Method for preparing quercetin liposome
By using ethanol as a solvent and microfluidic control technology, and combining dialysis to prepare quercetin liposomes, the problem of using toxic organic solvents in the prior art is solved, a safe, environmentally friendly and low-cost preparation process is achieved, and the bioavailability and therapeutic effect of the product is improved.
Patent Information
- Application Number
- CN202411914769.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-03-21
AI Technical Summary
The prior art uses toxic and costly organic solvents when preparing quercetin liposomes, resulting in safety hazards, environmental pollution and high production costs in the production process.
Ethanol is used as a solvent, combined with microfluidic control technology and dialysis method to avoid the use of harmful organic solvents, simplify the preparation process, and improve production efficiency.
The safe and environmentally friendly preparation of quercetin liposomes is achieved, the production cost is reduced, and the bioavailability and therapeutic effect of the product is improved.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to a method for preparing quercetin liposomes. Background Art
[0002] Quercetin is widely distributed in nature and is a flavonoid compound of the flavonol class, which has antioxidant, anti-inflammatory, cardioprotective, antiviral and antibacterial effects. Although quercetin has a wide range of pharmacological effects, its poor water solubility greatly affects its dissolution and absorption in the body.
[0003] To overcome this shortcoming, the use of nanoliposome preparations based on quercetin can significantly improve its bioavailability and efficacy. Liposomes are artificial membrane structures, spherical vesicles of bilayer lipid molecules spontaneously formed by phospholipid molecules in an aqueous environment, and have been widely used as drug delivery systems. Quercetin liposomes are nanomedicines prepared using liposome technology, which are designed to improve the solubility, stability and bioavailability of quercetin.
[0004] Traditional quercetin liposomes are mainly obtained by evaporating chloroform and methanol to form a thin film, and then hydrating. However, there are problems such as high toxicity, cumbersome preparation, and high pollution. Specifically, the traditional mainstream process for producing quercetin liposomes is the "thin film method", which is to mix quercetin and lipid materials in a solvent composed of chloroform and methanol, remove the solvent using rotary evaporation technology, leave a thin film, and then add water to form a dispersion of liposomes, and then apply it after appropriate post-treatment. It can be seen that in the existing preparation technology, organic solvents chloroform and methanol are usually required to prepare quercetin liposomes, and this process brings a series of problems. First, these organic solvents are toxic, not only posing a threat to the health of operators, but also may have adverse effects on the human body through solvent vapor. Secondly, the use of these solvents may also cause environmental pollution, because they may release volatile organic compounds during the production process, causing pollution to the atmosphere and water resources, and if not handled properly, they may also cause secondary pollution to soil and water bodies. In addition, the "thin film dispersion method" used in the prior art requires the use of an organic reagent containing chloroform as a solvent, and forms an organic film after vacuum rotary evaporation, and then needs to be hydrated and emulsified at a speed of 6000-8000r / min for 30 minutes and stabilized for 2-3 hours. Its operation is cumbersome, time-consuming, costly, and highly toxic, and requires additional safety facilities and operating procedures, which increases production costs and complexity.
[0005] Therefore, developing a quercetin liposome preparation technology that is efficient, safe, and environmentally friendly is of great significance for improving the application value of quercetin. Summary of the invention
[0006] In view of this, the present invention aims to solve the limitations of the prior art through an innovative method, achieve the safe and environmentally friendly preparation of quercetin liposomes, and reduce related costs, so as to enhance the application prospects of quercetin in the medical field and broaden its scope of use in the fields of treatment and health care.
[0007] To this end, the present invention provides a method for preparing quercetin liposomes, which avoids the use of harmful and expensive organic solvents such as chloroform and methanol. Compared with the existing preparation technology on the market, the present invention is not only safer, more environmentally friendly, and less time-consuming, but also has more advantages in production cost.
[0008] The present invention is achieved by adopting the following technical solutions.
[0009] The present invention provides a method for preparing quercetin liposomes, which comprises the following steps:
[0010] (1) adding phospholipids, cholesterol, phospholipid polyethylene glycol and quercetin into ethanol and dissolving them to obtain an ethanol phase solution;
[0011] (2) pumping the ethanol phase solution and pure water obtained in step (1) into the microfluidic chip from different channel inlets, respectively, and performing micromixing to obtain a crude quercetin liposome;
[0012] (3) dialyzing the crude quercetin liposome obtained in step (2) using an aqueous medium to obtain.
[0013] The preparation method of the present application uses the green solvent ethanol, avoiding the use of organic reagents containing toxic and costly chloroform in the prior art. The preparation method of the present application uses microfluidic technology, which has precise control and eliminates the time for preparing the film and the time for pretreatment in the prior art, and the reaction time is short. In the preparation method of the present application, by controlling some reaction conditions, such as selecting a suitable solvent, controlling the ratio of the ethanol phase and the pure water solution, microfluidic parameter conditions, feed ratio, etc., liposomes with consistent particle size and excellent product quality are obtained.
[0014] Preferably, in step (1), the phospholipid is selected from one or more of soybean lecithin (SPC), egg yolk lecithin (EPC), hydrogenated lecithin (HSPC), distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoylphosphatidylcholine (DOPC), preferably SPC, EPC, DSPC, DPPC and DOPC.
[0015] Preferably, in step (1), the phospholipid polyethylene glycol is selected from one or both of DSPE-PEG and DMG-PEG.
[0016] Preferably, in step (1), the number average molecular weight of the phospholipid polyethylene glycol is 500-5000.
[0017] Preferably, in step (1), the phospholipid polyethylene glycol is selected from one or more phospholipid polyethylene glycols having a number average molecular weight of 500, 1000, 2000, and 5000.
[0018] Preferably, in step (1), the molar ratio of the phospholipid, the cholesterol, the phospholipid polyethylene glycol and the quercetin is 1:0.2-1:0-0.05:0.1-0.3, preferably 1:0.5-1:0.015-0.05:0.1-0.2.
[0019] Preferably, in step (2), the molar concentration of phospholipids in the mixed solution formed by the ethanol phase solution and the pure water is 4-12 mmol / L, preferably 5-10 mmol / L.
[0020] Preferably, in step (2), the volume ratio between the ethanol phase solution obtained in step (1) and the pure water is 1:3-12, preferably 1:3-10.
[0021] Preferably, in step (2), the flow rate ratio between the ethanol phase solution obtained in step (1) and the pure water is 1:3-12, preferably 1:3-10.
[0022] Preferably, in step (2), the control parameters of the channel are as follows: the total flow rate is 2-15 mL / min, preferably 2.75-12 mL / min.
[0023] Preferably, in step (2), when obtaining the crude quercetin liposome product, the pre-waste liquid is removed, and the collected amount of the pre-waste liquid is not less than 100-300 μL, preferably 100-200 μL.
[0024] The aforementioned pre-waste liquid refers to the liquid that initially flows out of no less than 100-300 μL, preferably 100-200 μL, when liposomes are generated after microfluidic mixing.
[0025] Preferably, in step (3), the aqueous medium is selected from a pure water solution, a glucose aqueous solution with a mass concentration of 5-10%, a sodium chloride aqueous solution with a mass concentration of 0.9%, or a phosphate buffered saline solution with a pH value of 7.4.
[0026] Preferably, in step (3), the dialysis is performed in a dialysis bag or a dialysis card.
[0027] Preferably, in step (3), the molecular weight cutoff of the dialysis bag or the dialysis card is 1000-10000Da.
[0028] In a specific embodiment, the preparation method of the present invention operates as follows:
[0029] (a) adding phospholipids, cholesterol, phospholipid polyethylene glycol and quercetin into a centrifuge tube containing ethanol, dissolving all the raw materials under water bath ultrasound to obtain an ethanol phase solution;
[0030] (b) preparing a pure aqueous solution;
[0031] (c) preparing an aqueous medium;
[0032] (d) The ethanol phase solution from step (a) is drawn into a syringe, and after the air bubbles are removed, it is installed in a channel of the microfluidic system.
[0033] (e) Use a syringe to draw up the pure aqueous solution from step (b), expel air bubbles, and transfer it to another channel of the microfluidic system;
[0034] (f) setting channel parameters of the ethanol phase solution and the pure water solution, including total flow rate, flow rate ratio, front waste liquid and other parameters;
[0035] (g) confirming the start, discarding the waste liquid and collecting the product;
[0036] (h) dialyzing the product with an aqueous medium to obtain a quercetin liposome solution.
[0037] The quercetin liposomes prepared in this application can be applied to the following fields:
[0038] (1) Pharmaceutical field: The preparation method of the quercetin liposome of the present application can be applied to the pharmaceutical manufacturing industry to develop drugs containing quercetin, covering various drug forms such as treating inflammation, anti-oxidative stress and inhibiting tumors.
[0039] (2) Scientific research: This production technology can improve the water solubility and bioavailability of quercetin and is suitable for scientific research to explore the biological activity of quercetin in cell and animal models and enhance the accuracy and application value of scientific research.
[0040] Compared with the prior art, this application has at least the following beneficial technical effects:
[0041] The quercetin liposomes developed by this application have significantly improved the application value of quercetin by virtue of their antioxidant, anti-inflammatory, anti-tumor and cardiovascular protection effects in the fields of medicine and health care, and have huge market development space. At the same time, this technology optimizes the production process of quercetin liposomes, eliminates the use of toxic organic solvents, and adopts microfluidic technology, which not only ensures the consistency of the particle size of the liposomes, but also supports large-scale production. This innovation solves the shortcomings of traditional production methods and achieves cost reduction and improved production efficiency.
[0042] As a bioactive ingredient derived from nature, quercetin has a variety of biological effects and has been widely used in the fields of medicine and health products. The market demand for its efficient and stable preparations continues to rise. The present invention breaks through the limitations of the traditional thin film dispersion method, solves the problems of high cost and low production efficiency, and focuses on safety and environmental friendliness during the production process, enhancing the application prospects of quercetin in the field of biomedicine and opening up a new path for the development of the industry. This preparation technology provides a more efficient solution for the manufacture of liposomes, shows great practical value in both scientific research and manufacturing, and helps to further explore and study the biological effects of quercetin.
[0043] The present invention does not need to use chloroform, an organic solvent with high toxicity and high cost, thereby reducing potential harm to operators and the environment during the production process. By using ethanol as a solvent and combining it with an aqueous medium, the preparation process is simplified, the complicated emulsification and high-speed rotation steps are avoided, and the complexity and time cost of the operation are reduced.
[0044] The present invention abandons the harmful organic solvents used in traditional processes and selects ethanol as a solvent, which not only makes the preparation process safer, more reliable and environmentally friendly, but also improves the preparation efficiency and effectively reduces the cost. The technology involves mixing phospholipids, cholesterol, phospholipid polyethylene glycol and quercetin in ethanol and dissolving them, mixing them with the aqueous phase at a specific flow rate ratio and volume ratio through microfluidic technology, and then using dialysis to obtain an aqueous phase solution of quercetin liposomes. The prepared liposomes have excellent encapsulation efficiency and are suitable for the pharmaceutical and health product industries. This technology simplifies the preparation steps, reduces costs and improves preparation efficiency, and enhances the market competitiveness of quercetin liposomes. The quercetin liposomes of the present application have a high encapsulation rate of more than 90%, a uniform particle size, and a particle size distribution index below 0.3. BRIEF DESCRIPTION OF THE DRAWINGS
[0045] Figure 1 The electron microscopy characterization results of the quercetin liposomes in Example 1 are shown.
[0046] Figure 2 This is the particle size characterization result of quercetin liposomes in Example 2.
[0047] Figure 3 The stability test results of quercetin liposomes in Example 3 are shown in FIG. DETAILED DESCRIPTION
[0048] The present invention is further described below in conjunction with embodiments, which are as follows:
[0049] In the following examples, particle size characterization: The particle size and particle size distribution index of quercetin liposomes were measured using a laser particle size analyzer.
[0050] Transmission electron microscopy was used to observe the size and shape of quercetin liposomes.
[0051] Stability test: The stability of quercetin liposomes was determined using a laser particle size analyzer, and the particle size was measured once a day for one week.
[0052] Example 1
[0053] EPC (0.18 mmol), cholesterol (0.09 mmol), DSPE-PEG (0.0027 mmol) and quercetin (0.018 mmol) were added to a centrifuge tube, and 1.64 mL of ethanol was added and dissolved by ultrasound to obtain an ethanol phase solution of the mixture, which was then transferred to channel 1 of the microfluidic system after being sucked up by a syringe; another syringe was used to suck up 16.36 mL of pure water and transferred to channel 2 of the microfluidic system; the microfluidic parameters were set as follows: the total flow rate was 2.75 mL / min n, the flow rate of channel 1 is 0.25mL / min, the flow rate of channel 2 is 2.5mL / min, and the front waste liquid is 100uL; after confirmation of the start, the quercetin liposomes in the mixed solution of ethanol and water are collected after 6.56 minutes; the obtained quercetin liposomes are transferred to a dialysis bag with a molecular weight cutoff of 3000Da, and dialyzed with 5% glucose aqueous solution for 3 hours to remove ethanol, and a quercetin liposome aqueous solution is obtained, with a particle size of 115 nanometers, a particle size distribution index of 0.18, and a quercetin encapsulation rate of 95% after measurement. The electron microscopy characterization results of the quercetin liposomes in this embodiment are shown in Figure 1 shown.
[0054] Example 2
[0055] SPC (0.18 mmol), cholesterol (0.09 mmol), DMG-PEG (0.009 mmol) and quercetin (0.018 mmol) were added to a centrifuge tube, and 1.64 mL of ethanol was added and ultrasonicated to dissolve them completely to obtain an ethanol phase solution of the mixture, which was then transferred to channel 1 of the microfluidic system after being aspirated by a syringe; another syringe was used to aspirate 16.36 mL of pure water and transferred to channel 2 of the microfluidic system; the microfluidic parameters were set as follows: the total flow rate was 2.75 mL / min, and the flow rate of channel 1 was 0.25 mL / min, the flow rate of channel 1 is 2.5mL / min, and the front waste liquid is 100uL; after confirming the start, quercetin liposomes in a mixed solution of ethanol and water are collected after 6.56 minutes; the obtained quercetin liposomes are transferred to a dialysis bag with a cut-off molecular weight of 3000Da, and dialyzed with a sodium chloride aqueous solution with a mass fraction of 0.9% for 3.5 hours to remove ethanol, to obtain a quercetin liposome aqueous solution, the encapsulation rate of quercetin is measured to be 92%, the particle size is 110 nanometers, the particle size distribution index is 0.22, and the encapsulation rate of quercetin is measured to be 93%.
[0056] The particle size characterization results of the quercetin liposomes in this example are as follows Figure 2 shown.
[0057] Example 3
[0058] DSPC (0.18 mmol), cholesterol (0.18 mmol), DSPE-PEG (0.009 mmol) and quercetin (0.036 mmol) were added to a centrifuge tube, and 1.64 mL of ethanol was added and then sonicated to dissolve them completely to obtain an ethanol phase solution of the mixture, which was then transferred to channel 1 of the microfluidic system after being aspirated by a syringe; another syringe was used to aspirate 16.36 mL of pure water and then transferred to channel 2 of the microfluidic system; the microfluidic parameters were set as follows: the total flow rate was 2.75 mL / m in, wherein the flow rate of channel 1 is 0.25mL / min, the flow rate of channel 2 is 2.5mL / min, and the front waste liquid is 200uL; after confirmation of the start, the quercetin liposomes in the mixed solution of ethanol and water are collected after 6.56 minutes; the obtained quercetin liposomes are transferred to a dialysis bag with a molecular weight cutoff of 3000Da, and the ethanol is removed by dialysis with a pure aqueous solution for 4 hours to obtain a quercetin liposome aqueous solution with a particle size of 120 nanometers and a particle size distribution index of 0.20. The encapsulation rate of quercetin is measured to be 90%. The stability test results of the quercetin liposomes in this embodiment are shown as follows Figure 3 shown.
[0059] Example 4
[0060] DPPC (0.18 mmol), cholesterol (0.09 mmol), DMG-PEG (0.009 mmol) and quercetin (0.036 mmol) were added to a centrifuge tube, and 9 mL of ethanol was added and dissolved by ultrasound to obtain an ethanol phase solution of the mixture, which was then transferred to channel 1 of the microfluidic control system after being sucked up by a syringe. Another syringe was used to suck up 27 mL of pure water and then transferred to channel 2 of the microfluidic control system. The microfluidic parameters were set as follows: the total flow rate was 12 mL / min, and the channel 1 flow rate is 3mL / min, channel 2 flow rate is 9mL / min, and the front waste liquid is 200uL; after confirmation of the start, quercetin liposomes in a mixed solution of ethanol and water are collected after 3 minutes; the obtained quercetin liposomes are transferred to a dialysis bag with a cut-off molecular weight of 10000Da, and dialyzed with a phosphate buffered saline solution with a pH value of 7.4 for 3 hours to remove ethanol, to obtain a quercetin liposome aqueous solution with a particle size of 117 nanometers, a particle size distribution index of 0.27, and a quercetin encapsulation rate of 90% after measurement.
[0061] Comparative Example 1
[0062] DPPC (0.18mmol), cholesterol (0.09mmol), DMG-PEG (0.009mmol) and quercetin (0.036mmol) were added to a centrifuge tube, 9mL of dichloromethane was added and then sonicated to dissolve them completely to obtain an ethanol phase solution of the mixture, which was then transferred to channel 1 of the microfluidic control after being aspirated through a syringe; another syringe was used to aspirate 27mL of pure water and transferred to channel 2 of the microfluidic control; the microfluidic parameters were set as follows: the total flow rate was 12mL / min, of which the flow rate of channel 1 was 3mL / min, the flow rate of channel 2 was 9mL / min, and the front waste liquid was 200uL; after confirming the start, an emulsion with upper and lower layers was obtained, indicating that quercetin liposomes could not be obtained.
[0063] Comparative Example 2
[0064] DPPC (0.18mmol), cholesterol (0.09mmol), DMG-PEG (0.009mmol) and quercetin (0.036mmol) were added to a centrifuge tube, 9mL of ethanol was added and then sonicated to dissolve them completely to obtain an ethanol phase solution of the mixture, which was then transferred to channel 1 of the microfluidic system after being aspirated through a syringe; another syringe was used to aspirate 27mL of methanol and transferred to channel 2 of the microfluidic system; the microfluidic parameters were set as follows: the total flow rate was 12mL / min, of which the flow rate of channel 1 was 3mL / min, the flow rate of channel 2 was 9mL / min, and the front waste liquid was 200uL; after confirming the start, a clear and transparent solution was obtained, indicating that quercetin liposomes could not be obtained.
[0065] Comparative Example 3
[0066] DSPC (0.18mmol), cholesterol (0.18mmol), DSPE-PEG (0.009mmol) and quercetin (0.036mmol) were added to a centrifuge tube, 9mL of ethanol was added and ultrasonicated to dissolve them completely to obtain an ethanol phase solution of the mixture, which was then transferred to channel 1 of the microfluidic control after being aspirated through a syringe; another syringe was used to aspirate 18mL of pure water and transferred to channel 2 of the microfluidic control; the microfluidic parameters were set as follows: the total flow rate was 2.2mL / min, of which the flow rate of channel 1 was 0.2mL / min, and the flow rate of channel 2 was 2mL / min, and 200uL of waste liquid was discarded before and after; after confirming the start, quercetin liposomes could not be obtained, indicating that quercetin liposomes could not be obtained when the volume ratio of ethanol to water was greater than 1:3.
[0067] Comparative Example 4
[0068] SPC (0.18mmol), cholesterol (0.09mmol), DMG-PEG (0.009mmol) and quercetin (0.018mmol) were added to a centrifuge tube, 1.64mL of ethanol was added and then ultrasonically dissolved to obtain an ethanol phase solution of the mixture, which was then transferred to channel 1 of the microfluidic control after being sucked through a syringe; another syringe was used to suck 16.36mL of pure water and then transferred to channel 2 of the microfluidic control; the microfluidic parameters were set as follows: the total flow rate was 1mL / min, the flow rate of channel 1 was 0.1mL / min, the flow rate of channel 2 was 0.9mL / min, and the front waste liquid was 100uL; after confirming the start, quercetin liposomes in a mixed solution of ethanol and water were collected after 16.4 minutes; the obtained quercetin liposomes were transferred to a dialysis bag with a molecular weight cutoff of 3000Da, and dialyzed with a sodium chloride aqueous solution with a mass fraction of 0.9% for 3.5 hours to remove ethanol to obtain a quercetin liposome aqueous solution. The particle size is 170 nanometers, the encapsulation efficiency of quercetin is measured to be 85%, and the particle size distribution index is 0.32.
[0069] Comparative Example 5
[0070] EPC (0.18mmol), cholesterol (0.36mmol), DSPE-PEG (0.0027mmol) and quercetin (0.018mmol) were added to a centrifuge tube, 1.64mL of ethanol was added and then ultrasonically dissolved to obtain an ethanol phase solution of the mixture, which was then transferred to channel 1 of the microfluidic control after being aspirated by a syringe; another syringe was used to aspirate 16.36mL of pure water and then transferred to channel 2 of the microfluidic control; the microfluidic parameters were set as follows: the total flow rate was 2.75mL / min, the flow rate of channel 1 was 0.25mL / min, the flow rate of channel 2 was 2.5mL / min, and the front waste liquid was 100uL; after confirming the start, the quercetin liposomes in the mixed solution of ethanol and water were collected after 6.56 minutes; the obtained quercetin liposomes were transferred to a dialysis bag with a molecular weight cutoff of 3000Da, and dialyzed with a 5% glucose aqueous solution for 3 hours to remove ethanol to obtain a quercetin liposome aqueous solution. The particle size is 300 nanometers, the particle size distribution index is 0.35, and the encapsulation rate of quercetin is measured to be 89%.
Claims
1. A method for preparing quercetin liposomes, the method comprising the following steps: (1) adding phospholipids, cholesterol, phospholipid polyethylene glycol and quercetin into ethanol and dissolving them to obtain an ethanol phase solution; (2) pumping the ethanol phase solution and pure water obtained in step (1) into the microfluidic chip from different channel inlets, respectively, and performing micromixing to obtain a crude quercetin liposome; (3) dialyzing the crude quercetin liposome obtained in step (2) using an aqueous medium to obtain.
2. The method according to claim 1, wherein: In step (1), the phospholipid is selected from one or more of soybean lecithin (SPC), egg yolk lecithin (EPC), hydrogenated lecithin (HSPC), distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoylphosphatidylcholine (DOPC), preferably SPC, EPC, DSPC, DPPC and DOPC.
3. The method according to claim 1 or 2, wherein: In step (1), the phospholipid polyethylene glycol is selected from one or both of DSPE-PEG and DMG-PEG; Preferably, in step (1), the number average molecular weight of the phospholipid polyethylene glycol is 500-5000; Preferably, in step (1), the phospholipid polyethylene glycol is selected from one or more phospholipid polyethylene glycols having a number average molecular weight of 500, 1000, 2000, and 5000.
4. The method according to any one of claims 1 to 3, wherein: In step (1), the molar ratio of the phospholipid, the cholesterol, the phospholipid polyethylene glycol and the quercetin is 1:0.2-1:0-0.05:0.1-0.3, preferably 1:0.5-1:0.015-0.05:0.1-0.
2.
5. The method according to any one of claims 1 to 4, wherein: In step (2), the molar concentration of phospholipids in the mixed solution formed by the ethanol phase solution and the pure water is 4-12 mmol / L, preferably 5-10 mmol / L.
6. The method according to any one of claims 1 to 5, wherein: In step (2), the volume ratio between the ethanol phase solution obtained in step (1) and the pure water is 1:3-12, preferably 1:3-10.
7. The method according to any one of claims 1 to 6, wherein: In step (2), the flow rate ratio between the ethanol phase solution obtained in step (1) and the pure water is 1:3-12, preferably 1:3-10.
8. The method according to any one of claims 1 to 7, wherein: In step (2), the control parameters of the channel are as follows: the total flow rate is 2-15 mL / min, preferably 2.75-12 mL / min.
9. The method according to any one of claims 1 to 8, wherein: In step (2), when obtaining the crude quercetin liposome product, the pre-waste liquid is removed, and the collected amount of the pre-waste liquid is not less than 100-300 μL, preferably 100-200 μL.
10. The method according to any one of claims 1 to 9, wherein: In step (3), the aqueous medium is selected from a pure water solution, a glucose aqueous solution with a mass concentration of 5-10%, a sodium chloride aqueous solution with a mass concentration of 0.9%, or a phosphate buffered saline solution with a pH value of 7.4; Preferably, in step (3), the dialysis is performed in a dialysis bag or a dialysis card; Preferably, in step (3), the molecular weight cutoff of the dialysis bag or the dialysis card is 1000-10000Da.