HK1235705B - Hybrid-type multi-lamellar nanostructure of epidermal growth factor and liposome and method for manufacturing same - Google Patents

Description

Hybrid multilayer nanostructure of epidermal growth factor and liposome and method for producing the same

Technical Field

The present invention relates to a hybrid multilamellar nanostructure of epidermal growth factor (EGF) and liposomes and a method for manufacturing the same. More specifically, the present invention relates to novel protein-lipid hybrid multilamellar nanostructures and a method for manufacturing the same. The protein-lipid hybrid multilamellar nanostructures are formed by multivalent electrostatic interactions, in addition to hydrophobic interactions, between empty vesicles formed by cationic lipids and anionic epidermal growth factor proteins.

Background Art

As interest in beauty grows, physiologically active proteins with specific mechanisms of action and excellent efficacy have attracted attention as materials for functional cosmetic products. However, due to their high molecular weight, short half-life, and structural instability, physiologically active proteins do not easily penetrate the skin. Consequently, interest in the skin delivery of these physiologically active proteins has grown rapidly.

Among physiologically active proteins, epidermal growth factor (EGF) is known to play a key role in skin regeneration and is therefore used as a functional cosmetic ingredient. Currently, EGF is listed in the International Cosmetic Ingredient Dictionary (ICID) of the Cosmetic, Toiletries & Fragrance Association (CTFA) of the United States, and is also approved for use as a cosmetic raw material by the Ministry of Food and Drug Safety (MDFS) of South Korea. Consequently, it has been officially used as a cosmetic raw material in South Korea and other countries (Korean Food and Drug Administration Notice No. 2006-12, April 12, 2006).

However, the biggest problem in the skin delivery of physiologically active protein components (such as epidermal growth factor) is that these protein components have a low skin absorption rate (permeability). A typical method to overcome this problem is to deliver physiologically active proteins such as epidermal growth factor to the skin by using liposomes as carriers. Liposomes are lipid bilayer vesicles (vesicles) mainly formed by amphiphilic phospholipids (phospholipids) as components of cell membranes. Hydrophilic substances can be encapsulated in the internal aqueous compartment (aqueous compartment) of the liposome, or hydrophobic substances can be loaded in the lipid bilayer. The layer structure of the liposome is similar to that of the cell membrane, so the liposome has low toxicity and can deliver substances by fusion with cells or by endocytosis. In particular, because liposomes have excellent biocompatibility, research on the use of liposomes as carriers has been actively carried out (Bangham, A.D.; Torchilin, V.P., 2005, Nat. Rev. Drug Discov., 4: 145).

Despite such advantages, liposomes are difficult to be widely used due to some problems. One problem of liposomes is that the efficiency of encapsulation in liposomes is low. In particular, hydrophilic substances can only be encapsulated in the internal aqueous compartment of the liposome, and because the volume of the internal aqueous compartment is small, the efficiency of encapsulation is also necessarily low. It usually shows an encapsulation efficiency of about 10-20%, and shows serious technical limitations because the amount of encapsulated protein is very low relative to the gross weight of the liposome (Martins, Susana et al., 2007, Int. J. Nanomed., 2.4, 595). On the other hand, lipophilic substances are encapsulated with relatively high efficiency because they are solubilized in the lipid bilayer. However, in some cases, lipophilic substances can make the lipid bilayer unstable to reduce the stability of the liposome. Therefore, liposome technology is commercially applied to some lipophilic substances, but its use for hydrophilic substances is very insignificant.

Another problem with using liposomes as protein delivery vehicles is that physiologically active proteins are severely denatured during the general liposome preparation process to lose their characteristic physiological activity. As a general liposome preparation method, the Bangham method (Bangham et al., 1965 J. Mol. Biol. 13: 238-252) or high-pressure homogenization method is widely used. The Bangham method includes: adding and dissolving a surfactant in a solvent in a glass apparatus; evaporating the solution to form a surfactant (i.e., phospholipid) layer on the glass wall; introducing a solution of the material to be encapsulated; and vigorously stirring or ultrasonically homogenizing the solution to prepare liposomes. In the high-pressure homogenization method, the liposome components are mixed with each other and passed through a cartridge-type flow cell or valve (also referred to as an "interaction chamber") with a submicron-sized micropore. In this article, the size of the micropore is about 50-300 μm. Due to the high shear stress generated during passage, a lipid bilayer composed of surfactants is formed, and the drug is encapsulated in the phospholipid bilayer. If these methods are used to capture physiologically active proteins, they are exposed to the harsh conditions caused by the shear stress in the micropores (such as high pressure, high temperature, frictional heat), and due to the use of organic solvents, the proteins may aggregate, denature, oxidize, or degrade, and thus are likely to lose their characteristic physiological activity.

For example, Korean Patent No. 0752990 relates to a nanoliposome composed of a liposome layer containing esterified lecithin as a neutral lipid, and discloses a composition for preventing or treating skin diseases, comprising: nanoliposomes encapsulating epidermal growth factor; and a natural extract having anti-inflammatory activity. Korean Patent No. 0962566 discloses a nanoliposome containing human growth hormone as an active ingredient, wherein the nanoliposome is prepared using soybean lecithin as a neutral lipid through a high-pressure homogenization method. However, the liposomes containing physiologically active proteins disclosed in the above-described patent documents have the following problems: the encapsulation efficiency of the active ingredient is very low, and because the liposomes are prepared under high temperature and high pressure, the physiological activity of the protein is severely reduced.

Therefore, it is necessary to develop a method that can ensure a high encapsulation efficiency while stably maintaining the physiological activity of epidermal growth factor during the preparation of the carrier structure. According to the analysis of the previous research results, there are many attempts to use different additives or develop new process methods to improve the efficiency of encapsulating physiologically active proteins (such as epidermal growth factor) in the internal aqueous compartment of liposomes (Pisal, Dipak S. et al., 2010, J. Pharm. Sci., 99.6, 2557-2575). However, all attempts are characterized by their focus on improving the efficiency of encapsulating proteins in the internal aqueous compartment of liposomes.

Summary of the Invention

Technical issues

Therefore, the present inventors have endeavored to develop a novel method that can effectively incorporate or capture epidermal growth factor into liposomes while maintaining the high physiological activity of epidermal growth factor. In particular, breaking the old convention of methods that encapsulate proteins in the internal aqueous compartment of liposomes, the present inventors have studied methods for preparing new nanostructures by inducing more positive interactions between liposomes and proteins. In particular, the present inventors have noted that studies have shown that nucleic acids, which are anionic biopolymers, are conjugated to cationic liposomes to form new nanostructures (Safinya, C.R. et al., 1997, Science, 275.5301, 810-814), and therefore the present inventors have studied methods that do not encapsulate proteins during liposome preparation, but instead generate new structures through liposome-protein interactions after liposome preparation. Therefore, the present inventors have discovered that when a combination of multivalent electrostatic interactions and hydrophobic interactions is used between empty vesicles formed of cationic lipids and epidermal growth factor as an anionic protein under suitable conditions, a novel protein-lipid hybrid multilayer nanostructure can be prepared in which epidermal growth factor is captured with very high efficiency while maintaining its physiological activity, thereby completing the present invention.

Technical Solution

Therefore, one of the objects of the present invention is to provide a hybrid multilayer nanostructure made by the interaction between epidermal growth factor and empty vesicles formed by cationic lipids.

Another object of the present invention is to provide a method for manufacturing a protein-lipid mixed multilayer nanostructure by mixing epidermal growth factor and empty vesicles formed by cationic lipids through a spontaneous self-assembly process occurring at room temperature and pressure.

Another object of the present invention is to provide a cosmetic composition comprising a hybrid multilayer nanostructure.

Beneficial effects

The novel hybrid multilayer nanostructure according to the present invention exhibits not only high encapsulation efficiency but also a simple fabrication process, thereby allowing epidermal growth factor to be delivered to the body or cells with high physiological activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing a process of forming an EGF-DOTAP hybrid multilayer nanostructure according to one embodiment of the present invention.

FIG2 shows the measurement results of the particle size and surface charge of the EGF-DOTAP hybrid multilayer nanostructure prepared according to one embodiment of the present invention.

FIG3 shows the transmittance measurement results of an EGF-DOTAP hybrid multilayer nanostructure prepared according to one embodiment of the present invention.

FIG4 shows the measurement results of the encapsulation efficiency of the EGF-DOTAP hybrid multilayer nanostructure prepared according to one embodiment of the present invention.

FIG5 shows the actual structure of the EGF-DOTAP hybrid multilayer nanostructure prepared according to one embodiment of the present invention observed by Cryo-TEM.

FIG6 shows the analysis results of the stability of the EGF-DOTAP hybrid multilayer nanostructure prepared according to one embodiment of the present invention.

FIG7 shows the measurement results of the skin penetration properties of the EGF-DOTAP hybrid multilayer nanostructure prepared according to one embodiment of the present invention ( FIG7a : skin surface; and FIG7b : skin section).

DETAILED DESCRIPTION

The present invention provides a hybrid multilayer nanostructure formed by the interaction between epidermal growth factor and empty vesicles formed by cationic lipids; a method for producing a protein-lipid hybrid multilayer nanostructure by self-assembly between epidermal growth factor and empty vesicles formed by cationic lipids; and a cosmetic composition containing the hybrid multilayer nanostructure.

Hereinafter, the present invention is described in detail.

The present invention relates to an epidermal growth factor-liposome mixed multilayer nanostructure, comprising:

(a) Empty unilamellar liposomes composed of a cationic lipid bilayer;

(b) one or more unilamellar liposomes surrounding the empty unilamellar liposomes and consisting of a cationic lipid bilayer; and

(c) epidermal growth factor,

The epidermal growth factor is combined with the monolayer liposomes through electrostatic interaction and is located between the monolayer liposomes.

The cationic lipid may be selected from 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EDMPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (EDMPC), (1,2-distearoyl-sn-glycero-3-ethylphosphocholine, SPC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (EDPPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride chloride (DOTMA), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), dioleoyl glutamine, distearoyl glutamide, dipalmitoyl glutamide, dioleoylaspartamide and dimethyldioctadecylammonium bromide (DDAB) One or more of, but not limited to, these.

The empty unilamellar liposomes may have a zeta potential (or surface charge) of +1 to 100 mV, preferably +30 mV or higher.

The hybrid multilayer nanostructure may have a particle size of 50-900 nm, preferably 50-500 nm, and more preferably 100-200 nm. If the particle size of the nanostructure is greater than 900 nm, due to its large size, the nanostructure will have difficulty penetrating the skin and will therefore be unsuitable for delivering epidermal growth factor to the skin. If the particle size of the nanostructure is less than 50 nm, safety issues may arise, as the safety of nanostructures for human use has not yet been demonstrated.

The weight ratio (w/w) of epidermal growth factor to cationic lipid in the hybrid multilayer nanostructure can be 0.001 to 2.5:1, preferably 0.001 to 2.3:1, and more preferably 0.001 to 2.0:1. If the weight ratio (w/w) of epidermal growth factor to cationic lipid is less than 0.001:1, a multilayer nanostructure cannot be formed. If the weight ratio (w/w) of epidermal growth factor to cationic lipid is greater than 2.5:1, the nanostructure will have difficulty penetrating the skin due to its large size, and therefore it will not be suitable for delivering epidermal growth factor into the skin.

The encapsulation efficiency of the epidermal growth factor in the hybrid multilayer nanostructure may be at least 60%, preferably at least 80%, and more preferably at least 90%.

The epidermal growth factor used in the present invention may be a recombinant protein produced by South Korea Cetraon Co., Ltd., or may be a commercially available product.

The hybrid multi-lamellar nanostructures described herein are novel nanostructures formed through multivalent electrostatic and hydrophobic interactions between cationic lipid-derived vesicles and anionic epidermal growth factor. More specifically, as shown in Figure 1, the novel hybrid multi-lamellar nanostructures can be formed through spontaneous self-assembly between the cationic lipid-derived vesicles and the epidermal growth factor.

As used herein, the term "empty vesicle" refers to a vesicle with an empty interior space. More specifically, the term refers to a typical liposome composed of a lipid bilayer. Even more specifically, the term "empty vesicle" refers to an empty unilamellar liposome in the present invention.

The "one or more unilamellar liposomes" according to the present invention can be a multivesicular liposome (MVL) having multiple non-concentric internal aqueous compartments in the liposome particle, or a multivesicular liposome (MVL) having a series of generally spherical shells formed by lipid bilayers interspersed with aqueous layers, but is not limited thereto.

The present invention also relates to a method for preparing a hybrid multilayer nanostructure, comprising the following steps:

(1) preparing a solution containing empty unilamellar liposomes composed of cationic lipids and having uniform particle size;

(2) preparing an aqueous solution containing epidermal growth factor; and

(3) The solution containing the empty unilamellar liposomes obtained in step (1) is mixed with the aqueous solution containing the epidermal growth factor obtained in step (2).

The cationic lipid in step (1) can be selected from 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EDMPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (EPOPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EDMPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (EDMPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (EDOPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (EPOPC), 1,2-distearo ...EPOPC 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (SPC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (EDPPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride chloride (DOTMA), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), dioleoyl glutamine, distearoyl glutamide, dipalmitoyl glutamide, dioleoylaspartamide and dimethyldioctadecylammonium bromide (DDAB) One or more of, but not limited to, these.

The empty unilamellar liposomes in step (1) can be prepared by a general liposome preparation method. The liposome preparation method can be selected from the Bangham method, dry lipid hydration method, freeze and thaw method (in which a process of repeatedly freezing with liquid nitrogen and then thawing at room temperature), extrusion method, sonication method and microfluidizer method, but is not limited thereto.

The empty unilamellar liposomes obtained in step (1) may have a particle size of 100-300 nm, preferably 100-250 nm, more preferably 100-200 nm.

In step (3), the EGF and the empty unilamellar liposomes bind to each other through multivalent electrostatic and hydrophobic interactions to form a novel protein-lipid hybrid multilayer nanostructure. More specifically, as shown in FIG1 , the empty unilamellar liposomes and the EGF spontaneously self-assemble through multivalent electrostatic and hydrophobic interactions to form a hybrid multilayer nanostructure as shown in FIG5 .

The hybrid multilayer nanostructure contains:

(a) Empty unilamellar liposomes composed of a cationic lipid bilayer;

(b) one or more unilamellar liposomes surrounding the empty unilamellar liposomes and consisting of a cationic lipid bilayer; and

(c) epidermal growth factor,

The epidermal growth factor is located between the single-layer liposomes.

The hybrid multilayer nanostructure may have a particle size of 50-900 nm, preferably 50-500 nm, more preferably 100-200 nm.

The weight ratio (w/w) of epidermal growth factor:cationic lipid in the hybrid multilayer nanostructure may be 0.001 to 2.5:1, preferably 0.001 to 2.3:1, and more preferably 0.001 to 2.0:1.

The encapsulation efficiency of the epidermal growth factor in the hybrid multilayer nanostructure may be at least 60%, preferably at least 80%, and more preferably at least 90%.

Unlike conventional methods for preparing liposomes, the preparation method according to the present invention involves preparing empty lipid vesicles (empty unilamellar liposomes) of a desired size and mixing the prepared liposomes with a physiologically active ingredient. Therefore, the preparation method according to the present invention has the advantage of omitting the process of exposing the epidermal growth factor (i.e., the physiologically active ingredient) to high pressure, high temperature, or a strongly acidic solution during liposome preparation, thereby maintaining the physiological activity of the epidermal growth factor.

The present invention also relates to a cosmetic composition containing the hybrid multilayer nanostructure.

The hybrid multi-layer nanostructure may penetrate into the dermis of the skin, but is not limited thereto.

The type of cosmetic composition according to the present invention is not particularly limited, and depending on the formulation prepared, the cosmetic composition of the present invention may contain cosmetic composition components commonly used in the field of the present invention. The cosmetic composition of the present invention can be prepared into formulations including skin softeners, emulsions, nourishing creams, skin creams, beauty serums, essential oils, etc., and depending on the formulation prepared, the cosmetic composition of the present invention may further contain one or more selected from oils, water, surfactants, wetting agents, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc.

Inventive model

Hereinafter, the present invention will be described in more detail with reference to Examples, Experimental Examples and Formulation Examples. However, it should be understood that these Examples, Experimental Examples and Formulation Examples are only for illustrative purposes and are not intended to limit the scope of the present invention.

Example 1: Preparation of EGF-DOTAP hybrid multilayer nanostructures

1.1: Preparation of cationic empty unilamellar liposomes containing DOTAP

Cationic lipid DOTAP (20.96 mg, Avanti Polar Lipid, Inc.) was dissolved in 1 ml of chloroform and then mixed in a round glass flask. In a rotary evaporator, the lipid solution was flushed with nitrogen at a low speed to remove the chloroform, and the lipid was dried to form a thin lipid layer. The formed lipid layer was further dried in a vacuum for 12 hours to completely remove the remaining chloroform. 1 ml of purified water was added to the prepared lipid layer, and then stirred at 37 ° C for 2 hours to prepare empty lipid vesicles. The obtained empty lipid vesicles were extruded through a polycarbonate membrane (polycarbonate membrane) (Avanti Polar Lipid, Inc.) with a pore size of 100 nm several times to prepare cationic empty unilamellar liposomes containing DOTAP and having a uniform particle size.

1.2: Preparation of EGF-DOTAP hybrid multilayer nanostructures

The solution containing the DOTAP-containing cationic empty unilamellar liposomes prepared in Example 1.1 (500 μl) and the EGF solution (500 μl, Celltrion, Inc.) were mixed with each other in purified water at room temperature to prepare EGF-DOTAP hybrid multilamellar nanostructures. The prepared nanostructures were stored at 4°C until use.

Experimental Example 1: Evaluation of the Formation of EGF-DOTAP Hybrid Multilayer Nanostructures

1.1: Confirmation of the formation of cationic empty unilamellar vesicles

The particle size and zeta potential of the cationic empty unilamellar liposomes prepared in Example 1.1 were measured using dynamic light scattering (DLS, ELSZ-1000, Otsuka Electronics), and the measurement results are shown in the following Table 1. The measurement results showed that the prepared cationic empty unilamellar liposomes had a particle size of 200 nm and a positive surface charge.

[Table 1]

Empty unilamellar liposomes

Empty unilamellar liposomes Particle size (nm) Zeta potential (mV) DOTAP 197.7±4.9 56.5±2.5

1.2: Confirmation of the formation of EGF-DOTAP hybrid multilayer nanostructures

The particle size and zeta potential of the EGF-DOTAP hybrid multilayer nanostructure prepared in Example 1.2 were measured using DLS, and its transmittance was measured at 500 nm using a spectrophotometer (Jasco-815, Jasco, Inc.). The measurement results are shown in Figures 2 and 3.

As shown in Figure 2, when the EGF/DOTAP weight ratio was 2 or less, the particle size of the EGF-DOTAP hybrid multilamellar nanostructures was approximately 200 nm, similar to that of DOTAP empty unilamellar liposomes. However, the surface charge of the EGF-DOTAP hybrid multilamellar nanostructures was reduced compared to that of empty unilamellar liposomes. This is believed to be due to the electrostatic interaction between anionic EGF and cationic empty unilamellar liposomes, forming a novel structure.

As shown in Figure 3 , the transmittance remained constant when the EGF/DOTAP weight ratio was 2 or less, but decreased when the weight ratio was greater than 2. This may be because the amount of EGF that aggregated without being bound to the cationic empty unilamellar liposomes increased with increasing EGF concentration.

Experimental Example 2: Evaluation of EGF Encapsulation Efficiency in EGF-DOTAP Hybrid Multilayer Nanostructures

In order to measure the amount of EGF encapsulated in the EGF-DOTAP hybrid multilayer nanostructure, the EGF-DOTAP hybrid multilayer nanostructure (1 ml) prepared in Example 1.2 was centrifuged using an ultracentrifuge (200,000 x g, 2 hours, 4°C, Beckman) to separate the unencapsulated free EGF. The amount of the separated free EGF was measured using a micro BCA assay and an ELISA assay. The measurement results are shown in Figure 4.

As can be seen from Figure 4, high encapsulation efficiencies of 60% or more occur at most EGF/DOTAP weight ratios, which vary slightly depending on the method used to quantify EGF. Thus, it can be seen that the encapsulation efficiency in the structures according to the present invention is significantly higher than that in conventional liposomes (which is only 10-20%).

Experimental Example 3: Confirmation of the formation of a multilayer structure of an EGF-DOTAP hybrid multilayer nanostructure

In order to confirm the formation of the multilayer structure of the EGF-DOTAP hybrid multilayer nanostructure, observation was performed with Cryo-TEM using the plunge-dipping method, which enables accurate observation of particulate materials in aqueous solution. 4 μl of the EGF-DOTAP hybrid multilayer nanostructure prepared in Example 1.2 was placed on a lacey grid to form a thin layer. The thin aqueous layer was maintained at a suitable temperature and a humidity of 97-99% so that the solvent did not evaporate, and then it was quickly plunged into liquid ethane (about -170°C) to obtain a single-layer frozen sample. The frozen sample prepared as described above was observed using a transmission electron microscope (JEM-3011, JEOL Ltd.) at an accelerating voltage (300 kV), and the data were analyzed using the Gatan Digital Micrograph program.

As shown in FIG5 , the multilayer structure of the EGF-DOTAP hybrid multilayer nanostructure is shown to be formed by the electrostatic attraction between EGF and DOTAP.

Experimental Example 4: Investigation of the Stability of EGF-DOTAP Hybrid Multilayer Nanostructures

In order to investigate the stability of EGF protein by the structural change of EGF in the EGF-DOTAP hybrid multilayer nanostructure, circular dichroism (CD) was measured in the range of 180-260nm by using a Jasco-815CD spectropolarimeter (Jasco-815, Jasco Inc.). The EGF-DOTAP hybrid multilayer nanostructure prepared in Example 1.2 was placed in a 0.5mm path length flow cell and analyzed. Free EGF that was not trapped in the nanostructure was used as a control. The structural change of EGF was investigated at different temperatures for 100 days. The CD data are shown in Figure 6.

As can be seen from Figure 6, compared with the control (EGF), the EGF-DOTAP hybrid multilayer nanostructure stored at 4°C (EGF-DOTAP, 4°C) and the EGF-DOTAP hybrid multilayer nanostructure stored at room temperature (EGF-DOTAP, room temperature) did not undergo significant physical changes.

Experimental Example 5: Examination of the skin permeability of EGF-DOTAP hybrid multilayer nanostructure

To examine the skin permeability of EGF-DOTAP hybrid multilayer nanostructures, nude mice (SKH-1 Hairless, 5 weeks old, Orientbio, Korea) were used. A PDMS mold (diameter: 0.8 cm; height: 0.5 cm) was affixed to the back of each mouse. A 50 ml sample, obtained by reacting EGF, DOTAP, or the EGF-DOTAP hybrid multilayer nanostructure prepared in Example 1.2 with a fluorescent agent (FITC), was loaded into the PDMS mold and allowed to react with the mouse skin for 1 hour. The skin isolated from the mice was sectioned using a cryostat microtome (Leica CM1850, Leica Microsystems) and observed using a fluorescence microscope (Leica DMI 3000B, Leica Microsystems).

As shown in FIG7 a , it was observed that EGF or DOTAP alone was deposited on the skin surface without penetrating through the epidermis, but the EGF-DOTAP hybrid multilayer nanostructure did not remain on the skin surface.

Furthermore, as shown in FIG7 b , observation of skin sections obtained using a microtome revealed that the EGF-DOTAP hybrid multilayer nanostructure penetrated from the epidermis to the dermis of the skin.

Based on the results of the above experimental examples, formulation examples of cosmetic compositions containing the EGF-DOTAP hybrid multi-layered nanostructure of the present invention will now be described. However, the components of the present invention are not limited to these formulation examples.

Formulation Example 1: Skin Lotion

A skin lotion having the components shown in Table 2 below was prepared according to a conventional method.

[Table 2]

Skin lotion composition

Components Content (parts by weight) EGF-DOTAP hybrid multilayer nanostructure 0.5 1,3-Butanediol 6.0 glycerin 4.0 Oleyl alcohol 0.1 Polysorbate 20 0.5 ethanol 15.0 Benzophenone-9 0.05 Fragrances and preservatives appropriate amount purified water to 100

Formulation Example 2: Emulsion Lotion

Emulsion lotions having the components shown in Table 3 below were prepared according to a conventional method.

[Table 3]

Composition of emulsion lotion

Components Content (parts by weight) EGF-DOTAP hybrid multilayer nanostructure 1.0 Propylene glycol 6.0 glycerin 4.0 triethanolamine 1.2 Tocopheryl acetate 3.0 liquid paraffin 5.0 Squalane 3.0 macadamia oil 2.0 Polysorbate 60 1.5 Sorbitan sesquioleate 1.0 Carboxyvinyl polymer 1.0 Butylated hydroxytoluene 0.01 EDTA-2Na 0.01 Fragrances and preservatives appropriate amount purified water to 100

Formulation Example 3: Essential Oil

Essential oils having the composition shown in Table 4 below were prepared according to conventional methods.

[Table 4]

Composition of essential oils

Preparation Example 4: Nourishing Cream

A nourishing cream having the components shown in Table 5 below was prepared according to a conventional method.

[Table 5]

Composition of nourishing cream

Components Content (parts by weight) EGF-DOTAP hybrid multilayer nanostructure 2.0 Cedecanoic acid 2.0 Glyceryl Stearate 1.5 Tricaprylin 5.0 Polysorbate 60 1.2 Sorbitan stearate 0.5 Squalane 5.0 liquid paraffin 3.0 Cyclomethicone 3.0 Butylated hydroxytoluene 0.05 δ-Tocopherol 0.2 concentrated glycerin 4.0 1,3-Butanediol 2.0 Xanthan gum 0.1 EDTA-2Na 0.05 Fragrances and preservatives appropriate amount purified water to 100

Formulation Example 5: Skin Cream

Skin care creams having the components shown in Table 6 below were prepared according to conventional methods.

[Table 6]

Composition of skin care cream

Components Content (parts by weight) EGF-DOTAP hybrid multilayer nanostructure 1.0 Propylene glycol 2.0 glycerin 4.0 Carboxyvinyl polymer 0.3 ethanol 7.0 PEG-40 hydrogenated castor oil 0.8 triethanolamine 0.3 Butylated hydroxytoluene 0.01 EDTA-2Na 0.01 Fragrances and preservatives appropriate amount purified water to 100

Claims (9)

1. A hybrid multi-lamellarnanostructure comprising epidermal growth factor and liposomes, wherein: (a) Empty monolayer liposomes composed of cationic lipid bilayers; (b) One or more monolayer liposomes surrounding an empty monolayer liposome and consisting of a cationic lipid bilayer; and (c) Epidermal growth factor, The epidermal growth factor binds to monolayer liposomes through electrostatic interactions and is located between the monolayer liposomes. The hybrid multilayer nanostructure has a particle size of 50-900 nm and the weight ratio (w/w) of epidermal growth factor to cationic lipid is 0.001 to 2.5:1. 2. The nanostructure according to claim 1, wherein the cationic lipid is selected from 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EDMPC), 1,2-distearate-sn-glycero- 3-Ethylphosphocholine (1,2-distearoyl-sn-glycero-3-ethylphosphocholine, SPC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (EDPPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) One or more of the following: chloride (DOTMA), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), dioleoyl glutamide, distearoyl glutamide, dipalmitoyl glutamide, dioleoyl aspartamide, and dimethyldioctadecylammonium bromide (DDAB). 3. The nanostructure according to claim 1, wherein the hybrid multilayer nanostructure has a particle size of 50-500 nm. 4. The nanostructure according to claim 1, wherein the empty monolayer liposome has a zeta potential of +1 to 100 mV. 5. The nanostructure according to claim 1, wherein the encapsulation rate of epidermal growth factor in the hybrid multilayer nanostructure is 60% or higher. 6. A method for preparing a hybrid multilayer nanostructure according to any one of claims 1 to 5, the method comprising the following steps: (1) Prepare a solution containing empty monolayer liposomes with uniform particle size composed of cationic lipids; (2) Preparation of an aqueous solution containing epidermal growth factor; and (3) Mix the solution containing empty monolayer liposomes obtained in step (1) with the aqueous solution containing epidermal growth factor obtained in step (2). 7. The method according to claim 6, wherein the empty monolayer liposomes obtained in step (1) have a particle size of 100-300 nm. 8. A cosmetic composition comprising, as an active ingredient, a mixed multilayer nanostructure according to any one of claims 1 to 5. 9. The cosmetic composition according to claim 8, wherein the hybrid multilayer nanostructure penetrates into the dermis of the skin.