TWI804143B - Crosslinked hyaluronic acid acid and uses thereof - Google Patents

Abstract

The present disclosure provides a crosslinked hyaluronic acid and uses thereof. The crosslinked hyaluronic acid structure is formed by cross-linking an epoxy cross-linking agent and a hyaluronic acid and/or its derivatives. The hyaluronic acid and/or its derivatives before crosslinking are linear molecules with a weight average molecular weight (Mw) of 10 kDa to 2200 kDa, and the crosslinked hyaluronic acid structure has a three-dimensional network structure,, the crosslink degree of the crosslinked hyaluronic acid structure is 1-50%, and the viscosity of the crosslinked hyaluronic acid structure is 20 mPa·s to 25000 mPa·s.

Description

Cross-linked hyaluronic acid and its uses

The disclosure relates to a hyaluronic acid and its use, and in particular to a cross-linked hyaluronic acid formed by cross-linking under a specific condition and its use.

PM2.5 is solid particles or liquid droplets suspended in the air, and suspended particles with a particle size less than or equal to 2.5 microns, such as second-hand smoke. Suspended particles can stay in the atmosphere for a long time, while PM2.5 has a strong penetrability and is easy to carry toxic substances into the human body because of its small size, which is larger than viruses and smaller than bacteria. Inflamed and more likely to cause heart disease or other cardiovascular problems once it enters the body. However, the current method of preventing PM2.5 or heavy metal particles from contacting the skin is still only to protect the skin by wearing masks or jackets, and there is no other effective way to protect the skin from the damage of PM2.5 and other suspended particles.

Hyaluronic acid, also known as hyaluronic acid (Hyaluronic acid, HA), is a mucopolysaccharide widely present in connective tissue, epithelial tissue and nerve tissue. Hyaluronic acid contains uronic acid and amino sugar. It is a linear polymer with a molecular weight ranging from tens of thousands to millions, and its repeating unit is composed of D-glucuronic acid (D-glucuronic acid) and D-N-acetyl-glucosamine (D-N-acetyl- Glucosamine) is a dimer formed by β-(1-3) bonds, and then a linear polymer is formed by β-(1-4) bonds.

Because hyaluronic acid can naturally exist in the human body and does not cause immune reactions, it is often used in medicine, especially in injections, such as mainly used in eye surgery such as cataracts and corneal injuries, and for example, high molecular weight A solution of hyaluronic acid (in millions) is injected into the eye as a viscoelastic fluid, or into the skin to fill in facial wrinkles or tissue in certain parts of the body.

In addition, it has been reported in the past that a composition containing hyaluronic acid is disclosed, wherein polysaccharide materials are used together with injection products of local anesthetics such as mepivacaine or articaine, but these The composition is mainly used as an injection composition, so as to improve the safety and more effective anesthesia effect of injection products containing local anesthetics.

Overall, although hyaluronic acid has been used in many ways, it is mainly used as an injection composition. In addition, although there are many skin care products containing hyaluronic acid on the market, their properties and textures are limited. The original function of hyaluronic acid is lost, so the time it can exist on the skin is quite short, and it is easy to lose, so there is a problem that it is not easy to absorb.

Therefore, there is still an urgent need for a cross-linked hyaluronic acid that can not only reduce the damage of suspended particles such as PM2.5 to the skin, but also has better moisturizing properties and can effectively reduce wrinkles and smooth the skin.

The disclosure provides a cross-linked hyaluronic acid, wherein the cross-linked hyaluronic acid is formed by cross-linking an epoxy cross-linking agent and a hyaluronic acid and/or its derivatives, wherein the hyaluronic acid before cross-linking The acid and/or its derivatives are linear molecules with a weight average molecular weight (Mw) of 10 kDa to 2200 kDa, and the cross-linked hyaluronic acid has a three-dimensional network structure, and the cross-linking degree of the cross-linked hyaluronic acid is 1-50 %, the viscosity of the cross-linked hyaluronic acid is 20 mPa·s to 25000 mPa·s.

The present disclosure also provides a use of a biomedical composition for preparing a preparation for reducing the damage of fine suspended particles (PM2.5), wherein the biomedical composition is made of the above-mentioned cross-linked hyaluronic acid.

The present disclosure further provides an application of a biomedical composition for preparing a preparation for prolonging skin moisturizing and/or improving wrinkles, wherein the biomedical composition is made of the above-mentioned cross-linked hyaluronic acid.

In order to make the above and other purposes, features, and advantages of the present disclosure more comprehensible, the following specific examples are given together with the accompanying drawings, and the detailed description is as follows:

The present disclosure can be understood through various disclosure aspects disclosed in the following embodiments, examples and related descriptions listed in the table. Unless otherwise defined herein, terms (including technical and scientific terms) used in connection with this disclosure shall have the meanings understood by those of ordinary skill in the art to which this disclosure belongs. And it should be understood that, unless otherwise stated in the definitions provided herein, in the event of any potential ambiguity, the definitions of terms shall be consistent with such commonly used terms (as defined in dictionaries). It can be further understood that the terminology used in this application is only used for the purpose of describing a specific implementation, rather than limiting.

It must be noted that, unless there is a clear indication to the contrary, the singular forms "a", "an" and "the" used in the specification or patent claims also include plural expressions. Accordingly, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

As used herein, the terms "hyaluronic acid (HA)" and "hyaluronic acid" are polysaccharides, which can be various hyaluronic acids in vivo or artificially synthesized hyaluronic acid and its derivatives. The hyaluronic acid derivative can be, for example, any form of hyaluronate, but the present disclosure is not limited thereto. The salts include, for example, alkali metal, alkaline earth metal salts, ammonium salts, and hydrochlorides.

As used herein, the term "pharmaceutical grade" means, within the scope of sound medical judgment, suitable for use in contact with human and animal tissue without undue toxicity, irritation, allergic reaction or other problems or complications, and reasonable benefits/risks Ratio of matching materials, excipients and/or compositions. In some embodiments, pharmaceutical grade means that a material or excipient complies with a monograph detailing testing and acceptance criteria, such as specifications approved by the Food and Drug Administration.

The cross-linked hyaluronic acid disclosed in the present disclosure and its application are described in detail below.

The present disclosure provides a cross-linked hyaluronic acid formed by cross-linking an epoxy cross-linking agent and a hyaluronic acid and/or its derivatives, wherein the cross-linked hyaluronic acid has a three-dimensional network structure, three-dimensional The network structure refers to a structure in which hyaluronic acid or its derivatives are cross-linked and repeatedly arranged in a three-dimensional manner to form multiple layers and have pores in them.

In a specific embodiment, the degree of cross-linking between the hyaluronic acid and the epoxy cross-linking agent in the above-mentioned cross-linked hyaluronic acid with a three-dimensional network structure may be about 1.0-50.0%, for example, about 1.5%, 2.0% %, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 21.0%, 22.0%, 23.0%, 24.0%, 25.0%, 26.0%, 27.0% , 28.0%, 29.0%, 30.0%, 31.0%, 32.0%, 33.0%, 34.0%, 35.0%, 36.0%, 37.0%, 38.0%, 39.0%, 40.0%, 41.0%, 42.0%, 43.0%, 44.0 %, 45.0%, 46.0%, 47.0%, 48.0%, 49.0%, etc., but the present disclosure is not limited thereto.

In yet another specific embodiment, the viscosity of the above-mentioned cross-linked hyaluronic acid may be less than 25000 mPa·s, such as about 10 mPa·s, 25 mPa·s, 50 mPa·s, 75 mPa·s, 100 mPa·s s, 250 mPa·s, 500 mPa·s, 750 mPa·s, 1000 mPa·s, 1500 mPa·s, 2000 mPa·s, 2500 mPa·s, 2500 mPa·s, 3000 mPa·s, 3500 mPa·s s, 4000 mPa s, 4500 mPa s, 5000 mPa s, 5500 mPa s, 6000 mPa s, 6500 mPa s, 7000 mPa s, 7500 mPa s, 8000 mPa s, 8500 mPa s s, 9000 mPa s, 9500 mPa s, 10000 mPa s, 11000 mPa s, 12000 mPa s, 13000 mPa s, 14000 mPa s, 15000 mPa s, 16000 mPa s, 17000 mPa s s, 18000 mPa·s, 19000 mPa·s, 20000 mPa·s, 21000 mPa·s, 22000 mPa·s, 23000 mPa·s, 24000 mPa·s, 25000 mPa·s, etc., but the disclosure is not limited to this.

Referring to Figure 1, Figure 1 is a step for manufacturing the cross-linked hyaluronic acid proposed in this disclosure, which includes: (S101) dissolving hyaluronic acid or its derivatives in a solvent to configure hyaluronic acid aqueous solution; (S120) adding an epoxy cross-linking agent to carry out a cross-linking reaction; (S130) purifying and concentrating the cross-linked hyaluronic acid aqueous solution; and (S140) filtering the obtained cross-linked hyaluronic acid aqueous solution Sterilize. The cross-linked hyaluronic acid formed by the preparation method of the disclosed cross-linked hyaluronic acid can effectively delay the degradation rate of hyaluronic acid.

Specifically, the weight average molecular weight (Mw) of the hyaluronic acid and/or its derivatives before cross-linking used in the present disclosure may be a linear molecule of about 10 kDa to 2200 kDa, for example, it may be about 20 kDa to 2000 kDa , 50 kDa to 1800 kDa, 100 kDa to 1500 kDa, 200 kDa to 1200 kDa, 300 kDa to 1000 kDa, 500 kDa to 700 kDa linear molecules, and for example can be about 500 kDa, 510 kDa, 520 kDa, 530 kDa , 540 kDa, 550 kDa, 560 kDa, 570 kDa, 580 kDa, 590 kDa, 600 kDa, 610 kDa, 620 kDa, 630 kDa, 640 kDa, 650 kDa, 660 kDa, 670 kDa, 680 kDa, 690 kDa, 700 weight average molecular weight in kDa etc., but the present disclosure is not limited thereto. If hyaluronic acid and/or its derivatives with a weight-average molecular weight of less than 10 kDa are used, it is difficult to form a film and the degree of crosslinking is not good. In addition, if hyaluronic acid and/or its derivatives with a weight-average molecular weight exceeding 2200 kDa are used, the viscosity will be too high and it will be difficult to shape, resulting in poor post-processing and low absorption.

In addition, in the step of producing the cross-linked hyaluronic acid, the solvent used for the production of the cross-linked hyaluronic acid may be an aqueous solvent, for example, an alkali Aqueous solvents are not limited thereto as long as they can dissolve hyaluronic acid and/or derivatives thereof.

In a specific embodiment, the epoxy crosslinking agent used in this disclosure can be a pharmaceutical grade epoxy crosslinking agent, and the pharmaceutical grade epoxy crosslinking agent can include 1,4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether, BDDE). The use of pharmaceutical-grade epoxy cross-linking agents can easily control the synthesis parameters, so that the cross-linked hyaluronic acid can have an appropriate viscosity and structure, so the cross-linked hyaluronic acid can have high flow properties and excellent Excellent resistance to degradation, so it is easy to spread evenly on the surface of the skin, and can be used as skin care products and other products in dosage forms for external use. Furthermore, since the pharmaceutical-grade epoxy cross-linking agent has high biocompatibility, it will not cause skin irritation, allergic reactions and other problems. However, the pharmaceutical grade epoxy crosslinking agent of the present disclosure is not limited to the epoxy crosslinking agent listed above, that is, if the epoxy crosslinking agent complies with the monograph which describes the testing and acceptance criteria of the pharmaceutical grade material in detail, For example, the epoxy cross-linking agent approved by the Food and Drug Administration (Food and Drug Administration), and after being cross-linked, can obtain the above-mentioned similar characteristics to the present disclosure and can also be applied to the cross-linked hyaluronic acid of the present disclosure.

In yet another specific embodiment, in the above-mentioned crosslinking step, the molar equivalent ratio of hyaluronic acid and/or its derivatives and epoxy crosslinking agent used may be about 1.00:0.10~1.00, for example, About 1.00: 0.15, 1.00: 0.20, 1.00: 0.25, 1.00: 0.30, 1.00: 0.35, 1.00: 0.40, 1.00: 0.45, 1.00: 0.50, 1.00: 0.55, 1.00: 0.60, 1.00: 0.65, 1.00: 0.70, 1.00 : 0.75, 1.00: 0.80, 1.00: 0.85, 1.00: 0.90, 1.00: 0.95, but the disclosure is not limited thereto.

In another specific embodiment, in the above purification and concentration steps, Tangential Flow Filtration (TFF) can be used for dialysis purification, and then the cross-linked hyaluronic acid can be treated to an appropriate concentration by vacuum concentration. However, the purification and concentration treatment methods are not limited thereto, as long as the cross-linked hyaluronic acid aqueous solution is properly purified and concentrated into cross-linked hyaluronic acid that can be used as a formulation.

In another specific embodiment, in the above filter sterilization step, a two-stage sterilization method can be used for filtration. Specifically, in the two-stage process, the filter used for the first-stage filtration may have a pore size of about 20 μm to 50 μm, for example, a pore size of about 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, etc., but this The disclosure is not limited to this. In addition, the filter used for the second-stage filtration may have a pore size of about 0.1 μm to 2 μm, such as about 0.2 μm, 0.4 μm, 0.6 μm, 0.8 μm, etc., but the disclosure is not limited thereto.

In another specific embodiment, the disclosure provides a biomedical composition for preparing a preparation for reducing the damage of fine suspended particles (PM2.5), wherein the biomedical composition is made of the above-mentioned cross-linked hyaluronic acid be made of. Specifically, the preparation made of the cross-linked hyaluronic acid of the present disclosure can be applied to the skin in the form of external application, so as to slow down the degradation of hyaluronic acid and protect the skin from external air for a certain period of time. Pollutants in the environment, such as fine suspended particles (PM2.5) and other damage.

In another specific embodiment, the disclosure provides a biomedical composition for the preparation of a preparation for prolonging skin moisturizing and/or improving wrinkles, wherein the biomedical composition is made of the above-mentioned cross-linked hyaluronic acid . Specifically, the preparation made of the cross-linked hyaluronic acid disclosed herein can be applied to the skin externally or injected subcutaneously, so as to prolong the moisturizing function of the skin or achieve the effect of improving skin wrinkles.

In addition, within the scope of not damaging the effect of the present disclosure, the preparation may further contain, for example, water, spices, moisturizers, cosmetic ingredients, medicinal ingredients, thickeners, bactericides, pH regulators, antioxidants, Preservatives, and other ingredients commonly used in formulations.

Furthermore, the biomedical composition of the present disclosure can be designed into various dosage forms based on the ease of use and feeling of use. For example, the above biomedical composition can be formulated into external preparations, such as cream, lotion, ointment, gel, etc. Glue, patch, spray and other dosage forms.

The following examples are provided to assist those skilled in the art in implementing the present disclosure. Even so, these examples should not be considered limitations of the present disclosure, since modifications and variations of the embodiments discussed herein can be made by persons of ordinary skill in the art to which this disclosure pertains without departing from the spirit or scope of the present disclosure. changes and still fall within the scope of this disclosure.

Example preparation

Embodiment 1-9: Preparation of cross-linked hyaluronic acid (HyaSmooth)

According to the ratio and crosslinking degree in Table 1 below, the preparation of Examples 1-9 was carried out according to the following method. First, dissolve hyaluronic acid powder (purchased from Kewpie, molecular weight about 10-2200kDa) in 0.4N NaOH (purchased from HONEYWELL, 1.0N) aqueous solution, and stir at room temperature for 18 to 22 hours until completely dissolved, while Form a hyaluronic acid aqueous solution, and then add 1,4-butanediol diglycidyl ether (BDDE) (purchased from ALDRICH, 1,4-Butanediol diglycidyl ether ≧95%) to the hyaluronic acid aqueous solution at 35°C for cross-linking After reacting with the reagents for about 3 hours, add 1N HCl and stir for about 1 hour, then dilute to adjust the pH value to 7.0±0.5 to obtain a cross-linked hyaluronic acid aqueous solution. Next, the hyaluronic acid aqueous solution was subjected to tangential flow filtration (TFF) dialysis purification, and then the hyaluronic acid aqueous solution was concentrated to a solid content of 4.5±1 wt % by vacuum concentration, and then the hyaluronic acid aqueous solution was purified by two-stage filtration. After filtration sterilization, the obtained product is the cross-linked hyaluronic acid prepared in the example, hereinafter referred to as HyaSmooth. In the above two-stage filtration step, the filter pore size of the first stage filter is 20 µm, and the filter pore size of the second stage filter is 0.2 µm. Finally, the sterilized aqueous solution of cross-linked hyaluronic acid was stored at 4°C in a refrigerator.

The molecular weight of hyaluronic acid used in each example, the molar equivalent ratio of hyaluronic acid to cross-linking agent, the degree of cross-linking and the results of viscosity are shown in Table 1 below.

[Table 1] Example number Molecular weight of hyaluronic acid before cross-linking (kDa) Molar equivalence ratio nature HA BDDE Cross-linking degree (%) Viscosity (mPa·s) Example 1 38 1 1 42 373 Example 2 510 1 0.1 1.3 24.3 Example 3 510 1 0.5 12 7,400 Example 4 510 1 0.6 13 4,236 Example 5 510 1 1 31 541 Example 6 650 1 0.4 13 6,040 Example 7 650 1 0.45 15 3,498 Example 8 650 1 0.6 twenty one 9,290 Example 9 2000 1 0.4 10 20,460

Furthermore, refer to Figure 2, which is the result of NMR and GC-MS determination of the cross-linked hyaluronic acid in Example 6. From the NMR figure, it can be seen that the structure of the cross-linked hyaluronic acid is complete, and from the GC - The MS figure shows that the BDDE component is not detected, showing that there is no 1,4-butanediol diglycidyl ether (BDDE) residue in the cross-linked hyaluronic acid. Moreover, the above-mentioned cross-linked hyaluronic acid was subjected to ISO-10993-5 standard method for in vitro cytotoxicity test (Test for in vitro cytotoxicity) and bacterial count test, showing that the cross-linked hyaluronic acid of the present disclosure is non-toxic to cells and passed Bacterial count test.

Comparative example preparation

Comparative example 1

Hyaluronic acid (purchased from Kewpie, molecular weight about 2000 kDa), which is a solution of uncrosslinked hyaluronic acid, was used.

Comparative example 2

Hyaluronic acid (purchased from Kewpie, molecular weight about 650 kDa), which is a solution of uncrosslinked hyaluronic acid, was used.

Comparative example 3

Commercially available hyaluronic acid elastomer TL100 (purchased from Huaxi Freda Biomedical Co., Ltd. (China), INCI name: sodium hyaluronate crosslinked polymer/1, 2-pentanediol/water (Sodium Hyaluronate Crosspolymer/ Pentylene Glycol /Aqua)) (CAS No.: 105524-32-1/5343-92-0/ 7732-18-5), its physical properties are as follows: molecular weight > 1.8MDa, pH 5.0~7.5, viscosity ≥ 250,000mPa ·s.

Experimental evaluation

1. Hyaluronic Acid Enzyme Decomposition Evaluation Test

Weighed about 2 mg of the cross-linked hyaluronic acid of the examples of the present disclosure and the samples of the comparative example, and put them into a 15 ml centrifuge tube for subsequent use. In addition, 10 mg of hyaluronidase (Hyaluronidase) (Merck) was added to 0.1 M PBS solution to prepare a 250 units/ml hyaluronidase solution. Add 10 ml of the hyaluronidase solution into the above 15 ml centrifuge tube containing the sample, and react in a water bath at 37°C for 24 hours. Then, after taking out the centrifuge tube, centrifuge at 4000g for 20 minutes at 25°C, and absorb 0.5ml of the supernatant, and use the ELISA Reader instrument for uronic acid assay .

Based on the results in Figure 3, it can be seen that the amount of uronic acid released in Comparative Example 1 is about 3.25 times that of Example 3 and Example 4, showing that the cross-linked hyaluronic acid of the present disclosure is higher than that of the uncross-linked hyaluronic acid. Molecular hyaluronic acid can more effectively achieve anti-degradation, thereby making the film formation more stable, and can effectively protect the skin from environmental pollution such as air pollution for a long time.

In addition, based on the results in Figure 4, it can be seen that the amount of uronic acid released in Comparative Example 2 is about 3.9 times that of the cross-linked hyaluronic acid (HyaSmooth) in Example 6, and the amount of uronic acid released in Comparative Example 3 The amount is about 6.5 times that of the cross-linked hyaluronic acid (HyaSmooth) in Example 6, showing that the cross-linked hyaluronic acid of the present disclosure not only has a stronger Degradation resistance, compared with the commercially available cross-linked hyaluronic acid elastomer (Comparative Example 3), it also has a more excellent anti-degradation effect.

2. Scanning Electron Microscopy (SEM) Image Evaluation

After the samples were dried in an oven (50 ^o C) to form thin films, the films formed from the samples were observed with a scanning electron microscope (SEM) at magnifications of 100 times and 300 times to confirm the structure of each film.

Refer to Figure 5, where (a) and (b) in Figure 5 are the SEM images of HyaSmooth in this disclosure at 100 times and 300 times respectively, and (c) and (d) in Figure 5 are Comparative Example 3 SEM images at 300x and 1000x. As shown in Figure 5, the HyaSmooth disclosed in this disclosure obviously has a three-dimensional network structure. However, even after cross-linking treatment in Comparative Example 3, its structure is only a two-dimensional planar network structure, so the HyaSmooth disclosed in this disclosure has a denser structure. structure, and thus achieve better protection for the skin.

3. PM2.5 skin cell viability assessment

Human dermal fibroblasts HSF were implanted in a 24-well plate at a cell count of 5× ¹⁰ cells/well, and cultured in a cell incubator for 18 hours, then the culture medium was removed, and the culture solution containing PM2.5 (purchased from PM2.5( Diesel particulate matter NIST 1650b; CAS Number: 1333-86-4, product number: NIST1650B) and/or the culture solution of different concentrations of test samples were added to the above cells for co-culture for 12 hours, the culture solution was removed and buffered with PBS Fluid rinse. Next, add MTT solution (tetrazole dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analytical reagent (3-(4, 5-dimethylthiazol-z-yl)-2,5-diphenyltetrazolium bromide assay)) was put into a cell culture box to react for 4 hours, and the absorbance value was measured at 540nm wavelength by using an enzyme immunoassay analyzer (ELISA reader) for quantitative analysis. Quantitative results are shown in (e) of FIG. 6 and (g) of FIG. 7 and Table 2.

Refer to Figure 6, Figure 6 (a) is the image of the original appearance of the cells in the blank group. The cells are clear and smooth as a whole, showing that the cell membrane of the cells is complete and presents the elongated state that fibroblasts should have. (b) of Figure 6 is the image of cells treated with PM2.5. The cells are shrunken and deformed, and the boundaries of the cells are not clear, indicating that the cell membrane of the cells has been damaged. However, (c) and (d) in Figure 6 are images of cells treated with PM2.5 using 0.5% HyaSmooth and 0.1% HyaSmooth respectively. It can be seen from the images that only 0.1% HyaSmooth can keep the cells elongated type and the overall shape of the cells is intact. (e) of Figure 6 shows the quantitative analysis of the cell survival rate of the above groups. The results show that the cell survival rate of the PM2.5 group is significantly reduced, but the cells treated with 0.5% and 0.1% HyaSmooth can maintain the same level as the blank In other words, compared with the PM2.5 group, those treated with 0.5% and 0.1% HyaSmooth can increase the cell survival rate by more than 20%, even up to 25-30%, showing that the HyaSmooth disclosed in this disclosure has an effect on PM2. 5 and other suspended particles have excellent defense.

[Table 2] group %( average ) blank group 100.00% PM2.5 84.08% PM2.5+ Comparative Example 2 0.1% 91.53% PM2.5+ Comparative Example 2 0.5% 91.40% PM2.5 + Comparative Example 3 0.1% 84.21% PM2.5 + Comparative Example 3 0.5% 68.04%

Refer to Figure 7 and Table 2. Figure 7 (a) is the image of the original appearance of the cells in the blank group. The cells are clear and smooth as a whole, showing that the cell membranes of the cells are complete and present the elongated state that fibroblasts should have. (b) of Figure 7 is the image of cells treated with PM2.5. The cells have shrinkage and deformation, and the boundaries of the cells are not clear, indicating that the cell membrane of the cells has been damaged. However, (c) and (d) in Figure 7 are images of cells treated with PM2.5 using concentrations of 0.1% and 0.5% of Comparative Example 2 respectively. It can be seen from the images that only using concentrations of 0.1% and 0.5% of Comparative Example 2 The overall shape of the cells will be slightly deformed and shrunk. (e) and (f) in Figure 7 are images of cells treated with PM2.5 using 0.1% and 0.5% of Comparative Example 3, respectively. It can be seen from the images that even when using 0.5% of Comparative Example 3, the cells still shrink and deform The phenomenon. Furthermore, (g) of Figure 7 shows the quantitative analysis of the cell survival rates of the above groups. From the quantitative analysis results, it can be seen that compared with the cell survival rate of the blank group, the cell survival rate of the PM2.5 group was significantly reduced. However, compared with the cell survival rate of the blank group, although the cells treated with 0.1% and 0.5% of Comparative Example 2 can slightly increase the cell survival rate, they still cannot recover to the cell survival rate comparable to the blank group. Furthermore, compared with the cell survival rate of the blank group, the cells treated with 0.1% and 0.5% Comparative Example 3 were almost equivalent to the cell survival rate of the PM2.5 group, showing that the defense effect of Comparative Example 3 on PM2.5 was not significant. good. Based on the above results, it can be known that the HyaSmooth disclosed in the present disclosure has superior defense against suspended particles such as PM2.5 compared with non-cross-linked hyaluronic acid or commercially available cross-linked hyaluronic acid.

4. Evaluation of skin moisture content

No skin care products or medicines were applied to the skin of the subjects 24 hours before the experiment. During the experiment, the subject first washed the skin on the inner side of the arm with detergent, dried it after washing, and rested for 30 minutes under a constant temperature and humidity (22±1°C, 50±5%RH), and marked the test position (diameter 3 cm). The HyaSmooth disclosed in this disclosure and Comparative Example 3 were prepared into a 1.0% aqueous solution, and 0.03 g of the above aqueous solution was taken out and applied to the test position on the subject's arm, and the position without application was used as the control group. After the application was uniform, every 60 minutes, continuous After 5 hours, use a multi-function skin detector (Multi Probe Adapter, MPA) with a skin moisture meter (Corneometer CM825) to measure the moisture content of the skin stratum corneum at the test site and the non-smeared site, and calculate the moisture content of the skin stratum corneum in each group. percentage.

[table 3] Skin moisture content rate (%) Measurement time (min) 60 120 180 240 300 control group 42.9% 42.9% 42.8% 43.1% 43.3% 1% HyaSmooth 60.8% 56.4% 56.3% 56.5% 51.9% 1% Comparative Example 3 55.2% 51.4% 51.5% 51.4% 47.1%

Based on the results in Figure 8 and Table 3, compared with the control group and Comparative Example 3, the HyaSmooth disclosed in this disclosure has a better water retention rate, and can maintain a water retention rate of about 57% for at least 240 minutes, showing a long-term Effective water retention effect.

5. Evaluation of Skin Wrinkles

No skin care products or medicines were applied to the skin of the subjects 24 hours before the experiment. During the experiment, the subject first washed the skin on the inner side of the arm with detergent, dried it after washing, and rested for 30 minutes under a constant temperature and humidity (22±1°C, 50±5%RH), and marked the test position (diameter 3 cm). The HyaSmooth disclosed in this disclosure and Comparative Example 3 were formulated into a 5.0% aqueous solution, and 0.03 g of the above aqueous solution was taken out, and applied to the test position on the subject's arm, with the unsmeared position as the control group, and every 60 minutes after the application was even, continuous For 5 hours, the skin was irradiated with a ring-shaped UVA light source, and the skin roughness of each group was measured with a roughness analyzer Visioscan VC98 using high-resolution photomicrography, and roughness calculation and analysis were performed. The change of skin roughness in the measurement area is calculated by the program to compare the number of horizontal and vertical wrinkles, and the reduction rate of skin wrinkles in each group is calculated.

[Table 4] Reduction rate of skin wrinkles (%) Smudge time (min) 60 120 180 240 300 5% HyaSmooth ↓6.3% ↓2.8% ↓3.3% ↓2.8% ↓0.7% 5% Comparative Example 3 ↓7.9% ↓6.1% ↓1.7% ↓0.9% 1.0%

Referring to Fig. 9, (a) in Fig. 9 is the skin wrinkle image taken every 60 minutes up to 300 minutes by HyaSmooth disclosed in this disclosure. (b) of Figure 9 is the skin wrinkle image taken every 60 minutes up to 300 minutes in Comparative Example 3, and (c) of Figure 9 is the quantitative reduction rate of skin wrinkles in each group, as can be seen from the image results and Table 4, The subjects who applied the HyaSmooth of the present disclosure and Comparative Example 3 significantly reduced the skin wrinkles within 120 minutes, but the HyaSmooth of the present disclosure still had a significant effect after 240 minutes of application. In contrast, Comparative Example 3 can only be maintained for 180 minutes, and then the skin wrinkle reduction effect is significantly reduced, which shows that HyaSmooth of the present disclosure has better maintenance than Comparative Example 3.

6. Assessment of Collagen Hyperplasia

The HSF cell line of human skin fibroblast purchased from the Agricultural Technology Research Institute, Animal Technology Laboratories was used for the collagen proliferation test. First, the human dermal fibroblast cell line HSF was inoculated in a culture dish at ^3x105 cells/well. After the cells were attached, 1% and 5% HyaSmooth were added to the cells for co-cultivation. as a control group. After 7 days of co-cultivation, the supernatant of each well was collected, and the collagen secretion was analyzed by using a collagen analysis kit (purchased from Biocolor, Collagen Assay Kit, specification S1000) according to the manufacturer's instructions.

Refer to Figure 10, which is based on the collagen hyperplasia test results of 1% and 5% HyaSmooth compared with the control group. From the results in Figure 10, it can be seen that the collagen secretion of the 1% and 5% HyaSmooth groups increased by 48% and 55% respectively compared with the control group, showing that the cross-linked hyaluronic acid of the present disclosure can promote at least about 1.5 times The above collagen secretion has a significant effect of promoting collagen hyperplasia.

As the above experimental results show, the cross-linked hyaluronic acid disclosed in this disclosure can form a three-dimensional network structure that protects cells, and compared with the commercially available cross-linked hyaluronic acid, it can better protect the skin from particulate pollution such as PM2.5 infringement of things. Furthermore, compared with commercially available cross-linked hyaluronic acid, the cross-linked hyaluronic acid of the present disclosure has longer-lasting moisturizing properties, wrinkle improvement, and collagen hyperplasia effects, thereby maintaining skin smoothness and inhibiting aging for a longer period of time. The generation of wrinkles.

Although the present disclosure has been described with the above-mentioned embodiments, it is not intended to limit the present disclosure. Without departing from the spirit and scope of the present invention, changes and modifications can be made by those skilled in the art. Therefore, the protection of the present invention The scope shall be defined by the appended patent application scope.

S110~S140: steps

FIG. 1 shows a flowchart of a method for manufacturing cross-linked hyaluronic acid according to the present disclosure. Figure 2 shows the measurement results of cross-linked hyaluronic acid according to the present disclosure by nuclear magnetic resonance (nuclear magnetic resonance, NMR) and combined gas chromatography/mass spectrometry (gas chromatography/mass spectrometry, GC-MS), wherein (a) is the NMR measurement result graph of the cross-linked hyaluronic acid disclosed in the present disclosure; (b-1) is the GC-MS measurement result of 1,4-butanediol diglycidyl ether (BDDE) as a standard, ( b-2) is the GC-MS measurement result of the cross-linked hyaluronic acid disclosed in the present disclosure. FIG. 3 shows the evaluation results of hyaluronic acid enzymatic decomposition of the cross-linked hyaluronic acid example according to the present disclosure compared with the comparative example. FIG. 4 shows the evaluation results of enzymatic decomposition of hyaluronic acid according to the cross-linked hyaluronic acid embodiment of the present disclosure compared with other comparative examples. Figure 5 shows the scanning electron microscope (scanning election microscopy, SEM) images of the cross-linked hyaluronic acid according to the present disclosure and the comparative example, wherein (a) is the SEM of the cross-linked hyaluronic acid of the present disclosure at 100 times Image, (b) is the SEM image of the cross-linked hyaluronic acid of the present disclosure at 300 times, (c) is the SEM image of the cross-linked hyaluronic acid of the comparative example at 300 times, and (d) is the cross-linked hyaluronic acid of the comparative example SEM image of hyaluronic acid at 1000X. Figure 6 shows the microscope images and cell viability evaluation results of cross-linked hyaluronic acid according to the present disclosure, wherein (a) is the cell image of the blank group, (b) is the cell image of the PM2.5 group, and (c) is Image of cells treated with 0.5% HyaSmooth and PM2.5, (d) image of cells treated with 0.1% HyaSmooth and PM2.5, and (e) quantitative analysis results of cell viability in each group. Figure 7 shows the microscope images and cell viability evaluation results of cross-linked hyaluronic acid according to the present disclosure, wherein (a) is the cell image of the blank group, (b) is the cell image of the PM2.5 group, and (c) is Image of cells treated with PM2.5 using concentration 0.1% of Comparative Example 2, (d) image of cells using concentration of 0.5% Comparative Example 2 and treated with PM2.5, (e) using concentration of 0.1% of Comparative Example 3 and The image of cells treated with PM2.5, (f) is the image of cells treated with PM2.5 using concentration 0.5% of Comparative Example 3 and (g) is the quantitative analysis results of the cell survival rate of each group. Fig. 8 shows the results of skin moisture measurement of cross-linked hyaluronic acid according to the present disclosure. Figure 9 shows the skin wrinkle improvement results of cross-linked hyaluronic acid according to the present disclosure, where (a) is the skin wrinkle images taken every 60 minutes up to 300 minutes by HyaSmooth of the present disclosure, and (b) is the image of skin wrinkles every 60 minutes of the comparative example The skin wrinkle images were taken every minute until 300 minutes, and (c) is the quantitative analysis result of the skin wrinkle reduction rate of each group. Fig. 10 shows the results of the collagen proliferation test of the cross-linked hyaluronic acid embodiment according to the present disclosure compared with the control group.

Claims (12)

A cross-linked hyaluronic acid, which is formed by cross-linking an epoxy cross-linking agent and a hyaluronic acid and/or its derivatives, wherein the hyaluronic acid and/or its derivatives before cross-linking are weight average A linear molecule with a molecular weight (Mw) of 20kDa to 1200kDa, and the cross-linked hyaluronic acid has a three-dimensional network structure, the cross-linking degree of the cross-linked hyaluronic acid is 10-42%, and the viscosity of the cross-linked hyaluronic acid is 250mPa． s to 12000mPa. s. The cross-linked hyaluronic acid as described in Claim 1, wherein the viscosity of the cross-linked hyaluronic acid is 3000~9500mPa. s. The cross-linked hyaluronic acid as described in Claim 1, wherein the hyaluronic acid and/or its derivatives and the epoxy cross-linking agent form the cross-linked hyaluronic acid at a molar equivalent ratio of 1.00:0.10~1.00 . The cross-linked hyaluronic acid according to claim 1, wherein the epoxy-based cross-linking agent includes 1,4-butanediol glycidyl ether (BDDE). The cross-linked hyaluronic acid according to Claim 1, wherein the cross-linked hyaluronic acid is sterilized cross-linked hyaluronic acid. A biomedical composition is used to prepare a preparation for reducing the damage of fine suspended particles (PM2.5), wherein the biomedical composition is composed of cross-linked hyaluronic acid as described in any one of claims 1-5 made. The use as described in Claim 6, wherein after using the biomedical composition, the cell survival rate of cells treated with PM2.5 suspended particles is increased by more than 20%. A biomedical composition is used to prepare a preparation for prolonging skin moisturizing and/or improving wrinkles, wherein the biomedical composition is made of cross-linked hyaluronic acid as described in any one of claims 1-5 have to. The use as described in Claim 6 or 8, wherein the dosage form of the biomedical composition is cream, lotion, ointment, gel, patch or spray. The use as described in Claim 8, wherein the moisture content of the skin is maintained above 50% for at least 300 minutes after using the biomedical composition. The use as described in Claim 8, wherein the number of skin wrinkles after using the biomedical composition is reduced by 2-7%. The use as described in Claim 8, wherein within 240 minutes after using the biomedical composition, the number of skin wrinkles is maintained at a reduction of more than 2%.

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