Hyaluronate nanofabric sheet and method of making same

Abstract

The present invention relates to a hyaluronate nanofabric sheet and a method for preparing the same, and more particularly, to a hyaluronate nanofabric sheet having excellent transdermal absorption rate and physical properties and a method for preparing the same. The method for preparing the hyaluronate nanofabric sheet of the present invention is characterized by comprising the steps of: (a) preparing a hyaluronate aqueous solution by mixing and dissolving a hyaluronate having a weight average molecular weight of 5 x 104 Da to 5 x 105 Da and a hyaluronate having a weight average molecular weight of 1 x 104 Da to 1 x 105 Da in water; and (b) electrospinning the hyaluronate aqueous solution. The hyaluronate nanofabric sheet of the present invention uses ultra-low molecular weight hyaluronic acid as a main component, and is not only excellent in transdermal absorption rate, but also excellent in mechanical properties due to the mixing of a certain amount of medium molecular weight hyaluronic acid, and thus can be used as a variety of purposes such as a mask patch for a skin external agent, a carrier for an ophthalmic or mucosal drug delivery system, etc. 3 4 5 6 ​​​​

Description

Technical Field

[0001] This invention relates to a hyaluronic acid nanofiber fabric sheet and its preparation method, and more specifically, to a hyaluronic acid nanofiber fabric sheet with excellent transdermal absorption and physical properties and its preparation method. Background Technology

[0002] Hyaluronic acid or hyaluronic acid salts are evenly distributed in the connective tissue, epithelium, and nerve tissue of the human body. As a biocompatible material with various physiological activities, it can contain a large amount of water. Due to its excellent viscoelasticity, and with the confirmation of its effects on skin regeneration, moisturizing, maintaining elasticity, and improving wrinkles, the demand for fillers containing it in anti-aging cosmetics, food, pharmaceuticals, and medical devices is rapidly increasing.

[0003] Currently, the finished products using hyaluronic acid salts sold on the market are all in liquid form, containing a low concentration of hyaluronic acid salts. That is, the main component is not hyaluronic acid salts but mostly water, which limits their use. Of course, since hyaluronic acid salts themselves have low safety to microorganisms, they must be prepared in sterile facilities when used as pharmaceuticals or medical devices, and when used as cosmetics, preservatives with potential safety risks must be used.

[0004] Therefore, research is underway on solidifying hyaluronic acid or hyaluronic acid salts into non-liquid films, fibers, etc.

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

[0006] However, the preparation process of hyaluronic acid membranes is somewhat complicated when mass-produced, and when used as face masks, they have problems such as not being able to adhere well to the skin or having low transdermal absorption rates.

[0007] To improve the preparation process of this hyaluronic acid membrane, Korean Patent No. 2338355 discloses a method for preparing wet hyaluronic acid nonwoven fabric, which can be used as a cosmetic patch, wet wound dressing, and tissue adhesion prevention membrane. Korean Patent No. 1224882 discloses a nanofiber sheet made of hyaluronic acid. However, because Korean Patent No. 2338355 uses hyaluronic acid with a molecular weight of 50,000 to 100,000 Da, while Korean Patent No. 1224882 uses hyaluronic acid with a molecular weight of 100,000 to 1,600,000 Daltons (Da), there is a problem of low transdermal absorption rate.

[0008] Hyaluronic acid or hyaluronic acid salts can be classified into ultra-low molecular weight, low molecular weight, medium molecular weight, and high molecular weight based on their molecular weight.

[0009] While medium and high molecular weight hyaluronic acid and hyaluronic acid salts have excellent mechanical properties and are easy to form into films, they are difficult to penetrate and absorb into the skin. On the other hand, the lower the molecular weight of hyaluronic acid or hyaluronic acid salts, the easier they are to penetrate and absorb into the skin, but due to their low mechanical properties, they are difficult to form into films.

[0010] Furthermore, in the case of hyaluronic acid salts, as high-molecular-weight polysaccharides containing many hydroxyl groups, electrospinning faces many limitations due to the positive charge inherent in the molecule. Therefore, when preparing hyaluronic acid salt nonwovens via existing electrospinning methods, alkaline solvents such as sodium hydroxide or potassium hydroxide are used (Castro, KC, Campos, MGN, & Mei, LHI (2021). International Journal of Biological Macromolecules, 173, 251-266.), or toxic organic solvents are used (Ewelina et al. Advances in Polymer Technology, 37.6 (2018): 1929-1940.), but the use of toxic organic solvents or salts is unavoidable. Moreover, the practical use is limited due to concerns that these organic solvents or salts may remain after the nonwoven fabric is formed.

[0011] Therefore, in order to prepare hyaluronic acid sheets that do not contain organic solvents and salts, are easily penetrated and absorbed into the skin, and have excellent mechanical properties for commercialization, the inventors, through their efforts, have identified a hyaluronic acid sheet with a mass of 5,000 to 5 × 10⁻⁶ that is easily penetrated and absorbed into the skin. 4 Da's ultra-low molecular weight hyaluronic acid is the main ingredient, with the addition of 1×10⁻⁶ hydroxyl groups that impart excellent processability and mechanical properties for productization. 5 ~1×10 6 The medium-molecular-weight hyaluronic acid salt of Da, after being dissolved in water, can be used to prepare hyaluronic acid salt nanofiber sheets with excellent transdermal absorption and mechanical properties through electrospinning, thus completing the present invention. Summary of the Invention

[0012] Technical problems to be solved

[0013] The purpose of this invention is to provide a hyaluronic acid nanofiber fabric sheet with excellent transdermal absorption rate and physical properties.

[0014] Another objective of this invention is to provide a method for preparing a hyaluronic acid nanofiber fabric sheet with excellent transdermal absorption and tensile strength.

[0015] Solution to the problem

[0016] To achieve the above objectives, the present invention provides a hyaluronic acid nanofiber sheet, which is made of hyaluronic acid fibers with a diameter of 0.3 to 5 μm prepared by electrospinning using pure water as a solvent alone.

[0017] To achieve the above objectives, the present invention provides a hyaluronic acid nanofiber fabric sheet with a transdermal absorption rate of 77.70-86.08% and a tensile strength of 5.32-10.91 MPa.

[0018] In this invention, the hyaluronic acid salt has a weight-average molecular weight of 5 × 10⁻⁶ wt% at 88.9–98.1% wt%. 3 ~5×10 4 Da's hyaluronic acid salt and 1.9–11.1% by weight of a weight-average molecular weight of 1 × 10⁻⁶ 5 ~1×10 6 Da is composed of hyaluronic acid salts.

[0019] In this invention, the hyaluronic acid sheet is characterized as a carrier for topical skin masks, ophthalmic or mucosal drug delivery systems.

[0020] This invention also provides a method for preparing hyaluronic acid nanofiber fabric sheets, the preparation method comprising the following steps: (a) using a weight-average molecular weight of 5 × 10⁻⁶ nanofibers... 3 ~5×10 4 Da's hyaluronic acid salt and weight-average molecular weight are 1×10⁻⁶. 5 ~1×10 6 (a) preparing an aqueous solution of hyaluronic acid salts by mixing and dissolving the hyaluronic acid salts in water; and (b) electrospinning the aqueous solution of hyaluronic acid salts.

[0021] In this invention, the viscosity of the aqueous hyaluronic acid salt solution is 1,000 to 15,000 cPs.

[0022] The effects of the invention

[0023] The hyaluronic acid nanofiber fabric sheet of the present invention uses ultra-low molecular weight hyaluronic acid as the main component. It not only has excellent transdermal absorption rate, but also has excellent mechanical properties due to the mixing of a certain amount of medium molecular weight hyaluronic acid. Attached Figure Description

[0024] Figure 1These are photographs of hyaluronic acid nanofiber fabric sheets prepared by electrospinning according to an embodiment of the present invention. (A: Nanofiber sheet, B: Dot sheet, C: Micrograph of nanofiber sheet, D: Micrograph of dot sheet)

[0025] Figure 2 This is a permeation experiment photograph of a hyaluronic acid nanofiber fabric sheet prepared according to an embodiment of the present invention, using a fluorescence microscope. Detailed Implementation

[0026] The best way to implement an invention

[0027] In this invention, "hyaluronic acid nanofiber sheet" refers to a sheet containing nano-hyaluronic acid fibers prepared by electrospinning.

[0028] The lower the molecular weight of hyaluronic acid or hyaluronic acid salts, the easier it is to penetrate and be absorbed into the skin. However, there is a problem that it is difficult to prepare sheets with high physical properties through electrospinning.

[0029] Korean Patent No. 2338355 uses hyaluronic acid salts with a molecular weight of 50,000 to 100,000 Da to prepare hyaluronic acid nonwoven fabrics, while Korean Patent No. 1224882 uses hyaluronic acid salts with a molecular weight of 100,000 to 1,600,000 Da to prepare hyaluronic acid nanofiber sheets. Because these patents use high molecular weight hyaluronic acid salts, alkaline solvents such as sodium hydroxide and potassium hydroxide are used to reduce viscosity. Due to the low transdermal absorption rate, this presents a problem where various physiological activities of hyaluronic acid cannot be effectively delivered to the skin.

[0030] In this invention, the aim is to confirm that when at 5×10 3 ~5×10 4 Add 1×10 to Da's ultra-low molecular weight hyaluronic acid 5 ~1×10 6 When Da produces medium-molecular-weight hyaluronic acid salts, it is possible to uniformly and mass-produce hyaluronic acid salt nanofiber fabric sheets that not only have excellent transdermal absorption rates but also excellent mechanical properties with high processability.

[0031] In this invention, after mixing ultra-low molecular weight hyaluronic acid salts and medium molecular weight hyaluronic acid salts, no alkaline solvent is added, and only pure water is used to dissolve them to prepare an aqueous solution of hyaluronic acid salts with a viscosity of 1,000 to 15,000 cPs (25°C). Hyaluronic acid salt nanofiber sheets are then prepared by electrospinning.

[0032] The results confirmed that hyaluronic acid nanofiber sheets can be prepared in large quantities, and the transdermal absorption rate of the prepared hyaluronic acid nanofiber sheets is 77.70-86.08%.

[0033] Therefore, on one hand, the present invention relates to a hyaluronic acid nanofiber sheet, which is made of hyaluronic acid fibers with a diameter of 0.3 to 5 μm prepared by electrospinning using pure water as a solvent alone.

[0034] In addition, the present invention relates to a hyaluronic acid nanofiber fabric sheet with a transdermal absorption rate of 77.70-86.08% and a tensile strength of 5.32-10.91 MPa.

[0035] The hyaluronic acid salt constituting the hyaluronic acid salt nanofiber fabric sheet is characterized by having a weight-average molecular weight of 5 × 10⁻⁶ wt%, comprising 88.9–98.1% of the total weight. 3 ~5×10 4 Da's hyaluronic acid salt and 1.9–11.1% by weight of a weight-average molecular weight of 1 × 10⁻⁶ 5 ~1×10 6 Da is composed of hyaluronic acid salts.

[0036] That is, the hyaluronic acid nanofiber fabric sheet of this invention cannot be formed into a sheet with high physical properties by electrospinning using only hyaluronic acid with a molecular weight equivalent to ultra-low molecular weight. Therefore, hyaluronic acid with a molecular weight equivalent to medium molecular weight is used in order to form the skeleton of the sheet.

[0037] In this invention, the molecular weight of the ultra-low molecular weight hyaluronic acid salt is 5 × 10⁻⁶. 3 ~5×10 4 Da, equivalent to a medium-sized hyaluronic acid salt with a molecular weight of 1×10⁻⁶. 5 ~1×10 6 Da.

[0038] When the molecular weight is 5×10 3 ~5×10 4 When the content of hyaluronic acid in Da is less than 88.9% by weight, it is impossible to prepare sheets or the transdermal absorption rate is low. When it exceeds 98.1% by weight, although sheets are formed, there are problems such as reduced physical properties such as tensile strength.

[0039] In this invention, hyaluronic acid salts are substances in which hyaluronic acid is combined with salts. Examples of hyaluronic acid salts include sodium hyaluronate, calcium hyaluronate, and potassium hyaluronate, but they are not limited to these.

[0040] On the other hand, the present invention relates to a method for preparing a hyaluronic acid nanofiber fabric sheet, the preparation method comprising the following steps: (a) using a weight-average molecular weight of 5 × 10⁻⁶ nanofibers... 3 ~5×10 4 Da's hyaluronic acid salt and weight-average molecular weight are 1×10⁻⁶. 5 ~1×10 6(a) preparing an aqueous solution of hyaluronic acid salts by mixing and dissolving the hyaluronic acid salts in water; and (b) electrospinning the aqueous solution of hyaluronic acid salts.

[0041] In this invention, the weight-average molecular weight is 5 × 10⁻⁶. 3 ~5×10 4 Da's hyaluronic acid has a weight-average molecular weight of 1×10⁻⁶. 5 ~1×10 6 The preferred weight ratio of hyaluronic acid in Da is 88.9–98.1% by weight: 1.9–11.1% by weight.

[0042] In this invention, the viscosity of the hyaluronic acid solution containing ultra-low and medium molecular weight hyaluronic acid is preferably 1,000–15,000 cPs (25°C) (Brookfield DV2RTVJO, spindle number 5, rpm 12). The viscosity of the solution can vary depending on the HA molecular weight (Da), HA content, and the ratio to the solvent. When the viscosity is less than 1,000 cPs, it is difficult to form sheets. When it exceeds 15,000 cPs, the high viscosity can cause nozzle clogging, thus raising concerns about the smooth progress of electrospinning.

[0043] In this invention, the electrospinning is preferably carried out under the following conditions: tension: 30-35 kV, flow rate: 10-20 mL / h, and distance: 10-15 cm.

[0044] In this invention, the hyaluronic acid nanofiber sheet prepared by electrospinning can be composed of hyaluronic acid fibers with a diameter of 0.3 μm to 5 μm.

[0045] In this invention, the solvent is used to dissolve hyaluronic acid salts, preferably purified water.

[0046] Furthermore, in the case of the hyaluronic acid nanofiber sheet prepared by electrospinning used in this invention, hyaluronic acid can be used alone for preparation. However, depending on the application field, water-soluble and low molecular weight carrier or excipient components commonly used in the art can be added, and their types and content ranges are not particularly limited.

[0047] Methods of implementing the invention

[0048] The present invention will now be described in more detail through embodiments. These embodiments are merely illustrative of the invention, and it will be apparent to those skilled in the art that the scope of the invention should not be construed as limited to these embodiments.

[0049] Example 1: Preparation of hyaluronic acid nanofiber sheets by electrospinning

[0050] Using an electrospinning apparatus (NE300, Inonovenso, Turkey), after setting the tension, flow rate, and distance as shown in Table 1 below, spinning was performed by changing the molecular weight of hyaluronic acid salts and the concentration of the aqueous solution.

[0051] Table 1

[0052]

[0053]

[0054] Viscometers (DV2TRV TJO, Brookfield Engineering Labs Inc., USA) were used to measure the viscous properties at 12 rpm, 30 seconds, and 25°C. Table 1 confirms that nanofibers with molecular weights ranging from 5 kDa to 50 kDa can be electrospun into sheets. This indicates that, depending on the hyaluronic acid salt concentration or molecular weight, sheets in the form of high-performance nanofibers can be prepared, as well as sheets containing some dot-like structures but with weaker physical properties. Figure 1 ).

[0055] Example 2: Preparation of hyaluronic acid nanofiber sheets by electrospinning

[0056] Using the aqueous hyaluronic acid salt composition shown in Table 2, electrospinning was performed in the same manner as in Example 1 under the conditions of tension: 30 (kV), flow rate: 10-30 (mL / h), and distance: 10-15 (cm).

[0057] Table 2

[0058]

[0059]

[0060] As shown in Table 2, when using 5×10 alone... 3 Da, 1×10 4 Da and 5×10 4 When using Da hyaluronic acid salts, or when using 88.9–98.1% by weight of a weight-average molecular weight of 5 × 10⁻⁶, 3 ~5×10 4 Da's hyaluronic acid salts, with a content of 1.9–11.1% by weight and a weight-average molecular weight of 1 × 10⁻⁶, have a molecular weight of 1 × 10⁻⁶. 5 ~1×10 6 When Da's hyaluronic acid salt has a solution viscosity of 1,000 to 15,000 cPs, it can form hyaluronic acid salt nanofiber sheets.

[0061] Experimental Example 1. Confirmation of the microstructure of hyaluronic acid nanofiber fabric sheets

[0062] The microstructure of the hyaluronic acid nanofiber fabric sheets prepared by electrospinning was confirmed using a microscope (CX33RTFS2, Olympus Corporation, South Korea). Figure 1 In the case of hyaluronic acid nanofiber sheets, high-performance nanofibers (Examples 2-4) or low-performance dot structures (Examples 2-6, 2-11) can be identified based on the molecular weight and concentration of hyaluronic acid.

[0063] Experimental Example 2. Evaluation of transdermal absorption rate and determination of tensile strength

[0064] The test to evaluate transdermal absorption rate was conducted according to the "Guidelines for Testing Skin Absorption in Organisms," using a Franz Diffusion Cell (effective area: 0.64 cm²). 2 The skin penetration test was performed using a device (Logan Instruments, New Jersey, USA) with a receiving chamber volume of 5 ml under sink conditions.

[0065] The skin permeation assay used phosphate-buffered saline (PBS) at pH 7.4. A 1.5cm × 1.5cm section of prepared human skin was placed in the recipient chamber with the stratum corneum facing upwards. The donor chamber was then covered and secured with clamps. The aqueous phase was then filled into a Franz diffusion cell and maintained at 32±1°C. 100ml of hyaluronic acid sheet solution was evenly applied to the donor site to perform the permeation assay. After 1 hour, 3 hours, 6 hours, 9 hours, 12 hours, and 24 hours, 5ml of the aqueous phase was collected for analysis, and fresh PBS was added.

[0066] Furthermore, for further qualitative analysis, following the methods of Fudala, Rafal et al. and de Belder, Anthony N. et al. (Photobiology B: Biology 104.3 (2011), Carbohydrate Research 44.2 (1975)), fluorescence microscopy was used to confirm that hyaluronic acid nonwoven fabrics (Examples 2-4) penetrated into the skin after 3 hours under the same permeation test conditions. Figure 2 ).

[0067] In determining tensile strength, the tensile strength of the hyaluronic acid sheet prepared in Example 2 was measured, and the results are shown in Table 3. A sample with a length of 15 cm and a width of 2.5 cm was mounted on a test fixture, and the tensile strength was determined using a universal testing machine with the crosshead speed set to 2 mm / min.

[0068] Table 3

[0069]

[0070]

[0071] Although using 5×10 alone 3 Da, 1×10 4 Da or 5×10 4 Hyaluronic acid nanofiber sheets prepared from Da's hyaluronic acid have a transdermal absorption rate of 87.34%–92.81%, which is high. However, their tensile strength is only 2.14–3.08 MPa, indicating poor physical properties. Therefore, there is a problem of sheet damage during post-processing such as punching and packaging. On the other hand, hyaluronic acid nanofiber sheets prepared by mixing ultra-low 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. They not only have excellent transdermal absorption but also excellent physical properties, making them suitable for use as carriers in topical skin masks, ophthalmic drug delivery systems, or mucosal drug delivery systems.

[0072] In particular, it was confirmed that the lower the molecular weight of the mixed medium-molecular-weight hyaluronic acid salt, the higher the transdermal absorption rate, but the lower the tensile strength.

[0073] The foregoing has provided a detailed description of specific aspects of this invention. For those skilled in the art, these specific techniques are merely preferred embodiments, and it is obvious that the scope of this invention is not limited thereto. Therefore, the actual scope of this invention is defined by the claims and their equivalents.

[0074] Industrial applicability

[0075] Because the hyaluronic acid nanofiber fabric sheet of the present invention also has excellent mechanical properties, it can be used for a variety of purposes, such as as a mask for topical skin agents, a carrier for ophthalmic or mucosal drug delivery systems.

Claims

1. A transparent hyaluronic acid nanofiber fabric sheet, wherein, The hyaluronic acid nanofiber sheet is made of hyaluronic acid fibers with a diameter of 0.3–5 μm, prepared by electrospinning using pure water as the solvent alone. The transdermal absorption rate of the hyaluronic acid nanofiber fabric sheet is 77.70–86.08%, and the tensile strength is 5.32–10.91 MPa. The hyaluronic acid salt has a weight-average molecular weight of 5 × 10⁻⁶ wt% at 88.9–98.1% of its weight. 3 ~5×10 4 Da's hyaluronic acid salt and 1.9–11.1% by weight of a weight-average molecular weight of 1 × 10⁻⁶ 5 ~1×10 6 Da is composed of hyaluronic acid salts.

2. The hyaluronic acid nanofiber fabric sheet according to claim 1, characterized in that, The hyaluronic acid nanofiber fabric sheet is used as a carrier for topical skin masks, ophthalmic or mucosal drug delivery systems.

3. A method for preparing a hyaluronic acid nanofiber fabric sheet, wherein, The preparation method includes the following steps: (a) A weight-average molecular weight of 5 × 10 3 ~5×10 4 Da's hyaluronic acid salt and weight-average molecular weight are 1×10 5 ~1×10 6 A hyaluronic acid salt aqueous solution was prepared by mixing and dissolving Da's hyaluronic acid salt in water, wherein the weight-average molecular weight was 5 × 10⁻⁶. 3 ~5×10 4 Da's hyaluronic acid salt and the weight-average molecular weight of 1×10 5 ~1×10 6 The weight ratio of Da's hyaluronic acid salts is 88.9–98.1% by weight: 1.9–11.1% by weight; and (b) Electrospinning the aqueous solution of the hyaluronic acid salt to prepare a hyaluronic acid salt nanofiber sheet made of hyaluronic acid salt fibers with a diameter of 0.3 to 5 μm.

4. The method for preparing the hyaluronic acid nanofiber fabric sheet according to claim 3, characterized in that, The viscosity of the aqueous hyaluronic acid salt solution is 1,000 to 15,000 cPs.

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