A kind of small particle size agarose gel, preparation method and application thereof

Abstract

The present invention discloses a small-particle agarose gel, a preparation method and application thereof, and belongs to the field of medical cosmetology and biomedical technology. The agarose gel of the present invention is prepared by the following method: non-crosslinked agarose microspheres, a crosslinking agent and a dispersant are mixed, an alkaline solution is added, and non-static crosslinking is performed at 55 to 65° C. The crosslinking time is 7 to 15 hours. After the crosslinking is completed, the agarose gel is washed to obtain the agarose gel. The agarose gel prepared by the present invention has a small particle size, low residue, soft texture, good viscoelasticity, and good biocompatibility. Therefore, it can be prepared into a cosmetic injection for facial filling plasticity to increase the cosmetic effect, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of medical cosmetology and biomedical technology, and specifically relates to a small-particle agarose gel, a preparation method and application thereof. Background Art

[0002] Facial fillers have been widely used in maxillofacial beauty in recent years for wrinkle removal and facial contour modification. The cosmetic effects provided by micro-plastic surgery are mostly non-permanent, and the treatment plan is generally continuous rather than one-time. The price of a regular-sized hyaluronic acid ranges from 800 to 10,000 yuan, and substances such as hyaluronic acid and botulinum toxin are metabolized by the human body, so they need to be continuously injected to maintain the filling effect. Generally, they need to be re-injected every 6 to 12 months, which is very expensive. Because the commonly used sodium hyaluronate gel degrades too quickly, and patients have also put forward more demands on the efficacy of beauty products, most researchers have been trying to use other fillable substances to achieve longer-lasting filling effects.

[0003] Polymer facial fillers are divided into two categories: absorbable and non-absorbable. Absorbable fillers include collagen, hyaluronic acid, and poly-L-lactic acid, while non-absorbable fillers include polymethyl methacrylate, polyacrylamide, silicone, and agarose. The biggest advantage of non-absorbable fillers is that they can be permanently plasticized, once and for all, without the need for additional injections.

[0004] Agarose is a natural polysaccharide extracted from algae such as red algae. It has good biocompatibility and biodegradability. Considering the source of raw materials, agarose has higher biosafety than other non-absorbable fillers. The similarity of this natural carbohydrate polymer to the extracellular matrix has led to a strong interest in its use in the field of dermal fillers. Agarose is a biocompatible polysaccharide that can produce immediate and lasting clinical effects. Unlike sodium hyaluronate, agarose is stable in the body and can maintain a more lasting effect after tissue implantation.

[0005] Patent CN109897202A discloses a method for preparing agarose microspheres, but the agarose microspheres prepared by this method have a large particle size (400-1000 μm) and a wide particle size distribution range, and the amount of organic reagent and cross-linking agent used is large, which increases the difficulty of removal and may lead to a large degree of cross-linking. If the agarose microspheres or gels prepared by this method are used for filling, the patient may experience inflammation such as redness and swelling, increasing the risk of infection.

[0006] Patent CN105713212A provides a method for preparing agarose gel microspheres by cross-linking while balling. However, in this method, the amount of cross-linking agent epichlorohydrin added is 3 times the mass of agarose solid, and a large amount of cross-linking agent will make the agarose gel microspheres have a large degree of cross-linking, and the cross-linking agent is difficult to remove cleanly. If this method is used to prepare agarose gel for injection, the probability of inflammation in patients will increase.

[0007] Patent CN104774337A provides a method for preparing a cross-linked sodium hyaluronate gel containing agarose microspheres for injection. In this method, a large amount of cross-linking agent is used, resulting in a high degree of cross-linking of the gel, and there may be more cross-linking agent residues, which will increase the probability of inflammatory reactions. Moreover, according to the patent, the gel is used for filling the superficial skin in plastic surgery. Summary of the invention

[0008] The present invention provides a method for preparing agarose gel, which uses non-crosslinked agarose microspheres and a crosslinking agent for crosslinking, and the prepared agarose gel has a small particle size, a small amount of crosslinking agent, and low residue. Specifically, the technical solution of the present invention is as follows:

[0009] A method for preparing agarose gel, comprising the following steps:

[0010] The non-crosslinked agarose microspheres, the crosslinking agent and the dispersant are mixed, and an alkaline solution is added to perform non-static crosslinking at 55-65° C. for 7-15 hours. After the crosslinking is completed, the microspheres are washed to obtain an agarose gel.

[0011] In the above technical solution, the cross-linking agent is selected from epichlorohydrin or BDDE, or a mixture of the two.

[0012] In the above technical solution, the dispersant is selected from DMSO or anhydrous ethanol, or a mixture of the two.

[0013] In the above technical solution, the alkaline solution is selected from sodium hydroxide solution or potassium hydroxide solution, or a mixed solution of the two; the concentration of the alkaline solution is selected from 0.5 to 2.0M, preferably 0.8 to 1.5M.

[0014] In the above technical solution, the non-static cross-linking is selected from cross-linking under cyclotron oscillation or reciprocating oscillation conditions.

[0015] In the above technical solution, a salt solution is used to clean the cross-linked product; the salt solution is selected from disodium hydrogen phosphate solution, sodium dihydrogen phosphate solution or sodium chloride solution, or a mixed solution of any two or three thereof; the pH of the salt solution is selected from 6.8 to 7.6, and the osmotic pressure is selected from 280 to 350 mOsmol / L.

[0016] In the above technical solution, the cleaning may further include the steps of centrifugation or filtration.

[0017] In the above technical solution, the mass concentration of the prepared agarose gel is selected from 2 to 6%.

[0018] In the above technical solution, the mass ratio of non-cross-linked agarose microspheres, cross-linking agent and dispersant is selected from 100:0.6-1.2:10-20.

[0019] In the above technical solution, the mass volume ratio of non-cross-linked agarose microspheres to the alkaline solution is selected from 1:0.5 to 1:1.5, g / mL; preferably 1:1.

[0020] In the above technical solution, the non-cross-linked agarose microspheres are prepared by the following method:

[0021] The agarose is placed in water, heated at 95-130°C to dissolve, to form an agarose aqueous solution as the water phase; the emulsifier is placed in liquid paraffin, heated at 65-75°C, and stirred evenly to form an oil phase; the oil phase and the water phase are mixed, homogenized at 65-75°C for 30s-20min, the homogenate is cooled to below 10°C, and placed in a low temperature environment of 0-4°C to solidify for 1-2h, and after washing, non-cross-linked agarose microspheres are obtained.

[0022] In the above technical solution, the mass fraction of the agarose aqueous solution is 2-8%.

[0023] In the above technical solution, the emulsifier is selected from Span 80, Span 85 or Tween 80, or a mixture of any two.

[0024] In the above technical solution, the volume ratio of the emulsifier to the liquid paraffin is 1:20 to 1:25.

[0025] In the above technical solution, the volume ratio of the oil phase to the water phase is 1:1 to 2:1.

[0026] In the above technical solution, the homogenization temperature is selected from 65 to 75° C.; and the homogenization time is selected from 7 to 15 minutes.

[0027] In the above technical solution, the cooling means can be cooling by water bath or cooling by ice water bath.

[0028] In the above technical solution, the cleaning is selected from the group consisting of cleaning the solidified material with ethanol, dimethyl sulfoxide aqueous solution, ethyl acetate aqueous solution or salt solution. The salt solution is selected from the group consisting of sodium chloride solution, sodium dihydrogen phosphate solution or disodium hydrogen phosphate solution, or a mixture of any two or three thereof; the osmotic pressure of the salt solution is selected from the group consisting of 280 to 350 mOsmol / L.

[0029] To facilitate the cleaning of the solidified material, the solidified material can be centrifuged once before cleaning, that is, without adding any substances, it can be directly centrifuged, and most of the oil phase and emulsifier can be poured out, and the filter cake can be retained. After that, the filter cake can be cleaned by centrifugation or suction filtration until the oil phase, emulsifier, ethanol and other substances are washed away, and the mass fraction of agarose microspheres is ensured to be kept within a reasonable range.

[0030] The agarose gel prepared by the above method has small particle size, low residue, soft texture, good viscoelasticity and good biocompatibility, so it can be prepared into a cosmetic injection for facial plastic filling to increase the cosmetic effect.

[0031] The beneficial effects of the present invention are:

[0032] Unmodified agarose solution will remain in a semi-solid gel state at room temperature, with high hardness and poor viscosity. It is difficult to manipulate during medical or cosmetic injections, and the injection site has a strong sense of unevenness.

[0033] The present invention makes the prepared agarose microspheres smaller in size by homogenizing and adjusting the oil-water ratio, and the particle size range is 0 to 200 μm, wherein D 90 ≤55μm; and there is no cross-linking step in the preparation process; at the same time, in the process of preparing agarose gel, the particle size of agarose microspheres has not changed, and still maintains a small and excellent state. In the preparation process, less cross-linking agent is used and it is not easy to remain, which fully improves the biocompatibility and effectiveness of agarose gel, making the agarose gel soft and having good viscoelasticity. It can be continuously extruded when pushed, which is easy to operate, so that the effect after filling appears more natural and the plastic effect is long-lasting and effective.

[0034] At the same time, compared with similar products, the agarose gel prepared by the present invention is not only suitable for filling plasticity of nasolabial folds, nasolabial folds, neck lines, hand lines, etc., but also has a particle size D 90 ≤55μm, more suitable for filling fine wrinkles, with extensive and effective cosmetic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a microscope image of the non-cross-linked agarose microspheres described in Example 1;

[0036] Figure 2 This is a graph showing the particle size detection result of the laser particle size analyzer of the non-cross-linked agarose microspheres in the agarose gel described in Example 1;

[0037] Figure 3 This is a graph showing the particle size detection result of the laser particle size analyzer of the non-cross-linked agarose microspheres in the agarose gel described in Example 2;

[0038] Figure 4This is a graph showing the particle size detection result of the laser particle size analyzer of the non-cross-linked agarose microspheres in the agarose gel described in Example 3;

[0039] Figure 5 This is a graph showing the particle size detection result of the laser particle size analyzer of the non-cross-linked agarose microspheres in the agarose gel described in Example 4;

[0040] Figure 6 This is a graph showing the particle size detection result of the laser particle size analyzer of the non-cross-linked agarose microspheres in the agarose gel described in Comparative Example 4;

[0041] Figure 7 This is a graph showing the particle size detection result of the laser particle size analyzer of the non-cross-linked agarose microspheres in the agarose gel described in Comparative Example 5;

[0042] Figure 8 This is a graph showing the particle size detection results of the non-cross-linked agarose microspheres in the agarose gel described in Comparative Example 6 using a laser particle size analyzer. DETAILED DESCRIPTION

[0043] In the present invention, D 90 It refers to the particle size corresponding to when the cumulative particle size distribution percentage of a sample reaches 90%. Its physical meaning is that the particles with a particle size smaller than this value account for 90%. 10 , D 50 And so on.

[0044] 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.

[0045] Example 1

[0046] Preparation of non-cross-linked agarose microspheres:

[0047] Place 9.6g of agarose in 160mL of water, and heat and dissolve it in an autoclave (121°C, 30min). After dissolution, take it out and place it in a 65°C water bath to keep warm as the water phase. Place 14g of Span 85 in 300mL of liquid paraffin, and stir and mix at 65°C for 30min as the oil phase. Mix the oil phase and the water phase, and homogenize them with a homogenizer for 10min. Place the homogenate in an ice water bath to cool to below 10°C, and transfer it to a 4°C refrigerator to solidify for 1h. After solidification, wash the solid with a certain concentration of ethyl acetate aqueous solution and purified water in turn to obtain non-cross-linked agarose microspheres. The non-cross-linked agarose microspheres prepared in this example are observed under a microscope as shown in the following figure. Figure 1 shown.

[0048] Prepare agarose gel:

[0049] 10 g of non-cross-linked agarose microspheres, 0.1 g of epichlorohydrin and 2 g of dimethyl sulfoxide were mixed, 10 mL of sodium hydroxide solution (1 M) was added, and the mixture was shaken and cross-linked at 60° C. for 10 h. After the cross-linking was completed, the microspheres were washed with PBS solution (pH=7.2) to obtain agarose gel.

[0050] The physicochemical properties of the above agarose gel are shown in Table 1:

[0051] Table 1

[0052]

[0053] Example 2

[0054] Preparation of non-cross-linked agarose microspheres:

[0055] Place 9.6g of agarose in 160mL of water and heat and dissolve it in an autoclave (121°C, 30min). After dissolution, take it out and place it in a 65°C water bath to keep warm as the water phase. Place 14g of Span 85 in 300mL of liquid paraffin and stir and mix at 65°C for 30min as the oil phase. Mix the oil phase and the water phase, homogenize them with a homogenizer for 7min, place the homogenate in an ice water bath to cool below 10°C, and transfer it to a 4°C refrigerator to solidify for 1h. After solidification, wash the solid with a certain concentration of dimethyl sulfoxide aqueous solution and purified water in turn to obtain non-cross-linked agarose microspheres.

[0056] Prepare agarose gel:

[0057] 10 g of non-cross-linked agarose microspheres, 0.1 g of epichlorohydrin and 2 g of dimethyl sulfoxide were mixed, 10 mL of sodium hydroxide solution (1 M) was added, and the mixture was shaken and cross-linked at 60° C. for 10 h. After the cross-linking was completed, the microspheres were washed with PBS solution (pH=7.2) to obtain agarose gel.

[0058] The physicochemical properties of the above agarose gel are shown in Table 2:

[0059] Table 2

[0060]

[0061] Example 3

[0062] Preparation of non-cross-linked agarose microspheres:

[0063] Place 9.6g of agarose in 160mL of water and heat and dissolve it in an autoclave (121°C, 30min). After dissolution, take it out and place it in a 65°C water bath to keep warm as the water phase. Place 14g of Span 85 in 300mL of liquid paraffin and stir and mix at 65°C for 30min as the oil phase. Mix the oil phase and the water phase, homogenize them with a homogenizer for 15min, place the homogenate in an ice water bath to cool below 10°C, and transfer it to a 4°C refrigerator to solidify for 1h. After solidification, wash the solid with a certain concentration of ethyl acetate aqueous solution and purified water in turn to obtain non-cross-linked agarose microspheres.

[0064] Prepare agarose gel:

[0065] 10 g of non-cross-linked agarose microspheres, 0.1 g of epichlorohydrin and 2 g of dimethyl sulfoxide were mixed, 10 mL of sodium hydroxide solution (1 M) was added, and the mixture was shaken and cross-linked at 60° C. for 10 h. After the cross-linking was completed, the microspheres were washed with PBS solution (pH=7.2) to obtain agarose gel.

[0066] The physicochemical properties of the above agarose gel are shown in Table 3:

[0067] Table 3

[0068]

[0069] Example 4

[0070] Preparation of non-cross-linked agarose microspheres:

[0071] Place 9.6g of agarose in 160mL of water and heat and dissolve it in an autoclave (121°C, 30min). After dissolution, take it out and place it in a 65°C water bath to keep warm as the water phase. Place 14g of Span 85 in 300mL of liquid paraffin and stir and mix at 65°C for 30min as the oil phase. Mix the oil phase and the water phase, homogenize them with a homogenizer for 7min, place the homogenate in an ice water bath to cool to below 10°C, and transfer it to a 4°C refrigerator to solidify for 1h. After solidification, wash the solid with a certain concentration of ethyl acetate aqueous solution and purified water in turn to obtain non-cross-linked agarose microspheres.

[0072] Prepare agarose gel:

[0073] 10 g of non-cross-linked agarose microspheres, 0.06 g of epichlorohydrin and 2 g of dimethyl sulfoxide were mixed, 10 mL of sodium hydroxide solution (1 M) was added, and the mixture was shaken and cross-linked at 60° C. for 7 h. After the cross-linking was completed, the microspheres were washed with PBS solution (pH=7.2) to obtain agarose gel.

[0074] The physicochemical properties of the above agarose gel are shown in Table 4:

[0075] Table 4

[0076]

[0077] Comparative Example 1

[0078] Preparation of non-cross-linked agarose microspheres:

[0079] Place 9.6g of agarose in 160mL of water and heat and dissolve it in an autoclave (121°C, 30min). After dissolution, take it out and place it in a 65°C water bath to keep warm as the water phase. Place 14g of Span 85 in 300mL of liquid paraffin and stir and mix at 65°C for 30min as the oil phase. Mix the oil phase and the water phase, homogenize them for 10min using a homogenizer, place the homogenate in an ice water bath to cool to below 10°C, and transfer it to a 4°C refrigerator to solidify for 1h. After solidification, wash the solid with a certain concentration of ethyl acetate aqueous solution and purified water in turn to obtain non-cross-linked agarose microspheres.

[0080] Preparation of agarose-sodium hyaluronate gel:

[0081] 10g of non-crosslinked agarose microspheres and 2g of dimethyl sulfoxide were mixed, 10mL of sodium hydroxide solution (1M) was added, and the mixture was shaken and crosslinked at 60°C for 10h. After the crosslinking was completed, the mixture was washed with PBS solution (pH=7.2) to obtain an agarose gel. 1g of sodium hyaluronate was added to the agarose gel and stirred and dissolved to obtain an agarose-sodium hyaluronate gel. In this embodiment, sodium hyaluronate can increase the lubricity of the finished product.

[0082] When the agarose-sodium hyaluronate gel was tested for pushing force, both the 30G needle and the 27G needle were installed and the needle burst, and the test could not be performed normally. When some gel was scooped out of the syringe, it was found that the gel was in block shape and hard in texture, which was not suitable for injection and cosmetic filling.

[0083] Comparative Example 2

[0084] Preparation of non-cross-linked agarose microspheres:

[0085] Place 9.6g of agarose in 160mL of water and heat and dissolve it in an autoclave (121°C, 30min). After dissolution, take it out and place it in a 65°C water bath to keep warm as the water phase. Place 14g of Span 85 in 300mL of liquid paraffin and stir and mix at 65°C for 30min as the oil phase. Mix the oil phase and the water phase, homogenize them for 10min using a homogenizer, place the homogenate in an ice water bath to cool to below 10°C, and transfer it to a 4°C refrigerator to solidify for 1h. After solidification, wash the solid with a certain concentration of ethyl acetate aqueous solution and purified water in turn to obtain non-cross-linked agarose microspheres.

[0086] Prepare agarose gel:

[0087] 10 g of non-cross-linked agarose microspheres, 0.02 g of epichlorohydrin and 2 g of dimethyl sulfoxide were mixed, 10 mL of sodium hydroxide solution (1 M) was added, and the mixture was shaken and cross-linked at 60° C. for 10 h. After the cross-linking was completed, the microspheres were washed with PBS solution (pH=7.2) to obtain agarose gel.

[0088] The agarose gel above has lumps, indicating that the cross-linking is insufficient and the thermal stability of the gel is poor.

[0089] Comparative Example 3

[0090] Preparation of non-cross-linked agarose microspheres:

[0091] Place 9.6g of agarose in 160mL of water and heat and dissolve it in an autoclave (121°C, 30min). After dissolution, take it out and place it in a 65°C water bath to keep warm as the water phase. Place 14g of Span 85 in 300mL of liquid paraffin and stir and mix at 65°C for 30min as the oil phase. Mix the oil phase and the water phase, homogenize them for 10min using a homogenizer, place the homogenate in an ice water bath to cool to below 10°C, and transfer it to a 4°C refrigerator to solidify for 1h. After solidification, wash the solid with a certain concentration of ethyl acetate aqueous solution and purified water in turn to obtain non-cross-linked agarose microspheres.

[0092] Prepare agarose gel:

[0093] 10 g of non-cross-linked agarose microspheres, 0.05 g of epichlorohydrin and 2 g of dimethyl sulfoxide were mixed, 10 mL of sodium hydroxide solution (1 M) was added, and the mixture was shaken and cross-linked at 60° C. for 10 h. After the cross-linking was completed, the microspheres were washed with PBS solution (pH=7.2) to obtain agarose gel.

[0094] The agarose gel above showed stratification, indicating that the cross-linking was insufficient and the thermal stability of the gel was poor.

[0095] Comparative Example 4

[0096] Preparation of non-cross-linked agarose microspheres:

[0097] Place 9.6g of agarose in 160mL of water and heat and dissolve it in an autoclave (121°C, 30min). After dissolution, take it out and place it in a 65°C water bath to keep warm as the water phase. Place 14g of Span 85 in 300mL of liquid paraffin and stir and mix at 65°C for 30min as the oil phase. Mix the oil phase and the water phase, homogenize them with a homogenizer for 7min, place the homogenate in an ice water bath to cool to below 10°C, and transfer it to a 4°C refrigerator to solidify for 1h. After solidification, wash the solid with a certain concentration of ethyl acetate aqueous solution and purified water in turn to obtain non-cross-linked agarose microspheres.

[0098] Prepare agarose gel:

[0099] 10 g of non-cross-linked agarose microspheres, 0.2 g of epichlorohydrin and 2 g of dimethyl sulfoxide were mixed, 10 mL of sodium hydroxide solution (1 M) was added, and the mixture was shaken and cross-linked at 60° C. for 10 h. After the cross-linking was completed, the microspheres were washed with PBS solution (pH=7.2) to obtain agarose gel.

[0100] The physicochemical properties of the above agarose gel are shown in Table 5:

[0101] Table 5

[0102]

[0103] Comparative Example 5

[0104] Preparation of non-cross-linked agarose microspheres:

[0105] Place 9.6g of agarose in 160mL of water and heat and dissolve it in an autoclave (121°C, 30min). After dissolution, take it out and place it in a 65°C water bath to keep warm as the water phase. Place 18.7g of Span 85 in 400mL of liquid paraffin and stir and mix at 65°C for 30min as the oil phase. Mix the oil phase and the water phase, homogenize them for 10min using a homogenizer, place the homogenate in an ice water bath to cool to below 10°C, and transfer it to a 4°C refrigerator to solidify for 1h. After solidification, wash the solid with a certain concentration of ethyl acetate aqueous solution and purified water in turn to obtain non-cross-linked agarose microspheres.

[0106] Prepare agarose gel:

[0107] 10 g of non-cross-linked agarose microspheres, 0.2 g of epichlorohydrin and 2 g of dimethyl sulfoxide were mixed, 10 mL of sodium hydroxide solution (1 M) was added, and the mixture was shaken and cross-linked at 60° C. for 10 h. After the cross-linking was completed, the microspheres were washed with PBS solution (pH=7.2) to obtain agarose gel.

[0108] The physicochemical properties of the above agarose gel are shown in Table 6:

[0109] Table 6

[0110]

[0111] Comparative Example 6

[0112] Preparation of non-cross-linked agarose microspheres:

[0113] Place 9.6g of agarose in 160mL of water and heat and dissolve it in an autoclave (121°C, 30min). After dissolution, take it out and place it in a 65°C water bath to keep warm as the water phase. Place 14g of Span 85 in 300mL of liquid paraffin and stir and mix at 65°C for 30min as the oil phase. Mix the oil phase and the water phase, stir for 2h using a polytetrafluoroethylene stirring paddle, place the reactants in an ice water bath to cool to below 10°C, and transfer to a 4°C refrigerator to solidify for 1h. After solidification, wash the solid with ethyl acetate and purified water in turn to obtain non-cross-linked agarose microspheres.

[0114] Prepare agarose gel:

[0115] 10 g of non-cross-linked agarose microspheres, 0.2 g of epichlorohydrin and 2 g of dimethyl sulfoxide were mixed, 10 mL of sodium hydroxide solution (1 M) was added, and the mixture was shaken and cross-linked at 60° C. for 10 h. After the cross-linking was completed, the microspheres were washed with PBS solution (pH=7.2) to obtain agarose gel.

[0116] The physicochemical properties of the above agarose gel are shown in Table 7:

[0117] Table 7

[0118]

[0119]

[0120] (I) Cytotoxicity test

[0121] The agarose gels prepared in Examples 1-4 and Comparative Examples 4-5 were selected for cytotoxicity test, and the test method was as follows:

[0122] Weigh 1.0g of sample respectively and place it in 30mL cell culture medium. Under 37℃, shake and extract at 60rpm for 24h to prepare the extract. Centrifuge the extract and take the supernatant as the test solution. Mouse fibroblast L929 was used, which was purchased from the Kunming Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences. The experiment used cells that were vigorously grown for 48-72h to prepare the cell suspension, and incubated in the incubator for 24h to form a nearly confluent monolayer of cells to ensure that the cell growth in each plate well was relatively equal. The original culture medium was aspirated, and 100μL of the test solution was added to each of the 6 wells in each column. The wells were placed in a cell culture incubator and cultured for 24h. The cell morphology of each well was observed under a microscope. After removing the culture medium, 50μL of MTT culture medium was added, and isopropanol was added after continuing to culture in the cell culture incubator for 2h. The absorbance was measured with an enzyme marker according to the formula: Survival rate (%) = (100×OD 样品 ) / OD 介质 , calculate the survival rate.

[0123] Set up negative control, positive control and medium control. The positive control group is 10% DMSO solution, which is prepared and used immediately; the negative control group is polyethylene, which is prepared every 6 cm 2 The negative control was added with 1 mL of cell culture medium and extracted at 37°C for 24 h at 60 rpm; the medium control group was cell culture medium without the test sample and was treated in the same way as the test group.

[0124] The test results are shown in Table 8:

[0125] Table 8

[0126] sample Survival rate Example 1 95％ Example 2 96％ Example 3 95％ Example 4 97％ Comparative Example 4 86％ Comparative Example 5 79％ Medium control group - Negative control group 98％ Positive control group 7％

[0127] (II) Intradermal reaction test

[0128] The agarose gel of Example 1 and Comparative Example 4 was prepared into an injection, and the preparation method was as follows: the agarose gel was filled into a prefilled syringe and sterilized. The obtained injection was subjected to a maximum dose test in rabbits according to GB / T 14233.2-2005 and GB / T 16886.10-2005 "Biological Evaluation of Medical Devices Part 10: Irritation and Delayed Hypersensitivity Tests" to evaluate the potential intradermal reaction of the product.

[0129] The scoring criteria for erythema and edema are shown in Table 9:

[0130] Table 9

[0131] reaction Scoring erythema Scoring No erythema 0 Very slight erythema (barely visible) 1 Clear erythema 2 Moderate erythema 3 Severe erythema (purple red) to eschar formation 4 Edema Scoring No edema 0 Very slight edema (barely visible) 1 Clear edema (swelling that does not extend beyond the edge of the area) 2 Moderate edema (swelling about 1mm) 3 Severe edema (swelling greater than 1 mm and beyond the contact area) 4 Stimulate the highest score 8

[0132] Note: Record and report any other abnormalities at the injection site

[0133] After scoring at (72±2)h, all erythema and edema scores of each animal's test sample or blank control at (24±2)h, (48±2)h and (72±2)h were added together, and then divided by 15 [3 (scoring time point) × 5 (test sample or blank control injection point)] to calculate the score of each animal's test sample or blank control. The scores of the three animals were added together and divided by 3 to obtain the total average score of each test sample and the corresponding blank control. The final score of the test sample was obtained by subtracting the blank control score from the test sample score.

[0134] The scoring results are shown in Table 10:

[0135] Table 10

[0136]

[0137]

[0138] It can be seen from Table 10 that the intradermal reaction of the gel sample prepared in Example 1 was 0 points, which met the national standard requirements, and the gel prepared in Comparative Example 4 was scored 3.5 points, which did not meet the standard requirements.

[0139] 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 agarose gel, characterized in that: Here are the steps: The non-cross-linked agarose microspheres, the cross-linking agent and the dispersant are mixed, and an alkaline solution is added to perform non-static cross-linking at 55-65°C for 7-15 hours. After the cross-linking is completed, the microspheres are washed to obtain an agarose gel. The mass ratio of the non-cross-linked agarose microspheres, the cross-linking agent and the dispersant is selected from 100:0.6~1.2:10~20; The cross-linking agent is selected from one or both of epichlorohydrin and BDDE; the dispersant is selected from one or both of DMSO and anhydrous ethanol; The non-cross-linked agarose microspheres are prepared by the following method: The agarose is placed in water, heated at 95-130°C to dissolve, and an agarose aqueous solution is formed as the water phase; the emulsifier is placed in liquid paraffin, heated at 65-75°C, and stirred evenly to form the oil phase; the oil phase and the water phase are mixed, homogenized at 65-75°C for 30s-20min, the homogenate is cooled to below 10°C, and placed in a low temperature environment of 0-4°C for solidification for 1-2h, and non-cross-linked agarose microspheres are obtained after washing; The volume ratio of the oil phase to the water phase is 1:1 to 2:

1.

2. The preparation method according to claim 1, characterized in that: The mass volume ratio of the non-cross-linked agarose microspheres to the alkaline solution is selected from 1:0.5 to 1:1.

5.

3. The preparation method according to claim 1, characterized in that: The alkaline solution is selected from one or both of a sodium hydroxide solution and a potassium hydroxide solution.

4. The preparation method according to claim 1, characterized in that: The volume ratio of emulsifier to liquid paraffin is 1:20~1:

25.

5. The preparation method according to claim 1, characterized in that: The emulsifier is selected from one or more of Span 80, Span 85 and Tween 80.

6. Agarose gel prepared by the method according to any one of claims 1 to 5.

7. Use of the agarose gel according to claim 6 in the preparation of cosmetic injections.

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