separating small extracellular vesicles from low-density lipoproteins

Abstract

The application belongs to the technical field of biological nano detection and separation and purification, and provides a method for distinguishing and separating small extracellular vesicles and low-density lipoprotein. The method comprises the following steps: dividing a to-be-tested sample into three groups; adding PBS buffer solution to the first group of to-be-tested sample to maintain normal osmotic pressure; adding ultrapure water to the second group of to-be-tested sample to construct a low-osmotic environment; adding a sodium chloride solution with a mass fraction of 1.8% to the third group of to-be-tested sample to construct a high-osmotic environment; after the three groups of to-be-tested sample are left to stand for 20 min, the morphology and particle size of the to-be-tested sample are detected; and according to the detection results of the morphology and particle size of the to-be-tested sample under normal, low and high osmotic pressure, it is determined whether the to-be-tested sample is a small extracellular vesicle or a low-density lipoprotein. Under low-osmotic or high-osmotic treatment, the small extracellular vesicles will swell or shrink, but the membrane structure remains intact; and the low-density lipoprotein will not change. The method can avoid the use of destructive reagents and retain the natural morphology and contents of the extracellular vesicles and the low-density lipoprotein.

Description

Technical Field

[0001] This invention belongs to the field of bio-nano detection and separation purification technology, and particularly relates to a method for distinguishing and separating small extracellular vesicles and low-density lipoprotein. Background Technology

[0002] Low-density lipoprotein (LDL) is a lipoprotein particle that carries cholesterol into peripheral tissue cells. It plays a crucial role not only in substance transport but also participates extensively in various physiological activities. This lipoprotein is rich in cholesterol and easily deposits on the inner walls of blood vessels, promoting the development of atherosclerosis; hence, it is commonly known as "bad cholesterol." Under normal physiological conditions, LDL can complete the body's routine cholesterol transport; however, once oxidized, it transforms into oxidized low-density lipoprotein (ox-LDL). ox-LDL has a significantly enhanced pathogenicity, being targeted and taken up by macrophages in the vascular wall, causing excessive intracellular lipid accumulation and prompting macrophages to transform into foam cells. These cells continuously accumulate under the vascular endothelium, gradually forming lipid streaks, which then develop into fibrous plaques, damaging vascular structure and ultimately inducing atherosclerosis and various related cardiovascular diseases. Sample purity is crucial for physiological and pathological studies of LDL, and sEVs (similar to microalbuminuron-ethyl) represent a significant challenge in achieving sample purity.

[0003] Small extracellular vesicles (sEVs) are cell-derived membrane vesicles composed of a lipid bilayer. They are important mediators of intercellular communication and are widely used in pathology, physiology, treatment, diagnosis, and as mechanistic components. These two substances highly overlap in size, shape, and density, and may even interact to form complexes. However, current separation techniques, such as ultracentrifugation and size exclusion chromatography, are based on physical properties, and both yield and purity are affected by contaminants. Furthermore, identification techniques, such as Western blot, dynamic light scattering, and morphological characterization, are insufficient to eliminate the presence of contaminants. Moreover, existing methods for purifying lipoproteins, primarily ultracentrifugation and affinity chromatography, suffer from high equipment or material requirements, low throughput, or long processing times. Therefore, achieving the differentiation and separation of small extracellular vesicles and low-density lipoproteins remains challenging and crucial. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method for distinguishing and separating small extracellular vesicles and low-density lipoprotein, the purpose of which is to solve the problems mentioned in the background art.

[0005] In a first aspect, the present invention provides a method for distinguishing between small extracellular vesicles and low-density lipoprotein, comprising: dividing the test samples into three groups; adding PBS buffer solution to the first group of test samples to maintain normal osmotic pressure; adding ultrapure water to the second group of test samples to construct a hypotonic environment; adding a 1.8% sodium chloride solution to the third group of test samples to construct a hypertonic environment; and after the three groups of test samples are allowed to stand for 20 minutes, their morphology and particle size are detected respectively. Based on the detection results of the morphology and particle size of the sample under normal, low and high osmotic pressure, the sample is determined to be a small extracellular vesicle or a low-density lipoprotein.

[0006] Furthermore, the hypotonic solution is ultrapure water, and the hypertonic solution is a 1.8% sodium chloride solution.

[0007] Furthermore, when the morphology and particle size of the test sample do not change under different osmotic pressures, the test sample is determined to be low-density lipoprotein; when the test sample expands and increases in particle size under low osmotic pressure, and shrinks and decreases in particle size under high osmotic pressure, the test sample is determined to be small extracellular vesicles.

[0008] Extracellular vesicles are primarily composed of a phospholipid bilayer, similar to a cell membrane. The phospholipid bilayer has hydrophilic heads on both sides and a hydrophobic tail in the middle. When the osmotic pressure of the external environment changes, water molecules move in and out of the extracellular vesicles along the concentration gradient (osmotic pressure gradient) due to the concentration difference between the inside and outside of the vesicle. Low-density lipoprotein (LDL) has a hydrophobic central region and a hydrophilic outer layer. This structure acts like a protective shell; when the external osmotic pressure changes, the hydrophilic outer layer can buffer the effects of the external environment to some extent. In other words, when the osmotic pressure in the external environment changes, the size and particle size of the extracellular vesicles change accordingly, while LDL remains unaffected by osmotic pressure.

[0009] Secondly, the present invention provides a method for separating low-density lipoprotein, comprising the following steps: Step 1: Add the sample to be extracted to a hypotonic solution, adjust the osmotic pressure to a low osmotic pressure, let it stand, and then filter. Step 2: Collect the filtered solution, add hypertonic solution, and adjust the osmotic pressure to normal osmotic pressure to obtain the solution after osmotic pressure adjustment; Step 3: After adjusting the osmotic pressure, the solution is separated by differential ultracentrifugation, and the precipitate is collected to obtain low-density lipoprotein.

[0010] Furthermore, in step one, the hypotonic solution is ultrapure water.

[0011] Furthermore, in step one, the mixture is left to stand for 60 minutes.

[0012] Furthermore, in step one, a sterile 0.1μm filter membrane is used for filtration.

[0013] Furthermore, in step two, the hypertonic solution is a 1.8% sodium chloride solution.

[0014] Furthermore, in step three, the differential ultracentrifugation method specifically involves: centrifuging the solution after adjusting the osmotic pressure at 4°C and 10,000 × g for 60 min, collecting the supernatant, and then centrifuging it again at 4°C and 250,000 × g for 70 min.

[0015] The present invention has the following beneficial effects: (1) Maintaining structural integrity: Under hypotonic or hypertonic treatment, small extracellular vesicles will expand or shrink, but the membrane structure remains intact; while low-density lipoprotein does not change. This method avoids the use of destructive reagents and preserves the natural morphology and contents of extracellular vesicles and low-density lipoprotein.

[0016] (2) Avoiding exogenous labeling or antibody dependence: Traditional methods rely on affinity methods, but may introduce heterologous antibodies or non-specific binding. Osmotic pressure regulation is a purely physical means without chemical modification, which is more conducive to the standardized processing of clinical samples.

[0017] (3) High efficiency: The operation process is simple, eliminating complex steps such as incubation, washing membrane, and antibody binding. It can achieve differential sorting of extracellular vesicles and low-density lipoprotein in a short time and can quickly achieve high-purity separation.

[0018] (4) Low cost: By using the principle of osmotic pressure physical regulation, there is no need for expensive biochemical reagents, special kits and high-end supporting consumables, which greatly reduces the cost of sample separation and identification. Attached Figure Description

[0019] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures: Figure 1 The atomic force microscopy observation of the changes in sEV and LDL under different osmotic pressures in Example 1 of the present invention, wherein: Figure 1 In this context, A represents the change in sEV; Figure 1 B in the equation represents the change in LDL.

[0020] Figure 2 To illustrate the in-situ dynamic observation of sEV changes under different osmotic pressures using atomic force microscopy in Embodiment 1 of the present invention, wherein: Figure 2 In this context, A is a two-dimensional surface topography image of the sEV; Figure 2In this context, B represents the three-dimensional shape of the sEV; Figure 2 C in the figure represents the particle height profile curve of the sEV.

[0021] Figure 3 To illustrate the in-situ dynamic observation of LDL changes under different osmotic pressures using atomic force microscopy in Embodiment 1 of the present invention, wherein: Figure 3 In this context, A is a two-dimensional surface topography image of the LDL; Figure 3 In this context, B represents the three-dimensional morphology of the LDL. Figure 3 C in the figure represents the particle height profile curve of the LDL.

[0022] Figure 4 This invention provides an example of dynamic light scattering observation to observe the changes in sEV and LDL under different osmotic pressures, wherein: Figure 4 In this context, A represents the size change of sEV under different osmotic pressures; Figure 4 In this context, B represents the size change of LDL under different osmotic pressures.

[0023] Figure 5 This is a schematic diagram illustrating the principle of LDL separation and purification using osmotic pressure regulation combined with ultracentrifugation in Example 2 of the present invention. Figure 6 For the Western blot detection of LDL protein expression in Example 3 of the present invention, wherein: Figure 6 In this context, A represents the apoB marker protein extracted from LDL. Figure 6 B in the figure represents the purity of the extracted LDL.

[0024] Figure 7 This is an example of an in-situ dynamic observation using atomic force microscopy to observe the changes of extracted LDL under different osmotic pressures in Example 4 of the present invention. Figure 8 This is an example of dynamic light scattering observation of the changes in extracted LDL under different osmotic pressures in Example 4 of the present invention. Detailed Implementation

[0025] To make the technical problems, solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are merely illustrative and are not intended to limit the scope of the invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0027] Example 1: Differentiation between small extracellular vesicles (sEVs) and low-density lipoprotein (LDL) The test samples, sEV (150 μL, 10 μg / mL) and LDL (150 μL, 2 μg / mL), were divided into three groups. The first group of test samples was treated with 100 μL of PBS buffer solution to maintain normal osmotic pressure. The second group of test samples was treated with 100 μL of ultrapure water to create a hypotonic environment. The third group of test samples was treated with 100 μL of 1.8% sodium chloride solution to create a hypertonic environment. After standing for 20 min, the morphology and particle size of all three groups of test samples were measured, and the experimental results of the three groups were observed and compared.

[0028] Atomic force microscopy observation of changes in small extracellular vesicles and low-density lipoproteins under different osmotic pressures, such as... Figure 1 As shown, after the test samples were treated with isotonic solution (PBS buffer), hypotonic solution (ultrapure water), and hypertonic solution (1.8% sodium chloride solution), respectively, the morphology and size changes were observed by AFM scanning. The results showed that under normal osmotic pressure, the diameter distribution of RAW264.7 sEV was approximately 30 to 100 nm; under low osmotic pressure, the liquid in the external environment entered the sEV through the phospholipid bilayer, causing significant expansion, with a diameter distribution of approximately 50 to 150 nm. Under high osmotic pressure, the internal liquid flowed out, causing the sEV to shrink, with a size distribution of approximately 30 nm. Figure 1 (A) The morphological and size changes of LDL under different osmotic pressures show that LDL under normal osmotic pressure, LDL under low osmotic pressure (LDL-LO), and LDL under high osmotic pressure (LDL-HO) all maintain a relatively uniform spherical structure, showing no significant differences in morphology. Figure 1 (B in the middle).

[0029] In-situ dynamic observation of sEV changes under different osmotic pressures using atomic force microscopy, as follows: Figure 2As shown in the figure, the results indicate that RAW264.7 sEVs exhibit a relatively uniform structure and smooth surface in an isotonic environment, with an average diameter of 32.31 nm. After treatment with a hypotonic solution, the particles show significant water absorption and swelling, resulting in a more rounded and fuller overall profile, with a significant increase in particle size to 46.91 nm. In a hypertonic solution, however, the particles exhibit significant shrinkage and collapse, with a corresponding decrease in particle size to 27.98 nm. This demonstrates that sEVs possess a significant structural response to changes in external osmotic pressure.

[0030] In-situ dynamic observation of LDL changes under different osmotic pressures using atomic force microscopy, as follows: Figure 3 As shown in the figure. The results show that the diameter of the LDL changed from 36.55nm to 36.18nm and finally to 35.59nm, all within the error range; in addition, the area and volume of the LDL did not change significantly.

[0031] Dynamic light scattering observation shows the changes in sEV and LDL under different osmotic pressures, as follows: Figure 4 As shown in the figure. The results show that the particle size of sEV under normal osmotic pressure is 50.16±11.58 nm, which increases sharply to 250.15±39.38 nm under low osmotic pressure, and decreases to 12.66±3.42 nm under high osmotic pressure; while the particle size of LDL remains relatively stable under different osmotic pressure conditions, which are 25.81±7.37 nm, 23.94±8.30 nm and 22.21±7.14 nm under normal, low and high osmotic pressures, respectively, with no significant difference.

[0032] In summary, the method for distinguishing between small extracellular vesicles (sEVs) and low-density lipoproteins (LDL) according to this invention demonstrates that the particle size and height of sEVs are highly sensitive to changes in osmotic pressure. Their size parameters change significantly under different osmotic pressure conditions, with marked differences between groups. However, LDL is not sensitive to changes in osmotic pressure, and its size parameters remain essentially stable under different osmotic pressure environments.

[0033] Example 2: Isolation of low-density lipoprotein (LDL) (1) Adjust the external environment of the sample to be extracted to low osmotic pressure (ultrapure water) and let it stand for 60 min; (2) Filter the solution after standing using a sterile 0.1 μm filter membrane, collect the filtered solution into a centrifuge tube, and then adjust the osmotic pressure to normal using a hyperosmotic solution (1.8% sodium chloride solution); (3) Centrifuge the centrifuge tube at 4℃ and 10000×g for 60 min, collect the supernatant and transfer it to an ultra-high speed centrifuge tube, then centrifuge at 4℃ and 250000×g for 70 min; collect the precipitate to obtain low-density lipoprotein, and store it in a refrigerator at 4℃.

[0034] A schematic diagram illustrating the principle of LDL purification using osmotic pressure regulation combined with ultracentrifugation is shown below. Figure 5 As shown.

[0035] Experimental Example 3: Identification of Isolated Low-Density Lipoprotein (LDL) (1) SDS-PDGE gel electrophoresis: 6% separating gel and 5% stacking gel, the loading volume (sample and loading buffer) of each sample well is kept consistent, electrophoresis conditions: constant voltage 80V electrophoresis for 30 minutes, then switch to 120V, electrophoresis for about 90 minutes.

[0036] (2) Transfer: Use cellulose acetate membrane (NC membrane) under the following conditions: constant pressure 200 V, 4℃, 150 minutes.

[0037] (3) Washing the membrane: After the transfer, cut off the NC membrane containing the target band (including apoB, TSG101, Alix, HSP70), put them into the incubation box, and wash them three times with TBS-T solution for 10 minutes each time.

[0038] (4) Sealing: Sealing with TBS-T solution containing 5% skim milk powder at room temperature for 1 hour.

[0039] (5) Primary antibody incubation: Add primary antibody containing blocking solution (TBS-T solution containing 5% skim milk powder), incubate overnight at 4°C on a shaker, and place at room temperature for 1 hour the next day.

[0040] (6) Secondary antibody incubation: Wash the NC membrane three times with TBS-T solution for 10 minutes each time, add secondary antibody containing blocking solution, and incubate at room temperature for 2 hours.

[0041] (7) Development: Clean the NC membrane three times with TBS-T solution for 10 minutes each time, and develop it on a developer with chemiluminescent solution.

[0042] The experimental results of Western blot detection of LDL protein expression isolated in Example 2 are as follows: Figure 6 As shown, the results indicate that the method for separating low-density lipoprotein (LDL) according to the present invention can successfully extract LDL, and the protein does not undergo degradation or denaturation. Figure 6 In addition, TSG101, Alix, and HSP70 were all positively expressed in the sEV samples; the OP-LDL (oxidized low-density lipoprotein) component was consistent with the LDL standard, expressing only the LDL marker apoB (…). Figure 6 (B in the example). This indicates that the LDL isolated in Example 2 has a high purity.

[0043] Experiment Example 4: Changing osmotic pressure to distinguish and verify isolated low-density lipoprotein (LDL) The LDL separated in Example 2 was divided into three groups. The first group of LDL was added with PBS buffer solution to maintain normal osmotic pressure. The second group of LDL was added with ultrapure water to create a hypotonic environment. The third group of LDL was added with 1.8% sodium chloride solution to create a hypertonic environment. After the three groups of samples were allowed to stand for 20 minutes, their morphology and particle size were detected. The experimental results of the three groups of LDL were observed and compared.

[0044] In-situ dynamic observation of the changes of extracted LDL under different osmotic pressures using atomic force microscopy is as follows: Figure 7 As shown; the changes in extracted LDL under different osmotic pressures observed by dynamic light scattering are as follows. Figure 8 As shown.

[0045] The results showed that under normal osmotic pressure, LDL particles were regular spheres with a diameter of approximately 24.31 nm; under low osmotic pressure, the morphology of LDL did not change significantly, with a diameter of approximately 24.2 nm; under high osmotic pressure, LDL still maintained a spherical shape with a diameter of approximately 23.89 nm; and after changing the osmotic pressure, the particle size of LDL particles was not changed. This method verified LDL with low cost and effectively eliminated the interference and contamination of sEV.

[0046] The above description is merely an 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 within the protection scope of the present invention.

Claims

1. A method for distinguishing between small extracellular vesicles and low-density lipoprotein, characterized in that, include: The samples to be tested were divided into three groups; The first group of samples to be tested were added with PBS buffer solution to maintain normal osmotic pressure; The second group of samples to be tested was added with ultrapure water to create a hypotonic environment; The third group of test samples was added to a sodium chloride solution with a mass fraction of 1.8% to create a hypertonic environment; after all three groups of test samples were allowed to stand for 20 minutes, their morphology and particle size were measured. Based on the detection results of the morphology and particle size of the sample under normal, low and high osmotic pressure, the sample is determined to be a small extracellular vesicle or a low-density lipoprotein.

2. The method for distinguishing between small extracellular vesicles and low-density lipoprotein as described in claim 1, characterized in that, The hypotonic solution is ultrapure water, and the hypertonic solution is a 1.8% sodium chloride solution.

3. The method for distinguishing between small extracellular vesicles and low-density lipoprotein as described in claim 2, characterized in that, When the morphology and particle size of the test sample do not change under different osmotic pressures, the test sample is determined to be low-density lipoprotein; when the test sample expands and increases in particle size under low osmotic pressure, and shrinks and decreases in particle size under high osmotic pressure, the test sample is determined to be small extracellular vesicles.

4. A method for separating low-density lipoprotein, characterized in that, Includes the following steps: Step 1: Add the sample to be extracted to a hypotonic solution, adjust the osmotic pressure to a low osmotic pressure, let it stand, and then filter. Step 2: Collect the filtered solution, add hypertonic solution, and adjust the osmotic pressure to normal osmotic pressure to obtain the solution after osmotic pressure adjustment; Step 3: After adjusting the osmotic pressure, the solution is separated by differential ultracentrifugation, and the precipitate is collected to obtain low-density lipoprotein.

5. The method for separating low-density lipoprotein as described in claim 4, characterized in that, In step one, the hypotonic solution is ultrapure water.

6. The method for separating low-density lipoprotein as described in claim 4, characterized in that, In step one, let it stand for 60 minutes.

7. The method for separating low-density lipoprotein as described in claim 4, characterized in that, In step one, a sterile 0.1μm filter membrane is used for filtration.

8. The method for separating low-density lipoprotein as described in claim 4, characterized in that, In step two, the hypertonic solution is a 1.8% sodium chloride solution.

9. The method for separating low-density lipoprotein as described in claim 4, characterized in that, In step three, the differential ultracentrifugation method specifically involves: centrifuging the solution after adjusting the osmotic pressure at 4°C and 10,000 × g for 60 min, collecting the supernatant, and then centrifuging it again at 4°C and 250,000 × g for 70 min.

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