CN111848991A

CN111848991A - Preparation method of crosslinked hyaluronic acid microspheres

Info

Publication number: CN111848991A
Application number: CN202010704796.4A
Authority: CN
Inventors: 苏江伟; 吴万福; 潘存才; 张燕; 刘建建; 郭学平
Original assignee: Bloomage Biotech Co Ltd
Current assignee: Bloomage Biotech Co Ltd
Priority date: 2020-07-21
Filing date: 2020-07-21
Publication date: 2020-10-30
Anticipated expiration: 2040-07-21
Also published as: CN111848991B

Abstract

The invention discloses a preparation method of cross-linked hyaluronic acid microspheres, which comprises the following steps of uniformly mixing and dispersing hyaluronic acid or salt thereof, a cross-linking agent, alkali and water at a low temperature to obtain a water phase; adding the water phase into the oil phase containing the emulsifier, and fully emulsifying to obtain an emulsion; and (3) crosslinking the emulsion at a crosslinking temperature, removing the oil phase after crosslinking, and performing post-treatment to obtain the crosslinked hyaluronic acid microspheres. The invention prepares the crosslinked hyaluronic acid microspheres with uniform particles and strong mechanical property by using a small amount of cross-linking agent through a low-temperature control reverse-phase emulsification cross-linking technology, has controllable particle size and certain expansibility, and can be used in the fields of drug-loaded microspheres and cosmetic filling.

Description

Preparation method of crosslinked hyaluronic acid microspheres

Technical Field

The invention relates to a preparation method of cross-linked hyaluronic acid microspheres, belonging to the technical field of biomedical high polymer materials.

Background

Hyaluronic acid is an acidic mucopolysaccharide composed of D-glucuronic acid and N-acetylglucosamine, has good biocompatibility, has been applied to the fields of medicine and cosmetics, and is a hot point of research in the future. Because the natural sodium hyaluronate hydrogel has the defects of poor stability, sensitivity to hyaluronidase and free radicals, short in-vivo retention time, poor mechanical strength and the like, the natural sodium hyaluronate hydrogel is usually subjected to chemical modification and crosslinking, so that a series of novel sodium hyaluronate gels with bioactivity and functionality are obtained, the mechanical strength, stability and degradation resistance of the gels are enhanced, the gels can be maintained for a longer time in vivo, and the application of hyaluronic acid in the fields of biomedicine and tissue engineering is expanded. In addition to the applications in the fields of traditional ophthalmic surgery and joint diseases, postoperative adhesion prevention and soft tissue filling, sodium hyaluronate gel is gradually applied to the fields of medicine and protein carrier systems, cell culture scaffolds, postoperative wound healing and the like in recent years due to the unique viscoelasticity of the sodium hyaluronate gel.

The cross-linked hyaluronic acid gel has been studied at home and abroad for many years, and the main preparation method is that hyaluronic acid reacts with a cross-linking agent under an alkaline condition to form gel, the obtained block gel is generally obtained by mechanically crushing or pressing through a screen to obtain different particle size specifications, the cross-linked hyaluronic acid gel prepared by the method has the problems of nonuniform cross-linking degree, irregular particle shape, nonuniform particle size and the like, and is easy to cause red swelling and other side reactions due to irregular surface when being used for facial filling, and the development of the cross-linked hyaluronic acid gel in the aspect of drug carriers is also limited. The problem is solved by the crosslinked hyaluronic acid microspheres, the crosslinked hyaluronic acid microspheres are round and smooth in shape, the surfaces of the crosslinked hyaluronic acid microspheres are smooth and have no edges and corners, and the occurrence of side reactions of facial filling is greatly reduced.

CN10318902A discloses a preparation method of a composite cross-linked sodium hyaluronate gel microsphere for facial injection, the method comprises the steps of crushing, washing and filtering sodium hyaluronate gel subjected to secondary cross-linking to obtain the cross-linked sodium hyaluronate microsphere, the operation difficulty is high, and the microsphere preparation is difficult to realize. CN 103848995 a discloses a method for preparing hyaluronic acid nano-microspheres, which requires first preparing two kinds of functionalized hyaluronic acid, i.e. thymine functionalized hyaluronic acid and adenine functionalized hyaluronic acid, and then performing cross-linking to prepare microspheres, the operation is complicated, and the preparation process involves various organic solvents, so that the industrial production is difficult. CN 103333351 a and CN 104387600 a disclose a process for preparing crosslinked sodium hyaluronate microspheres capable of being used as an embolic agent from sodium hyaluronate and a method for preparing composite crosslinked sodium hyaluronate gel microspheres for facial injection, both of which are similar in that hyaluronic acid gel is added into an emulsion for emulsification and then a crosslinking agent is added into the emulsion for crosslinking, and this method needs a large amount of crosslinking agent, and the crosslinking agent is difficult to permeate into the gel, resulting in low crosslinking efficiency, nonuniform crosslinking, low mechanical properties of microspheres, and easy breakage.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the preparation method of the crosslinked hyaluronic acid microspheres, the crosslinking agent is added before emulsification, the crosslinking of hyaluronic acid before emulsification is inhibited by reducing the temperature, the preparation method has the characteristics of simple preparation steps, mild reaction conditions, small dosage of the crosslinking agent, good crosslinking uniformity and the like, and the prepared crosslinked hyaluronic acid microspheres have higher mechanical properties.

The specific technical scheme of the invention is as follows:

a method for preparing crosslinked hyaluronic acid microspheres, comprising the steps of:

(1) mixing hyaluronic acid or salt thereof, a cross-linking agent, alkali and water at low temperature, and uniformly dispersing to obtain a water phase;

(2) adding the water phase into an oil phase containing an emulsifier, and fully emulsifying to obtain an emulsion;

(3) and (3) crosslinking the emulsion at a crosslinking temperature, removing the oil phase after crosslinking, and performing post-treatment to obtain the crosslinked hyaluronic acid microspheres.

Further, in step (1), the preparation of the aqueous phase is carried out at a low temperature, which reduces the reaction rate to inhibit or reduce the cross-linking of the hyaluronic acid, and the temperature is preferably controlled so that the aqueous phase does not form large and hard gel lumps. Generally, when the temperature is controlled to be 2-8 ℃, the effect is better.

Further, in the preparation of the aqueous phase, the mixing order of the components is arbitrary, and the components may be added in the form of a pure product or in the form of an aqueous solution. Preferably, the hyaluronic acid or the salt thereof and the cross-linking agent are uniformly mixed in water, and then the alkali is added, so that the uniform contact between the cross-linking agent and the hyaluronic acid or the salt thereof is facilitated, the uniformity and the cross-linking efficiency of cross-linking are improved, and the probability of cross-linking of a water phase at low temperature can be reduced.

Further, before the aqueous phase is added into the oil phase, the aqueous phase is uniformly dispersed by means of shear dispersion, the shear speed is generally 1000-. The gel can be broken up by shearing dispersion, the dispersion pressure in the subsequent emulsification process is reduced, and the microspheres with smaller particle sizes can be obtained by matching with the subsequent emulsification process and carrying out high-speed shearing homogenization twice.

Further, the crosslinking agent and the base are various crosslinking agents and bases suitable for hyaluronic acid reported in the field of hyaluronic acid crosslinking. For example, the crosslinking agent may be one or more of carbodiimide, divinyl sulfone, ethylene glycol diglycidyl ether, 1, 4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, epichlorohydrin, and the like. The base may be one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, and the like.

Furthermore, the cross-linking agent is added before emulsification, can be well contacted and mixed with hyaluronic acid or salt thereof, and compared with the cross-linking agent added after emulsification, the cross-linking agent is less in dosage, and the content of the cross-linking agent in a water phase is 1-100 mg/mL.

Further, the amount of the base of the present invention may be selected according to the report of the prior art, for example, the content of the base in the aqueous phase is 0.5 to 5 wt%.

Further, the concentration of hyaluronic acid or a salt thereof in the aqueous phase may be adjusted according to the reports of the prior art, and the concentration of hyaluronic acid or a salt thereof in the aqueous phase is generally 5 to 25 wt%.

Further, the hyaluronic acid salt refers to medically acceptable salts of hyaluronic acid, such as sodium salt, potassium salt, zinc salt, calcium salt, and the like. The molecular weight of hyaluronic acid or a salt thereof is not particularly required, and can be selected according to the field of use.

Furthermore, the invention adopts a reverse phase emulsification method, the water phase is added into the oil phase for emulsification, the oil phase consists of an emulsifier and an oil phase matrix, the emulsifier can be selected from the available emulsifiers disclosed in the prior art, such as the emulsifier with the hydrophilic-hydrophobic balance value of 3-9, and the oil phase matrix can be selected from the oil phase matrix capable of coating the water phase to form small droplets, such as vegetable oil, mineral oil, silicone oil, liquid paraffin, dodecane, n-octane, cyclohexane and the like.

Further, the emulsifier may be sorbitan monooleate (Span 80), sorbitan monostearate (Span 60), or the like.

Further, in the oil phase, the content of the emulsifier is 2 to 10 wt%. The volume ratio of the water phase to the oil phase is 1: 5-20.

Further, after the aqueous phase and the oil phase are mixed, they are sufficiently emulsified by high-speed shearing, preferably at a shearing speed of 500-. After the emulsion is uniform, the emulsion generally needs to be kept still for a period of time to remove bubbles.

Further, after emulsification is finished, heating to a crosslinking temperature for crosslinking reaction, wherein the crosslinking temperature is 30-50 ℃, and the crosslinking time is 2-24 h.

Further, after the crosslinking is completed, microspheres are formed, and the microspheres are obtained through further post-treatment. The work-up can be carried out in some manner reported in the prior art, for example by centrifugation, washing, drying, etc. In one embodiment of the present invention, the following post-processing method is employed: after crosslinking is finished, removing the oil phase by centrifugation and the like, then washing the microspheres with an organic solvent to remove the oil phase on the surfaces of the microspheres, then washing the microspheres with water to remove the organic solvent on the surfaces of the microspheres, alkali liquor in the microspheres, unreacted BDDE and the like, finally dehydrating and precipitating the microspheres with ethanol, and drying in vacuum to obtain the dried microspheres. The organic solvent may be ethyl acetate, ethanol, acetone, etc.

The invention prepares the crosslinked hyaluronic acid microspheres by low-temperature control inverse emulsification crosslinking technology. The obtained microsphere has certain expansibility, strong mechanical property and controllable particle size and mechanical strength, and can be used in the fields of drug-loaded microspheres and cosmetic filling.

Furthermore, the size of the microsphere obtained by the invention is 20-620 μm, and the adjustable range is large. The size of the particle diameter of the microsphere and the mechanical strength can be adjusted by controlling the content of hyaluronic acid or salt thereof, the dosage of the cross-linking agent, the shearing speed during emulsification, the proportion of the water phase and the oil phase and other conditions.

The invention has the following advantages:

1. according to the invention, through low-temperature control of the water phase, the cross-linking agent is added before emulsification, the added cross-linking agent amount is far smaller than the amount of the cross-linking agent added in the emulsification process, the cost is reduced, the pressure of later-stage purification is reduced, and the preparation method is suitable for preparation of drug-loaded microspheres and preparation of cosmetic filler products.

2. According to the invention, the cross-linking agent is added before emulsification, so that the cross-linking uniformity is better and the cross-linking efficiency is higher.

3. According to the invention, the adding sequence of the materials in the water phase is optimized, the cross-linking agent and the alkali are added separately, the cross-linking of the cross-linking agent before emulsification is further reduced, the water phase is subjected to high-speed shearing homogenization before emulsification, the gel in the water phase is fully dispersed, the dispersing pressure in the subsequent emulsification process is reduced, and the microspheres with smaller particle sizes can be obtained.

4. The microsphere obtained by the invention has controllable particle size and mechanical strength and strong enzymolysis resistance, is beneficial to maintaining in vivo for a longer time, and can be used in the fields of drug-loaded microspheres and cosmetic filling.

Detailed Description

The present invention will be further described with reference to specific examples for better illustrating the objects, technical solutions and advantages of the present invention. It should be noted that the process parameters, raw materials and the like which are not described in detail in the present invention are performed according to the conventional technical means in the art.

In the examples described below, sodium hyaluronate was used from Huaxi Biotech Ltd.

Example 1

In this example, the influence of the content of sodium hyaluronate in the aqueous phase on the crosslinked sodium hyaluronate microspheres is mainly studied, and the preparation method is as follows:

1. maintaining the low temperature environment at 2-8 ℃, adding 0.1g of BDDE into 16mL of water, uniformly mixing, then adding sodium hyaluronate (molecular weight 2000 KDa) with different masses, uniformly mixing to obtain gel 1, adding 20 mL of 1 wt% NaOH solution into the gel 1, scattering the gel by using a high-shear dispersion emulsifying homogenizer at the rotation speed of 5000 rpm for 10min, and obtaining gel 2, namely a water phase.

2. Adding gel 2 into 400mL of n-octane containing 2wt% Span80, emulsifying at 10000 rpm for 10min by using a high-shear dispersion emulsifying homogenizer, and standing to remove bubbles after uniform emulsification.

3. And controlling the temperature of the emulsified liquid after emulsification to be 30 ℃, and stirring for 12 hours for crosslinking.

4. After the crosslinking was completed, the oil phase was removed by centrifugation, and washed twice with ethanol and distilled water in this order.

5. Precipitating the microspheres with ethanol, and vacuum drying to obtain dried microspheres.

The amount of sodium hyaluronate used is shown in table 1 below.

Example 2

In this example, the influence of the amount of the cross-linking agent in the aqueous phase on the cross-linked sodium hyaluronate microspheres is mainly studied, and the preparation method is as follows:

1. maintaining the low temperature environment at 2-8 ℃, adding BDDE with different masses into 16mL water, mixing uniformly, then adding 4 g sodium hyaluronate (molecular weight 2000 KDa), mixing uniformly to obtain gel 1, adding 20 mL 1 wt% NaOH solution into the gel 1, scattering the gel by using a high-shear dispersion emulsifying homogenizer at the rotation speed of 5000 rpm for 10min, and obtaining gel 2, namely a water phase.

2. Adding gel 2 into 400mL cyclohexane containing 2wt% Span80, emulsifying with high shear dispersion emulsifying homogenizer at 10000 rpm for 10min, standing to remove bubbles after uniform emulsification.

3. The same as in example 1.

4. The same as in example 1.

5. The same as in example 1.

The amount of cross-linking agent used is shown in table 2 below.

Example 3

In this embodiment, the influence of the crosslinking temperature and the crosslinking time on the crosslinked sodium hyaluronate microspheres is mainly studied, and the preparation method is as follows:

1. Maintaining the low temperature environment at 2-8 ℃, adding 0.1g of BDDE into 16mL of water, uniformly mixing, then adding 4 g of sodium hyaluronate (molecular weight 2000 KDa), uniformly mixing to obtain gel 1, adding 20 mL of 1 wt% NaOH solution into the gel 1, scattering the gel by using a high-shear dispersion emulsifying homogenizer at the rotation speed of 5000 rpm for 10 min, and obtaining gel 2, namely a water phase.

2. The same as in example 1.

3. And controlling the temperature and time of the emulsified liquid after the emulsification is finished, and performing crosslinking.

4. The same as in example 1.

5. The same as in example 1.

The crosslinking temperature and crosslinking time are shown in table 3 below.

Example 4

In this example, the influence of the emulsifying and stirring speed and time on the crosslinked sodium hyaluronate microspheres is mainly studied, and the preparation method is as follows:

2. Gel 2 was added to 400mL of n-octane containing 2wt% Span60, emulsified with a high shear dispersion emulsifying homogenizer, homogenized, and allowed to stand to remove air bubbles.

3. The same as in example 1.

4. The same as in example 1.

5. The same as in example 1.

The emulsification stirring speed and time are shown in table 4 below.

Example 5

In this example, the influence of the water phase stirring speed and time on the crosslinked sodium hyaluronate microspheres is mainly studied, and the preparation method is as follows:

1. maintaining the low temperature environment at 2-8 ℃, adding 0.1g of BDDE into 16mL of water, uniformly mixing, then adding 4 g of sodium hyaluronate (molecular weight 2000 KDa), uniformly mixing to obtain gel 1, adding 20 mL of 1 wt% NaOH solution into the gel 1, and scattering the gel by using a high-shear scattering emulsifying homogenizer at different speeds to obtain gel 2, namely a water phase.

2. The same as in example 1.

3. The same as in example 1.

4. The same as in example 1.

5. The same as in example 1.

The aqueous phase stirring speed and time are shown in table 5 below.

Example 6

In this example, the influence of the content of the emulsifier on the crosslinked sodium hyaluronate microspheres is mainly studied, and the preparation method is as follows:

2. Adding gel 2 into 400mL of n-octane containing Span80, emulsifying at 10000 rpm for 10min by a high-shear dispersion emulsifying homogenizer, and standing to remove bubbles after uniform emulsification.

3. The same as in example 1.

4. The same as in example 1.

5. The same as in example 1.

The emulsifier content is shown in table 6 below:

example 7

In this example, the influence of the ratio of the water phase to the oil phase on the crosslinked sodium hyaluronate microspheres is mainly studied, and the preparation method is as follows:

1. maintaining the low temperature environment at 2-8 ℃, adding 0.1g of BDDE into 16mL of water, uniformly mixing, then adding 4g of sodium hyaluronate (molecular weight 2000 KDa), uniformly mixing to obtain gel 1, adding 20 mL of 1 wt% NaOH solution into the gel 1, scattering the gel by using a high-shear dispersion emulsifying homogenizer at the rotation speed of 5000 rpm for 10min, and obtaining gel 2, namely a water phase.

2. Adding gel 2 into n-octane containing 2wt% Span80, emulsifying with high shear dispersion emulsifying homogenizer at 10000 rpm for 10min, standing to remove air bubbles after uniform emulsification.

3. The same as in example 1.

4. The same as in example 1.

5. The same as in example 1.

The water phase compared to the oil is shown in table 7 below, for example:

comparative example 1

1. Maintaining the low temperature environment at 2-8 ℃, adding 16mL of water into 4g of sodium hyaluronate (molecular weight 2000 KDa), uniformly mixing to obtain gel 1, adding 20 mL of 1 wt% NaOH solution into the gel 1, scattering the gel by using a high-shear dispersion emulsifying homogenizer at the rotation speed of 5000 rpm for 10min, and obtaining gel 2, namely a water phase.

3. Adding 0.1g BDDE into the emulsified liquid after the emulsification is finished, controlling the temperature to be 30 ℃, and stirring for 12 hours for crosslinking.

Comparative example 2

1. Adding 0.1g of BDDE into 16mL of water at normal temperature, uniformly mixing, then adding 4g of sodium hyaluronate (molecular weight 2000 KDa), uniformly mixing to obtain gel 1, adding 20 mL of 1 wt% NaOH solution into the gel 1, scattering the gel by using a high-shear dispersion emulsifying homogenizer at the rotating speed of 5000 rpm for 10min, and obtaining gel 2, namely a water phase.

2. The same as in example 1.

3. The same as in example 1.

4. The same as in example 1.

5. The same as in example 1.

Efficacy test

1. Evaluation of particle diameter and expansion ratio of microspheres

The particle size distribution of the dried crosslinked sodium hyaluronate microspheres prepared in each example and comparative example was measured using a laser particle size analyzer. Air is used as a carrier, and a dry measurement mode is adopted for measurement.

And (3) fully swelling the dried microspheres by using distilled water, measuring the particle size distribution of the microspheres, and calculating the expansion ratio. Expansion rate = (average particle size of microspheres after expansion-average particle size of dry microspheres)/average particle size of dry microspheres × 100%.

The average particle diameter and the expansion ratio of each example and comparative example are shown in the following tables 8 and 9:

from the results in the table, the crosslinked sodium hyaluronate microspheres with different crosslinking degrees and different particle sizes can be obtained by controlling the crosslinking conditions, and the expansion rate of the microspheres is closely related to the crosslinking degree and the particle size of the microspheres. From examples 1-1 to 1-3, it can be seen that the concentration of sodium hyaluronate has an influence on both the particle size and the swelling ratio of the finally obtained microspheres, and the higher the concentration, the smaller the particle size of the microspheres, the higher the degree of crosslinking, and thus the lower the swelling ratio of the microspheres. From examples 2-1 to 3-8, the amount of the crosslinking agent used and the crosslinking temperature and time are closely related to the degree of crosslinking, and have a small influence on the particle diameter of the microspheres. The higher the degree of crosslinking, the lower the expansion of the microspheres, with the higher expansion of examples 3-7 and 3-8, because sodium hyaluronate, when crosslinked at higher alkaline temperatures for too long a time, results in more degradation. From examples 4-1 to 5-5, the shear stirring speed and time mainly affected the particle size of the microspheres, did not greatly affect the expansion ratio of the microspheres, and the expansion ratio tended to decrease as the particle size of the microspheres decreased. From examples 6-1 to 7-2, the emulsifier to oil ratio mainly affects the particle size of the microspheres, and does not greatly affect the expansion ratio of the microspheres.

From comparative example 1, the crosslinked sodium hyaluronate microspheres prepared by adding the same amount of crosslinking agent as that in example 1 in the emulsification process have small crosslinking degree and high expansion rate. From the comparative example 2, the sodium hyaluronate is mixed with the cross-linking agent and the alkali liquor at normal temperature, so that the cross-linking reaction is generated in the uniform mixing process, the subsequent stirring and dispersing process is influenced, and the obtained cross-linked sodium hyaluronate microspheres have large particle size, large particle size distribution and non-concentrated particles.

Test 2: evaluation of viscoelasticity

After swelling the crosslinked sodium hyaluronate microspheres prepared in each example and comparative example, viscoelasticity was measured using a Haake RS6000 (seimer feishel (china)) apparatus under the following conditions: a rotor: p20 Ti; temperature: 25 ℃; gap value: 1.00 mm; measurement mode: oscillating frequency scan CD; stress: 1 percent; frequency range: 0.01-1 Hz. The storage modulus (G ') and loss modulus (G ' ') at a frequency of 0.1 Hz were recorded.

The storage modulus (G') and loss modulus (G ") at 0.1 Hz of the crosslinked sodium hyaluronate microspheres of each example and comparative example are shown in tables 10 and 11 below:

experimental data show that the crosslinked sodium hyaluronate microspheres prepared in comparative example 1 are low in elastic modulus, which indicates that the crosslinked sodium hyaluronate microspheres prepared by the method are insufficient in mechanical properties and consistent with the result of expansion rate. The crosslinked hyaluronic acid microspheres prepared by the embodiment can still keep good viscoelasticity after water absorption and expansion, which shows that the preparation method is effective, the dosage of the crosslinking agent can be reduced, the crosslinking efficiency is improved, the expanded gel can meet the requirement of mechanical property, and the crosslinked hyaluronic acid microspheres can be used in the fields of drug-loaded microspheres or cosmetic filling and the like.

Claims

1. A preparation method of crosslinked hyaluronic acid microspheres is characterized by comprising the following steps:

2. The method of claim 1, wherein: in the step (1), the aqueous phase is prepared at a low temperature of 2-8 ℃ to reduce the crosslinking of hyaluronic acid or a salt thereof.

3. The method according to claim 1 or 2, characterized in that: in the step (1), hyaluronic acid or a salt thereof and a crosslinking agent are mixed with water at low temperature, and then alkali is added.

4. The method according to claim 1 or 2, characterized in that: before the aqueous phase is added into the oil phase, the aqueous phase is dispersed uniformly by means of shear dispersion, and preferably, the aqueous phase is treated for 10 to 20min at a shear rate of 1000-20000 rpm.

5. The method according to claim 1 or 2, characterized in that: the concentration of hyaluronic acid or its salt in water phase is 5-25wt%, the content of cross-linking agent in water phase is 1-100 mg/mL, and the content of alkali in water phase is 0.5-5 wt%; preferably, the crosslinking agent is one or more of carbodiimide, divinyl sulfone, ethylene glycol diglycidyl ether, 1, 4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether and epichlorohydrin; preferably, the base is one or more of sodium hydroxide, potassium hydroxide and sodium carbonate.

6. The method of claim 1, wherein: the content of emulsifier in oil phase is 2-10 wt%.

7. The method according to claim 1 or 5, wherein: the volume ratio of the water phase to the oil phase is 1: 5-20.

8. The method according to claim 1 or 5, wherein: the oil phase consists of an emulsifier and an oil phase matrix, the oil phase matrix comprises vegetable oil, mineral oil, silicone oil, liquid paraffin, dodecane, n-octane or cyclohexane, the hydrophilic-hydrophobic balance value of the emulsifier is 3-9, and sorbitan monooleate or sorbitan monostearate is preferred.

9. The method of claim 1, wherein: in the step (2), after the water phase and the oil phase are mixed, the mixture is fully emulsified at a shear rate of 500-.

10. The method of claim 1, wherein: in the step (3), the crosslinking is carried out at 30-50 ℃ for 2-24 h.