CN-121987851-A - Injectable recombinant III type collagen hydrogel and preparation method thereof

CN121987851ACN 121987851 ACN121987851 ACN 121987851ACN-121987851-A

Abstract

The embodiment of the application provides an injectable recombinant III type collagen hydrogel and a preparation method thereof, and relates to the field of biomedical materials. The preparation method of the injectable recombinant type III collagen hydrogel comprises the following steps of uniformly mixing recombinant type III collagen, glutamine transaminase and distilled water to form a mixed protein solution, wherein the mass concentration of the recombinant type III collagen in the mixed protein solution is not lower than 1.5%, the mass ratio of the glutamine transaminase to the recombinant type III collagen is 0.5% -13%, and the recombinant type III collagen in the mixed protein solution is subjected to enzyme catalytic crosslinking reaction under the action of the glutamine transaminase to form the hydrogel. The preparation method can reduce the problems of local quick crosslinking and phase separation, and the prepared hydrogel has uniform and transparent structure, and realizes the adjustability of the rheological property and mechanical property of the hydrogel.

Inventors

SUN HUAIQING
ZHANG YUN
GUO CHAOWAN
WU JIATING
SUN TAOLIN
Lu mengze

Assignees

广东丸美生物医学有限公司

Dates

Publication Date: 20260508
Application Date: 20260403

Claims (10)

1. The preparation method of the injectable recombinant type III collagen hydrogel is characterized by comprising the following steps of: Uniformly mixing recombinant type III collagen, glutamine transaminase and water to form a mixed protein solution, wherein the mass concentration of the recombinant type III collagen in the mixed protein solution is not lower than 1.5%, and the mass ratio of the glutamine transaminase to the recombinant type III collagen is 0.5% -13%; and (3) carrying out enzyme catalytic crosslinking reaction on the recombinant type III collagen in the mixed protein solution under the action of the glutamine transaminase to form hydrogel.
2. The method of preparing an injectable recombinant type III collagen hydrogel according to claim 1, wherein the mixed protein solution meets one of the following requirements: The mass concentration of the recombinant III type collagen is 2% -5%, and the mass ratio of the glutamine transaminase relative to the recombinant III type collagen is 0.5% -3%; The mass concentration of the recombinant III type collagen is 5% -13%, and the mass ratio of the glutamine transaminase to the recombinant III type collagen is 3% -13%.
3. The method for preparing injectable recombinant type III collagen hydrogel according to claim 1, wherein the pH is 6.5-7.0 and the temperature is 25-28 ℃ during the enzyme-catalyzed crosslinking reaction.
4. The method for preparing injectable recombinant type III collagen hydrogel according to claim 1 or 3, wherein the method for preparing the mixed protein solution comprises: Mixing the recombinant type III collagen with water to form a collagen solution, and mixing the glutamine transaminase with water to form an enzyme solution; and firstly, regulating the pH of the collagen solution to 6.5-7.0, and then uniformly mixing the collagen solution with the enzyme solution to form the mixed protein solution.
5. The method for preparing injectable recombinant type III collagen hydrogel according to claim 4, wherein the mass concentration of the collagen solution is 2.5% -10%, optionally 2.5% -7%; And/or the mass concentration of the enzyme solution is 1% -5%, and optionally 2% -4%; And/or the volume ratio of the collagen solution to the enzyme solution is 1.4-60.
6. The method for preparing the injectable recombinant type III collagen hydrogel according to claim 4, wherein the enzyme activity of the glutamine transaminase is 1050U/g-1100U/g, and the addition amount of the glutamine transaminase relative to the mixed protein solution is 0.5U/mL-3U/mL, optionally 0.54U/mL-2.71U/mL.
7. The method of preparing an injectable recombinant type III collagen hydrogel according to claim 1 or 3, wherein the method of preparing the hydrogel comprises: and placing the mixed protein solution in an environment of 25-28 ℃ for reaction for 10-30 minutes until the system is converted from a solution state to a gel state.
8. The method of preparing injectable recombinant type III collagen hydrogel according to claim 1, further comprising the step of purifying the hydrogel by soxhlet extraction; alternatively, the Soxhlet extraction method is to flush the hydrogel with a continuous circulation of PBS buffer.
9. An injectable recombinant type III collagen hydrogel, characterized in that it is prepared by the method for preparing an injectable recombinant type III collagen hydrogel according to any one of claims 1 to 8.
10. The injectable recombinant type III collagen hydrogel according to claim 9, wherein the injectable recombinant type III collagen hydrogel has a storage modulus of 100pa to 39000pa, a loss modulus of 200pa to 1500pa when tested in step-strain alternation, and the loss modulus is lower than the storage modulus.

Description

Injectable recombinant III type collagen hydrogel and preparation method thereof Technical Field The application relates to the field of biomedical materials, in particular to an injectable recombinant III-type collagen hydrogel and a preparation method thereof. Background Collagen is used as the main structural protein in connective tissue of human body and is widely applied to soft tissue repair, medical filling and injectable scaffold materials. The III type collagen has rich content in skin, blood vessels and visceral tissues, has good biocompatibility and cell adhesion activity, and is considered to be an ideal matrix for constructing injectable hydrogel. However, the existing natural collagen materials generally have the problems of poor structural stability, low mechanical strength, too fast degradation and the like, and are difficult to maintain the supporting function in vivo for a long time, so that the defects of the natural collagen materials are overcome, and the injectable hydrogel based on the collagen is applied. At present, the preparation method of the injectable hydrogel based on collagen mainly depends on two technologies, namely physical crosslinking, such as temperature induction and pH adjustment, the hydrogel formed by the method is weak in mechanical strength and poor in stability and is easy to swell or degrade rapidly in a physiological environment, and chemical crosslinking, such as glutaraldehyde, carbodiimide, genipin and other reagents, can improve mechanical properties, but residual crosslinking agent can cause cytotoxicity and inflammatory reaction, and can cause uncontrollable material degradation behavior and long-term biosafety doubt. In addition, collagen, whether extracted naturally or of animal origin, has problems of large batch variation, potential pathogen risk, immunogenicity, and the like. In recent years, enzyme catalytic crosslinking is focused as a bio-friendly strategy, however, in the prior art, the enzyme catalytic crosslinking is mostly used as an auxiliary means or used for modifying a mixed system, an exogenous thickening component and a supporting component are needed, the preparation process is complex, and the phenomena of local quick crosslinking (sudden aggregation), non-uniformity, blushing and the like caused by phase separation are easy to occur when the pure collagen system is adopted for preparing the hydrogel through enzyme catalytic crosslinking, so that the transparent injectable hydrogel with a uniform network structure is difficult to stably obtain, and the application scene requirement on high requirements on biosafety and mechanical suitability cannot be met. Disclosure of Invention The embodiment of the application aims to provide an injectable recombinant III type collagen hydrogel and a preparation method thereof, wherein the preparation method can reduce the problems of local quick crosslinking and phase separation, and the prepared hydrogel has a uniform and transparent structure and realizes adjustability of rheological property and mechanical property of the hydrogel. In a first aspect, the present application provides a method for preparing an injectable recombinant type III collagen hydrogel, comprising the steps of: Uniformly mixing recombinant type III collagen, glutamine transaminase (TGase) and water to form a mixed protein solution, wherein the mass concentration of the recombinant type III collagen in the mixed protein solution is not lower than 1.5%, and the mass ratio of the glutamine transaminase to the recombinant type III collagen is 0.5% -13%; and (3) carrying out enzyme catalytic crosslinking reaction on the recombinant type III collagen in the mixed protein solution under the action of the glutamine transaminase to form hydrogel. In the technical scheme, the preparation method of the application takes single and definite recombinant III-type collagen as the only structural element, uses glutamine transaminase to carry out enzyme catalytic crosslinking to construct a complete network, does not need to introduce an exogenous chemical crosslinking agent, can prepare the injectable hydrogel under mild conditions, avoids the risk of chemical crosslinking agent residue, and improves the biosafety of the hydrogel. Meanwhile, the problems of local rapid crosslinking (sudden aggregation) and phase separation can be effectively reduced by precisely controlling the concentration of collagen and the enzyme addition proportion, uniform and transparent hydrogel can be obtained, and the adjustability of the rheological property and the mechanical property of the hydrogel can be realized. In one possible implementation, the mixed protein solution meets one of the following requirements: The mass concentration of the recombinant III type collagen is 2% -5%, and the mass ratio of the glutamine transaminase relative to the recombinant III type collagen is 0.5% -3%; The mass concentration of the recombinant III type collagen is 5% -13%