CN111714680B - Recombinant human-derived collagen membrane and preparation method and application thereof - Google Patents
Recombinant human-derived collagen membrane and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological pharmacy, and particularly relates to a recombinant human collagen membrane and a preparation method and application thereof. The recombinant human collagen film comprises: preparing a methacryl modified recombinant human collagen sample; and (3) performing fluorescence irradiation on the recombinant human collagen sample to induce a crosslinking reaction to form a recombinant human collagen film. The recombinant human collagen membrane is formed by introducing methacryloyl into the recombinant human collagen and inducing a cross-linking reaction through fluorescent irradiation, has the advantages of simple preparation process, controllable thickness, stable property and the like, can be used for repairing wound surfaces, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to a recombinant human collagen membrane and a preparation method and application thereof.
Background
The wound surface is the damage of normal tissues (skin or mucosa) caused by external injury factors such as surgical operation, external force, heat, current, chemical substances, low temperature and internal factors of the body such as local blood supply disorder, and is often accompanied by the damage of tissue integrity and the loss of a certain amount of normal tissues, and the normal physiological functions of the tissues are damaged to a certain extent, and the repair of the wound surface not only comprises the repair of the integrity of the damaged tissues, but also comprises the repair of the physiological functions of the tissues.
Collagen is the main component of extracellular matrix, and is the most abundant and widely distributed protein in animals. The collagen has unique tissue structure and biological function, plays an important role in maintaining normal physiological functions of cells, tissues and organs and repairing damage, has the characteristics of low immunogenicity, biodegradability, good biocompatibility, good mechanical property and the like, and is a good wound repair material.
The recombinant human collagen is a recombinant protein which is obtained by modifying or re-synthesizing a section of gene sequence of human collagen and expressing a new gene sequence through a host protein expression system. The recombinant human collagen has the advantages of antigenicity, biodegradability, good biocompatibility and the like of collagen, does not have the defects of virus hidden trouble, immunological rejection and the like of animal collagen, and is widely applied to the field of biological materials.
Disclosure of Invention
The invention provides a recombinant human collagen membrane and a preparation method and application thereof.
In order to solve the technical problems, the invention provides a preparation method of a recombinant human collagen membrane, which comprises the following steps: preparing a methacryl modified recombinant human collagen sample; and (3) performing fluorescence irradiation on the recombinant human collagen sample to induce a crosslinking reaction to form a recombinant human collagen film.
In a second aspect, the invention also provides a recombinant human collagen membrane prepared by the preparation method.
In a third aspect, the invention also provides an application of the recombinant human collagen membrane in wound repair.
The recombinant human-derived collagen membrane and the preparation method and application thereof have the advantages of simple preparation process, controllable thickness, stable property and the like, can be used for repairing wound surfaces, and have good application prospect.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail below.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a flow chart of the preparation process of the recombinant human collagen membrane of the present invention;
FIG. 2 is a schematic diagram of the methacryloyl group-modified recombinant human collagen of the present invention;
FIG. 3 is a spectral detection chart of the methacryloyl group-modified recombinant human collagen of the present invention.
Detailed Description
To make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A first part: elucidating the specific technical scheme
The patent of application No. 201911000883.5 discloses a photo-crosslinking recombinant collagen hydrogel, a preparation method and application thereof in 3D bioprinting, which includes dissolving methyl propyl anhydride modified recombinant collagen in a culture medium, adding a photoinitiator aqueous solution, uniformly mixing to obtain a hydrogel precursor solution, and carrying out photo-reaction crosslinking and curing to obtain the recombinant collagen hydrogel. The high-content photoinitiator can be remained in the recombinant collagen hydrogel after crosslinking and curing, the specific weight of the recombinant human collagen modified by the methacryloyl group is seriously influenced, the purity of the recombinant human collagen film is reduced, the wound repair effect is poor, and the recombinant human collagen film is difficult to use in clinical research because the photoinitiator has certain toxicity and can influence wound healing after being used in large quantities. Therefore, referring to fig. 1, the present invention provides a method for preparing a recombinant human collagen membrane, comprising: preparing a methacryl modified recombinant human collagen sample; and (3) performing fluorescence irradiation on the recombinant human collagen sample to induce a crosslinking reaction to form a recombinant human collagen film.
Optionally, the mass of the recombinant human collagen sample accounts for 20-70% of the total mass of the recombinant human collagen membrane, and is 30%, 45% or 60%.
Optionally, the irradiation may be performed by blue light, the vertical irradiation distance of the blue light is 1-5cm, and the irradiation time of the blue light is 1-30 min.
According to the preparation method of the recombinant human collagen membrane, the recombinant human collagen sample is irradiated by fluorescence to induce a crosslinking reaction, so that the recombinant human collagen membrane is formed, the mass ratio of the recombinant human collagen sample can be greatly improved, and the effective components of the recombinant human collagen membrane are improved.
As an alternative embodiment of the methacryl group modified recombinant human collagen sample.
The preparation of the methacryl group modified recombinant human collagen sample comprises the following steps: adding the recombinant human collagen and methacryloyl chloride into an alkaline aqueous solution at a certain temperature for modification reaction; dialyzing the modified reaction product; centrifuging; drying to obtain a methacryl modified recombinant human collagen sample (shown in the picture of FIG. 2).
Alternatively, the recombinant human collagen is, for example, but not limited to, human-like collagen prepared by a biological fermentation method by a genetic recombination technique. The concentration of the recombinant human collagen is 0.025-0.2g/mL, optionally 0.05g/mL and 0.08 g/mL; the mass ratio of the recombinant human collagen to the methacryloyl chloride is (2-10): 1, optionally 5: 1 or 8: 1.
optionally, the pH of the alkaline aqueous solution is-7.5-10, and the alkaline solute of the alkaline aqueous solution is Na2CO3，NaHCO3，K2CO3，NaOH，KOH，KH2PO4，NaH2PO4，Na2HPO4And one or more of inorganic bases; the reaction temperature of the modification reaction is 25-70 ℃, and the reaction time is 12-72 h.
Can be used for dialysis without limitation, such as a fiber membrane dialysis bag, and the residual molecular weight of the dialysis bag is 8000-14000Da, and the dialysis time is 1-7 days; the rotating speed of the centrifuge is 5000-; the drying mode is natural air drying or freeze drying, and the freeze drying condition is as follows: pre-freezing at-20 deg.C for 24-48h, and freeze-drying at-80 deg.C for 24-72 h.
The reaction formula of the modification reaction is:
adopt methacryloyl chloride to modify recombinant human collagen in the present case, methacryloyl chloride has high reactivity, is more favorable for the efficient implementation of modification reaction, and reaction by-product M+Cl-NaCl or KCl, and water as another byproduct, and has low toxicity and greener reaction. After the reaction, dialyzing, centrifuging and freeze-drying are carried out to obtain the recombinant human collagen sample modified by the methacryloyl group, and the use amount of a photoinitiator can be reduced under the irradiation of blue light by utilizing the high activity of the methacryloyl group, so that the crosslinking reaction of the recombinant human collagen sample modified by the methacryloyl group is induced.
As an alternative embodiment of the crosslinking reaction.
The photoinitiator used in the crosslinking reaction comprises a mixed system of camphorquinone and diphenyl iodonium hexafluorophosphate; the mass of the photoinitiator accounts for 0.05-1% of the total mass of the recombinant human collagen film, and 0.1%, 0.5% and 0.8% can be selected. Compared with the conventional photoinitiator, the photoinitiator mainly can induce a crosslinking reaction under blue light, is safer and more efficient than ultraviolet light, is a novel visible light polymerization technology, and can effectively avoid the problems that high-energy ultraviolet light easily deactivates bioactive substances (such as proteins and the like), the curing depth is shallow, radiation and ozone pollution are caused, and the like.
The crosslinking reaction of the embodiment adopts a mixed system of camphorquinone and diphenyl iodonium hexafluorophosphate, and adopts blue light irradiation to induce, so that the dosage of the photoinitiator can be reduced, the toxicity introduced by the initiator is reduced, and the components in the recombinant human collagen film are controlled on the raw material, so that the methacryloyl modified recombinant human collagen sample is kept to have higher mass ratio, and the wound repair effect is improved. Because the single camphorquinone is used as the initiator, the crosslinking efficiency is low, the dosage is large, and the addition of the auxiliary initiator diphenyl iodonium hexafluorophosphate can obviously increase the crosslinking efficiency and reduce the dosage of the photoinitiator.
Further, the invention provides a recombinant human-derived collagen membrane prepared by the preparation method.
The mixed system of camphorquinone and diphenyl iodonium hexafluorophosphate is used as the photoinitiator, so that the dosage can be reduced, the recombinant human collagen film can show a certain color, is generally a faint yellow film and is relatively close to the color of skin, and the application and the repair of a wound surface are facilitated.
Further, the invention provides an application of the recombinant human collagen membrane in wound repair.
A second part: some examples are given below
Example 1
(1) Preparation of methacryloyl group-modified recombinant human collagen sample
1g of recombinant human collagen was dissolved in 10mL of an alkaline aqueous solution (pH 8), and then 0.2mL of a pure methacryloyl chloride solution was added dropwise over a period of 10 min. The reaction mixture was reacted at 25 ℃ for 3 hours, and then 20mL of an aqueous alkaline solution (pH 8) was added to terminate the reaction. And (3) putting the diluted reaction solution into a 10KDa dialysis bag, dialyzing for 72 hours by using deionized water, and changing water once every 24 hours. After dialysis, the solution is put into a centrifuge tube, centrifuged for 5min at 10000rpm, and the supernatant is taken for freeze-drying operation, wherein the freeze-drying conditions are as follows: pre-freezing at-20 ℃ for 24h, and freeze-drying at-80 ℃ for 72h to obtain a methacryloyl modified recombinant human collagen white solid, namely a methacryloyl modified recombinant human collagen sample.
(2) Blue light irradiation induced crosslinking reaction
Dissolving 1g of the white solid of the recombinant human collagen prepared in the step (1) in 5mL of a mixed solution of camphorquinone with the concentration of 0.001g/mL and diphenyliodonium hexafluorophosphate with the concentration of 0.001g/mL, putting 0.5mL of the solution on a clean glass slide, and irradiating the clean glass slide for 5min by using blue light, wherein the irradiation distance is kept at 2 cm. And then naturally drying to obtain the recombinant human collagen membrane.
Example 2
(1) Preparation of methacryloyl group-modified recombinant human collagen sample
1g of recombinant human collagen was dissolved in 10mL of an alkaline aqueous solution (pH 8), and then 0.5mL of a pure methacryloyl chloride solution was added dropwise over a period of 10 min. The reaction mixture was reacted at 25 ℃ for 5 hours, and then 20mL of an aqueous alkaline solution (pH 8) was added to terminate the reaction. And (3) putting the diluted reaction solution into a 10KDa dialysis bag, dialyzing for 72 hours by using deionized water, and changing water once every 24 hours. After dialysis, the solution is put into a centrifuge tube, centrifuged for 5min at 10000rpm, and the supernatant is taken for freeze-drying operation, wherein the freeze-drying conditions are as follows: prefreezing at-20 deg.C for 24 hr, and lyophilizing at-80 deg.C for 72 hr to obtain white solid of recombinant human collagen modified by methacryloyl.
(2) Blue light irradiation induced crosslinking reaction
Dissolving 1g of the white solid of the recombinant human collagen prepared in the step (1) in 5mL of a mixed solution of camphorquinone with the concentration of 0.0002g/mL and diphenyliodonium hexafluorophosphate with the concentration of 0.0002g/mL, placing 0.5mL of the solution on a clean glass slide, and irradiating the glass slide with blue light for 5min, wherein the irradiation distance is kept at 2 cm. And then naturally drying to obtain the recombinant human collagen membrane.
Example 3
(1) Preparation of methacryloyl group-modified recombinant human collagen sample
1g of recombinant human collagen was dissolved in 10mL of an alkaline aqueous solution (pH 8), and then 0.1mL of a pure methacryloyl chloride solution was added dropwise over a period of 10 min. The reaction mixture was reacted at 25 ℃ for 5 hours, and then 20mL of an aqueous alkaline solution (pH 8) was added to terminate the reaction. And (3) putting the diluted reaction solution into a 10KDa dialysis bag, dialyzing for 72 hours by using deionized water, and changing water once every 24 hours. After dialysis, the solution is put into a centrifuge tube, centrifuged for 5min at 10000rpm, and the supernatant is taken for freeze-drying operation, wherein the freeze-drying conditions are as follows: prefreezing at-20 deg.C for 24 hr, and lyophilizing at-80 deg.C for 72 hr to obtain white solid of recombinant human collagen modified by methacryloyl.
(2) Blue light irradiation induced crosslinking reaction
Dissolving 1g of the white solid of the recombinant human collagen prepared in the step (1) in 5mL of camphorquinone with the concentration of 0.002g/mL and diphenyl iodonium hexafluorophosphate solution with the concentration of 0.002g/mL, putting 0.5mL of the solution on a clean glass slide, and irradiating for 2min by using a blue light lamp, wherein the irradiation distance is kept at 2 cm. And then naturally drying to obtain the recombinant human collagen membrane.
Example 4
(1) Preparation of methacryloyl group-modified recombinant human collagen sample
1g of recombinant human collagen was dissolved in 10mL of an alkaline aqueous solution (pH 8), and then 0.25mL of a pure methacryloyl chloride solution was added dropwise over a period of 10 min. The reaction mixture was reacted at 25 ℃ for 5 hours, and then 20mL of an aqueous alkaline solution (pH 8) was added to terminate the reaction. And (3) putting the diluted reaction solution into a 10KDa dialysis bag, dialyzing for 72 hours by using deionized water, and changing water once every 24 hours. After dialysis, the solution is put into a centrifuge tube, centrifuged for 5min at 10000rpm, and the supernatant is taken for freeze-drying operation, wherein the freeze-drying conditions are as follows: prefreezing at-20 deg.C for 24 hr, and lyophilizing at-80 deg.C for 72 hr to obtain white solid of recombinant human collagen modified by methacryloyl.
(2) Blue light irradiation induced crosslinking reaction
Dissolving 1g of the white solid of the recombinant human collagen prepared in the step (1) in 5mL of a mixed solution of camphorquinone with the concentration of 0.001g/mL and diphenyliodonium hexafluorophosphate with the concentration of 0.001g/mL, putting 0.5mL of the solution on a clean glass slide, and irradiating for 2min by using a blue light lamp, wherein the irradiation distance is kept at 2 cm. And naturally drying to obtain the recombinant human collagen membrane.
Example 5
(1) Preparation of methacryloyl group-modified recombinant human collagen sample
0.8g of recombinant human collagen was dissolved in 10mL of an alkaline aqueous solution (pH 8), and then 0.1mL of a pure methacryloyl chloride solution was added dropwise over a period of 10 min. The reaction mixture was reacted at 50 ℃ for 5 hours, and then 20mL of an aqueous alkaline solution (pH 8) was added to terminate the reaction. And (3) putting the diluted reaction solution into a 10KDa dialysis bag, dialyzing for 72 hours by using deionized water, and changing water once every 24 hours. After dialysis, the solution is put into a centrifuge tube, centrifuged for 5min at 10000rpm, and the supernatant is taken for freeze-drying operation, wherein the freeze-drying conditions are as follows: prefreezing at-20 deg.C for 24 hr, and lyophilizing at-80 deg.C for 72 hr to obtain white solid of recombinant human collagen modified by methacryloyl.
(2) Blue light irradiation induced crosslinking reaction
Dissolving 1g of the white solid of the recombinant human collagen prepared in the step (1) in 5mL of a mixed solution of camphorquinone with the concentration of 0.0016g/mL and diphenyliodonium hexafluorophosphate with the concentration of 0.0016g/mL, putting 0.5mL of the solution on a clean glass slide, and irradiating for 2min by using a blue lamp, wherein the irradiation distance is kept at 2 cm. And then naturally drying to obtain the recombinant human collagen membrane.
And a third part: comparative analysis of performance parameters
In this section, the magnetic resonance spectrum of the recombinant human collagen samples modified by methacryloyl groups prepared in examples 1 to 5 is detected, as shown in fig. 3, the magnetic resonance spectrum curves of the recombinant human collagen prepared in examples 1, 2, 3, 4 and 5 are sequentially arranged from top to bottom, the characteristic peaks are shown as the enclosed diagram in the square frame indicated by the characteristic peak in fig. 2, and the success of the recombinant human collagen sample in the present application in modifying the conventional recombinant human collagen by the methacryloyl chloride with high activity can be determined through the characteristic peaks.
In conclusion, the recombinant human-derived collagen membrane and the preparation method thereof modify the recombinant human-derived collagen by utilizing methacryloyl chloride and are matched with a blue light irradiation method to realize the crosslinking reaction of a recombinant human-derived collagen sample to generate the recombinant human-derived collagen membrane, so that the dosage of a photoinitiator can be reduced, the quality of the recombinant human-derived collagen membrane is ensured, the membrane thickness is controllable, the preparation process is simple, the recombinant human-derived collagen membrane is suitable for industrial production, and the recombinant human-derived collagen membrane and the preparation method thereof have wide application prospects in clinic particularly in the field of wound repair.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (7)
1. A preparation method of a recombinant human collagen membrane is characterized by comprising the following steps:
preparing a methacryl modified recombinant human collagen sample;
performing fluorescence irradiation on the recombinant human collagen sample to induce a crosslinking reaction to form a recombinant human collagen film;
the preparation of the methacryl group modified recombinant human collagen sample comprises the following steps:
adding the recombinant human collagen and methacryloyl chloride into an alkaline aqueous solution at a certain temperature for modification reaction;
dialyzing the modified reaction product;
centrifuging;
and drying to obtain a methacryloyl modified recombinant human collagen sample.
2. The production method according to claim 1,
the fluorescent light comprises blue light; and
the vertical irradiation distance of the blue light is 1-5cm, and the irradiation time of the blue light is 1-30 min.
3. The production method according to claim 1,
the concentration of the recombinant human collagen is 0.025-0.2 g/mL;
the mass ratio of the recombinant human collagen to the methacryloyl chloride is (2-10): 1.
4. the production method according to claim 1,
the pH value of the alkaline aqueous solution is 7.5-10;
the solute of the alkaline aqueous solution is an inorganic base including Na2CO3、NaHCO3、K2CO3、NaOH、KOH、KH2PO4、NaH2PO4、Na2HPO4One or more of the components are mixed;
the reaction temperature of the modification reaction is 25-70 ℃, and the reaction time is 12-72 h.
5. The production method according to claim 1,
the photoinitiator used in the crosslinking reaction comprises a mixed system formed by camphorquinone and diphenyl iodonium hexafluorophosphate;
the mass of the photoinitiator accounts for 0.05-1% of the total mass of the recombinant human collagen film.
6. The production method according to claim 1,
the mass of the recombinant human collagen sample accounts for 20-70% of the total mass of the recombinant human collagen membrane.
7. A recombinant human-derived collagen membrane produced by the production method according to claim 1.
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| CN106009011A (en) * | 2016-06-06 | 2016-10-12 | 陕西科技大学 | Method for preparing vinyl modified collagen membrane |
| CN110790950A (en) * | 2019-10-21 | 2020-02-14 | 南京理工大学 | Photo-crosslinking recombinant collagen hydrogel, preparation method and application thereof in 3D bioprinting |
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| WO2005110505A2 (en) * | 2004-04-30 | 2005-11-24 | Advanced Cardiovascular Systems, Inc. | Hyaluronic acid based copolymers |
| CN106009011A (en) * | 2016-06-06 | 2016-10-12 | 陕西科技大学 | Method for preparing vinyl modified collagen membrane |
| CN110790950A (en) * | 2019-10-21 | 2020-02-14 | 南京理工大学 | Photo-crosslinking recombinant collagen hydrogel, preparation method and application thereof in 3D bioprinting |
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