MULTI-COATED NANOPARTICLES COMPRISING MULTIPLE COATING LAYERS OF CHITOSAN AND POLYGLUTAMIC ACID, COMPOSITION FOR SKIN CARE COMPRISING THE SAME AND METHOD FOR MANUFACTURING THE SAME

Abstract

Multi-coated nanoparticles including multiple coating layers of chitosan and polyglutamic acid, composition for skin care including the same, and method of manufacturing the same are provided. The nanoparticles have a liposome core layer and coating layers. The core layer includes a bioactive particle and a core hydrogel. Not only is it easy to mass-produce, but also has the effect of steadily releasing bioactive particles for a long period of time due to its high sealing rate and high stability.

Claims

1. A multi-coated nanoparticle comprising: a liposome core layer comprising a bioactive particle and a core hydrogel having a charge opposite to a charge of the bioactive particle, the liposome core layer that is surrounded by a phospholipid bilayer; a first coating layer comprising chitosan of 0.05 wt % to 0.15 wt % with respect to a total weight of the nanoparticle, the first coating layer located on the core layer; a second coating layer comprising polyglutamic acid of 0.06 wt % to 0.15 wt % with respect to the total weight of the nanoparticle, the second coating layer located on the first coating layer; and a third coating layer including chitosan of 0.005 wt % to 0.03 wt % with respect to the total weight of the nanoparticle, the third coating layer located on the second coating layer.

2. The multi-coated nanoparticle of claim 1, wherein the bioactive particle is at least one substance selected from a group of peptides, natural extracts, and vitamins.

3. The multi-coated nanoparticle of claim 1, wherein the core hydrogel is at least one substance selected from a group of hyaluronic acid, alginate, pectin, carrageenan, dextran sulfate, guar gum, gum arabic, xanthan gum, polyacrylic acid, polygalaturonic acid, carboxymethylcellulose, polyglutamic acid, agar, collagen peptide, starch, hydrolyzed starch, crosslinked starch, modified starch, dextrin, gamma polyglutamic acid, gelatin, alginic acid, and chitosan.

4. The multi-coated nanoparticle of claim 1, wherein the bioactive substance is mixed with the core hydrogel or combined with the core hydrogel to exist in the core layer as a hydrogel-bioactive substance complex and wherein the multi-coated nanoparticle is configured to release the bioactive particle in a sustained-release manner.

5. The multi-coated nanoparticle of claim 1, wherein the liposome is nanoparticle, multi-layer liposome, elastic liposome or etosome.

6. The multi-coated nanoparticle of claim 1, wherein a size of the multi-coated nanoparticle is 10 nm to 1000 nm.

7. The multi-coated nanoparticle of claim 1, wherein the volume ratio of the core layer and the first layer is 1:0.0001 to 1:10.

8. The multi-coated nanoparticle of claim 1, wherein the volume ratio of the first layer and the second layer may be 1:0.0001 to 1:10.

9. Composition for skin care comprising: multi-coated nanoparticles; and purified water that is configured to contain the multi-coated nanoparticles; and wherein at least one of the multi-coated nanoparticles comprises: a liposome core layer comprising a bioactive particle and a core hydrogel, the liposome core layer that is surrounded by a phospholipid bilayer; a first coating layer comprising chitosan of 0.05 wt % to 0.15 wt % with respect to a total weight of the nanoparticle; a second coating layer comprising polyglutamic acid of 0.06 wt % to 0.15 wt % with respect to the total weight of the nanoparticle; a third coating layer including chitosan of 0.005 wt % to 0.03 wt % with respect to the total weight of the nanoparticle and wherein the core hydrogel is configured to electrostatically bind to the bioactive particle.

10. The composition for skin care of claim 9, wherein the core hydrogel having a charge opposite to a charge of the bioactive particle.

11. The composition for skin care of claim 9, wherein the bioactive particle is at least one substance selected from a group of peptides, natural extracts, and vitamins.

12. The composition for skin care of claim 9, wherein an amount of the bioactive particle is 0.01 wt % to 50 wt % with respect to total weight of the composition.

13. The composition for skin care of claim 9, wherein an amount of the core hydrogel is 0.0001 wt % to 1 wt % with respect to total weight of the composition.

14. The composition for skin care of claim 9, wherein the core hydrogel is at least one substance selected from a group of hyaluronic acid, alginate, pectin, carrageenan, dextran sulfate, guar gum, gum arabic, xanthan gum, polyacrylic acid, polygalaturonic acid, carboxymethylcellulose, polyglutamic acid, agar, collagen peptide, starch, hydrolyzed starch, crosslinked starch, modified starch, dextrin, gamma polyglutamic acid, gelatin, alginic acid, and chitosan.

15. A method for manufacturing a multi-coated nanoparticle comprising: providing a liposome core layer comprising a bioactive particle and a core hydrogel, the liposome core layer that is surrounded by a phospholipid bilayer; providing a first coating layer comprising chitosan of 0.05 wt % to 0.15 wt % with respect to a total weight of the nanoparticle on the core layer; providing a second coating layer comprising polyglutamic acid of 0.06 wt % to 0.15 wt % with respect to the total weight of the nanoparticle on the first coating layer; and providing a third coating layer comprising chitosan of 0.005 wt % to 0.03 wt % with respect to the total weight of the nanoparticle on the second coating layer.

16. The method of claim 15 further comprising high-pressure homogenizing the nanoparticle after the third coating layer is provided.

17. The method of claim 16, wherein the high-pressure homogenization is performed under 10 bar to 5000 bar.

18. The method of claim 15, wherein in the providing the first or the third coating layer, the first coating layer, the second coating layer or the third coating layer is surface-modified by the chitosan.

19. The method of claim 15, wherein in the providing the first, the second or the third coating layer, the first coating layer, the second coating layer or the third coating layer is formed to be electrostatically bonded.

20. The method of claim 15, wherein providing the liposome core layer comprising: providing peptide-hydrogel solution adding and dissolving copper tripeptide-1 and polyglutamic acid in purified water; providing phospholipid solution by dissolving hydrogenated lecithin in ethanol; providing core-phospholipid mixed solution by mixing peptide-hydrogel solution with the phospholipid solution; adding purified water to the core-phospholipid mixed solution to form the nanoparticle; and removing ethanol from the core-phospholipid mixed solution by degassing.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG. 1 is a schematic diagram of a multi-layered hydrogel nanoparticle including three coatings according to an embodiment of the present invention.

[0055] FIG. 2 is a diagram schematically showing a coating process of multilayed hydrogel nanoparticles according to an embodiment of the present invention.

[0056] FIG. 3 is a graph showing the analysis of the size and polydispersity index of multi-layered hydrogel nanoparticles in each coating step.

[0057] FIG. 4 is a diagram showing the long-term stability of the composition 30 days after preparation of the composition according to Experimental Example 1 and Comparative Example 1.

[0058] FIG. 5 is a graph showing the sustained-release behavior over time of nanoparticles including a hydrogel according to an embodiment of the present invention.

[0059] FIG. 6 is a graph showing DPPH radical scavenging activity over time of a multi-layer liposome according to an embodiment of the present invention.

DETAILED DESCRIPTION

[0060] Embodiments of the present invention will be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention.

[0061] To clearly describe embodiments of the present invention, parts that are irrelevant to the description are omitted, and like numerals refer to like or similar constituent elements throughout the specification.

[0062] In addition, unless explicitly described to the contrary, the word “comprise” and variations such as “comprises” or “comprising” will be understood to imply the inclusion of stated elements but not the exclusion of any other elements.

[0063] Hereinafter, a chirp noise generation device and method for a compressed pulse signal according to an embodiment of the present invention will be described with reference to FIG. 1 to FIG. 6.

Embodiment 1. Preparation of Experimental and Comparative Groups of Nanoparticles

1.1. Setting Experimental Examples and Comparative Examples

[0064] In order to confirm a difference in the effect of each of the coating stages and amounts of the nanoparticles, nanoparticles with the first coating of Experimental Example 1, nanoparticles with the second coating of Experimental Example 2, nanoparticles with the third coating of Experimental Example 3, uncoated nanoparticles of Experimental Example 4, and no hydrogel was added to the core of Experimental Example 5 were used.

[0065] In addition, as shown in Table 1, in order to compare the effect of the nanoparticles for each amount of hydrogel, Comparative Example 1 and Comparative Example 2 were set by varying the amount of compositions in each coating layer.

[0066] Table 1 shows the coating and amount of compositions according to the Experimental and Comparative examples.

TABLE-US-00001 TABLE 1 Coating layer (wt %) hydrogel (wt %) Nano particle Experimental Experimental Experimental Experimental Experimental Comparative Comparative Structure Example 1 Example 2 Example 3 Example 4 Example 5 Example 1 Example 2 Core Peptide 0.05% 0.05% 0.05% 0.05% 0.05% 0.05 0.05 Hydrogel 0.005% 0.005% 0.005% 0.005% — 0.005% 0.005% Liposome Phospholipid 2.0% 2.0% 2.0% 2.0% 2.0% 2.0% 2.0% Coating Chitosan 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% agent Poly — 0.1% 0.1% 0.1% 0.1% 0.05% Glutamic acid Chitosan — — 0.01% 0.01% 0.15% 0.01%

1.2. Preparation of Uncoated Nano Liposome (Experimental Example 4)

[0067] In order to prepare uncoated nanoparticles, a peptide-hydrogel solution and a phospholipid solution were prepared.

[0068] The peptide-hydrogel solution was prepared by adding and dissolving Copper TriPeptide-1 (CTP) and polyglutamic acid in purified water. The bioactive substance was adjusted to 0.05 wt % and the polyglutamic acid of the core layer was adjusted to 0.005 wt % with respect to the total weight of the nanoparticle solution.

[0069] Phospholipid solution was prepared by dissolving hydrogenated lecithin in ethanol. More specifically, ethanol was prepared in 10 wt % and hydrogenated lecithin was prepared in 2.0 wt % with respect to the total amount of the nanoparticle solution.

[0070] The peptide-hydrogel solution was mixed with the phospholipid solution and was stirred to prepare a core-phospholipid mixed solution. 10.0 wt % of purified water was added to the core-phospholipid mixed solution and was stirred to form a double-layered nanoparticle. Then, purified water was added to be 70.0 wt % of the total amount of nanoparticle solution and was stirred to remove ethanol through a degassing process. Finally, the solution was gradually cooled to room temperature and stirred to prepare nanoparticles of Experimental Example 4 in which there was no coating layer and the peptide was entrapped.

1.3. Preparation of Primary Coated Nano-Liposome (Experimental Example 1)

[0071] Nanoparticles with a positive first coating of Experimental Example 1 having an amount of composition shown in Table 1 was prepared.

[0072] More specifically, 1.0 wt % chitosan aqueous solution was prepared by dissolving chitosan, which is a cationic hydrogel, in purified water, and was added to the nanoparticles of Example 1.2 for first coating. Next, purified water was further added so that the amount thereof became 100%, thereby the primary coated nanoparticles of Experimental Example 1 was prepared.

1.4. Preparation of Secondary Coated Nano-Liposome (Experimental Example 2)

[0073] Nanoparticles with a positive second coating of Experimental Example 2 having an amount of composition shown in Table 1 was prepared.

[0074] More specifically, 1 wt % polyglutamic acid aqueous solution was added to the primary coated nanoparticles of Example 1.3, thereby the second coated nanoparticles of Experimental Example 2 were prepared.

1.5. Preparation of Third Coated Nano Liposome (Experimental Example 3)

[0075] Nanoparticles of Experimental Example 3 coated with the amount of composition shown in Table 1 were prepared.

[0076] A 0.1 wt % chitosan aqueous solution was prepared and added to the secondary coated nano-liposome of Example 1.4, followed by being thirdly coated. The coated nanoparticles were homogenized under high pressure through a microfluidizer. Specifically, the pressure of the high-pressure homogenization was set to 1000 bar, and the number of passes was set to 3 times.

[0077] FIG. 1 is a schematic diagram of nanoparticles including a hydrogel according to an embodiment of the present invention and FIG. 2 is a schematic diagram showing a coating process of nanoparticles including a hydrogel according to an embodiment of the present invention.

[0078] As shown in FIGS. 1 and 2, the nanoparticle of Experimental Example 3 includes the core layer containing a hydrogel-bioactive substance and a hydrogel multi-layer coated on the core layer. Therefore, it was confirmed that the drug entrapment rate is high and a sustained-release effect appears.

1.6. Preparation of Thirdly Coated Nano-Liposome without Core Hydrogel (Experimental Example 5)

[0079] Nanoparticles of Experimental Example 5 were prepared with a third coating of the amount shown in Table 1 without the addition of the core hydrogel.

[0080] The peptide-hydrogel solution entrapped in the core of Example 1.2 was not prepared, and only an aqueous peptide solution was provided to prepare a core-phospholipid solution. Then, it was prepared in the same manner as the that of third coated nano-liposome.

1.7. Preparation of Third-Coated Nano-Liposomes (Comparative Examples 1 and 2) with Different Amounts of Compositions

[0081] Nanoparticles of Comparative Examples 1 and 2 were third coated in the amount of the compositions shown in Table 1 above.

[0082] Comparative Example 1 was prepared in the same manner, except that 0.15 wt % chitosan aqueous solution was prepared and added to the second coated nano-liposome of Experimental Example 2 to perform the third coating.

[0083] Comparative Example 2 was prepared in the same manner, except that the primary coated nanoparticles of Experimental Example 1 were secondarily coated by adding a 0.05 wt % polyglutamic acid aqueous solution.

Embodiment 2. Comparison of Particle Size, Zeta Potential, and Stability According to Coating Steps of Nanoparticles and Amount of Hydrogel

2.1. Comparison of Particle Size and Zeta Potential of Nanoparticles by Amount

[0084] In order to compare the particle size and zeta potential of the nanoparticles according to the coating layer structure and the amount of hydrogel, the size and zeta potential of the nanoparticles of Embodiment 1 were measured using a nanoparticle analyzer. Zeta potential refers to the electrostatic potential of the cross section of particles, and is a numerical value of the degree of attraction or repulsion between adjacent particles. If the zeta potential is equal to or less than 0.3, the nanoparticles exist in a stable monodisperse form.

[0085] Table 2 is a graph showing the particle size and zeta potential of nanoparticles according to the coating layer structure and amount of hydrogel. FIG. 3 is a graph showing a particle size and zeta potential analysis of nanoparticles according to a coating step and amount of hydrogel.

TABLE-US-00002 TABLE 2 Experimental Experimental Experimental Experimental Experimental Comparative Comparative Example 1 Example 2 Example 3 Example 4 Example 5 Example 1 Example 2 Particle 134.31 ± 142.69 ± 153.91 ± 106.95 ± 157.45 ± 2265.98 ± 2221.66 ± size 3.57 3.06 4.48 5.06 0.79 316.9 418.7 (nm) Poly 0.229 0.194 0.157 0.254 0.154 0.713 0.427 Dispersity index Zete 29.07 ± −43.26 ± −38.51 ± −40.98 ± −37.63 ± 17.86 ± −15.13 ± Potential 2.24 2.01 0.82 2.3 1.35 0.28 0.92 (mV)

[0086] As shown in Table 2 and FIG. 3, the average size of the nanoparticles without coating hydrogel in Experimental Example 4 was 106.95±5.06 nm. However, in Experimental Examples 1 to 3 in which the hydrogel was coated once, twice, and third times, the size of nanoparticle size gradually increased as the nanoparticles included more multiple coating layers.

[0087] In addition, although nanoparticles were formed in Comparative Example 1 and Comparative Example 2, the binding ratio between chitosan and polyglutamic acid did not match, so it could be confirmed that chitosan and polyglutamic acid form a complex through self-assembly to be polydispersed.

[0088] However, the nanoparticles of Experimental Example 3 had a size of 153.91±4.48 nm, and the PDI was also 0.3 or less, indicating that they were monodispersed. These results imply that the third-coated nanoparticles of Experimental Example 4 were more stable than those of Experimental Examples 1 to 3.

[0089] In addition, in the Experimental Example 4, the zeta potential of the nanoparticles was −40.98±2.3 mV, but it was confirmed that the zeta potential of the nanoparticles in Experimental Example 1 was changed to 29.07±2.24 mV. This change in the zeta potential means that an electrostatic bond is formed between the cationic hydrogel and the anionic hydrogel during hydrogel coating.

2.2. Comparison of Long-Term Stability of Nanoparticles

[0090] In order to compare the long-term stability of the nanoparticles according to the structure of the coating layer and the amount of hydrogel, the long-term dispersion stability of the nanoparticles in Experimental Examples 1 to 5 and Comparative Examples 1 to 2 was measured.

[0091] Specifically, each nanoparticle solution was placed in a constant temperature chamber of 45° C., and changes in shape were observed with the naked eye after 1, 5, 10, 20, and 30 days. The change in shape was evaluated by discoloration and layer separation over time.

[0092] Table 3 shows the long-term stability of nanoparticles according to the coating layer structure and amount of hydrogel.

[0093] FIG. 4 is a photograph of a sample measuring the long-term stability of nanoparticles after 30 days of Experimental Example 1 and Comparative Example 1. In FIG. 4, Experimental Example 1 is denoted by (A), and Comparative Example 1 is denoted by (B).

TABLE-US-00003 TABLE 3 Experimental Experimental Experimental Experimental Experimental Comparative Comparative Example 1 Example 2 Example 3 Example 4 Example 5 Example 1 Example 2 1 day Stable Stable Stable Stable Stable Stable Stable 5 day3 Stable Stable Stable Stable Stable Precipitated Precipitated 10 day3 Stable Stable Stable Stable Stable Layer Layer Separated Separated 20 day3 Stable Stable Stable Stable Stable Layer Layer Separated Separated 30 day3 Stable Stable Stable Stable Agglomerated Layer Layer Separated Separated

[0094] As a result, as shown in Table 3, it was confirmed that the nanoparticles of Experimental Examples 1 to 4 had excellent dispersion stability even without a surfactant. In particular, in the nanoparticles of Experimental Example 5 in which the hydrogel was not included in the core, aggregation of the particles was confirmed after 30 days. Among the nanoparticles of Comparative Example 1 and Comparative Example 2, a layer was separated by sinking of particles. Therefore, it was confirmed that the stability of the nanoparticles was very low. These results mean that the long-term stability of the nanoparticles varies depending on whether the core hydrogels or coating layers exist or not, the number of coating layers, and the amount of the hydrogel.

2.3. Comparison of Entrapment Efficiency of Experimental Examples 1 to 5 Nano-Liposomes

[0095] The entrapment efficiency of the bioactive component was measured using the nanoparticles of Experimental Examples 1 to 5.

[0096] First, a bioactive substance that was not entrapped from the nanoparticle solution was separated using an MWCO bag (Molecular weight cut-off 10,000 dalton, Amicon). Next, the entrapment rate of each nanoparticle was calculated using Equation 1 below.

EE.sub.Free=(W.sub.Total−W.sub.Free)/W.sub.Total×100  [Equation 1]

[0097] In Equation 1, EE.sub.Free is the entrapment efficiency of the bioactive substance encapsulated in the multi-layer hydrogel nanoliposome, W.sub.Total is the total concentration of the added bioactive substance, and W.sub.Free is the concentration of the bioactive substance not entrapped in the multi-layered hydrogel nanoparticles.

[0098] Table 4 is a table showing the calculation results of the entrapment rate of each nanoparticle.

TABLE-US-00004 Experi- Experi- Experi- Experi- Experi- mental mental mental mental mental Example Example Example Example Example 1 2 3 4 5 Entrapment 93.46 97.86 99.56 83.57 91.5 Efficiency (%)

[0099] As a result, as shown in Table 4 and FIG. 4, the entrapment rate of the nanoparticles of Experimental Example 4 without hydrogel coating was as low as 83.57%, and the entrapment rate increased as the number of coating layer increased. Thus, the nanoparticles of Experimental Example 3 had the highest entrapment rate. In addition, the nanoparticles of Experimental Example 5 to which the hydrogel was not added to the core exhibited a relatively low entrapment efficiency. This result means that the binding force of the double layer of the nanoparticles multi-coated with the hydrogel of opposite charges is strengthened, so that the entrapment rate of the bioactive substance is high.

2.4. Comparison of Sustained-Release Effects of Experimental Examples 1 to 4 Nanoparticles

[0100] Using the nanoparticles of Experimental Examples 1 to 4, the amount of drug release in vitro of the bioactive ingredient was measured.

[0101] Specifically, 4 ml of the nanoparticle solutions of Experimental Examples 1 to 4 and 44 ml of a phosphate buffer of pH 7.4 were placed in a dialysis bag (molecular weight cut-off approximately 10,000 dalton, Thermal) and stirred by 200 RPM at 37° C. Next, 1 ml of samples were taken after 1, 2, 4, 6, 24, 48, 72 and 96 hours from the stirred solution, and the same amount of buffer solution was refilled. The collected samples were quantitatively analyzed by high performance liquid chromatography (HPLC) to measure the released bioactive substances over time.

[0102] Table 5 shows the amount of bioactive substances released by the nanoparticles extracted for each time period.

[0103] FIG. 5 is a graph showing the sustained-release behavior over time of nanoparticles including a hydrogel according to an embodiment of the present invention.

TABLE-US-00005 TABLE 5 Cumulative Expermental Expermental Expermental Expermental release (%) Example 1 Example 2 Example 3 Example 4  1 hr 30.04 4.45 0.00 23.11  2 hr 60.80 45.98 19.60 74.14  4 hr 72.81 66.90 37.64 92.30  6 hr 79.80 71.30 41.20 97.60  8 hr 81.40 72.82 53.30 100.00 24 hr 98.60 77.83 62.84 100.00 48 hr 100.00 98.91 91.17 100.00

[0104] As a result, as shown in Table 5 and FIG. 5, it was confirmed that most of the bioactive substances were released from the uncoated nanoparticles of Experimental Example 4 after 4 hours.

[0105] On the contrary, in the multi-layered nanoparticles of Experimental Examples 1 to 3, it was confirmed that initial release was suppressed and a sustained release behavior was shown. In particular, in the Experimental Example 3, it was confirmed that only 40% of the bioactive substance was released from the nanoparticles even after 6 hours. These results indicate that the nanoparticles have excellent entrapment efficiency and stability of the bioactive substance, and that the nanoparticles have a sustained-release behavior through inhibition of initial release.

Embodiment 3. Preparation of Experimental Group of Nanoparticles Encapsulating Natural Extracts

3.1. Preparation of Nanoparticles Encapsulating Natural Extracts and Coating by Step

[0106] In order to check the difference in shape according to the bioactive substances encapsulated in the nanoparticles, nanoparticles subjected to first coating by encapsulating natural extracts from existing peptides were set as Experimental Example 6, and nanoparticles subjected to be coated twice were set as Experimental Example 7, and the nanoparticles subjected to be coated three times were set as Experimental Example 8. Uncoated nanoparticles were set as Experimental Example 9.

[0107] Table 6 shows the amount of compositions for coatings used in the experiment.

TABLE-US-00006 TABLE 6 Coating layer Expermental Expermental Expermental Expermental Nano particle Structure Example 6 Example 7 Example 8 Example 9 Core Natural extracts 0.05% 0.05% 0.05% 0.05% Hydrogel 0.005% 0.005% 0.005% 0.005% Liposome Phospholipid 2.0% 2.0% 2.0% 2.0% Coating agent Chitosan 0.1% 0.1% 0.1% — Poly Glutamic acid — 0.1% 0.1% — Chitosan — — 0.01% —

3.2. Preparation of Uncoated Nano Liposome (Experimental Example 9)

[0108] In order to prepare uncoated natural extract-encapsulated nanoparticles, a natural extract-hydrogel solution and a phospholipid solution were prepared and mixed to provide a core-phospholipid mixed solution.

[0109] Natural extract-hydrogel solution was prepared by adding natural extracts, Pinus rigida Bark Extract, and polyglutamic acid to purified water and then dissolving them.

[0110] The following process was performed in the same manner as the process of manufacturing the uncoated nanoparticles of Experimental Example 4 shown in Example 1.2, and the nanoparticles of Experimental Example 9 in which the natural extract was encapsulated were prepared.

3.3 Preparation of Primary Coated Nano-Liposome (Experimental Example 6)

[0111] Nanoparticles of Experimental Example 6 coated with the amount shown in Table 6 were prepared.

[0112] Specifically, 1.0 wt % chitosan aqueous solution was prepared by dissolving chitosan, which is a cationic hydrogel, in purified water. Then, the chitosan aqueous solution was added to the nanoparticles of Example 3.2 for primary coating. Next, purified water was added for the total amount to be 100% to prepare the primary coated nanoparticles of Experimental Example 6.

3.4. Preparation of Secondary Coated Nano Liposome (Experimental Example 7)

[0113] Nanoparticles of Experimental Example 7 coated with the amount shown in Table 6 were prepared.

[0114] Specifically, to the primary coated nanoparticles of Example 3.3, 1 wt % polyglutamic acid aqueous solution was added to prepare the second coated nanoparticles of Experimental Example 7.

3.5. Preparation of Third Coated Nano Liposome (Experimental Example 8)

[0115] The nanoparticles of Experimental Example 8 coated with the amount shown in Table 6 were prepared.

[0116] A 0.1 wt % chitosan aqueous solution was prepared and added to the second coated nano-liposome of Example 3.4 to prepare the nanoparticles of Experimental Example 8 for the third coating. The coated nanoparticles were homogenized under high pressure through a microfluidizer. Specifically, the pressure condition of the high-pressure homogeneous step was set to 1000 bar, and the number of passes was set to 3 times.

Embodiment 4. Comparison of a Particle Size and Zeta Potential of the Nanoparticles According to by Coating Step and Amount of Hydrogel

4.1 Comparison of Particle Size and Zeta Potential of Nanoparticles by Amount

[0117] In order to compare the particle size and zeta potential of the nanoparticles according to the coating layer structure and the amount of hydrogel, the size and zeta potential of the nanoparticles encapsulated with the natural extract and the coated nanoparticles of Exemplary Example 3 were measured using a particle size analyzer.

[0118] Table 7 is a graph showing the particle size and zeta potential of nanoparticles according to the coating layer structure and amount of hydrogel.

TABLE-US-00007 TABLE 7 Experimental Experimental Experimental Experimental Example 6 Example 7 Example 8 Example 9 Particle size 125.94 ± 6.48   165.70 ± 06.05  177.83 ± 05.46  130.88 ± 00.48  (nm) Poly Dispersity 0.243 0.225 0.201 0.275 index Zete Potential 28.16 ± 01.76 −44.43 ± 01.66   −42.00 ± 00.87   −39.90 ± 04.17   (mV)

[0119] As shown in Tables 2 and 7, in the nanoparticles in which a bioactive substance was encapsulated in the core with a peptide of Experimental Example 4, the particle size was 106.95±5.06 nm. In contrast, the nanoparticles of Experimental Example 9 encapsulating natural extracts having various components increased in size of 130.88±0.48 nm due to the encapsulated components. The size of the hydrogel coating gradually increased as it included the multiple coating layers of the nanoparticles of Experimental Examples 6 to 8 in which the first, second, and third coating were performed, respectively.

4.2 Comparison of Entrapment Efficiency of Nano-Liposomes of Experimental Examples 6 to 9

[0120] Using the nanoparticles of Experimental Examples 6 to 9, the entrapment efficiency of Catechin among various components of the natural extract was measured. Analysis was carried out in the same manner as in Example 2.3.

[0121] Table 8 is a table showing the results of calculating the entrapment rate for each nanoparticle.

TABLE-US-00008 Experi- Experi- Experi- Experi- mental mental mental mental Example 6 Example 7 Example 8 Example 9 Entrapment 89.3 90.6 94.9 67.5 Efficiency (%)

[0122] As shown in Table 8, it was confirmed that the entrapment rate of the nanoparticles of Experimental Example 9 in which the hydrogel was not coated was 67.5%. However, as the coating layer increased, the entrapment rate increased, and thus the entrapment rate of the nanoparticles of Experimental Example 8 was the highest and the remarkable effect was exhibited. These results mean that in the nanoparticles coated with a multi-layered hydrogel of opposite charges, the binding force of the double coating layer is strengthened, so that the entrapment rate of the bioactive substance is high.

4.3 Measurement of DPPH Radical Scavenging Ability of Nanoparticles by Time

[0123] DPPH radical scavenging ability was measured to confirm the effect of antioxidant physiological activity according to the sustained-release release of nanoparticles according to the coating layer structure and amount of hydrogel.

[0124] 50 μL of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) was added to 100 ul of each sample solution. Then, it was stirred and reacted at room temperature for 30 minutes, and then the ELISA reader (Powerwave XS2, Biotek, USA) was used to measure the absorbance at 571 nm. The control groups were Butylated Hydroxynisole (BHA) and Catechin.

[0125] Next, using Equation 2 below, the DPPH radical scavenging ability of each nanoparticle was calculated.

DPPH radical scavenging ability (%)=(1−absorbance of sample added group/absorbance of no added group)×100  [Equation 2]

[0126] Table 9 shows the results of DPPH radical scavenging activity of Experimental Examples 6 to 9 and BHA and Catechin over time.

[0127] FIG. 6 is a graph showing DPPH radical scavenging activity over time of a multi-layer liposome according to an embodiment of the present invention.

TABLE-US-00009 TABLE 9 Experimental Experimental Experimental Experimental Example 6 Example 7 Example 8 Example 9 BHA Catechin 18 hr 91.0042 83.6820 66.1088 77.1967 93.2084 93.4211 24 hr 90.5882 77.6471 65.4118 76.7059 82.3362 87.4680 48 hr 87.4016 68.5039 50.3937 49.2126 47.0031 70.5607

[0128] As shown in Table 9 and FIG. 7, DPPH radical scavenging ability was not significantly reduced in Experimental Example 6, which proceeded to the third coating, as shown as 91.0042% at 18 hours and 87.4016% at 48 hours. However, in the case of BHA and Catechin, which were used as controls, 93.2084% and 93.4211%, respectively, were identified after 18 hours, but rapidly decreased to 47.0031% and 70.5607% after 48 hours. This result means that the anti-oxidation effect component in the hydrogel-coated nanoparticles is continuously released for a long period of time to exhibit a sustained-release effect.

[0129] While embodiments of the present invention have been particularly shown and described with reference to the accompanying drawings, the specific terms used herein are only for the purpose of describing the invention and are not intended to define the meanings thereof or be limiting of the scope of the invention set forth in the claims. Therefore, a person of ordinary skill in the art will understand that various modifications and other equivalent embodiments of the present invention are possible. Consequently, the true technical protective scope of the present invention must be determined based on the technical spirit of the appended claims.