CN116041727B - Synthesis preparation method and application of carbon-carbon double bond modified keratin injectable hydrogel - Google Patents
Synthesis preparation method and application of carbon-carbon double bond modified keratin injectable hydrogel Download PDFInfo
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- CN116041727B CN116041727B CN202310044571.4A CN202310044571A CN116041727B CN 116041727 B CN116041727 B CN 116041727B CN 202310044571 A CN202310044571 A CN 202310044571A CN 116041727 B CN116041727 B CN 116041727B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
- C08H1/00—Macromolecular products derived from proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Dermatology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Materials Engineering (AREA)
- Cell Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Developmental Biology & Embryology (AREA)
- Composite Materials (AREA)
- Dispersion Chemistry (AREA)
- Organic Chemistry (AREA)
- Urology & Nephrology (AREA)
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Abstract
The invention belongs to the technical field of biological materials, and particularly relates to a synthesis preparation method and application of a carbon-carbon double bond modified keratin injectable hydrogel. The carbon-carbon double bond modified keratin is obtained by nucleophilic addition reaction of isocyanate groups of isocyanate and amino groups of keratin, wherein the isocyanate also contains carbon-carbon double bond groups; the modification rate of the carbon-carbon double bond is more than or equal to 30 percent. The carbon-carbon double bond modified keratin has good biocompatibility and degradability, and can be crosslinked into hydrogel by initiating free radical polymerization of double bonds through a glucose/glucose oxidase/horseradish peroxidase/acetylacetone/N-vinylpyrrolidone enzyme cascade catalytic system, so that injectability is realized, mechanical strength is good, and mechanical supporting effect can be realized. The composite hydrogel prepared by wrapping bone marrow mesenchymal stem cell exosome with the injectable hydrogel provided by the invention has good treatment effect on intervertebral disc degeneration.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to a synthesis preparation method and application of a carbon-carbon double bond modified keratin injectable hydrogel for treating intervertebral disc degeneration.
Background
Along with the remarkable improvement of the life span of people, the occurrence rate of lower back pain and neck pain in the global scope is also greatly increased, and the image data show that the patients with the back pain and the neck pain commonly suffer from intervertebral disc degenerative diseases, thereby seriously reducing the living standard of the patients. At present, methods for clinically treating intervertebral disc degeneration (abbreviated as IVDD) are mainly divided into drug conservation treatment and surgical treatment, but some limitations still exist, such as the conservation treatment can not prevent the intervertebral disc from further degeneration, the surgical treatment is invasive and easy to relapse, and the like. Accordingly, in recent years, a repair therapy for regeneration of an intervertebral disc, which aims to control or reverse the progress of an IVDD disease, has been widely studied, wherein a biological scaffold material having both a structural support function and a sustained release capability has become a research hotspot.
The biological scaffold material for treating the IVDD is mainly a plastic prosthesis and a hydrogel material, and has the functions of mechanical support and medicine slow release on the degenerated intervertebral disc. The plastic prosthesis has the characteristics of loose structure, good mechanical strength and easy synthesis, but has poor biocompatibility and lacks hydrophilic water retention capacity, so that the plastic prosthesis is not beneficial to IVDD repair. The hydrogel material overcomes the defects, has strong hydrophilic water retention capacity, can effectively regulate the mechanical strength through chemical modification, and is an IVDD biological stent material with great potential.
Hydrogel materials can be divided into three types, natural, synthetic and semi-synthetic. The natural hydrogel is derived from the components of proteins, polysaccharides and the like of a biological extracellular matrix, and often has better biocompatibility and specific biological functions, including chitosan, hyaluronic acid, silk fibroin, sodium alginate and the like. The cross-linked network of the synthesized hydrogel is mainly chemically synthesized long-chain molecules including polyethylene glycol, poly isopropyl acrylamide polymer and the like. They have the advantages of definite structure, adjustable mechanical strength and adjustable function. The semisynthetic hydrogel is formed by modifying synthetic molecules and natural source molecules through chemical bonding, and can effectively combine the excellent biocompatibility of the natural molecules with the functional adjustability of the synthetic molecules, thereby being beneficial to tissue repair.
Although semisynthetic hydrogels have clinical therapeutic potential, their use in IVDD therapy is inadequate, and injectable, degradable and highly biocompatible cannot be achieved simultaneously.
Disclosure of Invention
The invention aims to provide a synthesis preparation method and application of a carbon-carbon double bond modified keratin injectable hydrogel for treating intervertebral disc degeneration. The carbon-carbon double bond modified keratin provided by the invention has good biocompatibility and degradability, and can initiate double bond free radical polymerization and crosslinking to form hydrogel through enzyme cascade catalytic system compounding, so that injectability is realized, and the hydrogel material is ensured to have the characteristics of injectability, degradability and high biocompatibility. The composite hydrogel material obtained by further wrapping bone marrow mesenchymal stem cell exosomes has good IVDD treatment effect.
Aiming at the design of the materials, the invention provides the following technical scheme:
The invention provides carbon-carbon double bond modified keratin, which is obtained by nucleophilic addition reaction of isocyanate groups of isocyanate and amino groups of keratin, wherein the isocyanate also contains carbon-carbon double bonds;
the carbon-carbon double bond modification rate of the carbon-carbon double bond modified keratin is more than or equal to 30 percent.
Preferably, the keratin is human hair keratin;
the isocyanate includes isocyanatoethyl methacrylate (IEM).
The invention provides a preparation method of the keratin modified by the carbon-carbon double bond, which comprises the following steps:
Mixing the keratin, an organic solvent and isocyanate, and heating to perform nucleophilic addition reaction to obtain the carbon-carbon double bond modified keratin.
Preferably, the temperature of the nucleophilic addition reaction is 60-70 ℃, and the heat preservation time of the nucleophilic addition reaction is 16-18 h.
The invention provides a carbon-carbon double bond modified keratin injectable hydrogel, which comprises a solution of the carbon-carbon double bond modified keratin and an enzyme cascade catalytic system; the carbon-carbon double bond modified keratin is the carbon-carbon double bond modified keratin prepared by the technical scheme or the preparation method;
The enzyme cascade catalytic system comprises N-vinyl pyrrolidone, acetylacetone, glucose oxidase solution, glucose solution and horseradish peroxidase solution.
Preferably, the mass volume fraction (w/v) of the carbon-carbon double bond modified keratin solution is 20%; the mass concentration of the glucose oxidase solution is 10mg/mL; the mass concentration of the horseradish peroxidase solution is 4mg/mL; the molar concentration of the glucose solution is 1.1mol/L;
The volume ratio of the solution of the carbon-carbon double bond modified keratin to the N-vinyl pyrrolidone is 100:0.11-1;
The volume ratio of the solution of the carbon-carbon double bond modified keratin to the acetylacetone is 100:0.07-1;
The volume ratio of the carbon-carbon double bond modified keratin solution to the glucose oxidase solution is 100:0.16-1;
The volume ratio of the carbon-carbon double bond modified keratin solution to the horseradish peroxidase solution is 100:2-4;
The volume ratio of the solution of the carbon-carbon double bond modified keratin to the glucose solution is 100:2-5.
The invention provides a carbon-carbon double bond modified keratin injectable composite hydrogel, which comprises a carbon-carbon double bond modified keratin injectable hydrogel and a stem cell exosome wrapped in the carbon-carbon double bond modified keratin injectable hydrogel; the carbon-carbon double bond modified keratin injectable hydrogel is the carbon-carbon double bond modified keratin injectable hydrogel in the technical scheme.
Preferably, the stem cell exosome is a bone marrow mesenchymal stem cell exosome.
Preferably, the mass ratio of the volume of the carbon-carbon double bond modified keratin injectable hydrogel to the stem cell exosomes is 100 μl:64ng.
The invention provides the carbon-carbon double bond modified keratin injectable hydrogel or the application of the carbon-carbon double bond modified keratin injectable composite hydrogel in preparation of biological scaffold materials for treating intervertebral disc degeneration.
The invention provides carbon-carbon double bond modified keratin, which is obtained by nucleophilic addition reaction of isocyanate groups of isocyanate and amino groups of keratin, wherein the isocyanate also contains carbon-carbon double bonds; the carbon-carbon double bond modification rate of the carbon-carbon double bond modified keratin is more than or equal to 30 percent. According to the invention, the isocyanate containing the carbon-carbon double bond is used for realizing the carbon-carbon double bond modification of keratin, and the obtained keratin modified by the carbon-carbon double bond not only has good biocompatibility and degradability, but also can realize double bond free radical polymerization crosslinking through an enzyme cascade catalytic system to form hydrogel, so that injectability is realized. Meanwhile, the modification rate of the carbon-carbon double bond modified keratin is more than or equal to 30%, and the hydrogel material with the same mechanical strength as the natural nucleus pulposus can be obtained after the polymerization and the cross-linking of the carbon-carbon double bond modified keratin, so that the mechanical supporting effect is achieved. Therefore, the hydrogel material prepared from the keratin modified by the carbon-carbon double bond has the characteristics of injectability, degradability and high biocompatibility, has good mechanical strength, and can realize mechanical supporting effect.
The invention provides a carbon-carbon double bond modified keratin injectable hydrogel, which comprises a solution of the carbon-carbon double bond modified keratin and an enzyme cascade catalytic system; the carbon-carbon double bond modified keratin is the carbon-carbon double bond modified keratin prepared by the technical scheme or the preparation method; the enzyme cascade catalytic system comprises N-vinyl pyrrolidone, acetylacetone, glucose oxidase solution, glucose solution and horseradish peroxidase solution. The invention uses an enzyme cascade catalytic system to initiate the polymerization and crosslinking of carbon-carbon double bonds of the keratin to form gel, so as to realize the injectability of the hydrogel.
The invention provides a carbon-carbon double bond modified keratin injectable composite hydrogel, which comprises a carbon-carbon double bond modified keratin injectable hydrogel and a stem cell exosome wrapped in the carbon-carbon double bond modified keratin injectable hydrogel; the carbon-carbon double bond modified keratin injectable hydrogel is the carbon-carbon double bond modified keratin injectable hydrogel in the technical scheme. The invention introduces the stem cell exosome into the composite hydrogel through loading the stem cell exosome, has the anti-inflammatory and nucleus pulposus repair promoting functions, and has application potential for treating intervertebral disc degeneration.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a structure of a carbon-carbon double bond modified Keratin, keratin (Keratin) and a carbon-carbon double bond modified Keratin (KeMA) prepared in example 1 of the present invention;
FIG. 2 is a plot of rheological properties of a KeMA hydrogel prepared in example 1 of the present invention at 20% w/v over time;
FIG. 3 shows an optimization process of the enzyme catalysis condition in the embodiment of the invention, wherein the nucleus pulposus cell of the catalysis condition M has good compatibility and is suitable for injectable hydrogel;
FIG. 4 is Western-Blot data of nucleus pulposus cells treated by each group of blank, IL-1. Beta., exosome, hydrogel extract, composite hydrogel extract according to an embodiment of the invention;
FIG. 5 is a graph of X-ray images of an IVDD rat model treated with each group of blank, disc degeneration, exosomes, hydrogels, composite hydrogels according to an embodiment of the present invention;
FIG. 6 is a graph showing a statistical distribution of disc heights measured from a plurality of sets of X-ray images on an animal model according to an embodiment of the present invention;
FIG. 7 is a graph of MRI images of an IVDD rat model treated with each group of blank, disc degeneration, exosomes, hydrogels, composite hydrogels according to an embodiment of the present invention;
FIG. 8 is a PFIRRMANN score chart obtained by analyzing and counting MRI images according to multiple sets of data according to an embodiment of the present invention.
Detailed Description
The invention provides carbon-carbon double bond modified keratin, which is obtained by nucleophilic addition reaction of isocyanate groups of isocyanate and amino groups of keratin, wherein the isocyanate also contains carbon-carbon double bonds;
the carbon-carbon double bond modification rate of the carbon-carbon double bond modified keratin is more than or equal to 30 percent.
In the present invention, all preparation materials/components are commercially available products well known to those skilled in the art unless specified otherwise.
In the present invention, the keratin is human hair keratin.
In the present invention, the method for preparing human hair keratin preferably comprises the steps of:
Mixing the sodium sulfide, urea, sodium Dodecyl Sulfate (SDS), water and human hair to obtain a keratin dissolving solution;
carrying out solid-liquid separation on the keratin dissolving solution to obtain a keratin solution;
and (3) sequentially dialyzing and drying the keratin solution to obtain the dry powder of the human hair keratin.
The invention mixes the sodium sulfide, urea, SDS, water and human hair to obtain the keratin dissolving solution. The water is preferably ultrapure water. In the present invention, the mass ratio of the sodium sulfide to the urea is preferably 1:4. The mass ratio of the sodium sulfide to the SDS is preferably 6:1.44. The ratio of the mass of sodium sulphide to the volume of water is preferably 6g:50ml. The mixing preferably comprises the steps of: ultrasonically mixing the sodium sulfide, urea, SDS and water for 10min to obtain an initial mixture; mixing the initial mixture and the human hair; the final mixing temperature is preferably 60 ℃, and the final mixing time is preferably 8h. The present invention preferably ensures that the keratin in the human hair is sufficiently dissolved by controlling the temperature and the holding time of the final mixing.
After the keratin dissolving solution is obtained, the keratin dissolving solution is subjected to solid-liquid separation to obtain the keratin solution. In the present invention, the solid-liquid separation is preferably centrifugation, the rotational speed of the centrifugation is preferably 9000rpm, and the number of times of the centrifugation is preferably 3. And (3) taking a liquid-phase product after the solid-liquid separation to obtain the keratin solution.
After the keratin solution is obtained, the keratin solution is dialyzed and dried in sequence to obtain the dry powder of the human hair keratin. In the present invention, the dialysis is preferably: filling the keratin solution into a dialysis bag, and immersing the dialysis bag filled with the keratin solution into water for dialysis; the molecular weight cut-off of the dialysis bag is preferably 3500Da; the dialysis time is preferably 3 days, and the present invention preferably changes the water used for dialysis every half day. In the present invention, the dialysis gives a purified keratin solution, which is preferably dried, preferably freeze-dried, preferably by: the purified keratin solution is frozen and then lyophilized in a freeze dryer, preferably for 3 days.
In the present invention, the isocyanate is preferably IEM.
In the invention, the carbon-carbon double bond modification rate of the carbon-carbon double bond modified keratin is more than or equal to 30%, preferably 30-40%.
In the present invention, the carbon-carbon double bond is modified in such a manner that the carbon-carbon double bond occupies a mole percentage of the carbon-carbon double bond modified keratin amino acid units.
The invention provides a preparation method of the keratin modified by the carbon-carbon double bond, which comprises the following steps:
Mixing the keratin, an organic solvent and isocyanate, and heating to perform nucleophilic addition reaction to obtain the carbon-carbon double bond modified keratin.
In the present invention, the organic solvent is preferably anhydrous dimethyl sulfoxide (DMSO).
In the present invention, the ratio of the mass of the keratin and the volume of the isocyanate is preferably a ratio at which the carbon-carbon double bond modified keratin having a target carbon-carbon double bond modification ratio can be synthesized.
As one or more embodiments of the present invention, the ratio of the mass of the keratin to the volume of the isocyanate is preferably 0.75 g:94. Mu.L.
In the present invention, the mixing of keratin, an organic solvent and isocyanate preferably comprises the steps of: dissolving the keratin in the organic solvent to obtain the keratin organic solution; mixing the keratin organic solution and the isocyanate; in the present invention, the ratio of the mass of the keratin and the volume of the organic solvent is preferably 0.75g:50mL.
In the present invention, the temperature of the nucleophilic addition reaction is preferably 60 to 70 ℃, more preferably 60 ℃; the heat preservation time of the nucleophilic addition reaction is preferably 16-18 h, more preferably 16h; in the present invention, the nucleophilic addition reaction is preferably performed in a protective gas atmosphere, and the protective gas is particularly preferably nitrogen.
In the invention, the mixed system forms a reaction solution after nucleophilic addition reaction, and the invention preferably carries out post-treatment on the reaction solution to obtain the carbon-carbon double bond modified keratin. In the present invention, the post-treatment preferably includes the steps of: and (3) sequentially dialyzing and drying the reaction liquid to obtain the carbon-carbon double bond modified keratin. In the present invention, the dialysis is preferably: filling the reaction liquid into a dialysis bag, and immersing the dialysis bag filled with the reaction liquid into water for dialysis; the water is preferably deionized water; the molecular weight cut-off of the dialysis bag is preferably 3500Da; the dialysis time is preferably 3 days, and the present invention preferably changes the water used for dialysis every half day during the dialysis. In the present invention, the dialysis is performed to obtain a purified modification reaction solution, and the purified modification reaction solution is preferably dried, preferably freeze-dried, preferably: the purified modification reaction solution is frozen and then placed in a freeze dryer for freeze-drying, and the freeze-drying time is preferably 3 days.
The invention provides a carbon-carbon double bond modified keratin injectable hydrogel, which comprises a solution of the carbon-carbon double bond modified keratin and an enzyme cascade catalytic system; the carbon-carbon double bond modified keratin is the carbon-carbon double bond modified keratin prepared by the technical scheme or the preparation method;
The enzyme cascade catalytic system comprises N-vinyl pyrrolidone, acetylacetone, glucose oxidase solution, glucose solution and horseradish peroxidase solution.
In the present invention, the mass volume fraction (w/v) of the solution of carbon-carbon double bond modified keratin is preferably 20%.
In the invention, the mass concentration of the glucose oxidase solution is 10mg/mL.
In the invention, the mass concentration of the horseradish peroxidase solution is 4mg/mL.
In the present invention, the molar concentration of the glucose solution is preferably 1.1mol/L.
In the invention, the glucose oxidase solution is a solution prepared by glucose oxidase and PBS buffer solution.
In the present invention, the glucose solution is a solution prepared from glucose and PBS buffer.
In the invention, the horseradish peroxidase solution is a solution prepared by horseradish peroxidase and PBS buffer solution.
In the present invention, the volume ratio of the solution of the carbon-carbon double bond modified keratin and the N-vinylpyrrolidone is preferably 100:0.11 to 1.
In the present invention, the volume ratio of the solution of the carbon-carbon double bond modified keratin and the acetylacetone is preferably 100:0.07 to 1.
In the present invention, the volume ratio of the solution of the carbon-carbon double bond modified keratin and the glucose oxidase solution is preferably 100:0.16-1.
In the invention, the volume ratio of the carbon-carbon double bond modified keratin solution to the horseradish peroxidase solution is preferably 100:2-4.
In the present invention, the volume ratio of the solution of the carbon-carbon double bond modified keratin to the glucose solution is preferably 100:2 to 5.
The invention provides a carbon-carbon double bond modified keratin injectable composite hydrogel, which comprises a carbon-carbon double bond modified keratin injectable hydrogel and a stem cell exosome wrapped in the carbon-carbon double bond modified keratin injectable hydrogel; the carbon-carbon double bond modified keratin injectable hydrogel is the carbon-carbon double bond modified keratin injectable hydrogel which is subjected to enzyme cascade reaction to initiate crosslinking according to the technical scheme.
In the present invention, the stem cell exosome is a mesenchymal stem cell exosome.
In the present invention, the mass ratio of the volume of the carbon-carbon double bond modified keratin injectable hydrogel to the stem cell exosomes is 100 μl:64ng.
The invention provides the carbon-carbon double bond modified keratin injectable hydrogel or the application of the carbon-carbon double bond modified keratin injectable composite hydrogel in preparation of biological scaffold materials for treating intervertebral disc degeneration.
The invention provides a novel method for realizing that injectable hydrogel is used for treating intervertebral disc degeneration after wrapping bone marrow mesenchymal stem cell exosomes by using keratin with double bond cross-linking initiated by an enzyme cascade catalytic system. According to the invention, disulfide bonds are opened through the reaction of sodium sulfide and human hair to obtain human hair keratin, and then isocyanate is used for modifying the human hair keratin to obtain the human hair keratin modified by carbon-carbon double bonds, and the carbon-carbon double bond modified keratin can be subjected to gel formation by an enzyme cascade catalytic system so as to realize injectability. The hydrogel provided by the invention and bone marrow mesenchymal stem cell exosome from rats are prepared into composite hydrogel, which has good anti-inflammatory and repair promoting effects in vitro and also has good treatment effect on a rat model with intervertebral disc degeneration. The invention provides a brand-new method for realizing injectable hydrogel based on keratin with carbon-carbon double bond cross-linking initiated by enzyme cascade reaction, which is successfully applied to intervertebral disc degeneration treatment after being compounded with exosomes, and provides a potential method for clinically treating the diseases.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
6G of sodium sulfide, 24g of urea and 1.44g of Sodium Dodecyl Sulfate (SDS) were taken and added to a flask, followed by 50mL of ultrapure water; after 10 minutes of sonication, a mixture was obtained; putting 5g of human hair into the mixture; subsequently, the flask was placed in an oil bath at 60 ℃ for reaction for 8 hours to sufficiently dissolve keratin in human hair, thereby obtaining a keratin lysate; afterwards, the keratin dissolved matter is centrifuged for 3 times in a centrifuge at 9000rpm to remove insoluble matters, and keratin solution is obtained; then, dialyzing the keratin solution in water for 3 days by using a dialysis bag with molecular weight cut-off of 3500Da, and changing water every half day; finally freezing the purified Keratin solution, and freeze-drying in a freeze dryer for 3 days to obtain human hair Keratin dry powder (Keratin);
0.75g of human hair keratin dry powder was dissolved in 50mL of anhydrous DMSO, followed by heating at 60℃for 45 minutes to dissolve the human hair keratin dry powder. After introducing high-purity nitrogen gas for 1 hour, 94 mu L of isocyanoethyl methacrylate (IEM) was added to the obtained human hair keratin solution, and the reaction was continued at 60 ℃ for 16 hours to allow the IEM to react with keratin sufficiently; after the reaction is completed, adding 2mL of deionized water to terminate the reaction, then dialyzing the reaction in deionized water for 3 days by using a dialysis bag with molecular weight cut-off of 3500Da, freezing the purified modified keratin solution, and freeze-drying the solution in a freeze dryer for 3 days to obtain double bond modified human hair keratin (KeMA), wherein the result of testing nuclear magnetic resonance hydrogen spectrum is shown in figure 1, the nuclear magnetic resonance hydrogen spectrum shows that the human hair keratin is successfully modified with carbon-carbon double bonds, the modification rate of isocyanoethyl methacrylate is calculated to be 30.3%, and good cross-linking property of the double bond modified human hair keratin is ensured.
Example 2
First, a 20% (w/v) modified keratin solution was prepared with PBS; preparing 4mg/mL horseradish peroxidase (HRP) mother liquor by PBS; preparing 10mg/mL glucose oxidase (GOx) mother liquor by PBS;
Taking 1mL of prepared double bond modified human hair keratin (KeMA) solution, adding 10 mu LN-vinyl pyrrolidone (NVP), 10 mu L acetylacetone (AcAc), 40 mu LHRP mother solution and 10 mu LGOx mother solution, uniformly mixing, adding 50 mu L1.1M glucose solution, and uniformly mixing again, wherein the double bond modified human hair keratin initiates free radical polymerization of carbon-carbon double bonds through enzyme cascade reaction of glucose/GOx/HRP/AcAc/NVP to obtain the carbon-carbon double bond modified keratin injectable hydrogel; observing the gel forming condition in the free radical polymerization reaction process, the mass volume fraction of KeMA in KeMA solutions is regulated according to the gel forming condition, so as to study the influence of KeMA component content change on the mechanical property of the hydrogel, the strength of the hydrogel is tested by using a rheometer, the mechanical strength of the KeMA hydrogel with 20%w/v is optimized to reach 5kPa, the strength is close to that of natural nucleus pulposus tissues, and the result is shown in figure 2.
Example 3
In the embodiment, the chemical reagent and GOx have certain toxicity, and on the premise of keeping the mass volume fraction of KeMA solution to be 20%, the contents of GOx, NVP and AcAc are adjusted, so that the proportioning relation between a proper enzyme cascade catalytic system and KeMA solution is obtained: 100 μL of 20% KeMA solution, 0.11 μ LNVP,0.07 μ LAcAc,0.16 μ LGOx,2 μ LHRP,2 μ L1.1M Glucose (Glucose) solution;
The KeMA hydrogel obtained in this example includes: 100 μL of 20% (w/v) KeMA solution, 0.11 μ LNVP,0.07 μ LAcAc,0.16 μ LGOx,2 μ LHRP,2 μ L1.1M Glucose (Glucose) solution;
Under the compounding condition of KeMA solution and enzyme cascade catalytic system (catalytic condition M in figure 3) of the embodiment, the obtained injectable hydrogel leaching solution has good compatibility with nucleus pulposus cells, and the result is shown in figure 3.
Example 4
Extraction of bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exo): exosomes were extracted from rat-derived bone marrow mesenchymal stem cells (BMSCs) using ultracentrifugation: firstly, culturing rat BMSCs in a serum-free stem cell culture medium for 48 hours, and then collecting the culture medium for separating exosomes; centrifuging the culture medium sequentially with a centrifugal force of 300g for 10 minutes, a centrifugal force of 2000g for 10 minutes and a centrifugal force of 10000g for 30 minutes to remove cells, dead cells and cell fragments respectively, and taking supernatant after each centrifugation for subsequent operation; then, the supernatant after centrifugation is subjected to ultracentrifugation with 100000g for 70 minutes, the supernatant is removed, and BMSC-Exo particles are reserved;
The BMSC-Exo particles were resuspended in 1mLPBS g and ultracentrifuged again at 100000g for 70 minutes, and the supernatant was removed to give purified BMSC-Exo particles; the purified BMSC-Exo particles were then resuspended with 100. Mu. LPBS to obtain a purified BMSC-Exo suspension; all centrifuges in this example were carried out at 4 ℃;
taking 100 mu L of 20% KeMA solution, 0.11 mu LNVP,0.07 mu LAcAc,0.16 mu LGOx,2 mu LHRP and 2 mu L1.1M Glucose (Glucose) solution to obtain a carbon-carbon double bond modified keratin injectable hydrogel;
And mixing the carbon-carbon double bond modified keratin injectable hydrogel with the purified BMSC-Exo suspension to obtain the carbon-carbon double bond modified keratin injectable composite hydrogel, namely KeMA composite hydrogel wrapped by the BMSC-Exo.
Test example 1
Leaching KeMA hydrogel obtained in example 3 with F12 medium to obtain leaching solution of KeMA hydrogel;
Leaching KeMA composite hydrogel which is obtained in example 4 and is coated with BMSC-Exo by using F12 culture medium to obtain leaching liquor of the composite hydrogel;
Dividing the rat nucleus pulposus cells into 5 groups, the first group being untreated; the second group was treated with 10 ng/mlIL-1. Beta. Induction; the third group was pretreated with 10 ng/mLIL-1. Beta. For 24 hours, followed by incubation with 0.64. Mu.g/mL BMSC-Exo; the fourth group was pretreated with 10 ng/mLIL-1. Beta. For 24 hours, followed by addition of KeMA hydrogel extract for incubation; the fifth group is pretreated for 24 hours with 10ng/mLIL-1 beta, and then the leaching solution of the composite hydrogel is added for culture; after the cells were cultured in the incubator for one day, the expression of inflammatory cytokines IL-6, TNF- α, matrix metal degrading enzyme MMP13, collagen type two collagenII was detected by Westernblot, and the results are shown in FIG. 4. The invention uses the hydrogel to wrap BMSC-Exo of rat source to obtain the injectable composite hydrogel, and in vitro experiments show that the composite hydrogel provided by the embodiment 4 of the invention has good capability of reducing nucleus pulposus cell inflammation and matrix degrading enzyme level and simultaneously regulating the expression level of type II collagen.
Test example 2
Construction of a rat model of disc degeneration: SD rats of 8 weeks old are selected, 4% chloral hydrate is injected into abdominal cavity for anesthesia, the rats are fixed on a molding table in a supine mode, tails of the rats are disinfected for 3 times by normal saline, iodophor solution, 75% alcohol and the like, 1 tail intervertebral disc segment (Co 6/7) is selected, after intervertebral disc positioning, a 26G needle head is used for needle feeding from the rear side of the intervertebral disc, the inner nucleus pulposus structure is punched and disturbed by the needle, and intervertebral disc injury is caused to induce intervertebral disc degeneration, and the intervertebral disc degeneration is kept for one minute. And then the needle is withdrawn, the tail of the rat is disinfected for 3 times by using an iodophor solution, 75% alcohol and the like in sequence, and the molding is finished.
Hydrogel injection treatment of rat disc: setting normal rats as blank groups, injecting 10 mu LPBS, 10 mu L of BMSC-Exo suspension prepared in example 4, 10 mu L of KeMA hydrogel prepared in example 3 or 10 mu L of composite hydrogel prepared in example 4 into the coccyx of the intervertebral disc degeneration rat respectively, killing the rats on the 30 th day of feeding, and taking out the coccyx for subsequent analysis experiments;
Testing the inflammation level and repair effect of the intervertebral disc: the inflammatory level was assessed by observing disc roughness by X-ray imaging, the results are shown in figure 5; further statistics of the disc height can quantitatively analyze the treatment effect of the IVDD, and the result is shown in figure 6; magnetic Resonance Imaging (MRI) results are shown in fig. 7; further, PFIRRMANN scores from each group were counted to evaluate IVDD severity, and the results are shown in fig. 8; the test example tests the treatment effect of the hydrogel prepared in the example 3 and the composite hydrogel prepared in the example 4 on the IVDD model of rats, and the in-vivo experiments in figures 5-8 show that the composite hydrogel provided by the invention has the treatment effect of anti-inflammatory and pro-repair on the IVDD rats.
In summary, the invention discloses a novel method for realizing injectable hydrogels by using keratins with double bond cross-linking induced by enzyme cascade reaction, which is used for IVDD treatment after wrapping BMSC-Exo exosomes.
Although the above embodiments have been described in detail, it is only a part of the embodiments of the present invention, but not all the embodiments, and other embodiments can be obtained according to the present embodiment without inventive effort, which fall within the scope of the present invention.
Claims (8)
1. A carbon-carbon double bond modified keratin injectable hydrogel, characterized by comprising a solution of carbon-carbon double bond modified keratin and an enzyme cascade catalytic system; the carbon-carbon double bond modified keratin is obtained by nucleophilic addition reaction of isocyanate groups of isocyanate and amino groups of keratin, and the isocyanate also contains carbon-carbon double bonds; the keratin is human hair keratin; the isocyanate comprises isocyanoethyl methacrylate;
the modification rate of the carbon-carbon double bond modified keratin is more than or equal to 30%;
The enzyme cascade catalytic system comprises N-vinyl pyrrolidone, acetylacetone, glucose oxidase solution, glucose solution and horseradish peroxidase solution.
2. The injectable keratin hydrogel modified by a carbon-carbon double bond according to claim 1, wherein the preparation method of the keratin modified by a carbon-carbon double bond comprises the following steps:
Mixing the keratin, an organic solvent and isocyanate, and heating to perform nucleophilic addition reaction to obtain the carbon-carbon double bond modified keratin.
3. The carbon-carbon double bond modified keratin injectable hydrogel according to claim 2, wherein the temperature of the nucleophilic addition reaction is 60-70 ℃, and the heat preservation time of the nucleophilic addition reaction is 16-18 h.
4. The carbon-carbon double bond modified keratin injectable hydrogel of claim 1, wherein the mass volume fraction of the solution of carbon-carbon double bond modified keratin is 20%; the mass concentration of the glucose oxidase solution is 10 mg/mL; the mass concentration of the horseradish peroxidase solution is 4 mg/mL; the molar concentration of the glucose solution is 1.1 mol/L;
the volume ratio of the solution of the carbon-carbon double bond modified keratin to the N-vinyl pyrrolidone is 100:0.11-1;
the volume ratio of the solution of the carbon-carbon double bond modified keratin to the acetylacetone is 100:0.07-1;
the volume ratio of the carbon-carbon double bond modified keratin solution to the glucose oxidase solution is 100:0.16-1;
the volume ratio of the carbon-carbon double bond modified keratin solution to the horseradish peroxidase solution is 100:2-4;
The volume ratio of the carbon-carbon double bond modified keratin solution to the glucose solution is 100:2-5.
5. A carbon-carbon double bond modified keratin injectable composite hydrogel, which is characterized by comprising a carbon-carbon double bond modified keratin injectable hydrogel and stem cell exosomes wrapped in the carbon-carbon double bond modified keratin injectable hydrogel; the carbon-carbon double bond modified keratin injectable hydrogel is the carbon-carbon double bond modified keratin injectable hydrogel according to any one of claims 1 to 4.
6. The carbon-carbon double bond modified keratin injectable composite hydrogel of claim 5, wherein the stem cell exosomes are bone marrow mesenchymal stem cell exosomes.
7. The carbon-carbon double bond modified keratin injectable composite hydrogel according to claim 5 or 6, wherein the mass ratio of the volume of the carbon-carbon double bond modified keratin injectable hydrogel to the stem cell exosomes is 100 μl:64 ng.
8. Use of the carbon-carbon double bond modified keratin injectable hydrogel according to any one of claims 1 to 4 or the carbon-carbon double bond modified keratin injectable composite hydrogel according to any one of claims 5 to 7 in the preparation of a biological scaffold material for treating disc degeneration.
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| CN104922734A (en) * | 2015-05-21 | 2015-09-23 | 东南大学 | Injectable chitosan composite hydrogel capable of promoting myocardium repair and preparation method of injectable chitosan composite hydrogel |
| CN114618010A (en) * | 2020-12-09 | 2022-06-14 | 四川大学 | Multifunctional keratin-based hydrogel and preparation method thereof |
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| CN104922734A (en) * | 2015-05-21 | 2015-09-23 | 东南大学 | Injectable chitosan composite hydrogel capable of promoting myocardium repair and preparation method of injectable chitosan composite hydrogel |
| CN114618010A (en) * | 2020-12-09 | 2022-06-14 | 四川大学 | Multifunctional keratin-based hydrogel and preparation method thereof |
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