阅读说明：本技术 一种目标粒径乳化交联透明质酸钠微粒的富集及交联剂清洗方法 (Enrichment and cross-linking agent cleaning method of emulsified cross-linked sodium hyaluronate particles with target particle size ) 是由 周奎 马矢徒 黄伟 孙伟庆 于 2020-05-27 设计创作，主要内容包括：本发明公开了一种目标粒径乳化交联透明质酸钠微粒富集及交联剂清洗方法,用于15～100μm乳化交联透明质酸钠微粒的富集及交联剂清洗。本方法以无水乙醇为溶剂,通过搅拌,静置分层,收集上层悬液的方法来富集粒径100μm以下的乳化交联透明质酸钠微粒,然后通过0.45μm负压过滤装置去除无水乙醇。同时采用无水乙醇清洗残余交联剂乙烯砜(DVS),真空干燥后获得目标产物。采用本发明可直接获得目标粒径的乳化交联透明质酸钠微粒,工艺简便,效率高,富集效果好；同时使用无水乙醇来清洗交联剂残留,不仅减少物料消耗,而且大幅减少清洗时间,DVS清洗效果极佳。本方法涉及医用材料技术领域,尤其在整形用注射交联透明质酸钠材料制备过程中具有重大应用前景。(The invention discloses a method for enriching and cleaning a cross-linking agent of emulsified and cross-linked sodium hyaluronate particles with a target particle size, which is used for enriching and cleaning the cross-linking agent of emulsified and cross-linked sodium hyaluronate particles with a particle size of 15-100 mu m. The method takes absolute ethyl alcohol as a solvent, and adopts the methods of stirring, standing for layering and collecting upper suspension to enrich emulsified crosslinked sodium hyaluronate particles with the particle size of less than 100 mu m, and then the absolute ethyl alcohol is removed by a negative pressure filtering device with the particle size of 0.45 mu m. And simultaneously, washing residual cross-linking agent vinyl sulfone (DVS) by using absolute ethyl alcohol, and drying in vacuum to obtain a target product. The method can directly obtain the emulsified cross-linked sodium hyaluronate particles with target particle size, and has the advantages of simple process, high efficiency and good enrichment effect; and meanwhile, the anhydrous ethanol is used for cleaning the residual cross-linking agent, so that the material consumption is reduced, the cleaning time is greatly reduced, and the DVS cleaning effect is excellent. The method relates to the technical field of medical materials, and particularly has great application prospect in the preparation process of the injection cross-linked sodium hyaluronate material for plastic surgery.)

1. A method for enriching emulsified cross-linked sodium hyaluronate particles with a target particle size and cleaning a cross-linking agent is characterized by comprising the following steps:

1) adding a certain amount of absolute ethyl alcohol into the emulsified cross-linked sodium hyaluronate particles, stirring until the particles are uniformly suspended,

2) stopping stirring, standing for sedimentation, after obvious layering, taking the upper suspension, transferring the upper suspension into a negative pressure filtering device, and carrying out vacuum filtration to obtain crosslinked sodium hyaluronate particles;

3) adding a certain amount of absolute ethyl alcohol into the rest part after the upper suspension is taken out, stirring until the particles are uniformly suspended, and repeating the collection process of the step 2);

4) vacuum drying the collected crosslinked sodium hyaluronate microparticles at a proper temperature for a certain time to obtain crosslinked sodium hyaluronate microparticle powder, and screening the crosslinked sodium hyaluronate microparticles with a target particle size by using a stainless steel screen with a proper mesh number;

5) transferring the cross-linked sodium hyaluronate particles with the target particle size screened in the step 4) to a negative pressure filtering device again, adding absolute ethyl alcohol, stirring and then carrying out suction filtration,

6) adding absolute ethyl alcohol into the crosslinked sodium hyaluronate particles obtained by suction filtration, stirring and then carrying out suction filtration;

7) transferring the obtained sample to a vacuum drying oven at a proper temperature for drying for a certain time, and removing the absolute ethyl alcohol.

2. The method as claimed in claim 1, wherein the mass ratio of the absolute ethyl alcohol to the cross-linked sodium hyaluronate particles in the step 1) is more than 10: 1.

3. the method according to claim 1, wherein the standing time in the step 2) is 10-60 min.

4. The method according to claim 1, wherein the step 3) is performed 2 to 6 times.

5. The method according to claim 1, wherein the number of times of performing in step 6) is 1 to 5 times.

6. The method as claimed in claim 1, wherein the filtration membrane in steps 5) and 6) is a negative pressure filtration device with a pore size of 0.22-15 μm.

7. The method according to claim 1, wherein the vacuum drying time in step 4) and step 7) is 6-24 hours and the temperature is 30-75 ℃.

Technical Field

The invention relates to an enrichment and cross-linking agent cleaning method of emulsified cross-linked sodium hyaluronate particles with a target particle size, belonging to the technical field of medical materials.

Background

Sodium Hyaluronate (Sodium Hyaluronate) has poor viscoelasticity and short half-life in the natural state, and when it is injected into dermal tissue as a facial dermal filler, its biomechanical properties and maintenance effect do not perform well. To achieve the effect of facial augmentation, chemical crosslinking/modification is required to improve its biomechanical properties while maintaining its biocompatibility and bioactivity.

The sodium hyaluronate dermal filler is divided into a single phase and a double phase according to the composition of the cross-linked sodium hyaluronate gel. Single phase sodium hyaluronate gel fillers are known as pure cross-linked and non-particulate sodium hyaluronate gels. In contrast, biphasic fillers (e.g. talc)Q-Med, sweden) consists of stable cross-linked sodium hyaluronate gel particles suspended in non-cross-linked sodium hyaluronate. The preparation of the cross-linked sodium hyaluronate gel particles is generally carried out by granulating through a stainless steel screen or homogenizing and granulating through a homogenizer, so as to form the cross-linked sodium hyaluronate gel with a certain particle size.

In order to obtain the cross-linked sodium hyaluronate gel with uniform particle size, the cross-linked sodium hyaluronate gel is prepared by an emulsion cross-linking method, has the characteristics of mild reaction conditions, simple preparation steps and convenience in operation, and can be used for preparing cross-linked sodium hyaluronate particles with corresponding particle size distribution ranges according to needs. Patent CN201310159737.3 adopts an emulsification crosslinking method to prepare a crosslinked sodium hyaluronate gel, and the process is as follows: preparing sodium hyaluronate alkaline solution gel with the concentration of 10-30% g/ml; adding the sodium hyaluronate alkaline solution gel into an oil phase containing an emulsifier, and then emulsifying at a high speed of 500-2000 rpm for 10-20 minutes by using a shearing machine; adding a certain amount of cross-linking agent, wherein the mass percentage of the cross-linking agent in the oil phase is 0.2-2%, stirring for 4-6 h at room temperature for cross-linking reaction, and standing overnight after completion; and (3) washing by using a water-soluble organic solvent to remove the residual oil phase on the surface of the microsphere, and finally drying to obtain the cross-linked sodium hyaluronate microsphere. The particle size of the cross-linked sodium hyaluronate gel prepared by the method is 80-2000 microns, and a stainless steel screen with a certain mesh number is used for screening sodium hyaluronate microspheres with target particle sizes. The disclosures of the above documents show that the sodium hyaluronate microparticles prepared by the emulsion crosslinking method often have a wide particle size distribution, the crosslinking agent is difficult to clean thoroughly, and the residue of the crosslinking agent may cause long-term hypersensitivity, which limits the application of the emulsified crosslinked sodium hyaluronate microparticles in the field of injection shaping to a certain extent.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a method for enriching emulsified cross-linked sodium hyaluronate gel particles (with the particle size distribution of 15-100 mu m) with target particle size and cleaning a cross-linking agent. In order to realize the purpose of the invention, the invention adopts the following technical method:

a method for enriching emulsified cross-linked sodium hyaluronate particles with a target particle size and cleaning a cross-linking agent is characterized by comprising the following steps:

1) adding a certain amount of absolute ethyl alcohol into the emulsified cross-linked sodium hyaluronate particles, stirring until the particles are uniformly suspended,

2) stopping stirring, standing for sedimentation, after obvious layering, taking the upper suspension, transferring the upper suspension into a negative pressure filtering device, and carrying out vacuum filtration to obtain crosslinked sodium hyaluronate particles;

3) adding a certain amount of absolute ethyl alcohol into the rest part after the upper suspension is taken out, stirring until the particles are uniformly suspended, and repeating the collection process of the step 2);

4) vacuum drying the collected crosslinked sodium hyaluronate microparticles at a proper temperature for a certain time to obtain crosslinked sodium hyaluronate microparticle powder, and screening the crosslinked sodium hyaluronate microparticles with a target particle size by using a stainless steel screen with a proper mesh number;

5) transferring the cross-linked sodium hyaluronate particles with the target particle size screened in the step 4) to a negative pressure filtering device again, adding absolute ethyl alcohol, stirring and then carrying out suction filtration,

6) adding absolute ethyl alcohol into the crosslinked sodium hyaluronate particles obtained by suction filtration, stirring and then carrying out suction filtration;

7) transferring the obtained sample to a vacuum drying oven at a proper temperature for drying for a certain time, and removing the absolute ethyl alcohol.

As a preferable scheme of the invention, the mass ratio of the addition amount of the absolute ethyl alcohol to the cross-linked sodium hyaluronate particles in the step 1) is more than 10: 1, more preferably 50: 1.

as a preferable scheme of the invention, the standing time in the step 2) is 10-60 min, and more preferably 20-30 min;

in a preferred embodiment of the present invention, the number of times of performing step 3) is 2 to 6, and more preferably 3.

As a preferable scheme of the invention, the number of times of performing in the step 6) is 1-5, and more preferably 3; in the steps 5) and 6), a negative pressure filtering device with the diameter of 0.22-15 mu m can be selected, and the preferable thickness is 0.45 mu m.

As a preferable scheme of the invention, the vacuum drying time in the step 4) and the step 7) is 6-24 h, and more preferably 12 h. The vacuum drying temperature is 30-75 ℃, and more preferably 60 ℃.

According to the technical scheme, the required crosslinked sodium hyaluronate particles can be obtained in a short time and in a material-saving manner by adopting common reagents and means such as ethanol and suction filtration without adopting means such as buffer solution and suction filtration.

Description of the drawings:

FIG. 1 is a graph showing the particle size distribution of a sample of the enriched emulsified cross-linked sodium hyaluronate microparticles of example 1;

FIG. 2 is a statistical chart of the enrichment effect of the emulsified cross-linked sodium hyaluronate microparticles with target particle size in examples 1, 2 and 3;

FIG. 3 shows the cleaning time and DVS residue after 3 and 5 times of cleaning with conventional crosslinking agent;

FIG. 4 shows the cleaning time and DVS residue in the case of cleaning 3 and 5 times in examples 4 and 5 of the present invention.

Detailed Description

In order to make the invention easier to understand and the technical solutions and advantages thereof more clear, the invention is further described in detail below with reference to examples and the accompanying drawings. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention, and that specific experimental procedures not mentioned in the following examples are generally conducted according to conventional experimental procedures.

The emulsified crosslinked sodium hyaluronate particles are prepared by adding a sodium hyaluronate water phase into an oil phase, stirring at a high speed to form a sodium hyaluronate microsphere emulsion, adding a crosslinking agent to perform a crosslinking reaction to form crosslinked sodium hyaluronate microspheres, cleaning to remove the oil phase, and drying. The emulsified crosslinked sodium hyaluronate microparticles used in the following examples of the present invention were prepared by the following method: and adding the sodium hyaluronate alkaline solution gel aqueous phase with the concentration of 10% g/ml into the oil phase, wherein the molecular weight of the sodium hyaluronate is 110-150 ten thousand molecular weight. The oil phase is a mixture of span80 and light liquid paraffin. The emulsifier Span80 accounts for 2% of the oil phase by mass. The mass ratio of the water phase to the oil phase is 2: 1. Emulsifying by an emulsifying machine at 4000r/min for 30 min; adding a certain amount of cross-linking agent, wherein the mass percentage of the cross-linking agent in the water phase is 3%, reacting for 2 hours at 45 ℃, and stirring for 1.5 hours at room temperature to perform cross-linking reaction. After the crosslinking reaction is finished, adding normal hexane according to the volume ratio of 1:5 for cleaning twice, pouring out supernatant after layering, and further adding ethyl acetate according to the volume ratio of 1:5 for cleaning twice; and finally, adding absolute ethyl alcohol according to the volume ratio of 1:1, and drying in vacuum to obtain the emulsified cross-linked sodium hyaluronate microspheres.

The emulsified cross-linked sodium hyaluronate particles obtained by the method have the following particle size distribution range as measured by a laser particle sizer: 5.0 to 150 microns.

Example 1 Collection of Cross-Linked sodium hyaluronate microparticles of target particle size

1) Adding 500ml of absolute ethyl alcohol into 10g of emulsified cross-linked sodium hyaluronate particles, and stirring until the particles are uniformly suspended;

2) stopping stirring, standing for settling, taking the upper suspension after 30 minutes, transferring the upper suspension into a negative pressure filtering device, and carrying out vacuum filtration to obtain sodium hyaluronate particles;

3) repeating the anhydrous ethanol collecting process for 3 times;

4) the collected crosslinked sodium hyaluronate microparticles were dried under vacuum at 60 ℃ for 12 hours, the microparticles were collected and weighed, and the mass of the enriched microparticles was recorded as 9.64g, which is 96.4% of the total mass (see fig. 2). The particle size distribution of the target sample is detected by using a Markov master 3000, the detection result is shown in figure 1, and as can be seen from figure 1, the Dv (90) of the crosslinked sodium hyaluronate particles recovered by the absolute ethanol method is 68.9 μm, and the Dv (50) is 35.2 μm. The residual amount of DVS in the samples was measured by gas chromatography to be 41.3. mu.g/g.

Example 2 Collection of Cross-Linked sodium hyaluronate microparticles of target particle size

1) Adding 400ml of absolute ethyl alcohol into 8.0g of emulsified cross-linked sodium hyaluronate particles, and stirring until the particles are uniformly suspended;

2) stopping stirring, standing for settling, taking the upper suspension after 30 minutes, transferring the upper suspension into a negative pressure filtering device, and carrying out vacuum filtration to obtain sodium hyaluronate particles;

3) the anhydrous ethanol collection process was repeated 5 times,

4) the resulting crosslinked sodium hyaluronate microparticles were collected, dried under vacuum at 45 ℃ for 12 hours, the microparticles were collected and weighed, and the mass of the enriched microparticles was recorded to be 9.35g, which is 93.5% of the total mass (see fig. 2). The residual amount of DVS in the samples was 40.7. mu.g/g as determined by gas chromatography.

Example 3 Collection of Cross-Linked sodium hyaluronate microparticles of target particle size

1) Adding 240ml of absolute ethyl alcohol into 6.0g of emulsified cross-linked sodium hyaluronate particles, and stirring until the particles are uniformly suspended;

2) stopping stirring, standing for settling, taking the upper suspension after 30 minutes, transferring the upper suspension into a negative pressure filtering device, and carrying out vacuum filtration to obtain sodium hyaluronate particles;

3) the anhydrous ethanol collection process was repeated 6 times,

4) the resulting crosslinked sodium hyaluronate microparticles were collected and then dried under vacuum at 75 ℃ for 12 hours, the microparticles were collected and weighed, and the mass of the enriched microparticles was recorded to be 9.35g, which is 93.5% of the total mass (see fig. 2). The residual amount of DVS in the samples was 39.5. mu.g/g as determined by gas chromatography.

The density of the absolute ethyl alcohol is matched with the target particle size, so that the cross-linked sodium hyaluronate particles with the target particle size can be suspended in the absolute ethyl alcohol within a certain time, and as can be seen from the examples 1, 2 and 3, 96.4% of emulsified cross-linked sodium hyaluronate particles with the target particle size can be collected in three enrichment processes, the target particle size particles are enriched in the process before cleaning, and the material consumption in the cleaning process is directly reduced by 50%.

Example 4 crosslinker cleaning Process

1) Weighing 2.0g of the sodium hyaluronate microspheres enriched in the embodiment 2, and screening sodium hyaluronate particles with target particle size by using a 800-mesh stainless steel screen;

2) transferring the sodium hyaluronate particles into a negative pressure filtering device, adding 100ml of absolute ethyl alcohol, stirring, performing suction filtration, repeating the process for 3 times, filling a sample after 24 hours of swelling by phosphate buffer solution, wherein the time required by the cleaning process is 32 hours, and detecting the residual quantity of DVS in the sample by adopting a gas chromatography, wherein the residual quantity of DVS is lower than the detection limit of the gas chromatography by 0.17 mug/g (shown in figure 4) and is far lower than the requirement of industry standard crosslinked sodium hyaluronate gel for YY/T0962-2014 plastic surgery by 2 mug/g.

Example 5 Cross-linker cleaning procedure

1) Weighing 4.0g of the sodium hyaluronate microspheres enriched in the embodiment 2, and screening sodium hyaluronate particles with target particle size by using a 800-mesh stainless steel screen;

2) transferring the sodium hyaluronate particles into a negative pressure filtering device, adding 200 anhydrous ethanol, stirring, performing suction filtration, repeating the process for 5 times, filling a sample after 24 hours of swelling by phosphate buffer solution, wherein the time required by the cleaning process is 34 hours, detecting the residual quantity of DVS in the sample by using a gas chromatography, wherein the residual quantity of DVS is lower than the detection limit of the gas chromatography by 0.17 mu g/g (shown in figure 4) and is far lower than the requirement of the industry standard crosslinked sodium hyaluronate gel for YY/T0962-2014 plastic surgery, namely 2 mu g/g.

Comparative example 1

1) Weighing 10.0g of emulsified crosslinked sodium hyaluronate particles, placing the particles on a 100-micron stainless steel screen, screening the particles by using a vibrating screen, and collecting the emulsified crosslinked sodium hyaluronate particles below the stainless steel screen;

2) placing the emulsified crosslinked sodium hyaluronate microparticles collected in the step 1) on a 15-micron stainless steel screen, screening by using a vibrating screen, and collecting the emulsified crosslinked sodium hyaluronate microparticles on the stainless steel screen;

3) weighing the emulsified and crosslinked sodium hyaluronate particles collected in the step 2), namely the emulsified and crosslinked sodium hyaluronate particles with the particle size range of 15-100 microns, wherein the mass is 4.62g, and the collection rate is 46.2%.

Comparative example 2

1) Weighing 4.0g of the sodium hyaluronate microspheres enriched in the above example 2, and selecting an 800-mesh stainless steel screen to screen emulsified crosslinked sodium hyaluronate microparticles with a target particle size;

2) and (2) preparing a PBS buffer solution, wherein the mass ratio of the disodium hydrogen phosphate to the sodium dihydrogen phosphate is 0.035%, the mass ratio of the sodium dihydrogen phosphate to the sodium chloride is 0.014%, and the mass ratio of the sodium chloride to the sodium hyaluronate microspheres obtained in the previous step is placed into the buffer solution, stands and swells overnight, and then the microspheres are collected by centrifugation, and each washing needs 24 hours.

3) And (3) repeating the buffer solution cleaning process in the step 2) for 3 times, wherein the time required by the cleaning process is 96 hours, and detecting the residual quantity of the cross-linking agent DVS in the sample by using a gas chromatography, wherein the detection result of the residual quantity of the DVS is 6.01 mu g/g, which is higher than the requirement of 2 mu g/g of cross-linked sodium hyaluronate gel for the orthopedic surgery of YY/T0962-2014 in the industry standard.

Comparative example 3

1) Weighing 4.0g of the sodium hyaluronate microspheres enriched in the above example 2, and selecting an 800-mesh stainless steel screen to screen emulsified crosslinked sodium hyaluronate microparticles with a target particle size;

2) and (2) preparing a PBS buffer solution, wherein the mass ratio of the disodium hydrogen phosphate to the sodium dihydrogen phosphate is 0.035%, the mass ratio of the sodium dihydrogen phosphate to the sodium chloride is 0.014%, and the mass ratio of the sodium chloride to the sodium hyaluronate microspheres obtained in the previous step is placed into the buffer solution, stands and swells overnight, and then the microspheres are collected by centrifugation, and each washing needs 24 hours.

3) And (3) repeating the buffer solution cleaning process in the step 2) for 5 times, wherein the time required by the cleaning process is 144 hours, and detecting the residual quantity of the cross-linking agent DVS in the sample by using a gas chromatography, wherein the detection result of the residual quantity of the DVS is 3.26 mu g/g, which is slightly higher than the requirement of 2 mu g/g of cross-linked sodium hyaluronate gel for the 2014 plastic surgery in the industry standard.

It is found from the experiments of examples 4 and 5 and comparative examples 1 and 2 that the cleaning time of the cross-linking agent cleaning method of the present invention is directly reduced by 110 hours, the cleaning effect of the cross-linking agent DVS is excellent, and the cross-linking agent DVS residue is not detected.