Microfluidic chip for producing lipid nanoparticles, method for producing lipid nanoparticles using same, and use of lipid nanoparticles produced thereby

Abstract

The present invention relates to: a microfluidic chip for producing lipid nanoparticles such as bicelles or liposomes; and a method for producing lipid nanoparticles using same. The microfluidic chip according to one aspect is characterized by including a rhombic lattice structure and is thus able to maximize distribution mixing between lipids and active ingredients used for bicelle production and thereby increase the loading efficiency of the active ingredients, and produce highly stable and uniform lipid nanoparticles having a high skin penetration rate.

Description

Microfluidic chip for manufacturing lipid nanoparticles, method for manufacturing lipid nanoparticles using the same, and use of lipid nanoparticles manufactured thereby

[0001] The present invention relates to a microfluidic chip for manufacturing lipid nanoparticles such as bicelles or liposomes, and a method for manufacturing lipid nanoparticles using the same.

[0002] In recent years, the importance of basic research in dermatology, the development of new materials, and formulation technologies that enhance the delivery, absorption, and durability of active ingredients within the skin has been increasingly emphasized in the cosmetics industry. Consequently, nanoparticle-based formulations are emerging to enhance skin friendliness and absorption. Research is steadily underway on lipid nanoparticles, such as liposomes, transfersomes, and ethosomes, and the development of delivery systems utilizing these materials. This, in turn, is fueling the need for new delivery systems.

[0003] Meanwhile, bicelles are novel, flat, disc-shaped delivery vehicles in which short-chain phospholipids surround long-chain phospholipids. They are known to have excellent penetration capabilities between the stratum corneum due to their coin-like shape with a diameter of approximately 10 to 50 nm. Despite their bilayer structure, bicelles are characterized by not containing water inside, unlike liposomes. In addition, bicelles can be used as delivery vehicles for lipid-soluble or oil-soluble active ingredients, as they can be loaded with hydrophobic active substances in the hydrophobic domains within the lipid layer. In addition, their synthesis through microtubules is more uniformly and densely structured.

[0004] Against this backdrop, the present inventors sought to develop a microfluidic chip for manufacturing bicelles or liposomes as lipid nanoparticle delivery vehicles, and a method for manufacturing lipid nanoparticles using the same. According to one aspect, the microfluidic chip comprises a rhombic lattice structure, which maximizes the distribution and mixing between lipids and active ingredients used in bicelles manufacturing, thereby enhancing the loading efficiency of active ingredients and producing lipid nanoparticles with high stability, uniformity, and rapid skin penetration.

[0005] One aspect provides a microfluidic chip for manufacturing lipid nanoparticles, comprising: a first injection portion into which a first sample containing an active substance and a lipid dissolved therein is injected; a second injection portion into which a second sample containing a buffer solution containing an active substance dissolved therein is injected; a focusing portion connected to the first injection portion and focusing the first sample; a mixing portion connected to the focusing portion and mixing the first sample and the second sample; and an outlet connected to the mixing portion.

[0006] Another aspect provides a method for manufacturing lipid nanoparticles using the above microfluidic chip.

[0007] Another aspect provides lipid nanoparticles manufactured by the above manufacturing method.

[0008] Another aspect provides a cosmetic composition for improving skin, comprising lipid nanoparticles manufactured by the above manufacturing method.

[0009] One aspect provides a microfluidic chip for manufacturing lipid nanoparticles, which includes a first injection portion (11) into which a first sample in which an active substance and lipid are dissolved is injected; a second injection portion (12) into which a second sample containing a buffer solution in which the active substance is dissolved is injected; a focusing portion (13) connected to the first injection portion and focusing the first sample; a mixing portion (14) connected to the focusing portion and mixing the first sample and the second sample; and an outlet (18) connected to the mixing portion. The microfluidic chip according to one aspect can maximize the distribution mixing between lipids and active ingredients used in manufacturing bicelles by including a lattice structure, thereby increasing the loading efficiency of active ingredients, and can produce lipid nanoparticles having high stability and uniformity and a fast skin penetration rate.

[0010] The above mixing unit may be characterized by having a backflow prevention shield (16) and a grid structure (17) formed therein. Specifically, the shield can prevent backflow of the injected sample.

[0011] The above grid structure may maximize the distributive mixing effect by repeatedly dividing and combining the flow of the injected first sample and second sample, and may be located behind the shield and in front of the outlet.

[0012] The pattern or shape of the above lattice structure is not particularly limited as long as it is a shape that can facilitate the process of repeatedly dividing and combining the flow of the sample, but specifically, it may be characterized by a rhombic lattice structure. In addition, the above lattice structure may be a three-dimensional shape (intaglio or relief), and in the case of a rhombic lattice structure, the length of one side of each rhombus may be 50 to 1000 μm, and the pitch may be 100 to 2000 μm.

[0013] The above mixing unit may additionally include a reflective membrane. The reflective membrane may be positioned in front of the grid structure so that the first sample injected through the first injection unit may bounce instead of flowing directly into the grid structure.

[0014] Lipid nanoparticles, depending on the aspect, may be bicelles or liposomes. As used herein, the term "bicelle" refers to a flat, disc-shaped carrier in which short-chain lipids (fatty acids or phospholipids) surround long-chain lipids, and may have a coin-like shape with a diameter of approximately 10 to 50 nm. Due to this, bicelles have excellent penetration capabilities between the stratum corneum, and unlike liposomes, they do not contain water inside, and hydrophobic active substances can be loaded into the hydrophobic domains within the lipid layer, enabling the loading and delivery of fat-soluble or oil-soluble active ingredients.

[0015] The above lipids contain 4 to 12 carbon atoms (C4~C 12 ) with a short carbon chain and 13 to 24 carbon atoms (C 13 ~C 24 ) may be a fatty acid, a phospholipid, or a mixture thereof containing a long carbon chain. Specifically, the short carbon chain lipid may be capric acid, and the long carbon chain lipid may be phosphatidylcholine, but is not particularly limited thereto as long as the lipid nanoparticle can form a bicellar or liposome shape.

[0016]

[0017] Another aspect provides a method for manufacturing lipid nanoparticles using the microfluidic chip. The same aspects described above also apply to the method for manufacturing lipid nanoparticles.

[0018] The above microfluidic chip may include a first injection portion into which a first sample containing an active substance and a lipid dissolved therein is injected; a second injection portion into which a second sample containing a buffer solution containing the active substance dissolved therein is injected; a focusing portion connected to the first injection portion and focusing the first sample; a mixing portion connected to the focusing portion and mixing the first sample and the second sample; and an outlet connected to the mixing portion, wherein the mixing portion is characterized in that a backflow prevention screen and a grid structure are formed therein.

[0019] The term “active substance” as used herein may mean a substance that can impart a useful activity to an object, such as skin regeneration, skin anti-aging, skin wrinkle improvement, skin whitening, skin moisturizing, or skin antioxidant effect, and may be, for example, titrated extract of Centella Asiatica (TECA), retinol, retinal, idebenone, tocopherol, melatonin, bakuchiol, alpha bisabolol, or oil-soluble licorice extract, but is not particularly limited thereto. An active substance according to an aspect may be appropriately selected according to the purpose by a person skilled in the art.

[0020] Specifically, the fatty acids or phospholipids including the short carbon chain lipid and the long carbon chain lipid may be mixed at a concentration ratio of 2:1 to 8:1. In one embodiment, a first sample was prepared by dissolving a solution in which the active substance (TECA, 1%) and capric acid as a short carbon chain lipid and phosphatidylcholine as a long carbon chain lipid were mixed at a ratio of 4:1 in a solvent.

[0021] When injecting the first sample and the second sample into the first injection port and the second injection port, respectively, the first sample and the second sample may be injected at a concentration ratio of 1:2 to 1:20. Specifically, in one embodiment, a buffer solution (pure deionized water) was used as the second sample, and the first sample and the second sample were injected into the first injection port and the second injection port, respectively, at a concentration ratio of 1:10 (30 ml / min: 300 ml / min). Thereafter, the first sample and the second sample were distributed and mixed, and the manufactured bicell nanoparticles were obtained through the outlet at about 330 ml / min.

[0022]

[0023] Another aspect provides lipid nanoparticles manufactured by the above manufacturing method. The same aspects described above also apply to the lipid nanoparticles.

[0024] The lipid nanoparticles according to the aspect may be bicell nanoparticles, and in one embodiment, the bicell nanoparticles manufactured were measured to have a diameter of about 24 nm and a PDI of 0.08 (Fig. 2), and it was confirmed that they exhibited a clear and transparent appearance.

[0025]

[0026] Another aspect provides a cosmetic composition for skin improvement, comprising lipid nanoparticles manufactured by the above-described manufacturing method. The same aspects described above also apply to the cosmetic composition.

[0027] Lipid nanoparticles according to one aspect and a cosmetic composition for skin improvement comprising the same may include the above active substance.

[0028] The term “skin improvement” in this specification may be any one or more selected from the group consisting of suppressing skin inflammation, improving skin troubles, regenerating skin, anti-oxidation, anti-aging, improving skin texture, increasing skin elasticity, and whitening, but is not particularly limited thereto.

[0029] Lipid nanoparticles according to one aspect and a cosmetic composition for skin improvement comprising the same may be characterized by excellent skin penetration ability and active substance delivery ability. Specifically, the lipid nanoparticles according to one aspect have excellent ability to penetrate the stratum corneum of the skin and can load hydrophobic active substances into hydrophobic domains within the lipid layer, and thus, as a loading and delivery vehicle for fat-soluble or oil-soluble active ingredients, they exhibit a skin penetration effect at a significantly improved speed compared to general liposomes, and thus, the skin improvement effect of the cosmetic composition comprising the lipid nanoparticles according to one aspect can be enhanced.

[0030] In one embodiment, it was confirmed that the Bicelle TECA treatment group containing lipid nanoparticles according to one aspect had an effect of alleviating epidermal inflammation by significantly reducing the expression level of the TSLP gene compared to the TECA treatment group for cells in which an inflammatory response was induced (Fig. 4). In addition, the Bicelle TECA treatment group according to one aspect had a superior wound healing effect compared to the TECA treatment group (Fig. 5), and as a result of confirming the degree of cell migration into the gap through a microscope, it was confirmed that the effect of reducing the gap area was superior to that of the TECA treatment group (Fig. 6).

[0031] The formulation of the above cosmetic composition may be, but is not particularly limited to, a solution, an external ointment, an emulsion, a cream, a foam, a nourishing toner, an emollient toner, a perfume, a pack, an emollient, a milky lotion, a makeup base, an essence, a soap, a liquid cleanser, a bath additive, a sunscreen cream, a sun oil, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing cleansing, an oil, a powder foundation, an emulsion foundation, a wax foundation, a patch, or a spray. Specifically, the formulation of the above cosmetic composition may be any one selected from the group consisting of an emulsion, a cream, a lotion, a solution, a suspension, a gel, a patch, a mask, a pack, a paste, a spray, a foam, and cleansing water.

[0032] The above cosmetic composition may further include functional additives and components included in general cosmetic compositions, and may further include conventional auxiliary ingredients and carriers such as commonly used antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances, etc. For example, the above cosmetic composition may further include auxiliary ingredients such as glycerin, butylene glycol, polyoxyethylene hydrogenated castor oil, tocopheryl acetate, citric acid, squalane, sodium citrate, allantoin, etc., and the solvent may include hexanediol, purified water, etc.

[0033] In addition, the functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular peptides, high molecular polysaccharides, sphingolipids, and seaweed extracts. In addition, the compounding components included may include, but are not particularly limited to, fat components, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, etc.

[0034] The microfluidic chip according to the aspect is characterized by being able to increase the loading efficiency of the active ingredient by maximizing the distribution mixing between the lipid and the active ingredient used in the manufacture of the bicelles, including a diamond-shaped lattice structure, and being able to produce lipid nanoparticles with high stability and uniformity and a fast skin penetration rate.

[0035] Figure 1 illustrates the structure of a microfluidic chip for manufacturing a bicelle according to one aspect.

[0036] Figure 2 shows the results of analyzing the shape and size of bicellar particles manufactured according to various aspects.

[0037] Figure 3 shows the results of analyzing the skin permeability of bicell nanoparticles manufactured according to one aspect.

[0038] Figure 4 shows the results of confirming the transparency of TECA 1% bicelles manufactured according to the daily aspect.

[0039] Figure 5 shows the results of confirming the anti-inflammatory factor (IL-1β) inhibitory effect of TECA 1% Vicelle manufactured according to the daily aspect.

[0040] Figures 6 and 7 show the results of confirming the cell regeneration promotion effect of TECA 1% bicelle manufactured according to the daily aspect.

[0041]

[0042] [Explanation of symbols]

[0043] 10: Microfluidic chip

[0044] 11: First injection unit

[0045] 12: Second injection unit

[0046] 13: Focus

[0047] 14: Mixed section

[0048] 15: Semi-desert

[0049] 16: Backflow prevention screen

[0050] 17: Lattice structure

[0051] 18: Exhaust

[0052]

[0053] The present invention will be described in more detail below with reference to the attached drawings and examples. However, these drawings and examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0054] Additionally, terms such as “comprises” or “includes” used in the present embodiments should not be construed to necessarily include all of the components or steps described in the specification, and should be construed to mean that some of the components or steps may not be included, or that additional components or steps may be included.

[0055]

[0056] Example 1: Fabrication of a microfluidic chip

[0057] A microfluidic chip was manufactured as shown in Fig. 1.

[0058] Specifically, a microfluidic chip (10) according to one aspect includes a first injection portion (11) into which a first sample containing an active substance and a lipid dissolved therein is injected; a second injection portion (12) into which a second sample containing a buffer solution containing an active substance dissolved therein is injected; a focusing portion (13) connected to the first injection portion and focusing the first sample; a mixing portion (14) connected to the focusing portion and mixing the first sample and the second sample; and an outlet (18) connected to the mixing portion. More specifically, the mixing portion is characterized in that a reflective film (15); a backflow prevention shield (16) and a diamond-shaped lattice structure (17) are formed. The reflective film prevents the injected sample from flowing directly into the diamond-shaped lattice structure, the backflow prevention shield prevents backflow of the injected sample, and the diamond-shaped lattice structure can increase the distributive mixing effect of the first sample and the second sample.

[0059]

[0060] Example 2: Fabrication and particle size analysis of bicellar and liposome nanoparticles

[0061] Bicell nanoparticles were produced using the microfluidic chip manufactured in Example 1 above, and their characteristics were analyzed.

[0062] Specifically, a solution containing capric acid as a short carbon chain lipid and phosphatidylcholine as a long carbon chain lipid in a 4:1 ratio was dissolved in a solvent to prepare the first sample, and a buffer solution (pure deionized water) was used as the second sample. Thereafter, the first sample and the second sample were injected into the first inlet and the second inlet, respectively, at a concentration ratio of 1:10 (30 ml / min: 300 ml / min), and the first and second samples were distributedly mixed to obtain the prepared bicellar nanoparticles at approximately 330 ml / min through the outlet.

[0063] As a result, the manufactured bisel nanoparticles were measured to have a diameter of approximately 24 nm and a PDI of 0.08 (Fig. 2).

[0064]

[0065] Example 3: Confirmation of skin penetration efficacy of Bicell nanoparticles

[0066] The extent of penetration into human skin tissue was examined over a 6-hour period after treatment of liposomes produced using a high-pressure homogenizer and bi-cell nanoparticles loaded with the fluorescent substance Rhodamine B. Samples were collected after 0.5, 1, 3, and 6 hours, and the amount of penetration was examined using a Multimode Microplate Reader. The human skin tissue was recovered and fixed, and tissue slides were prepared and observed under a fluorescence microscope.

[0067] As a result, it was confirmed that the biocell nanoparticles manufactured according to the daily pattern had a significantly higher rate and amount of penetration into the skin tissue after the same period of time compared to the control group (general liposomes) (Fig. 3).

[0068]

[0069] Experimental Example 1: Confirmation of the properties of bicell nanoparticles

[0070] When manufacturing bicell nanoparticles loaded with an insoluble active substance (TECA, 1%), it was confirmed that they exhibited a clear and transparent appearance, unlike TECA dissolved in water (Fig. 4).

[0071]

[0072] Experimental Example 2: Confirmation of the anti-inflammatory effect of Vicelle nanoparticles.

[0073] HaCaT cells, which are stratum corneum cells, were treated (+) with 10 μg / ml of Poly I:C, which induces inflammation, and 10 μg / ml of IL-4 protein, which induces inflammation. An experimental group was prepared in which dexamethasone (DEXA) 1 μM (Dexa), TECA, and Bicelle TECA, which are control inflammation-reducing substances, were treated at 10 ppm each for 4 hours while simultaneously inducing inflammation. An untreated group (-) was prepared as a negative control group.

[0074] Afterwards, the expression level of the TSLP gene, an inflammation-related factor expressed in skin keratinocytes, was measured through qRT-PCR analysis and compared by converting it to the level of actin expression. As a result, it was confirmed that the Bicelle TECA-treated group had the effect of alleviating epidermal inflammation by significantly reducing the expression level of the TSLP gene compared to the TECA-treated group in cells where an inflammatory response was induced (Fig. 5).

[0075]

[0076] Experimental Example 3: Confirmation of the Cell Regeneration Promotion Effect of Bicell Nanoparticles

[0077] After artificially creating a void in HDF cells, which are dermal fibroblasts, we prepared an experimental group treated with TECA and Bicelle TECA at 10 ppm each for 24 hours to examine the recovery effect after tissue damage. An untreated group (-) was prepared as a negative control group.

[0078] The degree of damage recovery was measured using WoundMaker at 2-hour intervals from immediately after the material treatment (0 hours) up to 14 hours. As a result, the Bicelle TECA-treated group showed 52.4% recovery within 12 hours, while the TECA-treated and untreated groups showed 43.6% and 39.6% recovery, respectively, confirming the excellent wound healing effect of Bicelle TECA (Fig. 6). In addition, to compare the recovery effect with the naked eye, the degree of cell migration into the void was confirmed under a microscope 24 hours after the material treatment. As a result, the Bicelle TECA-treated group was confirmed to have a greater effect on reducing the void area than the TECA-treated group (Fig. 7).

[0079]

[0080] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A microfluidic chip for manufacturing lipid nanoparticles, comprising: a first injection unit into which a first sample containing an active substance and a lipid dissolved therein is injected; a second injection unit into which a second sample containing a buffer solution containing an active substance dissolved therein is injected; a focusing unit connected to the first injection unit and focusing the first sample; a mixing unit connected to the focusing unit and mixing the first sample and the second sample; and an outlet connected to the mixing unit. A microfluidic chip characterized in that the above mixing section is formed with a backflow prevention screen and a grid structure.

2. A microfluidic chip according to claim 1, wherein the mixing portion further includes a reflective film.

3. A microfluidic chip according to claim 1, characterized in that the lattice structure is a rhombic lattice structure.

4. A microfluidic chip in the third paragraph, wherein the diamond-shaped lattice structure increases the distributive mixing effect of the first sample and the second sample.

5. A microfluidic chip according to claim 1, wherein the lipid nanoparticle is a bicelle or liposome.

6. In a method for manufacturing lipid nanoparticles using a microfluidic chip, The microfluidic chip comprises a first injection portion into which a first sample containing an active substance and a lipid dissolved therein is injected; a second injection portion into which a second sample containing a buffer solution containing the active substance dissolved therein is injected; a focusing portion connected to the first injection portion and focusing the first sample; a mixing portion connected to the focusing portion and mixing the first sample and the second sample; and an outlet connected to the mixing portion. A method for producing lipid nanoparticles, characterized in that the above mixing unit is formed with a reflux prevention screen and a lattice structure.

7. In paragraph 6, the lipid has 4 to 12 carbon atoms (C4~C 12 ) short carbon chain lipids and 13 to 24 carbon atoms (C 13 ~C 24 ) A method for producing lipid nanoparticles, which are fatty acids or phospholipids containing long carbon chain lipids.

8. A method for producing lipid nanoparticles in claim 7, wherein the short carbon chain lipid is capric acid.

9. A method for producing lipid nanoparticles in claim 7, wherein the long carbon chain lipid is phosphatidylcholine.

10. A method for producing lipid nanoparticles, wherein in paragraph 7, the fatty acids or phospholipids including the short carbon chain lipid and the long carbon chain lipid are mixed at a concentration ratio of 2:1 to 8:

1.

11. A method for producing lipid nanoparticles, wherein the first sample and the second sample are injected at a concentration ratio of 1:2 to 1:20 in the 7th paragraph.

12. A lipid nanoparticle manufactured by the manufacturing method of any one of claims 6 to 11.

13. A lipid nanoparticle according to claim 12, characterized in that the lipid nanoparticle has excellent skin penetration ability and active substance delivery ability.

14. A cosmetic composition for improving skin, comprising lipid nanoparticles manufactured by the manufacturing method of any one of claims 6 to 11.

15. A cosmetic composition according to claim 14, wherein the skin improvement is at least one selected from the group consisting of suppression of skin inflammation, improvement of skin trouble, skin regeneration, anti-oxidation, anti-aging, improvement of skin texture, increase in skin elasticity, and whitening.

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