Method for extracting exosome from microporous gel particles and application of exosome
By preparing microporous gel particles with controllable pore size combined with an osmotic pressure gradient buffer system, the complexity and low efficiency of existing exosome extraction methods have been solved, realizing high-purity, high-efficiency exosome extraction and large-scale application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-18
- Publication Date
- 2026-03-13
AI Technical Summary
Existing exosome extraction methods suffer from problems such as complex operation, low purity, poor efficiency, and high cost, especially in high-throughput screening and large-scale applications.
A method for extracting exosomes using microporous gel particles was developed. Polyethylene glycol diacrylate was used as the backbone monomer. Three-dimensional porous hydrogel particles with pore sizes of 100-800 nm were prepared by photo-initiated polymerization and low-temperature freezing. Combined with an osmotic pressure gradient buffer system, selective capture and reversible release of exosomes were achieved.
It achieves efficient, simple and low-cost exosome extraction, ensuring high purity and structural integrity of exosomes, making it suitable for large-scale applications, and avoiding cross-contamination and damage to exosomes from high shear forces.
Smart Images
Figure CN121653045A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of life science technology, specifically relating to a method for extracting exosomes from microporous gel particles and its application. Background Technology
[0002] Exosomes are small, membranous vesicles secreted by cells, typically between 30 and 150 nm in diameter, and widely distributed in body fluids such as blood, saliva, urine, and cerebrospinal fluid. Exosomes are considered important mediators of intercellular communication, containing abundant biomolecules such as proteins, RNA, and miRNAs, thus possessing significant application potential in disease diagnosis, drug delivery, cancer monitoring, and immune regulation. The extraction and purification of exosomes are crucial steps in their application. Existing exosome extraction methods suffer from problems such as complex operation, low purity, poor efficiency, and high cost.
[0003] Traditional methods for exosome extraction mainly include: ultracentrifugation, immunoaffinity assay, microfiltration, and density gradient centrifugation.
[0004] Ultracentrifugation is the most commonly used method for extracting exosomes, separating them through high-speed centrifugation. However, this method requires sophisticated equipment, is complex to operate, and takes a long time, and it is difficult to guarantee high purity of the extracted exosomes. Furthermore, ultracentrifugation can damage some exosomes, affecting their subsequent analysis and applications.
[0005] Immunoaffinity assays achieve separation by binding specific antibodies to exosome surface proteins. This method offers high selectivity but requires specific antibodies and is susceptible to sample complexity. Furthermore, it is inefficient for processing large-scale samples and is costly.
[0006] Microfiltration separates exosomes from samples using microporous membranes. This method is simple to operate and fast, but its separation efficiency is relatively low and easily affected by the pore size of the filter membrane, making it impossible to achieve high-purity exosome separation.
[0007] Density gradient centrifugation separates exosomes by centrifuging samples with solutions of different densities. Although this method can effectively separate different types of exosomes, it is cumbersome, time-consuming, and requires specific experimental conditions.
[0008] Despite the progress made in existing exosome extraction methods, numerous challenges remain, such as low efficiency, high cost, and operational complexity. The limitations of traditional methods are particularly evident in high-throughput screening and large-scale applications. Therefore, developing an efficient, simple, and low-cost exosome extraction method has become an important research topic in the life sciences. Summary of the Invention
[0009] The purpose of this invention is to solve the problems of complex operation, low purity, poor efficiency and high cost of existing exosome extraction methods.
[0010] To achieve the above objectives, the present invention provides a method for extracting exosomes from microporous gel particles, comprising the following steps: S1. Preparation of microporous gel particles, wherein the microporous gel particles are three-dimensional porous hydrogel particles with a pore size of 100-800 nm and a volume of 10-60 μL, and are prepared by combining photoinitiated polymerization with low-temperature freezing pore-forming with polyethylene glycol diacrylate with a molecular weight of 600-750 as the main skeleton monomer, and photoinitiator and deionized water. S2. Prepare an osmotic gradient buffer system, wherein the buffer system comprises: Hypertonic capture buffer: HEPES buffer containing 2.5-3.5 M NaCl, pH 7.0-8.0, preferably 7.4; Meso-osmotic washing buffer: HEPES buffer containing 1.0-2.0 M NaCl, pH 7.0-8.0, preferably 7.4; Isotonic harvest buffer: NaCl-free HEPES buffer with a pH of 7.0-8.0, preferably 7.4; S3. The microporous gel particles and the exosome sample are mixed and incubated in the hypertonic capture buffer to allow the exosomes to enter and adsorb into the pores of the microporous gel particles; S4. Separate the microporous gel particles and wash them with the mesoosmotic washing buffer to remove non-specifically adsorbed impurities; S5. The washed microporous gel particles are mixed with the isotonic harvesting buffer and incubated to allow exosomes to desorb and release from the pores of the microporous gel particles, thereby obtaining purified exosomes.
[0011] Preferably, the preparation of the microporous gel particles specifically includes the following steps: S1. Polyethylene glycol diacrylate with a molecular weight of 700-750, deionized water and photoinitiator are mixed in the dark to form a transparent and uniform precursor solution; S2. The precursor solution is injected into a mold for cryogenic freezing; S3. In the frozen state, the precursor solution is irradiated with ultraviolet light to initiate a photopolymerization reaction, causing polyethylene glycol diacrylate to crosslink and solidify, forming a hydrogel; S4. Thaw the hydrogel, wash away ice crystals, residual photoinitiator and unreacted monomers to obtain microporous gel particles with a three-dimensional interconnected porous structure, wherein the average pore size of the microporous gel particles is 100-800 nm.
[0012] Preferably, in step S1, the volume fraction of polyethylene glycol diacrylate is 8-12%, the volume fraction of photoinitiator is 1-5%, and the remainder is deionized water.
[0013] Preferably, the photoinitiator in step S1 is one of 2-hydroxy-2-methylphenylacetone, 1-hydroxycyclohexylphenyl ketone, or lithium phenyl (2,4,6-trimethylbenzoyl)phosphate.
[0014] Preferably, in step S2, the low-temperature freezing temperature is -20°C to -196°C, and the freezing time is 10-60 minutes; the wavelength of the ultraviolet light is 365 nm, and the irradiation time is 2 minutes.
[0015] Preferably, in step S2, the mold is a PDMS mold with a hole diameter of 5 mm and a depth of 2 mm, and the volume of the microporous gel particles is 40 μL.
[0016] Preferably, the exosome sample is one of serum, plasma, saliva, urine, cerebrospinal fluid, or cell culture supernatant.
[0017] Preferably, the incubation conditions for the capture step are incubation at 2-8°C for 10-60 minutes; and the incubation conditions for the release step are incubation at 2-8°C for 10-20 minutes.
[0018] The present invention also provides a method for extracting exosomes from microporous gel particles, which has applications in the preparation of reagent kits or in the development of drug delivery carriers.
[0019] Compared with the prior art, the advantages of this invention are: The gel particles of this invention use polyethylene glycol diacrylate (PEG) as a cross-linking backbone. The PEG diacrylate backbone does not react with proteins or RNA, avoiding interference with exosome components. It exhibits good water solubility and biocompatibility, is mild, requires no high-speed centrifugation or organic solvents, and its molecular weight is controlled within the range of 600–750, ensuring stable gel structure and controllable pore size. During preparation, PEG diacrylate and a water-soluble photoinitiator are easily handled using standard centrifuge tubes or microtubes, and are suitable for single-use applications to avoid cross-contamination. It offers low cost and high throughput, is easy to prepare, and is suitable for large-scale production. It also boasts high biosafety, with non-toxic and residue-free materials, making it suitable for subsequent detection. Attached Figure Description
[0020] Figure 1 1. Overall drawing of the manufactured PDMS mold; Figure 2 The diagram shows the state of the precursor solution dispensed into each PDMS well. Detailed Implementation
[0021] The terms used in this invention, unless otherwise stated, generally have the meanings commonly understood by those skilled in the art.
[0022] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art.
[0023] The reagents used in the following examples were obtained through common commercial channels. Experimental procedures and conditions not specified are in accordance with conventional procedures and conditions in the art.
[0024] The specific implementation of the present invention will be described below with reference to the embodiments.
[0025] This embodiment provides a method for extracting exosomes from microporous gel particles.
[0026] I. Technical Principles By utilizing the spatial screening effect and reversible osmotic pressure regulation effect of the porous three-dimensional network formed by photocrosslinking of polyethylene glycol diacrylate, selective capture and gentle release of exosomes can be achieved.
[0027] Polyethylene glycol diacrylate (PEG) is a double-bond modified polyethylene glycol compound that can be rapidly cross-linked and cured under ultraviolet light with the aid of a photoinitiator, exhibiting tunable mechanical properties. The molecular weight of PEG is adjustable, primarily depending on the molecular weight of the polyethylene glycol (PEG) used in its synthesis. Common PEG diacrylates have molecular weights of 400, 700, 6000, 8000, etc. This compound is non-toxic, non-immunogenic, has excellent blood compatibility, and excellent hydrophilicity. Based on these characteristics, this invention selects this compound as the basic structural material for exosome separation media.
[0028] 1. Formation Mechanism of Porous Gel: Polyethylene glycol diacrylate (PEG) is miscible with water in any proportion. When a certain amount of water-soluble photoinitiator is added to a mixture of PEG and water, phase separation occurs during freezing at -80°C. Water acts as an opening agent, forming ice crystals in the frozen state. After the ice crystal template is formed, cross-linking and curing are completed under ultraviolet light. After the ice crystals melt, uniformly distributed interconnected channels are left, forming a three-dimensional porous network with an average pore size of 400 nm. This structure ensures that exosomes can be retained within the pores under high osmotic pressure conditions, while larger particles are excluded.
[0029] 2. Exosome capture and release mechanism: Under high-salt conditions (3 M NaCl), Na... +Ions replace water molecules near the polyethylene glycol (PEG) chains, reducing the electrostatic repulsion between exosomes and making them easier to enter and aggregate within the gel pores. When the concentration of sodium chloride (NaCl) in the external solution decreases (to 0 M), osmotic pressure reversal causes water molecules to re-enter the inter-chain spaces of the PEG chains, and the exosomes gradually desorb from the gel pores and are released into the external solution, achieving gentle recovery. This dynamic equilibrium process achieves "controlled adsorption-reversible desorption" of exosomes, ensuring purity and structural integrity.
[0030] 3. Comprehensive Mechanism of Action Size-exclusion effect: The 400 nm pore size enables selective retention of exosomes ranging from 30 to 150 nm. Osmotic Modulation: Reversible capture and release of exosomes is achieved through a NaCl concentration gradient; PEG spatial shielding effect: The three-dimensional flexible mesh formed by PEG chains forms local encapsulation and charge regulation of exosomes, enhancing adsorption efficiency.
[0031] The gel particles of this invention use polyethylene glycol diacrylate (PEG) as the crosslinking backbone. PEG diacrylate molecules have good water solubility and biocompatibility, and their molecular weight is controlled within the range of 600–750, ensuring a stable gel structure and controllable pore size. During preparation, PEG diacrylate is mixed with a water-soluble photoinitiator, such as 2-hydroxy-2-methylphenylacetone, 1-hydroxycyclohexylphenyl ketone, or lithium phenyl (2,4,6-trimethylbenzoyl)phosphate, and purified water to form a homogeneous precursor solution. After freezing at -80°C, the solution is cured and crosslinked under 365 nm ultraviolet light. The ice crystals formed during freezing act as temporary templates; after photocuring, they melt to form a connected porous network structure, thus obtaining hydrogel particles with an average pore size of approximately 400 nm. The volume of the gel particles is controlled between 10–40 μL, which is convenient for handling in standard centrifuge tubes or microtubes and suitable for single-use applications to avoid cross-contamination. The exosome separation process is based on a dual mechanism of "pore size screening effect + osmotic pressure gradient drive". The 400 nm micropores uniformly distributed inside the gel can selectively adsorb exosomes in the diameter range of 30–150 nm, and achieve controlled release through changes in ionic strength.
[0032] II. Extraction Process Preparation of polyethylene glycol diacrylate hydrogel particles (taking the preparation of 40 μL gel particles as an example) S1. Mold Preparation: Using Dow Corning Sylgard 184 PDMS (organic silicone) photocurable silicone as the substrate, the degassed PDMS and curing agent were mixed at a mass ratio of 10:1 and poured into a milled hard aluminum master mold (each hole is 5 mm in diameter and 2 mm in depth, equivalent to a volume of 40 microliters per particle), to form a mold as shown. Figure 1 A PDMS mold with multiple pore arrays was prepared. After curing the PDMS mixture at 60°C for 3 hours, the PDMS replica was peeled off, with each pore having a volume of approximately 40 μL.
[0033] S2. Preparation of precursor solution: 10% (v / v) polyethylene glycol diacrylate (Mn700 or Mn750) as gel structure, 88% (v / v) deionized water as pore opener and 2% (v / v) 2-hydroxy-2-methylphenylacetone as photoinitiator. Keep the solution away from light during preparation and stir until transparent and homogeneous.
[0034] S3. Freezing and light curing: See [link] Figure 2 40 µl of precursor solution was dispensed into each PDMS well. The mold prepared for filling with the precursor solution was cooled at 4 °C for 10 minutes. After adding the precursor solution, it was frozen at -80 °C for 10 minutes, and then immediately irradiated with 365 nm UV light in a chamber for 2 minutes to initiate the curing process. The resulting cryo-photocrosslinked polyethylene glycol diacrylate particles were thawed by rinsing with excess deionized water for about 1 minute. This step also helps to wash away the photoinitiator and uncrosslinked polyethylene glycol diacrylate, and allows for the harvesting of free microporous polyethylene glycol diacrylate particles. (Particle pore size 100–800 nm (preferably 300–500 nm, most preferably ≈400 nm)) S4. Thawing and Washing: To achieve efficient capture and controlled release of exosomes in porous polyethylene glycol diacrylate gels, this invention employs three HEPES buffer systems with different ionic strengths. All buffers were prepared under sterile conditions. Analytical grade HEPES was dissolved in ultrapure water, and the pH was adjusted to 7.4 using 1 M NaOH. Sodium chloride was weighed out according to the required NaCl concentration, dissolved, and the total volume was made up. Ultrapure water (resistivity ≥ 18.2 MΩ·cm) was used, filtered through a 0.22 μm filter for sterilization, and stored at 4 °C. If used for RNA or protein analysis, a mixture of 1 mM EDTA and 1× protease inhibitor should be added before use. name composition pH effect Hypertonic capture buffer (Buffer A) 25 mM HEPES + 3.0 M NaCl 7.4 In a hypertonic environment, exosomes are driven into the gel pores and adsorbed. Intermediate osmotic washing buffer (Buffer B) 25 mM HEPES + 1.5 M NaCl 7.4 Balance osmotic pressure to elute non-specifically adsorbed proteins and impurity particles. Isotonic harvest buffer (Buffer C) 25 mM HEPES (NaCl-free) 7.4 Lowering the osmotic pressure promotes the gentle desorption and release of exosomes from the pores.
[0035] In the extraction stage (capture), a hypertonic 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer at pH 7.4 and 3 M NaCl is used. Under high ionic strength conditions, exosomes are driven into the gel pores by osmotic pressure, forming local aggregation and spatial shielding effects with the PEG chains, thus achieving efficient capture. In the washing stage (impurity removal), a neutral-osmotic HEPES buffer at pH 7.4 and 1.5 M NaCl is used. This step washes away non-specifically adsorbed proteins, lipids, and impurity particles through osmotic equilibrium, retaining the physically retained exosomes. In the harvest stage (release), a pH 7.4 buffer at 0 M NaCl (i.e., salt-free HEPES) is used. Lowering the osmotic pressure allows the exosomes to be gently desorbed from the gel pores and released into the solution, obtaining a high-purity, highly intact exosome component.
[0036] Through a three-step osmotic pressure regulation process, exosome capture and release are completed under mild conditions without the need for high shear forces or chemical lysis, which greatly protects the integrity of the exosome membrane structure and its contents.
[0037] S5. Storage: The prepared particles can be stored at 4 °C for more than 30 days in sterile pH 7.4 HEPES buffer or purified water, and the pore structure and adsorption performance remain stable.
[0038] Example 1 Extraction and purification of serum exosomes (taking human serum as an example) S1. Sample pretreatment: Take 1 mL of fresh human serum and centrifuge at 3000g for 10 min to remove cells and debris.
[0039] S2. Capture phase: Add one polyethylene glycol diacrylate hydrogel particle to 1 mL of serum, use pH 7.4, 3 M NaCl HEPES buffer, mix gently, and mix at 4°C for 30 min to capture exosomes.
[0040] S3. Washing stage: Wash three times (500 μL each) with pH 7.4, 1.5M NaCl HEPES buffer to remove non-specific adsorbed impurities.
[0041] S4. Release phase: Add 500 μL of pH 7.4, 0M NaCl (HEPES-free) buffer and incubate at 4°C for 15 min. Exosomes are gently released from the gel.
[0042] S5. Detection results: NanoSight analysis showed that the obtained exosomes were concentrated in the range of 80–120 nm, and the concentration was about 2.4 times that of the traditional ultracentrifugation method; Western blotting showed that the CD63 and TSG101 signals were significantly enhanced, indicating that the exosomes had higher purity and integrity.
[0043] Example 2 Extraction and purification of salivary exosomes (using saliva as an example) S1. Sample processing: Collect 1 mL of fresh saliva and centrifuge at 3000g for 15 min to remove mucin and impurities.
[0044] S2. Capture step: Add 1 mL of centrifuged saliva to 1 polyethylene glycol diacrylate hydrogel particle, add 1 mL of pH 7.4, 3M NaCl HEPES buffer, and mix for 30 min.
[0045] S3. Washing procedure: Wash 3 times with 500 μL of 1.5M NaCl HEPES buffer.
[0046] S4. Harvesting procedure: Release exosomes by standing for 10 min with 500 μL of 0M NaCl HEPES buffer.
[0047] S5. Result Detection: Exosomes were observed to be morphologically intact and uniform in size by TEM scanning electron microscopy; high-quality miRNAs (such as miR-21, miR-155, etc.) were obtained by qPCR detection, verifying that this method is suitable for rapid extraction of salivary exosomes.
[0048] III. Specific Applications Its function varies depending on the sample it is extracted from: 1. Serum / plasma exosomes: used for tumor markers, immune regulation, and detection of metabolic diseases; 2. Salivary exosomes: Suitable for non-invasive screening of sleep disorders, depression, and neurodegenerative diseases; 3. Urinary exosomes: used for assessing kidney disease and metabolic status; 4. Cerebrospinal fluid exosomes: used for research on biomarkers of nervous system diseases; 5. Cell culture supernatant: used for cell secretion analysis, drug stimulation experiments, and exosome vector development.
[0049] The exosomes obtained by this method are suitable for a variety of subsequent experiments, including: Electron microscopy (TEM) morphological observation; NTA particle size / concentration analysis; Western blotting detection of exosome marker proteins (CD9, CD63, TSG101, etc.); RNA extraction and qPCR or RNA-seq analysis; proteomics and metabolomics studies. As a standard exosome extraction module, it can be integrated into diagnostic kits, microarray platforms, or automated sample processing systems.
[0050] The above is a detailed description of the embodiments, which is intended to enable those skilled in the art to correctly understand and use the present invention. Any improvements or modifications to technical solutions obtained by those skilled in the art based on the present invention and on the existing technology, without innovative effort but only through analysis, analogy, or limited enumeration, should be within the scope of protection defined by the claims.
Claims
1. A method for extracting exosomes from microporous gel particles, characterized in that, Includes the following steps: S1. Preparation of microporous gel particles, wherein the microporous gel particles are three-dimensional porous hydrogel particles with a pore size of 100-800 nm and a volume of 10-60 μL, and are prepared by combining photoinitiated polymerization with low-temperature freezing pore-forming with polyethylene glycol diacrylate with a molecular weight of 600-750 as the main skeleton monomer, and photoinitiator and deionized water. S2. Prepare an osmotic gradient buffer system, wherein the buffer system comprises: Hypertonic capture buffer: HEPES buffer containing 2.5-3.5 M NaCl, pH 7.0-8.0, preferably 7.4; Meso-osmotic washing buffer: HEPES buffer containing 1.0-2.0 M NaCl, pH 7.0-8.0, preferably 7.4; Isotonic harvest buffer: NaCl-free HEPES buffer, pH 7.0-8.0, preferably 7.4; S3. The microporous gel particles and the exosome sample are mixed and incubated in the hypertonic capture buffer to allow the exosomes to enter and adsorb into the pores of the microporous gel particles; S4. Separate the microporous gel particles and wash them with the mesoosmotic washing buffer to remove non-specifically adsorbed impurities; S5. The washed microporous gel particles are mixed with the isotonic harvesting buffer and incubated to allow exosomes to desorb and release from the pores of the microporous gel particles, thereby obtaining purified exosomes.
2. The method for extracting exosomes from microporous gel particles as described in claim 1, characterized in that, The preparation of the microporous gel particles specifically includes the following steps: S1. Polyethylene glycol diacrylate with a molecular weight of 700-750, deionized water and photoinitiator are mixed in the dark to form a transparent and uniform precursor solution; S2. The precursor solution is injected into a mold for cryogenic freezing; S3. In the frozen state, the precursor solution is irradiated with ultraviolet light to initiate a photopolymerization reaction, causing polyethylene glycol diacrylate to crosslink and solidify, forming a hydrogel; S4. Thaw the hydrogel, wash away ice crystals, residual photoinitiator and unreacted monomers to obtain microporous gel particles with a three-dimensional interconnected porous structure, wherein the average pore size of the microporous gel particles is 100-800 nm.
3. The method for extracting exosomes from microporous gel particles as described in claim 2, characterized in that, In step S1, the volume fraction of polyethylene glycol diacrylate is 8-12%, the volume fraction of photoinitiator is 1-5%, and the remainder is deionized water.
4. The method for extracting exosomes from microporous gel particles as described in claim 2, characterized in that, In step S1, the photoinitiator is one of 2-hydroxy-2-methylphenylacetone, 1-hydroxycyclohexylphenyl ketone, or lithium phenyl (2,4,6-trimethylbenzoyl)phosphate.
5. The method for extracting exosomes from microporous gel particles as described in claim 2, characterized in that, In step S2, the cryogenic freezing temperature is -20°C to -196°C, and the freezing time is 10-60 minutes; the wavelength of the ultraviolet light is 365 nm, and the irradiation time is 2 minutes.
6. The method for extracting exosomes from microporous gel particles as described in claim 2, characterized in that, In step S2, the mold is a PDMS mold with a hole diameter of 5 mm and a depth of 2 mm, and the volume of the microporous gel particles is 40 μL.
7. The method for extracting exosomes from microporous gel particles as described in claim 1, characterized in that, The exosome sample is one of serum, plasma, saliva, urine, cerebrospinal fluid, or cell culture supernatant.
8. The method for extracting exosomes from microporous gel particles as described in claim 1, characterized in that, The incubation conditions for the capture step are 10-60 minutes at 2-8°C; the incubation conditions for the release step are 10-20 minutes at 2-8°C.
9. The application of the method for extracting exosomes from microporous gel particles as described in claim 1 in the preparation of reagent kits or in the development of drug delivery carriers.