CN115819802A - A kind of colorless transparent collagen hydrogel and preparation method thereof - Google Patents

Abstract

The invention relates to the field of biological materials, in particular to a colorless and transparent collagen hydrogel and a preparation method thereof. The collagen hydrogel disclosed by the invention has the characteristic of high transparency, and can be used in the fields of visual repair materials, cornea repair and the like.

Description

A kind of colorless transparent collagen hydrogel and preparation method thereof

technical field

The invention relates to a biological material and a preparation method thereof, in particular to a colorless transparent collagen hydrogel and a preparation method thereof.

Background technique

Collagen is the main structural protein of connective tissue. It is widely distributed in tissues and organs such as human bones, skin, blood vessels, ligaments, cartilage, muscles and tendons, accounting for 25% to 30% of the total protein in animals. As the main component of the extracellular matrix, collagen plays an important role in maintaining the biological properties and structural integrity of the extracellular matrix. Collagen itself has a special quaternary structure, which can spontaneously aggregate to form collagen fibers under certain circumstances, and further form hydrogels. However, from the perspective of color tone, conventional collagen hydrogels have obvious "white turbidity", that is, collagen hydrogels are white and opaque, which is not conducive to the application of collagen hydrogels in visibility materials, artificial corneas, etc. .

The invention mainly solves the "white turbidity" problem of the collagen hydrogel, can prepare the colorless and transparent collagen hydrogel, and enhances the light transmittance of the collagen hydrogel.

Contents of the invention

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

A preparation method of a colorless transparent collagen hydrogel, wherein, comprising the following steps in sequence:

(1) Dissolving the solid collagen material in water, fully dissolving, centrifuging, and taking the supernatant;

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

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

(4) adding a crosslinking agent in the solution obtained in step (3);

(5) Adding protective molecules to the solution obtained in step (4);

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

A method for preparing a colorless transparent collagen hydrogel of the present invention, wherein, in the step (1), the solid collagen material is selected from one or more of type I, type II, type III, and type V collagen Combination; the total concentration of various types of collagen in the supernatant of step (1) is greater than or equal to 0.01wt.% but less than or equal to 10wt.%. If the total collagen concentration is less than 0.01wt.%, the collagen solution cannot form a hydrogel; if the total collagen concentration is greater than 10wt.%, the collagen molecules in the collagen solution are easy to form collagen fibers, and "white turbidity" cannot form a colorless transparent collagen water gel.

A kind of preparation method of colorless transparent collagen hydrogel of the present invention, wherein, the acidic solution described in step (2) comprises salicylic acid, lactic acid, sulfuric acid, tartaric acid, citric acid, phosphoric acid, oxalic acid, acetic acid, oxalic acid, At least one of succinic acid, hydrochloric acid, maleic acid, benzoic acid, nitric acid, malic acid, nicotinic acid, sodium dihydrogen phosphate and formic acid; the alkaline solution includes tetramethylethylenediamine, sodium hydroxide, tris At least one of ethylamine, sodium carbonate, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia water, disodium hydrogen phosphate and sodium bicarbonate. Collagen aqueous solution is prone to "white turbidity" phenomenon under neutral pH conditions. The pH value of collagen aqueous solution is precisely adjusted through acidic or alkaline solutions, such as pH=6.9 or pH=7.9, to ensure that collagen molecules do not aggregate to form collagen fibers. Acidic or alkaline solutions range in concentration from 0.01M–2M.

A method for preparing a colorless transparent collagen hydrogel of the present invention, wherein the specific ion/molecular pair in the step (3) includes 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-EDTA 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 at least one of the Regulate the ionic environment of collagen aqueous solution through specific ion/molecule pairs, prevent collagen molecules from self-assembling to form collagen fibers, and maintain the homogeneous fluidity of collagen aqueous solution, thereby preventing the occurrence of "white turbidity". The concentration range of specific ion/molecule pairs is 0.1M–10M.

A preparation method of a colorless transparent collagen hydrogel of the present invention, wherein, in the step (4), the crosslinking agent includes NHS ester, sulfonyl chloride, acid anhydride, fluorophenyl ester, proanthocyanidin, genipin, aldehyde, iso At least one of thiocyanates, isocyanates, acyl azides, epoxides, aryl halides, imide esters, and carbodiimides. The concentration of the crosslinking agent ranges from 0.0001% to 5%.

A kind of preparation method of colorless transparent collagen hydrogel of the present invention, wherein, in described step (5), protection molecule comprises sodium lauryl sulfate, sodium dodecylbenzenesulfonate, Tween, triton, Ethylene glycol, phenylalanine, proline, lecithin, glutamic acid, lysine, cysteine, mangiferin, glycine, aspartic acid, n-butanol, propylene glycol, EDTA At least one of sodium salt, stearic acid, span, glycerin and gelatin. The main purpose of adding protective molecules is to weaken the impact of irradiation treatment on the activity of collagen molecules. The concentration of the protective molecule ranges from 1% to 50%.

A method for preparing a colorless transparent collagen hydrogel of the present invention, wherein the low temperature range of the low temperature irradiation treatment in the step (6) is -20-5°C. The low temperature condition is mainly used to weaken the influence of irradiation treatment on the activity of collagen molecules.

The colorless and transparent collagen hydrogel prepared by using any one of the preparation methods of the colorless and transparent collagen hydrogel of the present invention.

The present invention is 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.

Beneficial effects of the present invention: in the process of collagen molecules forming collagen hydrogels, collagen molecules self-assemble to form collagen fibers, and the formed collagen hydrogels are usually white and opaque, which makes collagen hydrogels unable to be used as visible materials , artificial cornea, etc. In order to enhance the transparency of the collagen hydrogel, in the preparation method of a colorless and transparent collagen hydrogel of the present invention, the pH value of the collagen solution and the addition of specific ion/molecule pairs are coordinated to balance the surface charge of the collagen molecules. , enhance the ionic strength in the solution, etc., so as to reduce the self-assembly behavior of collagen molecules as much as possible; on this basis, add a suitable cross-linking agent to make the collagen molecules cross-link slowly and gently, and avoid rapid cross-linking to form more collagen fibers. Thereby avoiding the reduction of the transparency of the collagen hydrogel. In order to further meet the sterilization and inactivation requirements of the colorless and transparent collagen hydrogel in medical devices, and avoid the influence of radiation treatment on the collagen molecules in the colorless and transparent collagen hydrogel to reduce the transparency, further increase in the preparation method of the present invention Steps of adding protective molecules and low temperature treatment were added to minimize the impact of irradiation treatment on collagen molecules. Through the above steps, a stable colorless and transparent collagen hydrogel can be prepared.

Description of drawings

Fig. 1 is a collagen hydrogel light transmittance effect figure (Fig. 1A is the sample of embodiment 1, Fig. 1B is the sample of embodiment 2, Fig. 1C is the sample of embodiment 3, Fig. 1D is the sample of embodiment 4, Fig. 1E is the sample of embodiment 5 samples, Fig. 1F is the sample of comparative example 1, Fig. 1G is the sample of comparative example 2, Fig. 1H is the sample of comparative example 3, Fig. 1I is the sample of comparative example 4, Fig. 1J is the sample of comparative example 5).

Detailed ways

Example 1

A kind of preparation of colorless transparent collagen hydrogel, wherein, comprises the following steps successively:

(1) Dissolve 1g type I collagen in 100mL water, fully dissolve, centrifuge, and take the supernatant;

(2) adjusting the pH value of the supernatant obtained in step (1) to 7.8 by 1M magnesium hydroxide solution or 0.1M acetic acid solution;

(3) Add a specific ion/molecule pair H ₂ PO ₄ ^- -citric acid to the solution obtained in step (2) at a concentration of 0.1M;

(4) Add 0.01% proanthocyanidins to the solution obtained in step (3);

(5) add 5% n-butanol in the solution gained in step (4);

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

Example 2

A kind of preparation of colorless transparent collagen hydrogel, wherein, comprises the following steps successively:

(1) Dissolve 0.1g type II collagen and 0.4g type III collagen in 100mL water, fully dissolve, centrifuge, and take the supernatant;

(2) adjusting the pH value of the supernatant obtained in step (1) to 7.1 by 0.5M ammonia solution or 1M oxalic acid solution;

(3) Adding a specific ion/molecule pair Tris-H ₂ PO ₄ ⁻ to the solution obtained in step (2) at a concentration of 1M;

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

(5) add 1% glycerol and 2% Tween in step (4) gained solution;

(6) The solution obtained in step (5) was irradiated with Co ⁶⁰ at -5°C.

Example 3

A kind of preparation of colorless transparent collagen hydrogel, wherein, comprises the following steps successively:

(1) Dissolve 20g of type I collagen, 0.6g of type III collagen and 5g of type V collagen in 1000mL of water, fully dissolve, centrifuge, and take the supernatant;

(2) adjusting the pH value of the supernatant obtained in step (1) to 6.5 by 0.2M sodium hydroxide solution or 1M hydrochloric acid solution;

(3) Add specific ion/molecule pair Tris-lysine hydrochloride-ethylenediamine tetraacetate ion to the solution obtained in step (2), the concentration is 2M;

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

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

(6) The solution obtained in step (5) is irradiated with gamma rays at 5°C.

Example 4

A kind of preparation of colorless transparent collagen hydrogel, wherein, comprises the following steps successively:

(1) Dissolve 5g type II collagen and 5g type II collagen in 100mL water, fully dissolve, centrifuge, and take the supernatant;

(2) adjust the pH value of the supernatant obtained in step (1) to 5.0 by 0.2M sodium carbonate, 0.01M potassium hydroxide or 2M oxalic acid, 1M succinic acid solution;

(3) Add specific ion/molecule pairs PO ₄ ^3- -HPO ₄ ^2- , H ₂ PO ₄ ^- -citric acid, and Tris-H ₂ PO ₄ ^- to the solution obtained in step (2) at a concentration of 0.5M respectively , 10M, 1M;

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

(5) add 2% phenylalanine, 1% triton and 50% lysine in step (4) gained solution;

(6) The solution obtained in step (5) is subjected to Co ⁶⁰ irradiation treatment at -20°C.

Example 5

A kind of preparation of colorless transparent collagen hydrogel, wherein, comprises the following steps successively:

(1) Dissolve 0.1g type II collagen in 1000mL water, fully dissolve, centrifuge, and take the supernatant;

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

(3) Add specific ion/molecule pairs HAc-Ac ^- , H ₂ PO ₄ ^- -CO ₃ ^2- , and NH ₄ ⁺ -NH ₃ to the solution obtained in step (2) 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) add 1% sodium dodecylbenzenesulfonate and 5% propylene glycol in step (4) gained solution;

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

Example 6

Comparative experiment on the effect of colorless transparent collagen hydrogel

1. Preparation of comparative collagen hydrogels

Comparative example 1: the preparation of a collagen hydrogel, wherein, including the following steps in sequence:

(1) Dissolve 1g type I collagen in 100mL water, fully dissolve, centrifuge, and take the supernatant;

(2) Adjust the pH value of the supernatant obtained in step (1) to 7.4 to be neutral by using 1M magnesium hydroxide solution or 0.1M acetic acid solution.

Comparative example 2: the preparation of a collagen hydrogel, wherein, including the following steps in sequence:

(1) Dissolve 1g type I collagen in 100mL water, fully dissolve, centrifuge, and take the supernatant;

(2) adjusting the pH value of the supernatant obtained in step (1) to 7.8 by 1M magnesium hydroxide solution or 0.1M acetic acid solution;

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

Comparative example 3

A kind of preparation of colorless transparent collagen hydrogel, wherein, comprises the following steps successively:

(1) Dissolve 20g of type I collagen, 0.6g of type III collagen and 5g of type V collagen in 1000mL of water, fully dissolve, centrifuge, and take the supernatant;

(2) Add 1% NHS ester to the solution obtained in step (1);

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

(4) The solution obtained in step (3) is irradiated with gamma rays at 5°C.

Comparative example 4

A kind of preparation of colorless transparent collagen hydrogel, wherein, comprises the following steps successively:

(1) Dissolve 5g type II collagen and 5g type II collagen in 100mL water, fully dissolve, centrifuge, and take the supernatant;

(2) adjust the pH value of the supernatant obtained in step (1) to 5.0 by 0.2M sodium carbonate, 0.01M potassium hydroxide or 2M oxalic acid, 1M succinic acid solution;

(3) Add specific ion/molecule pairs PO ₄ ^3- -HPO ₄ ^2- , H ₂ PO ₄ ^- -citric acid, and Tris-H ₂ PO ₄ ^- to the solution obtained in step (2) at a concentration of 0.5M respectively , 10M, 1M;

(4) The solution obtained in step (3) was subjected to Co ⁶⁰ irradiation treatment at -20°C.

Comparative example 5

A kind of preparation of colorless transparent collagen hydrogel, wherein, comprises the following steps successively:

(1) Dissolve 0.1g type II collagen in 1000mL water, fully dissolve, centrifuge, and take the supernatant;

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

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

2. Effect test comparison

(1) Collagen hydrogel turbidity test: The turbidity of collagen hydrogel was observed on a turbidimeter (TZD-BZ-905, Suzhou DiagVita Technology Co., Ltd.) with a wavelength of 625 nm. The test results are shown in Table 1. Collagen hydrogel light transmittance effect diagram is shown in Figure 1, wherein, Figure 1A is the sample of Example 1, Figure 1B is the sample of Example 2, Figure 1C is the sample of Example 3, Figure 1D is the sample of Example 4, and Figure 1E is the sample of Example 2 Example 5 sample, Figure 1F is the sample of Comparative Example 1, Figure 1G is the sample of Comparative Example 2, Figure 1H is the sample of Comparative Example 3, Figure 1I is the sample of Comparative Example 4, and Figure 1J is the sample of Comparative Example 5.

Table 1 Collagen hydrogel turbidity test

sample Turbidity (×10⁴NTU) sample Turbidity (×10⁴NTU) 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

It can be seen from Table 1 that the turbidity of the collagen hydrogel in Examples 1-5 is very low, and the collagen hydrogel in Example 1 in Figure 1A is very transparent, and the text at the bottom of the material can be clearly observed. Similarly, Figure 1B- The transparency of the collagen hydrogel in 1E is also very high, and the scale value of the syringe can be seen through the collagen hydrogel; while the turbidity of the collagen hydrogel in Comparative Examples 1-5 is very large, and the collagen hydrogel in Figure 1F is very turbid , the text on the bottom of the material cannot be observed. Similarly, the collagen hydrogel in Fig. 1G-1J is very blurred, and the light transmission is very poor, and the scale value of the syringe can hardly be seen.

In the preparation method of a colorless and transparent collagen hydrogel of the present invention, the pH value of the collagen solution and the addition of specific ions/molecules are coordinated to balance the surface charge of the collagen molecules and enhance the ionic strength in the solution. In order to reduce the self-assembly behavior of collagen molecules as much as possible; on this basis, adding a suitable cross-linking agent makes the collagen molecules undergo a slow and gentle cross-linking reaction, avoiding rapid cross-linking to form more collagen fibers, thereby avoiding the reduction of the transparency of the collagen hydrogel. In order to further meet the sterilization and inactivation requirements of the colorless and transparent collagen hydrogel in medical devices, and avoid the impact of radiation treatment on the collagen molecules in the colorless and transparent collagen hydrogel, resulting in a decrease in transparency, the preparation method of the present invention further increases Steps of adding protective molecules and low temperature treatment were added to minimize the impact of irradiation treatment on collagen molecules. Through the above steps, a stable colorless and transparent collagen hydrogel can be prepared.

The above-mentioned embodiment is only a description of the preferred implementation of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those skilled in the art to the technical solution of the present invention shall fall within the scope of the present invention. Into the scope of protection determined by the claims of the present invention.

Claims (10)

1. The preparation method of the colorless and transparent collagen hydrogel is characterized by sequentially comprising the following steps:

(1) Dissolving solid collagen material in water, fully dissolving, centrifuging, and taking supernatant; the total concentration of each type of collagen in the supernatant is 0.01wt.% or more but 10wt.% or less;

(2) Adjusting the pH value of the supernatant obtained in the step (1) to 5.0-9.0 by using an alkaline solution or an acidic solution;

(3) Adding specific ion/molecule pairs into the solution obtained in the step (2); specific ion/molecule pairs include 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-ethylenediamine tetraacetic acid radical 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 At least one of (a);

(4) Adding a cross-linking agent into the solution obtained in the step (3); the cross-linking agent comprises at least one of NHS ester, sulfonyl chloride, acid anhydride, fluorophenyl ester, procyanidin, genipin, aldehyde, isothiocyanate, isocyanate, acyl azide, epoxide, aryl halide, imide ester and carbodiimide;

(5) Adding a protective molecule into the solution obtained in the step (4); the protective molecule comprises at least one of sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, tween, triton, ethylene glycol, phenylalanine, proline, lecithin, glutamic acid, lysine, cysteine, mangiferin, glycine, aspartic acid, n-butanol, propylene glycol, sodium ethylene diamine tetracetate, stearic acid, span, glycerol and gelatin;

(6) And (5) carrying out low-temperature irradiation treatment on the solution obtained in the step (5).

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 types selected from type I, type II, type III and type V collagen.

3. The method for preparing a colorless and transparent collagen hydrogel according to claim 1, wherein said acidic solution in the step (2) comprises 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; the alkaline solution comprises 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. The method for preparing a colorless and transparent collagen hydrogel according to claim 3, wherein the concentration of the acidic solution or the alkaline solution in the step (3) is in the range of 0.01M to 2M.

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

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

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

8. The method for preparing a colorless and transparent collagen hydrogel according to any one of claims 1 to 7, wherein the low temperature irradiation treatment in the step (6) is performed at a low temperature ranging from-20 ℃ to 5 ℃.

9. The colorless and transparent collagen hydrogel prepared by using a method for preparing a colorless and transparent collagen hydrogel according to any one of claims 1 to 8.

10. The colorless and transparent collagen hydrogel according to claim 9, wherein said colorless and transparent collagen hydrogel has a turbidity ranging from 0.01 x 10 ⁴ -2×10 ⁴ NTU。

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