A colorless, transparent collagen hydrogel and its preparation method.

Abstract

The present invention relates to the field of biomaterials, and in particular to a colorless and transparent collagen hydrogel and a method for preparing the same. The collagen hydrogel of the present invention has high transparency and can be used in fields such as visual regeneration materials and corneal repair.

Description

[Technical Field]

[0001] The present invention relates to a biomaterial and a method for preparing the same, and more particularly to a colorless, transparent collagen hydrogel and a method for preparing the same. [Background technology]

[0002] Collagen is the main structural protein of connective tissue, widely distributed in human tissues and organs such as bone, skin, blood vessels, ligaments, cartilage, muscles, and tendons, accounting for 25% to 30% of all animal proteins. As the main component of the extracellular matrix, collagen plays a crucial role in maintaining the biological properties and structural integrity of the extracellular matrix. Collagen itself has a special quaternary structure and can spontaneously aggregate under specific conditions to form collagen fibers, and further form hydrogels. However, conventional collagen hydrogels exhibit a clear "whitening" phenomenon, i.e., a white, opaque state, making them unsuitable for applications such as visual materials and artificial corneas.

[0003] This invention primarily solves the problem of "cloudiness" in collagen hydrogels, enabling the preparation of colorless and transparent collagen hydrogels and improving the light transmittance of collagen hydrogels. [Overview of the Initiative]

[0004] This invention provides a colorless, transparent collagen hydrogel and a method for preparing the same. A method for preparing a colorless and transparent collagen hydrogel, (1) A step of thoroughly dissolving the solid collagen material in water, centrifuging it, and collecting the supernatant, (2) The supernatant obtained in step (1) is adjusted to a pH of 5.0 to 9.0 with an alkaline solution or an acidic solution. (3) A step of adding a specific ion / molecular pair to the solution obtained in step (2), (4) A step of adding a crosslinking agent to the solution obtained in step (3), (5) A step of adding protective molecules to the solution obtained in step (4), (6) A step of performing a low-temperature irradiation treatment on the solution obtained in step (5), A method for preparing a colorless, transparent collagen hydrogel, characterized by sequentially containing the following.

[0005] In the method for preparing a colorless and transparent collagen hydrogel of the present invention, the solid collagen material in step (1) above is selected from one or more combinations of type I, type II, type III, and type V collagen, and the total concentration of the various collagens in the supernatant in step (1) above is 0.01 wt.% or more and 10 wt.% or less. If the total concentration of collagen is less than 0.01 wt.%, the collagen solution cannot form a hydrogel, and if the total concentration of collagen exceeds 10 wt.%, the collagen molecules in the collagen solution are more likely to form collagen fibers, causing "turbidity" and making it impossible to form a colorless and transparent collagen hydrogel.

[0006] In the method for preparing a colorless, transparent collagen hydrogel of the present invention, the acidic solution in step (2) comprises at least one of salicylic acid, lactic acid, sulfuric acid, tartaric acid, citric acid, phosphoric acid, oxalic acid, acetic acid, ethanedioic acid, succinic acid, hydrochloric acid, maleic acid, benzoic acid, nitric acid, malic acid, nicotinic acid, sodium dihydrogen phosphate, and formic acid, and the alkaline solution comprises at least one of tetramethylethylenediamine, sodium hydroxide, triethylamine, sodium carbonate, potassium hydroxide, calcium hydroxide, magnesium hydroxide, aqueous ammonia, disodium hydrogen phosphate, and sodium bicarbonate. Since the collagen aqueous solution is prone to "turbidity" under neutral pH conditions, the pH is precisely adjusted to a pH of 6.9 or 7.9 using an acidic or alkaline solution to prevent collagen molecules from aggregating and forming collagen fibers. The concentration of the acidic or alkaline solution is in the range of 0.01M to 2M.

[0007] In the method for preparing a colorless, transparent collagen hydrogel of the present invention, the specific ion / molecular pair in step (3) above is HPO4 2‐-citric acid, HCO3 ‐ -citric acid, CO3 2‐ -citric acid, H2PO4 ‐ -citric acid, PO4 3‐ -citric acid, Ac ‐ -citric acid, Ac ‐ -H + -NH4 + , HAc-Ac ‐ , HAc-Ac ‐ -NH4 + , PO4 3‐ -H + -triethylamine, HPO4 2‐ -H + -triethylamine, H2PO4 ‐ -H + -triethylamine, PO4 3‐ -HPO4 2‐ , Tris-HAc, Tris-H2PO4 ‐ , Tris-NH4 + , Tris-H + , Tris-lysine hydrochloride, Tris-lysine hydrochloride-ethylenediaminetetraacetic acid ion, PO4 3‐ -H2PO4 ‐ , HPO4 2‐ -H2PO4 ‐ , HPO4 2‐ -NH4 + , H2PO4 ‐ -HCO3 ‐ , H2PO4 ‐ -CO3 2‐ , NH4 + -NH3. The composition comprises at least one selected from the above. By adjusting the ionic environment of the aqueous collagen solution with specific ion / molecule pairs, the formation of collagen fibers caused by self-assembly of collagen molecules is inhibited, the homogeneous fluidity of the aqueous collagen solution is ensured, and the "white turbidity" phenomenon is prevented. The concentration of the specific ion / molecule pairs ranges from 0.1 M to 10 M.

[0008] In the method for preparing the colorless and transparent collagen hydrogel of the present invention, the crosslinking agent in the above step (4) comprises at least one of NHS esters, sulfonyl chloride, acid anhydrides, fluorophenyl esters, proanthocyanidins, genipin, aldehydes, isothiocyanates, isocyanates, acyl azides, epoxides, aryl halides, imide esters, and carbodiimides. The concentration of the crosslinking agent ranges from 0.0001% to 5%.

[0009] In the method for preparing the colorless and transparent collagen hydrogel of the present invention, the protective molecule in the above step (5) comprises at least one of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, Tween, Triton, ethylene glycol, phenylalanine, proline, lecithin, glutamic acid, lysine, cysteine, mangiferin, glycine, aspartic acid, n-butanol, propylene glycol, ethylenediaminetetraacetic acid sodium salt, stearic acid, Span, glycerin, and gelatin. The addition of protective molecules is mainly for reducing the influence of irradiation treatment on the activity of collagen molecules. The concentration of the protective molecule ranges from 1% to 50%.

[0010] In the method for preparing the colorless and transparent collagen hydrogel of the present invention, the low temperature range of the low-temperature irradiation treatment in the above step (6) is -20 to 5°C. The low-temperature condition is mainly adopted to mitigate the influence of the irradiation treatment on the activity of collagen molecules.

[0011] A colorless and transparent collagen hydrogel prepared by any of the methods for preparing a colorless and transparent collagen hydrogel of the present invention.

[0012] In the colorless and transparent collagen hydrogel of the present invention, the turbidity range of the above colorless and transparent collagen hydrogel is 0.01×10 4 ~2×10 4 NTU.

[0013] The beneficial effects of the present invention are as follows. In the process by which collagen molecules form collagen hydrogel, the collagen molecules self-assemble to form collagen fibers, and the formed collagen hydrogel usually exhibits a white, opaque state, making it difficult to use in visual materials and artificial corneas. To improve the transparency of collagen hydrogel, the method for preparing a colorless and transparent collagen hydrogel of the present invention mainly reduces the self-assembly behavior of collagen molecules as much as possible by balancing the surface charge of collagen molecules and increasing the ionic strength of the solution through the synergistic effect of precisely adjusting the pH of the collagen solution and adding specific ion / molecular pairs. Based on this, an appropriate crosslinking agent is added to slowly induce the crosslinking reaction of collagen molecules, avoiding an increase in collagen fibers due to rapid crosslinking and thus avoiding a decrease in the transparency of the collagen hydrogel. To further satisfy the sterilization and inactivation requirements of colorless and transparent collagen hydrogel in medical devices and to avoid a decrease in transparency due to the effects of irradiation on collagen molecules of the colorless and transparent collagen hydrogel, the preparation method of the present invention further adds steps of adding protective molecules and low-temperature treatment to reduce the effects of irradiation on collagen molecules as much as possible. Through the above process, a stable, colorless, and transparent collagen hydrogel can be prepared. [Brief explanation of the drawing]

[0014] [Figure 1] This figure shows the light transmittance effect of collagen hydrogel (Figure 1A is the sample from Example 1, Figure 1B is the sample from Example 2, Figure 1C is the sample from Example 3, Figure 1D is the sample from Example 4, Figure 1E is the sample from Example 5, Figure 1F is the sample from Comparative Example 1, Figure 1G is the sample from Comparative Example 2, Figure 1H is the sample from Comparative Example 3, Figure 1I is the sample from Comparative Example 4, and Figure 1J is the sample from Comparative Example 5). [Modes for carrying out the invention]

[0015] Example 1 A method for preparing a colorless and transparent collagen hydrogel, (1) Dissolve 1 g of type I collagen thoroughly in 100 mL of water, centrifuge, and collect the supernatant. (2) The supernatant obtained in step (1) is adjusted to pH 7.8 with a 1 M magnesium hydroxide solution or a 0.1 M aqueous acetic acid solution, (3) Add a specific ion / molecular pair, H2PO4, to the solution obtained in step (2). ‐ - A step of adding citric acid at a concentration of 0.1M, (4) A step of adding 0.01% proanthocyanidin to the solution obtained in step (3), (5) Adding 5% n-butanol to the solution obtained in step (4), (6) The solution obtained in step (5) is subjected to electron beam irradiation at 0°C, This is a method for preparing a colorless, transparent collagen hydrogel containing the following in sequence.

[0016] Example 2 A method for preparing a colorless and transparent collagen hydrogel, (1) Dissolve 0.1 g of type II collagen and 0.4 g of type III collagen thoroughly in 100 mL of water, centrifuge, and collect the supernatant. (2) The supernatant obtained in step (1) is adjusted to pH 7.1 with a 0.5 M ammonia aqueous solution or a 1 M oxalic acid aqueous solution,

[0017] (3) Add the specific ion / molecule pair Tris-H2PO4 to the solution obtained in step (2). ‐ The process involves adding it at a concentration of 1M, (4) Adding 0.001% genipin and 1% isocyanate to the solution obtained in step (3), (5) The step of adding 1% glycerin and 2% tween to the solution obtained in step (4), (6) Add Co to the solution obtained in step (5) at -5°C 60 The process of applying irradiation treatment, This is a method for preparing a colorless, transparent collagen hydrogel containing the following in sequence.

[0018] Example 3 A method for preparing a colorless and transparent collagen hydrogel, (1) Dissolve 20g of type I collagen, 0.6g of type III collagen, and 5g of type V collagen thoroughly in 1000mL of water, centrifuge, and collect the supernatant. (2) The supernatant obtained in step (1) is adjusted to pH 6.5 with a 0.2 M sodium hydroxide solution or a 1 M hydrochloric acid aqueous solution. (3) Adding the Tris-lysine hydrochloride-ethylenediaminetetraacetate ion, a specific ion / molecular pair, to the solution obtained in step (2) at a concentration of 2M, (4) Adding 1% NHS ester to the solution obtained in step (3), (5) Adding 5% ethylene glycol and 10% mangiferin to the solution obtained in step (4), (6) The solution obtained in step (5) is subjected to gamma ray irradiation at 5°C, A method for preparing a colorless, transparent collagen hydrogel containing the following in sequence.

[0019] Example 4 A method for preparing a colorless and transparent collagen hydrogel, (1) Dissolve 5g of type I collagen and 5g of type II collagen thoroughly in 100mL of water, centrifuge, and collect the supernatant. (2) The supernatant obtained in step (1) is adjusted to pH 5.0 with 0.2 M sodium carbonate, 0.01 M potassium hydroxide, or 2 M ethanedioic acid and 1 M succinic acid aqueous solution. (3) Add a specific ion / molecular pair, PO4, to the solution obtained in step (2). 3‐ - HPO4 2‐ H2PO4 ‐ - Citric acid, Tris-H2PO4 ‐ The process involves adding these at concentrations of 0.5M, 10M, and 1M, respectively. (4) Adding 0.2% isothiocyanate, 5% NHS ester and 0.5% acyl azide to the solution obtained in step (3), (5) Adding 2% phenylalanine, 1% triton, and 50% lysine to the solution obtained in step (4), (6) Add Co to the solution obtained in step (5) at -20°C 60 The process of applying irradiation treatment, This is a method for preparing a colorless, transparent collagen hydrogel containing the following in sequence.

[0020] Example 5 A method for preparing a colorless and transparent collagen hydrogel, (1) Dissolve 0.1 g of type II collagen thoroughly in 1000 mL of water, centrifuge, and collect the supernatant. (2) The supernatant obtained in step (1) is adjusted to pH 9.0 with a 2M tetramethylethylenediamine, 1M disodium hydrogen phosphate solution, or 0.1M citric acid aqueous solution. (3) Add a specific ion / molecular pair, HAc-Ac, to the solution obtained in step (2). ‐ H2PO4 ‐ -CO3 2‐ NH4 + - A step of adding NH3 at concentrations of 1M, 1M, and 5M, respectively. (4) Adding 0.5% sulfonyl chloride and 0.5% imide ester to the solution obtained in step (3), (5) Adding 1% sodium dodecylbenzenesulfonate and 5% propylene glycol to the solution obtained in step (4), (6) The solution obtained in step (5) is subjected to gamma ray irradiation at 4°C, This is a method for preparing a colorless, transparent collagen hydrogel containing the following in sequence.

[0021] Comparative study on the effects of colorless, transparent collagen hydrogel I. Preparation of comparative collagen hydrogels

[0022] Comparative Example 1 A method for preparing collagen hydrogel, (1) Dissolve 1 g of type I collagen thoroughly in 100 mL of water, centrifuge, and collect the supernatant.

[0023] (2) The supernatant obtained in step (1) is adjusted to a neutral pH of 7.4 with a 1M magnesium hydroxide solution or a 0.1M aqueous acetic acid solution. This is a method for preparing a collagen hydrogel, comprising the following steps in sequence.

[0024] Comparative Example 2 A method for preparing collagen hydrogel, (1) Dissolve 1 g of type I collagen thoroughly in 100 mL of water, centrifuge, and collect the supernatant.

[0025] (2) The supernatant obtained in step (1) is adjusted to pH 7.8 with a 1 M magnesium hydroxide solution or a 0.1 M aqueous acetic acid solution,

[0026] (3) The solution obtained in step (2) is subjected to electron beam irradiation at 0°C, This is a method for preparing a collagen hydrogel, comprising the following steps in sequence.

[0027] Comparative Example 3 A method for preparing a colorless and transparent collagen hydrogel, (1) Dissolve 20g of type I collagen, 0.6g of type III collagen, and 5g of type V collagen thoroughly in 1000mL of water, centrifuge, and collect the supernatant.

[0028] (2) A step of adding 1% NHS ester to the solution obtained in step (1),

[0029] (3) Adding 5% ethylene glycol and 10% mangiferin to the solution obtained in step (2),

[0030] (4) The solution obtained in step (3) is subjected to gamma ray irradiation at 5°C, This is a method for preparing a colorless, transparent collagen hydrogel containing the following in sequence.

[0031] Comparative Example 4 A method for preparing a colorless and transparent collagen hydrogel, (1) Dissolve 5g of type I collagen and 5g of type II collagen thoroughly in 100mL of water, centrifuge, and collect the supernatant.

[0032] (2) The supernatant obtained in step (1) is adjusted to pH 5.0 with 0.2 M sodium carbonate, 0.01 M potassium hydroxide, or 2 M ethanedioic acid and 1 M succinic acid aqueous solution.

[0033] (3) Add a specific ion / molecular pair, PO4, to the solution obtained in step (2). 3‐ - HPO4 2‐ H2PO4 ‐ - Citric acid, Tris-H2PO4 ‐ The process involves adding these at concentrations of 0.5M, 10M, and 1M, respectively.

[0034] (4) Add Co to the solution obtained in step (3) at -20°C 60 The process of applying irradiation treatment, A method for preparing a colorless, transparent collagen hydrogel containing the following in sequence.

[0035] Comparative Example 5 A method for preparing a colorless and transparent collagen hydrogel, (1) Dissolve 0.1 g of type II collagen thoroughly in 1000 mL of water, centrifuge, and collect the supernatant.

[0036] (2) The supernatant obtained in step (1) is adjusted to pH 9.0 with a 2M tetramethylethylenediamine, 1M disodium hydrogen phosphate solution, or 0.1M citric acid aqueous solution.

[0037] (3) The solution obtained in step (2) is subjected to gamma ray irradiation at 4°C, This is a method for preparing a colorless, transparent collagen hydrogel containing the following in sequence.

[0038] II. Comparison of Efficacy Tests (1) Collagen hydrogel turbidity test: The turbidity of the collagen hydrogel was observed at a wavelength of 625 nm using a turbidimeter (TZD-BZ-905, Suzhou DiagVita Technology Co., Ltd.). The test results are shown in Table 1. Figure 1 shows the light transmittance effect of the collagen hydrogel, with Figure 1A being the sample from Example 1, Figure 1B being the sample from Example 2, Figure 1C being the sample from Example 3, Figure 1D being the sample from Example 4, Figure 1E being the sample from Example 5, Figure 1F being the sample from Comparative Example 1, Figure 1G being the sample from Comparative Example 2, Figure 1H being the sample from Comparative Example 3, Figure 1I being the sample from Comparative Example 4, and Figure 1J being the sample from Comparative Example 5.

[0039] Table 1. Turbidity test of collagen hydrogel JPEG0007919767000001.jpg52170

[0040] As can be seen from Table 1, the turbidity of the collagen hydrogels in Examples 1-5 is very low. The collagen hydrogel in Example 1 (Figure 1A) is very transparent, and the letters at the bottom of the material can be clearly observed. Similarly, the transparency of the collagen hydrogels in Figures 1B-1E is also very high, and the markings on the syringe can be seen through the collagen hydrogel. In contrast, the turbidity of the collagen hydrogels in Comparative Examples 1-5 is very high. The collagen hydrogel in Figure 1F is very cloudy, and the letters at the bottom of the material cannot be observed. Similarly, the collagen hydrogels in Figures 1G-1J are very blurry, have poor light transmittance, and the markings on the syringe can hardly be seen.

[0041] The present invention's method for preparing a colorless, transparent collagen hydrogel primarily involves a synergistic effect of precisely adjusting the pH of the collagen solution and adding specific ion / molecular pairs to balance the surface charge of the collagen molecules and increase the ionic strength of the solution, thereby minimizing the self-assembly behavior of the collagen molecules. Based on this, an appropriate crosslinking agent is added to slowly induce the crosslinking reaction of the collagen molecules, avoiding a rapid increase in collagen fibers due to crosslinking and thus preventing a decrease in the transparency of the collagen hydrogel. To further satisfy the sterilization and inactivation requirements for colorless, transparent collagen hydrogels in medical devices and to avoid a decrease in transparency due to the effects of irradiation on the collagen molecules, the preparation method of the present invention further adds steps of adding protective molecules and low-temperature treatment to minimize the effects of irradiation on the collagen molecules. Through these steps, a stable, colorless, transparent collagen hydrogel can be prepared.

[0042] The embodiments described above merely illustrate preferred embodiments of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the invention shall be included within the scope of protection as defined by the claims of the present invention. [Industrial applicability]

[0043] The present invention provides a method for preparing a colorless collagen hydrogel that solves the problem of "cloudiness" in collagen hydrogels, enhances the light transmittance of collagen hydrogels, and makes it possible to prepare a colorless and transparent collagen hydrogel, which is advantageous for the application of collagen hydrogels in fields such as visual materials and artificial corneas.

Claims

1. A method for preparing a colorless and transparent collagen hydrogel, (1) A process in which the solid collagen material is thoroughly dissolved in water, centrifuged, and the supernatant is collected, and the total concentration of various collagens in the supernatant is 0.01 wt.% or more and 10 wt.% or less, (2) A step of adjusting the pH of the supernatant obtained in step (1) to 5.0 to 9.0 with an alkaline solution or an acidic solution, (3) To the solution obtained in step (2), add HPO ４ ２‐ -citric acid, HCO ３ ‐ -citric acid, CO ３ ２‐ -citric acid, H ２ PO ４ ‐ -citric acid, PO ４ ３‐ -citric acid, Ac ‐ -citric acid, Ac ‐ -H ＋ -NH ４ ＋ , HAc-Ac ‐ , HAc-Ac ‐ -NH ４ ＋ , PO ４ ３‐ -H ＋ -triethylamine, HPO ４ ２‐ -H ＋ -triethylamine, H ２ PO ４ ‐ -H ＋ -triethylamine, PO ４ ３‐ -HPO ４ ２‐ , Tris-HAc, Tris-H ２ PO ４ ‐ , Tris-NH ４ ＋ , Tris-H ＋ , Tris-lysine hydrochloride, Tris-lysine hydrochloride-ethylenediaminetetraacetate ion, PO ４ ３‐ -H ２ PO ４ ‐ , HPO ４ ２‐ -H ２ PO ４ ‐ , HPO ４ ２‐ -NH ４ ＋ , H ２ PO ４ ‐ - HCO ３ ‐ , H ２ PO ４ ‐ -CO ３ ２‐ NH ４ ＋ -NH ３ A step of adding a specific ion / molecular pair containing at least one of the following, (4) Adding a crosslinking agent to the solution obtained in step (3), which includes at least one of the following: NHS ester, sulfonyl chloride, acid anhydride, fluorophenyl ester, proanthocyanidin, genipin, aldehyde, isothiocyanate, isocyanate, acyl azide, epoxide, aryl halide, imide ester, and carbodiimide. (5) A step of adding a protective molecule to the solution obtained in step (4), which includes at least one of the following: sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, tween, triton, ethylene glycol, phenylalanine, proline, lecithin, glutamic acid, lysine, cysteine, mangiferin, glycine, aspartic acid, n-butanol, propylene glycol, sodium ethylenediaminetetraacetate, stearic acid, span, glycerin, and gelatin. (6) A step of performing a low-temperature irradiation treatment on the solution obtained in step (5), This includes, in order, The low-temperature range for the low-temperature irradiation treatment in step (6) is -20 to 5°C. A method for preparing a colorless, transparent collagen hydrogel, characterized by the following features.

2. The method for preparing a colorless, transparent collagen hydrogel according to claim 1, characterized in that the solid collagen material in step (1) is selected from one or more combinations of type I, type II, type III, and type V collagen.

3. The method for preparing a colorless, transparent collagen hydrogel according to claim 1, characterized in that the acidic solution in step (2) comprises at least one of salicylic acid, lactic acid, sulfuric acid, tartaric acid, citric acid, phosphoric acid, oxalic acid, acetic acid, ethanedioic acid, succinic acid, hydrochloric acid, maleic acid, benzoic acid, nitric acid, malic acid, nicotinic acid, sodium dihydrogen phosphate, and formic acid, and the alkaline solution comprises at least one of tetramethylethylenediamine, sodium hydroxide, triethylamine, sodium carbonate, potassium hydroxide, calcium hydroxide, magnesium hydroxide, aqueous ammonia, disodium hydrogen phosphate, and sodium bicarbonate.

4. The method for preparing a colorless, transparent collagen hydrogel according to claim 3, characterized in that the concentration of the acidic or alkaline solution in step (2) is in the range of 0.01 M to 2 M.

5. The method for preparing a colorless, transparent collagen hydrogel according to claim 1, characterized in that the concentration of the specific ion / molecular pair in step (3) is in the range of 0.1 M to 10 M.

6. The method for preparing a colorless, transparent collagen hydrogel according to claim 1, characterized in that the concentration of the crosslinking agent in step (4) is in the range of 0.0001% to 5%.

7. The method for preparing a colorless, transparent collagen hydrogel according to claim 1, characterized in that the concentration of the protective molecule in step (5) is in the range of 1% to 50%.

8. A colorless, transparent collagen hydrogel, characterized by being prepared using the method for preparing a colorless, transparent collagen hydrogel described in any one of claims 1 to 7.

9. The turbidity range of the aforementioned colorless and transparent collagen hydrogel is 0.01 × 10⁻⁶. ４ ~2 x 10 ４ The colorless, transparent collagen hydrogel according to claim 8, characterized in that it is NTU.

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