KR20230013682A - Hyaluronic acid filler crosslinked by PEGDE and method for preparing the same - Google Patents

Abstract

Provided is a hyaluronic acid hydrogel filler with high safety and excellent physical properties and durability by using PEGDE, which has high biocompatibility, at a molar ratio lower than that of the existing widely used BDDE crosslinking agent, and a manufacturing method thereof.

Description

Hyaluronic acid filler crosslinked by PEGDE and method for preparing the same {Hyaluronic acid filler crosslinked by PEGDE and method for preparing the same}

The present invention relates to a hyaluronic acid filler crosslinked with PEGDE and a method for preparing the same.

Hyaluronic acid is a biomolecular substance in which repeating units composed of N-acetyl-D-glucosamine and D-glucuronic acid are linearly connected. Since hyaluronic acid has high biocompatibility, it is widely used as a soft tissue augmentation material for medical and cosmetic purposes, a so-called dermal filler.

Hyaluronic acid has a very short half-life of several hours and lacks elasticity when injected into the body, but it can increase durability and elasticity in the body through crosslinking. Existing hyaluronic acid filler products are mainly manufactured using chemical crosslinking agents such as 1,4-butanediol diglycidyl ether (BDDE) and divinyl sulfone (DVS). Recently, issues regarding side effects and safety of chemical crosslinking agents have been raised.

Therefore, a hyaluronic acid filler having excellent physical properties and durability while increasing safety by using a crosslinking agent with lower toxicity than conventional crosslinking agents at a lower molar ratio is required.

Republic of Korea Publication No. 10-2020-0032103 (2020.03.25)

According to one embodiment, a hyaluronic acid hydrogel filler having excellent physical properties and durability while using a low mol% of PEGDE relative to 1 mole of hyaluronic acid unit is provided, and a manufacturing method thereof.

One aspect provides a hyaluronic acid hydrogel filler comprising hyaluronic acid crosslinked with polyethylene glycol diglycidyl ether (PEGDE).

The polyethylene glycol diglycidyl ether (PEGDE) has an advantage of being less toxic than previously used crosslinking agents such as BDDE (1,4-butanediol diglycidyl ether) and DVS (divinyl sulfone). The half-lethal dose (LD ₅₀ ) for rats is 32 mg/kg for DVS and 1,134 mg/kg for BDDE, while PEGDE is over 5000 mg/kg, which is relatively safe.

According to one embodiment, the concentration of the polyethylene glycol diglycidyl ether is 0.05 to 0.2 mol%, 0.075 to 0.15 mol%, 0.09 to 0.11 mol% or 0.1 mol% of hyaluronic acid unit 1 mol, hyaluronic acid hydrogel Provide filler. The unit unit is composed of N-acetyl-D-glucosamine and D-glucuronic acid, and the average molecular weight of the unit unit may be about 378 or 378.3, and according to one embodiment, based on the Na-conjugated salt, about It could be 403.

According to one embodiment, the hyaluronic acid hydrogel may have a complex viscosity of 5 to 200 Pa.s or 30 to 60 Pa.s at 1 Hz as measured by a rheometer.

The filler may further include uncrosslinked hyaluronic acid or a salt thereof. The salt of hyaluronic acid may be, for example, sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, or cobalt hyaluronate, but is not limited thereto.

The average molecular weight of the hyaluronic acid may be 100,000 Da to 2,500,000 Da, for example, about 1,000,000 Da.

According to one embodiment, the polyethylene glycol diglycidyl ether is represented by Formula 1 below, and n may be 200, 250, 300, 350, 400, 450, or 500 on average.

[Formula 1]

The polyethylene glycol diglycidyl ether (PEGDE) has a molecular weight of 400 to 20000, 500 to 20000, 1000 to 20000, 2000 to 20000, 3000 to 20000, 4000 to 20000, 5000 to 20000, 10000 to 20000, or 15000 can be The average molecular weight of the polyethylene glycol diglycidyl ether (PEGDE) may be about 17725 (n = about 400).

According to one embodiment, the filler may further include an anesthetic. The anesthetic may be a local anesthetic, for example, lidocaine, bupivacaine, mepivacaine, or salts thereof, but is not limited thereto.

The hyaluronic acid hydrogel filler may be used as a material for tissue repair, deep body cavity wound dressing, body cavity wound dressing, joint synovial fluid supplementation, and the like. The hyaluronic acid hydrogel filler is a material for tissue repair, which is used for biological tissue filling, wrinkle improvement, facial contour correction, or volume restoration or volume of soft tissues such as lips, nose, hips, cheeks or breasts. It can be used for enlargement, and can also be used for whitening, skin tone improvement and moisturizing purposes. The hyaluronic acid hydrogel filler may be used for the purpose of wound healing and prevention of wound adhesion as a deep body cavity wound dressing material or a body cavity wound dressing material. The hyaluronic acid hydrogel filler is used to treat skin wrinkles and wrinkles, glabellar lines, nasolabial folds, chin folds, marionette lines, and lower jaw lines. (jawline), buccal commissure, perioral wrinkles, crow's feet, skin depressions, scars, temples, subdermal supports of eyebrows, cheekbones and buccal fat pads, tear troughs trough), nose, lips, cheeks, chin, perioral area, suborbital area, and cosmetic use or treatment of facial asymmetry.

Another aspect provides a pre-filled syringe filled with the hyaluronic acid hydrogel filler. A pre-filled syringe filled with hyaluronic acid hydrogel filler may be used for the aforementioned medical and cosmetic purposes.

According to another aspect, a method for producing a hyaluronic acid hydrogel filler is provided, which includes mixing hyaluronic acid or a salt thereof, polyethylene glycol diglycidyl ether, and an aqueous alkali solution and crosslinking them.

According to one embodiment, the polyethylene glycol diglycidyl ether may be mixed by 0.05 to 0.2 mol%, 0.075 to 0.15 mol%, 0.09 to 0.11 mol% or 0.1 mol% based on 1 mol of hyaluronic acid unit unit.

The average molecular weight of the hyaluronic acid may be 100,000 Da to 2,500,000 Da, for example, about 1,000,000 Da.

The hyaluronic acid unit is composed of N-acetyl-D-glucosamine and D-glucuronic acid, and the average molecular weight of the unit unit may be about 378 or 378.3, and about 403 days based on the Na-conjugated salt. can

The polyethylene glycol diglycidyl ether is represented by Formula 1 below, and n may be 200, 250, 300, 350, 400, 450, or 500 on average.

[Formula 1]

The polyethylene glycol diglycidyl ether (PEGDE) has a molecular weight of 400 to 20000, 500 to 20000, 1000 to 20000, 2000 to 20000, 3000 to 20000, 4000 to 20000, 5000 to 20000, 10000 to 20000, or 15000 can be The average molecular weight of the polyethylene glycol diglycidyl ether (PEGDE) may be about 17725 (n = about 400).

The aqueous alkali solution may be an aqueous solution of NaOH, KOH, NaHCO ₃ , LiOH, or a combination thereof. When using an aqueous sodium hydroxide (NaOH) solution, the concentration may be, for example, about 0.25M (about 1% (w/w)), and the pH may be 12 to 15, 13 to 15, 14 to 15, 12 to 14, 13 to 14, or 12 to 13. An acid aqueous solution may be used instead of the alkali aqueous solution, and for example, an aqueous solution of HCl, H ₂ SO ₄ , H ₂ PO ₄ or a combination thereof may be used. The concentration of the alkali or acid aqueous solution may have a pH of 12 or more or 3 or less.

The concentration of hyaluronic acid or its salt is 10 to 50 w/w%, 10 to 40 w/w%, 10 to 30 w/w% as a weight ratio of hyaluronic acid or its salt to the total weight of the mixture of hyaluronic acid or its salt and aqueous alkali solution. w%, 15 to 25 w/w%, or about 20 w/w%.

The manufacturing method may further include coarsely pulverizing the crosslinked hyaluronic acid hydrogel after the crosslinking reaction.

The manufacturing method may further include washing and swelling the hyaluronic acid hydrogel using a buffer solution after the step of crosslinking or coarsely pulverizing. The buffer solution is, for example, citric acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate, acetic acid, diethyl barbituric acid, sodium acetate, sodium chloride, TAPS (tris (hydroxy) Methyl) methylamino) propanesulfonic acid), Bicine (2-bis (2-hydroxyethyl) amino) acetic acid), Tris (tris (hydroxymethyl) ammonium methane), Tricine (N- (2-hydroxy-1, 1-bis(hydroxymethyl)ethyl)glycine), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TES (2-[[1,3-dihydroxy-2-( hydroxymethyl) propan-2-yl] amino] methanesulfonic acid) and PIPES (piperazine-N, N'-bis (2-ethanesulfonic acid)).

The manufacturing method may further include grinding the swollen hyaluronic acid hydrogel after washing and swelling. Grinding the swollen hyaluronic acid can facilitate filling in a syringe and injection through a needle.

The manufacturing method includes defoaming the pulverized hyaluronic acid hydrogel; Mixing and defoaming the pulverized hyaluronic acid hydrogel and an anesthetic; Alternatively, the method may further include mixing the pulverized hyaluronic acid hydrogel, the mixture of the anesthetic agent and uncrosslinked hyaluronic acid, and defoaming the mixture.

The manufacturing method may further include filling a container with the hyaluronic acid hydrogel. The container may be, for example, a vial or syringe, and the material may include glass, rubber, or plastic, but is not limited thereto.

The manufacturing method may further include sterilizing the container filled with the hyaluronic acid hydrogel. However, the sterilization step may not be performed by using sterilized raw materials and performing each step in an aseptic facility.

The hyaluronic acid hydrogel filler according to one embodiment may have high safety by crosslinking using PEGDE, which has better biocompatibility than BDDE.

The hyaluronic acid hydrogel filler according to one embodiment may have equal or superior elasticity and durability while using a lower mol% crosslinking agent than conventional manufacturing methods.

The hyaluronic acid hydrogel filler according to one embodiment may have excellent cosmetic effects such as skin moisturizing and wrinkle improvement by further promoting collagen expression than the hyaluronic acid filler prepared by the conventional manufacturing method.

1 and 2 show the results of measuring the complex viscosity of hyaluronic acid hydrogels prepared by mixing hyaluronic acid and PEGDE at various molar ratios. Specifically, the concentrations of hyaluronic acid are 15 mg/ml, 17 mg/ml, and 20 mg/ml, and for each of these, 0.05 mol%, 0.075 mol%, 0.1 mol%, 0.15 mol% of PEGDE based on 1 mol of hyaluronic acid unit unit mol%, or 0.2 mol%.
3 shows a process of subcutaneously injecting a hyaluronic acid filler into a mouse.
4 shows a replica manufacturing process and cycle for observing wrinkle patterns.
Figure 5 shows the results confirming that the persistence and volume retention in the body of the hyaluronic acid hydrogel filler according to one embodiment are superior to those of the hyaluronic acid fillers prepared with a BDDE crosslinking agent.
FIG. 6 shows the results confirming that the hyaluronic acid hydrogel according to an embodiment is superior to hyaluronic acid fillers prepared with a BDDE cross-linking agent in terms of promoting collagen production.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of hyaluronic acid hydrogel crosslinked with 0.05 mol% of PEGDE based on 1 mole of hyaluronic acid unit

Hyaluronic acid (average molecular weight of about 1,000,000 Da) was dissolved in NaOH aqueous solution (0.25 M, about 1% (w/w), pH 13 or higher) at 15 mg/ml, 17 mg/ml, or 20 mg/ml, respectively. 0.05 mol% of polyethylene glycol diglycidyl ether (PEGDE) was added to each solution, based on 1 mol of hyaluronic acid unit (1 mol of unit unit has an average molecular weight of 403 based on Na salt) A cross-linked hyaluronic acid hydrogel was prepared by reacting at 30° C. for 20 hours. The n of the PEGDE used was about 400 and the average molecular weight was about 17725.

The cross-linked hyaluronic acid gel was coarsely pulverized. The crushed gel was washed and swelled in 50 mM phosphate buffer, pH 6.8. The washed and swollen hyaluronic acid gel was pulverized to a size that could pass through the inner diameter of a syringe needle and filled into a syringe. The filled syringe was sealed with a rubber seal and autoclaved steam sterilization was performed.

The prepared hyaluronic acid gels are Example 1-1 (15 mg/ml hyaluronic acid), Example 1-2 (17 mg/ml), and Example 1-3 (20 mg/ml) according to the content of hyaluronic acid. distinguished by

Example 2: Preparation of hyaluronic acid hydrogel crosslinked with 0.075 mol% of PEGDE based on 1 mole of hyaluronic acid unit

A hyaluronic acid gel was prepared in the same manner as in Example 1, except that the concentration of PEGDE was 0.075 mol% relative to 1 mole of hyaluronic acid unit.

The prepared hyaluronic acid gels were prepared according to the content of hyaluronic acid in Example 2-1 (15 mg/ml of hyaluronic acid), Example 2-2 (17 mg/ml), and Example 2-3 (20 mg/ml). distinguished by

Example 3: Preparation of hyaluronic acid hydrogel crosslinked with 0.1 mol% of PEGDE based on 1 mole of hyaluronic acid unit

A hyaluronic acid gel was prepared in the same manner as in Example 1, except that the concentration of PEGDE was 0.1 mol% relative to 1 mole of hyaluronic acid unit.

The prepared hyaluronic acid gels were prepared according to the content of hyaluronic acid in Example 3-1 (15 mg/ml of hyaluronic acid), Example 3-2 (17 mg/ml), and Example 3-3 (20 mg/ml). distinguished by

Example 4: Preparation of hyaluronic acid hydrogel crosslinked with 0.15 mol% of PEGDE based on 1 mole of hyaluronic acid unit

A hyaluronic acid gel was prepared in the same manner as in Example 1, except that the concentration of PEGDE was 0.15 mol% relative to 1 mole of hyaluronic acid unit.

The prepared hyaluronic acid gels were prepared according to the content of hyaluronic acid in Example 4-1 (15 mg/ml of hyaluronic acid), Example 4-2 (17 mg/ml), and Example 4-3 (20 mg/ml). distinguished by

Example 5: Preparation of hyaluronic acid hydrogel crosslinked with 0.2 mol% of PEGDE based on 1 mole of hyaluronic acid unit

A hyaluronic acid gel was prepared in the same manner as in Example 1, except that the concentration of PEGDE was 0.2 mol% based on 1 mole of hyaluronic acid unit.

The prepared hyaluronic acid gels were prepared according to the content of hyaluronic acid in Example 5-1 (15 mg/ml of hyaluronic acid), Example 5-2 (17 mg/ml), and Example 5-3 (20 mg/ml). distinguished by

Experimental Example 1: Measurement of complex viscosity of hyaluronic acid hydrogel crosslinked with PEGDE

The complex viscosity of the hyaluronic acid gels of Examples 1 to 5 was measured. Complex viscosity was measured with a rheometer under the following measurement conditions.

[Measuring conditions]

Equipment: Kinexus rotational rheometer from Malvern; Geometry: PU SC0033 SS (diameter 20 mm); Measurement temperature: 25 degrees Celsius; Gap 0.5 mm; Complex viscosity measurement at 1 Hz.

The complex viscosity measurement results are shown in FIGS. 1 and 2 and Table 1 below.

Example hyaluronic acid content
(mg/ml) Molar ratio of PEGDE to hyaluronic acid (%) average complex viscosity
(Pa.s.) Complex viscosity range
(Pa.s.) 1-1 15 0.050 5.097 5 to 8 1-2 17 5.219 1-3 20 8.082 2-1 15 0.075 16.98 15 to 26 2-2 17 19.70 2-3 20 26.23 3-1 15 0.100 31.17 30 to 60 3-2 17 47.95 3-3 20 60.67 4-1 15 0.150 56.42 60 to 105 4-2 17 82.34 4-3 20 104.10 5-1 15 0.200 54.99 55 to 200 5-2 17 141.1 5-3 20 192.4

As a result of the experiment, the hyaluronic acid gel crosslinked by adding 20 mg/ml of hyaluronic acid and 0.1 mol% of PEGDE relative to 1 mole of hyaluronic acid unit has viscoelasticity of more than 60 Pas.s despite the use of a low crosslinking agent. This was found to be excellent.

Experimental Example 2: Comparison of crosslinking agent content and complex viscosity of hyaluronic acid hydrogel crosslinked with BDDE

A hyaluronic acid hydrogel was prepared in the same manner as in Examples 1 to 5, except that BDDE was used as a crosslinking agent. The complex viscosity was measured in the same manner as in Experimental Example 1, and the content of BDDE to achieve the same complex viscosity as that of the hyaluronic acid gel crosslinked with PEGDE was confirmed.

According to the experimental results, it was confirmed that PEGDE can achieve the same complex viscosity even with a lower mol% of 1/8 to 1/10 of BDDE. (See Table 2)

No Physical properties (complex viscosity, Pa.s, at 1Hz) PEGDE ratio (mol%) BDDE ratio (mol%) One 5-8 0.05 4 2 15-26 0.075 8 3 30-60 0.1 8.5 4 60-105 0.15 10 5 55-200 0.2 14.5

Experimental Example 3: Evaluation of durability and collagen production of hyaluronic acid gel crosslinked with PEGDE

20-week-old female hairless mice (body weight: 28 ± 1 g), 10 per group, were administered with the prepared hyaluronic acid filler to the same abdominal cavity. Wrinkles were evaluated on the 1st, 7th, 30th, 60th, 120th and 180th days after administration, and on the last 180th day, the subject was sacrificed and a biopsy (H&E stain) and immunological test (Western blot) were performed. The specific test method is as follows.

3-1. Experimental animal preparation

The experimental animals used in this experiment were 50 20-week-old female hairless mice (28 ± 1g). All experimental animals were allowed to freely consume solid feed and water throughout the entire experimental period, and the temperature was maintained at 22-24 ° C and the humidity was 60%. bred Group classification was classified into a total of 5 groups by random sampling method, each with 10 animals. Group classification and administered hyaluronic acid filler are shown in Table 3 below.

group classification Filler administered Dosage (μl) Number of animals per group (lives) A 0.9% physiological saline 400 10 B Restylane 400 10 C PEGDE (Example 3-3) 400 10 D High Viscoelasticity-1 (Revolline HARA-L) 400 10 E High Viscoelasticity-2 (Bellenard) 400 10

Among them, Restylane, Revolin Hara-L, and Bellenar are commercially available products, and all three products contain 20 mg/ml of hyaluronic acid, and Restylane contains 1 to 3 mol% of BDDE, high viscoelasticity-1 and high viscoelasticity. Viscoelastic-2 is a filler crosslinked by adding 1.9 mol% of BDDE. This experiment was reviewed by the Animal Experimentation Ethics Committee of Kyung Hee University and complied with the Animal Protection Act of the National Veterinary Science and Quarantine Service and the Animal Welfare Guidelines of the World Organization for Animal Health.

3-2. drug injection

For drug administration, Zoletil 50 ^® (10 mg/kg, ip; Virbac Laboratories, Carros, France) was injected intraperitoneally to anesthetize. In order to maintain the same administration site for each individual, the administration site was marked with a dot at each corner of a square model with a size of 1.2 cm x 1.2 cm at the bottom of both sides. Using a syringe (31 G), 400 μl of the hyaluronic acid filler was administered subcutaneously in the center of the square area. (See Fig. 3)

3-3. Observation of filler's volume retention

After filler administration, wrinkles and volume maintenance were observed. If the persistence of the injected filler is low, the volume decreases and wrinkles increase. Therefore, the volume change and persistence of the filler can be confirmed by observing the volume and wrinkles. After filler administration, photographs were taken under the same conditions, and the volume and shape were observed with the naked eye. After that, a replica was collected at the site of administration using a silicone polymer (SILFLO impression material, Flexico, England). Replica collection using silicone polymer was first anesthetized by intraperitoneal injection of Zoletil 50 ^® (10 mg/kg, ip; Virbac Laboratories), and then the silicone polymer was applied to the injection site and waited until sufficiently solidified before collection. For wrinkle pattern observation, a second replica was collected 7 days after the first administration and the first replica was collected, and a third replica (about 1 month after drug administration) was collected 21 days after the second replica was collected. Thereafter, replicas were collected 3 more times until the end of the experiment (6 months after drug administration), for a total of 6 replicas. (See Fig. 4) Volume and wrinkle indexes were measured from replicas taken using Visioline VL650 (Courage & Khazaka, Koln, Germany). The measured volume and wrinkle index were combined to confirm the volume retention over time after filler injection.

3-4. Experimental animal sacrifice and tissue processing

After 6 months of drug administration (day 168), all experimental animals were sacrificed after making the last silicone replica. All experimental animals were anesthetized by intraperitoneal injection of Zoletil 50 ^® (Virbac Laboratories, 10 mg/kg, ip), and then skin and muscle layers were collected in a size of 1.5 cm × 1.5 cm from the drug administration site.

Among the excised tissues, some tissues to be evaluated for collagen fibers by Western blotting were stored at -80 ° C, and some were post-fixed after 24 hours in 4% paraformaldehyde (PFA) fixative dissolved in 100 mM phosphate buffer. . Then, they were dehydrated in 70%, 80%, 90%, and 100% ethanol, deposited in xylene, and paraffin infiltrated into the tissue through a three-step process in a hard paraffin (Leica, Nussloch, Germany) solution. Invasive tissues were made into blocks to prepare sections, and fixed paraffin tissues were sectioned with a thickness of 5 μm using a paraffin section (Shandon Finesse 325, Thermo Electron Co., Cambridge, England), and coated slides. After attaching to the slide oven at 37 ℃ overnight.

3-5. Hematoxylin & Eosin (H&E) staining

H&E staining was performed for histological evaluation. The tissue deposited on the slide was reacted with Mayer's hematoxylin (Mayer's hematoxylin, Sigma Chemical Co., St. Louis, MO, USA) solution for 30 seconds, and then washed in running water for 10 minutes. After that, it was reacted with an eosin (Eosin, Sigma Chemical Co,) solution for 3 seconds. The stained tissues were dehydrated and sealed with Permount ^® (Fischer Scientific, Fair Lawn, NJ, USA). For histological evaluation, the presence or absence of the drug administered between the skin layer and the muscle layer and the degree of adverse reactions were observed.

3-6. Collagen type Ⅰ, Ⅱ Western blot

After drug administration, Western blotting was performed to measure collagen fiber expression by type. First, the tissue was pulverized, and the protein was extracted with lysis buffer, followed by centrifugation to obtain a supernatant. The protein concentration of the supernatant was quantified using Bio-Rad reagent, and 50 μg of protein was obtained. The extracted proteins were electrophoresed on a 10% sodium dodecyl sulfate (SDS)-polyarylamide gel, and the gel proteins were blotted with a nitro cellulose membrane. After blocking overnight with 5% skin milk, collagen type Ⅰ and collagen type Ⅱ primary antibodies were incubated overnight at 4℃, and washed three times at 10-minute intervals with tris-buffered saline with 0.05% tween 20 (TBST). . Next, each secondary antibody was reacted at room temperature for 1 hour, and then washed three times with TBST at 15-minute intervals. Finally, it was developed by chemiluminescence.

3-7. Collagen type Ⅰ, Ⅱ immunohistochemical staining

Immunohistochemistry was performed to measure collagen fiber expression by type of drug administration site. After selecting the same area for each group, the tissues were washed three times for 5 minutes in 50 mM PBS. After washing, the mixture was reacted with a 30% H ₂ O ₂ solution for 20 minutes. After the reaction, the cells were washed again in 50 mM PBS and reacted with a blocking solution for one hour before reacting with the antibody. In each tissue, collagen type I and collagen type II primary antibodies were diluted in a primary antibody solution containing 0.5% BSA and 0.5% sodium azide in 50 mM PBS at a ratio of 1:500, and reacted for 48 hours or more. After washing with 50 mM PBS, each corresponding secondary antibody was diluted in a secondary antibody solution containing 0.3% Triton X-100 in 50 mM PBS at a ratio of 1:200 and reacted for 2 hours. made it After washing with 50 mM PBS, the mixture was reacted for 1 hour in an avidine-biotin complex (Vector Laboratories, Burlingame, CA, USA). After washing with 50 mM PBS, a color development reaction was performed with a color developing agent containing hydrogen peroxide and DAB in 50 mM Tris buffer, followed by washing. Tissues that had undergone color development were sealed with Permount ^® (Fischer Scientific) through dehydration.

3-8. data processing

Tissue observation for H&E staining and collagen fiber type I and II measurement was performed using Image-Pro ^® Plus software (Media Cybernetic, Siver Spring, MD, USA). The mean and standard error mean (SEM) for each item of the results measured in this experiment were calculated using SPSS (ver. 18.0) statistical program. One-way ANOVA was used for comparison between groups, Duncan's post hoc test was performed according to the results, and the statistical significance level was set at P = 0.05.

3-9. Persistence of the injected filler in the body

The volume observation results of the injected filler are shown in FIG. 5 . 5, 1st corresponds to 1 day, 2nd to 7 days, 3rd to 30 days, 4th to 60 days, 5th to 120 days, and 6th to 180 days. According to FIG. 5, Restylane, Ribolin Hara-L, and Bellenar significantly decreased in volume after the 5th, but Example 3-3 filler (hyaluronic acid 20 mg/ml and PEGDE 0.1 mol%) even at the 6th volume was maintained. Therefore, the hyaluronic acid gel of Example 3-3 was confirmed to have superior durability in the body compared to commercially available BDDE cross-linked hyaluronic acid filler products even though PEGDE, which is less toxic than BDDE, was used at a lower molar ratio.

3-10. Effects of injected fillers on collagen expression

The expression patterns of type 1 collagen and type 2 collagen in the subcutaneous area injected with the hyaluronic acid gel of the present invention were confirmed by immunohistochemistry and Western blotting.

In FIG. 6, (A) is saline, (B) is Restylane, (C) is Example 3-3 (20 mg/ml of hyaluronic acid and 0.1 mol% of PEGDE), and (D) is Ribolin. -L, (E) is Belenar. According to FIG. 6, it was confirmed that the hyaluronic acid gel of Example 3-3 significantly increased the expression of Type 1 and Type 2 collagens compared to other commercially available hyaluronic acid fillers. Based on this, it can be seen that the hyaluronic acid gel cross-linked with PEGDE is superior to the gel cross-linked with BDDE in improving skin wrinkles, moisturizing, and elasticity.

Summarizing the above experimental results, the hyaluronic acid gel crosslinked with PEGDE has excellent physical properties even with a lower molar ratio of PEGDE than BDDE, has a longer duration in the body, and has excellent collagen expression effect, so it has not only wrinkle improvement effect but also skin moisturizing and elasticity. It was confirmed that the improvement effect was also excellent.

Claims (6)

Contains hyaluronic acid crosslinked with polyethylene glycol diglycidyl ether (PEGDE),
The concentration of the polyethylene glycol diglycidyl ether is 0.05 to 0.2 mol% relative to 1 mol of hyaluronic acid unit,
Hyaluronic acid hydrogel filler. According to claim 1,
The hyaluronic acid hydrogel filler has a complex viscosity of 5 to 200 at an angular velocity of 1 Hz as measured by a rheometer,
Hyaluronic acid hydrogel filler. According to claim 1,
The polyethylene glycol diglycidyl ether is represented by Formula 1 below, and n is an average of 400,
Hyaluronic acid hydrogel filler.
[Formula 1]

According to claim 1,
The hyaluronic acid hydrogel filler further comprises an anesthetic,
Hyaluronic acid hydrogel filler. A pre-filled syringe filled with the hyaluronic acid hydrogel filler of claim 1. Mixing hyaluronic acid or a salt thereof, polyethylene glycol diglycidyl ether, and an aqueous alkali solution and cross-linking the mixture,
The polyethylene glycol diglycidyl ether is mixed by 0.05 to 0.2 mol% relative to 1 mol of hyaluronic acid unit unit,
Manufacturing method of hyaluronic acid hydrogel filler.

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