Colorless transparent collagen hydrogel and preparation method thereof

Abstract

The present invention relates to the field of biomaterials, and more specifically, to a colorless and transparent collagen hydrogel and a preparation method thereof. The collagen hydrogel of the present invention has the characteristic of high transparency and can be used in the fields of visual repair materials, corneal repair, etc.

Description

Technical Field

[0001] The present invention relates to a biomaterial and a preparation method thereof, in particular to a colorless and transparent collagen hydrogel and a preparation method thereof. Background Art

[0002] Collagen is the primary structural protein of connective tissue and is widely distributed in tissues and organs such as bone, skin, blood vessels, ligaments, cartilage, muscles, and tendons, accounting for 25% to 30% of total animal protein. As a major component of the extracellular matrix (ECM), collagen plays a crucial role in maintaining its biological properties and structural integrity. Collagen possesses a unique quaternary structure that, under certain conditions, can spontaneously aggregate into collagen fibrils and further form hydrogels. However, conventional collagen hydrogels exhibit a pronounced "white turbidity" (i.e., an opaque white color), hindering their application in visual materials and artificial corneas.

[0003] The present invention mainly solves the "white turbidity" problem of collagen hydrogel, can prepare colorless and transparent collagen hydrogel, and enhances the light transmittance of collagen hydrogel. Summary of the Invention

[0004] The invention provides a colorless and transparent collagen hydrogel and a preparation method thereof.

[0005] A method for preparing a colorless and transparent collagen hydrogel, comprising the following steps in sequence:

[0006] (1) Dissolve the solid collagen material in water, fully dissolve it, centrifuge it, and collect the supernatant;

[0007] (2) adjusting the pH value of the supernatant obtained in step (1) to 5.0-9.0 using an alkaline solution or an acidic solution;

[0008] (3) adding a specific ion / molecule pair to the solution obtained in step (2);

[0009] (4) adding a crosslinking agent to the solution obtained in step (3);

[0010] (5) adding a protective molecule to the solution obtained in step (4);

[0011] (6) The solution obtained in step (5) is subjected to low-temperature irradiation treatment.

[0012] The present invention provides a method for preparing a colorless and transparent collagen hydrogel, 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; and the total concentration of various types of collagen in the supernatant of step (1) is greater than or equal to 0.01 wt.% but less than or equal to 10 wt.%. If the total collagen concentration is less than 0.01 wt.%, the collagen solution cannot form a hydrogel; and if the total collagen concentration is greater than 10 wt.%, the collagen molecules in the collagen solution easily form collagen fibers, resulting in "white turbidity" and unable to form a colorless and transparent collagen hydrogel.

[0013] The present invention provides a method for preparing a colorless and transparent collagen hydrogel, wherein the acidic solution in step (2) includes at least one of salicylic acid, lactic acid, sulfuric acid, tartaric acid, citric acid, phosphoric acid, oxalic acid, acetic acid, oxalic 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 includes at least one of tetramethylethylenediamine, sodium hydroxide, triethylamine, sodium carbonate, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia water, disodium hydrogen phosphate, and sodium bicarbonate. Collagen aqueous solution is prone to "white turbidity" under neutral pH conditions. The pH value of the collagen aqueous solution is precisely controlled by using an acidic or alkaline solution, such as pH = 6.9 or pH = 7.9, to ensure that collagen molecules do not aggregate to form collagen fibers. The concentration range of the acidic or alkaline solution is 0.01M–2M.

[0014] The present invention provides a method for preparing a colorless transparent collagen hydrogel, wherein the specific ion / molecule pair in step (3) includes 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 anion, PO4 3- -H2PO4 - 、HPO4 2- -H2PO4 - 、HPO4 2- -NH4 + 、H2PO4 - -HCO3 - 、H2PO4 - -CO3 2- NH4 + -NH3. By regulating the ionic environment of the collagen solution through specific ion / molecule pairs, collagen molecules are prevented from self-assembling into collagen fibers, maintaining the homogeneous fluidity of the collagen solution and preventing the occurrence of "white turbidity." The concentration range of the specific ion / molecule pairs is 0.1M–10M.

[0015] The present invention provides a method for preparing a colorless and transparent collagen hydrogel, wherein the crosslinking agent in step (4) comprises 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 crosslinking agent ranges from 0.0001% to 5%.

[0016] The present invention provides a method for preparing a colorless and transparent collagen hydrogel, wherein the protective molecule in step (5) comprises 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, sodium ethylenediaminetetraacetic acid, stearic acid, Span, glycerol, and gelatin. The protective molecule is added primarily to reduce the effect of irradiation on the activity of the collagen molecules. The concentration of the protective molecule ranges from 1% to 50%.

[0017] The present invention provides a method for preparing a colorless and transparent collagen hydrogel, wherein the low temperature range of the low temperature irradiation treatment in step (6) is -20-5° C. The low temperature condition is mainly used to reduce the effect of the irradiation treatment on the activity of collagen molecules.

[0018] A colorless and transparent collagen hydrogel prepared by any one of the methods for preparing a colorless and transparent collagen hydrogel of the present invention.

[0019] The present invention provides a colorless and transparent collagen hydrogel, wherein the turbidity range of the colorless and transparent collagen hydrogel is 0.01×10 4 -2×10 4 NTU.

[0020] The present invention has the following beneficial effects: During the process of collagen hydrogel formation, collagen molecules self-assemble into collagen fibers. The resulting collagen hydrogel is typically white and opaque, making it unsuitable for use in visual materials, artificial corneas, and the like. To enhance the transparency of the collagen hydrogel, the present invention provides a method for preparing a colorless, transparent collagen hydrogel. This method primarily involves precisely controlling the pH of the collagen solution and adding specific ion / molecule pairs to coordinate the effects, balancing the surface charge of the collagen molecules and enhancing the ionic strength of the solution, thereby minimizing the self-assembly behavior of the collagen molecules. Furthermore, a suitable crosslinking agent is added to allow the collagen molecules to undergo a slow and gentle crosslinking reaction, avoiding rapid crosslinking to form more collagen fibers, thereby preventing a decrease in the transparency of the collagen hydrogel. To further meet the sterilization and inactivation requirements of colorless, transparent collagen hydrogels in medical devices and prevent the effects of irradiation on the collagen molecules in the colorless, transparent collagen hydrogel, which can lead to a decrease in transparency, the present invention further includes the steps of adding protective molecules and performing 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect diagram of collagen hydrogel light transmittance ( Figure 1 A is the sample of Example 1, Figure 1 B is the sample of Example 2, Figure 1 C is the sample of Example 3, Figure 1 D is the sample of Example 4, Figure 1 E is the sample of Example 5, Figure 1 F is the sample of comparative example 1, Figure 1 G is the sample of Example 2, Figure 1 H is the sample of comparative example 3, Figure 1 1 is the sample of comparative example 4, Figure 1 J is the sample of Comparative Example 5). DETAILED DESCRIPTION

[0022] Example 1

[0023] A preparation method of a colorless and transparent collagen hydrogel comprises the following steps in sequence:

[0024] (1) Dissolve 1 g of type I collagen in 100 mL of water, centrifuge, and collect the supernatant;

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

[0026] (3) Adding a specific ion / molecule pair H2PO4 to the solution obtained in step (2) --Citric acid, concentration 0.1 M;

[0027] (4) adding 0.01% proanthocyanidins to the solution obtained in step (3);

[0028] (5) adding 5% n-butanol to the solution obtained in step (4);

[0029] (6) The solution obtained in step (5) was subjected to electron beam irradiation treatment at 0°C.

[0030] Example 2

[0031] A preparation method of a colorless and transparent collagen hydrogel comprises the following steps in sequence:

[0032] (1) Dissolve 0.1 g of type II collagen and 0.4 g of type III collagen in 100 mL of water, centrifuge, and collect the supernatant;

[0033] (2) adjusting the pH of the supernatant obtained in step (1) to 7.1 with 0.5 M aqueous ammonia solution or 1 M oxalic acid solution;

[0034] (3) Adding a specific ion / molecule pair Tris-H2PO4 to the solution obtained in step (2) - , concentration is 1M;

[0035] (4) adding 0.001% genipin and 1% isocyanate to the solution obtained in step (3);

[0036] (5) adding 1% glycerol and 2% Tween to the solution obtained in step (4);

[0037] (6) The solution obtained in step (5) was subjected to Co 60 Irradiation treatment.

[0038] Example 3

[0039] A preparation method of a colorless and transparent collagen hydrogel comprises the following steps in sequence:

[0040] (1) Dissolve 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, centrifuge, and collect the supernatant;

[0041] (2) adjusting the pH of the supernatant obtained in step (1) to 6.5 using 0.2 M sodium hydroxide solution or 1 M hydrochloric acid solution;

[0042] (3) adding a specific ion / molecule pair Tris-lysine hydrochloride-ethylenediaminetetraacetic acid ion to the solution obtained in step (2) at a concentration of 2 M;

[0043] (4) adding 1% NHS ester to the solution obtained in step (3);

[0044] (5) adding 5% ethylene glycol and 10% mangiferin to the solution obtained in step (4);

[0045] (6) The solution obtained in step (5) was irradiated with gamma rays at 5°C.

[0046] Example 4

[0047] A preparation method of a colorless and transparent collagen hydrogel comprises the following steps in sequence:

[0048] (1) Dissolve 5 g of type II collagen and 5 g of type II collagen in 100 mL of water, centrifuge, and collect the supernatant;

[0049] (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 oxalic acid, 1 M succinic acid solution;

[0050] (3) Adding specific ion / molecule pair PO4 to the solution obtained in step (2) 3- -HPO4 2- 、H2PO4 - -Citric acid, Tris-H2PO4 - , the concentrations are 0.5M, 10M, and 1M respectively;

[0051] (4) adding 0.2% isothiocyanate, 5% NHS ester and 0.5% acyl azide to the solution obtained in step (3);

[0052] (5) adding 2% phenylalanine, 1% Triton X-ray distillate, and 50% lysine to the solution obtained in step (4);

[0053] (6) The solution obtained in step (5) was subjected to Co 60 Irradiation treatment.

[0054] Example 5

[0055] A preparation method of a colorless and transparent collagen hydrogel comprises the following steps in sequence:

[0056] (1) Dissolve 0.1 g of type II collagen in 1000 mL of water, centrifuge, and collect the supernatant;

[0057] (2) adjusting the pH of the supernatant obtained in step (1) to 9.0 using 2 M tetramethylethylenediamine, 1 M disodium hydrogen phosphate solution, or 0.1 M citric acid solution;

[0058] (3) Adding a specific ion / molecule pair HAc-Ac to the solution obtained in step (2)- 、H2PO4 - -CO3 2- NH4 + -NH3, concentrations of 1M, 1M, and 5M;

[0059] (4) adding 0.5% sulfonyl chloride and 0.5% imide ester to the solution obtained in step (3);

[0060] (5) adding 1% sodium dodecylbenzenesulfonate and 5% propylene glycol to the solution obtained in step (4);

[0061] (6) The solution obtained in step (5) was irradiated with gamma rays at 4°C.

[0062] Example 6

[0063] Comparative test of the effects of colorless and transparent collagen hydrogel

[0064] 1. Preparation of comparative collagen hydrogels

[0065] Comparative Example 1: Preparation of a collagen hydrogel, comprising the following steps in sequence:

[0066] (1) Dissolve 1 g of type I collagen in 100 mL of water, centrifuge, and collect the supernatant;

[0067] (2) The pH value of the supernatant obtained in step (1) was adjusted to neutral at 7.4 using 1 M magnesium hydroxide solution or 0.1 M acetic acid solution.

[0068] Comparative Example 2: Preparation of a collagen hydrogel, comprising the following steps in sequence:

[0069] (1) Dissolve 1 g of type I collagen in 100 mL of water, centrifuge, and collect the supernatant;

[0070] (2) adjusting the pH of the supernatant obtained in step (1) to 7.8 using a 1 M magnesium hydroxide solution or a 0.1 M acetic acid solution;

[0071] (3) The solution obtained in step (2) was subjected to electron beam irradiation treatment at 0°C.

[0072] Comparative Example 3

[0073] A preparation method of a colorless and transparent collagen hydrogel comprises the following steps in sequence:

[0074] (1) Dissolve 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, centrifuge, and collect the supernatant;

[0075] (2) adding 1% NHS ester to the solution obtained in step (1);

[0076] (3) adding 5% ethylene glycol and 10% mangiferin to the solution obtained in step (2);

[0077] (4) The solution obtained in step (3) was irradiated with gamma rays at 5°C.

[0078] Comparative Example 4

[0079] A preparation method of a colorless and transparent collagen hydrogel comprises the following steps in sequence:

[0080] (1) Dissolve 5 g of type II collagen and 5 g of type II collagen in 100 mL of water, centrifuge, and collect the supernatant;

[0081] (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 oxalic acid, 1 M succinic acid solution;

[0082] (3) Adding specific ion / molecule pair PO4 to the solution obtained in step (2) 3- -HPO4 2- 、H2PO4 - -Citric acid, Tris-H2PO4 - , the concentrations are 0.5M, 10M, and 1M respectively;

[0083] (4) The solution obtained in step (3) was subjected to Co 60 Irradiation treatment.

[0084] Comparative Example 5

[0085] A preparation method of a colorless and transparent collagen hydrogel comprises the following steps in sequence:

[0086] (1) Dissolve 0.1 g of type II collagen in 1000 mL of water, centrifuge, and collect the supernatant;

[0087] (2) adjusting the pH of the supernatant obtained in step (1) to 9.0 using 2 M tetramethylethylenediamine, 1 M disodium hydrogen phosphate solution, or 0.1 M citric acid solution;

[0088] (3) The solution obtained in step (2) was irradiated with gamma rays at 4°C.

[0089] 2. Comparison of effect tests

[0090] (1) Turbidity test of collagen hydrogel: The turbidity of collagen hydrogel was observed on a turbidimeter (TZD-BZ-905, Suzhou DiagVita Technology Co., Ltd.) at a wavelength of 625 nm. The test results are shown in Table 1. Figure 1 ,in, Figure 1 A is the sample of Example 1, Figure 1 B is the sample of Example 2, Figure 1 C is the sample of Example 3, Figure 1 D is the sample of Example 4, Figure 1 E is the sample of Example 5, Figure 1 F is the sample of comparative example 1, Figure 1 G is the sample of Example 2, Figure 1 H is the sample of comparative example 3, Figure 1 1 is the sample of comparative example 4, Figure 1 J is the sample of Comparative Example 5.

[0091] Table 1 Collagen hydrogel turbidity test

[0092] sample <![CDATA[Turbidity (×10 4 NTU)]]> sample <![CDATA[Turbidity (×10 4 NTU)]]> Example 1 0.15±0.02 Comparative Example 1 10.27±1.37 Example 2 0.09±0.01 Comparative Example 2 15.63±2.46 Example 3 1.11±0.02 Comparative Example 3 18.03±0.73 Example 4 2.00±0.63 Comparative Example 4 20.34±1.77 Example 5 0.01±0.002 Comparative Example 5 7.83±1.54

[0093] As shown in Table 1, the turbidity of the collagen hydrogel in Examples 1-5 is very low. Figure 1 The collagen hydrogel of Example 1 in A is very transparent, and the text on the bottom of the material can be clearly seen. Figure 1 The transparency of the collagen hydrogel in B-1E is also very high, and the syringe scale value can be seen through the collagen hydrogel; while the turbidity of the collagen hydrogel in Comparative Example 1-5 is very large. Figure 1 The collagen hydrogel in F is very turbid and the text at the bottom of the material cannot be seen. Similarly, Figure 1 The collagen hydrogel in G-1J is very blurry and has poor light transmittance, and the syringe scale values ​​are basically invisible.

[0094] The preparation method of a colorless and transparent collagen hydrogel of the present invention mainly reduces the self-assembly behavior of the collagen molecules as much as possible by precisely controlling the pH value of the collagen solution and adding specific ion / molecule pairs to coordinate, balance the surface charge of the collagen molecules, enhance the ionic strength in the solution, etc.; on this basis, a suitable cross-linking agent is added to allow the collagen molecules to undergo a slow and gentle cross-linking reaction, avoiding rapid cross-linking to form more collagen fibers, thereby avoiding a decrease in the transparency of the collagen hydrogel. In order to further meet the sterilization and inactivation requirements of colorless and transparent collagen hydrogels in medical devices and avoid the influence of irradiation treatment on the collagen molecules in the colorless and transparent collagen hydrogel, which leads to a decrease in transparency, the preparation method of the present invention further adds the steps of adding protective molecules and low-temperature treatment to minimize the influence of irradiation treatment on the collagen molecules. Through the above steps, a stable colorless and transparent collagen hydrogel can be prepared.

[0095] The embodiments described above are merely descriptions of preferred implementations of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A method for preparing a colorless and transparent collagen hydrogel, characterized in that: The following steps are included in sequence: (1) Dissolve 1 g of type I collagen in 100 mL of water, centrifuge, and collect the supernatant; (2) adjusting the pH of the supernatant obtained in step (1) to 7.8 using a 1 M magnesium hydroxide solution or a 0.1 M acetic acid solution; (3) Adding a specific ion / molecule pair H2PO4 to the solution obtained in step (2) - -Citric acid, concentration 0.1 M; (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 at 0°C; The turbidity of the prepared colorless and transparent collagen hydrogel was (0.15±0.02)×10 4 NTU.

2. A method for preparing a colorless and transparent collagen hydrogel, characterized in that: The following steps are included in sequence: (1) Dissolve 0.1 g of type II collagen and 0.4 g of type III collagen in 100 mL of water, centrifuge, and collect the supernatant; (2) adjusting the pH of the supernatant obtained in step (1) to 7.1 with 0.5 M aqueous ammonia solution or 1 M oxalic acid solution; (3) Adding a specific ion / molecule pair Tris-H2PO4 to the solution obtained in step (2) - , concentration is 1M; (4) adding 0.001% genipin and 1% isocyanate to the solution obtained in step (3); (5) adding 1% glycerol and 2% Tween to the solution obtained in step (4); (6) The solution obtained in step (5) was subjected to Co 60 irradiation treatment; The turbidity of the prepared colorless and transparent collagen hydrogel was (0.09±0.01)×10 4 NTU.

3. A method for preparing a colorless and transparent collagen hydrogel, characterized in that: The following steps are included in sequence: (1) Dissolve 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, centrifuge, and collect the supernatant; (2) adjusting the pH of the supernatant obtained in step (1) to 6.5 using 0.2 M sodium hydroxide solution or 1 M hydrochloric acid solution; (3) adding a specific ion / molecule pair Tris-lysine hydrochloride-ethylenediaminetetraacetic acid ion to the solution obtained in step (2) at a concentration of 2 M; (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) irradiating the solution obtained in step (5) with gamma rays at 5° C.; The turbidity of the prepared colorless and transparent collagen hydrogel was (1.11±0.02)×10 4 NTU.

4. A method for preparing a colorless and transparent collagen hydrogel, characterized in that: The following steps are included in sequence: (1) Dissolve 5 g of type II collagen and 5 g of type II collagen in 100 mL of water, centrifuge, and collect 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 oxalic acid, 1 M succinic acid solution; (3) Adding specific ion / molecule pair PO4 to the solution obtained in step (2) 3- -HPO4 2- 、H2PO4 - -Citric acid, Tris-H2PO4 - , the concentrations are 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 X-ray distillate, and 50% lysine to the solution obtained in step (4); (6) The solution obtained in step (5) was subjected to Co 60 irradiation treatment; The turbidity of the prepared colorless and transparent collagen hydrogel was (2.00±0.63)×10 4 NTU.

5. A method for preparing a colorless and transparent collagen hydrogel, characterized in that: The following steps are included in sequence: (1) Dissolve 0.1 g of type II collagen in 1000 mL of water, centrifuge, and collect the supernatant; (2) adjusting the pH of the supernatant obtained in step (1) to 9.0 using 2 M tetramethylethylenediamine, 1 M disodium hydrogen phosphate solution, or 0.1 M citric acid solution; (3) Adding a specific ion / molecule pair HAc-Ac to the solution obtained in step (2) - 、H2PO4 - -CO3 2- NH4 + -NH3, concentrations of 1M, 1M, and 5M; (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) irradiating the solution obtained in step (5) with gamma rays at 4°C; The turbidity of the prepared colorless and transparent collagen hydrogel was (0.01±0.002)×10 4 NTU. 6 . A colorless and transparent collagen hydrogel prepared by the method for preparing a colorless and transparent collagen hydrogel according to claim 1 .

Images