KR20210102602A - Hyaluronic acid hydrogel stabilizing a bio-active substance and the method for preparing the same - Google Patents

Abstract

The present invention relates to a hydrogel capable of stably maintaining the activity of a bioactive material for a long period of time by encapsulating the bioactive material, and a method for producing the same, and specifically, to a hyaluronic acid hydrogel that is environmentally friendly by producing the hydrogel by using minimal organic solvents and can control a particle size by controlling the concentration of hyaluronic acid and the bioactive material used in the synthesis of the hydrogel, and a method for producing the same.

Description

Hyaluronic acid hydrogel stabilizing a bioactive material and a method for preparing the same

The present invention relates to a hydrogel capable of encapsulating a bioactive material so that the activity of the material can be stably maintained for a long period of time and a method for producing the same. It relates to a hyaluronic acid hydrogel capable of controlling the particle size by controlling the concentration of hyaluronic acid and bioactive material used for synthesis, and a method for preparing the same.

Representative methods among conventional hydrogel synthesis techniques include a synthesis method using desolvation, a synthesis method using organic solvent evaporation, and the like. In particular, gelatin-based hydrogels through desolvation and albumin-based hydrogels through organic solvent evaporation can easily encapsulate bioactive molecules during the hydrogel synthesis process, and the size of the prepared hydrogel is several hundred nanometers. It has the advantage that it can be synthesized in various sizes with 1 micrometer. However, these hydrogels are unstable particles and tend to be easily decomposed by external stimuli, and since a large amount of organic solvent is used in the manufacturing process, the synthesis process of these hydrogels is not suitable for the manufacturing process of cosmetics. .

As another hydrogel synthesis method, there is an ion pairing method using the inherent charge of hyaluronic acid. can do. However, hyaluronic acid has an amorphous surface form, an additional crosslinking agent is required during hydrogel synthesis, and it is inconvenient to have to adjust the pH during the reaction process.

Therefore, a minimum amount of organic solvent is used in the manufacturing process to be applicable to cosmetics, or more preferably, without using an organic solvent, the process is simple, the particle size control is easy, the availability is high, and the material inside is stable. There is a need for a method for producing a hydrogel that can be supported as

1. US Patent Publication 2018/0147318 A1 (published on May 31, 2018) 2. Korean Patent Laid-Open Patent No. 10-2015-0048238 (published on May 6, 2015)

Accordingly, the present inventors limit the reaction conditions such as the concentration of the aqueous solution of hyaluronic acid used in the preparation of the hydrogel, the molecular weight and concentration of polyethylene glycol, and the hydrogel of desired physical properties without using a small amount of organic solvent or using an organic solvent. It has been found that an intermediate can be easily obtained, and the present invention has been completed.

Accordingly, it is an object of the present invention to provide a hydrogel capable of controlling the size of particles and stably supporting an active material therein, and a method for preparing the same, without requiring a large amount of organic solvent in the manufacturing process.

In order to achieve the above object, the hyaluronic acid hydrogel according to an embodiment of the present invention includes a hyaluronic acid-based intermediate and a biocompatible polymer monomer having a vinyl group, and the hyaluronic acid-based intermediate is hyaluronic acid and polyethylene It contains glycol diacrylate, and the biocompatible polymer monomer having a vinyl group is vinylcaprolactone and bisacrylamide, and the hyaluronic acid-based intermediate is 2:2 in an aqueous solution with vinylcaprolactone and bisacrylamide: It is mixed in a volume ratio of 1 to form hyaluronic acid hydrogel particles with improved stability, and the particle size of the prepared hyaluronic acid hydrogel is in the range of 100 to 2000 nm.

A method for producing a hyaluronic acid hydrogel according to another embodiment of the present invention,

1) synthesizing a hyaluronic acid-based intermediate by adding a photoinitiator to an aqueous solution of hyaluronic acid and an aqueous solution of polyethylene glycol diacrylate (PEG-diacrylate) and irradiating UV;

2) obtaining a hyaluronic acid hydrogel solution by mixing a biocompatible polymer monomer having a vinyl group and potassium persulfate, which is a radical reaction initiator, with the intermediate of step 1); and

3) centrifuging the hyaluronic acid hydrogel solution and then filtering to remove impurities to obtain a colloidal hyaluronic acid hydrogel

It is characterized in that it includes.

The hydrogel according to an embodiment of the present invention can be easily synthesized without using a large amount of organic solvent, and has a simple manufacturing process, is useful for cosmetic applications, and encapsulates bioactive materials including enzymes. It makes it possible to keep the activity of a substance stable for a long period of time.

1 shows the synthesis process of a hyaluronic acid hydrogel according to the present invention.
Figure 2 shows the analysis results of the physicochemical properties of the synthesized hyaluronic acid hydrogel particles.
3 shows the colloidal stability test results under various storage conditions.
4 shows the results of evaluating the stability of hydrogel particles in various cosmetic formulations.
5 shows the activity of EGCG supported inside the hydrogel particles and the result of stimuli-sensitive release by the enzyme.
6 shows the results of monitoring the activity and long-term stability of the antioxidant enzyme (SOD) supported inside the hydrogel particles.

The hydrogel according to an embodiment of the present invention is made by polymerizing hyaluronic acid and a biocompatible polymer monomer, and it is characterized in that the bioactive material can be encapsulated and the material can be supported therein.

More specifically, the hydrogel according to an embodiment of the present invention is a hyaluronic acid hydrogel, prepared by mixing a hyaluronic acid-based intermediate (or referred to herein as a “hyaluronic acid intermediate”) and a biocompatible polymer monomer. .

In one embodiment of the present invention, the hyaluronic acid used has a molecular weight in the range of 100,000 to 200,000 Da.

In one embodiment of the present invention, the hyaluronic acid-based intermediate is to include hyaluronic acid and polyethylene glycol diacrylate.

In one embodiment of the present invention, the biocompatible polymer monomer used is vinylcaprolactone (NVCPL) and bisacrylamide (more specifically, N,N-methylenebisacrylamide (MBA)).

In one embodiment of the present invention, vinyl caprolactone is prepared as an aqueous solution having a concentration of 0.5 to 2% (w/v), preferably 1% (w/v), and bisacrylamide is 0.005 to 0.2% (w) /v), preferably prepared as an aqueous solution of 0.01 ~ 0.1% (w / v) concentration.

In one embodiment of the present invention, the hyaluronic acid-based intermediate, vinyl caprolactone and bisacrylamide are mixed in an aqueous solution in a volume ratio of 2: 2: 1.

In one embodiment of the present invention, vinylcaprolactone and bisacrylamide are mixed in a weight ratio of 1:0.01-0.1, preferably 1:0.05-0.1, more preferably 1:0.05.

In one embodiment of the present invention, hyaluronic acid, vinyl caprolactone and bisacrylamide are mixed in a weight ratio of 0.0001 to 0.5: 1: 0.01 to 0.1, preferably 0.002 to 0.05: 1: 0.05.

The hyaluronic acid hydrogel according to an embodiment of the present invention has a particle size of 100 to 2000 nm, preferably 100 to 1000 nm, more preferably 200 to 300 nm.

One embodiment of the present invention relates to a method for preparing a hyaluronic acid hydrogel made by polymerization of hyaluronic acid and a biocompatible polymer monomer (FIG. 1), the method comprising:

1) synthesizing a hyaluronic acid-based intermediate by adding a photoinitiator to an aqueous solution of hyaluronic acid and an aqueous solution of polyethylene glycol diacrylate (PEG-diacrylate) and irradiating UV;

2) obtaining a hyaluronic acid hydrogel solution by mixing a biocompatible polymer monomer having a vinyl group and potassium persulfate, which is a radical reaction initiator, with the intermediate of step 1); and

3) centrifuging the hyaluronic acid hydrogel solution and then filtering to remove impurities to obtain a colloidal hyaluronic acid hydrogel

includes

The aqueous solution of hyaluronic acid used in step 1) has a hyaluronic acid concentration of 0.05 to 0.005% (w/v).

The polyethylene glycol diacrylate used in step 1) has an average molecular weight of 575 Da.

The polyethylene glycol diacrylate aqueous solution used in step 1) has a polyethylene glycol diacrylate concentration of 1 to 10% (v/v).

In one embodiment of the present invention, in step 1), hyaluronic acid and polyethylene glycol diacrylate are mixed in an aqueous solution in a volume ratio of 0.5 to 2: 0.5 to 2, preferably 1:1.

In one embodiment of the present invention, in step 1), hyaluronic acid and polyethylene glycol diacrylate are mixed in a weight ratio of 0.005: 1.

The photoinitiator used in step 1) may be Darocur 1173, benzophenone, Irgacure 500, 2959, etc., preferably Darocur 1173.

The UV used in step 1) has a wavelength range of 250 nm to 370 nm, preferably 365 nm.

The biocompatible polymer monomer having a vinyl group used in step 2) is vinylcaprolactone (NVCPL) and bisacrylamide (more specifically, N,N-methylenebisacrylamide (MBA)), wherein the polymer monomer is Most of the vinyl groups are crosslinked with each other, and in some cases, the COO ^- group of hyaluronic acid is a molecule that serves as a linker connecting the vinyl group.

In step 2), potassium persulfate, which is the radical reaction initiator, is mixed in a volume ratio of 1-5:25, preferably 1:12.5, with respect to the total volume of the intermediate and vinyl group-containing biocompatible polymer monomers in step 1). do.

In step 2), potassium persulfate is mixed, and then the solution may be stirred so that the reaction occurs well. At this time, stirring is performed until the color of the solution becomes cloudy, and stirring is usually performed for about 30 to 40 minutes. is done while

One embodiment of the present invention relates to a cosmetic composition comprising a hyaluronic acid hydrogel having an active ingredient supported therein.

The hyaluronic acid hydrogel prepared according to an embodiment of the present invention is very useful for encapsulating a bioactive material by using the excellent water absorption ability of hyaluronic acid. The bioactive material capable of being supported on the hydrogel according to the present invention may be a protein including enzymes, an antioxidant material, a drug, and the like, but is not limited thereto.

The hyaluronic acid hydrogel according to an embodiment of the present invention can support the active ingredient therein with an efficiency of 70 to 80% with respect to the total amount of the active ingredient mixed with the hydrogel at the time of manufacture.

The cosmetic composition comprising the hydrogel according to an embodiment of the present invention is not particularly limited in its formulation, and may be a solubilized formulation, an emulsified formulation, or the like. Specifically, the cosmetic composition according to an embodiment of the present invention may be a cosmetic composition having a formulation of a flexible lotion, nutrient lotion, massage cream, nutrient cream, pack, gel or skin adhesion type cosmetic, and also a lotion, ointment, gel, It may be a transdermal dosage form such as a cream or a patch.

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples. However, these Examples and Test Examples are provided only for the purpose of illustration to help the understanding of the present invention, and the scope and scope of the present invention are not limited by these Examples and Test Examples.

[Example 1] Synthesis of hyaluronic acid hydrogel particles

0.005% (w/v) aqueous solution of hyaluronic acid (molecular weight 100,000 to 200,000 Da) and 1% (v/v) aqueous solution of polyethylene glycol diacrylate (PEG-diacrylate, PEG-DA) having a molecular weight of 575 Da After mixing at a ratio of :1 (v/v), 30 μL of Darocur 1173 (photoinitiator, present in 10% EtOH) was added per 1 mL of the mixed solution, and UV light of 365 nm wavelength was irradiated for 10 minutes. An intermediate based on hyaluronic acid was synthesized (yield: about 79%). The obtained hyaluronic acid-based intermediate was used as it is in the next step without going through a separate separation procedure.

Next, an aqueous solution (1% (w/v)) of vinylcaprolactone, a biocompatible polymer monomer having a vinyl group and a hyaluronic acid-based intermediate synthesized in aqueous solution state, and N,N-methylenebisacrylamide (MBA) of an aqueous solution (0.1% (w/v)) was mixed in a volume ratio of 2: 2: 1 and the temperature of the solution was raised to at least 50 °C. For the radical reaction, potassium persulfate was mixed in a ratio of 5:1 (v/v) compared to the volume of the intermediate, and stirred until the color of the solution became turbid, at which time the required time was approximately 30 to 40 it was a minute Finally, the completed solution was collected by centrifugation (10,000 rpm, 20 minutes), and impurities were removed using a 0.8 μm syringe filter, and a colloidal hyaluronic acid hydrogel was obtained.

[Test Example 1] Analysis of the physicochemical properties of the synthesized hydrogel particles

The size of the particles according to the synthesis conditions of the hydrogel synthesized in Example 1 was confirmed through dynamic light scattering (DLS, ELS-Z2, Otsuka electronics) method.

Figure 2a shows that the size of the intermediate can be controlled by adjusting the amount of PEG-DA during the synthesis of the intermediate. When 1% of PEG-DA is used, the size range of the intermediate is 5% of PEG-DA. It is shown that it is wider than the case of use, and the median diameter appears smaller (see Table 1 below). In addition, when 10% of PEG-DA was used, it was found that a hydrogel in the form of a film was formed (top right photo in FIG. 2 ).

0.005% (w/v) HA + 1% PEG-Da 0.005% (w/v) HA + 5% PEG-Da Diameter (nm) burglar(%) Diameter (nm) burglar(%) 129.4557 0 188.673 0 140.5789 0.32377 198.8338 0 152.6577 1.54355 209.5419 0 165.7743 3.63215 220.8266 0.48134 180.018 6.23053 232.719 2.23399 195.4855 8.85096 245.2519 5.1494 212.2821 11.02209 258.4597 8.61756 230.5218 12.38326 272.3789 11.84739 250.3287 12.7358 287.0476 14.11105 271.8375 12.05853 302.5064 14.91339 295.1943 10.49509 318.7976 14.08861 320.558 8.31951 335.9662 11.82646 348.101 5.8864 354.0594 8.63087 378.0106 3.57086 373.127 5.21427 410.4901 1.70288 393.2214 2.33113 445.7602 0.4998 414.3981 0.55455 484.0609 0 436.7151 0 525.6523 0 460.2341 0

Figure 2b shows that the size of the final hydrogel particles (HAG) can be changed by controlling the amount of MBA used when crosslinking a polymer having a vinyl group, when using a 0.1% concentration of MBA 0.01 It shows that the median diameter of the final hydrogel particles appears smaller than when using the % concentration of MBA (see Table 2 below).

0.1% MBA 0.01% MBA Diameter (nm) burglar(%) Diameter (nm) burglar(%) 148.324 0 192.3538 0 162.4669 0 220.0235 0 177.9585 3.79845 251.6733 4.39385 194.9272 7.59633 287.8759 9.88567 213.5139 12.06801 329.2863 14.71177 233.8728 13.36054 376.6534 15.71605 256.1731 14.1165 430.8341 15.12131 280.5997 14.11566 492.8086 14.5738 307.3554 12.84337 563.6981 12.88135 336.6624 11.20735 644.7847 8.84797 368.7638 7.19401 737.5356 3.86823 403.9261 3.69978 843.6284 0 442.4413 0 964.9825 0 484.629 0 1103.793 0 530.8392 0 1262.571 0

Figure 2c shows how much the difference occurs as the size and polydispersity index of the intermediate and HAG change as the concentration of hyaluronic acid is changed (-: intermediate; +: HAG) (see Table 3 below) . As a result of the measurement, it was confirmed that both the polydispersity index and size of the intermediate were relatively larger than those of HAG. This means that the size of the hydrogel particles can be easily controlled by changing the composition of the monomers, etc. during the synthesis process.

Concentration of HA Kinds Average diameter (nm) standard deviation (mean diameter) Polydispersity Index (a.u.) 0.001% (w/v) intermediate 259.9 67.5 0.25 HAG 237.9 55.5 0.193 0.005% (w/v) intermediate 283.4 77 0.27 HAG 250.1 31 0.06 0.01% (w/v) intermediate 388.9 114.5 0.254 HAG 266.3 60 0.18 0.05% (w/v) intermediate 551.3 172 0.366 HAG 332.3 45.5 0.075

* Intermediate: X% HA + 1% PEG

** HAG: Intermediate + 1% NVCPL + 0.1% MBA

2d shows the results of measuring the surface charge of hyaluronic acid, the intermediate, and HAG (see Table 4 below). Through this, it was confirmed that the surface of the hydrogel particles had a negative charge as intended, which means that the charge of hyaluronic acid and materials used for synthesis is almost maintained.

Zeta potential (mV) Standard Deviation HA -23.7 0.46072 intermediate -10.50333 1.07717 HAG -29.75333 0.41899

* Intermediate: 0.005% HA + 1% PEG

** HAG: Intermediate + 1% NVCPL + 0.1% MBA

2 e and f show the results of observation of the morphology of the hydrogel particles using an electron microscope (e: TEM, LIBRA120, Carl Zeiss; f: SEM, SU8010, HITACHI). From the electron microscopic analysis results obtained using TEM and SEM, it was confirmed that the hydrogel particles had a spherical uniform size (e, f in Fig. 2). It was observed to be sharp (Fig. 2e). This can be said to be observed more clearly on the electron microscope because the vinyl group more firmly crosslinked the intermediates in the second process of synthesis.

[Test Example 2] Stability analysis of synthesized hydrogel particles

The stability in the colloidal state was tested by analyzing how well the hydrogel particles synthesized in Example 1 maintain their size in various storage conditions (eg, solvent, pH change). In addition, for comparison, the intermediate obtained after primary crosslinking was also tested for stability in the same colloidal state.

Specifically, the hydrogel particles and intermediates synthesized in Example 1 were each prepared in a phosphate buffer (PBS) of pH 4, PBS of pH 7.4, PBS of pH 10, Tris-HCl, NaCl aqueous solution (10 mM) and 10% ethanol. Whether the particle size was maintained in various pH and buffer conditions by leaving it in an aqueous solution for at least 12 hours was confirmed through the dynamic light scattering (DLS, ELS-Z2, Otsuka electronics) method. The results are presented in FIG. 3 .

3a shows the observation results for the intermediate (see Table 5 below). In the case of intermediates, under various storage conditions, the diameter measurement results showed that the peak appeared in a broad form over a fairly wide range from about 100 nm to more than 1000 nm, indicating that the size deviation was large and not constant. It can be seen that the stability of

PBS (pH 7.4) PBS (pH 4) PBS (pH 10) diameter
(nm) burglar
(%) diameter
(nm) burglar
(%) diameter
(nm) burglar
(%) 88.5952896.57413
105.2715
114.7522
…
322.9711
352.0577
…
1080.106
1177.38
1283.414
1398.998 0
0
0.55713
0.89491
…
5.91184
5.90992
…
0.74835
0.46677
0
0 85.81857
93.49007
101.8474
110.9517
…
309.9881
337.6986
…
1027.838
1119.719
1219.813
1328.854 0
0
0.40136
0.69888
…
6.08102
6.12195
…
0.65174
0.37079
0
0 72.63251
78.9994
85.92442
93.45647
…
256.1666
277.6219
…
903.4706
982.668
1068.808
1162.498 0
0
0.33018
0.6237
…
5.89529
5.92406
…
0.62633
0.38099
0
0 10 mM NaCl Tris-HCl 10% (v/v) EtOH diameter
(nm) burglar
(%) diameter
(nm) burglar
(%) diameter
(nm) burglar
(%) 81.27563
88.59528
96.57413
105.2715
…
271.8086
296.2876
…
1080.106
1177.38
1283.414
1398.998 0
0
0.54421
0.91712
…
5.85696
5.89144
…
0.50972
0.30856
0
0 99.81724
109.0566
119.1511
130.18
…
344.7095
376.6167
…
1300.61
1420.998
1552.529
1696.235 0
0
0.54147
0.91088
…
6.08053
6.11361
…
0.55088
0.32343
0
0 105.3529
115.0296
125.5951
137.1311
…
393.635
429.7906
…
1233.714
1347.032
1470.757
1605.847 0
0
0.36802
0.66355
…
6.24422
6.27216
…
0.87122
0.52711
0
0

3 b shows the observation results for the hydrogel (HAG) finally synthesized in Example 1 (see Table 6 below). In the hydrogel, the diameter measurement results are concentrated in a narrow range of about 150 to 400 nm under various pH and buffer conditions except for the ethanol atmosphere, and the peak appears in a sharp form, indicating that the size deviation is narrow and constant. It can be confirmed that the stability of the colloid is well maintained through this. This is because the stability of the hydrogel particles is improved by wrapping the outer periphery of the intermediate using a monomer having a vinyl group.

PBS (pH 7.4) PBS (pH 4) PBS (pH 10) diameter
(nm) burglar
(%) diameter
(nm) burglar
(%) diameter
(nm) burglar
(%) 119.2127 0 117.2053 0 117.2053 0 129.4557 0 127.2385 0 127.2385 0 140.5789 0.32377 138.1307 0 138.1307 0 152.6577 1.54355 149.9553 0.89447 149.9553 1.40332 165.7743 3.63215 162.7921 2.46521 162.7921 3.47565 180.018 6.23053 176.7277 4.65478 176.7277 6.09388 195.4855 8.85096 191.8564 7.12015 191.8564 8.7546 212.2821 11.02209 208.2801 9.45586 208.2801 10.97455 230.5218 12.38326 226.1097 11.28318 226.1097 12.38797 250.3287 12.7358 245.4657 12.31635 245.4657 12.79597 271.8375 12.05853 266.4785 12.40421 266.4785 12.17834 295.1943 10.49509 289.2902 11.54746 289.2902 10.67706 320.558 8.31951 314.0547 9.8929 314.0547 8.55967 348.101 5.8864 340.9391 7.70719 340.9391 6.16921 378.0106 3.57086 370.1249 5.33314 370.1249 3.86661 410.4901 1.70288 401.8091 3.13216 401.8091 1.96984 445.7602 0.4998 436.2056 1.41671 436.2056 0.69334 484.0609 0 473.5467 0.37623 473.5467 0 525.6523 0 514.0842 0 514.0842 0 570.8176 0 558.092 0 558.092 0 619.8633 0 605.867 0 605.867 0 10 mM NaCl 10% (v/v) EtOH Tris-HCl diameter
(nm) burglar
(%) diameter
(nm) burglar
(%) diameter
(nm) burglar
(%) 115.1733 0 117.2053 0 130.7918 0 124.9949 0 127.2385 0 142.2569 0 135.654 0 138.1307 0 154.727 0 147.2222 0.84538 149.9553 1.40332 168.2902 0 159.7768 2.57697 162.7921 3.47565 183.0423 0.43076 173.4021 4.9365 176.7277 6.09388 199.0876 1.75442 188.1893 7.48601 191.8564 8.7546 216.5395 3.9069 204.2375 9.77757 208.2801 10.97455 235.5211 6.52553 221.6542 11.4471 226.1097 12.38797 256.1666 9.12993 240.5562 12.26446 245.4657 12.79597 277.6219 11.25696 261.0701 12.15027 266.4785 12.17834 303.0457 12.55236 283.3333 11.16823 289.2902 10.67706 329.6104 12.8231 307.4951 9.5002 314.0547 8.55967 358.5037 12.05524 333.7173 7.40996 340.9391 6.16921 389.9298 10.40289 362.1757 5.20043 370.1249 3.86661 424.1107 8.15397 393.0609 3.16788 401.8091 1.96984 461.2878 5.67836 426.5799 1.55619 436.2056 0.69334 501.7238 3.3637 462.9573 0.51284 473.5467 0 545.7044 1.54356 502.4368 0 514.0842 0 593.5403 0.42232 545.283 0 558.092 0 645.5695 0 591.7831 0 605.867 0 702.1595 0

In addition, colloidal stability was indirectly confirmed through the Tyndall's effect, which is a principle in which clear bands are observed due to scattering by particles when a laser is irradiated to an aqueous colloidal solution containing several hundred nanometers of particles. The results are shown in Fig. 3c.

3 c, a band that is not observed in the blank (the solution without hyaluronic acid of Example 1) is observed under the condition in which hyaluronic acid is present, and the band is clearly observed even under various pH and buffer conditions. , it can be seen indirectly that the hydrogel prepared according to the present invention is not significantly affected by pH or organic solvents and stably maintains its shape.

[Test Example 3] Evaluation of the stability of hydrogel particles in various cosmetic formulations

In order to find out whether the hydrogel (HAG) particles prepared in Example 1 are stably maintained in various cosmetic formulations, the hydrogel particles are put in a solubilization formulation, an emulsion formulation, and an essence formulation (Tables 7 to 9) The following colloidal stability was confirmed through dynamic light scattering analysis (DLS, ELS-Z2, Otsuka electronics).

emulsified formulation ingredient content (wt%) deionized water 87.3 E.D.T.A.-2NA 0.01 Glycerin (Won-Ki Jang, KP) 3 Butylene Glycol (1,3) UK Grade 5 KELTROL - F 0.1 Phenoxyethanol P5 (phenoxetol) 0.3 Sensiva SC 50 0.05 Vegetable (Pripure 3759, Phytosqualane) 2.5 Dermofeel BGC (Miglyol 8810) 1.5 CARBOPOL ETD 2020 0.14 Tris Amino Ultra PC 0.1 total 100

Essence Formulation 1 ingredient content (wt%) Purified water To 100 propanediol 7 dipropylene glycol 2 glycerin 2 1,2-Hexanediol 1.5 betaine 1.5 Polyglycerin-3 One Glyceryl Polymethacrylate One Carbomer 0.2 tromethamine 0.14 EDTA-2Na 0.02

Essence Formulation 2 ingredient content (wt%) Purified water To 100 1,3-butylene glycol 4.5 ethanol 3 glycerin 2 niacinamide 2 Glyceres-26 1.5 betaine 0.5 Phenoxyethanol 0.4 Carbomer 0.17 KOH 0.1 PEG-60 Hydrogenated Castor Oil 0.1

Specifically, the used cosmetic formulation and hydrogel particles were prepared in a volume ratio of 10:1. In addition, for comparison, distilled water was used instead of the cosmetic formulation.

The measurement results are shown in FIG. 4 .

As shown in Fig. 4, when observed using a laser, not only a very clear band is observed, but also a peak appears in the form of a sharp peak as a result of measuring the particle diameter. It can be seen that can be stably maintained.

[Example 2] Encapsulation of bioactive materials using hydrogel particles

The hyaluronic acid hydrogel prepared and stored in Example 1 was centrifuged at 10000 rpm for 20 minutes to recover the hydrogel in the form of a pellet, and then a solution of EGCG (Epigallocatechin gallate), an antioxidant, dissolved in water at a concentration of 400 μM. By adding to the hydrogel and using a 150 rpm shaking incubator for 10 hours or more, EGCG was supported inside the hydrogel, and then the unencapsulated EGCG was separated through centrifugation to obtain a hydrogel capsule carrying a bioactive molecule therein. did.

[Example 3] Encapsulation of protein using hydrogel particles

Through the same procedure as in Example 2, except that an antioxidant enzyme SOD (superoxide dismutase, MW 32 kDa) was used as a material to be supported on the hydrogel capsule, a hydrogel capsule having SOD loaded therein was obtained.

[Test Example 4] Activity test of antioxidant EGCG

The activity of EGCG supported in the hydrogel capsule prepared in Example 2 was stored for 0 to 24 hours in phosphate buffer (PBS), and then ABTS (2,2'-azino-bis(3-ethylbenzothiazoline) -6-sulfonic acid))) was reacted with the aqueous solution for 30 minutes to confirm the absorbance value at 730 nm. At this time, the darker the green, the more active oxygen species in the aqueous solution (Fig. 5a). For comparison, the activity was also measured for EGCG stored in PBS in an unencapsulated state. The results are shown in FIG. 5 .

Figure 5b shows the activity of EGCG stored in PBS in an unencapsulated state (see Table 10 below). From this, the activity of unencapsulated EGCG decreases with time in PBS conditions, and the presence of salt (NaCl) or enzyme (HAase) does not significantly affect the activity of EGCG, but when heat (50°C) is applied It can be seen that the decrease in the activity of EGCG was significantly increased.

EGCG EGCG+heat EGCG+NaCl EGCG+HAase hour activation(%) Standard Deviation activation(%) Standard Deviation activation(%) Standard Deviation activation(%) Standard Deviation 0 61.705 0 61.705 0 61.705 0 61.705 0 One 58.42568 2.22466 55.49989 0 65.01216 1.78432 53.58165 1.72472 2 57.84188 0.81994 45.85148 2.92307 62.11307 1.17705 51.74898 3.19693 4 55.39668 1.18308 42.45292 2.29632 55.37024 0.29653 52.05997 1.25242 6 55.79542 2.17588 41.64094 0.1783 56.66913 0.23137 52.83177 0.06702 10 53.07538 0.28474 34.736 0.93892 53.50816 0.20534 49.14859 0.29935 24 48.50992 0.83369 27.1281 0.45035 49.67513 0.05368 48.1567 0.33474

*EGCG: 30 μM

heat: 50℃

NaCl: 10 mM

HAase: 400 U/mL

On the other hand, looking at FIG. 5 c showing the activity of EGCG (EGCG@HAG) encapsulated in a hydrogel, it can be seen that the activity does not differ from the initial state even under stimulation conditions by heat (50 ° C) or salt (NaCl). (see Table 11 below), suggesting that EGCG is protected by the hydrogel. In addition, when an enzyme (hyaluronidase, HAase) capable of decomposing the hyaluronic acid hydrogel was treated, the activity of EGCG was shown to gradually increase, which was caused by the decomposition of the hydrogel by the enzymatic reaction. It means that the supported material is selectively released.

EGCG EGCG+heat EGCG+NaCl hour activation(%) Standard Deviation activation(%) Standard Deviation activation(%) Standard Deviation 0 10.90909 0 10.90909 0 10.90909 0 One 11.48445 1.69698 13.71615 2.21955 18.70612 0.68945 2 12.68806 1.66103 13.84152 1.1414 19.25777 1.06385 4 14.91976 2.71218 16.32397 4.7351 22.99398 2.45827 6 14.11013 3.4101 13.71681 1.60745 21.53392 1.13611 10 15.39024 1.72792 14.90244 1.59173 23.14634 3.38761 24 17.07317 3.16058 15.85366 1.51534 24.70732 2.49628 EGCG+HAase1 EGCG+Haase2 hour activation(%) Standard Deviation activation(%) Standard Deviation 0 10.90909 0 10.90909 0 One 14.34303 2.08592 36.65998 2.8017 2 15.39619 2.09974 43.30491 2.42842 4 16.37412 3.10072 44.20762 1.64716 6 17.74828 2.27487 40.97837 3.82179 10 16.97561 1.31255 43.90244 2.40661 24 18.97561 1.85943 45.87805 1.75389

* EGCG@HAG: 30 μM

heat: 50℃

NaCl: 10 mM

HAase 1: 400 U/mL

HAase2: 4000 U/mL

In addition, while the EGCG supported on the hydrogel was stored in PBS, stimulation by heat (50° C.), salt (NaCl, 10 mM), or enzyme (HAase; 400 U/mL or 4000 U/mL) was applied thereto, 24 After storage for a period of time, it was reacted with ABTS aqueous solution for 30 minutes, and the absorbance value at 730 nm was compared with the control to evaluate the activity of EGCG. The results are shown in Fig. 5d (see Table 12 below).

EGCG EGCG+heat EGCG+NaCl hour activation(%) Standard Deviation activation(%) Standard Deviation activation(%) Standard Deviation 24 17.07317 3.16058 15.85366 1.51534 24.70732 2.49628 EGCG+HAase1 EGCG+Haase2 hour activation(%) Standard Deviation activation(%) Standard Deviation 24 18.97561 1.85943 45.87805 1.75389

Referring to d of FIG. 5 , it can be seen that the EGCG activity was significantly increased when the enzyme was treated, and it can be confirmed that there is a statistically significant difference in each condition by comparing the results of the EGCG activity measured in each condition.

[Test Example 5] Activity test of antioxidant enzyme SOD

To test the protein loading efficiency and activity, an antioxidant enzyme SOD (superoxide dismutase, MW 32 kDa) was used.

The size of the hydrogel carrying SOD was measured using DLS in the same manner as in Example 2, which is shown in FIG. 6 a.

Even if the enzyme is supported through a of FIG. 6, it can be confirmed that there is no significant effect on the size of the hyaluronic acid hydrogel.

In addition, after mixing the SOD-supported hydrogel and Bradford indicator in a volume ratio of 10:1 using the Bradford's assay, by checking the absorbance value at 525 nm wavelength, it was confirmed that the SOD is supported on the hydrogel. It was confirmed (FIG. 6 b).

The activity of SOD was determined by measuring the degree of change of the indicator turning yellow through the reaction of WST-1 and xanthine oxidase. For example, when the concentration of SOD is high, the color of the indicator changes slowly by inhibiting the reaction of the protein substance to be evaluated. The results for SOD activity are shown in FIG. 6 c (see Table 13 below).

hour HAG SOD@HAG 0 -0.00232 -0.00358 One 0.07419 0.02772 3 0.16798 0.05487 6 0.33885 0.07315 10 0.57228 0.10107 15 0.84856 0.14438 20 1.00559 0.17018

In the case of the hydrogel not carrying SOD, the color of the indicator changed rapidly, but in the case of the hydrogel carrying SOD, the color change width was significantly reduced. From this, it can be seen that the enzyme activity is maintained even if the SOD is supported inside the hydrogel.

Then, in order to confirm that the SOD supported on the hydrogel can be stored for a long time, the hydrogel loaded with 200 U/mL of SOD was diluted in PBS, solubilized formulation and essence, respectively, and the activity of SOD was observed for about 30 days. . The results are shown in d of FIG. 6 (see Table 14 below).

EGCG EGCG+heat EGCG+NaCl EGCG+HAase hour
(Work) activation(%) Standard Deviation activation(%) Standard Deviation activation(%) Standard Deviation activation(%) Standard Deviation 0 91.0473 0 62.6689 0 62.6689 0 62.6689 0 One 79.92581 0.758 65.27778 0.49441 62.79331 0.42443 58.35059 2.83262 2 81.49836 2.03833 67.25871 3.823 67.06573 0.66862 60.37414 5.46059 4 88.60043 0.82429 72.24359 3.13557 72.13675 2.28489 67.78846 4.22351 7 83.22627 1.52866 63.3833 2.99956 61.02784 2.93778 58.88651 3.84778 10 86.05769 1.66928 70.27244 4.84273 67.54808 5.32958 57.05128 8.39653 20 88.67224 3.08789 76.32312 6.90223 80.50139 2.75752 64.80966 12.83623 30 83.55457 1.44889 64.45428 6.41805 65.26549 4.1665 50.81121 5.34139

As shown in d of FIG. 6 , the activity of the SOD supported in the hydrogel according to the storage conditions was small, and the initial activity remained largely unchanged even after approximately 30 days.

Claims (11)

As a hyaluronic acid hydrogel comprising a hyaluronic acid-based intermediate and a biocompatible polymer monomer having a vinyl group,
The hyaluronic acid-based intermediate is to include hyaluronic acid and polyethylene glycol diacrylate, and the biocompatible polymer monomer having a vinyl group is vinylcaprolactone and bisacrylamide,
The hyaluronic acid-based intermediate is mixed with vinyl caprolactone and bisacrylamide in a volume ratio of 2: 2: 1,
The particle size of the prepared hyaluronic acid hydrogel is in the range of 100 to 2000 nm, hyaluronic acid hydrogel. According to claim 1, wherein the hyaluronic acid has a molecular weight range of 100,000 ~ 200,000 Da or more, hyaluronic acid hydrogel. According to claim 1, wherein the hyaluronic acid hydrogel has a particle size in the range of 200 ~ 300 nm, hyaluronic acid hydrogel. The hyaluronic acid hydrogel according to claim 1, wherein the hyaluronic acid hydrogel carries the active ingredient therein with an efficiency of 70 to 80%. 1) synthesizing a hyaluronic acid-based intermediate by adding a photoinitiator to an aqueous solution of hyaluronic acid and an aqueous solution of polyethylene glycol diacrylate and irradiating UV;
2) obtaining a hyaluronic acid hydrogel solution by mixing a biocompatible polymer monomer having a vinyl group and potassium persulfate, which is a radical reaction initiator, with the intermediate of step 1); and
3) centrifuging the hyaluronic acid hydrogel solution and then filtering to remove impurities to obtain a colloidal hyaluronic acid hydrogel
includes,
The biocompatible polymer monomer having the vinyl group is vinylcaprolactone and bisacrylamide,
The potassium persulfate is mixed with the intermediate of step 1) and the biocompatible polymer monomer having a vinyl group in a volume ratio of 1:12.5,
A method for preparing the hyaluronic acid hydrogel of any one of claims 1 to 4. The method of claim 5, wherein the polyethylene glycol diacrylate aqueous solution has a concentration of 1 to 10% (v/v). The method of claim 6, wherein the aqueous solution of hyaluronic acid and the aqueous solution of polyethylene glycol diacrylate are mixed in a volume ratio of 0.5-2: 0.5-2. The method of claim 5, wherein the UV has a wavelength range of 250 nm to 370 nm. The method according to claim 5, wherein the vinyl caprolactone is prepared as an aqueous solution having a concentration of 0.5 to 2% (w/v), and the bisacrylamide is prepared as an aqueous solution having a concentration of 0.005 to 2% (w/v), hyaluronic acid A method for preparing a hydrogel. The method of claim 5, wherein the hyaluronic acid-based intermediate, vinyl caprolactone and bisacrylamide are mixed in a volume ratio of 2: 2: 1 in an aqueous solution. A cosmetic composition comprising the hyaluronic acid hydrogel of any one of claims 1 to 4, wherein the active ingredient is supported therein.

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