Method of preparing bioactive substance-encapsulated ethosome, ethosome composition, and cosmetic composition including ethosome composition

Abstract

Provided is a method of preparing a bioactive substance-encapsulated ethosome, the method including: preparing an aqueous solution of a bioactive substance, preparing a lipid-dissolved solution by dissolving lipids in ethanol, preparing a hydrated liquid crystalline phase by hydrating the lipid-dissolved solution by mixing and agitating the bioactive substance aqueous solution together with the lipid-dissolved solution, and preparing an ethosome solution by adding purified water to the hydrated liquid crystalline phase to produce a mixture and agitating the mixture.

Claims

1. A method of preparing a bioactive substance-encapsulated ethosome, the method comprising: preparing an aqueous solution of a bioactive substance comprising a peptides-hydrogel complex formed by binding an amine group of the peptides and a carboxyl group of the hydrogel, wherein the hydrogel is a carbomer; preparing a lipid-dissolved solution by dissolving lipids in ethanol, wherein the lipids comprise phospholipids; preparing a hydrated liquid crystalline phase by hydrating the lipid-dissolved solution by mixing and agitating the aqueous solution of the bioactive substance together with the lipid-dissolved solution; and preparing a bioactive substance-encapsulated ethosome solution by adding purified water to the hydrated liquid crystalline phase to produce a mixture and agitating the mixture, wherein in the step of preparing the ethosome solution, the peptides-hydrogel complex is encapsulated inside the ethosome to generate the bioactive substance-encapsulated ethosome, wherein a content of the phospholipids in the ethosome solution is 0.1 wt % to 10 wt %, and wherein a content of the ethanol in the ethosome solution is 2 wt % to 8 wt %, and wherein a content of the hydrogel in the ethosome solution is 0,005 wt %.

2. The method of preparing the bioactive substance-encapsulated ethosome of claim 1, wherein a content of the bioactive substance in the ethosome solution is 0.0001 wt % to 10 wt %.

3. The method of preparing the bioactive substance-encapsulated ethosome of claim 1, wherein a weight ratio of the peptides and the hydrogel in the peptides-hydrogel complex is 1:0.0001 to 1:0.5.

4. The method of preparing the bioactive substance-encapsulated ethosome of claim 1, wherein a weight ratio of the ethanol and the lipids in the lipid-dissolved solution is 1:0.1 to 1:0.3.

5. The method of preparing the bioactive substance-encapsulated ethosome of claim 1, wherein, in the hydrated liquid crystalline phase, a ratio of the aqueous solution of the bioactive substance to the lipid-dissolved solution is 1:0.1 to 1:10 by weight.

6. The method of preparing the bioactive substance-encapsulated ethosome of claim 1, further comprising adding an alkaline aqueous solution to the hydrated liquid crystalline phase to adjust pH of the ethosome from 6.0 to 7.5.

7. The method of preparing the bioactive substance-encapsulated ethosome of claim 1, further comprising homogenizing the ethosome solution.

8. The method of preparing the bioactive substance-encapsulated ethosome of claim 7, wherein the homogenization comprises primarily homogenizing the ethosome solution by using a homo mixer at 3,000 rpm to 5,000 rpm for 5 minutes to 10 minutes and further homogenizing the primarily homogenized solution by using a high-pressure homogenizer.

9. The method of preparing the bioactive substance-encapsulated ethosome of claim 1, wherein the peptides are acetyl hexapeptide and palmitoyl pentapeptide.

10. The method of preparing the bioactive substance-encapsulated ethosome of claim 9, wherein a ratio of the acetyl hexapeptide to the palmitoyl pentapeptide is 1:1 by weight.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) These and/or other aspects will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings in which:

(2) FIG. 1 illustrates example structures of a known liposome (a) and a novel nanoethosome (b);

(3) FIG. 2 shows experimental results of analysis of particle sizes of prepared ethosomes;

(4) FIG. 3 shows photographs showing experimental results of a stability test of the prepared ethosomes after 30 days;

(5) FIG. 4 is a graph showing experimental results of an encapsulation efficiency test of the prepared ethosomes;

(6) FIG. 5 is a graph showing experimental results of a collagen production test of the prepared ethosomes;

(7) FIG. 6 is a photograph showing results of evaluation of wrinkle improvement before and at 4 weeks after application of the nanoethosome solution; and

(8) FIG. 7 is respectively show graphs showing results of evaluation of wrinkle improvement (a) and elasticity improvement (b) of subjects before and at 2 weeks and 4 weeks after application of the nanoethosome solution.

MODE FOR THE INVENTION

(9) Hereinafter, an ethosome delivery system prepared by the above-described processes will be described with reference to Examples. The following Examples describes a peptide, a hydrogel, a lipid, a solvent, etc., which is limited to a specific substance. However, as described above, various other types of the peptide, hydrogel, lipid, solvent, etc. may be used.

EXAMPLE 1

(10) Palmitoyl pentapeptide-4 was added to and dissolved in purified water. Since palmitoyl pentapeptide-4 was hardly dissolved in purified water, a small amount of lactic acid was added thereto to easily dissolve palmitoyl pentapeptide-4 (preparation of a peptide aqueous solution). The peptide was dissolved such that a content thereof in all ethosome components was 0.1 wt %.

(11) To the dissolved peptide aqueous solution, 1 wt % of a carbomer aqueous solution was added, and dispersed with a magnetic stirrer of 700 rpm under environments at 70° C. A content of the carbomer in all ethosome components was 0.005 wt %. Through these processes, a peptide-hydrogel complex dispersion solution was prepared.

(12) Ethanol was added such that its content in the ethosome solution was 4 wt %. Cholesterol and dicetyl phosphate were added to ethanol and dissolved with a magnetic stirrer of 300 rpm under environments at 50° C. Hydrogenated lecithin was added to the dissolved solution, and dissolved with a magnetic stirrer of 300 rpm under environments at 70° C. to prepare a transparent solution phase (a lipid-dissolved solution).

(13) The peptide-hydrogel complex dispersion solution was added to the lipid-dissolved solution, and mixed well for 5 minutes or more to dehydrate the lipid solution phase, and as a result, a hydrated liquid crystalline phase was prepared.

(14) To the hydrated liquid crystalline phase, purified water was added and stirred for 1 hour or more, and pH thereof was adjusted from 6.0 to 7.5 by using a 1 N NaOH aqueous solution to prepare a peptide-hydrogel-encapsulated nanoethosome solution.

(15) The prepared solution was homogenized by using a homo mixer at 5000 rpm for 5 minutes, and then further homogenized by using a high pressure homogenizer at a pressure of 1000 bar and the number of passage of three times. After completing the homogenization, the solution was slowly cooled in a water bath.

EXAMPLE 2

(16) In Example 2, ethosomes were prepared in the same manner as in Example 1, except that the content of ethanol was changed to 2 wt %.

EXAMPLE 3

(17) Example 3 is an example of using acetyl hexapeptide-8 and palmitoyl pentapeptide-4 at a ratio of 1:1. Acetyl hexapeptide-8 and palmitoyl pentapeptide-4 were added in an amount of 0.5 wt %, respectively and other procedures were performed in the same manner as in Example 1.

EXAMPLE 4

(18) In Example 4, ethosomes were prepared in the same manner as in Example 3, except that carbomer which is a hydrogel was not added.

EXAMPLE 5

(19) Cosmetic ingredient compositions were prepared by adding the cosmetic compositions prepared in Examples 1 to 4.

EXAMPLE 6

(20) A cosmetics including the cosmetic ingredient compositions prepared in Example 5 was prepared.

(21) In the following Table 1, the above-described components of Examples 1 to 4 and homogenization are summarized.

(22) TABLE-US-00001 TABLE 1 (Unit: wt %) Component Example 1 Example 2 Example 3 Example 4 Lecithin 1 1 1 1 Cholesterol 0.1 0.1 0.1 0.1 Dicetyl 0.05 0.05 0.05 0.05 phosphate Palmitoyl 0.1 0.1 0.05 0.05 pentapeptide-4 Acetyl — — 0.05 0.05 hexapeptide-8 Carbomer 0.005 0.005 0.005 — Ethanol 4 2 4 4 purified water Up to 100 Up to 100 Up to 100 Up to 100 Homogenization ◯ ◯ ◯ ◯

COMPARATIVE EXAMPLE 1

(23) In Comparative Example 1, ethosomes were prepared in the same manner as in Example 1, except that the content of ethanol was changed to 20 wt %.

COMPARATIVE EXAMPLE 2

(24) In Comparative Example 2, the homogenization process was omitted from the procedures of Example 3. In other words, the peptide-encapsulated ethosome solution was used as it is without homogenization by the high pressure homogenizer.

(25) In the following Table 2, the composition ratios of Comparative Examples 1 to 2 and homogenization are summarized.

(26) TABLE-US-00002 TABLE 2 (Unit: wt %) Component Comparative Example 1 Comparative Example 2 Lecithin 1 1 Cholesterol 0.1 0.1 Dicetyl phosphate 0.05 0.05 Palmitoyl 0.05 0.05 pentapeptide-4 Acetyl 0.05 0.05 hexapeptide-8 Carbomer 0.005 0.005 Ethanol 20 4 purified water Up to 100 Up to 100 Homogenization X X

(27) The nanoethosomes of Examples and Comparative Examples were subjected to pre-determined experiments.

(28) Experimental results will be described in Experimental Examples below.

(29) Experimental Example 1: Ethosome Particle Size according to Formulation Composition Ratio and High Pressure Homogenization

(30) To analyze average particle sizes of the nanoethosomes prepared in Examples and Comparative Examples, a nanoparticle analyzer (NICOMP 380ZLS, Santa Barbara) was used to measure their particle sizes, and results are shown in Table 3.

(31) FIG. 2 shows results of analysis of particle sizes. FIG. 2(a) shows the result of Comparative Example 2, and FIG. 2(b) shows the result of Example 3. The size of Examples 1 to 4 was approximately 250 nm or less. The preferred size of ethosome particle is 50 nm to 1000 nm.

(32) Example 1 and Example 2 suggest that as the content of ethanol is lower, the particle size is larger, and Example 3 and Example 4 suggest that the particle size is increased by addition of hydrogen. As Example 3 is compared with Comparative Example 2, the particle size was decreased by homogenization through the high pressure homogenizer. When 20 wt % of ethanol was added in Comparative Example 1, aggregation occurred and no ethosomes were formed.

(33) TABLE-US-00003 TABLE 3 Particle size (nm) Example 1 161.1 Example 2 220.3 Example 3 248.2 Example 4 195.9 Comparative Example 1 Aggregation Comparative Example 2 853.1

(34) Experimental Example 2: Stability According to Formulation Composition Ratio and High Pressure Homogenization

(35) First, stabilities of the prepared ethosome solutions were tested. Stabilities of the prepared nanoethosomes were measured to examine discoloration, phase separation, etc. under particular conditions over time. FIG. 3 shows results of a stability test of the prepared ethosomes over time. FIG. 3(a) is a photograph showing the peptide-hydrogel complex ethosome of Example 3 after 30 days. FIG. 3(b) is a photograph showing the peptide ethosome of Example 4 after 30 days.

(36) Experimental conditions are as follows. The ethosome solutions prepared in Examples and Comparative Examples were placed in a constant temperature chamber maintained at 45° C. Changes of these ethosome solutions were observed at time points of 1 day, 10 days, 20 days, and 30 days. Discoloration, phase separation, etc. of the ethosome solutions were examined over time. Experimental results are as in the following Table 4.

(37) TABLE-US-00004 TABLE 4 1 day 10 days 20 days 30 days Example 1 Stable Stable Stable Stable Example 2 Stable Stable Stable Stable Example 3 Stable Stable Stable Stable Example 4 Stable Stable Stable Precipitation Comparative Precipitation Precipitation Precipitation Precipitation Example 1 Comparative Opaque Precipitation Precipitation Precipitation Example 2

(38) Referring to the results of Table 4, Example 1 to Example 3 showed a stable shape in a transparent solution phase at day 1, and no discoloration and no phase separation for 30 days. Furthermore, Example 4 showed a transparent solution phase at day 1, but precipitation was observed and stability was reduced at about 20 days. Comparative Example 2 showed an opaque solution phase at day 1, but precipitation was observed at about 10 days.

(39) Experimental Example 3: Measurement of Encapsulation Efficiency of Formulated Nanoethosome

(40) The ethosome solutions prepared in Examples and Comparative Examples were centrifuged at 4° C. and 17,000 rpm for 1 hour to separate supernatants and precipitates. Thereafter, the supernatants were collected and concentrations of peptides not encapsulated were measured. Encapsulation efficiency was calculated by the following Equation:
Encapsulation efficiency (%)=(E.sub.1−E.sub.A)/E.sub.1*100

(41) E.sub.A: Concentration of peptide of supernatant after centrifugation

(42) E.sub.1: Concentration of initially added peptide

(43) FIG. 4 is a graph showing experimental results of an encapsulation efficiency test of the prepared ethosomes. Table 5 shows numerical values of encapsulation efficiencies measured as the experimental results.

(44) TABLE-US-00005 TABLE 5 Encapsulation efficiency (%) Example 1 67.1 Example 2 77.2 Example 3 92.7 Example 4 92.4 Comparative Example 1 Not measurable Comparative Example 2 54.2

(45) Referring to Table 5, Example 1 to Example 2 showed encapsulation efficiency of 60% or more, and Example 3 to Example 4 showed encapsulation efficiency of 90% or more, indicating that higher encapsulation efficiency was observed when encapsulation was performed by using both palmitoyl pentapeptide-4 acetyl and hexapeptide-8, as compared with use of palmitoyl pentapeptide-4 alone. This is because the total content of peptides was the same at 0.1 wt % but the content of palmitoyl pentapeptide-4 was decreased. The ethosome of Comparative Example 2 which was not passed through the high pressure homogenizer showed low encapsulation efficiency, which was caused by some unstable ethosomes which were not homogenized.

(46) Experimental Example 4: Cell Experiment for Collagen Producing Effect of Nanoethosome

(47) The nanoethosome of Example 3 which was prepared by adding acetyl hexapeptide-8 and palmitoyl pentapeptide-4 at a ratio of 5:5 was used to perform an in vitro collagen production test. Normal human dermal fibroblasts (nHDFs) were used, and to determine a concentration of a test solution, normal human dermal fibroblasts were treated with the sample material for 24 hours, and a range of the concentration at which cytotoxicity was not observed was confirmed by a cytotoxicity test. Normal human dermal fibroblasts were treated with the test solution at the determined concentration in the same manner, and mRNA expression levels of COL1A1 which is a collagen gene and MMP1 were analyzed by Real Time-PCR (RT-PCR). FIG. 5 shows results of the collagen production test.

(48) As a result, COL1A1 mRNA expression was increased to 38.44±3.81% at a concentration of 0.05%, as compared with a negative control, indicating increased collagen production, and MMP1 mRNA expression was decreased to 21.05±0.62% at a concentration of 0.05%, as compared with the negative control, indicating decreased collagenase production.

(49) Experimental Example 5: Human Body Application Test of Nanoethosome for Wrinkle and Elasticity Improvement

(50) The nanoethosome of Example 3 which was prepared by adding acetyl hexapeptide-8 and palmitoyl pentapeptide-4 at a ratio of 5:5 was applied to humanbodies to test wrinkle and elasticity improvement.

(51) The nanoethosome solution was evenly applied and absorbed to 23 women aged 39 to 61 years after morning and evening cleansing for 4 weeks, and improvement of wrinkles and elasticity was evaluated at 2 and 4 weeks. FIG. 6 is a photograph showing the result of testing wrinkle improvement before and at 4 weeks after application of the nanoethosome solution. FIG. 7 is a graph showing the result of testing wrinkle and elasticity improvement of the subjects before and at 2 weeks and 4 weeks after application of the nanoethosome solution. FIG. 7(a) is a graph showing the result of testing wrinkle improvement of the subjects and FIG. 7(b) is a graph showing the result of testing elasticity improvement of the subjects.

(52) To measure wrinkles medium values indicating skin's wrinkles, ANTERA 3D (Miravex, Ireland) was used for evaluation, and wrinkles were reduced to 9.99% at 2 weeks after application and 10.82% at 4 weeks after application, as compared with wrinkles before application.

(53) To measure CoR values indicating skin's elasticity, Ballistometer (Ballistometer BLS780, Dia-Stroh Ltd., UK) was used for evaluation, and elasticity was increased to 1.79% at 2 weeks after application and 3.36% at 4 weeks after application, as compared with elasticity before application.

(54) Referring to the results of Examples and Experimental Examples, nanoethosomes with low irritation were formulated by optimizing the addition amounts of phospholipid and lipid and reducing the content of ethanol by the above-described preparation method. High encapsulation efficiency and stability, and nanosize were confirmed and collagen production effect was also confirmed, and as a result, it can be seen that the nanoethosomes may be applied as a functional cosmetic delivery system.

(55) It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments.

(56) While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.