CN115894969A - Agarose-based hydrogel, and preparation method and application thereof - Google Patents

Abstract

The invention discloses an agarose-based hydrogel, a preparation method and application thereof, and belongs to the technical field of injection filling and beauty treatment. The agarose-based hydrogel is prepared by the following method: uniformly mixing a curing agent and a dispersing agent to prepare a curing system; and placing the agarose solution and the curing system in an alkaline solution, carrying out curing reaction, and stripping and cleaning to obtain the agarose-based hydrogel. According to the invention, the hydrophilic agarose-based hydrogel suitable for facial filling is obtained by directly solidifying agarose, so that the problems of difficulty in purification, obvious granular sensation and the like caused by using modified agarose microspheres are avoided, and the safety of injection gel is improved. The injection gel prepared by the invention is uniform, fine and smooth, has no granular sensation, has better cohesion, can support skin and tissues delicately, and is not easy to move and dissociate; moreover, the medicinal composition has no stimulation to skin, long local retention time, good plasticity and less side effect, and can realize long-acting filling.

Description

Agarose-based hydrogel, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of injection filling beauty, and particularly relates to an agarose-based hydrogel, and a preparation method and application thereof.

Background

Beauty medicine shows vigorous vigor in China. The facial soft tissue filling is one of the minimally invasive treatment means for treating facial volume tissue loss, contour change and static wrinkles at present, and the ideal soft tissue filling material has good biocompatibility, effectiveness and safety. At present, the facial soft tissue filling material mainly comprises non-autologous tissue injections, such as Hyaluronic Acid (HA), polycaprolactone (PCL) and poly (L-lactic acid) (PLLA), and in addition, some filling materials derived from autologous tissues, such as fat grafts and platelet-rich plasma, and the like. Although hyaluronic acid is still the most popular degradable injection product, the product has short degradation period, and frequent multiple injections are needed to maintain the filling effect. Therefore, finding an effective and safe tissue filler has been a continuing challenge for orthopedic and cosmetic procedures. In order to achieve the long-term filling effect, people try to use biodegradable materials to prepare microspheres as fillers, such as polyvinyl alcohol (PVA), polymethyl methacrylate (PMMA) and the like. Although the filling effect of these fillers is maintained for a significantly longer time, these materials remain in the body too long, releasing harmful substances and thus causing a series of side reactions with high later safety risks.

Agarose is a biodegradable polymer, and the similarity of the natural polymer and extracellular matrix makes people strongly interested in the application of the natural polymer in the field of dermal fillers. Like HA, agarose is a biocompatible polysaccharide with immediate and long-lasting clinical results. However, it differs from HA in that it is stable in vivo and provides a long-lasting filling effect after tissue implantation.

The reason why the prior art can not directly prepare a hydrogel product suitable for facial filling by using agarose powder as a raw material is that agarose is generally heated to more than 90 ℃ in water to be dissolved, and when the temperature is reduced to 35-40 ℃, a good semisolid gel is formed, the gel strength is high, and the agarose gel can not be pushed and injected by a syringe, which is also the reason that the agarose gel can only be generally used as an electrophoresis support. Therefore, the current agarose filling products need to prepare agarose powder into agarose microspheres firstly, and then prepare hydrogel products suitable for facial filling by taking the agarose microspheres as raw materials; for example, in chinese patent CN104774337A, a preparation method of agarose microsphere-containing cross-linked sodium hyaluronate gel for injection is provided.

The preparation method of agarose microspheres includes an emulsion-solidification method, a spray method, a membrane emulsification method and a microfluidic method, and the above methods have a series of problems, for example, the organic solvent is consumed during the preparation, the complete removal cannot be guaranteed, or the prepared microspheres have a wide particle size distribution and non-smooth particles. Therefore, agarose microspheres are selected as raw materials to prepare or to be mixed and crosslinked with sodium hyaluronate to prepare gel, and the defects of excessive addition of organic reagents, nonuniform particle size distribution, obvious granular sensation and the like exist, so that the probability of inflammatory reaction is increased (Zhang Sai. Preparation modification and application of agarose gel microspheres).

The invention aims to overcome the problems of complex process, obvious granular sensation, uneven particle size and the like of agarose microsphere gel, improve seamless fusion of agarose and muscle base fluid, enable the prepared hydrogel to have a finer pushing effect, and improve the safety and effectiveness of the agarose microsphere gel as a facial filling product.

Disclosure of Invention

The technical scheme of the invention is as follows:

the invention provides a preparation method of agarose-based hydrogel, which comprises the following steps:

uniformly mixing a curing agent and a dispersing agent to prepare a curing system; and placing the agarose solution and the curing system in an alkaline solution, carrying out curing reaction, and stripping and cleaning to obtain the agarose-based hydrogel.

In the 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.

In the preparation method, the dispersant is selected from dimethyl sulfoxide or absolute ethyl alcohol.

In the preparation method, the curing agent accounts for 0.5 to 10 percent of the mass of the agarose solution; the dispersant accounts for 1 to 30 percent of the mass of the agarose solution.

In the preparation method, the curing reaction conditions are as follows: the reaction temperature is 50-70 ℃, and the reaction time is 2-72 h.

In the 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 0.5-3M. The volume of the alkaline solution is 0.1-10 times of that of the agarose solution.

In the above preparation method, the agarose solution may be prepared by the following method: putting the agarose powder into water, and dissolving the agarose powder for 15min to 5h at the temperature of between 80 and 125 ℃ to obtain an agarose solution with the concentration of between 0.5 and 20 percent.

In the preparation method, the steps of demoulding and cleaning are as follows: placing the reactant obtained by the curing reaction into purified water, stirring, cleaning, and filtering to obtain the agarose-based hydrogel; the cleaning speed is 50-400 rpm/min, the cleaning time is 4-48 h, and the cleaning solution is replaced every 2-6 h.

The present invention provides the agarose-based hydrogel prepared by the above method.

The invention provides the application of the agarose-based hydrogel in facial filling and beauty treatment.

The invention provides an application of the agarose-based hydrogel in the preparation of a facial filler.

The invention provides an agarose-based hydrogel facial filler, which consists of muscle base solution and agarose-based hydrogel; wherein, the dosage ratio of the agarose-based hydrogel to the muscle base fluid is selected from 1.01-1. Preferably from 2.

The muscle base solution can be one or more selected from phosphate buffer solution, physiological saline, hyaluronic acid, collagen, chitosan, amino acid, cellulose and sodium alginate; wherein the concentration of the muscle base solution is selected from 0.1 to 10 percent

The preparation method of the agarose-based hydrogel facial filler is simple, and the muscle base solution is fully dispersed in the agarose-based hydrogel. After the preparation is finished, conventional filling sterilization can be carried out.

The invention has the beneficial effects that:

the invention overcomes the prejudice in the prior art, obtains the hydrophilic agarose-based hydrogel suitable for facial filling by directly curing agarose, avoids the problems of difficult purification, obvious granular sensation and the like caused by using modified agarose microspheres, and improves the safety of injection gel. The injection gel prepared by the invention is uniform, fine and smooth, has no granular feeling, has better cohesion, can support skin and tissues delicately, and is not easy to move and dissociate; moreover, the gel has no stimulation to skin, long local retention time, good plasticity and few side effects, and can realize long-acting filling. The invention has the advantages of easy control of process conditions, few operation steps and stable product quality, and is suitable for large-scale production.

Drawings

FIG. 1 is the results of rheological testing;

FIG. 2 shows the result of the squeezing force test;

FIG. 3 is a tissue section HE X20 after 1 week of implantation;

FIG. 4 is a tissue section after 4 weeks of implantation, HE X20;

fig. 5 is a tissue section, HE x 20, 30 weeks after implantation.

Detailed Description

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

Example 1

Preparation of agarose-based hydrogel:

5g agarose powder in a beaker, adding 100mL purified water, 121 degrees C high temperature dissolution for 1h, obtain agarose solution. 2g of epichlorohydrin and 15g of dimethyl sulfoxide are placed in a centrifuge tube and are uniformly mixed to form a curing system. And adding the agarose solution and the curing system into 80mL of sodium hydroxide solution (1.2M), curing and reacting for 24h at 60 ℃, taking out a reaction product after the reaction is finished, putting the reaction product into purified water, stirring and cleaning, wherein the stirring speed is 100rpm/min, changing water every 4-8 h, and cleaning for 24h to obtain the agarose-based hydrogel.

And (3) preparing 20mL of phosphate buffer solution, adding the phosphate buffer solution into the 150g of the agarose-based hydrogel, uniformly dispersing, filling the gel into a pre-filled syringe, and performing moist heat sterilization to obtain the agarose-based hydrogel for injection and filling.

Example 2

Preparation of agarose-based hydrogel:

2g of the agarose powder was placed in a beaker, 50mL of purified water was added, and the mixture was dissolved at 121 ℃ for 30 hours to obtain an agarose solution. 0.7g of epichlorohydrin and 10g of dimethyl sulfoxide are put into a centrifugal tube and mixed evenly to form a curing system. And adding the agarose solution and the curing system into 50mL of sodium hydroxide solution (0.7M), curing and reacting for 24h at 60 ℃, taking out a reaction product after the reaction is finished, putting the reaction product into purified water, stirring and cleaning, wherein the stirring speed is 100rpm/min, changing water every 4-8 h, and cleaning for 24h to obtain the agarose-based hydrogel.

Preparing 50mL of 1% sodium hyaluronate solution, adding the solution into 100g of the agarose-based hydrogel, uniformly dispersing, filling the gel into a pre-filled syringe, and performing moist heat sterilization to obtain the agarose-based hydrogel for injection and filling.

Example 3

Preparing the agarose-based hydrogel:

10g agarose powder in a beaker, adding 200mL purified water, 121 degrees C high temperature solution for 30h, obtain agarose solution. 5g of epichlorohydrin and 50g of dimethyl sulfoxide are placed in a centrifuge tube and are uniformly mixed to form a curing system. And adding the agarose solution and the curing system into 160mL of sodium hydroxide solution (1M), curing and reacting for 12h at 60 ℃, taking out a reaction product after the reaction is finished, putting the reaction product into purified water, stirring and cleaning, wherein the stirring speed is 200rpm/min, changing water every 4-8 h, and cleaning for 24h to obtain the agarose-based hydrogel.

Preparing 70mL of 2% algal polysaccharide solution, adding the algal polysaccharide solution into 300g of the agarose-based hydrogel, uniformly dispersing, filling the gel into a pre-filled syringe, and performing moist heat sterilization to obtain the agarose-based hydrogel for injection and filling.

Example 4

Preparation of agarose-based hydrogel:

5g agarose powder in a beaker, adding 200mL purified water, 100 degrees C high temperature dissolution for 4h, obtain the agarose solution. Placing 2g of 1, 4-butanediol diglycidyl ether and 10g of dimethyl sulfoxide into a centrifugal tube, and uniformly mixing to form a curing system. And adding the agarose solution and the curing system into 100mL of sodium hydroxide solution (1M), curing and reacting for 6h at 55 ℃, taking out a reaction product after the reaction is finished, putting the reaction product into purified water, stirring and cleaning, wherein the stirring speed is 100rpm/min, changing water twice, and cleaning for 6h in total to obtain the agarose-based hydrogel.

And (2) adding 2g of sodium hyaluronate, 2g of collagen and 1g of amino acid into 50mL of physiological saline, dissolving and stirring, adding the mixture into 250g of the agarose-based hydrogel after complete dissolution, uniformly dispersing, filling the gel into a pre-filled syringe, and performing moist heat sterilization to obtain the agarose-based hydrogel for injection filling.

Comparative example 1

Placing 5g of agarose powder in 150mL of purified water, dissolving at the high temperature of 121 ℃ for 30min, taking out after dissolving, placing in a water bath at the temperature of 65 ℃ for heat preservation, and obtaining a water phase; placing 14g of span 80 in 300mL of liquid paraffin, stirring and mixing for 30min at 65 ℃ to obtain an oil phase; dissolving the water phase, taking out, adding into the oil phase, emulsifying and homogenizing for 10min; curing the homogenized solution for 1h at normal temperature; and after solidification, washing with ethanol, and performing suction filtration to obtain the agarose microspheres. Preparing 20mL of 1% sodium hyaluronate solution, adding the solution into the 100g of agarose microspheres for uniform dispersion, filling the gel into a pre-filled syringe, and sterilizing. The gel is agglomerated into a block after sterilization, and when the extrusion force is detected, the phenomenon of needle explosion occurs, so that the normal test cannot be performed, and the gel is not suitable for injection and cosmetic filling.

Comparative example 2

Adding 25g of agarose powder and 5g of non-crosslinked hyaluronic acid into 1000mL of phosphate buffer solution (pH 6.8), stirring and dissolving at 80 deg.C for 30min, cooling to room temperature, bottling, and sterilizing. Two-phase demixing phenomenon is found in the syringe after sterilization, which indicates that the product has low uniformity and is not suitable for injection and cosmetic filling.

(I) viscoelastic test

The agarose-based hydrogels prepared in examples 1 to 4 above were subjected to rheological (viscoelasticity) tests. The rheological properties are related to the ability of the filling to withstand different types of deformation and forces when implanted in different surface areas and planes. The disclosure of the rheological properties of the filled product described above will guide the clinician in selecting the desired filling for each area of the face, based on the pathology and the deformation forces acting on the area of interest. Therefore, the selection of dermal fillers with the correct rheological properties is a key factor in achieving the desired aesthetic effect of natural appearance, long-lasting.

The test conditions were as follows: the name of the instrument: a rheometer; testing a clamp: an upper 20mm flat plate; the lower plate is a 60mm flat plate; clearance: 1mm; description of the test: constant temperature 10 deg.c test and amplitude scanning.

The test results are shown in fig. 1:

the figure shows that the linear viscoelastic regions of the 4 groups of products are similar, the elastic modulus is about 500Pa, the viscous modulus is about 90Pa, the elastic modulus and the viscous modulus are moderate, the gel is soft and smooth, the shaping and lifting effect after injection is obvious, and no convex feeling exists. As is known, the ratio between G 'and G' can reflect the change between the viscosity and the elasticity of the gel, and the graph shows that the ratio of loss modulus G '/elastic modulus G' of four groups of hydrogel is more than 1, which indicates that the hydrogel prepared by the invention has good elastic performance, can be firmly remained at an injection site, maintains firm existing structural strength and is beneficial to the maintenance of the shaping effect after injection.

(II) Pushing force test

The hydrogel products prepared in example 1, example 2, comparative example 1 and comparative example 2 were subjected to a squeezing force test under the following conditions: the name of the instrument: a universal material testing machine; and (3) testing temperature: room temperature; and (3) testing distance: setting the test distance as a full scale; extrusion speed: the extrusion speed was set at 30mm/min.

The test results are shown in fig. 2:

as can be seen from the figure, the extrusion force of the gel in the example 1 and the example 2 is about 10N, the gel meets the standard requirement of injection filling products, the experience feeling is good, and the excellent injectability is shown; in the extrusion of comparative example 1, after the deformation of 10mm, the extrusion force is linearly reduced, which indicates that the needle explosion phenomenon occurs in the test process, and indicates that the gel has poor fluidity and the ductility is almost zero, so that the gel is not suitable for injection filling; the extrusion results of comparative example 2 show that the range of extrusion force fluctuates widely, indicating that the product has low continuity and poor uniformity, and is also not conducive to injection filling.

(III) physical and chemical Properties

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:

TABLE 1

As can be seen from Table 1, in terms of properties, the two hydrogels have great difference in shape, and the hydrogel prepared by the agarose microspheres is semitransparent milk white and has great viscosity; the hydrogel prepared by directly utilizing the agarose powder is transparent and has high elasticity, so that the hydrogel is high in deformation resistance and not easy to deform. Therefore, the agarose-based hydrogel prepared by the invention is more suitable for parts needing lasting plasticity, such as the chin, the nose bridge and the like. In the aspect of particle size, the hydrogel is prepared by directly taking the agarose powder as a raw material, so the hydrogel does not contain granular components, has fine texture and granular feeling, embodies superior cohesive force, is smoother to inject, is more natural to fill and is more uniform to degrade. In the aspect of curing agent residue, the hydrogel of the invention has no epoxy chloropropane residue detected, has higher safety, and can not cause inflammatory reaction by in vivo injection. In the aspect of pushing force, the pushing force of the two hydrogels meets the requirements of ergonomics and standards, so that an operator can perform better pushing operation. The hydrogel of the present invention, which has a degree of crosslinking of 2.1% which is lower than 10.22% of the reference example, shows that it has an excellent plastic effect, and a low degree of crosslinking (modification) reflects high safety in application and can reduce the allergic rate.

Comparative example 3

Effect of curing reaction temperature on agarose-based hydrogels:

5g agarose powder in a beaker, adding 100mL purified water, 121 degrees C high temperature dissolution for 1h, obtain the agarose solution. 2g of epichlorohydrin and 15g of dimethyl sulfoxide are placed in a centrifugal tube and are uniformly mixed to form a curing system. Adding the agarose solution and the curing system into 80mL of sodium hydroxide solution (1.2M), respectively carrying out curing reaction at 37 ℃, 38 ℃, 39 ℃ and 40 ℃ for 24h, after the reaction is finished, taking out the reaction product, finding that the uniform curing fails, and putting the reaction product in a centrifuge tube into purified water to be completely dispersed, wherein the reaction product still is liquid with high fluidity and cannot be cleaned and separated.

Comparative example 4

Effect of type of curing agent on agarose-based hydrogels:

2g agarose powder in a beaker, adding 50mL purified water, 121 degrees C high temperature solution for 30h, obtain agarose solution. 0.5g of adipic acid dihydrazide, 1g of carbodiimide and 10g of dimethyl sulfoxide are placed in a centrifuge tube and uniformly mixed to form a curing system. And adding the agarose solution and the curing system into 50mL of sodium hydroxide solution (0.7M), curing and reacting for 24h at 60 ℃, taking out a reaction product after the reaction is finished, putting the reaction product into purified water, stirring and cleaning, wherein the stirring speed is 100rpm/min, changing water every 4-8 h, and cleaning for 24h to obtain the agarose-based hydrogel.

Preparing 50mL of 1% sodium hyaluronate solution, adding the solution into 100g of the agarose-based hydrogel, uniformly dispersing, filling the gel into a pre-filled syringe, performing moist heat sterilization, finding that the product is hydrated after sterilization and loses gel characteristics, and solidifying and hardening the hydrated gel after the temperature is reduced to room temperature, so that the hydrated gel cannot be injected. The reason for this is that adipic Acid Dihydrazide (ADH) and carbodiimide (EDC) are commonly used crosslinking agents for carboxyl sites, and there is no carboxyl site in the molecular structure of agarose, so the carboxyl crosslinking method is not suitable for preparing agarose hydrogel, and the crosslinking effect is not good.

Comparative example 5

Influence of curing agent dosage on agarose-based hydrogels:

5g agarose powder in a beaker, adding 200mL purified water, 100 degrees C high temperature dissolution for 4h, obtain the agarose solution. 0.8g of 1, 4-butanediol diglycidyl ether and 10g of dimethyl sulfoxide are placed in a centrifugal tube and uniformly mixed to form a curing system. Adding the agarose solution and the curing system into 100mL of sodium hydroxide solution (1M), curing and reacting at 55 ℃ for 6h, and after the reaction is finished, taking out the reaction product, and finding that no gel is formed and the reaction system is yellow aqueous liquid. After the mixture is placed at room temperature, the fluidity is still strong, and the properties are not changed. Therefore, under the preparation parameters, the gel cannot be used as an injection filling gel because the gel has the supporting force and viscoelasticity required for filling an injection product.

On the basis of the preparation method, the dosage of the curing agent is continuously adjusted to 0.5g, 0.6g and 0.7g, and the curing reaction is carried out for 6 hours. After the reaction was completed, it was found that the reaction system was also a yellow aqueous liquid, and no gel was formed. After the mixture is placed at room temperature, the fluidity is still strong, and the properties are not changed. This indicates that, if the agarose powder is used as a raw material to directly prepare an agarose gel suitable for face-filling, the amount of the curing agent to be used needs to be increased.

Application example 1

In vivo implantation degradation test:

the injection gel prepared in example 1 was subjected to an in vivo rabbit implantation degradation test. Before the test, the rabbit hair on both sides of the spine of the rabbit is cut off, the rabbit hair is anesthetized by intravenous injection with or without the use of glucose according to the animal reaction condition during the test, the skin of an operation area is disinfected by iodine tincture and 75% ethanol according to the requirement of a conventional surgical operation, 4 implantation points are respectively selected at equal intervals of 2.5cm on both sides of the spine of the rabbit, each point is spaced by 2.5cm, and each point is implanted with 0.2mL of a sample. After implantation, animals were euthanized at 1 week, 4 weeks, and 30 weeks, respectively, the skin on both sides of the spinal column was separated, the subcutaneous material was exposed, the size and shape of the implanted sample were observed, whether the surrounding tissues were suppurative or not and the secretion was observed, and the surrounding tissue site specimen containing the material was taken, the tissue of about 0.5 to 1cm surrounding the wrapped specimen was excised, fixed with 10% formalin solution, dehydrated and transparent, paraffin-embedded, and serially sectioned to a thickness of 5 μm, stained with hematoxylin-eosin, and the histopathological conditions were observed under a microscope.

The test results are shown in fig. 3 to 5:

pathological results of 1 week (figure 3) and 4 weeks (figure 4) of implantation show that no obvious abnormality is seen around the sample, which indicates that the injection gel has better compatibility with surrounding tissues and shows good biocompatibility and tissue adaptability. When the section implanted for 30 weeks (fig. 5) was observed, it was found that the sample was still present in vivo, indicating that the hydrogel product of the present invention had excellent in vivo degradability and long-lasting filling property.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Claims (10)

1. The preparation method of the agarose-based hydrogel is characterized by comprising the following steps:

uniformly mixing a curing agent and a dispersing agent to prepare a curing system; and (3) placing the agarose solution and the curing system in an alkaline solution, carrying out curing reaction, and stripping and cleaning to obtain the agarose-based hydrogel.

2. The method according to claim 1, wherein the curing agent is selected from one or more of epichlorohydrin, 1,2,7, 8-diepoxyoctane, divinyl sulfone, and 1, 4-butanediol diglycidyl ether.

3. The method of claim 1, wherein the dispersant is selected from the group consisting of dimethyl sulfoxide and absolute ethanol.

4. The method of claim 1, wherein the curing agent accounts for 0.5-10% of the mass of the agarose solution; the dispersant accounts for 1 to 30 percent of the mass of the agarose solution.

5. The method of claim 1, wherein the curing reaction conditions are: the reaction temperature is 50-70 ℃, and the reaction time is 2-72 h.

6. The method according to claim 1, wherein the alkaline solution is selected from the group consisting of sodium hydroxide solution, potassium hydroxide solution, calcium hydroxide solution, barium hydroxide solution, and the concentration of the alkaline solution is selected from the group consisting of 0.5 to 3M.

7. An agarose-based hydrogel prepared by the method of any one of claims 1 to 6.

8. Use of the agarose-based hydrogel of claim 7 for facial filling and cosmetic and/or preparation of facial fillers.

9. An agarose-based hydrogel facial filler is characterized by consisting of muscle base fluid and agarose-based hydrogel; wherein, the dosage ratio of the agarose-based hydrogel to the muscle base fluid is selected from 1.01-1.

10. The facial filler according to claim 9, wherein the muscle base fluid is selected from one or more of phosphate buffer, physiological saline, hyaluronic acid, collagen, chitosan, amino acid, cellulose and sodium alginate; wherein the concentration of the muscle base fluid is selected from 0.1-10%.

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