Colorless and transparent collagen hydrogel and method for preparing same

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 and transparent collagen hydrogel and a method for preparing the same. [Background technology]

[0002] Collagen is the major structural protein of connective tissue and is widely distributed in human tissues and organs, including bone, skin, blood vessels, ligaments, cartilage, muscle, and tendons, accounting for 25% to 30% of all animal proteins. Collagen, the main component of the extracellular matrix, plays an important role in maintaining the biological properties and structural integrity of the extracellular matrix. Collagen itself has a unique quaternary structure and, under certain conditions, can spontaneously aggregate to form collagen fibers and further hydrogels. However, due to their color, conventional collagen hydrogels exhibit a distinct "cloudy" (white, opaque) state, making them unsuitable for applications such as visual materials and artificial corneas.

[0003] The present invention mainly solves the problem of "cloudiness" of collagen hydrogel, prepares a colorless and transparent collagen hydrogel, and improves the light transmittance of the collagen hydrogel. Summary of the Invention

[0004] The present invention provides a colorless and transparent collagen hydrogel and a method for preparing the same. A method for preparing a colorless and transparent collagen hydrogel, comprising: (1) thoroughly dissolving a solid collagen material in water, centrifuging the solution, and collecting the supernatant; (2) adjusting the pH of the supernatant obtained in step (1) to 5.0 to 9.0 with an alkaline or acidic solution; (3) adding a specific ion / molecule pair to the solution obtained in step (2); (4) adding a crosslinker to the solution obtained in step (3); (5) adding a protecting molecule to the solution obtained in step (4); (6) subjecting the solution obtained in step (5) to low-temperature irradiation treatment; A method for preparing a colorless and transparent collagen hydrogel, comprising the steps of:

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

[0006] In the method for preparing a colorless and transparent collagen hydrogel of the present invention, the acidic solution in step (2) contains 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. The alkaline solution contains at least one of tetramethylethylenediamine, sodium hydroxide, triethylamine, sodium carbonate, potassium hydroxide, calcium hydroxide, magnesium hydroxide, aqueous ammonia, disodium hydrogen phosphate, and sodium bicarbonate. Because collagen aqueous solutions tend to become cloudy under neutral pH conditions, the pH is precisely adjusted to, for example, pH 6.9 or 7.9 with an acidic or alkaline solution to prevent collagen molecules from aggregating into 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 and transparent collagen hydrogel of the present invention, the specific ion / molecule pair in the above step (3) 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-ethylenediaminetetraacetate ion, PO4 3‐ -H2PO4 ‐ , HPO4 2‐ -H2PO4 ‐ , HPO4 2‐ -NH4 + , H2PO4 ‐ -HCO3 ‐ , H2PO4 ‐ -CO3 2‐ , NH4 + -NH3. By adjusting the ionic environment of the collagen solution using a specific ion / molecule pair, the formation of collagen fibers due to the self-assembly of collagen molecules is suppressed, the collagen solution maintains uniform fluidity, and the "cloudiness" phenomenon is prevented. The concentration of the specific ion / molecule pair is in the range of 0.1M to 10M.

[0008] In the method for preparing a colorless and transparent collagen hydrogel of the present invention, the crosslinker used in step (4) includes at least one of NHS ester, sulfonyl chloride, acid anhydride, fluorophenyl ester, proanthocyanidin, genipin, aldehyde, isothiocyanate, isocyanate, acyl azide, epoxide, aryl halide, imide ester, and carbodiimide. The concentration of the crosslinker is in the range of 0.0001% to 5%.

[0009] In the method for preparing a colorless and transparent collagen hydrogel of the present invention, the protective molecule used in step (5) includes at least one of sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, 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, spanish, glycerin, and gelatin. The addition of the protective molecule is primarily intended to reduce the effect of irradiation on the activity of the collagen molecules. The concentration of the protective molecule ranges from 1% to 50%.

[0010] In the method for preparing a colorless and transparent collagen hydrogel of the present invention, the low temperature range for the low-temperature irradiation treatment in the above step (6) is −20 to 5° C. The low temperature condition is adopted mainly to reduce the effect 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-mentioned 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: During the collagen hydrogel formation process, collagen molecules self-assemble to form collagen fibers. The resulting collagen hydrogel typically exhibits a white, opaque state, making it difficult to use in visual materials, artificial corneas, and other applications. To enhance the transparency of collagen hydrogels, the preparation method for colorless and transparent collagen hydrogels of the present invention primarily involves precisely adjusting the pH of the collagen solution and adding specific ion / molecule pairs to balance the surface charge of collagen molecules and increase the ionic strength of the solution, thereby minimizing the self-assembly behavior of collagen molecules. Based on this, an appropriate crosslinking agent is added to slowly induce the crosslinking reaction of collagen molecules, avoiding the formation of collagen fibers due to rapid crosslinking and the resulting loss of collagen hydrogel transparency. To better meet the sterilization and inactivation requirements of colorless and transparent collagen hydrogels for medical devices and avoid the loss of transparency due to the effects of irradiation on the collagen molecules, the preparation method of the present invention further includes the steps of adding protective molecules and low-temperature treatment to minimize the effects of irradiation on the collagen molecules. Through the above steps, a stable, colorless, and transparent collagen hydrogel can be prepared. [Brief explanation of the drawings]

[0014] [Figure 1] 1A and 1B show the optical transparency effect of collagen hydrogels (FIG. 1A shows the sample of Example 1, FIG. 1B shows the sample of Example 2, FIG. 1C shows the sample of Example 3, FIG. 1D shows the sample of Example 4, FIG. 1E shows the sample of Example 5, FIG. 1F shows the sample of Comparative Example 1, FIG. 1G shows the sample of Comparative Example 2, FIG. 1H shows the sample of Comparative Example 3, FIG. 1I shows the sample of Comparative Example 4, and FIG. 1J shows the sample of Comparative Example 5). DETAILED DESCRIPTION OF THE INVENTION

[0015] Example 1 A method for preparing a colorless and transparent collagen hydrogel, comprising: (1) thoroughly dissolving 1 g of type I collagen in 100 mL of water, centrifuging the solution, and collecting the supernatant; (2) adjusting the pH of the supernatant obtained in step (1) to 7.8 with 1 M magnesium hydroxide solution or 0.1 M acetic acid aqueous solution; (3) The solution obtained in step (2) is treated with a specific ion / molecule pair, H2PO4 ‐ - adding citric acid at a concentration of 0.1M; (4) adding 0.01% proanthocyanidins to the solution obtained in step (3); (5) adding 5% n-butanol to the solution obtained in step (4); (6) subjecting the solution obtained in step (5) to electron beam irradiation treatment at 0°C; The method for preparing a colorless and transparent collagen hydrogel comprises the steps of:

[0016] Example 2 A method for preparing a colorless and transparent collagen hydrogel, comprising: (1) thoroughly dissolving 0.1 g of type II collagen and 0.4 g of type III collagen in 100 mL of water, centrifuging the mixture, and collecting the supernatant; (2) adjusting the pH of the supernatant obtained in step (1) to 7.1 with a 0.5 M aqueous ammonia solution or a 1 M aqueous oxalic acid solution;

[0017] (3) The solution obtained in step (2) is treated with a specific ion / molecule pair, Tris-H2PO4 ‐ at a concentration of 1M; (4) adding 0.001% genipin and 1% isocyanate to the solution obtained in step (3); (5) adding 1% glycerin and 2% Tween to the solution obtained in step (4); (6) The solution obtained in step (5) was cooled to -5°C. 60 A step of performing an irradiation treatment; The method for preparing a colorless and transparent collagen hydrogel comprises the steps of:

[0018] Example 3 A method for preparing a colorless and transparent collagen hydrogel, comprising: (1) thoroughly dissolving 20 g of type I collagen, 0.6 g of type III collagen, and 5 g of type V collagen in 1000 mL of water, centrifuging the mixture, and collecting the supernatant; (2) adjusting the pH of the supernatant obtained in step (1) to 6.5 with 0.2 M sodium hydroxide solution or 1 M hydrochloric acid solution; (3) adding a specific ion / molecule pair, Tris-lysine hydrochloride-ethylenediaminetetraacetate ion, 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) subjecting the solution obtained in step (5) to gamma irradiation treatment at 5°C; A method for preparing a colorless and transparent collagen hydrogel, comprising the steps of:

[0019] Example 4 A method for preparing a colorless and transparent collagen hydrogel, comprising: (1) thoroughly dissolving 5 g of type I collagen and 5 g of type II collagen in 100 mL of water, centrifuging the mixture, and collecting the supernatant; (2) adjusting the pH of the supernatant obtained in step (1) to 5.0 with 0.2 M sodium carbonate, 0.01 M potassium hydroxide or 2 M ethanedioic acid, 1 M succinic acid aqueous solution; (3) The solution obtained in step (2) is treated with a specific ion / molecule pair, PO4 3‐ -HPO4 2‐ , H2PO4 ‐ - Citric acid, Tris-H2PO4 ‐ 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) The solution obtained in step (5) was cooled to -20°C. 60 A step of performing an irradiation treatment; The method for preparing a colorless and transparent collagen hydrogel comprises the steps of:

[0020] Example 5 A method for preparing a colorless and transparent collagen hydrogel, comprising: (1) thoroughly dissolving 0.1 g of type II collagen in 1000 mL of water, centrifuging the solution, and collecting the supernatant; (2) adjusting the pH of the supernatant obtained in step (1) to 9.0 with 2 M tetramethylethylenediamine, 1 M disodium hydrogen phosphate solution, or 0.1 M citric acid aqueous solution; (3) The solution obtained in step (2) is treated with a specific ion / molecule pair, HAc-Ac. ‐ , H2PO4 ‐ -CO3 2‐ , NH4 + - adding NH3 at concentrations of 1M, 1M and 5M, respectively; (4) adding 0.5% sulfonyl chloride and 0.5% imidoester to the solution obtained in step (3); (5) adding 1% sodium dodecylbenzenesulfonate and 5% propylene glycol to the solution obtained in step (4); (6) subjecting the solution obtained in step (5) to gamma irradiation at 4°C; The method for preparing a colorless and transparent collagen hydrogel comprises the steps of:

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

[0022] Comparative Example 1 A method for preparing a collagen hydrogel, comprising: (1) thoroughly dissolving 1 g of type I collagen in 100 mL of water, centrifuging the solution, and collecting the supernatant;

[0023] (2) adjusting the supernatant obtained in step (1) to a neutral pH of 7.4 with a 1 M magnesium hydroxide solution or a 0.1 M aqueous acetic acid solution; The method for preparing a collagen hydrogel comprises the steps of:

[0024] Comparative Example 2 A method for preparing a collagen hydrogel, comprising: (1) thoroughly dissolving 1 g of type I collagen in 100 mL of water, centrifuging the solution, and collecting the supernatant;

[0025] (2) adjusting the pH of the supernatant obtained in step (1) to 7.8 with 1 M magnesium hydroxide solution or 0.1 M acetic acid aqueous solution;

[0026] (3) subjecting the solution obtained in step (2) to electron beam irradiation treatment at 0°C; The method for preparing a collagen hydrogel comprises the steps of:

[0027] Comparative Example 3 A method for preparing a colorless and transparent collagen hydrogel, comprising: (1) thoroughly dissolving 20 g of type I collagen, 0.6 g of type III collagen, and 5 g of type V collagen in 1000 mL of water, centrifuging the mixture, and collecting the supernatant;

[0028] (2) 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) subjecting the solution obtained in step (3) to gamma irradiation treatment at 5°C; The method for preparing a colorless and transparent collagen hydrogel comprises the steps of:

[0031] Comparative Example 4 A method for preparing a colorless and transparent collagen hydrogel, comprising: (1) thoroughly dissolving 5 g of type I collagen and 5 g of type II collagen in 100 mL of water, centrifuging the mixture, and collecting the supernatant;

[0032] (2) adjusting the pH of the supernatant obtained in step (1) to 5.0 with 0.2 M sodium carbonate, 0.01 M potassium hydroxide or 2 M ethanedioic acid, 1 M succinic acid aqueous solution;

[0033] (3) The solution obtained in step (2) is treated with a specific ion / molecule pair, PO4 3‐ -HPO4 2‐ , H2PO4 ‐ - Citric acid, Tris-H2PO4 ‐ at concentrations of 0.5M, 10M, and 1M, respectively;

[0034] (4) The solution obtained in step (3) was cooled to -20°C. 60 A step of performing an irradiation treatment; A method for preparing a colorless and transparent collagen hydrogel, comprising the steps of:

[0035] Comparative Example 5 A method for preparing a colorless and transparent collagen hydrogel, comprising: (1) thoroughly dissolving 0.1 g of type II collagen in 1000 mL of water, centrifuging the solution, and collecting the supernatant;

[0036] (2) adjusting the pH of the supernatant obtained in step (1) to 9.0 with 2 M tetramethylethylenediamine, 1 M disodium hydrogen phosphate solution, or 0.1 M citric acid aqueous solution;

[0037] (3) subjecting the solution obtained in step (2) to gamma irradiation at 4°C; The method for preparing a colorless and transparent collagen hydrogel comprises the steps of:

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

[0039] Table 1. Collagen hydrogel turbidity test JPEG2026502826000002.jpg52170

[0040] As can be seen from Table 1, the turbidity of the collagen hydrogels in Examples 1 to 5 was very low. The collagen hydrogel in Example 1 in Figure 1A was very transparent, with the letters on the bottom of the material clearly visible. Similarly, the transparency of the collagen hydrogels in Figures 1B to 1E was also very high, with the scale readings on the syringe visible through the collagen hydrogel. Meanwhile, the turbidity of the collagen hydrogels in Comparative Examples 1 to 5 was very high. The collagen hydrogel in Figure 1F was very cloudy, with the letters on the bottom of the material not visible. Similarly, the collagen hydrogels in Figures 1G-1J were very blurry, with poor light transmittance, and the scale readings on the syringe were barely visible.

[0041] The method for preparing a colorless and transparent collagen hydrogel of the present invention primarily relies on the synergistic effects of precisely adjusting the pH of the collagen solution and adding specific ion / molecule 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 the increase in collagen fibers caused by rapid crosslinking and reducing the transparency of the collagen hydrogel. To better meet the sterilization and inactivation requirements of colorless and transparent collagen hydrogels for medical devices and avoid the loss of transparency due to the effects of irradiation on the collagen molecules, the preparation method of the present invention further includes the 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 and transparent collagen hydrogel can be prepared.

[0042] The above-mentioned examples merely illustrate the preferred embodiments of the present invention, and various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope determined by the claims of the present invention. [Industrial Applicability]

[0043] The method for preparing a colorless collagen hydrogel of the present invention solves the problem of "cloudiness" in collagen hydrogels, improves the optical transparency of collagen hydrogels, and makes it possible to prepare colorless and transparent collagen hydrogels, 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, comprising: (1) a step of sufficiently dissolving a solid collagen material in water, centrifuging the resulting solution, and collecting the supernatant, the total concentration of various collagens in the supernatant being 0.01 wt.% or more and 10 wt.% or less; (2) adjusting the pH of the supernatant obtained in step (1) to 5.0 to 9.0 with an alkaline or acidic solution; (3) The solution obtained in step (2) is added with HPO ４ ２‐ - Citric acid, HCO ３ ‐ - Citric acid, CO ３ ２‐ - Citric acid, H ２ P.O. ４ ‐ - Citric acid, PO ４ ３‐ - Citric acid, Ac ‐ - Citric acid, Ac ‐ -H ＋ -NH ４ ＋ , HAc‐Ac ‐ , HAc‐Ac ‐ -NH ４ ＋ , P.O. ４ ３‐ -H ＋ - Triethylamine, HPO ４ ２‐ -H ＋ - Triethylamine, H ２ P.O. ４ ‐ -H ＋ - Triethylamine, PO ４ ３‐ -HPO ４ ２‐ , Tris-HAc, Tris-H ２ P.O. ４ ‐ , Tris-NH ４ ＋ , Tris-H ＋ , Tris-lysine hydrochloride, Tris-lysine hydrochloride-ethylenediaminetetraacetate ion, PO ４ ３‐ -H ２ P.O. ４ ‐ , H.P.O. ４ ２‐ -H ２ P.O. ４ ‐ , H.P.O. ４ ２‐ -NH ４ ＋ , H ２ P.O. ４ ‐ -HCO ３ ‐ , H ２ P.O. ４ ‐ -CO ３ ２‐ , N.H. ４ ＋ -NH ３ adding a specific ion / molecule pair comprising at least one of: (4) adding a cross-linking agent to the solution obtained in step (3), the cross-linking agent including at least one of an NHS ester, a sulfonyl chloride, an acid anhydride, a fluorophenyl ester, a proanthocyanidin, a genipin, an aldehyde, an isothiocyanate, an isocyanate, an acyl azide, an epoxide, an aryl halide, an imidoester, and a carbodiimide; (5) adding a protective molecule to the solution obtained in step (4), the protective molecule comprising at least one of sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, 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; (6) subjecting the solution obtained in step (5) to low-temperature irradiation treatment; A method for preparing a colorless and transparent collagen hydrogel, comprising the steps of:

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

3. 2. The method for preparing a colorless and transparent collagen hydrogel according to claim 1, wherein the acidic solution in step (2) contains 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 contains at least one of tetramethylethylenediamine, sodium hydroxide, triethylamine, sodium carbonate, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia water, disodium hydrogen phosphate, and sodium bicarbonate.

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

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

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

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

8. 8. The method for preparing a colorless and transparent collagen hydrogel according to claim 1, wherein the low temperature range of the low-temperature irradiation treatment in step (6) is -20 to 5°C.

9. A colorless and transparent collagen hydrogel, characterized in that it is prepared by the method for preparing a colorless and transparent collagen hydrogel according to any one of claims 1 to 8.

10. The turbidity range of the colorless and transparent collagen hydrogel is 0.01 × 10 ４ ~2 x 10 ４ The colorless and transparent collagen hydrogel according to claim 9, which is NTU.

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