Agarose-based hydrogel, preparation method and application thereof

Abstract

The present invention discloses an agarose-based hydrogel, a preparation method and application thereof, and belongs to the field of injection filling beauty technology. The agarose-based hydrogel of the present invention is prepared by the following method: a curing agent and a dispersant are mixed evenly to prepare a curing system; an agarose solution and a curing system are placed in an alkaline solution, a curing reaction is carried out, and the film is removed and cleaned to obtain an agarose-based hydrogel. The present invention obtains a hydrophilic agarose-based hydrogel suitable for facial filling by directly curing agarose, avoiding the problems of purification difficulties and obvious granularity caused by the use of modified agarose microspheres, thereby improving the safety of the injection gel. The injection gel prepared by the present invention is uniform and delicate, has no granularity, has good cohesion, can delicately support the skin and tissue, is not easy to move and free; moreover, it has no irritating effect on the skin, has a long local retention time, good plasticity, and few side effects, and can achieve long-term filling.

Description

Technical Field

[0001] The present invention belongs to the technical field of injection filling cosmetics, and in particular relates to an agarose-based hydrogel, a preparation method and application thereof. Background Art

[0002] Aesthetic medicine is booming in my country. Facial soft tissue filling is one of the minimally invasive treatments for facial volume tissue loss, contour changes and static wrinkles. The ideal soft tissue filling material should have good biocompatibility, effectiveness and safety. At present, facial soft tissue filling materials are mainly non-autologous tissue injections, such as hyaluronic acid (HA), polycaprolactone (PCL), poly-lactic acid (PLLA), and some filling materials derived from autologous tissue, such as fat grafts and platelet-rich plasma. Although hyaluronic acid is still the most popular degradable injection product, its product degradation period is short and requires frequent and multiple injections to maintain its filling effect. Therefore, finding effective and safe tissue fillers has always been a continuous challenge for plastic and cosmetic surgery. In order to achieve long-term filling effects, people have tried to use biological non-degradable materials to make microspheres as fillers, such as polyvinyl alcohol (PVA) and polymethyl methacrylate (PMMA). Although the filling effects of these fillers last significantly longer, if these materials remain in the body for too long, they will release harmful substances and trigger a series of side effects, posing a high safety risk in the later stages.

[0003] Agarose is a biodegradable polymer. This natural polymer has similarities to the extracellular matrix, which has led to strong interest in its use in the field of dermal fillers. Like HA, agarose is a biocompatible polysaccharide with immediate and long-lasting clinical results. However, unlike HA, it is stable in vivo and can provide long-term filling effects after tissue implantation.

[0004] The current prior art cannot directly use agarose powder as a raw material to prepare hydrogel products suitable for facial filling. The reason is that agarose is generally dissolved in water when heated to above 90°C. When the temperature drops to 35-40°C, a good semi-solid gel is formed. The gel is strong and cannot be pushed and injected through a syringe. This is also the reason why agarose gel can only be used as an electrophoresis support. Therefore, the current agarose filling products need to first prepare agarose powder into agarose microspheres, and then use agarose microspheres as raw materials to prepare hydrogel products suitable for facial filling; for example, in Chinese patent CN104774337A, a method for preparing an injectable cross-linked sodium hyaluronate gel containing agarose microspheres is provided.

[0005] The preparation methods of agarose microspheres include emulsification-solidification method, injection method, membrane emulsification method and microfluidics method. The above methods have a series of problems, for example, the organic solvent is consumed a lot during preparation, and it cannot be guaranteed to be completely removed, or the particle size distribution of the prepared microspheres is too wide, and the particles are not smooth, etc. Therefore, when agarose microspheres are selected as raw materials to prepare or mix and cross-link with sodium hyaluronate to prepare gel, there are defects such as excessive addition of organic reagents, uneven particle size distribution of the gel, and obvious granularity, thereby increasing the probability of inflammatory reaction (Zhang Fan. Preparation, modification and application of agar gel microspheres).

[0006] In order to overcome the problems of complex process, obvious granularity and uneven particle size of agarose microsphere gel, improve the seamless fusion of agarose and base fluid, make the prepared hydrogel have a more delicate pushing effect, and improve its safety and effectiveness as a facial filling product, the present invention is committed to using agarose powder as raw material, overcoming the prejudices in the prior art, and exploring a simpler, more effective and safer agarose hydrogel and its preparation process. Summary of the invention

[0007] The technical solution of the present invention is as follows:

[0008] The present invention provides a method for preparing an agarose-based hydrogel, comprising the following steps:

[0009] The curing agent and the dispersant are mixed evenly to prepare a curing system; the agarose solution and the curing system are placed in an alkaline solution to carry out a curing reaction, and the film is removed and cleaned to obtain an agarose-based hydrogel.

[0010] In the above preparation method, the curing agent is selected from one or more of epichlorohydrin, 1,2,7,8-diepoxyoctane, divinyl sulfone, and 1,4-butanediol diglycidyl ether.

[0011] In the above preparation method, the dispersant is selected from dimethyl sulfoxide or anhydrous ethanol.

[0012] In the above preparation method, the solidifying agent accounts for 0.5-10% of the mass of the agarose solution; and the dispersing agent accounts for 1-30% of the mass of the agarose solution.

[0013] In the above preparation method, the conditions for the curing reaction are: reaction temperature is 50-70° C., and reaction time is 2-72 hours.

[0014] In the above preparation method, the alkaline solution is selected from sodium hydroxide solution, potassium hydroxide solution, calcium hydroxide solution, and barium hydroxide solution, and the concentration of the alkaline solution is selected from 0.5 to 3 M. The volume of the alkaline solution can be selected to be 0.1 to 10 times that of the agarose solution.

[0015] In the above preparation method, the agarose solution can be prepared by the following method: placing agarose powder in water, dissolving it at 80-125° C. for 15 min-5 h, and obtaining an agarose solution with a concentration of 0.5-20%.

[0016] In the above preparation method, the steps of demolding and cleaning are as follows: the reactants obtained by the solidification reaction are placed in purified water, stirred and cleaned, and filtered after cleaning to obtain an agarose-based hydrogel; the cleaning speed is 50 to 400 rpm / min, the cleaning time is 4 to 48 hours, and the cleaning solution is changed every 2 to 6 hours.

[0017] The present invention provides an agarose-based hydrogel prepared by the above method.

[0018] The present invention provides application of the agarose-based hydrogel in facial filling and beautification.

[0019] The present invention provides use of the agarose-based hydrogel in preparing a facial filler.

[0020] The present invention provides an agarose-based hydrogel facial filler, which is composed of a muscle base solution and an agarose-based hydrogel; wherein the amount ratio of the agarose-based hydrogel to the muscle base solution is selected from 1:0.01 to 1:1, g:mL, and preferably from 2:1 to 15:2, g:mL.

[0021] The above-mentioned base solution can be selected from one or more of phosphate buffer, physiological saline, hyaluronic acid, collagen, chitosan, amino acids, cellulose, and sodium alginate; wherein the concentration of the base solution is selected from 0.1 to 10%

[0022] The preparation method of the agarose-based hydrogel facial filler is relatively simple, and the base fluid is fully dispersed in the agarose-based hydrogel. After the preparation is completed, conventional filling and sterilization can be performed.

[0023] The beneficial effects of the present invention are:

[0024] The present invention overcomes the prejudice in the prior art, obtains a hydrophilic agarose-based hydrogel suitable for facial filling by directly solidifying agarose, avoids the problems of purification difficulty and obvious granularity caused by the use of modified agarose microspheres, and improves the safety of injection gel. The injection gel prepared by the present invention is uniform and delicate, has no granularity, has good cohesion, can delicately support the skin and tissue, and is not easy to move and free; moreover, it has no irritating effect on the skin, has a long local retention time, good plasticity, and few side effects, and can achieve long-term filling. The process conditions of the present invention are easy to control, the operation steps are few, the product quality is stable, and it is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1is the rheological test result;

[0026] Figure 2 This is the result of the pushing force test;

[0027] Figure 3 This is the tissue section after 1 week of implantation, HE×20;

[0028] Figure 4 This is the tissue section 4 weeks after implantation, HE×20;

[0029] Figure 5 This is a tissue section 30 weeks after implantation, HE×20. DETAILED DESCRIPTION

[0030] Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0031] Example 1

[0032] Preparation of agarose-based hydrogels:

[0033] Place 5g of agarose powder in a beaker, add 100mL of purified water, and dissolve at 121℃ for 1h to obtain an agarose solution. Place 2g of epichlorohydrin and 15g of dimethyl sulfoxide in a centrifuge tube and mix well to form a solidification system. Add the agarose solution and the solidification system to 80mL of sodium hydroxide solution (1.2M), and solidify at 60℃ for 24h. After the reaction is completed, take out the reaction product, place it in purified water, stir and wash it, and the stirring speed is 100rpm / min. Change the water every 4 to 8h, and wash it for a total of 24h to obtain an agarose-based hydrogel.

[0034] Prepare 20 mL of phosphate buffer, add it to the above 150 g of agarose-based hydrogel, disperse it evenly, then fill the gel into a prefilled syringe and sterilize it with wet heat to obtain the agarose-based hydrogel for injection filling.

[0035] Example 2

[0036] Preparation of agarose-based hydrogels:

[0037] Place 2g of agarose powder in a beaker, add 50mL of purified water, and dissolve at 121℃ for 30h to obtain an agarose solution. Place 0.7g of epichlorohydrin and 10g of dimethyl sulfoxide in a centrifuge tube and mix well to form a solidification system. Add the agarose solution and the solidification system to 50mL of sodium hydroxide solution (0.7M), and solidify at 60℃ for 24h. After the reaction is completed, take out the reaction product, place it in purified water, stir and wash it, and the stirring speed is 100rpm / min. Change the water every 4 to 8h, and wash it for a total of 24h to obtain an agarose-based hydrogel.

[0038] Prepare 50 mL of 1% sodium hyaluronate solution, add it to the above 100 g agarose-based hydrogel and disperse it evenly. Then fill the gel into a prefilled syringe and sterilize it with wet heat to obtain the agarose-based hydrogel for injection filling.

[0039] Example 3

[0040] Preparation of agarose-based hydrogels:

[0041] Place 10g of agarose powder in a beaker, add 200mL of purified water, and dissolve at 121℃ for 30h to obtain an agarose solution. Place 5g of epichlorohydrin and 50g of dimethyl sulfoxide in a centrifuge tube and mix well to form a solidification system. Add the agarose solution and the solidification system to 160mL of sodium hydroxide solution (1M), and solidify at 60℃ for 12h. After the reaction is completed, take out the reaction product, place it in purified water, stir and wash it, and the stirring speed is 200rpm / min. Change the water every 4 to 8h, and wash it for a total of 24h to obtain an agarose-based hydrogel.

[0042] Prepare 70 mL of 2% seaweed polysaccharide solution, add it to the above 300 g agarose-based hydrogel, disperse it evenly, then fill the gel into a pre-filled syringe and sterilize it with wet heat to obtain the agarose-based hydrogel for injection filling.

[0043] Example 4

[0044] Preparation of agarose-based hydrogels:

[0045] Place 5g of agarose powder in a beaker, add 200mL of purified water, and dissolve at 100℃ for 4h to obtain an agarose solution. Place 2g of 1,4-butanediol diglycidyl ether and 10g of dimethyl sulfoxide in a centrifuge tube and mix well to form a solidification system. Add the agarose solution and the solidification system to 100mL of sodium hydroxide solution (1M) and solidify at 55℃ for 6h. After the reaction is completed, take out the reaction product, place it in purified water, stir and wash it, the stirring speed is 100rpm / min, change the water twice, and wash it for a total of 6h to obtain an agarose-based hydrogel.

[0046] Take 2g of sodium hyaluronate, 2g of collagen, and 1g of amino acid, add them to 50mL of normal saline, dissolve and stir, and after complete dissolution, add them to the above 250g of agarose-based hydrogel and disperse them evenly. Then fill the gel into a prefilled syringe and sterilize it with wet heat to obtain the agarose-based hydrogel for injection filling.

[0047] Comparative Example 1

[0048] Place 5g of agarose powder in 150mL of purified water, dissolve at 121℃ for 30min, take out after dissolution and place in a 65℃ water bath to obtain the aqueous phase; place 14g of Span 80 in 300mL of liquid paraffin, stir and mix at 65℃ for 30min to obtain the oil phase; take out the aqueous phase after dissolution and add it to the oil phase, emulsify and homogenize for 10min; solidify the homogenized solution at room temperature for 1h; after solidification, wash with ethanol and filter to obtain agarose microspheres. Prepare 20mL of 1% sodium hyaluronate solution, add it to the above 100g agarose microspheres and disperse evenly, then fill the gel into a prefilled syringe and sterilize. After sterilization, the gel agglomerates into blocks. When the pushing force is tested, the needle bursts and cannot be tested normally. It is not suitable for injection and cosmetic filling.

[0049] Comparative Example 2

[0050] 25g agarose powder and 5g non-cross-linked hyaluronic acid were added to 1000mL phosphate buffer solution (pH 6.8), stirred and dissolved at 80℃ for 30min, cooled to room temperature, filled and sterilized. After sterilization, two phases were found in the syringe, indicating that the product had low uniformity and was not suitable for injection and cosmetic filling.

[0051] (I) Viscoelasticity test

[0052] The agarose-based hydrogels prepared in Examples 1 to 4 above were subjected to rheological (viscoelastic) testing. Rheological properties are related to the ability of a filler to withstand different types of deformations and forces when implanted in different facial areas and planes. The revelation of the rheological properties of the above filling products will guide clinicians in selecting the ideal filler for each area of ​​the face based on the pathology and deformation forces acting on the area of ​​interest. Therefore, selecting a dermal filler with the correct rheological properties is a key factor in achieving a natural-looking, long-lasting, desired aesthetic effect.

[0053] The test conditions are as follows: Instrument name: Rheometer; Test fixture: upper plate 20mm flat plate; lower plate 60mm flat plate; Gap: 1mm; Test description: Constant temperature 10℃ test with amplitude scanning.

[0054] The test results are as follows Figure 1 As shown:

[0055] It can be seen from the figure that the linear viscoelastic regions of the four groups of products are similar, the elastic modulus is about 500Pa, the viscous modulus is about 90Pa, the elastic modulus and viscous modulus are moderate, the gel is soft and smooth, and the shaping and lifting effect after injection is significant without bulging. It is well known that the ratio between G' and G" can reflect the change between gel viscosity and elasticity. It can be seen from the figure that the ratio of loss modulus G' / elastic modulus G" of the four groups of hydrogels is greater than 1, which shows that the hydrogel prepared by the present invention has good elastic properties, can be firmly retained at the injection site, maintain a firm current structural strength, and help maintain the shaping effect after injection.

[0056] (II) Pushing force test

[0057] The hydrogel products prepared in Example 1, Example 2, Comparative Example 1 and Comparative Example 2 were subjected to a pushing force test, and the test conditions were as follows: instrument name: universal material testing machine; test temperature: room temperature; test distance: set the test distance to full scale; pushing speed: set the pushing speed to 30 mm / min.

[0058] The test results are as follows Figure 2 As shown:

[0059] As can be seen from the figure, the pushing force of the gel in Example 1 and Example 2 is about 10N, which meets the standard requirements of injection filling products, has a good experience, and exhibits excellent injectability; when pushing Example 1, the pushing force drops sharply after a deformation of 10mm, indicating that a needle burst occurs during the test process, indicating that the gel has poor fluidity and almost zero ductility, and is not suitable for use as injection filling; the pushing results of Example 2 show that the pushing force range fluctuates greatly, indicating that the product has low continuity and poor uniformity, which is also not conducive to injection filling.

[0060] (III) Physical and chemical properties

[0061] The physicochemical properties of the agarose-based hydrogel prepared in Example 1 were tested and compared with the agarose gel prepared using agarose microspheres in Example 1 of patent CN115260545A (reference example), as shown in Table 1:

[0062] Table 1

[0063]

[0064] As can be seen from Table 1, in terms of properties, the two hydrogels have a large difference in shape. The hydrogel prepared using agarose microspheres is translucent milky white and has high viscosity; while the hydrogel directly prepared using agarose powder is transparent and has high elasticity, so that it has strong deformation resistance and is not easy to deform. Therefore, the agarose-based hydrogel prepared by the present invention is more suitable for parts such as the chin and the bridge of the nose that require lasting plasticity. In terms of particle size, since the present invention realizes the direct preparation of hydrogel using agarose powder as raw material, its hydrogel does not contain granular components, has a fine texture, and has no granular feeling, which reflects a more superior cohesive force, smoother injection, more natural filling, and more uniform degradation. In terms of curing agent residue, the hydrogel of the present invention does not detect epichlorohydrin residue, has higher safety, and injection in vivo does not cause inflammatory reaction. In terms of pushing force, the pushing force of the two hydrogels meets ergonomic and standard requirements, allowing operators to better push injection operations. In terms of cross-linking degree, the hydrogel of the present invention has a cross-linking degree of 2.1%, which is lower than 10.22% of the reference example, indicating that the hydrogel has excellent plasticity. The low cross-linking degree (modification degree) reflects the high safety in application and can reduce the allergy rate.

[0065] Comparative Example 3

[0066] Effect of curing reaction temperature on agarose-based hydrogels:

[0067] Place 5g of agarose powder in a beaker, add 100mL of purified water, and dissolve at 121℃ for 1h to obtain an agarose solution. Place 2g of epichlorohydrin and 15g of dimethyl sulfoxide in a centrifuge tube and mix well to form a curing system. Add the agarose solution and the curing system to 80mL of sodium hydroxide solution (1.2M), and cure at 37℃, 38℃, 39℃, and 40℃ for 24h. After the reaction is completed, take out the reaction products, and find that they all fail to cure. The reaction products in the centrifuge tube do not form a jelly gel, but are still a highly fluid liquid. They are completely dispersed in purified water and cannot be washed and separated.

[0068] Comparative Example 4

[0069] Effect of solidifying agent type on agarose-based hydrogels:

[0070] Place 2g of agarose powder in a beaker, add 50mL of purified water, and dissolve at 121℃ for 30h to obtain an agarose solution. Place 0.5g of adipic acid dihydrazide, 1g of carbodiimide, and 10g of dimethyl sulfoxide in a centrifuge tube and mix evenly to form a solidification system. Add the agarose solution and the solidification system to 50mL of sodium hydroxide solution (0.7M), and solidify at 60℃ for 24h. After the reaction is completed, take out the reaction product, place it in purified water, stir and wash it, and the stirring speed is 100rpm / min. Change the water every 4 to 8h, and wash it for a total of 24h to obtain an agarose-based hydrogel.

[0071] Prepare 50mL of 1% sodium hyaluronate solution, add it to the above 100g agarose hydrogel, disperse it evenly, then fill the gel into a prefilled syringe and sterilize it with wet heat. After sterilization, it is found that the product is hydrated and loses its gel properties. After the temperature drops to room temperature, the hydrated gel solidifies and hardens and cannot be injected. The reason is that adipic acid dihydrazide (ADH) and carbodiimide (EDC) are commonly used crosslinkers at carboxyl sites, and there is no carboxyl site in the agarose molecular structure, so the carboxyl crosslinking method is not suitable for preparing agarose hydrogel, and the crosslinking effect is poor.

[0072] Comparative Example 5

[0073] Effect of curing agent dosage on agarose-based hydrogel:

[0074] Place 5g of agarose powder in a beaker, add 200mL of purified water, and dissolve at 100℃ for 4h to obtain an agarose solution. Place 0.8g of 1,4-butanediol diglycidyl ether and 10g of dimethyl sulfoxide in a centrifuge tube and mix evenly to form a solidification system. Add the agarose solution and the solidification system to 100mL of sodium hydroxide solution (1M), and solidify at 55℃ for 6h. After the reaction is completed, take out the reaction product and find that it has not formed a gel, and the reaction system is a yellow watery liquid. After being placed at room temperature, the fluidity is still very strong and the properties have not changed. Therefore, under this preparation parameter, it is impossible to obtain the support force and viscoelasticity required for filling injection products, and it cannot be used as an injection filling gel.

[0075] On the basis of the above preparation method, the amount of curing agent was further adjusted to 0.5g, 0.6g, and 0.7g, and the curing reaction was continued for 6h. After the reaction was completed, it was found that the reaction system was also a yellow aqueous liquid and failed to form a gel. After being placed at room temperature, the fluidity was still very strong and the properties did not change. This shows that if agarose gel suitable for facial filling is to be directly prepared using agarose powder as a raw material, the amount of curing agent used needs to be increased.

[0076] Application Example 1

[0077] In vivo implantation degradation test:

[0078] The injection gel prepared by Example 1 was subjected to a degradation test by implantation in rabbits. Before the test, the rabbit hair on both sides of the rabbit spine was cut off. During the test, according to the animal reaction, anesthesia was performed by intravenous injection of ulex or not. The skin of the surgical area was disinfected with iodine tincture and 75% ethanol according to the requirements of conventional surgery. Four implantation points were selected at equal distances of about 2.5 cm on both sides of the rabbit spine, and each point was separated by 2.5 cm, and 0.2 mL of sample was implanted at each point. After implantation, the animals were euthanized at 1 week, 4 weeks, and 30 weeks, respectively, and the skin on both sides of the spine was separated to expose the material implanted subcutaneously. The size, form, and surrounding tissue of the implanted sample were observed to be suppurated and secretory, etc., and the surrounding tissue position specimens containing the material were taken, and the tissue of about 0.5 to 1 cm around the sample was cut and wrapped, fixed with 10% formalin solution, dehydrated and transparent, and embedded in paraffin. Continuous sections were cut, and the thickness was 5 μm. Hematoxylin-eosin staining was performed to observe the pathological conditions of the tissue under a microscope.

[0079] The test results are as follows Figure 3 to Figure 5 As shown:

[0080] 1 week after implantation ( Figure 3 )、4 weeks( Figure 4 ) showed that no obvious abnormalities were found around the sample, which indicates that the injection gel has good compatibility with the surrounding tissues, reflecting good biocompatibility and tissue adaptability. Figure 5 ) slices were observed and it was found that there were still samples in the body, which shows that the hydrogel product of the present invention has excellent in vivo degradability and long-lasting filling performance.

[0081] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing an agarose-based hydrogel, characterized in that: Here are the steps: The curing agent and the dispersant are mixed evenly to prepare a curing system; the agarose solution and the curing system are placed in an alkaline solution to perform a curing reaction, and the film is removed and cleaned to obtain an agarose-based hydrogel; The curing agent is selected from one or more of epichlorohydrin, 1,2,7,8-diepoxyoctane, and 1,4-butanediol diglycidyl ether; The dispersant is selected from dimethyl sulfoxide or anhydrous ethanol; The solidifying agent accounts for 0.5-10% of the mass of the agarose solution; the dispersing agent accounts for 1-30% of the mass of the agarose solution; The curing reaction conditions are: reaction temperature is 50-70°C, reaction time is 2-72h; The preparation method of the agarose solution is as follows: agarose powder is placed in water, and dissolved at 80-125° C. for 15 min-5 h to obtain an agarose solution with a concentration of 0.5-20%.

2. The method according to claim 1, characterized in that The alkaline solution is selected from sodium hydroxide solution, potassium hydroxide solution, calcium hydroxide solution, and barium hydroxide solution, and the concentration of the alkaline solution is selected from 0.5 to 3M.

3. An agarose-based hydrogel prepared by the method according to any one of claims 1 to 2.

4. Use of the agarose-based hydrogel according to claim 3 in the preparation of facial fillers.

5. An agarose-based hydrogel facial filler, characterized in that: It is composed of a muscle base solution and the agarose-based hydrogel according to claim 3; wherein the dosage ratio of the agarose-based hydrogel to the muscle base solution is selected from 1:0.01 to 1:1, g:mL.

6. The facial filler according to claim 5, characterized in that The base fluid is selected from one or more of phosphate buffer, physiological saline, hyaluronic acid, collagen, chitosan, amino acids, cellulose, and sodium alginate; wherein the concentration of the base fluid is selected from 0.1 to 10%.

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