KR101356320B1 - A long-lasting injectable hyraluronic acid dermal filler developed by pullulan microsphere - Google Patents

Abstract

The present invention relates to a skin filler composition and, more specifically, to a skin filler composition containing hyaluronic acid gel, comprising pullulan acetate microspheres, as an active ingredient. Since the skin filler composition in the present invention contains the hyaluronic acid gel comprising the pullulan acetate microspheres, the degradation speed of the hyaluronic acid gel is much slower than hyaluronic acid gel itself, thereby being stable in vivo for a long time. Also, the hyaluronic acid gel comprising the pullulan acetate microspheres gives viscoelasticity to the skin since it is more viscous than the existing hyaluronic acid gels, improving roles of cushioning a blow and buffer, promoting the production of main components including collagen, and helping the components to maintain skin tissues. Thus, the skin filler composition in the present invention will be very valuable as a semipermanent filler material with biocompatibility. [Reference numerals] (AA) Cell viability(%); (BB) Hyaluronic acid; (CC) Hyaluronic acid bridge gel; (DD) Hyaluronic acid bridge gel including PA microspheres

Description

A long-lasting injectable hyraluronic acid dermal filler developed by pullulan microsphere}

The present invention relates to a skin filler composition, and more particularly to a skin filler composition comprising a hyaluronic acid gel containing pullulan acetate microspheres as an active ingredient.

Aging begins at birth, and wrinkles on the skin are one of the signs of life aging. The causes of skin aging are largely divided into external factors and intrinsic factors. External factors include sunlight, gravity, and smoking. Intrinsic factors include genetic factors.

Many procedures to supplement the skin aging and to have a young appearance have been performed. Among them are surgery to remove wrinkles, botox, and fillers. In the case of surgery, scars remain and take a long time, and Botox uses neurotoxins, which cause a lot of side effects. On the other hand, the filler (fiiler) using the polymer present in the living body has been used a lot because it is possible to save time with less side effects and simple procedures.

Fillers are applied to dermis in the skin to improve wrinkles and are mainly used to improve nasolabial folds, vertical folds on the mouth, wrinkles on the lips, contours of the lips, and acne scars. It is estimated that the filler market will grow by more than 10% every year as the number of fillers increases significantly.The domestic market has grown by nearly 20% from 60 billion won in 2009 to 70 billion won in 2010, and will increase to 100 billion won. It is a prospect.

The filler was first performed by autologous lithography in the 1890s. Since then, paraffin and liquid silicone have been used, but due to serious side effects, it is now illegal in many countries including the United States and Korea. Subsequently, a filler using collagen was invented, and Zyderm and Zyplast are representative collagen fillers. However, these collagen fillers require allergic testing one month before the procedure because animal-derived collagen is used, and has a relatively short lifespan of 3 to 6 months (Baumann, L., J. Kaufman, and S. Saghari, Collagen). fillers.Dermatologic therapy, 2006. 19 (3): p. 134-140.).

To supplement this, a hyaluronic acid-based filler was developed. Representatives include Hylaform and Restylane / Perlane, and their lifespan is twice as long as 6 to 12 months compared to collagen.

Hyaluronic acid is a natural polymer that is present in many skins such as animals. In addition, hyaluronic acid is present in the living body as epidermis, cartilage, vitreous fluid of eye, and lubricant of joint. It is hydrophilic because of its high number of hydroxyl groups and can contain water 1000 times its own weight. 7 ~ 8g hyaluronic acid is present in the human body, and plays an important role in maintaining the structure of the skin and normal operation. The high molecular weight hyaluronic acid has a high viscosity, which imparts viscoelasticity to the skin and acts as a shock absorber and lubricant. It also promotes the production of major substances such as collagen and helps maintain skin tissue.

The hyaluronic acid having the above characteristics is widely used for medical materials, medical devices, cosmetics, etc., because of excellent biocompatibility and viscoelasticity, such as ophthalmic surgical aids, joint function improving agents, drug delivery materials and eye drops.

However, hyaluronic acid itself is limited in its use because it is easily degraded in vivo or under conditions such as acids and alkalis (half life in vivo is about 1 to 2 days). Therefore, efforts to develop structurally stable hyaluronic acid derivatives have been widely progressed. As one of the hyaluronic acid derivatives, hyaluronic acid crosslinked products which covalently bond hyaluronic acids with a crosslinking agent have excellent biocompatibility, physical stability and biodegradation, and are described in US Pat. Nos. 4,716,224, 4,963,666, 5,017,229, 5,356, 883 and the like disclose methods for their preparation.

However, the hyaluronic acid crosslinked materials prepared by these methods also have relatively low stability against hyaluronic acid degrading enzymes and heat, and it is difficult to remove unreacted chemicals, and thus there is a limit to use them as high biocompatible materials.

Korean Patent Publication No. 10-2007-0004159 Korean Patent Publication No. 10-2011-0138765

Accordingly, the present inventors have made diligent efforts to develop a composition for long-term stable skin filling by including biocompatible beads in a hyaluronic acid crosslinked product. As a result, the hyaluronic acid crosslinked product is made using pullulan acetate microspheres having biocompatibility and excellent biodegradability. In the case of forming, the present invention was completed by confirming that the decomposition is well delayed over time while being suitable for a living body compared with a conventional hyaluronic acid crosslinked product.

Accordingly, an object of the present invention is to provide a skin filler composition having biocompatibility and excellent long-term persistence.

In order to achieve the above object of the present invention, the present invention provides a skin filler composition comprising a hyaluronic acid gel containing pullulan acetate microspheres as an active ingredient.

 In one embodiment of the invention, the pullulan acetate microspheres are (a) adding pyridine and acetic anhydride to the solution dissolved by adding pullulan to formamide; (b) dropping the finished solution in water to obtain a precipitate and purifying it; And (c) lyophilizing the purified final reactant.

 In one embodiment of the invention, the pullulan acetate microspheres may have a size of 20-100 micrometers.

 In one embodiment of the present invention, the hyaluronic acid gel containing the pullulan acetate microspheres is dissolved by adding and dissolving pullulan acetate microspheres and hyaluronic acid in an aqueous alkali solution, followed by stirring and mixing, and then adding and reacting a crosslinking agent. Forming a primary crosslinked gel; And (ii) mixing the primary crosslinked gel prepared in step (iii) with an aqueous alkali solution dissolved by adding pullulan acetate microspheres and hyaluronic acid, and then reacting with a crosslinking agent to form a second crosslinked gel. It may be prepared through a process comprising a.

 In one embodiment of the invention, the alkaline aqueous solution may be a sodium hydroxide solution having a concentration of 0.2N.

 In one embodiment of the invention, the hyaluronic acid may be added to the aqueous alkali solution at a concentration of 50mg / ml to 100mg / ml.

 In one embodiment of the invention, the pullulan acetate microspheres may be added to the aqueous alkali solution at a concentration of 15mg / ml to 25mg / ml.

 In one embodiment of the invention, the crosslinking agent may be used butanediol diglycidyl ether (1,4-butandiol diglycidyl ether).

 In one embodiment of the invention, the present invention may be a filler (filler) that can be injected into the skin soft tissue.

 The skin filler composition of the present invention contains pullulan acetate microspheres in the hyaluronic acid gel, so that the rate of decomposition is significantly delayed compared to the hyaluronic acid gel itself, and a long-term stable effect can be expected in vivo. In addition, the viscosity is higher than the conventional hyaluronic acid gel to impart viscoelasticity to the skin to improve the role of impact relief and lubricating oil, promote the production of major substances such as collagen and helps maintain skin tissue. Therefore, the skin filler composition of the present invention is expected to be very valuable as a semi-permanent filler material with biocompatibility.

1 is a graph confirmed by 1 H-NMR for structural analysis of pullulan acetate.
2 is a scanning electron micrograph of pullulan acetate microspheres.
Figure 3 is a schematic diagram showing the preparation of hyaluronic acid gel crosslinked using butanediol diglycidyl ether (1,4-butandiol diglycidyl ether).
Figure 4 (a) is a photograph showing the viscosity of the gel according to the concentration of the hyaluronic acid addition, cross-linking agent added, Figure 4 (b) is a photograph showing the hyaluronic acid gel prepared through the primary cross-linking and secondary cross-linking (Reference FIG. 4 shows a hyaluronic acid gel not containing pullulan acetate fine particles).
Figure 5 shows a simplified schematic diagram of a cross-linked (a) hyaluronic acid gel and (b) hyaluronic acid gel containing microspheres.
Figure 6 is a graph showing the measurement of the viscosity of the hyaluronic acid gel containing pullulan acetate microspheres prepared in Example 2 of the present invention.
Figure 7 is a graph showing the molecular weight change over time after the treatment of hyaluronidase in the hyaluronic acid gel containing the pullulan acetate microspheres prepared in Example 2 of the present invention.
8 is a graph showing the cytotoxicity of hyaluronic acid gels containing pullulan acetate microspheres prepared in Example 2 of the present invention by MTT assay.
9 (a) is a photograph taken at 5 and 7 days after injection of hyaluronic acid gel containing pullulan acetate microspheres prepared in Example 2 of the present invention into BALB / c nude mice and the like. 9 (b) shows tissues at 5 days after 7 days of injecting hyaluronic acid gel containing pullulan acetate microspheres prepared in Example 2 of the present invention to BALB / c nude mice and the like. It was taken after dyeing with H & E staining method.

Hereinafter, the present invention will be described in detail.

The present invention is characterized by providing a skin filler composition comprising a hyaluronic acid gel containing pullulan acetate microspheres as an active ingredient.

In the present invention, 'pululan' refers to a neutral and linear extracellular polysaccharide, in which glucose produced by the Aureobasidium pullulans strain is linked to α (1 → 4) or (1 → 6). it means. These flulans are edible natural polysaccharides with no toxicity and mutagenicity, and are characterized by being easily degraded by flulanase, isofluranase, and the like.

In the present invention, "pullulan acetate microspheres" means microspheres composed of pullulan acetate having the following formula (1).

The pullulan acetate microspheres of the present invention comprises the steps of: (a) adding pyridine and acetic anhydride to the solution dissolved by adding pullulan to formamide; (b) dropping the finished solution in water to obtain a precipitate and purifying it; And (c) lyophilizing the purified final reactant.

Step (a) of the present invention is a step of acetate to pullulan in order to introduce amphiphilic to the pullulan, in detail pullulan is added to formamide and dissolved by vigorous stirring at 45 ℃ to 55 ℃ , Pyridine and acetic anhydride are added and reacted at 45 ° C. to 55 ° C., respectively.

Step (b) of the present invention is a step of dropping the solution of the reaction of step (a) in water to obtain a precipitate to purify, step (c) is a reactant purified through the step (b) Freezing and drying to finally obtain pullulan acetate microspheres.

In one embodiment of the invention, the pullulan acetate microspheres may have a size of 20-100 micrometers.

The skin filler composition of the present invention may include the hyaluronic acid gel containing the pullulan acetate microspheres as an active ingredient.

That is, the skin filler composition of the present invention comprises a hyaluronic acid gel as an active ingredient, hyaluronic acid as an active ingredient of the composition is characterized in that it contains pullulan acetate microspheres.

In the present invention, 'hyaluronic acid' is a concept including both hyaluronic acid itself and hyaluronic acid salt, the hyaluronic acid salt is sodium hyaluronate, potassium hyaluronic acid, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronic acid, cobalt hyaluronic acid And hyaluronic acid may be one or more selected from the group consisting of tetrabutylammonium.

The molecular weight of hyaluronic acid is not particularly limited, but may be in the range of 100,000 to 5,000,000 Daltons to implement various physical properties and biocompatibility.

In one embodiment of the present invention, the hyaluronic acid gel containing the pullulan acetate microspheres of the present invention is dissolved by adding pullulan acetate microspheres and hyaluronic acid to an aqueous alkali solution, followed by stirring and mixing, and then adding a crosslinking agent. Reacting to form a primary crosslinked gel; And (ii) mixing the primary crosslinked gel prepared in step (iii) with an aqueous alkali solution dissolved by adding pullulan acetate microspheres and hyaluronic acid, and then reacting with a crosslinking agent to form a second crosslinked gel. It may be prepared through a process comprising a.

The step (iii) of the present invention is a step of forming a primary crosslinked gel, in detail, by adding and dissolving pullulan acetate microspheres and hyaluronic acid in an aqueous alkali solution, stirring and mixing, and then adding and reacting a crosslinking agent. Forming a crosslinked gel.

As the alkaline aqueous solution that can be used in the step (iii) of the present invention, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia water, and the like may be used, and preferably sodium hydroxide solution. Moreover, it is preferable for dissolution and crosslinking reaction of hyaluronic acid that concentration of aqueous alkali solution is 0.1N-10N.

In the step (iii) of the present invention, the concentration of hyaluronic acid added to the aqueous alkali solution may be 50 mg / ml to 100 mg / ml, and the pullulan acetate microsphere may be 15 mg / ml to 25 mg / ml.

The crosslinking agent that can be used in the step (iii) of the present invention is not particularly limited as long as it is a compound containing two or more epoxy functional groups, but preferred examples thereof are butanediol diglycidyl ether (1,4-butandiol). diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), hexanediol diglycidyl ether (1,6-hexanediol diglycidylether), propylene glycol diglycidyl ether ), Polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neoopentyl glycol diglycidyl ether, polyglycol poly Glycidyl ether (polyglycerol polyglycidyl ether), diglycerol polyglycidyl ether (glycerol polyglycidyl ether), glycerol polyglycidyl ether Glycerol polyglycidyl ether, trimethylpropane polyglycidyl ether, bisepoxypropoxy ethylene (1,2- (bis (2,3-epoxypropoxy) ethylene), pentaerythritol poly Glycidyl ether (pentaerythritol polyglycidyl ether) and sorbitol polyglycidyl ether (sorbitol polyglycidyl ether) may be selected, most preferably butanediol diglycidyl ether (BDDE) is preferred. .

In the step (iii) according to the present invention, the primary crosslinked gel is formed by reacting an epoxy functional group of the crosslinker with an alcohol group of hyaluronic acid.

Step (ii) of the present invention is a step of forming a secondary crosslinked gel, in detail, the primary crosslinked gel prepared through the step (iii) in an aqueous alkali solution dissolved by adding pullulan acetate microspheres and hyaluronic acid. After mixing, the crosslinking agent is added to form a secondary crosslinked gel.

The pullulan acetate microspheres, hyaluronic acid, aqueous alkali solution and crosslinking agent used in step (ii) of the present invention are the same as those used in step (iii).

Therefore, step (ii) of the present invention may be performed in the same manner as step (iii) except for mixing the first crosslinked gel prepared in step (iii) with an aqueous alkali solution.

The hyaluronic acid concentration of the gel finally formed through the steps (iii) and (ii) of the present invention may be 20mg / ml to 50mg / ml.

The hyaluronic acid gel of the present invention, prepared through the above process, has excellent long-term persistence (half-life due to long decomposition half-life) as well as biocompatibility, such as wrinkle improving agent, molding aid, joint function improving agent, anti-adhesion agent, etc. It can be used as a medicine, cosmetic or cosmetic material to maintain a certain form without any side effects in the introduced state, preferably can be useful as a filler (filler) material that can be injected into the skin soft tissue.

This fact was confirmed through the following experimental example of the present invention, the viscosity of the hyaluronic acid gel containing the pullulan acetate microspheres of the present invention was measured, compared with a gel crosslinked using only hyaluronic acid (Comparative Example 1) It was confirmed that the viscosity is further enhanced (see FIG. 6).

In addition, in another experimental example of the present invention, as a result of measuring the resolution by hyaluronidase affecting the fast half-life of hyaluronic acid, the pullul of the present invention was compared with a gel crosslinked using only hyaluronic acid (Comparative Example 1). In the case of the hyaluronic acid gel containing lan acetate microspheres, it was confirmed that the decomposition time was significantly increased (see FIG. 7).

In another experimental example of the present invention, the cytotoxicity of the hyaluronic acid gel containing the pullulan acetate microspheres of the present invention was evaluated, and it was confirmed that no toxicity was observed in the In Vitro experiment (see FIG. 8).

In another experimental example of the present invention, as a result of measuring the stability and intracellular inflammatory response of the hyaluronic acid gel containing the pullulan acetate microspheres of the present invention (In Vivo), a gel crosslinked using only hyaluronic acid (Comparative Example 1 In case of), hyaluronic acid cross-linked gels containing pullulan acetate microspheres retained their structure even after 7 days. It has been shown to induce proliferation of fibroblasts such as blast cells (see FIGS. 8 and 9 below).

Through these results, the present inventors have experimentally demonstrated that hyaluronic acid gel containing pullulan acetate microspheres can be usefully used as a filler material that can be injected into skin soft tissues.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

The Pullulan  acetate Microsphere  Produce

To prepare the pullulan acetate microspheres of the present invention, 1 g of pullulan (molecular weight 100,000 Da, Tokyo Chemical Industry) was dissolved in 30 mL formamide, followed by reaction for 24 hours by adding 7 mL of pyridine and 9 mL of acetic anhydride. The reaction solution was purified by dropping a drop into 200ml of water to obtain a precipitate, and the final reactant (pullulan acetate microspheres) was obtained through lyophilization.

The obtained reactant (pullulan acetate microspheres) was confirmed to have been synthesized by structural analysis through 1H-NMR (see FIG. 1), and the prepared microspheres were confirmed by scanning electron microscopy (see FIG. 2 below). ).

< Example  2>

The Pullulan  acetate Microspheres  Containing hyaluronic acid gel

Hyaluronic acid having a molecular weight of 330 kDa was added to a concentration of 0.2 mg sodium hydroxide at a concentration of 100 mg / mL, and vortexing was performed by adding pullulan acetate microspheres prepared in Example 1 at a concentration of 20 mg / mL. After thoroughly mixing through, butanediol diglycidyl ether (1,4-butandiol diglycidyl ether: BDDE) as a crosslinking agent was added to each concentration (2, 10, 20% by weight) was mixed sufficiently. The material was then stored in a 40 ° C. dry oven for 2 hours and then dialyzed with PBS to titrate to pH 6.9-7.4. The degree of crosslinking was confirmed by gel formation in the photograph, and a small particle was formed by breaking the gel with a homogenizer.

The primary crosslinked gel formed through the above process was again vortexed by addition to hyaluronic acid (500 mg) and the fluan acetate microspheres (20 mg / mL) prepared in Example 1 to a 0.2 N sodium hydroxide solution. After thorough mixing through (voltexing), finally, the pullulan acetate microspheres of the present invention are added through a process of sufficiently adding 50 μl of a crosslinking agent, butanediol diglycidyl ether (BDDE). A hyaluronic acid gel containing was prepared. The degree of crosslinking was confirmed by gel formation in a photograph, and the gel was broken by a homogenizer to form small particles, which was used as a sample in the following experiment.

< Comparative Example  1>

Hyaluronic Acid Gel Preparation

Hyaluronic acid gel to be used as a comparative example was carried out in the same manner as in Example 2, it was prepared without the addition of the pullulan acetate microspheres prepared in Example 1 above.

< Experimental Example  1>

The Pullulan  acetate Microparticle  Containing hyaluronic acid Gel  Viscosity measurement

The viscosity of the hyaluronic acid gel containing the pullulan acetate microspheres of the present invention prepared in Example 2 was measured according to the shear rate of 0.025s ^-1 using a DV-II + P viscometer.

As a result, as shown in FIG. 6, the gel crosslinked using only hyaluronic acid (Comparative Example 1) had a viscosity of 233,000 cps, whereas the hyaluronic acid gel containing the pullulan acetate microspheres of the present invention was 335,000 cps. It was confirmed that the viscosity was significantly increased compared to the comparative example.

< Experimental Example  2>

The Pullulan  acetate Microparticle  Containing hyaluronic acid Gel Hyaluronidaa My( Hyaluronidase Decomposition degree measurement by

The degree of hydrolysis of the hyaluronic acid gel containing the pullulan acetate microspheres of the present invention prepared in Example 2 was measured. In detail, 100 mg of hyaluronic acid gel of the present invention was added to a PBS buffer in which hyaluronidase was dissolved at a concentration of 150 units / mL, and the degree of hyaluronic acid degradation was examined over time. At this time, the degree of decomposition of hyaluronic acid was confirmed as a relative comparison with the existing hyaluronic acid 300kDa by measuring the molecular weight using GPC.

As a result, as shown in Fig. 7, the hyaluronic acid gel containing the pullulan acetate microspheres of the present invention is slow to decompose over time compared to the gel crosslinked using only hyaluronic acid (Comparative Example 1). I could confirm it. That is, the graph of Figure 7 shows that the molecular weight is different depending on the time appearing on the x-axis, the faster the molecular weight is higher and the slower the molecular weight is smaller. Therefore, in the case of microsphere-containing hyaluronic acid gel, the peak can be confirmed at a slow time, and it can be seen that the decomposition rate is slower than that of the gel with only hyaluronic acid compared to the same time.

< Experimental Example  3>

The Pullulan  acetate Microparticle  Containing hyaluronic acid Gel  Toxicity Assessment

Crosslinking agents added to improve the viscoelasticity of hyaluronic acid can be toxic in vivo when high doses are added. Therefore, in this experiment, the cytotoxicity of the hyaluronic acid gel containing the pullulan acetate microspheres of the present invention prepared in Example 2 was evaluated.

First, 1 × 104 cells / well of NIH3T3 cells were placed on a 96 well plate and incubated for 24 hours. Then, 100 mg of the hyaluronic acid gel of the present invention prepared in Example 2 was eluted in PBS for one day, and then Treated and incubated for 24 hours. After incubation, MTT assay was performed. At this time, as a control group, a gel crosslinked using only hyaluronic acid itself treatment group and hyaluronic acid (Comparative Example 1) was used.

As a result, as shown in Figure 8, it was confirmed that the hyaluronic acid gel containing the pullulan acetate microspheres of the present invention as compared to the control group also has no cytotoxicity.

< Experimental Example  4>

The Pullulan  acetate Microparticle  Hyaluronic acid crosslinking Gel  Stability assessment ( in vivo )

In order to examine the stability and action in vivo of the hyaluronic acid gel containing the pullulan acetate microspheres of the present invention prepared in Example 2, after the injection of the hyaluronic acid gel of the present invention in animal models H & E (Hematoxylin Inflammation was confirmed by staining.

Briefly, the tissue at 5 and 7 days after injecting 50 μl of hyaluronic acid gel containing pullulan acetate microspheres of the present invention prepared in Example 2 into BAL / c nude mice, which are animal models Was extracted and H & E (Hematoxylin-Eosin) staining was confirmed through photographs and micrographs.

As a result, as shown in Fig. 9 (a), in the case of the gel crosslinked using only hyaluronic acid (Comparative Example 1) after 7 days, the structure was not maintained by water absorption, but containing pullulan acetate microspheres It was confirmed that the hyaluronic acid crosslinked gel retained its structure even after 7 days.

In addition, as shown in Fig. 9 (b), in the treatment group of the hyaluronic acid gel containing the pullulan acetate microspheres of the present invention, it was found that the macrophage and neutrophils were not observed when 5 days and 7 days elapsed. It was found that no reaction occurred. And it was confirmed that the action of hyaluronic acid induced the proliferation of fibroblasts, such as fibroblasts.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (9)

A skin filler composition comprising a hyaluronic acid gel containing pullulan acetate microspheres as an active ingredient. The method of claim 1,
The pullulan acetate microspheres are reacted by adding (a) pyridine and acetic anhydride to a solution obtained by adding pullulan to formamide; (b) dropping the finished solution in water to obtain a precipitate and purifying it; And (c) lyophilizing the purified final reactant. The method of claim 1,
Wherein said pullulan acetate microspheres have a size of 20-100 micrometers. The method of claim 1,
The hyaluronic acid gel containing the pullulan acetate microspheres is (i) dissolved by adding the pullulan acetate microspheres and the hyaluronic acid to an aqueous alkali solution, followed by stirring and mixing, followed by addition reaction to form a crosslinked gel. step; And (ii) mixing the primary crosslinked gel prepared in step (iii) with an aqueous alkali solution dissolved by adding pullulan acetate microspheres and hyaluronic acid, and then reacting with a crosslinking agent to form a second crosslinked gel. Skin filler composition, characterized in that it is prepared through a process comprising a. 5. The method of claim 4,
The alkaline aqueous solution is a skin filler composition, characterized in that the sodium hydroxide solution having a concentration of 0.2N. 5. The method of claim 4,
The hyaluronic acid is a skin filler composition, characterized in that added to the aqueous solution at a concentration of 50mg / ml to 100mg / ml. 5. The method of claim 4,
The pullulan acetate microspheres are added to the aqueous alkaline solution at a concentration of 15mg / ml to 25 mg / ml skin filler composition. 5. The method of claim 4,
The crosslinking agent is a butanediol diglycidyl ether (1,4-butandiol diglycidyl ether) skin filler composition, characterized in that. The method according to any one of claims 1 to 8,
The composition is a skin filler composition, characterized in that the filler (filler) that can be injected into the skin soft tissue.

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