Hyaluronic acid nanofabric sheet and method for manufacturing the same

Abstract

The present invention relates to a hyaluronate nanofabric sheet and a method for producing the same, and more particularly to a hyaluronate nanofabric sheet having excellent percutaneous absorption rate and physical properties and a method for producing the same. The method for producing the hyaluronate nanofabric sheet of the present invention comprises: (a) a hyaluronate nanofabric sheet having a weight average molecular weight of 5×10 3 ~5×10 4 Da hyaluronate and weight-average molecular weight 1×10 5 ~1×10 6 The method includes the steps of (a) mixing hyaluronic acid salt of 1000 Da with hyaluronic acid salt of 1000 Da and dissolving it in water to prepare an aqueous solution of hyaluronic acid salt, and (b) electrospinning the aqueous solution of hyaluronic acid salt. The hyaluronic acid salt nanofabric sheet of the present invention is mainly composed of ultra-low molecular weight hyaluronic acid, and has excellent percutaneous absorption rate. In addition, since a certain amount of medium molecular weight hyaluronic acid is mixed, it also has excellent mechanical properties, and therefore can be applied to various applications such as a mask patch for skin topical preparations and a carrier for ophthalmic or mucosal drug delivery systems.

Description

[Technical Field]

[0001] This invention relates to a hyaluronic acid nanofabric sheet and a method for producing the same, and more particularly to a hyaluronic acid nanofabric sheet with excellent transdermal absorption rate and physical properties, and a method for producing the same. [Background technology]

[0002] Hyaluronic acid or hyaluronic acid salts are uniformly distributed in the connective tissue, epithelium, and nerve tissue of the human body. As a biocompatible material with diverse physiological activities, it can contain large amounts of water and possesses excellent viscoelasticity. As its effects on skin regeneration, moisturizing, elasticity maintenance, and wrinkle improvement have been proven, demand for anti-aging cosmetics, foods, pharmaceuticals, and medical device fillers containing it has recently surged.

[0003] Most commercially available hyaluronic acid-based products are in liquid form and contain hyaluronic acid at a dilute concentration. In other words, the main component is water, not hyaluronic acid. This limits their applications, and due to the low safety of hyaluronic acid itself against microorganisms, they must be manufactured in sterile facilities when used in pharmaceuticals or medical devices, and preservatives that raise safety concerns must be used when used in cosmetics.

[0004] Therefore, research is being conducted to solidify hyaluronic acid or hyaluronic acid salts not in liquid form, but as films, fibers, and other materials.

[0005] Korean Published Patent No. 2019-0138907 discloses a mask sheet containing a hyaluronic acid film, and Korean Published Patent No. 2019-0106091 discloses a method for producing a hyaluronic acid film using solvent casting or automatic coating machine casting.

[0006] However, hyaluronic acid films have a somewhat complex manufacturing process during mass production, and when used in products such as face masks, they have problems such as not adhering well to the skin or having a low transdermal absorption rate.

[0007] To improve the manufacturing process of such hyaluronic acid films, Korean Patent No. 2338355 discloses a method for manufacturing a wet hyaluronic acid nonwoven fabric that can be used as a cosmetic patch, a moist wound dressing, or a tissue adhesion prevention membrane, and Korean Patent No. 1224882 discloses a nanofiber sheet made of hyaluronic acid. However, Korean Patent No. 2338355 uses hyaluronic acid with a molecular weight of 50,000 to 100,000 Da, and Korean Patent No. 1224882 uses hyaluronic acid with a molecular weight of 100,000 to 1,600,000 Datons (Da), resulting in the problem of low transdermal absorption rates.

[0008] Hyaluronic acid or hyaluronic acid salts are classified into very low molecular weight, low molecular weight, medium molecular weight, and high molecular weight based on their molecular weight.

[0009] Medium and high molecular weight hyaluronic acid salts have excellent mechanical properties, making them easy to manufacture into films, but they have difficulty penetrating and absorbing through the skin. Conversely, the lower the molecular weight of hyaluronic acid or hyaluronic acid salt, the easier it is for the skin to penetrate and absorb, but it has the problem of having poor mechanical properties, making it difficult to manufacture into films.

[0010] Furthermore, hyaluronic acid salts are high-molecular-weight polysaccharides with many hydroxyl groups in their molecules, and because the molecules themselves carry a positive charge, there are many limitations to electrospinning. Therefore, in the conventional electrospinning method for producing hyaluronic acid nonwoven fabrics (Castro, KC, Campos, MGN, & Mei, LHI (2021). International Journal of Biological Macromolecules, 173, 251-266.), alkaline solvents such as sodium hydroxide or potassium hydroxide, or toxic organic solvents (Ewelina, et al. Advances in Polymer Technology 37.6 (2018):1929-1940.) were used to produce hyaluronic acid nonwoven fabrics. However, the use of toxic organic solvents or salts is unavoidable, and there is a risk that these organic solvents or salts may remain after the formation of the nonwoven fabric, thus limiting its practical use.

[0011] Therefore, the inventors have strived to produce a hyaluronic acid sheet that is easily absorbed and penetrates the skin without the addition of organic solvents or salts, and has excellent mechanical properties for commercialization. As a result, they have produced a hyaluronic acid sheet with a density of 5,000 to 5 × 10 that is easily absorbed and penetrates the skin. 4 The main component is ultra-low molecular weight hyaluronic acid of Da, and it has excellent mechanical properties that allow for processability for product development, resulting in a 1 x 10 5 ~1 × 10 6 We confirmed that by adding a medium-molecular-weight hyaluronic acid salt (Da) and dissolving it in water, and then using electrospinning, we could produce a hyaluronic acid nanofabric sheet with excellent transdermal absorption and mechanical properties, thus completing the present invention. [Overview of the project] [Problems that the invention aims to solve]

[0012] The object of the present invention is to provide a hyaluronic acid nanofabric sheet with excellent transdermal absorption rate and mechanical properties.

[0013] Another object of the present invention is to provide a method for producing a hyaluronate nanofabric sheet excellent in percutaneous absorption rate and tensile strength.

Means for Solving the Problems

[0014] In order to achieve the above object, the present invention provides a hyaluronate nanofabric sheet made of hyaluronate fibers having a diameter of 0.3 to 5 μm, produced by electrospinning using a solvent of pure water alone.

[0015] In order to achieve the above object, the present invention provides a hyaluronate nanofabric sheet having a percutaneous absorption rate of 77.70 to 86.08% and a tensile strength of 5.32 to 10.91 MPa.

[0016] In the present invention, the hyaluronate is composed of 88.9 to 98.1% by weight of hyaluronate having a weight average molecular weight of 5 × 10 3 ~5 × 10 4 Da and 1.9 to 11.1% by weight of hyaluronate having a weight average molecular weight of 1 × 10 5 ~1 × 10 6 Da.

[0017] =]] In the present invention, the hyaluronate nanofabric sheet is characterized in that it is for use as a mask patch for external skin preparations, a carrier for ophthalmic or mucosal drug delivery systems.

[0018] Further, the present invention provides a method for producing a hyaluronate nanofabric sheet, comprising: (a) mixing hyaluronate having a weight average molecular weight of 5 × 10 3 ~5 × 10 4 Da and hyaluronate having a weight average molecular weight of 1 × 10 5 ~1 × 10 6 Da, dissolving them in water to produce an aqueous hyaluronate solution; and (b) electrospinning the aqueous hyaluronate solution.

[0019] In the present invention, the viscosity of the hyaluronic acid aqueous solution is characterized by being 1,000 to 15,000 cPs. [Effects of the Invention]

[0020] The hyaluronic acid nanofabric sheet of the present invention is primarily composed of ultra-low molecular weight hyaluronic acid, and not only does it have excellent transdermal absorption rates, but it also has excellent mechanical properties because it is mixed with a certain amount of medium molecular weight hyaluronic acid. [Brief explanation of the drawing]

[0021] [Figure 1] These are photographs of hyaluronic acid nanofabric sheets produced by electrospinning according to one embodiment of the present invention. (A: Sheet in nanofiber form, B: Sheet in dot form, C: Microscopic image of the nanofiber form sheet, D: Microscopic image of the dot form sheet) [Figure 2] This is a transmission image of a hyaluronic acid nanofabric sheet produced according to one embodiment of the present invention, taken using a fluorescence microscope. [Modes for carrying out the invention]

[0022] In the present invention, "hyaluronic acid nanofabric sheet" means a sheet containing nanohyaluronic acid fibers manufactured by electrospinning.

[0023] Hyaluronic acid or hyaluronic acid salts are more easily absorbed and penetrated by the skin the lower their molecular weight, but this presents a problem in that it is difficult to manufacture sheets with high physical properties by electrospinning.

[0024] Korean Registered Patent No. 2338355 uses hyaluronic acid with a molecular weight of 50,000 to 100,000 Da to manufacture hyaluronic acid nonwoven fabric, and Korean Registered Patent No. 1224882 uses hyaluronic acid with a molecular weight of 100,000 to 1,600,000 Da to manufacture hyaluronic acid nanofiber sheets. Because these use high molecular weight hyaluronic acid, alkaline solvents such as sodium hydroxide and potassium hydroxide are used to reduce viscosity, which has the problem of low transdermal absorption and the inability to properly transmit the various physiological activities of hyaluronic acid to the skin.

[0025] In this invention, 5 × 10 3 ~5×10 4 1 x 10 5 ~1 × 10 6 We confirmed that by adding medium-molecular-weight hyaluronic acid (Da), it is possible to uniformly mass-produce hyaluronic acid nanofabric sheets that not only have excellent transdermal absorption rates but also possess mechanical properties that allow for processing.

[0026] In this invention, ultra-low molecular weight hyaluronic acid and medium molecular weight hyaluronic acid were mixed, and then dissolved in purified water only without adding an alkaline solvent to produce an aqueous hyaluronic acid solution with a viscosity of 1,000 to 15,000 cPs (25°C). This solution was then electrospinned to produce a hyaluronic acid nanofabric sheet.

[0027] As a result, we were able to manufacture hyaluronic acid nanofabric sheets in large quantities, and we were able to confirm that the transdermal absorption rate of the manufactured hyaluronic acid nanofabric sheets was 77.70-86.08%.

[0028] Accordingly, the present invention relates, in one aspect, to a hyaluronic acid nanofabric sheet comprising hyaluronic acid fibers with a diameter of 0.3 to 5 μm, manufactured by electrospinning using purified water as the sole solvent.

[0029] Furthermore, from another perspective, the present invention relates to a hyaluronic acid nanofabric sheet having a transdermal absorption rate of 77.70 to 86.08% and a tensile strength of 5.32 to 10.91 MPa.

[0030] The hyaluronic acid that constitutes the hyaluronic acid nanofabric sheet has a weight-average molecular weight of 5 × 10 3 ~5×10 4 88.9-98.1% by weight of hyaluronic acid of Da, and a weight-average molecular weight of 1 × 10⁻⁶ 5 ~1 × 10 6 It is characterized by being composed of 1.9 to 11.1% by weight of Da hyaluronic acid.

[0031] In other words, the hyaluronic acid nanofabric sheet of the present invention is characterized by using a hyaluronic acid with a medium molecular weight together with hyaluronic acid, since hyaluronic acid with an ultra-low molecular weight alone cannot form a sheet with high physical properties by electrospinning, in order to form the framework of the sheet.

[0032] In this invention, the molecular weight of the hyaluronic acid, which corresponds to an ultra-low molecular weight, is 5 × 10⁻⁶. 3 ~5×10 4 The molecular weight of hyaluronic acid, which is Da and corresponds to a medium molecule, is 1 × 10⁻⁶ 5 ~1 × 10 6 It is Da.

[0033] Molecular weight 5×10 3 ~5×10 4 If the hyaluronic acid content of Da is less than 88.9% by weight, the sheet may not be manufactured or the transdermal absorption rate may decrease. If it exceeds 98.1% by weight, the sheet may be formed, but physical properties such as tensile strength may decrease.

[0034] In the present invention, hyaluronic acid salts are those to which a salt is bound, and examples include sodium hyaluronate, calcium hyaluronate, potassium hyaluronate, etc., but are not limited to these.

[0035] From another perspective, the present invention relates to (a) a weight-average molecular weight of 5 × 10 3 ~5×10 4 Da hyaluronic acid and weight-average molecular weight 1 × 10 5 ~1 × 10 6 The present invention relates to a method for producing a hyaluronic acid nanofabric sheet, comprising the steps of (b) mixing hyaluronic acid with Da and dissolving it in water to produce an aqueous hyaluronic acid solution, and (b) electrospinning the aqueous hyaluronic acid solution.

[0036] In the present invention, the weight-average molecular weight is 5 × 10 3 ~5×10 4 Da hyaluronic acid and weight-average molecular weight 1 × 10 5 ~1 × 10 6 The weight ratio of Da to hyaluronic acid is preferably 88.9-98.1% by weight:1.9-11.1% by weight.

[0037] In the present invention, the viscosity of the hyaluronic acid solution, which is a mixture of ultra-low molecular weight and medium molecular weight hyaluronic acid, is preferably 1,000 to 15,000 cPs (25℃) (Brookfield DV2RTVJO, Spindle No. 5, rpm 12). The viscosity of the solution varies depending on the HA molecular weight (Da), HA content, and ratio with the solvent, but if the viscosity is less than 1,000 cPs, sheet formation is difficult, and if it exceeds 15,000 cPs, it is highly viscous, and there is a risk that the nozzle will clog and electrospinning will not proceed smoothly.

[0038] In the present invention, the electrospinning is preferably carried out under conditions of a voltage (Tension) of 30 to 35 (kV), a flow rate of 10 to 20 (mL / h), and a distance of 10 to 15 (cm).

[0039] In the present invention, the hyaluronic acid nanofabric sheet produced by electrospinning may be composed of hyaluronic acid fibers with a diameter of 0.3 μm to 5 μm.

[0040] In the present invention, the solvent is for dissolving the hyaluronic acid salt, and it is preferable to use purified water.

[0041] Furthermore, in the production of hyaluronic acid nanofabric sheets by electrospinning used in the present invention, hyaluronic acid alone can be used for production. However, depending on the field of application, water-soluble carriers or excipients with low molecular weights, commonly used in this industry, may be further included, and the types and content ranges of such excipients are not particularly limited. [Examples]

[0042] The present invention will be described in more detail below with reference to examples. It will be obvious to those skilled in the art that these examples are merely illustrative and that the scope of the present invention is not limited by these examples.

[0043] <Example 1: Production of hyaluronic acid nanofabric sheet by electrospinning> Using an electrospinning apparatus (NE300, Innovenso Co., Turkey), the voltage (Tension), flow rate (Flow Rate), and distance (Distance) were set as shown in Table 1 below, and then spinning was performed by changing the conditions for hyaluronic acid molecular weight and aqueous solution concentration.

[0044] [Table 1]

[0045] *Viscosity values ​​were measured using a viscometer (DV2TRV TJO, Brookfield Engineering Labs Inc., USA) at 12 rpm, 30 sec, and 25°C. From Table 1, it was confirmed that sheets with molecular weights from 5 kDa to 50 kDa can be manufactured by electrospinning, and it was found that depending on the concentration or molecular weight of hyaluronic acid, sheets ranging from high-performance nanofiber sheets to sheets containing some dots and having weaker properties can be manufactured (Figure 1).

[0046] <Example 2: Production of hyaluronic acid nanofabric sheet by electrospinning> Electrospinning was performed in the same manner as in Example 1, using the hyaluronic acid aqueous solution compositions shown in Table 2, under the conditions of a voltage (Tension) of 30 (kV), a flow rate of 10-30 (mL / h), and a distance of 10-15 (cm).

[0047] [Table 2]

[0048] From Table 2, 5 × 10 3 Da, 1 × 10 4 Da, and 5×10 4 Da hyaluronic acid can be used alone, or with a weight-average molecular weight of 5 × 10 3 ~5×10 4 The hyaluronic acid content of Da is 88.9-98.1% by weight, and the weight-average molecular weight is 1 × 10⁻⁶. 5 ~1 × 10 6 It was found that hyaluronic acid nanofabric sheets can be formed when the hyaluronic acid content of Da is 1.9 to 11.1% by weight and the viscosity of the solution is 1,000 to 15,000 cPs.

[0049] <Experimental Example 1. Confirmation of the microstructure of hyaluronic acid nanofabric sheets> The microstructure of the hyaluronic acid nanofabric sheet produced by electrospinning was confirmed using a microscope (CX33RTFS2, Olympus Corporation, Korea) (Figure 1). In the case of the hyaluronic acid nanofabric sheet, depending on the molecular weight and concentration of the hyaluronic acid, high-quality nanofibers (Examples 2-4) or low-quality dot structures (Examples 2-6, 2-11) could be confirmed.

[0050] <Experimental Example 2: Evaluation of transdermal absorption rate and measurement of tensile strength> The transdermal absorption rate evaluation test was conducted in accordance with the "Guidelines for Skin Absorption Tests in Living Organisms" using Franz diffusion cells (effective area: 0.64 cm²). 2 A skin permeability test was performed under sink conditions using a receptor chamber with a volume of 5 mL (Logan Instruments, New Jersey, USA).

[0051] The receptor phase used in the skin permeability test was phosphate-buffered saline (PBS) with a pH of 7.4. A 1.5 cm x 1.5 cm epidermal layer of prepared human skin was positioned on the receptor chamber with the stratum corneum facing upwards. The donor chamber was then covered and secured with clamps. The receptor phase was then packed into a Franz-type diffusion cell and maintained at 32 ± 1°C. Permeability experiments were conducted by applying 100 μL of hyaluronic acid sheet dissolving solution to the donor site at a constant rate. 5 ml of the receptor phase was collected and analyzed after 1, 3, 6, 9, 12, and 24 hours of the permeability experiment, and then replenished with fresh phosphate-buffered saline.

[0052] Furthermore, for further qualitative analysis, referring to the methods of Fudala, Rafal, et al. and deBelder, Anthony N. et al. (Photobiology B:Biology 104.3 (2011), Carbohydrate Research 44.2 (1975)), we used a fluorescence microscope to confirm that the hyaluronic acid nonwoven fabric (Examples 2-4) penetrated the skin after 3 hours under the same penetration test conditions (Figure 2).

[0053] For tensile strength measurement, the tensile strength of the hyaluronic acid sheet manufactured in Example 2 was measured, and the results are shown in Table 3. The tensile strength was measured using a universal material testing machine with a sample measuring 15 cm in length and 2.5 cm in width, mounted on a test fixture, and the crosshead speed set to 2 mm / min.

[0054] [Table 3]

[0055] 5 x 10 3 Da, 1 × 10 4 Da, or 5×10 4 Hyaluronic acid nanofabric sheets manufactured using hyaluronic acid (Da) alone have a high transdermal absorption rate of 87.34-92.81%, but their tensile strength is only 2.14-3.08 MPa, resulting in poor physical properties and the problem of the sheets being damaged during post-processing such as punching and packaging. In contrast, hyaluronic acid nanofabric sheets manufactured by mixing ultra-low molecular weight and medium molecular weight hyaluronic acid have a transdermal absorption rate of 77.70-86.08% and a tensile strength of 5.32-10.91 MPa. Because they are superior not only in transdermal absorption but also in physical properties, they can be usefully used in mask patches for topical skin preparations and as carriers for ophthalmic or mucosal drug delivery systems.

[0056] In particular, we confirmed that the lower the molecular weight of the high-molecular-weight hyaluronic acid mixed, the greater the transdermal absorption rate, while the lower the tensile strength.

[0057] Although specific parts of the present invention have been described in detail above, it will be clear to those with ordinary skill in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Therefore, the substantial scope of the invention is defined by the appended claims and their equivalents. [Industrial applicability]

[0058] The hyaluronic acid nanofabric sheet of the present invention also exhibits excellent mechanical properties, making it applicable to a variety of uses, such as mask patches for topical skin preparations and carriers for ophthalmic or mucosal drug delivery systems.

Claims

1. (a) Weight average molecular weight 5×10 ３ ~5 x 10 ４ 88.9–98.1% by weight of Da hyaluronic acid and a weight-average molecular weight of 1 × 10⁻⁶ ５ ~1 x 10 ６ A step of preparing an aqueous hyaluronic acid solution by dissolving a hyaluronic acid composed of 1.9 to 11.1% by weight of Da hyaluronic acid in water, (b) A method for producing a hyaluronic acid nanofabric sheet, comprising the step of forming a hyaluronic acid nanofabric sheet consisting of hyaluronic acid fibers with a diameter of 0.3 to 5 μm by electrospinning the hyaluronic acid aqueous solution.

2. The method for producing a hyaluronic acid nanofabric sheet according to Claim 1, characterized in that the viscosity of the hyaluronic acid aqueous solution is 1,000 to 15,000 cPs.

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