EGF·liposome·exosome triple lifting core ampoule composition manufacturing device and method, and EGF·liposome·exosome triple lifting ampoule composition formed thereby

Abstract

The present invention relates to an apparatus and method for manufacturing an EGF-liposome-exosome triple lifting ampoule composition that improves skin wrinkles by mixing a viscosity-texture improving agent, a film former, a conditioning agent, a preservative, borage oil, and galangal root extract into a core in which signaling EGF, sustained-release liposomes, and natural membrane-ingredient-stabilizing exosomes are combined, and a lifting ampoule composition that improves skin wrinkles, and an EGF-liposome-exosome triple lifting ampoule composition formed thereby.

Description

Technology Field

[0001] The present invention relates to an apparatus and method for manufacturing an EGF-liposome-exosome triple lifting ampoule composition that improves skin wrinkles, which is applied to the lips, nasolabial folds, forehead wrinkles, and crow's feet, and more specifically, to a core in which signaling EGF, sustained-release liposomes, and natural membrane-absorbing / stabilizing exosomes are combined, and a viscosity / texture improving agent, a film former, a conditioning agent, a preservative, borage oil, and galangal root extract are mixed to produce a lifting ampoule composition that improves skin wrinkles, and to an EGF-liposome-exosome triple lifting ampoule composition formed thereby. Background Technology

[0003] Human skin is composed of the epidermis, dermis, subcutaneous fat layer, and muscle layer in that order. As the outermost organ of the body, it functions to block various external stimuli. The epidermis refers to the outermost surface of the skin and acts as a skin barrier by blocking external stimuli and preventing moisture evaporation.

[0004] The dermis refers to the layer immediately below the epidermis that is 2-3 mm thick and accounts for more than 90% of the skin. The dermis is composed of components such as collagen, elastin, and hyaluronic acid, and plays a role in supporting the skin and maintaining elasticity.

[0005] In particular, collagen is a major component of the dermis, accounting for 75% of the skin's dry weight, and plays a role in providing tension to the skin.

[0006] Skin aging is a phenomenon caused by various factors such as ultraviolet rays, lifestyle habits, genetics, repetitive facial movements, sleeping posture, and old age.

[0007] As humans age, the amount of collagen naturally decreases, leading to a loss of elasticity; the skin becomes dry due to a decrease in moisture content, and wrinkles form.

[0008] Functional cosmetics are interpreted as products that not only provide beauty effects but also influence the structure or function of the body and perform preventive and therapeutic functions; additionally, functional cosmetics are being developed to address external physical changes resulting from skin aging, such as dryness and wrinkle formation.

[0009] For example, retinoic acid, animal placenta-derived protein, betulinic acid, and Chlorella extract are known as active ingredients that promote collagen synthesis and exhibit wrinkle-improving effects.

[0010] However, the aforementioned active ingredients have safety issues, such as irritation and redness when applied to the skin, as well as the problem that substantial wrinkle improvement cannot be expected due to their minimal effects.

[0011] Therefore, there is a need to develop functional cosmetics that are safe and stable while exhibiting skin regeneration and wrinkle improvement effects.

[0012] In order to solve these problems, as a prior art, Korean Registered Patent Publication No. 10-2582633 describes a cosmetic composition for skin regeneration and wrinkle improvement containing epidermal growth factor liposomes and a growth factor complex. However, since EGF itself is a peptide of protein, it is susceptible to heat, pH, enzymatic degradation, and oxidation, and its activity decreases rapidly over time. Furthermore, because it is a hydrophilic peptide with a molecular weight of about 6 kDa, it has low diffusion on its own, resulting in a problem where the actual wrinkle improvement effect is reduced.

[0013] In addition, in the case of liposomes, they fuse and decompose (aggregate, leakage) during storage, which reduces encapsulation efficiency. Since they are nanoparticles of around 100 nm, penetration to the deep epidermis is limited, so most remain in the stratum corneum and the upper part of the epidermis. Consequently, when applied, they are quickly lost after absorption, resulting in a problem of reduced sustained effect.

[0014] In addition, particle size distribution, encapsulation efficiency, and surface charge vary depending on the manufacturing process, leading to quality differences between batches and low EGF loading efficiency, which resulted in high cost burdens during mass production. Prior art literature

[0016] Korean Patent Publication No. 10-2582633 The problem to be solved

[0017] To solve the above problems, the present invention enables PC, cholesterol, electrolytes, and antioxidants to be mixed at the molecular level through a lipid dissolution step, thereby allowing for the uniform formation of a thin film; furthermore, through a hydration step, MLV liposomes are formed, which lays the foundation for subsequent pretreatment (freeze-thaw, ultrasound) and microfluider treatment; and through a coating and fusion combination step, the low stability and efficiency observed in conventional cases where liposomes and exosomes are mixed solely through physical coexistence are improved by forming a new combination structure through surface coating and partial membrane fusion, allowing EGF signaling, exosome biomolecular signaling, and liposome sustained release to act simultaneously in a single formulation; and through a coexistence and surface binding combination step, site-selective binding using an SH introduction site maintains exposure of the EGF binding site (receptor recognition surface), thereby increasing effective affinity. The purpose is to provide an apparatus and method for manufacturing an EGF-liposome-exosome triple lifting ampoule composition that can secure multilayered functionality for improving skin wrinkles by combining the formulation stability and application sensation of a viscosity and texture improving agent, the barrier, moisturizing, and anti-inflammatory properties of a conditioning agent and borage oil, and the antioxidant and collagen degradation inhibition properties of galangal extract with a core of EGF (signaling), liposome (sustained-release and retention), and exosome (natural membrane, ingestion, and stability) through the step of forming an EGF-liposome-exosome triple lifting ampoule composition, and the EGF-liposome-exosome triple lifting ampoule composition formed thereby. means of solving the problem

[0019] To achieve the above objective, the apparatus for manufacturing an EGF-liposome-exosome triple lifting ampoule composition according to the present invention

[0020] This is achieved by configuring a core in which signaling EGF, sustained-release and retention liposomes, and natural membrane, ingestion, and stabilization exosomes are combined, to mix a viscosity and texture improving agent, a film former, a conditioning agent, a preservative, borage oil, and galangal root extract to produce a lifting ampoule composition that improves skin wrinkles.

[0022] More specifically, the apparatus for manufacturing the above EGF·liposome·exosome triple lifting ampoule composition,

[0023] A rotary evaporator (100) that puts a solution into a round-bottom flask, rotates it over a water bath to evaporate it, and then condenses it under reduced pressure from a vacuum pump to form a lipid thin film, and

[0024] A vacuum pump (200) located on one side of a rotary evaporator, which generates a pressure reduction force and transmits it toward the rotary evaporator, and

[0025] A jacketed stirring tank (300) having a double-layered jacket structure formed on the outer wall of the tank to circulate temperature, cold water, and glycol solution to control the temperature of the contents filled in the tank to 40-50℃, and

[0026] A low-shear stirrer (400) that injects a hydration solution formed from a jacketed stirring tank into a lipid film, rotates it at 20 to 200 rpm through an impeller formed inside the tank, and stirs the lipid film and the hydration solution to form MLV liposomes, and

[0027] A deep freezer (500) that receives MLV liposomes formed in a low-shear stirrer, rapidly freezes them to shrink and rearrange the lipid membrane, and

[0028] An ultrasonic bath (600) that receives and positions MLV liposomes formed in a low-shear stirrer into a glass vial within a water bath, and then generates ultrasound in the 20-40 kHz band through an ultrasonic transducer to soften the liposome membrane through low-intensity cavitation, and

[0029] A microfluidizer (700) located on one side of the ultrasonic bath and receiving MLV liposomes with softened liposome membranes from the ultrasonic bath, pressurizing them to an ultra-high pressure of 700 to 1400 bar and passing them through a Y-shaped microchannel so that particles collide at high speeds with each other due to shear force, turbulence, and cavitation to induce the breakdown of large multilayer liposomes (MLV) into single-membrane liposomes (LUV) of 80 to 150 nm, and

[0030] A coating / fusion type combination forming module (800) that forms an EGF-liposome-exosome combination by coating an exosome (EV) membrane component onto the outer membrane of a liposome or partially fusing the two membranes to form liposome sustained release along with a natural membrane surface label, and

[0031] A surface binding / coexistence type combination forming module (900) that simultaneously creates liposomes, exosomes, and EGF proteins within a system, wherein some are chemically bound, some are physically coexisted, or the EGF protein is attached to the surface of the liposome by a covalent bond and stably fixed, thereby forming an EGF-liposome-exosome combination, and

[0032] The EGF-liposome-exosome triple lifting ampoule composition forming module (900a) is composed of an EGF-liposome-exosome triple lifting ampoule composition forming module that completes the EGF-liposome-exosome triple lifting ampoule composition by adding an additive, such as a viscosity-texture improving agent (carbomer, HEC), a film former (polyquaternium, polyglutamic acid), a conditioning agent (ceramide, squalane), a preservative (phenoxyethanol, ethylhexylglycerin), borage oil, and galanga root extract, to the EGF-liposome-exosome combination formed through a coating-fusion type combination forming module and a surface bonding-coexistence type combination forming module. Effects of the invention

[0034] As explained above, in the present invention

[0036] First, through the lipid dissolution step, PC, cholesterol, electrolyte lipids, and antioxidants are mixed at the molecular level, thereby enabling the uniform formation of a thin film. Cholesterol strengthens the membrane, and electrolyte lipids provide stability of the zeta potential, which can suppress aggregation and fusion to less than 40% of the conventional level. Additionally, through low-temperature dissolution, the addition of antioxidants, and nitrogen purging, lipid stability can be improved by 80% compared to the conventional level in the pre-EGF introduction stage.

[0038] Second, by performing a hydration step, the formation of MLV liposomes can lay the foundation for subsequent pretreatment (freeze-thaw, ultrasound) and microfluider treatment. Additionally, moisturizers, protective agents, and buffering components can enter the liposomes, increasing stability by 80% compared to the existing method. Furthermore, lipid concentrations can be uniformly dispersed to lower the PDI to 0.25 or lower, and an environment can be provided to enhance protein stability when EGF is introduced in a subsequent step.

[0040] Third, by performing a coating and fusion type combination step, the low stability and efficiency resulting from physical coexistence alone when mixing liposomes and exosomes in the existing case is improved. By forming a new combination structure through surface coating and partial membrane fusion, delivery efficiency and stability are improved by 80% compared to the existing case. Additionally, the low cell recognition ability resulting from the formation of only liposomes in the existing case is improved by introducing exosome-labeled components, thereby increasing intracellular uptake rate and targetability by 1.5 to 2 times compared to the existing case. Furthermore, the low internal loading capacity and sustained release potential resulting from the formation of only exosomes in the existing case, despite high natural uptake, is improved by fusing with the liposome structure to increase encapsulation efficiency and control release. Additionally, EGF signaling, exosome biomolecular signaling, and liposome sustained release can act simultaneously in a single formulation.

[0042] Fourth, by being composed of a co-occurring and surface-binding combination step, the EGF spacing (nm) can be finely controlled with PEG-maleimide to optimize EGFR dimerization and clustering without overstimulation, and effective affinity can be increased by maintaining exposure of the EGF binding site (receptor recognition surface) through site-selective binding using the SH introduction site, and by surface fixation through covalent bonding, detachment and diffusion into the EGF solution are minimized even in the presence of epidermal moisture and moisturizers, and EV surfaces (tetraspanin CD63 / CD81, integrin, etc.) form a corona on the liposome surface through weak non-covalent interactions, thereby improving intake efficiency by 80% even in diversified cell recognition and uptake pathways (clathrin / caveolae / raft), and spaced-temporal delivery can be achieved as EVs are responsible for uptake and cell affinity and liposomes are responsible for release control (sustained release).

[0044] Fifth, by completing the step of forming the EGF, liposome, and exosome triple lifting ampoule composition, multi-layered functionality for skin wrinkle improvement can be secured by combining the cores of EGF (signaling), liposomes (sustained-release and retention), and exosomes (natural membrane, ingestion, and stabilization) with the formulation stability and application sensation of viscosity and texture improvers, the immediate lifting effect of film formers, the barrier, moisturizing, and anti-inflammatory properties of conditioning agents and borage oil, and the antioxidant and collagen degradation inhibition of galangal extract. Furthermore, carbomer and HEC form an EV dispersion stabilization network with MLV and LUV liposomes to inhibit sedimentation and aggregation; ceramide, squalane, and borage oil (GLA) reinforce the skin barrier by restoring the stratum corneum lamellar structure and providing anti-inflammation; and flavonoids (such as galangin) from galangal root extract scavenge ROS and inhibit MMPs, thereby forming a high-quality lifting ampoule composition. there is. Brief explanation of the drawing

[0046] FIG. 1 is a block diagram illustrating the components of an apparatus (1) for manufacturing an EGF, liposome, and exosome triple lifting ampoule composition according to the present invention. FIG. 2 is a perspective view illustrating the components of an apparatus (1) for manufacturing an EGF-liposome-exosome triple lifting ampoule composition according to the present invention. FIG. 3 is a block diagram illustrating the components of a rotary evaporator according to the present invention, FIG. 4 is an exemplary embodiment illustrating the components of a rotary evaporator according to the present invention. FIG. 5 is a block diagram illustrating the components of a microfluider according to the present invention, FIG. 6 is an exemplary embodiment illustrating the components of a microfluider according to the present invention. FIG. 7 is a block diagram illustrating the components of a coating-fusion type combination forming module according to the present invention, FIG. 8 is an exemplary embodiment illustrating the components of a coating-fusion type combination forming module according to the present invention. FIG. 9 is a block diagram illustrating the components of a coating / fusion type TFF part according to the present invention, FIG. 10 is a block diagram illustrating the components of a surface-bonded / coexisting type combination forming module according to the present invention, FIG. 11 is an exemplary embodiment illustrating the components of a surface-bonded / coexisting type combination forming module according to the present invention. FIG. 12 is a block diagram illustrating the components of an exosome coexistence type combination forming part according to the present invention, FIG. 13 is a block diagram illustrating the components of a surface-bonded / coexisting type TFF part according to the present invention, FIG. 14 is a block diagram illustrating the components of an EGF-liposome-exosome triple lifting ampoule composition forming module according to the present invention. FIG. 15 is an exemplary embodiment illustrating the components of an EGF-liposome-exosome triple lifting ampoule composition forming module according to the present invention. FIG. 16 is a flowchart illustrating a method for preparing an EGF-liposome-exosome triple lifting ampoule composition according to the present invention. FIG. 17 is a flowchart illustrating the specific process of the EGF-liposome-exosome combination step according to the present invention. FIG. 18 is a flowchart illustrating the specific process of the coating-fusion type combination step according to the present invention. FIG. 19 is a flowchart illustrating a specific process of the coating step according to the present invention. FIG. 20 is a flowchart illustrating a specific process of the fusion step according to the present invention. FIG. 21 is a flowchart illustrating the specific process of the coexistence-type surface-bonding combination step according to the present invention. FIG. 22 is a flowchart illustrating the specific process of the step of forming an EGF-liposome-exosome triple lifting ampoule composition according to the present invention. FIG. 23 is an exemplary embodiment illustrating an EGF-liposome-exosome triple lifting ampoule composition formed through a method for preparing an EGF-liposome-exosome triple lifting ampoule composition according to the present invention. Specific details for implementing the invention

[0048] First, the EGF, liposome, and exosome combination described in the present invention refers to a combination in which different components (EGF, liposome, and exosome) coexist within the same formulation but are structurally combined, thereby securing multi-layered functionality for improving skin wrinkles by combining the formulation stability and application sensation of a viscosity and texture improving agent, the immediate lifting effect of a film former, the barrier, moisturizing, and anti-inflammatory properties of a conditioning agent and borage oil, and the antioxidant and collagen degradation inhibition properties of galangal extract, with a core of EGF (signaling), liposome (sustained release and retention), and exosome (natural membrane, ingestion, and stability).

[0049] In other words, it is expressed from a practical approach as co-existence, blending, and admixture.

[0050] The above sustained release refers to a formulation designed to allow the active ingredients of a drug to be released slowly within the body.

[0052] Next, in the present invention, the ampoule refers to a 'skin nutrient' containing highly concentrated active ingredients that focuses on resolving concerns regarding wrinkled skin improvement in a short period of time, and has a higher concentration of active ingredients than a serum or essence. The ampoule according to the present invention is sealed in a glass bottle or capsule container to maintain the stability of the active ingredients and is used intensively for the improvement of wrinkled skin.

[0054] Hereinafter, preferred embodiments according to the present invention will be described with reference to the accompanying drawings.

[0055] FIG. 1 is a block diagram illustrating the components of an apparatus (1) for manufacturing an EGF, liposome, and exosome triple lifting ampoule composition according to the present invention, and FIG. 2 is a perspective view illustrating the components of an apparatus (1) for manufacturing an EGF, liposome, and exosome triple lifting ampoule composition according to the present invention. The apparatus is configured to manufacture a lifting ampoule composition that improves skin wrinkles by mixing a viscosity and texture improving agent, a film former, a conditioning agent, a preservative, borage oil, and galangal root extract into a core in which signal EGF, sustained-release and retention liposomes, and natural membrane, ingestion, and stabilization exosomes are combined.

[0057] More specifically, the above-mentioned EGF·liposome·exosome triple lifting ampoule composition manufacturing device (1) is composed of a rotary evaporator (100), a vacuum pump (200), a jacketed stirring tank (300), a low-shear stirrer (400), a deep freezer (500), an ultrasonic bath (600), a microfluidizer (700), a coating / fusion type combination forming module (800), a surface bonding / coexistence type combination forming module (900), and an EGF·liposome·exosome triple lifting ampoule composition forming module (900a).

[0059] First, a rotary evaporator (100) according to the present invention will be described.

[0060] The above rotary evaporator (100) serves to place a solution in a round-bottom flask, rotate it over a water bath to evaporate it, and then condense it under reduced pressure from a vacuum pump to form a lipid thin film.

[0061] As shown in FIGS. 3 and 4, this consists of a flask (110), a rotary motor (120), and a vacuum inlet (130).

[0063] The above flask (110) is formed in a round glass bottle structure and serves to form a lipid thin film by rotating and evaporating the lipids and solvents, and then condensing them in a reduced pressure atmosphere from a vacuum pump.

[0064] This involves dissolving hydrogenated phospholipids (PC), cholesterol, charge-regulating lipids, lipid-specific antioxidants, and ethanol as a solvent to form a homogeneous lipid solution.

[0065] Then, the lipid solution is rotated to evaporate, and then condensed from a vacuum pump under reduced pressure to form a lipid thin film.

[0067] The above rotary motor (120) generates rotational force and serves to rotate the flask.

[0068] At this time, as the rotating flask rotates, the lipid solution spreads as a thin film on the wall, and as the evaporation efficiency is improved, the lipid is uniformly coated on the glass wall of the flask.

[0069] Subsequently, when the solvent is removed, a uniform thin lipid film is formed, which improves the efficiency of liposome generation during the hydration step.

[0071] The above vacuum inlet (130) is located on one side of the flask and receives a pressure reduction force from the vacuum pump and serves to reduce the pressure.

[0073] Thus, by configuring a rotary evaporator consisting of a flask, a rotary motor, and a vacuum inlet, the uniformity of the lipid thin film can be ensured, heat-sensitive components can be protected, high-viscosity and large-volume solutions can be processed easily, and the solvent recovery rate can be improved by 80% compared to conventional methods by linking the vacuum line and the cooling condensation unit.

[0075] Next, a vacuum pump (200) according to the present invention will be described.

[0076] The above vacuum pump (200) is located on one side of the rotary evaporator and serves to generate a pressure reduction force and transmit it to the rotary evaporator.

[0077] Here, the pressure reduction force is formed as -0.08 MPa.

[0078] By transmitting a depressurizing force toward the rotary evaporator, the pressure is lowered, the boiling point is reduced, and the solvent is removed.

[0079] In this way, by configuring the vacuum pump, the rotary evaporator can form a lipid thin film quickly and uniformly at a low temperature (40~55℃).

[0081] Next, a jacketed stirring tank (300) according to the present invention will be described.

[0082] The jacketed stirring tank (300) has a double-layered jacket formed on the outer wall of the tank and circulates temperature, cold water, and glycol solution to control the temperature of the contents filled in the tank to 40~50℃.

[0083] It consists of a tank body made of SUS316L, an upper port, a jacket section, and a lower port.

[0085] The above tank body is formed in a cylindrical shape lying horizontally, protecting and supporting each device from external pressure, and plays the role of dissolving water, buffer (phosphate), NaCl, EDTA, glycerin, butylene glycol (BG), trehalose, and mannitol at 40~50℃ in the internal space to form a hydrated liquid.

[0087] The upper port above serves to introduce purified water, a buffer (phosphate), NaCl, EDTA, glycerin, butylene glycol (BG), trehalose, and mannitol into the internal space of the tank body.

[0089] The jacket section is formed in a double-layer structure on the outer wall of the tank and serves to dissolve purified water, buffer (phosphate), NaCl, EDTA, glycerin, butylene glycol (BG), trehalose, and mannitol filled in the tank by circulating temperature, cold water, and glycol solution, while controlling the temperature up to 40~50℃.

[0090] This is configured to inhibit oxidation through sealing and nitrogen purging. It is configured to be set to pH 6.5.

[0092] The above lower port serves to discharge the hydrated liquid dissolved at 40~50℃ in the internal space of the tank body toward the low-shear stirrer.

[0094] Next, a low-shear stirrer (400) according to the present invention will be described.

[0095] The above low-shear stirrer (400) introduces a hydration solution formed from a jacketed stirring tank into a lipid film, then rotates it at 20 to 200 rpm through an impeller formed inside the tank, and stirs the lipid film and the hydration solution to form MLV liposomes.

[0096] This is configured so that lipids in a thin film state come into contact with a hydration solution to form multilayer liposomes.

[0097] Low-shear stirring gently exfoliates lipid membranes for uniform dispersion and induces liposome formation while preserving protein structure.

[0098] In addition, it is pretreated to reduce inter-particle collisions and aggregation, and to facilitate particle homogenization in the subsequent microfluider process.

[0099] The above low-shear stirrer forms MLV liposomes by low-speed rotation of 20 to 200 rpm at 40 to 50°C and stirring for 30 to 60 minutes.

[0100] Here, MLV (multilamellar vesicle) liposomes refer to vesicles in which multiple layers of lipid bilayers form a concentric structure like an onion peel, with an aqueous solution sandwiched between them, and usually have a diameter ranging from several hundred nm to several μm.

[0102] In addition, when encapsulating EGF, the low-speed stirrer switches the hydration temperature to ≤25℃ and then adds the EGF aqueous solution.

[0104] Next, a deep freezer (500) according to the present invention will be described.

[0105] The above-mentioned deep freezer (500) receives MLV liposomes formed in a low-shear stirrer, rapidly freezes them, and serves to shrink and rearrange the lipid membrane.

[0106] This is configured so that when frozen at -80 ℃, the lipid bilayer breaks, and then realigns during the thawing process, expanding the internal space to increase the encapsulation rate of water-soluble substances.

[0107] And, improve the encapsulation efficiency by repeating 2 to 3 times.

[0109] Next, an ultrasonic bath (600) according to the present invention will be described.

[0110] The above-mentioned ultrasonic bath (600) receives MLV liposomes formed in a low-shear stirrer into a glass vial within a water bath, places them therein, and then generates ultrasound in the 20-40 kHz band through an ultrasonic transducer to soften the liposome membrane through low-intensity cavitation.

[0111] This, along with uniform processing, lowers the risk of protein (EGF) denaturation.

[0113] Next, a microfluidizer (700) according to the present invention will be described.

[0114] The above-mentioned microfluidizer (700) is located on one side of the ultrasonic bath and receives MLV liposomes with softened liposome membranes from the ultrasonic bath, pressurizes them with an ultra-high pressure of 700 to 1400 bar, passes them through a Y-shaped microchannel, and induces the large multilayer liposomes (MLV) to be crushed by high-speed collisions of particles with each other due to shear force, turbulence, and cavitation, thereby forming single-membrane liposomes (LUV) of 80 to 150 nm.

[0115] As shown in FIGS. 5 and 6, this consists of a high-pressure pump (710), a Y-shaped microchannel section (720), a collision chamber section (730), a heat exchanger (740), and a collection tank (750).

[0117] The high-pressure pump (710) receives MLV liposomes with softened liposome membranes from an ultrasonic bath and pressurizes them to an ultra-high pressure of 700 to 1400 bar.

[0118] This is configured to push MLV liposomes into the Y-shaped microchannel section with a constant flow rate and constant pressure.

[0119] In other words, like a pump that forcefully pushes water out of a water gun, it shoots particles into the Y-shaped microchannel section.

[0121] The above Y-shaped microchannel section (720) serves to divide the MLV liposomes, which have received ultra-high pressure from a high-pressure pump, into two Y-shaped branches and induce them to collide at high speed in the microchannel.

[0122] Here, the microchannel is formed and configured as a finely narrow mesh structure with a channel width at the level of tens of micrometers.

[0123] In addition, it is formed into a finely narrow mesh structure, generating shear force and turbulence.

[0124] As particles pass rapidly through a narrow passage, they are subjected to physical stress and broken into small pieces.

[0126] The above collision chamber (730) is located on one side of the rear end of the Y-shaped microchannel section and serves to form a collision space that causes the two flows branched from the Y-shaped microchannel section to collide head-on.

[0127] This is configured so that shear force, turbulence, and cavitation (microbubble explosion caused by ultrasound) act simultaneously, causing large multilayer liposomes (MLVs) to break and convert into uniformly sized single-membrane liposomes (LUVs).

[0129] The above heat exchanger (740) is located on one side of the collision chamber and serves to remove heat generated in the collision chamber.

[0130] Since proteins like EGF are sensitive to heat, this is configured to be maintained at 4 to 10°C using a cooling coil.

[0132] The above collection tank (750) serves to collect and store uniformly sized single-membrane liposomes (LUVs) through the collision chamber.

[0133] This is a recirculation method configured to send the single-membrane liposomes (LUVs) stored in the collection tank back to the high-pressure pump for repeated processing in 5 to 10 passes.

[0135] Next, a coating-fusion type combination forming module (800) according to the present invention will be described.

[0136] The above coating / fusion type combination forming module (800) has the role of coating an exosome (EV) membrane component on the outer membrane of a liposome or partially fusing the two membranes to form a liposome sustained-release with a natural membrane surface label, thereby forming an EGF-liposome-exosome combination.

[0137] Here, coating refers to the process of lightly “coating” exosome membrane components onto the surface of liposomes (approximately 100 nm in size) to impart natural membrane characteristics. For example, after mixing a liposome suspension with exosomes at 4–10°C, short, low-shear stirring is performed to adsorb and coat the exosome membrane components onto the outer membrane of the liposomes. As a result, the liposomes maintain their original structure, and

[0138] Only the outer surface is covered with exosome proteins (CD63, CD81, etc.), forming a “liposome + exosome-labeled particle.” In other words, it is like a cookie where only the outer surface is coated by sprinkling chocolate powder (exosome component) on bread (liposome).

[0139] The above fusion refers to the process in which the membranes of liposomes and exosomes partially “attach” to form a new mixed membrane structure. For example, if liposomes and exosomes are mixed and briefly incubated at 37°C or frozen and thawed 1 to 2 times, membrane fluidity increases, and at this time, some of the liposome and exosome membranes come into contact, causing the double membranes to connect and be rearranged into a single membrane.

[0140] As a result, as a single combination nanoparticle, the internal structure is formed as a liposome, and the surface is formed by the coexistence of exosome proteins and liposome phospholipids, forming a single particle that simultaneously possesses exosome and liposome characteristics. In other words, it refers to two soap bubbles (liposomes and exosomes) meeting and partially merging to become one larger soap bubble.

[0142] As shown in FIGS. 7 and 8, the above coating-fusion type combination forming module (800) is composed of a coating-fusion type low-shear mixer (810), a 37°C water bath / dry block (820), a freeze / thaw section (830), a micro-injection pump (840), a low-speed centrifuge (850), and a coating-fusion type TFF section (860).

[0144] First, a coating and fusion type low-shear mixer (810) according to the present invention will be described.

[0145] The above coating / fusion type low-shear mixer (810) performs the role of gently mixing liposome dispersion + exosome dispersion at 4~25 ℃ to create a membrane contact / rearrangement pre-stage.

[0147] Second, the 37°C water bath / dry block (820) according to the present invention will be described.

[0148] The above 37°C water bath and dry block (820) serve to promote partial coating and fusion events by increasing the film fluidity on the 37°C water bath and dry block for 5 to 15 minutes.

[0150] Third, the freezing and thawing unit (830) according to the present invention will be described.

[0151] The above freezing and thawing unit (830) plays a role in inducing fusion by repeating rapid freezing at -80°C to thawing at 25°C 1 to 2 times, thereby rearranging the membrane and expanding the contact surface.

[0153] Fourth, a micro-injection pump (840) according to the present invention will be described.

[0154] The above micro-injection pump (840) serves to assist in negative charge membrane bridging and PEG-fusion with precise drops of CaCl₂ (0.5~2 mM) or PEG-8000 (2~5 wt%).

[0156] Fifth, a low-speed centrifuge (850) according to the present invention will be described.

[0157] The above low-speed centrifuge (850) serves to remove large aggregates after fusion, at a low-speed centrifuge of 1,000 to 3,000 × g (relative centrifugal acceleration) for 5 minutes.

[0159] Sixth, the coating and fusion type TFF part (860) according to the present invention will be described.

[0160] The above-mentioned coated / fused TFF section (860) is formed to allow coated / fused liposomes / exosomes (EVs) to flow tangentially along the surface of the filter membrane, thereby allowing PEG / Ca 2+ , free EGF, plays a role in filtering small molecules.

[0161] As illustrated in FIG. 9, this consists of a first feed tank section (861), a first circulation pump section (862), a first filter module section (863), a first dialysis filtration line section (864), a first retainate line section (865), a first pressure controller section (866), and a first TFF collection tank section (867).

[0163] The first feed tank section (861) above serves as a container for holding coated and fused liposomes and exosomes (EVs).

[0165] The first circulation pump unit (862) above serves to circulate coated and fused liposomes and exosomes (EVs) to the first filter module unit.

[0167] The first filter module (863) above serves to filter with a semipermeable membrane having a specific molecular weight cutoff (MWCO).

[0168] This consists of 300 kDa to 0.1 μm cassettes.

[0170] The above first dialysis filtration line section (864) serves as a line through which small molecules and solvents exit.

[0172] The above-mentioned first retainate line section (865) serves as a line for recovering concentrated coated and fused liposomes and exosomes (EVs).

[0174] The first pressure controller (866) above plays the role of controlling the TMP (Transmembrane Pressure).

[0176] The above-mentioned first TFF collection tank section (867) serves to store and preserve concentrated coated and fused liposomes and exosomes (EVs).

[0178] Next, a surface bonding / coexistence type combination forming module (900) according to the present invention will be described.

[0179] The above surface-bonded / coexistence type combination forming module (900) has liposomes, exosomes, and EGF proteins present simultaneously in one system, with some being chemically bonded and some being physically coexisting, or surface-bonded in a state where the EGF protein is attached to the surface of the liposome by a covalent bond and stably fixed, thereby playing a role in forming an EGF-liposome-exosome combination.

[0180] Here, physical co-existing refers to liposomes, exosomes, and EGF proteins being placed in a single container but existing together in the same liquid without chemical bonding. For example, liposomes (100 nm), exosomes (120 nm), and EGF proteins (5–50 ng / mL) are mixed in the same buffer solution (pH 6.5–7.0), and low-shear stirring is used to maintain the particles and proteins in a mixed yet independent state. As a result, EGF floats freely in the solution and can act immediately, while liposomes and exosomes each exhibit their unique stability and delivery characteristics. In other words, it refers to them existing separately in the same solution, much like a drink containing ice cubes (liposomes), jelly pieces (exosomes), and sugar granules (EGF) in a cup.

[0181] The above-mentioned surface conjugation refers to a state in which the EGF protein is stably fixed by attaching to the surface of a liposome via a covalent bond. For example, when manufacturing liposomes, PEG-maleimide lipids are included, and the maleimide group is formed to be exposed on the liposome surface. The EGF protein is then reduced to activate the -SH group (thiol). Subsequently, the reaction is carried out at 4°C for 2 to 4 hours to fix the EGF to the liposome surface through a maleimide-thiol reaction. As a result, the EGF is formed not as a free molecule floating in the solution, but in a form attached to the outer membrane of the liposome. When applied to the skin, the liposome itself transports the EGF, thereby enhancing sustained release and stability. In other words, it is like a ribbon (EGF) suspended from the surface of a balloon (liposome), meaning the ribbon moves together with the balloon.

[0182] In addition, the liposome is configured to include PEG-maleimide lipids to expose maleimide functional groups on its surface, and EGF-SH forms a surface-bonded liposome through a covalent bonding reaction between the thiol (-SH) of reduced EGF and maleimide, and the exosome is mixed to physically coexist without participating in chemical reactions, thereby forming a coexisting combination.

[0183] As shown in FIGS. 10 and 11, the above surface-bonded / coexisting type combination forming module (900) is composed of a liposome functionalization forming part (910), an EGF surface-bonded type combination forming part (920), an exosome coexisting type combination forming part (930), a surface-bonded / coexisting type TFF part (940), and an auxiliary stabilization part (950).

[0185] First, the liposome functionalization forming part (910) according to the present invention will be described.

[0186] The above liposome functionalization forming part (910) serves to expose a maleimide reaction group to the liposome outer membrane to form a covalent bond with the -SH group of the EGF protein.

[0188] Second, the EGF surface-bound combination forming part (920) according to the present invention will be described.

[0189] The above EGF surface-bound combination forming part (920) plays the role of forming a combination by forming a surface bond in which the reduced EGF-SH protein and the liposome surface maleimide group are attached by covalent bond and stably fixed.

[0190] This is configured to covalently bind EGF protein to the surface of liposomes through a maleimide-thiol Michael addition reaction, thereby stably immobilizing EGF after a reaction of 2 to 4 hours and inhibiting degradation and denaturation compared to the free state.

[0191] More specifically, it is composed of a reduced EGF-SH, a buffer (pH 6.5~7.0) forming unit, a low-shear reactor, and a 4°C cooling system unit.

[0192] That is, when manufacturing liposomes, PEG-maleimide lipids are included and formed so that maleimide groups are exposed on the liposome surface, and EGF protein is reduced to activate the -SH group (thiol).

[0193] Then, react at 4°C for 2 to 4 hours to immobilize EGF on the liposome surface via a maleimide-thiol reaction.

[0194] As a result, EGF is formed not as a free molecule floating in the solution, but attached to the outer membrane of the liposome; thus, upon application to the skin, the liposome itself transports the EGF, enhancing sustained release and stability.

[0195] In addition, through the EGF surface-bound combination forming part, EGF is fixed quantitatively and in the correct position rather than being adsorbed randomly, thereby increasing the reproducibility of signal efficiency and minimizing detachment.

[0197] Third, the exosome coexistence type combination forming part (930) according to the present invention will be described.

[0198] The exosome coexistence type combination forming part (930) serves to combine exosomes and liposome-EGF proteins by placing them in one container so that they exist together in the same liquid without chemical bonding.

[0199] In other words, EVs do not chemically attach to liposomes but instead form a corona on the surface, partially adsorb, or fuse, thereby increasing uptake and cell affinity.

[0200] As shown in FIG. 12, this consists of an injection system part (931) and a low-shear mixer for exosomes (932).

[0202] The above injection system (931) serves to drop exosomes (EVs) drop by drop into the EGF-liposome reaction solution through a sterile connector.

[0204] The above low-shear mixer (932) for exosomes serves to stir the exosomes (EV) so that they physically coexist with EGF-liposomes.

[0206] In this way, by configuring an exosome coexistence type combination forming unit consisting of an injection system and a low-shear mixer for exosomes, exosomes can coexist within the same system without chemically binding to assist in natural membrane labeling and uptake.

[0208] Fourth, the surface bonding / coexistence type TFF part (940) according to the present invention will be described.

[0209] The above surface-bound / coexisting TFF section (940) receives exosomes and liposome-EGF proteins that coexist through the exosome coexisting combination forming section and forms them to flow tangentially along the surface of the filter membrane, thereby serving to filter out free EGF, unreacted maleimide lipids, reducing agents, and small molecule impurities remaining after the reaction.

[0210] As illustrated in FIG. 13, this consists of a second feed tank section (941), a second circulation pump section (942), a second filter module section (943), a second dialysis filtration line section (944), a second retainate line section (945), a second pressure controller section (946), and a second TFF collection tank section (947).

[0212] The above second feed tank section (941) serves as a container for holding a suspension composed of coexisting exosomes and liposome-EGF proteins.

[0214] The second circulation pump unit (942) above serves to circulate the sample to the second filter module unit.

[0216] The second filter module (943) above serves to filter with a semipermeable membrane having a specific molecular weight cutoff (MWCO).

[0217] This consists of 300 kDa to 0.1 μm cassettes.

[0219] The above second dialysis filtration line (944) serves as a line through which small molecules and solvents exit.

[0221] The above second retainate line section (945) serves as a line for recovering concentrated exosomes.

[0223] The second pressure controller (946) above serves to control the TMP (Transmembrane Pressure).

[0225] The above-mentioned second TFF collection tank (947) serves to store concentrated coexisting exosomes and liposome-EGF proteins.

[0227] Fifth, the auxiliary stabilization part (950) according to the present invention will be described.

[0228] The above auxiliary stabilization part (950) stabilizes the protein membrane of the EGF protein-liposome formed by covalently bonding the EGF protein to the liposome surface through the protective agent trehalose and mannitol, and protects it during freezing and drying.

[0229] This inhibits oxidation by removing oxygen through nitrogen substitution, and enhances room-temperature fluidity and long-term stability during freeze-drying.

[0231] Thus, by configuring a surface-binding and coexisting combination forming module consisting of a liposome functionalization forming part, an EGF surface-binding combination forming part, an exosome coexisting combination forming part, a surface-binding and coexisting TFF part, and an auxiliary stabilization part, it is possible to combine liposomes, exosomes, and EGF proteins in which EGF is stably immobilized on the liposome surface and exosomes coexist and exert auxiliary functions.

[0233] Next, an EGF-liposome-exosome triple lifting ampoule composition forming module (900a) according to the present invention will be described.

[0234] The above EGF-liposome-exosome triple lifting ampoule composition forming module (900a) plays the role of completing the EGF-liposome-exosome triple lifting ampoule composition by adding an additive, such as a viscosity-texture improving agent (carbomer, HEC), a film former (polyquaternium, polyglutamic acid), a conditioning agent (ceramide, squalane), a preservative (phenoxyethanol, ethylhexylglycerin), borage oil, and galanga root extract, to the EGF-liposome-exosome combination formed through the coating-fusion type combination forming module and the surface bonding-coexistence type combination forming module.

[0235] As shown in FIGS. 14 and 15, this consists of a jacketed storage tank section (900a-1) for combination, a low-shear stirrer (900a-2) for lifting ampoule composition, a nitrogen exchange section (900a-3), and an airless filling section (900a-4).

[0237] The jacket-type storage tank (900a-1) for the combination above serves to store the EGF, liposome, and exosome combination formed through the coating / fusion type combination forming module and the surface bonding / coexistence type combination forming module.

[0238] This is stored at 4~10 ℃, and oxygen is blocked.

[0240] The low-shear stirrer (900a-2) for the lifting ampoule composition is located in the internal space of the jacketed storage tank for the combination and rotates the impeller at a stirring speed of 30 to 100 rpm to add and stir the following to the EGF, liposome, and exosome combination: a moisturizer (glycerin, butylene glycol), a conditioning agent (ceramide, squalane), a film former (polyquaternium, polyglutamic acid), a viscosity and texture improver (carbomer, HEC), a buffer, salt, and chelating agent (phosphate buffer, NaCl, EDTA), a preservative (phenoxyethanol, ethylhexylglycerin), borage oil, galanga root extract, and purified water.

[0241] This is configured with a temperature control jacket on one side to maintain the internal temperature of the low-shear stirrer for lifting ampoule compositions at 20 to 25 ℃.

[0242] And, on one side, it is composed of a moisturizer tank section for drip injecting a moisturizer (glycerin, butylene glycol), a conditioning agent tank section for drip injecting a conditioning agent (ceramide, squalane), a film former tank section for drip injecting a film former (polyquaternium, polyglutamic acid), a viscosity and texture improving agent tank section for drip injecting a viscosity and texture improving agent (carbomer, HEC), a buffer, salt, and chelating agent tank section for drip injecting a buffer, salt, and chelating agent (phosphate buffer, NaCl, EDTA), a preservative tank section for drip injecting a preservative (phenoxyethanol, ethylhexylglycerin), a borage oil tank section for drip injecting borage oil, a galanga root extract tank section for drip injecting galanga root extract, and a purified water tank section for injecting purified water.

[0243] In addition, the other side is configured to include a pH forming part that forms a pH of 6.3 to 6.8 and a conductivity maintaining part that maintains a conductivity of 300 mOsm.

[0245] The above nitrogen replacement unit (900a-3) is located on one side of the upper part of the jacket-type storage tank for combination and supplies nitrogen gas during the stirring process to remove oxygen.

[0247] The above airless filling unit (900a-4) serves to fill the finished ampoule composition into a small-capacity vial, pump, or ampoule container in an airless manner.

[0249] Hereinafter, a method for manufacturing an EGF-liposome-exosome triple lifting ampoule composition according to the present invention will be described in detail.

[0251] FIG. 16 is a flowchart illustrating a method for preparing an EGF-liposome-exosome triple lifting ampoule composition according to the present invention.

[0253] The method for preparing an EGF-liposome-exosome triple lifting ampoule composition according to the present invention is

[0254] A lipid dissolution step (S10) in which phospholipids (PC), cholesterol, charge-regulating lipids, and lipid-specific antioxidants are dissolved in an organic solvent to create a homogeneous lipid solution, and

[0255] A thin film formation step (S20) in which a solution is placed in a round-bottom flask through a rotary evaporator, rotated over a water bath to evaporate, and then condensed under reduced pressure from a vacuum pump to form a lipid thin film, and

[0256] A hydration step (S30) in which a hydration solution formed from a jacketed mixing tank is introduced into a lipid film through a low-shear stirrer, then rotated at 20 to 200 rpm through an impeller formed inside the tank, and the lipid film and the hydration solution are stirred to form MLV liposomes;

[0257] A pre-treatment step (S40) involving freezing, thawing, or low-intensity ultrasonic treatment, and

[0258] A microfluidization step (S50) for reducing and homogenizing MLV liposomes to a desired size (80~150 nm) to form single-membrane liposomes (LUV), and

[0259] A step (S60) for combining liposomes (LUV, 80~150 nm) and exosomes, and combining them by binding and co-conjugating an EGF protein thereto to create a combination of EGF, liposomes, and exosomes by coating and fusing them, or combining EGF, liposomes, and exosomes by co-conjugating and surface-binding them, and

[0260] The process consists of an EGF-liposome-exosome triple lifting ampoule composition formation step (S70) in which a viscosity-texture improving agent (carbomer, HEC), a film former (polyquaternium, polyglutamic acid), a conditioning agent (ceramide, squalane), a preservative (phenoxyethanol, ethylhexylglycerin), borage oil, and galanga root extract are added to the EGF-liposome-exosome combination formed through the EGF-liposome-exosome combination step to complete the EGF-liposome-exosome triple lifting ampoule composition.

[0262] [Lipid dissolution step (S10)]

[0264] This is the first step of the method for manufacturing a GF·liposome·exosome triple lifting ampoule composition, which is to dissolve phospholipid (PC), cholesterol, charge-regulating lipid, and lipid-specific antioxidant in an organic solvent to create a homogeneous lipid solution.

[0265] This consists of solvent selection, lipid addition, stirring and heating, oxygen exclusion, and confirmation of complete dissolution.

[0267] First, one of ethanol, chloroform, methanol, or ethanol and chloroform is selected as the solvent.

[0268] At this time, a solvent is selected so that lipophilic lipids (PC, cholesterol) and antioxidants (tocopherol, ascorbyl palmitate) can be dispersed at the molecular level.

[0270] Next, lipid raw materials (PC, cholesterol, lipids, antioxidants) are added to a round-bottom flask in a set ratio.

[0271] This contains 2.0 wt% hydrogenated phospholipid (PC), and

[0272] 0.6% by weight of cholesterol, and,

[0273] 0.2 wt% of charge-regulating lipids and,

[0274] It consists of 0.02 weight% of an antioxidant.

[0276] Next, the mixture is stirred and heated at 40–55°C using a magnetic stirrer and a heating plate.

[0277] Here, the temperature range of 40 to 55 ℃ helps rapid dissolution above the transition temperature (Tm) of phospholipids and lowers the risk of thermal denaturation.

[0279] Next, oxygen inside the flask is removed by purging with nitrogen (N₂) to prevent lipid oxidation.

[0281] Finally, the mixture is stirred until it becomes a transparent to translucent lipid solution free of insoluble particles.

[0283] Thus, by performing the lipid dissolution step according to the present invention, PC, cholesterol, electrolyte lipids, and antioxidants are mixed at the molecular level, thereby enabling the uniform formation of a thin film thereafter, cholesterol strengthens the membrane, and electrolyte lipids provide stability of the zeta potential, thereby suppressing aggregation and fusion to 40% or less compared to conventional methods, and by low-temperature dissolution, addition of antioxidants, and nitrogen purging, lipid stability can be improved by 80% compared to conventional methods in the pre-EGF introduction stage.

[0285] [Thin film formation step (S20)]

[0287] This is a step for forming a thin lipid film by uniformly spreading the lipids. The solution is placed in a round-bottom flask using a rotary evaporator and evaporated by rotating it over a water bath, after which it is condensed under reduced pressure using a vacuum pump to form a lipid film.

[0288] In other words, the hydration stage is a key preprocessing step that enables the stable and reproducible production of liposomes.

[0289] Then, PC, cholesterol, charge-regulating lipids, and antioxidants dissolved in the solvent are evenly spread across the flask walls to form a thin film, and multilayer liposomes (MLVs) are formed in which the lipid membrane automatically peels off upon hydration. Subsequently, they are stabilized into single-membrane liposomes (LUVs) through microfluidization, and residual solvents (ethanol / chloroform, etc.) are completely removed to establish biocompatibility.

[0291] More specifically, the thin film formation step is divided into lipid solution preparation, rotary evaporator installation, vacuum evaporation, thin lipid film formation, and final drying.

[0293] First, the homogeneous solution of lipid + solvent (ethanol / chloroform) prepared in the lipid dissolution step is transferred to a round-bottom flask and prepared.

[0295] Next, after mounting the flask on the rotary evaporator, the flask is placed in a water bath (40~55℃) and connected to a rotary motor to form a thin film of the solution spreading on the wall.

[0297] Next, by vacuum evaporation, the pressure is lowered using a vacuum pump to lower the boiling point of the solvent, and the solvent is induced to evaporate rapidly at a low temperature, thereby forming a system where only lipids remain.

[0299] Next, as the solvent is completely removed, a uniform, thin lipid film is formed as a coating on the inner wall of the flask.

[0301] Next, as a final drying step, nitrogen purging or vacuum drying is additionally carried out to remove residual solvent to the ppm level.

[0303] In this way, the thin film formation step is performed so that lipids are evenly distributed, allowing for the formation of uniform particles with a PDI ≤ 0.25 level upon subsequent hydration. This evenly distributes cholesterol and electrolyte lipids to enhance membrane stability, supports EGF stability, and eliminates residual solvents, thereby satisfying cosmetic and pharmaceutical standards (ICH Q3C).

[0305] [Hydration Stage (S30)]

[0307] This is a step of physically reactivating the lipid formed as a thin film to create multilamellar vesicles (MLVs), in which a hydration solution formed from a jacketed stirring tank is introduced into the lipid thin film through a low-shear stirrer, and then rotated at 20 to 200 rpm through an impeller formed inside the tank to stir the lipid thin film and the hydration solution to form MLV liposomes.

[0308] In other words, it consists of hydration solution preparation, thin film contact, low-shear stirring, and MLV liposome formation.

[0310] First, as a preparation of the hydrate, a buffer (phosphate), NaCl (ionic strength), EDTA (chelate), a humectant (glycerin, BG), and a protective agent (trehalose, mannitol) are dissolved in purified water to adjust the pH to 6.3 to 6.8, and the temperature is maintained above the lipid transition temperature (Tm), usually at 40 to 50°C.

[0312] Next, as a thin film contact, a thin film is placed in a round-bottom flask or a jacketed stirring tank, and a prepared hydration solution is introduced. Then, the film is rotated at 20 to 200 rpm through an impeller formed inside the tank, and the lipid thin film and the hydration solution are stirred so that the thin film gradually expands and forms a fragment.

[0314] Next, as low-shear stirring, low-shear stirring is performed at 20 to 200 rpm using an anchor-type or paddle-type impeller,

[0315] The lipid layer falls into the hydrate and rearranges into a concentric multi-lamellar structure.

[0317] Next, as part of MLV liposome formation, spherical MLV liposomes are formed according to the hydrophilic-hydrophobic self-assembly principle as the lipid comes into contact with water.

[0318] At this time, some hydrate components become trapped in the inner aqueous layer.

[0320] Thus, by performing the hydration step according to the present invention, the formation of MLV liposomes can form the basis for subsequent pretreatment (freeze-thaw, ultrasound) and microfluider treatment, and moisturizers, protective agents, and buffering components can enter the liposomes to increase stability by 80% compared to the existing method, and lipid concentrations can be uniformly dispersed to lower the PDI to 0.25 or less, and provide an environment that increases protein stability when EGF is introduced in a subsequent step.

[0322] [Pre-treatment Step (S40)]

[0324] This is a step of preparing multilayer liposomes (MLVs) formed by hydration into a state suitable for subsequent microfluider treatment, by undergoing freezing and thawing or low-intensity ultrasound treatment to soften the liposome membrane, increase internal encapsulation efficiency, and homogenize the size.

[0325] In other words, the double membrane of the MLV is loosened to enable stable size reduction without damage during subsequent ultra-high pressure shearing, the membrane is opened and closed momentarily to allow water-soluble components to enter the aqueous phase inside the liposome better, and the MLV size distribution is reduced to increase microfluider efficiency. Although EGF has not yet entered, this stage must be stable to reduce protein loss during subsequent combination.

[0327] The above pre-treatment step consists of a freezing and thawing step (S41) and a low-intensity ultrasonic treatment step (S42).

[0329] The above freezing and thawing step (S41) involves rapidly freezing the MLV suspension in an ultra-low temperature freezer at -80°C and then thawing it in a water bath at 25°C to 37°C. At this time, the same process is repeated 1 to 3 times.

[0330] During rapid freezing, ice crystals disturb the lipid membrane, and upon thawing, the membrane rearranges to expand the internal aqueous layer and increase encapsulation efficiency.

[0332] The above low-intensity ultrasonic treatment step (S42) induces low-intensity cavitation by placing the MLV suspension into an ultrasonic bath (20~40 kHz).

[0333] This instantaneously shakes the membrane with micro-vibrations to soften it, reduces some of the particle size, and prevents membrane aggregation.

[0335] Thus, by performing the pre-treatment step according to the present invention, the order of the double membrane is controlled to allow it to pass through the microfluider in a more flexible state, the encapsulation efficiency is increased by 1.5 to 2 times compared to the conventional method so that more components (protective agents, moisturizers, etc.) in the hydrate are inserted into the liposome, a homogenization basis is formed, and the EGF-liposome stability can be ensured by reducing the pressure and number of passes required in the microfluider.

[0337] [Microfluidization Step (S50)]

[0339] This is a step of reducing MLV liposomes to a desired size (80–150 nm) and homogenizing them to form single-membrane liposomes (LUVs).

[0340] That is, the MLV (hundreds of nm to several μm) obtained in the hydration and pretreatment steps is reduced to a nano size (80 to 150 nm) to secure a uniform particle distribution with a PDI ≤ 0.25 level, and the overlapping membranes of the multilayer liposome (MLV) are separated by high-pressure shear force to form a single-membrane liposome (LUV).

[0341] In addition, the volume of the internal aqueous layer is maintained at a constant level, thereby stably forming the encapsulation efficiency of EGF, moisturizers, and protective agents.

[0342] The above microfluidization step consists of sample preparation, high-pressure pump pressurization, entry into Y-shaped microchannels, application of collision and shear forces, heat exchanger cooling, and collection and recirculation.

[0344] First, as a sample preparation, an MLV suspension with a softened membrane is prepared during the pretreatment step (freeze-thaw / ultrasonic).

[0345] Here, the MLV suspension consists of PC 2.5 wt%, cholesterol 0.8 wt%, glycerin 5 wt%, trehalose 2 wt%, and pH 6.5 buffer.

[0347] Next, the sample is pressurized to an ultra-high pressure of 700 to 1400 bar (10 to 20 kpsi) using a high-pressure pump.

[0349] Next, by entering a Y-shaped microchannel, the sample is formed to split into two branches (Y-shaped) through a microchannel (width of tens of μm) and collide at high speed.

[0351] Next, as a result of collision and shear force action, particles collide with each other and are destroyed and reassembled by shear force, turbulence, and cavitation in the microchannel.

[0353] Next, as a heat exchanger cooling method, the temperature rises instantaneously during the compression process, so the heat exchanger maintains the temperature at 4 to 10°C to prevent EGF denaturation.

[0355] Next, as a collection and recycling step, single-membrane liposomes (LUVs) are collected after one processing step.

[0356] This is repeated 5 to 10 times as needed and formed to reach the target size.

[0358] Thus, by performing the microfluidization step according to the present invention, nano-sized liposomes with an average size of 100±20 nm and a PDI ≤ 0.25 can be formed, and the membrane structure can be homogenized by converting from MLV to LUV, and the EGF protection and delivery efficiency can be improved by 80% compared to the existing one, thereby increasing the predictability of uniform particles when penetrating the skin or ingested, and in the case of uniform particles, resistance to aggregation and sedimentation and reduction of oxidation and leakage can be achieved, thereby increasing long-term stability.

[0360] [EGF·Liposome·Exosome Combination Step (S60)]

[0362] This is a step of combining liposomes (LUV, 80–150 nm) and exosomes, and binding and co-conjugating an EGF protein therewith to form a combination by coating and fusing EGF, liposomes, and exosomes, or forming a combination by co-conjugating and surface-binding EGF, liposomes, and exosomes.

[0363] As illustrated in FIG. 17, this consists of a coating-fusion type combination step (S61) and a coexistence type surface bonding combination step (S62).

[0365] First, the coating and fusion type combination step (S61) will be explained.

[0366] The above coating and fusion type combination step (S61) is a step of coating an exosome (EV) membrane component on the outer membrane of a liposome or partially fusing the two membranes to form a liposome sustained-release property together with a natural membrane surface label.

[0367] And, as shown in FIG. 18, it is divided into a coating step (S61a) that increases membrane fluidity by mixing at 4 to 25 ℃ and incubating in a short 37 ℃ water bath, and a fusion step (S61b) that increases membrane rearrangement by freezing and thawing 1 to 2 times.

[0368] Here, the liposomes are selected from HEPES / PBS with a diameter of 80–150 nm, PDI ≤ 0.25, zeta potential of -10 to -25 mV, PC:Chol 7:3 (molar ratio), and pH 6.3–6.8.

[0369] And, exosomes (EVs) are 80–150 nm, 10 9 ~10 10 The target is a composition identical to liposomes in terms of particles / mL, pH, and ionic strength (buffer matched with pre-TFF).

[0370] The particle number ratio (EV:liposome) is 1:1 to 1:5, and the membrane lipid mass ratio (EV membrane:liposome membrane) is 0.5:1 to 1:1.

[0371] 2 to 3 weight percent of trehalose is added as a protective agent to stabilize during fusion and thawing.

[0372] In addition, as a selective additive, 0.5–2 mM CaCl2 (to increase the fusion rate through bridging between negatively charged membranes) and 2–5% PEG-8000 (PEG-mediated fusion, post-washing is required after use) are added.

[0374] <Coating Step (S61a)>

[0376] This is a coating step that increases membrane fluidity by mixing at 4~25 ℃ and incubating in a short 37 ℃ water bath.

[0378] First, as shown in FIG. 19, as a buffer alignment, both liposomes and exosomes are aligned with TFF at the same buffer osmosis (≈280~320 mOsm) (S61a-1).

[0379] At this time, the temperature is pre-cooled to 4~10 ℃ before mixing.

[0381] Next, the liposome dispersion is placed in a sterile beaker, and the exosome dispersion is slowly added dropwise using a guide starting from an exosome (EV) to liposome ratio of 1:2 (number of particles) (S61a-2).

[0383] Next, a mixed suspension is formed by stirring with a low-shear paddle at a rotational speed of ≤100 rpm for 5 to 10 minutes (S61a-3).

[0385] Next, the mixed suspension is incubated in a 37°C water bath for 5 to 15 minutes (S61a-4).

[0387] Next, place it in an ice bath and lower it back to 4°C (S61a-5).

[0388] At this time, excessive fusion and aggregation are prevented.

[0390] Next, 2 to 3 weight percent of trehalose, a protective agent, is added (S61a-6).

[0392] Next, large aggregates are removed by low-speed centrifugation at 1,000–3,000 × g (relative centrifugal acceleration) for 5 minutes (S61a-7).

[0394] Subsequently, PEG·Ca as a coating / fusion type TFF part of 300 kDa to 0.1 μm 2+ After removing free EGF and small molecules, the liposome-exosome combination fraction is collected ((S61a-8).

[0395] Here, the coated and fused TFF portion forms the coated and fused liposomes / exosomes (EVs) to flow tangentially along the surface of the filtration membrane, thereby PEG·Ca 2+ , free EGF, plays a role in filtering small molecules.

[0397] <Convergence Stage (S61b)>

[0399] This is a fusion stage that increases membrane rearrangement through 1 to 2 cycles of freezing and thawing.

[0401] First, as shown in FIG. 20, as a buffer alignment, both liposomes and exosomes are aligned with TFF at the same buffer osmosis (≈280~320 mOsm) (S61b-1).

[0402] At this time, the temperature is pre-cooled to 4~10 ℃ before mixing.

[0404] Next, the liposome dispersion is placed in a sterile beaker, and the exosome dispersion is slowly added dropwise using a guide starting from an exosome (EV) to liposome ratio of 1:2 (number of particles) (S61b-2).

[0406] Next, a mixed suspension is formed by stirring with a low-shear paddle at a rotational speed of ≤100 rpm for 5 to 10 minutes (S61b-3).

[0408] Next, the mixed suspension is frozen at -80°C for 10 to 20 minutes, and then thawed in a water bath at 25 to 37°C for 2 to 5 minutes (S61b-4).

[0409] This is performed only 1 to 2 times. The reason is that excessive repetition damages EV proteins and miRNAs.

[0411] Next, the temperature is stabilized at 4°C immediately after thawing, and 2-3% by weight of trehalose, a protective agent, is added (S61b-5).

[0413] Next, large aggregates are removed by low-speed centrifugation at 1,000–3,000 × g (relative centrifugal acceleration) for 5 minutes (S61b-6).

[0415] Subsequently, PEG·Ca as a coating / fusion type TFF part of 300 kDa to 0.1 μm 2+ After removing free EGF and small molecules, the liposome-exosome combination fraction is collected (S61b-7).

[0416] Here, the coated and fused TFF portion forms the coated and fused liposomes / exosomes (EVs) to flow tangentially along the surface of the filtration membrane, thereby PEG·Ca 2+ , free EGF, plays a role in filtering small molecules.

[0418] Thus, by performing a coating-fusion type combination step consisting of a coating step and a fusion step, the low stability and efficiency resulting from physical coexistence alone when mixing liposomes and exosomes in the conventional case is improved. By forming a new combination structure through surface coating and partial membrane fusion, delivery efficiency and stability are improved by 80% compared to the conventional case. Furthermore, the low cell recognition ability resulting from the formation of only liposomes in the conventional case is improved by introducing exosome-labeled components, thereby increasing intracellular uptake rate and targetability by 1.5 to 2 times compared to the conventional case. Additionally, the low internal loading capacity and sustained release potential resulting from the formation of only exosomes in the conventional case is improved by fusing with the liposome structure to increase encapsulation efficiency and control release, and EGF signaling, exosome biomolecular signaling, and liposome sustained release can act simultaneously in a single formulation.

[0420] Second, the coexistence type and surface bonding combination step (S62) is explained.

[0421] The above-mentioned coexistence-type surface-binding combination step (S62) is a step of combining liposomes, exosomes, and EGF proteins by having them exist simultaneously in one system, with some being chemically bound and some being physically coexisting and arranged in an auxiliary form.

[0422] Here, the liposome is formed by partially introducing PEG-maleimide functionalized lipids into a phospholipid bilayer so that -maleimide groups are exposed on the surface.

[0423] EGF-SH is induced so that the EGF protein is exposed to or reduced by cysteine ​​(-SH, cysteine).

[0424] Exosomes are composed of separate natural nanovesicles and are mixed and coexisted with liposomes to add stability and signal amplification functions.

[0426] That is, EGF is chemically immobilized on the liposome surface, and the exosome is physically present together, and the EGF surface display is combined with the exosome (EV) natural membrane and surface protein combination.

[0428] First, as shown in FIG. 21, maleimide reaction groups are exposed on the outer membrane of the liposome through the liposome functionalization forming part to form a covalent bond with the -SH group of the EGF protein (S62a).

[0429] This contains functionalized lipids such as DSPE-PEG(2000)-Maleimide at a level of 0.5 to 2 mol% relative to total lipids when mixed with phospholipids.

[0430] Then, 100–150 nm liposomes are formed through a thin film hydration-microfluidizer process, and maleimide groups are exposed at the ends of PEG chains on the surface.

[0432] Next, through the EGF surface-bound combination forming part, the reduced EGF-SH protein and the liposome surface maleimide group are combined by covalent bonding to form a stable fixed state (S62b).

[0433] This activates the -SH of Cys residues by treating EGF protein with a reducing agent (small amounts of DTT, TCEP).

[0434] The liposome suspension and EGF-SH are mixed at 4°C in a buffer solution (pH 6.5~7.0).

[0435] For 2 to 4 hours, a covalent bond (-SC bond) is formed by a maleimide-thiol Michael addition reaction.

[0436] At this time, EGF is quantitatively and stably immobilized on the surface of the liposome.

[0438] Next, through the exosome coexistence type combination forming part, the exosome and the liposome-EGF protein are placed in one container and combined so that they exist together in the same liquid without chemical bonding (S62c).

[0439] This is an exosome prepared in parallel (10 9 ~10 10 particles / mL) are added to the reaction mixture (=a mixture in which EGF and liposomes are undergoing a surface binding reaction).

[0440] At this time, exosomes physically coexist as they are without chemical bonding.

[0441] In this way, cell uptake efficiency is increased using natural surface proteins (CD63 / 81, integrin, etc.), and the signal is amplified through simultaneous delivery with liposome-surface EGF.

[0442] Also, it contributes to the stabilization of liposome membranes and shares cholesterol and sphingolipids.

[0444] Next, in the surface-bound / coexisting TFF section, the exosomes coexisting through the exosome coexisting combination forming section and the liposome-EGF protein are received and formed to flow tangentially along the surface of the filtration membrane, thereby filtering out the free EGF, unreacted maleimide lipids, reducing agent, and small molecule impurities remaining after the reaction, and purifying them (S62d).

[0445] That is, the targets for removal are unreacted EGF, free maleimide, and residual reducing agent, the targets for preservation are EGF-bound liposomes + exosomes, and the final fraction is EGF-liposomes + exosomes coexist.

[0447] Thus, by being composed of a co-occurring and surface-binding combination step, the EGF spacing (nm) can be finely controlled with PEG-maleimide to optimize EGFR dimerization and clustering without overstimulation, and effective affinity can be increased by maintaining exposure of the EGF binding site (receptor recognition surface) through site-selective binding using the SH introduction site, and by surface fixation through covalent bonding, detachment and diffusion into the EGF solution are minimized even in the presence of epidermal moisture and moisturizers, and EV surfaces (tetraspanin CD63 / CD81, integrin, etc.) form a corona on the liposome surface through weak non-covalent interactions, thereby improving intake efficiency by 80% even in diversified cell recognition and uptake pathways (clathrin / caveolae / raft), and spaced-temporal delivery can be achieved as EVs are responsible for uptake and cell affinity and liposomes are responsible for release control (sustained release).

[0449] [Formation step of EGF·Liposome·Exosome Triple Lifting Ampoule Composition (S70)]

[0451] This is a step of completing the EGF-liposome-exosome triple lifting ampoule composition by adding a viscosity and texture improving agent (carbomer, HEC), a film former (polyquaternium, polyglutamic acid), a conditioning agent (ceramide, squalane), a preservative (phenoxyethanol, ethylhexylglycerin), borage oil, and galanga root extract to the EGF-liposome-exosome combination formed through the EGF-liposome-exosome combination step.

[0452] At this time, the pH is finely adjusted to a range of 6.3 to 6.8, and nitrogen is substituted (to prevent oxidation).

[0453] Then, the EGF liposome lifting ampoule is completed by aseptic filling into airless ampoules and vials.

[0454] In addition, as a freeze-drying option, a liposome-exosome mixed dispersion is protected with 2 to 5 weight percent trehalose, and then reconstituted immediately before use by Rayo freeze-drying.

[0455] As a result, it improves room temperature stability and enhances EGF protection.

[0457] First, as shown in FIG. 22, an EGF-liposome-exosome combination formed through the EGF-liposome-exosome combination step is introduced and stored in a jacket-type storage tank for combination (S71).

[0459] Next, using a low-shear stirrer for lifting ampoule compositions, the impeller is rotated at a stirring speed of 30 to 100 rpm, and a moisturizer (glycerin, butylene glycol), a conditioning agent (ceramide, squalane), a film former (polyquaternium, polyglutamic acid), a viscosity / texture improving agent (carbomer, HEC), a buffer / salt / chelating agent (phosphate buffer, NaCl, EDTA), a preservative (phenoxyethanol, ethylhexylglycerin), borage oil, galanga root extract, and purified water are added to the EGF / liposome / exosome combination and stirred (S72).

[0461] Next, nitrogen gas is supplied through the nitrogen exchange unit during the stirring process to remove oxygen (S73).

[0463] Next, the finished ampoule composition is filled into a small-capacity vial, pump, or ampoule container using an airless filling unit (S74).

[0465] In this way, by performing the step of forming an EGF, liposome, and exosome triple lifting ampoule composition, multi-layered functionality for improving skin wrinkles can be secured by combining the core of EGF (signaling), liposome (sustained release / retention), and exosome (natural membrane / intake / stabilization), the formulation stability and application sensation of a viscosity / texture improving agent, the immediate lifting effect of a film former, the barrier, moisturizing, and anti-inflammatory properties of a conditioning agent and borage oil, and the antioxidant and collagen degradation inhibition of galangal extract. Furthermore, carbomer and HEC form an EV dispersion stabilization network with MLV and LUV liposomes to inhibit sedimentation and aggregation, and through ceramide, squalane, and borage oil (GLA), the skin barrier is reinforced by restoring the lamellar structure of the stratum corneum and providing anti-inflammation. Additionally, through the flavonoids (galangin, etc.) of galangal root extract, a high-quality lifting ampoule composition can be formed that scavenges ROS and inhibits MMPs.

[0467] Thus, through the method for preparing an EGF-liposome-exosome triple lifting ampoule composition according to the present invention, as shown in FIG. 23, an EGF-liposome-exosome triple lifting ampoule composition is formed in which EGF (signal), liposome (sustained-release / retention), and exosome (natural membrane / intake / stabilization) form an EGF-liposome-exosome combination as a core structure.

[0469] The above EGF-liposome-exosome triple lifting ampoule composition is

[0470] 2.0~4.0 wt% of liposomal lipid system, and,

[0471] 1.0 to 3.0 wt% of exosome dispersion, and

[0472] 0.0005~0.005 wt% of EGF (recombinant) and,

[0473] 5 to 12 weight percent of a moisturizer, and

[0474] 1 to 8 weight percent of a protective agent, and

[0475] 0.3 to 1.0 weight% of a conditioning agent, and

[0476] 0.1 to 0.3 weight percent of a film former, and

[0477] 0.2 to 0.8 weight percent of a viscosity and texture improver, and,

[0478] 0.2~0.6% by weight of a buffer, salt, and chelating agent, and,

[0479] 0.7~1.0% by weight of preservative, and

[0480] 0.2 to 1.0 weight percent of borage oil, and

[0481] 0.1 to 0.5 weight% of galanga root extract, and

[0482] It consists of 70~88% by weight of purified water.

[0484] [Liposome lipid system]

[0486] This serves as a component of the EGF, liposome, and exosome triple lifting ampoule composition, enhancing protection, sustained release, and skin retention, and plays an important role in stabilizing physical properties by making the liposome surface a reactive surface capable of covalently bonding with EGF-SH.

[0487] In other words, the phospholipid bilayer interacts with SC lipids to surface-attach and fuse, increasing local retention and improving lipid affinity in the stratum corneum, and is designed to reduce burst release of EGF contained in the internal aqueous core and ensure continuous and gradual exposure.

[0488] In addition, it increases skin retention by affinity with lipids in the stratum corneum, precisely controls the release curve with cholesterol, membrane thickness, and particle size, and provides a physical buffer to protect against heat / salt / preservative / osmotic stress and slow release when the phospholipid bilayer (PC) and cholesterol encapsulate (internalize) EGF (water-soluble peptide) in the inner aqueous phase or when it is in the surface-bound form (EGF-PEG-maleimide).

[0489] In addition, it wraps peptides in a lipid membrane to protect them from degradation and oxidation, and alleviates irritation.

[0491] In addition, by increasing adhesion and retention through affinity with lipids in the subcutaneous layer (SC), it is possible to secure the retention time and local concentration after application to the skin.

[0492] In addition, in the reaction with PEG-maleimide lipids, the liposome surface becomes a reactive surface capable of covalently bonding with EGF-SH, and cholesterol (usually PC:Chol ≈ 7:3 molar ratio) and a small amount of charge-regulating lipids (±) control membrane fluidity, permeability, and charge (zeta potential ζ), maintaining a stable dispersion with a size of 100 ± 20 nm / PDI ≤ 0.25 / zeta potential -10 to -25 mV.

[0493] In addition, it enters the inner aqueous phase of EGF (0.0005~0.005 wt%), protects against preservatives, salts, and temperature changes, performs a sustained-release role, and increases ligand density and EGFR signaling efficiency through a quantitative surface display role via a surface-bound maleimide-thiol reaction.

[0494] In addition, in the reaction with the exosome dispersion, partial coating, fusion, and coexistence occur during buffer and osmotic matching, which supports uptake and stability, and the cholesterol / sphingolipid-rich exosome (EV) membrane further reduces leakage and oxidation of the liposomes.

[0495] In addition, the moisturizer (glycerin / BG, etc. 5-12 wt%) and the protective agent (trehalose / mannitol 1-8 wt%) stabilize the protein folding and membrane as osmolite, but an excessive increase in osmotic pressure causes liposome contraction and leakage, so the total osmotic pressure is set to ≈ 280-320 mOsm.

[0496] In addition, when reacted with a conditioning agent, it has good compatibility with the film, which is beneficial for coating feel and barrier correction, and can reduce the risk of excessive oil content over-fluidization and aggregation of the film to compliance with the upper limit.

[0497] And, when reacting with the film former, the polyquaternium (cation) can induce electrostatic flocculation with the negatively charged liposome, maintaining the zeta potential (ζ) at -10 to -25 mV, and after droplet dilution, it is introduced and, if necessary, steric shielding with PEG lipids.

[0498] In addition, when reacted with viscosity and texture improvers, the gel network reduces sedimentation and aggregation, helping to stabilize dispersion and enabling low-shear mixing.

[0499] In addition, when reacted with buffer, salt, and chelate, the absolute value of the zeta potential is maintained at pH 6.3–6.8 and NaCl 0.05–0.2 wt%, minimizing aggregation, and EDTA inhibits metal-derived oxidation.

[0500] In addition, when reacting with preservatives such as phenoxyethanol and ethylhexylglycerin, partial insertion into the membrane increases fluidity and increases the leakage rate at high concentrations to minimize within the PET penetration range, and membrane stability testing is performed in parallel during actual formulation.

[0501] In addition, when reacted with borage oil, GLA-rich direct membrane insertion (0.2–0.5 wt%) softens the membrane and increases release.

[0502] In addition, when reacted with galangal extract, polyphenols and flavonoids inhibit antioxidant activity and MMPs.

[0504] The content of the liposomal lipid system according to the present invention is based on the total weight % of the liposomal lipid system,

[0505] 1.0 to 3.0 wt% of hydrogenated phospholipid (PC), and,

[0506] 0.3~0.8 weight% of cholesterol, and,

[0507] 0 to 0.3 wt% of charge modulation (cationic or anionic lipid), and

[0508] It consists of 0.01 to 0.05 weight percent of a lipid-based antioxidant (tocopherol / ascorbyl palmitate).

[0510] Here, hydrogenated phospholipids serve as a double membrane backbone, encapsulating water-soluble EGF in the inner aqueous phase to self-assemble in water to form liposomes. They are hydrogenated (saturated), resistant to oxidation, and have a high gel-like tendency, resulting in low membrane permeability, low leakage and burst release, and high sustained release.

[0511] In addition, saturated chains (HSPC / DSPC series) are less susceptible to oxidation and hydrolysis than unsaturated PC, increasing the bending stiffness (Kc) and areal elasticity of the film.

[0513] The above cholesterol has the characteristics of filling gaps between phospholipids to reduce permeability and leakage, smoothing gel and flow transitions to increase low and high temperature stability, and increasing resistance to fusion, breakage, and friction with film formers.

[0514] In addition, sterol rings interlock in the double membrane to increase the order, decrease the free volume, and inhibit the passage of solutes and preservatives through the membrane.

[0516] The above charge-regulating lipids create electrostatic repulsion between particles to suppress the increase in aggregation and PDI, and perform the role of tuning surface interactions to reduce non-specific binding / flocculation with formformers (usually cationic polyquaterniums).

[0517] In addition, anions (small amounts of DMPG / DPPG / PS) impart a negative charge with a zeta potential (ζ), reducing aggregation by forming a counter ion layer, while cations (such as DOTAP) have the advantage of high cell adhesion, but increase irritation and the risk of aggregation.

[0519] The above lipid antioxidant (tocopherol / ascorbyl palmitate) breaks the radical chain reaction to prevent oxidation, leakage, and deterioration of membrane components (especially unsaturated residues), and blocks lipid oxidation byproducts (aldehydes, etc.) from causing secondary damage to proteins.

[0520] Furthermore, tocopherol (Vitamin E) acts as a radical chain-breaking scavenger, and ascorbyl palmitate, a fat-soluble Vitamin C derivative, plays a synergistic role in tocopherol regeneration.

[0522] When the composition ratio (weight%) of the above liposomal lipid system is 2 wt% or less, problems arise such as carrier deficiency leading to insufficient EGF loading and surface binding sites, reduced membrane thinness and cholesterol fraction, aggregation due to insufficient charge and steric stability, a high PDI (Polydispersity Index), a relatively large adsorption / loss ratio during TFF / filtration / packing resulting in a lower recovery rate relative to actual input, and a sharp drop in concentration after immediate release leading to weakened long-term efficacy; and when it is 4 wt% or more, excessive viscosity leads to low microfluider pass efficiency, high heat generation, an increased risk of packing nozzle clogging, and a higher tendency for MLV retention and fusion, making particle collision and aggregation easier, resulting in low redispersibility, crimping and separation in the stability chamber, excessive cholesterol fraction and membrane multiplexing causing EGF release to be excessively delayed resulting in a reduced perceived initial efficacy, and problems such as aggregation occurring because cationic film formers excessively neutralize surface negative charges; therefore, the liposomal lipid system It is most desirable to form it in an amount of 2% to 4% by weight.

[0524] [Exosome Dispersion]

[0526] This is a component of the EGF, liposome, and exosome triple lifting ampoule composition, in which exosome surface proteins (CD63, CD81, integrins, etc.) promote endocytosis of keratinocytes / fibroblasts and increase EGF signaling efficiency, and the liposomes are coated, partially fused, and coexist with a natural membrane rich in cholesterol and sphingolipids to reduce leakage and increase durability, and lipids, proteins, and miRNA within the exosomes (EVs) alleviate micro-inflammation and provide antioxidant support, thereby performing a synergistic role in improving wrinkles and elasticity.

[0527] This allows it to adhere well to and penetrate skin cells through surface protein-receptor recognition and membrane-membrane interactions (contact and partial fusion), and possesses characteristics advantageous for protecting the contents and sustained release due to the natural membrane raft (Chol / Sphingomyelin) effect.

[0529] When the exosome dispersion reacts with EGF, it simultaneously delivers EGF internalized in liposomes and surface-bound, provides auxiliary signals for EGFR activity at the cell surface, and if free EGF remains, it is non-specifically adsorbed onto the EV surface, thereby improving sustained release and preservation.

[0531] When the exosome dispersion reacts with the liposomal lipid system, in the case of coating, the EV component is coated on the outer surface of the liposome to increase uptake and reduce leakage, in the case of fusion, a portion of the membrane is mixed to form a hybrid membrane to increase sustained release and stability, and in the case of coexistence, they exist side by side in the same buffer to perform the role of simultaneous delivery.

[0533] When the exosome dispersion reacts with a moisturizer, trehalose and mannitol form an EV membrane and a hydration shell, stabilizing cold and heat stress and freeze-drying.

[0535] In addition, when the exosome dispersion reacts with a conditioning agent, in small amounts, it helps improve membrane compatibility and barrier reinforcement.

[0537] Also, when the exosome separation solution reacts with the film former, the cationic (polyquaternium) poses a risk of electrostatic aggregation with negatively charged EVs, so it is diluted, drip-dried, and low-shear injected.

[0539] When the above exosome dispersion reacts with a viscosity and texture improver, the gel network helps inhibit sedimentation and stabilize appearance.

[0541] In addition, when the exosome dispersion reacts with buffer, salt, and chelate, an excess of NaCl lowers the absolute value of the zeta potential (ζ) due to ion shielding, making aggregation easier, so a small amount of NaCl is applied.

[0543] In addition, when the exosome dispersion reacts with a preservative, the hydrophobic preservative can be partially inserted into the EV membrane, so at high concentrations, the membrane fluidity is high and leakage is high, thereby maintaining the minimum concentration for PET acceptance.

[0545] In addition, when the exosome dispersion reacts with borage oil, it is introduced dropwise in the form of a nanoemulsion (100–200 nm).

[0547] In addition, when the exosome dispersion reacts with the galangal extract, the long-term efficacy of EV and EGF is supported by the antioxidant and MMP inhibition of polyphenols. After lowering the EtOH to < 0.5%, low-temperature drip infusion is performed.

[0549] When the composition ratio (weight%) of the exosome dispersion according to the present invention is 1.0 weight% or less, the EV:liposome particle ratio (recommended around 1:2) breaks down, resulting in incomplete coating / partial fusion and the mixing of dual populations (coated / uncoated), which leads to a higher PDI (Polydispersity Index, uniformity of particle size distribution) and lower consistency. Additionally, the number of hits for cell contact and internalization is statistically reduced, leading to a decrease in EGF efficiency, and the natural membrane (Chol / Sphingo)-based stability correction is weakened, resulting in vulnerability to leakage, oxidation, and aggregation. When it is 3.0 weight% or more, high-concentration EVs over-contact with liposomes, increasing PDI and turbidity, and posing a risk of pre-filter clogging. Furthermore, liposomes and EVs fuse excessively, causing size bias (>200 nm), resulting in an over-delay in sustained release, which lowers the initial perceived release rate and alters the leakage pattern. Since the amount of bio-derived components increases, the demand for preservatives rises, and the burden of managing endotoxins and nucleic acids increases, it is most desirable to have it in an amount of 1.0 to 3.0 weight%.

[0551] [EGF(recombinant)]

[0553] This serves as a component of the EGF, liposome, and exosome triple lifting ampoule composition, acting as a core element of the efficacy signal. It performs an important role in providing foundational signals that reduce wrinkle depth and increase elasticity by dimerizing receptors and phosphorylating tyrosine through EGFR binding, and activating the RAS / RAF / MEK / ERK and PI3K / AKT pathways to promote the proliferation, migration, and collagen / elastin / hyaluronic acid synthesis signals of keratinocytes and fibroblasts.

[0554] That is, EGF, a 53aa peptide, binds to EGFR in keratinocytes and fibroblasts, and is then activated into the RAS / ERK pathway and the PI3K / AKT pathway, thereby promoting collagen and elastin synthesis signals and helping to improve the wound microenvironment.

[0555] Here, the RAS / RAF / MEK / ERK paths are explained.

[0556] First, EGF binds to EGFR, and EGFR is activated, causing the receptor to dimerize and undergo autophosphorylation of tyrosine residues.

[0557] At this time, the phosphorylated site of EGFR becomes the docking point of the SH2 domain protein.

[0558] Next, as an adapter protein binding, Grb2 (Adaptor protein) and SOS (Son of Sevenless, GEF) bind to EGFR. At this time, SOS activates the RAS protein (GTPase) (RAS-GDP → RAS-GTP).

[0559] Subsequently, RAS-GTP is activated into RAF (serine / threonine kinase), RAF is phosphorylated into MEK (MAP kinase kinase), and MEK is phosphorylated and activated into ERK (Extracellular signal-regulated kinase).

[0560] Here, activated ERK moves to the cell nucleus and phosphorylates transcription factors (Elk-1, c-Fos, Myc, etc.).

[0561] As such, the RAS / ERK pathway promotes cell proliferation, differentiation, and the expression of genes for collagen and elastin synthesis, which increases fibroblast activity, enhances collagen synthesis, reduces wrinkles, and increases skin elasticity.

[0563] The above PI3K / AKT path is explained.

[0564] First, PI3K (Phosphoinositide 3-kinase) binds to and activates the EGFR phosphorylation site.

[0565] Subsequently, PI3K converts the membrane phospholipid PIP2 (phosphatidylinositol-4,5-bisphosphate) into PIP3 (phosphatidylinositol-3,4,5-trisphosphate).

[0566] Subsequently, PIP3 recruits AKT (Protein Kinase B) to the membrane, and PDK1 phosphorylates and activates AKT.

[0567] Here, AKT increases protein synthesis by activating the mTOR pathway for cell survival / anti-apoptosis, inhibits GSK-3β, and activates eNOS.

[0568] As such, the PI3K / AKT pathway enhances fibroblast viability, protects against oxidative stress, and promotes ECM protein synthesis, thereby assisting in strengthening skin elasticity and improving wrinkles.

[0570] The above RAS / ERK pathway is primarily centered on cell proliferation, differentiation, and collagen synthesis, and the above PI3K / AKT pathway is centered on cell survival, metabolism, and anti-aging signaling, so both pathways must be activated simultaneously to maximize the skin regeneration and wrinkle improvement effects of EGF.

[0572] When the above EGF (recombinant) reacts with the liposomal lipid system, it enters the inner aqueous phase to protect against preservatives, salts, and temperature spikes, lowers burst release, and increases sustained release. Through the covalent bonding of EGF-SH and maleimide-thiol, it quantifies ligand density and surface exposure, increases EGFR activity efficiency, and inhibits EGF leakage and aggregation by regulating membrane fluidity and zeta potential of cholesterol / electrolyte lipids.

[0574] When the above EGF (recombinant) reacts with an exosome dispersion, it supports the uptake and durability of the liposome-EGF complex through coating / partial fusion / coexistence, and allows a small amount of EGF to be non-specifically adsorbed onto the EV surface, which is advantageous for sustained release and protection.

[0576] When the above EGF (recombinant) reacts with a moisturizer / protector, it forms a protein hydration shell together with glycerin / BG + trehalose / mannitol, thereby stabilizing EGF folding under heat / freeze / drying stress.

[0578] When the above EGF (recombinant) reacts with a conditioning agent (ceramide / squalane), it reinforces the barrier and texture, and improves the environment for maintaining EGF signals.

[0580] When the above EGF (recombinant) reacts with borage oil or is administered dropwise as a nanoemulsion (100–200 nm), it performs the role of anti-inflammatory and barrier support without membrane disturbance.

[0582] When the above EGF (recombinant) reacts with film formers, in the case of cationic (polyquaternium) forms, there is a high risk of electrostatic aggregation with negatively charged liposomes / EVs; therefore, it is diluted, dropwise, and low-shear administered.

[0584] When the above EGF (recombinant) reacts with a viscosity and texture improver, it controls diffusion and sedimentation through a gel network to help stabilize the dispersion of EGF and particles.

[0586] When the above EGF (recombinant) reacts with a buffer, salt, and chelate, it maintains a pH of 6.3 to 6.8, sets the ionic strength with 0.05 to 0.2 wt% NaCl, and blocks the metal catalyst with 0.05 wt% EDTA, thereby inhibiting deactivation, aggregation, and oxidation.

[0588] When the above-mentioned EGF (recombinant) reacts with a preservative, the preservatives phenoxyethanol / ethylhexylglycerin interact with the hydrophobic sites of the EGF to achieve the PET pass mark at the minimum effective concentration, and perform the role of confirming the presence or absence of denaturation through DLS / activity (ELISA / cell test).

[0590] When the above EGF (recombinant) reacts with galangal root extract, it assists in signal maintenance and photoaging inhibition through the antioxidant and MMP inhibition of polyphenols; since residual EtOH places a burden on proteins and membranes, it is administered via low-temperature drip infusion after reducing EtOH to <0.5%.

[0592] When the composition ratio (weight%) of the EGF (recombinant) according to the present invention is 0.0005 weight% or less, in the case of surface-bound type, it lowers the EGF density on the liposome surface, thereby reducing EGFR binding probability and clustering, and degrading signal efficiency. Furthermore, if irreversibly adsorbed in packaging materials, film formers, gel networks, filtration, etc., the perceived effective concentration becomes even lower, and if consumed by preservation systems, trace proteases, or oxidation, problems arise in which the effective serving dose is prone to falling below the critical threshold. When it is 0.005 weight% or more, excessive exposure increases EGFR internalization and degradation, paradoxically reducing the signal, increasing the possibility of erythema, acne-like reactions, and oil fluctuations in sensitive skin, increasing self-aggregation and turbidity in the case of high-concentration proteins, and causing problems with turbidity and odor change due to partial denaturation caused by interactions with preservatives and salts. Additionally, it induces over-adsorption and surface rearrangement on the liposome / EV surface, increasing the PDI and the release curve Since destabilizing problems occur, it is most desirable that EGF (recombinant) be composed of 0.0005 to 0.005 weight%.

[0594] [Moisturizer]

[0596] This plays an important role as a component of the EGF, liposome, and exosome triple lifting ampoule composition, attracting and retaining moisture from the stratum corneum to increase moisture content, reducing dry fine lines, lowering the active water (aw) in the aqueous phase to assist in inhibiting microbial growth, maintaining a balance of viscosity and osmosis, and protecting EGF, liposome, and exosome through osmotic buffering and the formation of a hydration layer.

[0597] This maintains the moisture content within the stratum corneum by binding with water molecules through a hydrogen bond network, reduces dehydration and expansion stress of cells, liposomes, and exosomes from the external environment (low humidity, high salt) through osmotic buffering, and alleviates heat, dryness, and oxidative stress by forming a moisture film around EGF proteins as a hydration layer stabilization.

[0599] The above humectant manufacturing content is based on the total weight % of the humectant,

[0600] It consists of 3 to 6 weight percent glycerin and 2 to 4 weight percent butylene glycol.

[0602] Here, glycerin is a trihydric alcohol and has strong and long-lasting moisturizing properties.

[0604] The above butylene glycol (BG) has a refreshing texture, enhances solventability, and acts as an antimicrobial booster.

[0606] When the composition ratio (weight%) of the moisturizer according to the present invention is 5 weight% or less, the improvement of dry fine wrinkles is weak due to insufficient moisture reinforcement, and the protein membrane is vulnerable to temperature, salt, and preservative stress, which weakens the protective layer of EGF, liposomes, and exosomes, and causes the sensation of "moisture" to be rapidly lost. When it is 12 weight% or more, the osmotic pressure increases excessively (~>350 mOsm), causing liposomes and exosomes to contract, increasing aggregation and PDI, destabilizing the release curve, impairing EGF stability, delaying stickiness, peeling, and absorption, and causing the appearance to become turbid and the viscosity to become uneven due to the breakdown of the gel network. Therefore, it is most desirable to have a composition of 5 weight% to 12 weight%.

[0608] [Protectant]

[0610] This plays an important role as a component of the EGF, liposome, and exosome triple lifting ampoule composition, protecting EGF, stabilizing membranes (liposomes and exosomes), increasing resistance to refrigeration, acceleration, light, freeze-thaw, and filling stress, preventing cake collapse and stickiness during freeze-drying, and maintaining particles after reconstruction.

[0611] This is because sugars partially replace / reinforce water sites on the surface of proteins and lipids, stabilizing EGF folding and membrane bilayer arrangement, and in particular, trehalose forms a glassy matrix with a high glass transition temperature, inhibiting molecular motion and lowering the rate of chemical reactions, and small molecule protective agents such as mannitol help maintain isotonicity to buffer dehydration and swelling stress, and mannitol buffers a small amount of radicals (indirectly inhibits lipid peroxidation).

[0612] The composition of the protective agent is composed of 1 to 3 weight percent of trehalose and 0 to 1 weight percent of mannitol relative to the total weight percent of the protective agent in the case of the liquid-stabilized type, and 2 to 5 weight percent of trehalose and 1 to 3 weight percent of mannitol relative to the total weight percent of the protective agent in the case of the freeze-dried type.

[0613] Here, in the case of the liquid-stabilized type, trehalose is composed of 1 to 3 weight percent of the total composition and mannitol is composed of 0 to 1 weight percent of the total composition, and the total is 1 to 4 weight percent, which corresponds to the lower range of the total protective agent amount of 1 to 8 weight percent.

[0614] At this time, in the case of the trehalose:mannitol ratio (relative to the total composition), trehalose forms the main component (75~100%) and mannitol forms the auxiliary component (0~25%).

[0616] In the case of the freeze-dried type, trehalose is composed of 2 to 5 weight percent of the total composition, mannitol is composed of 1 to 3 weight percent of the total composition, and the total is 3 to 8 weight percent, which corresponds to the lower range of the total protective agent amount of 1 to 8 weight percent.

[0617] In this case, regarding the trehalose:mannitol ratio (relative to the total composition), trehalose constitutes the main component (accounting for 60–70%), and mannitol constitutes the auxiliary component (accounting for 30–40%). That is, the trehalose:mannitol is blended in a ratio of approximately 6:4.

[0619] The above trehalose plays a role in protecting proteins (EGF) and membranes (liposomes / exosomes) from heat, drying, and freezing stress, and forming a glassy matrix during freeze-drying.

[0620] That is, it replaces or reinforces water on the surface of proteins and lipids to stabilize folding and membrane arrangement, and as a non-reducing disaccharide, it has a low risk of saccharification / browning, raises the glass transition temperature to prevent the collapse and stickiness of the rayo cake, and maintains particle size after reconstitution.

[0622] The above mannitol acts as a bulking agent, osmotic buffer, and partial radical scavenger, provides mechanical strength to the Rayo-Cake, and corrects osmotic and isotonic properties in the liquid phase.

[0623] In other words, it crystallizes during freezing and drying to provide a framework, preventing the cake from shrinking or becoming distorted.

[0625] When the composition ratio (weight%) of the protective agent according to the present invention is 1 weight% or less, problems arise such as increased loss of activity under acceleration, light, and storage stress due to insufficient EGF protection, increased leakage, fusion, and aggregation of liposomes / exosomes due to particle instability, increased PDI, and delayed cake collapse and reconstruction due to insufficient glassiness / bulking; when it is 8 weight% or more, problems arise such as particle shrinkage and aggregation due to hyperosmosis and dehydration stress, increased or excessively delayed initial burst of the release curve, increased adhesion, peeling, and turbidity due to deterioration of physical properties / sensory properties, and disruption of the gel network; and problems arise such as bloating / layer separation due to mannitol hypercrystallization, increased particle size after reconstruction, and increased PDI. Therefore, it is most desirable to have a composition ratio of 1 weight% to 8 weight%.

[0627] [Conditioning Agent]

[0629] This serves as a component of the EGF, liposome, and exosome triple lifting ampoule composition, reinforcing the barrier, reducing moisture evaporation, providing slipperiness, lubrication, and shine, and creating a skin condition favorable for EGF signaling to function through moisture retention and inflammation relief.

[0630] The composition of the above conditioning agent consists of 0.05 to 0.2 weight percent of ceramide and 0.2 to 0.6 weight percent of squalane, based on the total weight percent of the conditioning agent.

[0631] Here, ceramide is a key component of the lipid lamellae (ceramide / cholesterol / free fatty acids) of the stratum corneum, and has the characteristics of repairing damaged barriers, retaining moisture, and restoring barrier function by increasing order and reducing permeability through long-chain lipids interlocking in the lamellar structure.

[0632] The above-mentioned squalane is a non-polar emollient that forms an occlusive film, inhibits moisture evaporation, and has the characteristics of a soft, slippery texture, where skin-friendly hydrocarbons create a surface oil film to reduce moisture evaporation and enhance the feel.

[0634] When the composition ratio (weight%) of the conditioning agent according to the present invention is 0.3 weight% or less, moisture evaporation increases, moisture retention is weak, the sensation of improvement in dry fine lines is reduced, slip sensation is insufficient, film former tightness is prominent, spreadability becomes rough, and the skin surface dries out easily, resulting in a decrease in EGF signal persistence. When it is 1 weight% or more, problems occur such as stickiness and greasiness due to over-occlusion and heavy texture, excessive fluidity and leakage of liposome / EV membranes due to excess squalane, excessive membrane rigidity and aggregation due to excess ceramide, increased PDI and turbidity causing the pre-filter to become clogged, and the initial sensation of EGF being dulled due to membrane rigidity and oil phase distribution. Therefore, it is most desirable to have a composition ratio of 0.3 to 1.0 weight%.

[0636] [Film Former]

[0638] This is a component of the EGF, liposome, and exosome triple lifting ampoule composition, and a thin, semi-permeable polymer film formed upon drying after application provides a firm, tightening sensation through micro-contraction and tension, creates a semi-occlusive film on the moisturizing layer to suppress moisture evaporation, thereby assisting in the alleviation of fine lines (dryness), and helps liposomes, exosomes, and EGF remain on the skin surface for a longer period, thereby increasing the residence time.

[0639] As moisture evaporates, the polymer chains approach and bond with each other (hydrogen bonding / electrostatic interaction), forming a continuous film.

[0640] In addition, the composition of the film former according to the present invention consists of 0.05 to 0.15 weight% of polyquaternium-51 and 0.05 to 0.15 weight% of polyglutamic acid (PGA) based on the total weight% of the film former.

[0641] Here, polyquaternium-51 has a cationic phosphate-like structure that increases electrostatic affinity for skin lipids / proteins and plays a role in forming a strong moisturizing film and a soft texture.

[0642] It is skin-friendly, has low stickiness, and forms a fresh moisturizing film.

[0644] The above polyglutamic acid (PGA) is an anionic polymer derived from glutamic acid, and provides strong moisture binding power (reported to be 4 to 5 times that of hyaluronic acid), film formation, and elasticity.

[0645] This forms a moisturizing and elastic film on the skin surface and reduces fine wrinkles.

[0647] The total amount of the above polyquaternium-51 and polyglutamic acid (PGA) is 0.1 to 0.3 weight% based on 100 weight% of the total composition, and the ratio is polyquaternium-51 : PGA = 1 : 1 to 6 : 4.

[0648] For example, when used in a total of 0.2 wt%, it consists of 0.1 wt% polyquaternium-51 and 0.1 wt% PGA, and

[0649] When used at a total of 0.25 wt%, it consists of 0.15 wt% polyquaternium-51 and 0.1 wt% PGA, and when used at a total of 0.15%, it consists of 0.07 wt% polyquaternium-51 and 0.08 wt% PGA.

[0651] When the composition ratio (weight%) of the film former according to the present invention is 0.1 weight% or less, the lifting sensation is weakened due to insufficient film continuity, the maintenance is short with only the moisturizing layer, the sensation of improvement in dry fine wrinkles is weak, and the effect of increasing the local residence time of liposomes, exosomes, and EGF is limited. When it is 0.3 weight% or more, lint is formed when rubbing due to excessive film formation, frictional whitening occurs, the PDI increases due to liposome / EV aggregation caused by electrostatic bridging, and problems such as pre-filter and nozzle clogging occur, and the film is over-densified, which lowers the initial EGF access and diffusion speed, thereby weakening the immediate sensation and reducing uniformity. Therefore, it is most desirable to have a composition of 0.1 to 0.3 weight%.

[0653] [Viscosity and Texture Improver]

[0655] This serves as a component of the EGF, liposome, and exosome triple lifting ampoule composition, providing a target viscosity (1,500–4,000 cP @ 25℃, 20 rpm) and yield stress to keep the product from flowing and to keep it on the skin, inhibiting the sedimentation, creaming, and aggregation of the liposome, exosome, and borage nanoemulsion to prevent an increase in PDI, controlling slipperiness, cushioning, and residual film, and controlling the diffusion rate of EGF particles through a gel network.

[0656] The manufacturing content of the above viscosity and texture improver is based on the total weight % of the viscosity and texture improver,

[0657] It consists of 0.1 to 0.3 wt% hydroxyethylcellulose (HEC), 0.05 to 0.2 wt% carbomer, and 0.05 to 0.2 wt% acrylate copolymer.

[0659] The above hydroxyethylcellulose (HEC) is a non-ionic cellulose-based thickener that plays a role in stably forming electrolytes, moisturizers, and EGF.

[0660] It has the characteristics of a clear solution, flexible gel texture, low stickiness, and shear-thinning, and is used as a primary thickener. In addition, its specific gravity accounts for about 30 to 50 percent of the viscosity and texture improver.

[0662] The above carbomer is an acrylic acid-based polymer that forms a strong 3D network upon neutralization (pH 6.3~6.8), thereby providing high viscosity and yield stress.

[0663] This strengthens the gel structure even in small amounts, reduces tackiness and transparency, and has characteristics sensitive to electrolyte / cation components. Also, its specific gravity accounts for about 20-30% of the viscosity and texture improver.

[0665] The above acrylate copolymer provides rheology control and film properties, offers a cushioning and soft feel upon application, and minimizes residue.

[0666] This has the characteristics of texture improvement and rheology tuning. Also, the specific gravity accounts for about 20-30% of the viscosity and texture improver.

[0668] When the composition ratio (weight%) of the viscosity and texture improver according to the present invention is 0.2 weight% or less, due to insufficient yield stress, it flows like water when pumped, and sagging and dripping occur after application. Additionally, due to the sedimentation and creaming of liposomes, exosomes, and oils, the PDI increases, the initial burst release increases, and the local residence time of EGF decreases, resulting in unstable perceived efficacy. When it is 0.8 weight% or more, the gel density increases, creating an over-wall due to EGF and particle diffusion, which dulls the initial sensation, increases shear sensitivity, and traps microbubbles, causing the filling nozzle pressure to increase and clogging to occur. When it is mainly carbomer, problems such as aggregation by salt and cations and increased turbidity occur. Therefore, it is most desirable to have a composition of 0.2 weight% to 0.8 weight%.

[0670] [Buffer, Salt, Chelate]

[0672] This serves as a component of the EGF, liposome, and exosome triple lifting ampoule composition, and plays an important role in determining EGF activity, liposome / exosome membrane stability, and overall dispersion stability by precisely managing pH, ionic strength, and metal ions.

[0673] This acts as a buffer, maintaining a constant pH of 6.3 to 6.8, stabilizing the folding and disulfide bonds of EGF, and stabilizing the charge and fluidity of liposome and exosome membranes.

[0674] Then, using salt, an appropriate ionic strength and osmotic pressure (≈280~320 mOsm) are formed, and aggregation and flocculation are achieved by controlling the thickness of the electric double layer.

[0675] In addition, as a chelating agent, Fe 3+ / Cu 3+ It captures metal catalysts to inhibit oxidation and radical-induced reactions, prevents lipid peroxidation and secondary damage to EGF, and supports the efficacy of preservatives.

[0677] The manufacturing content of the above buffer, salt, and chelating agent is based on the total weight % of the buffer, salt, and chelating agent,

[0678] It consists of 0.10~0.35 wt% phosphate buffer, 0.05~0.2 wt% NaCl, and 0.05 wt% EDTA.

[0680] Here, the phosphate buffer is maintained in the pH range of 6.3 to 6.8 to stabilize the structure of the EGF protein and stabilize the membrane charges of liposomes and exosomes.

[0682] The above NaCl compresses the electrical double layer by adjusting ionic strength, inhibits aggregation between liposomes and exosomes, and maintains osmotic balance.

[0684] The above EDTA (Ethylenediaminetetraacetic acid, disodium salt) is a metal ion (Fe 3+ , Cu 2+ , Ca 2+Etc.) As a chelator, it inhibits lipid peroxidation and EGF oxidation, and supports the efficacy of preservatives.

[0686] When the composition ratio (weight%) of the buffer, salt, and chelating agent according to the present invention is 0.2 wt% or less, problems arise due to insufficient buffering capacity, which causes pH fluctuations during manufacturing and storage, thereby reducing EGF activity, altering membrane fluidity and charge, leading to leakage and increased PDI, and due to low salt, excessive strengthening of electrostatic coupling between film formers (cations), PGA (anions), and negatively charged particles (liposomes / EVs), resulting in bridging flocs and increased turbidity, and insufficient chelation, which leads to the persistence of trace metal catalysts, promoting lipid peroxidation and EGF oxidation, and reducing long-term stability. When it is 0.6 wt% or more, problems arise due to excessive compression of the electric double layer, loss of inter-particle repulsion, increased aggregation, sedimentation, and creaming, and clogging of pre-filters and nozzles, high salt / phosphate concentrations, which stress protein solubility and folding, and a risk of reduced activity, and the interaction between polyquaternium / PGA and phosphate / salt, coacervates And since problems such as increased turbidity occur, it is most desirable to have it in an amount of 0.2 to 0.6 weight percent.

[0688] [Preservatives]

[0690] This serves as a component of the EGF, liposome, and exosome triple lifting ampoule composition, inhibiting the growth of bacteria, yeast, and mold during manufacturing, distribution, and consumption processes, lowering the risk of contamination of EGF, liposomes, exosomes (EV), moisturizing and natural extracts, and maintaining quality even with consumer contact and exposure to air.

[0691] The composition of the above preservative consists of 0.5 to 0.8 weight percent of phenoxyethanol and 0.1 to 0.3 weight percent of ethylhexylglycerin, based on the total weight percent of the preservative.

[0692] Here, phenoxyethanol (PE) is a broad-spectrum antimicrobial agent (Gram + , Gram -As a yeast, mold, etc., it penetrates the cell membrane to induce protein denaturation and is used as the active backbone of preservatives.

[0693] Additionally, while it has preservation capabilities on its own, it is used in combination with a booster (EHG) to enhance efficiency.

[0695] The above ethylhexylglycerin acts as a preservation booster, increasing the permeability of microbial cell membranes to improve the penetration power of PE, and has a weak antibacterial and preservative effect of its own, and plays a role in improving skin texture.

[0697] When the composition ratio (weight%) of the preservative according to the present invention is 0.7 weight% or less, the inhibition curve for negative and mold is unstable, the risk of regrowth is high, and re-contamination after contact with hands and air grows, causing changes in odor, discoloration, turbidity, and viscosity. Furthermore, problems arise such as lowering EGF activity through lipid peroxidation and proteolytic enzymes induced by trace contamination, as well as liposome and EV leakage and increased PDI. When it is 1.0 weight% or more, the risk of burning sensation, stinging, and odor increases at high PE concentrations, leakage and increased PDI occur due to excessive fluidization of liposome and EV membranes, partial denaturation and reduced activity due to PE interacting with the protein hydrophobic surface, and peeling, turbidity, and viscosity drift due to interaction with film formers / gels. Therefore, it is most desirable to have a composition ratio of 0.7 to 1.0 weight%.

[0699] Borage oil

[0701] This is a component of the EGF, liposome, and exosome triple lifting ampoule composition, and is rich in gamma-linolenic acid (GLA), which acts positively on the lipid resynthesis and prostaglandin (PGE₁) pathways of the stratum corneum to help improve dry and micro-inflammatory wrinkles, provides a thin oil film to reduce moisture evaporation, gives a slightly richer sensation compared to squalane, and plays a role in adjusting the balance of the application sensation.

[0702] In addition, as a highly unsaturated (linoleic acid / GLA), it is sensitive to oxidation, has a slight color and odor, changes odor upon oxidation, and is lipophilic, having high compatibility with lipid membranes and having the characteristic of affecting liposome and exosome membranes.

[0703] In addition, GLA, a component of borage oil, contributes to the synthesis of ceramides and free fatty acids, increasing lamellar order and reducing permeability; it assists in the alleviation of inflammatory aging via the DGLA→PGE₁ pathway, and the oil film forms a semi-occlusive film to retain moisture.

[0705] When the composition ratio (weight%) of the borage oil according to the present invention is 0.2 weight% or less, the barrier reinforcement and anti-inflammatory effects are weak due to insufficient GLA dose, the contribution to improving dry wrinkles is statistically weak, the moisture retention curve drops rapidly due to a lack of occlusion and the retention of the moisturizing and film former layer, and the overall user experience feels dry due to weak slip / gloss correction. When it is 1.0 weight% or more, the PDI is high and turbidity is high due to over-adsorption and bridging on the liposome / EV surface, which causes clogging of the pre-filter and nozzle, and the EGF leakage rate increases due to over-insertion into the liposome and EV membrane, the preservative is further distributed in the oil phase, lowering the effective concentration in the aqueous phase, and the peroxide increases due to excessive unsaturated fatty acids, causing long-term stability and odor problems. Therefore, it is most desirable to have a composition ratio of 0.2 to 1.0 weight%.

[0707] [Galanga Root Extract]

[0709] This serves as a component of the EGF, liposome, and exosome triple lifting ampoule composition, in which polyphenols and flavonoids remove ROS to alleviate oxidative stress caused by UV rays and pollution, and there is a reported MMP-1 / collagenase activity inhibition system that reduces collagen degradation to assist in the improvement of wrinkles and elasticity, and there is a reported tyrosinase and inflammatory signal inhibition system that indirectly contributes to alleviating dullness.

[0710] In addition, as a polyphenolic water-soluble / water-soluble glycol extract extracted from the root of galangal (Alpinia galanga), it has the characteristics of skin soothing and anti-inflammatory action, antioxidant effect, antibacterial action, and moisturizing effect, and accelerates browning and oxidation upon contact with metal (Fe / Cu), and helps stabilize when EDTA (0.05%) is used simultaneously.

[0712] When the composition ratio (weight%) of the galanga root extract according to the present invention is 0.1 weight% or less, the antioxidant and MMP inhibitory effects are statistically weak due to insufficient effective dose, resulting in a low contribution to wrinkle and elasticity perception, and the quantitative variation of indicator components (HPLC) increases, leading to lower QC sensitivity and a smaller improvement range in oxidation and elasticity indicators under accelerated / light conditions. When it is 0.5 weight% or more, polyphenols and solvents affect the membrane, causing problems such as increased PDI, increased turbidity, clogging of the pre-filter, accelerated browning and odor change, and a high risk of partial denaturation and leakage of EGF due to an excess of EtOH and polyphenols. Therefore, it is most desirable to have a composition of 0.1 to 0.5 weight%.

[0714] [Purified Water]

[0716] This serves as a component of the EGF, liposome, and exosome triple lifting ampoule composition, dissolves all water-soluble and dispersible ingredients (buffer, salt, moisturizer, and protective agent), acts as a dispersion medium for liposomes, exosomes, and borage oil, controls the release rate of EGF particles and skin retention through viscosity and diffusion coefficients where diffusion increases with higher water content, and determines the osmotic pressure (target approximately 280–320 mOsm) with the amount of purified water and the ratio of buffer / salt / moisturizer / protective agent to stabilize particle size, and performs the role of maintaining the pH at 6.3–6.8 as a medium for the phosphate buffer to operate.

[0718] If the composition ratio (weight%) of purified water according to the present invention is 70 weight% or less, the relative concentrations of moisturizing, protective, salt, and buffering agents increase, leading to problems such as liposomes and exosomes shrinking due to an excessive rise in osmotic pressure, increased PDI, increased leakage, and increased EGF folding stress. Additionally, the gel and polymer networks become tight, increasing the differential pressure of the filling, filtration, and pre-filter, causing bubble trapping, streaking, and peeling. Furthermore, the diffusion coefficient decreases, resulting in a slowdown in the initial release of EGF and particles, which weakens the immediate perceived effect (lifting / moisture sensation). If it is 88 weight% or more, the absolute amounts of moisturizing, protective, buffering agents, and salts decrease, leading to a decrease in osmotic pressure (low ionization). This causes increased electrostatic bridging between polymers and particles, resulting in increased flocs and turbidity. Additionally, the viscosity decreases, leading to problems such as an increased tendency for gravitational separation of nanoemulsions and particles. Moreover, the film and moisturizing layer become thin, resulting in a short duration of lifting and hydration, and a thin and light consistency. It may only leave a feeling of use, and because the absolute buffering amount is low, pH drift increases under external stress, causing problems that reduce EGF and membrane stability, it is most desirable to have it composed of 70% to 99% by weight.

[0720] ingredient Test Example (Existing EGF Liposome) (Weight%) Comparative Example A (excluding exosomes, borage oil, and galangal root extract) (weight%) Comparative Example B (excluding exosomes, protective agents, borage oil, and galangal root extract) (weight%) Example A (Standard Ampoule) (Weight%) Example B (Specialized Ampoule) (Weight%) Liposomal lipid system 3.5 3.5 3.5 3.2 3.5 Exosome dispersion - - - 2.5 3.0 EGF (recombinant) 0.005 0.005 0.005 0.005 0.005 moisturizer 6.0 8.0 8.0 10.0 11.0 protective agent 1.0 3.0 - 4.0 5.0 conditioning agent 0.4 0.5 0.5 0.6 0.7 Film former 0.2 0.2 0.2 0.3 0.3 Viscosity and texture improvers 0.3 0.5 0.5 0.6 0.7 Buffer, salt, and chelate 0.3 0.3 0.3 0.5 0.6 preservatives 0.7 0.8 0.8 1.0 1.0 Borage oil - - - 0.5 1.0 Galangal root extract - - - 0.3 0.5 purified water 87.595 83.195 86.195 76.495 72.695 total 100 100 100 100 100

[0722] Table 1 relates to the total composition weight ratio (100wt%) consisting of Test Example, Comparative Example A, Comparative Example B, Example A, and Example B.

[0723] More specifically, Test Example relates to a composition composed solely of conventional EGF liposomes, Comparative Example A relates to a composition not containing exosome dispersion, borage oil, and galangal root extract, Comparative Example B relates to a composition not containing exosome dispersion, protective agent, borage oil, and galangal root extract, Example A relates to a standard ampoule of the EGF·liposome·exosome triple lifting ampoule composition according to the present invention, and Example B relates to a specialized ampoule of the EGF·liposome·exosome triple lifting ampoule composition according to the present invention. Here, the specialized ampoule is an ampoule targeted for high-efficacy intensive care, particularly for deep wrinkles and aged skin.

[0725] item Test Example (Existing EGF Liposome) (Weight%) Comparative Example A (excluding exosomes, borage oil, and galangal root extract) (weight%) Comparative Example B (excluding exosomes, protective agents, borage oil, and galangal root extract) (weight%) Example A (Standard Ampoule) (Weight%) Example B (Specialized Ampoule) (Weight%) Wrinkle improvement +5% (Epidermal level improvement) +12% (Improvement due to moisturization) +18% (Partially improved due to fault infiltration) +28% (Significant recovery of elasticity) +38% (Significant improvement in eye and nasolabial folds) Skin penetration depth Approximately 30–40 µm (localized to the epidermal layer) Approximately 60㎛ (delivered to some upper dermal layer) Approximately 80㎛ (penetration into the upper dermis) Approximately 120㎛ (penetration into the dermis) Approximately 150㎛ or more (deposition of active ingredients within the dermis) Natural membrane / Intake / Stability (Stability index) 1.5 / 5.0 (Weak membrane formation) 2.0 / 5.0 (Temporary moisturizing barrier) 2.5 / 5.0 (Low retention) 4.0 / 5.0 (Liposome + Film Former Stabilization) 4.8 / 5.0 (Triple stabilizing film + protective film formation) Long-lasting preservation Concern over discoloration within 6 months 9 months (maintaining basic stability) 6~8 months (unstable) 12 months (maintained stability) 18 months or more (maintains stability at room temperature) Usability (average sensory evaluation score, 5-point scale) 3.0 / 5 points (sticky) 3.2 / 5 points (slightly stiff) 3.5 / 5 points (low moisture) 4.5 / 5 points (Moist, Light) 4.8 / 5 points (Smooth · High texture)

[0727] Table 2 is a comparative analysis table of functionality. In the case of the test example, it consists only of conventional EGF liposomes, and the degree of wrinkle improvement is +5%, which is an improvement at the epidermal level. The skin penetration depth is about 30~40㎛, which is limited to the epidermal layer. In terms of natural film / ingestion / stability, the film formation is weak at 1.5 / 5.0, there is a concern about discoloration within 6 months of long-term support, and the user experience is 3.0 / 5, indicating stickiness.

[0728] In the case of Comparative Example A, the composition was made without exosome dispersion, protective agent, borage oil, or galangal root extract, and the degree of wrinkle improvement was +12%, which is the level of improvement due to moisturization, the skin penetration depth was about 60㎛, and it was delivered to some upper dermal layers, and in terms of natural membrane / ingestion / stability, temporary moisturization was formed with a score of 2.0 / 5.0, and basic stability was maintained with a long-term supportive power of 9 months, and the user experience was 3.2 / 5 points, indicating that it was slightly stiff.

[0729] In the case of Comparative Example B, the composition was made without exosome dispersion, protective agent, borage oil, or galangal root extract, and the degree of wrinkle improvement was +18%, which is a partial improvement level due to single-layer penetration, and the skin penetration depth was about 80㎛, penetrating to the upper layer of the dermis, and in terms of natural membrane / ingestion / stability, the retention power was 2.5 / 5.0, the long-term support power was unstable at 6 to 8 months, and the user experience was 3.5 / 5 points, confirming that the moisturizing effect was weak.

[0730] In the case of Example A, as a standard ampoule of the EGF·liposome·exosome triple lifting ampoule composition, the degree of wrinkle improvement is +28%, showing clear elasticity recovery, the skin penetration depth is about 120㎛, penetrating to the dermis layer, the natural membrane / ingestion / stability is 4.0 / 5.0, stabilizing the liposome + film former, the long-term supportive power is maintained for 12 months, and the user experience is 4.5 / 5 points, confirming that it is moist and light.

[0731] In the case of Example B, as a specialized ampoule of an EGF, liposome, and exosome triple lifting ampoule composition, the degree of wrinkle improvement is +38%, showing a level of significant improvement in wrinkles around the eyes and nasolabial folds; the skin penetration depth is approximately 150㎛ or more, with the active ingredient being deposited in the dermal layer; regarding natural film / ingestion / stability, it forms a triple safety net + protective film with a score of 4.8 / 5.0; the long-term supportive power maintains room temperature stability for 18 months; and the user experience is 4.8 / 5, confirming a smooth and high-touch texture.

[0732] The sensory evaluation regarding the degree of wrinkle improvement was conducted on 30 adult women aged 35 to 60 with moderate to severe forehead wrinkles. The test period was 8 weeks, and the product was applied twice a day (morning and evening). For objective analysis, the depth and volume of forehead wrinkles were measured using a 3D skin analyzer (PRIMOS, VISIA), and the sensory evaluation was set on a 5-point scale (1 = no improvement at all, 5 = very significant improvement).

[0733] In addition, the sensory evaluation regarding the above skin penetration depth was conducted on 30 adult women aged 35 to 60 with moderate to severe forehead wrinkles, with a test period of 8 weeks and application twice a day (morning / evening). Specifically, the penetration depth was measured using confocal microscopy and skin bioimaging with nanoliposomes / exosomes combined with fluorescent markers, and at the same time, the subjects evaluated the sensation (absorption sensation, residue sensation) after application on a 5-point scale.

[0735] Through Tables 1 and 2 above, it can be confirmed that EGF induces skin epidermal regeneration, and that its effect alone is minimal.

[0736] In addition, it can be confirmed that liposomes are carriers that help penetrate the skin barrier, and exosomes deliver cell regeneration and antioxidant signals to the dermis layer. Accordingly, in the present invention, a high-quality lifting ampoule composition can be formed by forming a core consisting of EGF (signaling), liposomes (sustained-release and retention), and exosomes (natural membrane, ingestion, and stabilization).

[0737] In addition, it can be confirmed that borage oil and galangal extract provide sebum-control, anti-inflammatory, and antioxidant synergies. Explanation of the symbols

[0739] 1 : Device for manufacturing an EGF, liposome, and exosome triple lifting ampoule composition 100 : Rotary evaporator 200 : Vacuum pump 300 : Jacketed mixing tank 400 : Low-shear stirrer 500 : Deep Freezer 600 : Ultrasonic Bath 700 : Microfluidizer 800 : Coating / Fusion Type Combination Forming Module 900 : Surface-bonded / coexisting combination forming module 900a : EGF·Liposome·Exosome Triple Lifting Ampoule Composition Forming Module

Claims

Claim 1 In an apparatus for manufacturing an EGF-liposome-exosome triple lifting ampoule composition that improves skin wrinkles by mixing a viscosity-texture improving agent, a film former, a conditioning agent, a preservative, borage oil, and galangal root extract into a core in which signaling EGF, sustained-release / retention liposomes, and natural membrane / ingestion / stabilizing exosomes are combined, the apparatus comprises: a rotary evaporator in which a solution is placed in a round-bottom flask, rotated over a water bath to evaporate, and then condensed into a lipid thin film in a reduced-pressure atmosphere from a vacuum pump; a vacuum pump located on one side of the rotary evaporator to generate a reduced-pressure force and transmit it toward the rotary evaporator; and a tank in which a double-layer jacket structure is formed on the outer wall, and a temperature, cold water, and glycol solution are circulated to fill the tank A jacketed stirring tank that controls the temperature of the contents to 40–50℃; a low-shear stirrer that injects the hydration solution formed from the jacketed stirring tank into the lipid film, rotates it at 20–200 rpm through an impeller formed inside the tank, and stirs the lipid film and the hydration solution to form MLV liposomes; a deep freezer that receives the MLV liposomes formed in the low-shear stirrer, rapidly freezes them to shrink and rearrange the lipid film; an ultra-low temperature bath that receives the MLV liposomes formed in the low-shear stirrer in a glass vial within a water bath, places them therein, and then generates ultrasound in the 20–40 kHz band through an ultrasonic transducer to soften the liposome membrane through low-intensity cavitation; and an ultrasonic bath located on one side of the front of the ultrasonic bath, which receives the MLV liposomes with softened membranes from the ultrasonic bath, By pressurizing to an ultra-high pressure of 700–1400 bar and passing through a Y-shaped microchannel, particles collide at high speeds with each other due to shear force, turbulence, and cavitation, thereby inducing the fragmentation of large multilayer liposomes (MLVs),A microfluidizer that forms single-membrane liposomes (LUVs) of 80–150 nm; a coating / fusion type combination forming module that forms an EGF-liposome-exosome combination by coating an exosome (EV) membrane component onto the outer membrane of a liposome or partially fusing the two membranes to enable sustained liposome release along with natural membrane surface labeling; a surface binding / coexistence type combination forming module that forms an EGF-liposome-exosome combination by simultaneously allowing liposomes, exosomes, and EGF proteins to exist within a single system, with some chemically bound, some physically coexisting, or surface binding in which the EGF protein is stably fixed to the liposome surface via covalent bonding; and to the EGF-liposome-exosome combination formed through the coating / fusion type combination forming module and the surface binding / coexistence type combination forming module, an additive An apparatus for manufacturing an EGF-liposome-exosome triple lifting ampoule composition, characterized by comprising an EGF-liposome-exosome triple lifting ampoule composition forming module that completes the EGF-liposome-exosome triple lifting ampoule composition by additionally adding a viscosity-texture improving agent composed of hydroxyethylcellulose (HEC), carbomer, and acrylate copolymer; a film former agent composed of polyquaternium and polyglutamic acid; a conditioning agent composed of ceramide and squalane; a preservative composed of phenoxyethanol and ethylhexylglycerin; borage oil; and galanga root extract. Claim 2 delete Claim 3 An apparatus for manufacturing an EGF, liposome, and exosome triple lifting ampoule composition according to claim 1, wherein the microfluidizer comprises: a high-pressure pump that receives MLV liposomes with softened liposome membranes from an ultrasonic bath and pressurizes them to an ultra-high pressure of 700 to 1400 bar; a Y-shaped microchannel section that divides the MLV liposomes, which receive the ultra-high pressure force from the high-pressure pump, into two Y-shaped branches and induces them to collide at high speed in a microchannel; a collision chamber section located at one side of the rear end of the Y-shaped microchannel section and forms a collision space that causes the two flows branched from the Y-shaped microchannel section to collide head-on; a heat exchanger located at one side of the collision chamber section and removes heat generated in the collision chamber section; and a collection tank that collects and stores single-membrane liposomes (LUV) of uniform size through the collision chamber section. Claim 4 In claim 1, the coating-fusion type combination forming module comprises: a coating-fusion type low-shear mixer that performs the role of gently mixing liposome dispersion + exosome dispersion at 4–25°C to create a pre-stage of membrane contact and rearrangement; a 37°C water bath and dry block that promote a partial coating-fusion event by increasing membrane fluidity for 5–15 minutes on a 37°C water bath and dry block; a freeze-thaw unit that induces fusion by expanding the membrane rearrangement and contact surface by repeating rapid freezing at -80°C and thawing at 25–37°C 1–2 times; a micro-injection pump that assists negative charge inter-membrane bridging and PEG-fusion with precise drops of CaCl₂ (0.5–2 mM) or PEG-8000 (2–5 wt%); and, after fusion, removes large aggregates by low-speed centrifugation at 1,000–3,000 × g (relative centrifugal acceleration) for 5 minutes. An apparatus for manufacturing an EGF-liposome-exosome triple lifting ampoule composition, characterized by comprising a low-speed centrifuge and a coated-fused TFF section that forms coated-fused liposomes and exosomes (EVs) to flow tangentially along the surface of a filter membrane to filter out PEG-Ca2+, free EGF, and small molecules. Claim 5 An apparatus for manufacturing an EGF, liposome, and exosome triple lifting ampoule composition according to claim 4, wherein the coating and fusion type TFF section comprises a first feed tank section that serves as a container for coated and fused liposomes and exosomes (EVs), a first circulation pump section that circulates coated and fused liposomes and exosomes (EVs) to a first filter module section, a first filter module section that filters with a semipermeable membrane having a specific molecular weight cutoff (MWCO), a first dialysis filtration line section that serves as a line for small molecules and solvents to exit, a first retentate line section that serves as a line for recovering concentrated coated and fused liposomes and exosomes (EVs), a first pressure controller section that controls the Transmembrane Pressure (TMP), and a first TFF collection tank section that stores and preserves concentrated coated and fused liposomes and exosomes (EVs). Claim 6 In claim 1, the surface-binding / coexistence type combination forming module comprises: a liposome functionalization forming part that exposes maleimide reactive groups to the liposome outer membrane to form a covalent bond with the -SH group of the EGF protein; an EGF surface-binding type combination forming part that forms a combination by surface-binding the reduced EGF-SH protein and the liposome surface maleimide group to a stable, fixed state by covalent bonding; an exosome coexistence type combination forming part that combines exosomes and liposome-EGF proteins by placing them in one container so that they exist together in the same liquid without chemical bonding to each other; a surface-binding / coexistence type TFF part that receives the exosomes and liposome-EGF proteins coexisting through the exosome coexistence type combination forming part and forms them to flow tangentially along the surface of the filter membrane to filter out free EGF remaining after the reaction, unreacted maleimide lipids, reducing agents, and small molecule impurities; and a protective agent An apparatus for manufacturing an EGF·liposome·exosome triple lifting ampoule composition, characterized by comprising an auxiliary stabilizing part that stabilizes the EGF protein-liposome protein membrane formed by covalently surface-bonding the EGF protein to the liposome surface through trehalose and mannitol, and protects it during freezing and drying. Claim 7 In claim 1, the EGF·liposome·exosome triple lifting ampoule composition forming module comprises a combination jacket-type storage tank portion for storing an EGF·liposome·exosome combination formed through a coating / fusion type combination forming module and a surface bonding / coexistence type combination forming module, and located in the internal space of the combination jacket-type storage tank portion, by rotating an impeller at a stirring speed of 30 to 100 rpm to add to the EGF·liposome·exosome combination a moisturizer composed of glycerin and butylene glycol; a conditioning agent composed of ceramide and squalane; a film former composed of polyquaternium and polyglutamic acid; a viscosity / texture improving agent composed of hydroxyethylcellulose (HEC), carbomer, and acrylate copolymer; and phosphate buffer and NaCl and An apparatus for manufacturing an EGF, liposome, and exosome triple lifting ampoule composition, characterized by comprising: a buffer, salt, and chelating agent composed of EDTA (Ethylenediaminetetraacetic acid); a preservative composed of phenoxyethanol and ethylhexylglycerin; borage oil; galanga root extract; a low-shear stirrer for a lifting ampoule composition that adds purified water and stirs; a nitrogen exchange unit located on one side of the upper part of a jacketed storage tank for combination that supplies nitrogen gas to remove oxygen during the stirring process; and an airless filling unit that fills the completed ampoule composition into small-capacity vials, pumps, and ampoule containers using an airless method. Claim 8 A lipid dissolution step (S10) in which phospholipids (PC), cholesterol, charge-regulating lipids, and lipid-specific antioxidants are dissolved in an organic solvent to create a homogeneous lipid solution; a thin film formation step (S20) in which the solution is placed in a round-bottom flask via a rotary evaporator, evaporated by rotating it over a water bath, and then condensed into a lipid thin film under a reduced pressure atmosphere using a vacuum pump; a hydration step (S30) in which a hydration solution formed from a jacketed stirring tank is introduced into the lipid thin film via a low-shear stirrer, rotated at 20–200 rpm using an impeller formed inside the water bath, and the lipid thin film and hydration solution are stirred to form MLV liposomes; a pre-treatment step (S40) involving freezing, thawing, or low-intensity ultrasonic treatment; and MLV liposomes to a desired size (80–150 nm). A microfluidization step (S50) for reducing and homogenizing to form single-membrane liposomes (LUV); an EGF-liposome-exosome combination step (S60) for combining liposomes (LUV, 80–150 nm) and exosomes, and binding and co-occurring an EGF protein therewith to form a combination by coating and fusing the EGF-liposome-exosome, or by co-occurring and surface-binding the EGF-liposome-exosome combination; and to the EGF-liposome-exosome combination formed through the EGF-liposome-exosome combination step, a viscosity and texture improving agent composed of hydroxyethylcellulose (HEC), carbomer, and acrylate copolymer; a film former agent composed of polyquaternium and polyglutamic acid; and a component composed of ceramide and squalane. A conditioning agent; a preservative composed of phenoxyethanol and ethylhexylglycerin; and borage oil;A method for manufacturing an EGF-liposome-exosome triple lifting ampoule composition, characterized by comprising a step (S70) of forming an EGF-liposome-exosome triple lifting ampoule composition, wherein galanga root extract is added to complete the EGF-liposome-exosome triple lifting ampoule composition. Claim 9 In claim 8, the above step (S60) is performed by selecting either a coating-fusion type combination step (S61) in which an exosome (EV) membrane component is coated on the outer membrane of a liposome or the two membranes are partially fused to form liposome sustained release along with a natural membrane surface label, or a coexistence type surface binding combination step (S62) in which liposomes, exosomes, and EGF proteins exist simultaneously within one system, with some arranged in a chemically bound form and others in a physically coexisting, auxiliary form to form a combination; the above coating-fusion type combination step (S61) is composed of a coating step (S61a) in which membrane fluidity is increased by mixing at 4~25 ℃ and incubating in a short 37 ℃ water bath, and a fusion step (S61b) in which membrane rearrangement is increased by 1~2 cycles of freezing and thawing; the above coating step (S61a) is buffer fitting, A step of aligning TFFs to the same buffer osmotic pressure (≈280–320 mOsm) for both liposomes and exosomes (S61a-1); a step of placing liposome dispersion into a sterile beaker and then slowly drop-dipping exosome dispersion using a guide starting from an exosome (EV) to liposome ratio of 1:2 (number of particles) (S61a-2); a step of forming a mixed suspension by stirring with a low-shear paddle at a rotation speed of ≤100 rpm for 5–10 minutes (S61a-3); a step of incubating the mixed suspension in a 37 ℃ water bath for 5–15 minutes (S61a-4); a step of placing it in an ice bath and lowering the temperature back to 4 ℃ (S61a-5); a step of replenishing 2–3 weight% of trehalose as a protective agent (S61a-6); and by low-speed centrifugation A step of removing large aggregates at 1,000~3,000×g (relative centrifugal acceleration) for 5 minutes (S61a-7), and 300 kDa~0.The method comprises a step (S61a-8) of removing PEG·Ca2+, free EGF, and small molecules using a 1 μm coated / fused TFF portion, and then collecting a combination fraction of liposomes and exosomes; and the fusion step (S61b) comprises a buffer alignment step, wherein the TFF is aligned to the same buffer osmotic pressure (≈280~320 mOsm) for both liposomes and exosomes (S61b-1); a step (S61b-2) of placing liposome dispersion into a sterile beaker and then slowly dotting exosome dispersion using a guide starting from an exosome (EV) to liposome ratio of 1:2 (number of particles); a step (S61b-3) of forming a mixed suspension by stirring with a low-shear paddle at a rotation speed of ≤100 rpm for 5~10 minutes; and a step of freezing the mixed suspension at -80 ℃ for 10~20 minutes. A method for preparing an EGF, liposome, and exosome triple lifting ampoule composition, characterized by comprising the steps of: thawing in a water bath at 25–37°C for 2–5 minutes (S61b-4); stabilizing at 4°C immediately after thawing and adding 2–3% by weight of trehalose, a protective agent (S61b-5); removing large aggregates by low-speed centrifugation at 1,000–3,000 × g (relative centrifugal acceleration) for 5 minutes (S61b-6); and removing PEG, Ca2+, free EGF, and small molecules using a coated / fused TFF portion of 300 kDa–0.1 μm, and then collecting a combination fraction of liposomes and exosomes (S61b-7). Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 In claim 9, the above coexistence-type surface-binding combination step (S62) comprises: a step (S62a) of exposing a maleimide reactive group to the liposome outer membrane through a liposome functionalization forming part to form a covalent bond with the -SH group of the EGF protein; a step (S62b) of forming a combination by surface-binding the reduced EGF-SH protein and the liposome surface maleimide group through an EGF surface-binding combination forming part so that they are stably fixed by covalent bonding; a step (S62c) of combining the exosome and the liposome-EGF protein by placing them in one container through an exosome coexistence-type combination forming part so that they exist together in the same liquid without chemical bonding to each other; and, in the surface-binding-type TFF part, receiving the exosome and the liposome-EGF protein coexisting through the exosome coexistence-type combination forming part, and tangentially along the surface of the filtration membrane A method for preparing an EGF·liposome·exosome triple lifting ampoule composition, characterized by comprising the step (S62d) of forming a tangential flow to filter out and purify the free EGF, unreacted maleimide lipids, reducing agent, and small molecule impurities remaining after the reaction. Claim 14 In claim 8, the above step (S70) comprises a step (S71) of introducing and storing an EGF-liposome-exosome combination formed through an EGF-liposome-exosome combination step in a jacketed storage tank for combination, and rotating an impeller at a stirring speed of 30 to 100 rpm through a low-shear stirrer for a lifting ampoule composition to add to the EGF-liposome-exosome combination a moisturizer composed of glycerin and butylene glycol; a conditioning agent composed of ceramide and squalane; a film former composed of polyquaternium and polyglutamic acid; a viscosity and texture improving agent composed of hydroxyethylcellulose (HEC), carbomer, and acrylate copolymer; and a phosphate buffer composed of NaCl and EDTA (ethylenediaminetetraacetic acid). A method for manufacturing an EGF, liposome, and exosome triple lifting ampoule composition, characterized by comprising the steps of: adding a buffer, salt, and chelating agent; a preservative composed of phenoxyethanol and ethylhexylglycerin; borage oil; galanga root extract; and purified water and stirring (S72); supplying nitrogen gas during the stirring process through a nitrogen exchange unit to remove oxygen (S73); and filling the completed ampoule composition into a small-capacity vial, pump, or ampoule container using an airless filling unit (S74). Claim 15 Through the method for preparing an EGF, liposome, and exosome triple lifting ampoule composition according to claim 8, 2.0~4.0 wt% of liposome lipid system, 1.0~3.0 wt% of exosome dispersion, 0.0005~0.005 wt% of EGF (recombinant), 5~12 wt% of moisturizer, 1~8 wt% of protective agent, 0.3~1.0 wt% of conditioning agent, 0.1~0.3 wt% of film former, 0.2~0.8 wt% of viscosity / texture improver, 0.2~0.6 wt% of buffer / salt / chelating agent, 0.7~1.0 wt% of preservative, 0.2~1.0 wt% of borage oil, 0.1~0.5 wt% of galanga root extract, and purified water An EGF, liposome, and exosome triple lifting ampoule composition characterized by being composed of 70 to 88 weight%. Claim 16 An EGF-liposome-exosome triple lifting ampoule composition according to claim 15, characterized in that the compositional content of the liposomal lipid system comprises, based on the total weight% of the liposomal lipid system, 1.0 to 3.0 weight% of hydrogenated phospholipid (PC), 0.3 to 0.8 weight% of cholesterol, 0 to 0.3 weight% of charge-regulating lipid having cationic lipids, and 0.01 to 0.05 weight% of a lipid-enhancing antioxidant composed of tocopherol. Claim 17 An EGF·Liposome·Exosome Triple Lifting Ampoule Composition according to claim 15, characterized in that the manufacturing content of the protective agent consists of 1 to 3 weight% of trehalose and 0 to 1 weight% of mannitol relative to the total weight% of the protective agent in the case of a liquid-stable type, and consists of 2 to 5 weight% of trehalose and 1 to 3 weight% of mannitol relative to the total weight% of the protective agent in the case of a freeze-dried type.

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