Preparation method of luteolin nano slow-release dosage form

Abstract

A preparation method of a luteolin nano slow-release dosage form belongs to the field of food and medicine, and the specific scheme comprises the following steps: step 1, dissolving luteolin and carnosic acid in a mixed solvent of dichloromethane and methanol to obtain a solution A; step 2, adding the solution A into a polyvinyl alcohol aqueous solution I, and performing ultrasonic treatment to form an emulsion B; step 3, dropwise adding the emulsion B into a polyvinyl alcohol aqueous solution II, and stirring at room temperature to obtain a suspension C; and 4, carrying out centrifugal separation on the suspension C, and washing the precipitate to obtain the luteolin nano stable slow-release dosage form. The luteolin nano stable slow-release dosage form prepared by the invention has the advantages of good water solubility, high loading rate and the like, and can effectively improve the water solubility and slow-release performance of natural micromolecular luteolin in practical application.

Description

Technical Field

[0001] This invention belongs to the field of food and medicine, and specifically relates to a method for preparing a luteolin nano-sustained-release formulation. Background Technology

[0002] Luteolin (Lut) is a flavonoid compound widely found in natural plants such as carrots, celery, onion leaves, broccoli, and chrysanthemums. It possesses a wide range of biological activities, including antioxidant, anti-inflammatory, anti-diabetic, and neuroprotective effects, making it valuable for applications in functional foods and disease prevention. However, the polyhydroxy structure of Lut also leads to poor water solubility and low bioavailability, severely limiting its practical application. Therefore, improving the water solubility of Lut and enhancing its sustained-release properties are effective ways to improve its bioavailability and promote its practical application.

[0003] In recent years, studies have employed chitosan encapsulation and protein carrier delivery systems to load Lut, which can improve Lut's solubility and stability to some extent. However, macromolecular delivery systems have low loading rates and delivery efficiencies, and their application carries potential immunogenicity. In contrast, small molecule delivery systems exhibit unique advantages due to their well-defined chemical structures and green preparation methods of self-assembly / co-assembly. Some natural small molecule compounds with polyhydroxy structures can combine with each other through non-covalent interactions such as intermolecular hydrogen bonds and hydrophobic interactions to form nanostructures with spherical or rod-like microstructures that are hydrophilic on the outside and hydrophobic on the inside. This system can be used to load hydrophobic small molecule compounds to improve their water solubility and stability, while also exhibiting good sustained-release properties. Currently, some triterpenoid small molecule compounds (such as ursolic acid and oleanolic acid) have been used in the development of self-assembly / co-assembly nanodelivery systems due to their amphiphilic structures. However, the self-assembly / co-assembly properties of small molecule compounds are highly specific. Even with small differences in molecular structure, their self-assembly / co-assembly abilities vary significantly. The number of triterpenoids that can effectively form nanodelivery systems is relatively limited. Only a few carriers (such as oleanolic acid and ursolic acid) can form nanoparticles, which can improve the water solubility, stability, and sustained-release properties of hydrophobic small molecules to some extent. Meanwhile, betulinic acid, salicylic acid, and glycyrrhetinic acid, which have similar molecular structures, can only form filamentous, network, and sheet-like structures, and are not suitable for loading hydrophobic small molecule compounds. Therefore, these small molecule delivery systems still require extensive development, and suitable natural delivery carriers need to be continuously screened and expanded.

[0004] Based on the assembly mechanism of small molecule delivery systems, diterpenoids theoretically possess the potential to serve as carriers for small molecule nanoparticles, but related research reports are limited. Carnosic acid (CA) is a natural diterpenoid bioactive component with antioxidant, anti-inflammatory, and neuroprotective effects. Developing CA-based nanoparticle delivery systems can not only address issues such as the water solubility and stability of active small molecules but may also produce synergistic effects, offering unique application advantages. Therefore, this study innovatively prepared a CA nanoparticle delivery system that can effectively load Lut and improve its water solubility and sustained-release properties, promoting the practical application of Lut in the food and pharmaceutical fields and expanding the application of diterpenoid small molecules in nanoparticle delivery systems. Summary of the Invention

[0005] To address the issues of poor water solubility of Lut and insufficient loading rate of existing delivery systems, this invention provides a method for preparing Lut nano-sustained-release formulations.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention develops a method for preparing a nano-sustained-release delivery system (CA-Lut NPs) co-assembled with natural small molecule Lut and CA. This technique utilizes the hydrogen bonding and hydrophobic interactions between CA and Lut molecules to form nanoparticles through non-covalent co-assembly. The prepared CA-Lut NPs exhibit good water solubility and stability, effectively solving the problems of poor water solubility and easy degradation of Lut, and providing an effective delivery solution for the application of Lut in the food and pharmaceutical fields. The specific method is as follows:

[0008] A method for preparing a Lut nano-sustained-release formulation includes the following steps:

[0009] Step 1: Dissolve Lut and CA together in a mixed solvent of dichloromethane and methanol to obtain solution A, wherein the mass ratio of Lut to CA is 0.2:1-1:1, the mass-volume ratio of Lut to the mixed solvent of dichloromethane and methanol is 1-5 mg / mL, and the volume ratio of dichloromethane to methanol is 4:1-9:1.

[0010] Step 2: Add solution A to polyvinyl alcohol aqueous solution I, stir evenly, and use an ultrasonic cell disruptor to sonicate to form emulsion B. The concentration of polyvinyl alcohol aqueous solution I is 0.02-0.03 g / mL, and the volume ratio of solution A to polyvinyl alcohol aqueous solution I is 1:3-1:4.

[0011] Step 3: Add emulsion B dropwise to polyvinyl alcohol aqueous solution II and stir at room temperature to obtain suspension C. The concentration of polyvinyl alcohol aqueous solution II is 0.001-0.003 g / mL, and the volume ratio of emulsion B to polyvinyl alcohol aqueous solution II is 1:5-1:10.

[0012] Step 4: Centrifuge the suspension C, wash the precipitate, and obtain the Lut nano-sustained-release formulation.

[0013] Furthermore, in step two, the power of the ultrasonic cell disruptor is 50-100 W. During the ultrasonic process, it is turned on for 5 seconds and off for 5 seconds, alternating in this manner, for a total processing time of 1-2 minutes.

[0014] Furthermore, in step two, solution A is thoroughly mixed with polyvinyl alcohol aqueous solution I by vortexing for 1-2 minutes.

[0015] Furthermore, in step three, emulsion B is mixed evenly with polyvinyl alcohol aqueous solution II by stirring at 400 rpm for 12-16 h.

[0016] Furthermore, in step four, the centrifugation speed is 10000-12000 rpm, the time is 15-20 min, and the temperature is 4℃.

[0017] Furthermore, in step four, the precipitate is washed several times with distilled water.

[0018] Furthermore, 0.6-0.9 g of polyvinyl alcohol was dissolved in 30 mL of ultrapure water, and after heating and stirring, a polyvinyl alcohol aqueous solution I with a concentration of 0.02-0.03 g / mL was obtained; 0.3-0.9 g of polyvinyl alcohol was dissolved in 300 mL of ultrapure water, and after heating and stirring, a polyvinyl alcohol aqueous solution II with a concentration of 0.001-0.003 g / mL was obtained.

[0019] Currently, no studies have reported constructing Lut nanodelivery systems using small molecule co-assembly strategies to enhance their water solubility and sustained-release properties. Both CA and Lut possess multi-hydroxyl structures, providing a molecular structural basis for intermolecular hydrogen bonding and hydrophobic interactions, potentially improving Lut's water solubility and sustained-release performance. Therefore, this invention innovatively employs CA as a nanocarrier for co-assembly with Lut, aiming to improve Lut's water solubility and sustained-release properties, providing an efficient delivery system for its application in the food industry.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] 1. The Lut nano-release formulation CA-Lut NPs prepared by this invention mainly rely on non-covalent interactions such as hydrogen bonding and hydrophobic interactions between small molecules, and do not involve chemical bond modification.

[0022] 2. The Lut nano-release formulation CA-Lut NPs prepared by this invention has a particle size of less than 390 nm and good water solubility.

[0023] 3. The Lut nano-sustained-release formulation CA-Lut NPs prepared by this invention has the advantage of high loading rate compared with general drug delivery systems such as polysaccharides and proteins. Attached Figure Description

[0024] Figure 1 The graph shows the molecular simulation results of CA-Lut NPs;

[0025] Figure 2 The Fourier transform infrared (FT-IR) spectra of CA-Lut NPs are shown.

[0026] Figure 3 A schematic diagram showing the contact angles of CA, Lut, and CA-Lut NPs with aqueous solution;

[0027] Figure 4 To assess the sustained-release effect of Lut and CA-Lut NPs in simulated gastrointestinal digestion. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of the invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0029] Example 1:

[0030] A method for preparing a Lut nano-sustained-release formulation includes the following steps:

[0031] Step 1: Dissolve 5 mg CA and 5 mg Lut together in a mixed solution of 1.0 mL dichloromethane and methanol (V / V=8 / 2), and vortex to fully dissolve them to obtain solution A;

[0032] Step 2: Add 1 mL of solution A to 3 mL of 0.025 g / mL polyvinyl alcohol aqueous solution I, vortex for 1 min, and then sonicate for 1 min using an ultrasonic cell disruptor (5 s on, 5 s off, alternating, power 60 W) to form emulsion B;

[0033] Step 3: Add emulsion B dropwise to 30 mL of 0.003 g / mL polyvinyl alcohol aqueous solution II, and stir at 400 rpm for 12 h at room temperature to obtain suspension C;

[0034] Step 4: Centrifuge suspension C at 12000 rpm and 4℃ for 15 min. Wash the precipitate three times with distilled water to obtain Lut nano-release formulation CA-Lut NPs. The washed CA-Lut NPs can be suspended in a small amount of deionized water, freeze-dried and stored for subsequent detection.

[0035] 1.1 Particle size, PDI, encapsulation efficiency, and loading rate

[0036] 1.1.1 Detection of Z-average Size and PDI of Nanoparticles:

[0037] Take an appropriate amount of CA-Lut NPs nanoparticle suspension, dilute it with deionized water, vortex mix for 1 min, and then sonicate it for 15 min using an ultrasonic cleaner to ensure thorough dispersion of the nanoparticles. Place the filtered nanoparticle solution in a test dish and use a ZetasizerNano particle size analyzer to determine its Z-average size and PDI.

[0038] As shown in Table 1, the Z-average size of CA-Lut NPs was 375.83 ± 8.15 nm, and the PDI (particulate density index) was 0.31 ± 0.03. The low PDI value confirms the uniformity of CA-Lut NPs, indicating successful particle preparation and a uniform particle size distribution. During in vivo delivery, the small particle size of nanoparticles facilitates their crossing of various biological barriers, such as promoting intestinal mucosal penetration. Simultaneously, they can enter cells through multiple mechanisms of action, overcoming intracellular drug delivery obstacles. The small particle size of nanoparticles effectively enhances their bioavailability by improving membrane permeability, promoting tissue distribution, and prolonging in vivo residence.

[0039] Table 1. Basic characterization results of CA-Lut NPs

[0040]

[0041] 1.1.2 Encapsulation efficiency and loading rate of nanoparticles:

[0042] Weigh 1 mg of lyophilized CA-Lut NPs nanoparticle powder and add 1 mL of dimethyl sulfoxide to completely dissolve it, thereby disrupting the nanoparticle structure. The solution was then filtered through a 0.2 μm microporous membrane to remove any remaining particles. A portion of the filtrate was analyzed using a UV spectrophotometer to determine the Lut content in the nanoparticles. Simultaneously, the supernatant obtained from centrifugation in step four of Example 1 was analyzed under the same conditions to determine the free Lut content. Finally, the loading rate and encapsulation efficiency of the nanoparticles were determined using a calculation formula.

[0043] Encapsulation efficiency (EE) measurement:

[0044] Encapsulation ratio = (Initial weight of Lut - Weight of unencapsulated Lut) / Initial weight of Lut × 100%.

[0045] Load factor (LE) determination:

[0046] Loading rate = weight of Lut in nanoparticles / weight of nanoparticles × 100%.

[0047] As shown in Tables 1 and 2, CA-Lut NPs exhibit a high loading rate of 32.45 ± 0.34%. In contrast, previous studies have shown that the loading rate of Lut encapsulated with glycyrrhizic acid-modified bovine serum albumin (BSA) is only 1.09%, the loading rate of Lut encapsulated with zein and carboxymethyl chitosan is only 5.66%, and the loading rate of Lut encapsulated with chitosan is less than 20%. Compared to other delivery systems, CA-Lut NPs have a higher loading rate, which significantly improves drug delivery efficiency and ensures higher effective drug concentrations while reducing the amount of carrier material used.

[0048] Table 2 Current Status of Research on Lut Delivery Systems

[0049]

[0050] 1.2 Molecular Simulation of Nanoparticles

[0051] All-atom molecular dynamics simulations were performed using Materials Studio (MS) 2019 to calculate intermolecular forces. First, a box was created using the Amorphous Cell module, and CA and Lut molecules were added to the box. A suitable amount of TIP3P water molecules was also added to simulate an aqueous solution environment. A density of 1 g / cm³ was selected. 3 The final dimensions of the box are 34×34×34 Å. Then, the Forcite module is used to perform geometry optimization and annealing on the box. Finally, the Forcite module is used to perform Dynamics calculations on the optimized box, with a Berendsen temperature controller selected, the temperature set to 298K, the calculation time set to 1000 ps, ​​the NVT ensemble selected, and the time step set to 1.0 fs. The entire simulation process is carried out under the COMPASS force field.

[0052] like Figure 1Molecular dynamics simulations show that the initial conformations of CA and Lut exhibit a large distance between their hydrophilic ends, failing to form hydrogen bonds. After 1000 ps of simulation, the distance between their hydrophilic ends shortens, and intermolecular hydrogen bonds of 2.604 Å, 2.986 Å, and 3.373 Å are formed. Simultaneously, the proximity of the hydrophobic ends effectively reduces steric hindrance, promoting the formation of hydrophobic interactions. These results suggest that the molecular assembly of CA and Lut may be primarily driven by both hydrogen bonds and hydrophobic interactions.

[0053] 1.3 Fourier Transform Infrared Spectroscopy (FT-IR) Analysis

[0054] CA, Lut powder, and CA-Lut NPs lyophilized powder were respectively mixed with potassium bromide powder in a mortar at a mass ratio of 1:100. The powders were then carefully and slowly ground in the same direction with a pestle until they were uniform. The powders were then placed in a tableting device and pressed for about 3 minutes to obtain transparent, uniform tablets without cracks. The tablets were then placed in a Fourier transform infrared spectrometer (Perkin-Elmer, USA) for detection, and the results were analyzed using the infrared analysis software OMNIC8.0.

[0055] like Figure 2 As shown, in the FT-IR spectrum, CA is at 3533 cm⁻¹. -1 The nearby absorption peak corresponds to the stretching vibration of the OH group, at 1646 cm⁻¹. -1 Corresponding to the stretching vibration of the C=O group, 800-1600 cm⁻¹ -1 Multiple absorption peaks within the range are characteristic absorption peaks of the phenolic hydroxyl group in CA. In the infrared spectrum of Lut, 3423 cm⁻¹... -1 The absorption peak at 800-1600 cm⁻¹ corresponds to the stretching vibration of the OH group. -1 Multiple absorption peaks within the range are characteristic absorption peaks of the phenolic hydroxyl groups of Lut. No new characteristic absorption peaks appeared after CA and Lut assembled to form CA-Lut NPs, indicating that no new covalent bonds were formed between CA and Lut molecules. CA-Lut NPs are more likely formed through non-covalent interactions such as intermolecular hydrogen bonding or hydrophobic interactions. The infrared spectrum of CA-Lut NPs shows that after the formation of CA-Lut NPs, Lut exhibits a peak at 3423 cm⁻¹. -1 The absorption peak of CA at 1646 cm⁻¹ shifts, indicating that CA and Lut may form intermolecular hydrogen bonds through the OH group. Furthermore, CA at 1646 cm⁻¹... -1 The absorption peak of C=O stretching vibration at 1260 cm⁻¹-1 The CO stretching vibration absorption peak at 2956 cm⁻¹ and the absorption peak at 2956 cm⁻¹ -1 The CH stretching vibration absorption peaks at the locations all shifted, indicating that hydrophobic interactions may be involved in the formation of CA-Lut NPs.

[0056] 1.4 Contact Angle Measurement

[0057] CA, Lut, and CA-Lut NPs powders were evenly coated on glass slides measuring 20×20×2mm, and the contact angles were measured using a contact angle measuring instrument. The size of the water droplets was kept consistent throughout the test.

[0058] like Figure 3 As shown, CA-Lut NPs exhibit a smaller triphase contact angle than CA and Lut alone, indicating a significantly enhanced hydrophilicity. This result confirms that constructing a CA-Lut NPs nanodelivery system can significantly improve the hydrophilicity of Lut, leading to a significant increase in its solubility. In the aqueous environment of the gastrointestinal tract after oral administration, Lut can be released from the delivery system and dissolved in digestive fluids at a higher concentration. With the increase in solubility, the probability and area of ​​contact between drug molecules and intestinal epithelial cells also increase significantly. These soluble drug molecules can diffuse more effectively and penetrate the aqueous layer on the surface of the intestinal mucosa, directly contacting the cell membrane, thereby being efficiently taken up through mechanisms such as passive diffusion or active transport, further improving the efficiency of drug uptake by cells.

[0059] Example 2

[0060] A method for preparing a Lut nano-sustained-release formulation includes the following steps:

[0061] Step 1: Dissolve 4 mg Lut and 5 mg CA together in a mixed solution of 1.0 mL dichloromethane and methanol (V / V=8 / 2), and vortex to fully dissolve them to obtain solution A;

[0062] Step 2: Add 1 mL of solution A to 3 mL of 0.03 g / mL polyvinyl alcohol aqueous solution I, vortex for 2 min, and then sonicate for 1 min using an ultrasonic cell disruptor (5 s on, 5 s off, alternating, power 100 W) to form emulsion B;

[0063] Step 3: Add emulsion B dropwise to 30 mL of 0.001 g / mL polyvinyl alcohol aqueous solution II, and stir at 400 rpm for 16 h at room temperature to obtain suspension C;

[0064] Step 4: Centrifuge suspension C at 12000 rpm and 4℃ for 15 min. Wash the precipitate three times with distilled water to obtain Lut nano-release formulation CA-Lut NPs. The washed CA-Lut NPs can be suspended in a small amount of deionized water, freeze-dried and stored.

[0065] Under the conditions described in this embodiment, CA-Lut NPs were successfully prepared, exhibiting uniform and stable dispersion in aqueous solution and good water solubility. As shown in Table 3, the CA-Lut NPs prepared under these conditions had a particle size of 383.43±9.04 nm, a PDI of 0.27±0.03, and a loading rate of 37.40±0.34%. The low PDI value confirms the uniformity of the CA-Lut NPs, indicating successful nanoparticle preparation and uniform particle size distribution. The high loading rate demonstrates that this nanodelivery system can effectively load Lut, exhibiting stronger delivery efficiency. During in vivo delivery, the smaller particle size of the nanoparticles facilitates their crossing of various biological barriers and allows them to enter cells through multiple mechanisms of action, thus improving bioavailability.

[0066] Table 3. Basic characterization results of CA-Lut NPs

[0067]

[0068] The sustained-release effect of CA-Lut NPs on Lut during simulated gastrointestinal digestion.

[0069] First, prepare the simulated gastrointestinal fluid and release medium solution according to the following steps: Dissolve 100 mg NaCl and 160 mg pepsin in 50 mL of deionized water, and adjust the pH to 4.0 with 0.1 M HCl to obtain simulated gastric fluid for later use; Dissolve 704 mg NaCl, 544 mg KH2PO4, 160 mg pancreatin, and 400 mg bile salts in 80 mL of deionized water, and adjust the pH to 7.4 with 0.1 M NaOH to obtain simulated intestinal fluid for later use; Mix the simulated gastric / intestinal fluid with ethanol at a 1:1 (V / V) ratio to obtain the simulated gastric / intestinal fluid release medium solution for later use.

[0070] Lut content was detected during simulated gastrointestinal digestion: 3 mL of CA-Lut NPs suspension was mixed with 3 mL of simulated gastric juice and placed in a dialysis bag (3500 Da cutoff). The dialysis bag was then immersed in 60 mL of gastric juice release medium for 2 h. Next, 6 mL of simulated intestinal juice was added to the dialysis bag, and the bag was transferred to 120 mL of simulated intestinal juice release medium for 4 h. The entire experiment was incubated in a shaking incubator at 37 ℃ and a shaking speed of 120 rpm. Every 30 min, 1 mL of liquid was taken from the simulated gastric / intestinal juice release medium (replacing 1 mL of fresh release medium), and the Lut content was detected using ultraviolet spectrophotometry to calculate the Lut release rate.

[0071] like Figure 4 As shown, after 6 hours of simulated gastrointestinal digestion, free Lut was released at a rate of 79.07 ± 0.57% throughout the simulated gastrointestinal digestion process. In contrast, CA-Lut NPs released only 46.89 ± 1.34% of Lut, lower than the amount of free Lut, indicating that CA-Lut NPs exhibit good sustained-release performance in gastrointestinal digestion. This result proves that the Lut nano-sustained-release formulation CA-Lut NPs prepared in this invention can be slowly released in the gastrointestinal digestive environment, achieving a sustained-release effect and protecting Lut.

[0072] Example 3

[0073] A method for preparing a Lut nano-sustained-release formulation includes the following steps:

[0074] Step 1: Dissolve 1 mg Lut and 5 mg CA together in a mixed solution of 1.0 mL of dichloromethane and methanol (V / V=9 / 1), and vortex to fully dissolve them to obtain solution A;

[0075] Step 2: Add 1 mL of solution A to 4 mL of 0.02 g / mL polyvinyl alcohol aqueous solution I, vortex for 2 min, and then sonicate for 2 min using an ultrasonic cell disruptor (5 s on, 5 s off, alternating, power 50 W) to form emulsion B;

[0076] Step 3: Add emulsion B dropwise to 20 mL of 0.003 g / mL polyvinyl alcohol aqueous solution II, and stir at 400 rpm for 16 h at room temperature to obtain suspension C;

[0077] Step 4: Centrifuge suspension C at 10,000 rpm and 4°C for 20 min. Wash the precipitate three times with distilled water to obtain Lut nano-release formulation CA-Lut NPs. The washed CA-Lut NPs can be suspended in a small amount of deionized water.

[0078] Under the conditions of this embodiment, the mass ratio of Lut to CA in the raw materials for preparing nanoparticles is 0.2:1, and the emulsification and centrifugation conditions are slightly modified. The CA-Lut NPs prepared under these conditions can be stably suspended in deionized water, improving the water solubility of Lut and CA. CA-Lut NPs can be successfully prepared under these conditions.

[0079] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a luteolin nano-sustained-release formulation, characterized in that, Includes the following steps: Step 1: Dissolve luteolin and sarsaparilla acid together in a mixed solvent of dichloromethane and methanol to obtain solution A, wherein the mass ratio of luteolin to sarsaparilla acid is 0.2:1-1:1, the mass-to-volume ratio of luteolin to the mixed solvent of dichloromethane and methanol is 1-5 mg / mL, and the volume ratio of dichloromethane to methanol is 4:1-9:1; Step 2: Add solution A to polyvinyl alcohol aqueous solution I, stir evenly, and use an ultrasonic cell disruptor to sonicate to form emulsion B. The concentration of polyvinyl alcohol aqueous solution I is 0.02-0.03 g / mL, and the volume ratio of solution A to polyvinyl alcohol aqueous solution I is 1:3-1:

4. Step 3: Add emulsion B dropwise to polyvinyl alcohol aqueous solution II and stir at room temperature to obtain suspension C. The concentration of polyvinyl alcohol aqueous solution II is 0.001-0.003 g / mL, and the volume ratio of emulsion B to polyvinyl alcohol aqueous solution II is 1:5-1:

10. Step 4: Centrifuge the suspension C, wash the precipitate, and obtain the luteolin nano-sustained-release formulation.

2. The preparation method according to claim 1, characterized in that: In step two, the ultrasonic cell disruptor has a power of 50-100 W. During the ultrasonic process, it is turned on for 5 seconds and off for 5 seconds, alternating between the two, for a total of 1-2 minutes.

3. The preparation method according to claim 1, characterized in that: In step two, solution A is thoroughly mixed with polyvinyl alcohol aqueous solution I by vortexing for 1-2 minutes.

4. The preparation method according to claim 1, characterized in that: In step three, the emulsion B is stirred at 400 rpm for 12-16 hours to mix the polyvinyl alcohol aqueous solution II evenly.

5. The preparation method according to claim 1, characterized in that: In step four, the centrifugation speed is 10000-12000 rpm, the time is 15-20 min, and the temperature is 4 ℃.

6. The preparation method according to claim 1, characterized in that: In step four, the precipitate is washed several times with distilled water.

7. The preparation method according to claim 1, characterized in that: Dissolve 0.6-0.9 g of polyvinyl alcohol in 30 mL of ultrapure water, heat and stir to obtain a polyvinyl alcohol aqueous solution I with a concentration of 0.02-0.03 g / mL; dissolve 0.3-0.9 g of polyvinyl alcohol in 300 mL of ultrapure water, heat and stir to obtain a polyvinyl alcohol aqueous solution II with a concentration of 0.001-0.003 g / mL.

Images