CN114209889A - Modified chitosan/functional short peptide composite hydrogel and preparation method and application thereof - Google Patents
Modified chitosan/functional short peptide composite hydrogel and preparation method and application thereof Download PDFInfo
- Publication number
- CN114209889A CN114209889A CN202111603364.5A CN202111603364A CN114209889A CN 114209889 A CN114209889 A CN 114209889A CN 202111603364 A CN202111603364 A CN 202111603364A CN 114209889 A CN114209889 A CN 114209889A
- Authority
- CN
- China
- Prior art keywords
- short peptide
- functional
- chitosan
- solution
- modified chitosan
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 229920001661 Chitosan Polymers 0.000 title claims abstract description 139
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 97
- 239000000017 hydrogel Substances 0.000 title claims abstract description 87
- 239000002131 composite material Substances 0.000 title claims abstract description 53
- 238000002360 preparation method Methods 0.000 title claims abstract description 39
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 claims abstract description 48
- 150000001413 amino acids Chemical group 0.000 claims abstract description 35
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims abstract description 12
- 125000002883 imidazolyl group Chemical group 0.000 claims abstract description 10
- 208000020431 spinal cord injury Diseases 0.000 claims abstract description 9
- 238000013329 compounding Methods 0.000 claims abstract description 5
- 230000008439 repair process Effects 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 75
- 239000011259 mixed solution Substances 0.000 claims description 22
- 239000007864 aqueous solution Substances 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 13
- 238000002156 mixing Methods 0.000 claims description 13
- NUGAFWCHXXQOQJ-UHFFFAOYSA-N 1-hydroxypyrrolidine-2,5-dione;sodium Chemical compound [Na].ON1C(=O)CCC1=O NUGAFWCHXXQOQJ-UHFFFAOYSA-N 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 7
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 5
- 108010067520 RADA16-I Proteins 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 239000004475 Arginine Substances 0.000 claims description 3
- 239000011261 inert gas Substances 0.000 claims description 3
- 239000004471 Glycine Substances 0.000 claims description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 2
- 239000004472 Lysine Substances 0.000 claims description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000006143 cell culture medium Substances 0.000 claims description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical group OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 abstract description 4
- 238000006116 polymerization reaction Methods 0.000 abstract description 4
- 230000002441 reversible effect Effects 0.000 abstract description 4
- 230000009471 action Effects 0.000 abstract description 2
- 238000002324 minimally invasive surgery Methods 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 29
- 239000008367 deionised water Substances 0.000 description 27
- 229910021641 deionized water Inorganic materials 0.000 description 27
- 239000000499 gel Substances 0.000 description 17
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 14
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 14
- 238000004108 freeze drying Methods 0.000 description 10
- 238000003756 stirring Methods 0.000 description 10
- AZKVWQKMDGGDSV-BCMRRPTOSA-N Genipin Chemical compound COC(=O)C1=CO[C@@H](O)[C@@H]2C(CO)=CC[C@H]12 AZKVWQKMDGGDSV-BCMRRPTOSA-N 0.000 description 7
- 108010038807 Oligopeptides Proteins 0.000 description 7
- 102000015636 Oligopeptides Human genes 0.000 description 7
- 238000000502 dialysis Methods 0.000 description 7
- AZKVWQKMDGGDSV-UHFFFAOYSA-N genipin Natural products COC(=O)C1=COC(O)C2C(CO)=CCC12 AZKVWQKMDGGDSV-UHFFFAOYSA-N 0.000 description 7
- XQQUSYWGKLRJRA-RABCQHRBSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-6-amino-2-[[(2s,3s)-2-amino-3-methylpentanoyl]amino]hexanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-3-methylbutanoic acid Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O XQQUSYWGKLRJRA-RABCQHRBSA-N 0.000 description 6
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 6
- MHUWZNTUIIFHAS-CLFAGFIQSA-N dioleoyl phosphatidic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(O)=O)OC(=O)CCCCCCC\C=C/CCCCCCCC MHUWZNTUIIFHAS-CLFAGFIQSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 108010088381 isoleucyl-lysyl-valyl-alanyl-valine Proteins 0.000 description 6
- 229960004502 levodopa Drugs 0.000 description 6
- 210000000278 spinal cord Anatomy 0.000 description 6
- 230000008569 process Effects 0.000 description 5
- 239000000376 reactant Substances 0.000 description 5
- 239000012465 retentate Substances 0.000 description 5
- 208000027418 Wounds and injury Diseases 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 208000014674 injury Diseases 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 238000004132 cross linking Methods 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 210000004126 nerve fiber Anatomy 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 229920002101 Chitin Polymers 0.000 description 2
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 2
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- -1 cationic polysaccharide Chemical class 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000001125 extrusion Methods 0.000 description 2
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 210000005036 nerve Anatomy 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- MWOGMBZGFFZBMK-LJZWMIMPSA-N (2s)-2-[[(2s)-2-[[2-[[(2s,3s)-2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylpentanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-5-(diaminomethylideneamino)pentanoic acid Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 MWOGMBZGFFZBMK-LJZWMIMPSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 206010072170 Skin wound Diseases 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 230000028600 axonogenesis Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000000850 deacetylating effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 210000001951 dura mater Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea group Chemical group NC(=S)N UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 description 1
- 108010052768 tyrosyl-isoleucyl-glycyl-seryl-arginine Proteins 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N urea group Chemical group NC(=O)N XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 230000037314 wound repair Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
-
- 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
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
-
- 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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
-
- 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
- A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
-
- 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
-
- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
-
- 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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Dermatology (AREA)
- Veterinary Medicine (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Materials Engineering (AREA)
- Engineering & Computer Science (AREA)
- Composite Materials (AREA)
- Dispersion Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Materials For Medical Uses (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention provides a modified chitosan/functional short peptide composite hydrogel and a preparation method and application thereof, wherein the composite hydrogel is obtained by compounding modified chitosan and functional short peptide, and the modified chitosan is dopa grafted functionalized chitosan; the functional short peptide is a short peptide with one side connected with an amino acid sequence containing a benzene ring and an imidazolyl and the other side connected with a functional amino acid sequence. The invention utilizes the self-polymerization of catechol group on dopa and the reversible non-covalent bond action of catechol group and functional short peptide to obtain the injectable and self-repairing hydrogel which can be used as a tissue engineering scaffold, is injected to a spinal cord injury part through a minimally invasive surgery, can promote the repair of the spinal cord injury, and has very wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a modified chitosan/functional short peptide composite hydrogel as well as a preparation method and application thereof.
Background
The chitosan is derived from chitin, is obtained by deacetylating amino groups on natural polysaccharide chitin, has various physiological functions of biodegradability, biocompatibility, nontoxicity, bacteriostasis, immunity enhancement and the like, and is widely applied to the field of medical treatment. Chitosan is the only cationic polysaccharide in nature, has the advantages of rich content, low price, good biocompatibility and biodegradability and the like, and is often used for preparing hydrogel.
In recent years, with further research, injectable chitosan hydrogels with excellent properties have been developed by various chemical or physical modification and modification methods, introduction of various biofunctional molecules, or various crosslinking methods, and their application range has been expanded, playing more and more important roles in practical applications (Liuyang, Wang Ying, Wang Yuanjuan, etc. [ J ] preparation and application of injectable chitosan hydrogels, 2020(5):17-23 ]. For example, the chinese patent discloses a method for preparing an injectable composite chitosan hydrogel for skin wound repair, which is injectable, but requires the addition of an initiator for preparation, and has a risk of toxic auxiliary agent residue. The Chinese invention patent also discloses a self-repairable chitosan hydrogel and a preparation method thereof, wherein hydrogen bond groups such as thiourea group and urea group are introduced into the high molecular chain segment of the chitosan hydrogel, and self-repair of the material is realized by means of the breakage and recombination of hydrogen bonds in molecules or between molecules.
The injectable hydrogel has the advantages of non-invasive or minimally invasive implantation, can be used as a repair material for irregular-shaped injuries, and avoids risks brought by large-scale operations; however, hydrogels implanted in vivo are highly susceptible to stress cracking if subjected to external mechanical action, the further development of cracks can destroy the regularity of the internal structure and the functional integrity of the material, and the damaged gel matrix can easily cause rejection after entering body fluids, with the risk of causing inflammation. If the hydrogel has self-repairing performance and can be spontaneously recombined into a whole after being damaged by external force, the hydrogel has greater application value. In the existing research, no report of preparing injectable and self-repairing hydrogel by using chitosan is found.
Disclosure of Invention
The invention aims to overcome the defects of the existing chitosan hydrogel and provide a modified chitosan/functional short peptide composite hydrogel as well as a preparation method and application thereof, and the composite hydrogel has injectability and self-repairing performance. The preparation method comprises the steps of grafting dopa on chitosan molecules by a chemical synthesis method to prepare modified chitosan, compounding the modified chitosan with functional short peptides to prepare hydrogel, and obtaining the injectable and self-repairing hydrogel by utilizing the oxidative self-polymerization characteristic of dopa on the chitosan molecules and the dynamic and reversible pi-pi conjugation between the benzene ring of a dopa group and the benzene ring of the short peptides.
The invention aims to provide a modified chitosan/functional short peptide composite hydrogel.
The invention also aims to provide a preparation method of the modified chitosan/functional short peptide composite hydrogel.
The invention also aims to provide application of the modified chitosan/functional short peptide composite hydrogel in repairing spinal cord injury.
The above purpose of the invention is realized by the following technical scheme:
a modified chitosan/functional short peptide composite hydrogel is obtained by compounding modified chitosan and functional short peptide, wherein the modified chitosan is dopa grafted functionalized chitosan; the functional short peptide is a short peptide with one side connected with an amino acid sequence containing a benzene ring and an imidazolyl and the other side connected with a functional amino acid sequence.
The composite hydrogel adopts chitosan as one of the main components, utilizes dopa grafting reaction to carry out functionalization modification on the chitosan, and achieves the effects of increasing tissue viscosity and forming gel through the self-polymerization of catechol groups on dopa; meanwhile, a kind of functional short peptide is designed to form a dynamic reversible pi-pi conjugated noncovalent bond with the dopa modified chitosan, and the pi-pi conjugated noncovalent bond enables the crosslinking point of the composite hydrogel to be in a dynamic 'combination-dissociation' state, so that the hydrogel has injectability and self-repairability.
Preferably, the functional short peptide is (arginine-alanine-aspartic acid-alanine)4-I (RADA16-I, i.e. (RADA)4One side of the sequence of-I) is connected with an amino acid sequence containing a benzene ring and an imidazolyl, and the other side is connected with a short peptide of a functional amino acid sequence.
Further preferably, 1-3 positively charged amino acids are contained between the RADA16-I sequence and the functional amino acid sequence, and 1-3 achiral amino acids are contained between the amino acid sequence containing the benzene ring and the imidazolyl group.
Further preferably, the positively charged amino acids are lysine, arginine; the achiral amino acid is glycine.
Further preferably, the amino acid sequence containing a benzene ring and an imidazole group is histidine-glycine-phenylalanine (HGF).
Further preferably, the functional amino acid sequence comprises: amino acids that promote nerve cell adhesion, including but not limited to arginine-glycine-aspartic acid (RGD), tyrosine-isoleucine-glycine-serine-arginine (YIGSR), phenylalanine-aspartic acid-glycine-aspartic acid-arginine-glycine-aspartic acid-serine (FDGDRGDS); the functional amino acid sequence isoleucine-lysine-valine-alanine-valine (IKVAV) for promoting axon growth.
Preferably, the pH value of the composite hydrogel is 6-8.
Further preferably, the pH value of the composite hydrogel is 6-7.
Most preferably, the composite hydrogel has a pH of 7.
A preparation method of modified chitosan/functional short peptide composite hydrogel is characterized by comprising the following steps:
s1, preparation of modified chitosan
In an inert gas atmosphere, uniformly mixing an N-hydroxysuccinimide sodium (NHS) aqueous solution and a DOPA (DOPA) aqueous solution, then uniformly mixing the N-hydroxysuccinimide sodium (NHS) aqueous solution and a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) aqueous solution, fully and uniformly mixing the N-hydroxysuccinimide sodium (NHS) aqueous solution and the DOPA aqueous solution, and adjusting the pH of the mixed solution to 5.5-6; the pH value of the aqueous solution is 4.0-5.5; placing the fully and uniformly mixed solution at the temperature of 20-30 ℃ for reaction for 20-25 h, dialyzing, and drying to obtain modified chitosan; modified chitosan, namely dopa grafted functionalized chitosan;
s2, preparation of functional short peptide
Connecting an amino acid sequence containing a benzene ring and an imidazolyl on one side of the short peptide, and connecting a functional amino acid sequence on the other side to obtain the functional short peptide.
S3, preparation of modified chitosan/functional short peptide composite hydrogel
And (3) mixing the solution of the modified chitosan obtained in the step S1 and the solution of the functional short peptide obtained in the step S2, and then injecting the mixed solution into a buffer solution or a cell culture medium to obtain the composite hydrogel.
Preferably, the pH of the aqueous solution of N-hydroxysuccinimide sodium (NHS), the aqueous solution of DOPA (DOPA) and the aqueous solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 5-5.5; the pH value of the chitosan water solution is 4-5.
Preferably, the concentration of the chitosan solution in the step S1 is 1-8 mM.
Further preferably, the final concentration of the chitosan solution is 1-4 mM.
Most preferably, the final concentration of the chitosan solution is 4 mM.
Preferably, the molar ratio of chitosan, NHS, EDC and dopa in step S1 is 1: 1-4: 1-4: 1 to 4.
Further preferably, the molar ratio of chitosan, NHS, EDC and dopa in step S1 is 1: 2-4: 2-4: 1 to 2.
Most preferably, the molar ratio of chitosan, NHS, EDC and dopa in step S1 is 1: 4: 4: 2.
preferably, the dialysis in step S1 is performed by using deionized water in a dialysis bag with a molecular weight cut-off of 3000-4000.
Preferably, the drying condition in the step S1 is a pressure of 20-50 Pa, a temperature of-50 to-80 ℃ and a time of 36-72 h.
Further preferably, the drying condition in the step S1 is that the pressure is 45-50 Pa, the temperature is-50 to-55 ℃, and the time is 48-72 h.
Most preferably, the drying condition of step S1 is pressure 50Pa, temperature-50 deg.C, and time 72 h.
Preferably, the inert gas in step S1 is nitrogen or helium.
Preferably, the solvent of the solution of step S3 is deionized water.
Preferably, the solution of the modified chitosan in the step S3 has a mass percentage concentration of 5% -15%; the mass percentage concentration of the solution of the functional short peptide is 0.5-2%.
Further preferably, the solution of the modified chitosan in the step S3 has a mass percentage concentration of 10% to 15%; the mass percentage concentration of the solution of the functional short peptide is 1-2%.
Most preferably, the solution of the modified chitosan in the step S3 has a mass percentage concentration of 10%; the mass percentage concentration of the functional short peptide solution is 1%.
Preferably, the volume ratio of the modified chitosan solution to the functional short peptide solution in the mixed solution of step S3 is 1:0.5 to 2.
Further preferably, the volume ratio of the modified chitosan solution to the functional short peptide solution in the mixed solution of step S3 is 1: 1-2.
Most preferably, the volume ratio of the modified chitosan solution to the functional short peptide solution of the mixed solution in the step S3 is 1: 1.
Preferably, the pH of the mixed solution in the step S3 is 6-8.
Further preferably, the pH of the mixed solution in the step S3 is 6-7.
Most preferably, the pH of the mixed solution in step S3 is 7.
An application of a modified chitosan/functional short peptide composite hydrogel in preparing a spinal cord injury repair material.
Preferably, the application is to inject the modified chitosan/functional short peptide composite aqueous solution into the defect part after spinal cord injury, and form hydrogel in situ to be used as an artificial extracellular matrix to support nerve regeneration.
The dopa grafted functionalized chitosan is modified chitosan.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the chitosan is functionalized and modified by utilizing a dopa grafting reaction, and the effect of increasing tissue viscosity is achieved through self-polymerization of catechol groups on dopa; the functional short peptide and the dopa grafted functionalized chitosan form a dynamic reversible pi-pi conjugated noncovalent bond, and finally, the injectable and self-repairing composite hydrogel with stronger mechanical property and mechanical stability is obtained; meanwhile, the hydrogel is simple in preparation conditions, low in cost, free of additional additives, capable of effectively avoiding the problem of harmful influence caused by toxic additive residues, and high in biological safety.
Drawings
FIG. 1 is the observation result of modified chitosan/functional short peptide composite gel in example 1 of the present invention; wherein A is an injectable image, and B is a scanning electron microscope image.
FIG. 2 is a diagram showing the self-repairing effect of the modified chitosan/functional short peptide composite gel in example 1 of the present invention; a is the hydrogel block cut into 4 blocks, and B is the gel formed by the gel block after extrusion.
FIG. 3 shows the immunofluorescence staining of sagittal sections at the spinal cord transection in example 6 of the present invention; NF200 is nerve fiber, GFAP is astrocyte, and DAPI is nucleus.
FIG. 4 shows the splitting and extruding effects of the hydrogel formed by compounding genipin cross-linked phenylalanine graft-modified chitosan/RADA 16-RIKVAV short peptide in the comparative example of the invention. Wherein A is a hydrogel block cut into 4 blocks; b is the effect of being cut into 4 pieces and extruded without forming an integral gel.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Antibody NF200, Gibco (usa); antibody, GFAPGibco (USA)
Example 1 preparation of modified Chitosan/functional short peptide Complex gel
1. Preparation of modified chitosan
In N2Dissolving chitosan in deionized water in an atmosphere, and adjusting the pH to 5.0 by using 1M HCl to prepare a solution with a final concentration of 4 mM; NHS and EDC were dissolved in deionized water and pH adjusted to 5.5 with 1M HCl, dopa was dissolved in deionized water and pH adjusted to 5.5 with 1M HCl, respectively. Adding the NHS solution into the dopa solution, stirring for 15min, then adding the EDC solution, continuing stirring for 15min, and dropwise adding the chitosan solution to enable the molar ratio of chitosan, NHS, EDC and dopa in the mixed solution to be 1: 4: 4: 2, then adjusting the pH value to 5.5, reacting for 24 hours, and maintaining the whole process at N2The temperature in the atmosphere was 25 ℃. And (3) putting the reactant into a dialysis bag with the molecular weight cutoff of 3000-4000, dialyzing for 3 days by taking deionized water as dialysate, and freeze-drying the retentate to obtain the modified chitosan, namely the dopa grafted functionalized chitosan. The freeze drying condition is pressure of 50Pa, temperature of-50 deg.C, and time of 72 h.
2. Functional short peptide HGF- (RADA)4Preparation of RIKVAV
Using short peptide synthesizer (arginine-alanine-aspartic acid-alanine)4Connecting histidine-glycine-phenylalanine (HGF) at one end of the sequence and connecting functional amino acid sequence IKVAV at the other end to obtain functional short peptide HGF- (RADA)4-RIKVAV。
3. Modified chitosan/functional oligopeptide HGF- (RADA)4Preparation of-RIKVAV composite hydrogel
Grafting the dopa prepared in the step 1 with the functionalized chitosan and the functional short peptide HGF- (RADA) prepared in the step 24Dissolving RIKVAV in deionized water respectively to obtain a dopa grafted functionalized chitosan solution with the mass percentage concentration of 10%, and a functional short peptide HGF- (RADA) with the mass percentage concentration of 1%4-RIKVAV solution, mixing the two solutions, dopa grafted functionalized chitosan solution and functional short peptide HGF- (RADA)4The volume ratio of RIKVAV solution was 1:1, pH was adjusted to 7, and hydrogel was formed after injection into PBS.
Example 2 preparation of modified Chitosan/functional short peptide Complex gel
1. Preparation of modified chitosan
In N2Dissolving chitosan in deionized water in an atmosphere, and adjusting the pH to 5.0 by using 1M HCl to prepare a solution with a final concentration of 1 mM; NHS and EDC were dissolved in deionized water and pH adjusted to 5.5 with 1M HCl, dopa was dissolved in deionized water and pH adjusted to 5.5 with 1M HCl, respectively. Adding the NHS solution into the dopa solution, stirring for 15min, then adding the EDC solution, continuing stirring for 15min, and dropwise adding the chitosan solution to enable the molar ratio of chitosan, NHS, EDC and dopa in the mixed solution to be 1: 1: 1:1, then adjusting the pH value to 5.5, reacting for 24 hours, and maintaining the whole process at N2The temperature in the atmosphere was 25 ℃. And (3) putting the reactant into a dialysis bag with the molecular weight cutoff of 3000-4000, dialyzing for 3 days by taking deionized water as dialysate, and freeze-drying the retentate to obtain the modified chitosan, namely the dopa grafted functionalized chitosan. The freeze drying condition is pressure of 45Pa, temperature of-55 deg.C, and time of 48 h.
2. Functional short peptide HGF- (RADA)4Preparation of RIKVAV
Connecting histidine-glycine-phenylalanine (HGF) at one end of the (arginine-alanine-aspartic acid-alanine) 4 sequence and connecting functional amino acid sequence IKVAV at the other end by using a short peptide synthesizer to obtain functional short peptide HGF- (RADA)4-RIKVAV。
3. Modified chitosan/functional oligopeptide HGF- (RADA)4Preparation of-RIKVAV composite hydrogel
Grafting the dopa prepared in the step 1 with the functionalized chitosan and the functional short peptide HGF- (RADA) prepared in the step 24Respectively dissolving RIKVAV in deionized water to obtain a dopa grafted functionalized chitosan solution with the mass percentage concentration of 10%, and functional short peptide HGF- (RADA) with the mass percentage concentration of 1%4-RIKVAV solution, mixing the two solutions, dopa grafted functionalized chitosan solution and functional short peptide HGF- (RADA)4The volume ratio of RIKVAV solution was 1:1, pH adjusted to 6, and hydrogel was formed after injection into PBS.
Example 3 preparation of modified Chitosan/functional short peptide Complex gel
1. Preparation of modified chitosan
In N2Dissolving chitosan in deionized water in an atmosphere, and adjusting the pH to 5.0 by using 1M HCl to prepare a solution with a final concentration of 8 mM; NHS and EDC were dissolved in deionized water and pH adjusted to 5.5 with 1M HCl, dopa was dissolved in deionized water and pH adjusted to 5.5 with 1M HCl, respectively. Adding the NHS solution into the dopa solution, stirring for 15min, then adding the EDC solution, continuing stirring for 15min, and dropwise adding the chitosan solution to enable the molar ratio of chitosan, NHS, EDC and dopa in the mixed solution to be 1: 2: 2: 1, then adjusting the pH value to 5.5, reacting for 24 hours, and maintaining the whole process at N2The temperature in the atmosphere was 25 ℃. And (3) putting the reactant into a dialysis bag with the molecular weight cutoff of 3000-4000, dialyzing for 3 days by taking deionized water as dialysate, and freeze-drying the retentate to obtain the modified chitosan, namely the dopa grafted functionalized chitosan. The freeze drying condition is pressure of 50Pa, temperature of-50 deg.C, and time of 72 h.
2. Functional short peptide HGF- (RADA)4Preparation of RIKVAV
Using short peptide synthesizer on (arginine-alanine-aspartic acid-alanine))4, one end of the sequence is connected with histidine-glycine-phenylalanine (HGF), the other end is connected with a functional amino acid sequence IKVAV, and a functional short peptide HGF- (RADA) is obtained4-RIKVAV。
3. Modified chitosan/functional oligopeptide HGF- (RADA)4Preparation of-RIKVAV composite hydrogel
Grafting the dopa prepared in the step 1 with the functionalized chitosan and the functional short peptide HGF- (RADA) prepared in the step 24Respectively dissolving RIKVAV in deionized water to obtain a dopa grafted functionalized chitosan solution with the mass percentage concentration of 15%, and functional short peptide HGF- (RADA) with the mass percentage concentration of 2%4-RIKVAV solution, mixing the two solutions, dopa grafted functionalized chitosan solution and functional short peptide HGF- (RADA)4The volume ratio of RIKVAV solution was 1:0.5, pH adjusted to 8, and hydrogel was formed after injection into PBS.
Example 4 preparation of modified Chitosan/functional short peptide Complex gel
1. Preparation of modified chitosan
In N2Dissolving chitosan in deionized water in an atmosphere, and adjusting the pH to 5.0 by using 1M HCl to prepare a solution with a final concentration of 4 mM; NHS and EDC were dissolved in deionized water and pH adjusted to 5.5 with 1M HCl, dopa was dissolved in deionized water and pH adjusted to 5.5 with 1M HCl, respectively. Adding the NHS solution into the dopa solution, stirring for 15min, then adding the EDC solution, continuing stirring for 15min, and dropwise adding the chitosan solution to enable the molar ratio of chitosan, NHS, EDC and dopa in the mixed solution to be 1: 4: 4: 2, then adjusting the pH value to 5.5, reacting for 24 hours, and maintaining the whole process at N2The temperature in the atmosphere was 25 ℃. And (3) putting the reactant into a dialysis bag with the molecular weight cutoff of 3000-4000, dialyzing for 3 days by taking deionized water as dialysate, and freeze-drying the retentate to obtain the modified chitosan, namely the dopa grafted functionalized chitosan. The freeze drying condition is pressure of 50Pa, temperature of-50 deg.C, and time of 72 h.
2. Functional short peptide HGF- (RADA)4Preparation of RIKVAV
Connecting histidine-glycine-phenylalanine (HGF) to one end of (arginine-alanine-aspartic acid-alanine) 4 sequence and another end to the other end by short peptide synthesizerConnecting functional amino acid sequence IKVAV to obtain functional short peptide HGF- (RADA)4-RIKVAV。
3. Modified chitosan/functional oligopeptide HGF- (RADA)4Preparation of-RIKVAV composite hydrogel
Grafting the dopa prepared in the step 1 with the functionalized chitosan and the functional short peptide HGF- (RADA) prepared in the step 24Respectively dissolving RIKVAV in deionized water to obtain DOPA grafted functionalized chitosan solution with mass percentage concentration of 5%, functional short peptide HGF- (RADA) with mass percentage concentration of 0.5%4-RIKVAV solution, mixing the two solutions, dopa grafted functionalized chitosan solution and functional short peptide HGF- (RADA)4The volume ratio of RIKVAV solution was 1:2, pH was adjusted to 7, and hydrogel was formed after injection into PBS.
Example 5 Structure Observation and self-repair test of composite hydrogel
Carrying out observation and self-repairing tests on the composite hydrogel prepared in the embodiments 1 to 4, wherein the mixed solution prepared in the embodiments 1 to 4 can form hydrogel after being injected into PBS; observing that the hydrogel is in a porous structure by using a scanning electron microscope, wherein the pores are directly connected with one another; and the cut hydrogel block can be extruded to form integral hydrogel again, and the hydrogel block has a self-repairing function.
Wherein the observation result of the modified chitosan/functional short peptide composite hydrogel of example 1 is shown in fig. 1, fig. 1A is an injectable diagram of the modified chitosan/functional short peptide composite hydrogel, and the hydrogel can be formed after the mixed solution is injected into PBS; FIG. 1B is a scanning electron microscope image of a modified chitosan/functional short peptide composite gel, wherein the hydrogel has a porous structure, and pores are directly connected with pores; the self-repairing effect of the modified chitosan/functional short peptide composite hydrogel in example 1 is shown in fig. 2, wherein 2-a is a hydrogel block cut into 4 blocks, and 2-B is a gel block which is extruded and then reformed into an integral gel, that is, the separated hydrogels can be connected into the integral gel again, which indicates that the hydrogel has a self-repairing function.
Example 6 spinal cord injury repair
Modified Chitosan/functional oligopeptide HGF- (RADA) prepared in example 1 was used4-RIKVAV composite gel for spinal cord injuryAnd (5) repairing.
Construction of rat spinal cord T8-T9 Total transection (2mm defect) injury, modified Chitosan/functional oligopeptide HGF- (RADA) prepared in example 14Injecting the RIKVAV composite water solution into the injury part to form hydrogel. Dura mater, muscle and skin were sutured in order. After 2 months, the animals were perfused with 4% paraformaldehyde solution and spinal cord tissue was taken and frozen and sectioned. NF200 and GFAP were used as primary antibodies, incubated overnight at 4 ℃ and then incubated with fluorescently labeled secondary antibodies for 2h at room temperature. And (4) photographing by using a laser confocal microscope and detecting the distribution of the regenerated nerve fibers at the spinal cord transection.
The result of immunofluorescence staining of sagittal sections at the spinal cord cross section is shown in fig. 3, and it can be seen from fig. 3 that the rat spinal cord injury has fibers with positive NF200 at the near end, the middle and the far end, which indicates that regenerated nerve fibers grow to the injury area; the modified chitosan/functional short peptide Fmoc-RIKVAV compounded hydrogel has good safety and can promote nerve growth; the modified chitosan/functional oligopeptide HGF- (RADA)4The RIKVAV composite gel can be used for repairing spinal cord injury.
Preparation of comparative example genipin crosslinked phenylalanine graft modified chitosan/RADA 16-RIKVAV short peptide composite hydrogel
1. Experiment of
1.1 preparation of phenylalanine graft-modified Chitosan
In N2Dissolving chitosan in deionized water in an atmosphere, and adjusting the pH to 5.0 by using 1M HCl to prepare a solution with a final concentration of 4 mM; NHS and EDC were dissolved separately in deionized water and the pH was adjusted to 5.5 with 1M HCl, phenylalanine was dissolved in deionized water and the pH was adjusted to 5.5 with 1M HCl. Adding the NHS solution into the dopa solution, stirring for 15min, then adding the EDC solution, continuing stirring for 15min, and dropwise adding the chitosan solution to enable the molar ratio of chitosan, NHS, EDC and phenylalanine in the mixed solution to be 1: 4: 4: 2, then adjusting the pH value to 5.5, reacting for 24 hours, and maintaining the whole process at N2The temperature in the atmosphere was 25 ℃. Putting the reactant into a dialysis bag with the molecular weight cutoff of 3000-4000, dialyzing for 3 days by taking deionized water as dialysate, taking the retentate, freeze-drying,obtaining the modified chitosan, namely the phenylalanine grafted and functionalized chitosan. The freeze drying condition is pressure of 45Pa, temperature of-55 deg.C, and time of 48 h.
1.2(RADA)4Preparation of short-chain RiKVAV peptides
Using short peptide synthesizer (arginine-alanine-aspartic acid-alanine)4Sequence and functional amino acid sequence IKVAV to obtain functional short peptide (RADA)4-RIKVAV。
1.3 composite of phenylalanine grafted modified chitosan and RADA16-RIKVAV short peptide
Grafting the phenylalanine prepared in the step 1.1 with the modified chitosan and the functional short peptide (RADA) prepared in the step 1.24Dissolving RIKVAV in deionized water respectively to obtain phenylalanine graft modified chitosan solution with the mass percentage concentration of 10% and functional short peptide (RADA) with the mass percentage concentration of 1%4Mixing the RIKVAV solution and the two solutions, enabling the volume ratio of the phenylalanine grafted modified chitosan to the functional short peptide in the mixed solution to be 1:1, and adjusting the pH value to be 7 to obtain the phenylalanine grafted modified chitosan/RADA 16-RIKVAV short peptide composite solution.
1.4 preparation of genipin cross-linked phenylalanine graft-modified chitosan/RADA 16-RIKVAV short peptide composite hydrogel
And (3) adding genipin into the phenylalanine grafted and modified chitosan/RADA 16-RIKVAV short peptide composite solution prepared in the step 1.3 to enable the genipin to be 0.2 wt%, and standing to form hydrogel.
2. Results
The phenylalanine grafted and modified chitosan and the RADA16-RIKVAV short peptide are compounded to form hydrogel, and the hydrogel can be formed after being crosslinked with genipin, but the formed hydrogel cannot be self-repaired. As shown in fig. 4, the hydrogel block formed by crosslinking the phenylalanine-grafted modified chitosan/RADA 16-RIKVAV short peptide and genipin can not be extruded to form a complete hydrogel block after being divided into 4 blocks; the hydrogel block after extrusion is still broken after being pulled, which indicates that the hydrogel has no self-repairing function.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The modified chitosan/functional short peptide composite hydrogel is characterized by being obtained by compounding modified chitosan and functional short peptide, wherein the modified chitosan is dopa grafted functionalized chitosan; the functional short peptide is a short peptide with one side connected with an amino acid sequence containing a benzene ring and an imidazolyl and the other side connected with a functional amino acid sequence.
2. The composite hydrogel according to claim 1, wherein the functional short peptide is a short peptide which is formed by connecting an amino acid sequence containing a benzene ring and an imidazolyl on one side of a RADA16-I sequence and connecting a functional amino acid sequence on the other side of the RADA16-I sequence.
3. The composite hydrogel according to claim 2, wherein the RADA16-I sequence contains 1 to 3 positively charged amino acids with the functional amino acid sequence and 1 to 3 achiral amino acids with the amino acid sequence containing a benzene ring and an imidazole group.
4. The composite hydrogel according to claim 3, wherein the positively charged amino acid is lysine and/or arginine; the achiral amino acid is glycine.
5. The composite hydrogel according to any one of claims 1 to 4, wherein the composite hydrogel has a pH of 6 to 8.
6. A method for preparing the composite hydrogel according to claim 1, comprising the steps of:
s1, preparation of modified chitosan
In an inert gas atmosphere, uniformly mixing an N-hydroxysuccinimide sodium aqueous solution with a dopa aqueous solution, then uniformly mixing the N-hydroxysuccinimide sodium aqueous solution with a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride aqueous solution, fully and uniformly mixing the N-hydroxysuccinimide sodium aqueous solution with a chitosan aqueous solution, and adjusting the pH of the mixed solution to 5.5-6; the pH value of the aqueous solution is 4.0-5.5; placing the fully and uniformly mixed solution at the temperature of 20-30 ℃ for reaction for 20-25 h, dialyzing, and drying to obtain modified chitosan;
s2, preparation of functional short peptide
Connecting an amino acid sequence containing a benzene ring and an imidazolyl on one side of the short peptide, and connecting a functional amino acid sequence on the other side to obtain a functional short peptide;
s3, preparation of modified chitosan/functional short peptide composite hydrogel
And (3) mixing the solution of the modified chitosan obtained in the step S1 and the solution of the functional short peptide obtained in the step S2, and then injecting the mixed solution into a buffer solution or a cell culture medium to obtain the composite hydrogel.
7. The method according to claim 6, wherein the molar ratio of chitosan, sodium N-hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and dopa in step S1 is 1: 1-4: 1-4: 1 to 4.
8. The method according to claim 6, wherein the volume ratio of the modified chitosan solution to the functional short peptide solution in the mixed solution in step S3 is 1:0.5 to 2.
9. The method according to claim 6, wherein the pH of the mixed solution in step S3 is 6 to 8.
10. Use of the composite hydrogel according to any one of claims 1 to 5 in preparation of a spinal cord injury repair material.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111603364.5A CN114209889B (en) | 2021-12-24 | 2021-12-24 | Modified chitosan/functional short peptide composite hydrogel and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111603364.5A CN114209889B (en) | 2021-12-24 | 2021-12-24 | Modified chitosan/functional short peptide composite hydrogel and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114209889A true CN114209889A (en) | 2022-03-22 |
| CN114209889B CN114209889B (en) | 2023-03-10 |
Family
ID=80705768
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111603364.5A Active CN114209889B (en) | 2021-12-24 | 2021-12-24 | Modified chitosan/functional short peptide composite hydrogel and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114209889B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180085500A1 (en) * | 2015-04-03 | 2018-03-29 | Innotherapy Inc. | Hemostatic injection needle coated with crosslinked chitosan having catechol group and oxidized catechol group |
| CN108517006A (en) * | 2018-05-10 | 2018-09-11 | 暨南大学 | Improve carbon nanotube dispersed polypeptide material and its application in water under a kind of normal temperature and pressure conditions |
| CN109180970A (en) * | 2018-08-30 | 2019-01-11 | 武汉理工大学 | A kind of citric acid cross-linked chitosan and the hydrogel of dopamine and preparation method thereof |
-
2021
- 2021-12-24 CN CN202111603364.5A patent/CN114209889B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180085500A1 (en) * | 2015-04-03 | 2018-03-29 | Innotherapy Inc. | Hemostatic injection needle coated with crosslinked chitosan having catechol group and oxidized catechol group |
| CN108517006A (en) * | 2018-05-10 | 2018-09-11 | 暨南大学 | Improve carbon nanotube dispersed polypeptide material and its application in water under a kind of normal temperature and pressure conditions |
| CN109180970A (en) * | 2018-08-30 | 2019-01-11 | 武汉理工大学 | A kind of citric acid cross-linked chitosan and the hydrogel of dopamine and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| JINGHUA LUO等: ""An injectable and self-healing hydrogel with controlled release of curcumin to repair spinal cord injury"", 《BIOACTIVE MATERIALS》 * |
| 张玲等: "短肽水凝胶RADA16结构特征及在生物医学中的应用研究与进展", 《中国组织工程研究》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114209889B (en) | 2023-03-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Pandit et al. | Periodate oxidized hyaluronic acid-based hydrogel scaffolds for tissue engineering applications | |
| JP7014845B2 (en) | Preparation and / or formulation of proteins cross-linked by polysaccharides | |
| AU2004204465B2 (en) | Hydroxyphenyl cross-linked macromolecular network and applications thereof | |
| US5264214A (en) | Composition for bone repair | |
| AU638687B2 (en) | Collagen-polymer conjugates | |
| AU677789B2 (en) | Biocompatible polymer conjugates | |
| US8137688B2 (en) | Hydroxyphenyl cross-linked macromolecular network and applications thereof | |
| Wang et al. | Dual-enzymatically crosslinked and injectable hyaluronic acid hydrogels for potential application in tissue engineering | |
| CN110760103B (en) | Viscoelastic hydrogel and preparation method and application thereof | |
| JP2009529086A (en) | Hydrazide derivatives of hyaluronic acid | |
| CN105524472A (en) | Keratin/silk fibroin blended membrane and preparation method thereof | |
| CN114874455B (en) | Construction method of neutral-dissolution modified collagen and gel with self-assembly capability and photocrosslinking capability | |
| WO2021173698A9 (en) | Compositions and methods for in situ-forming gels for wound healing and tissue regeneration | |
| EP2529226A1 (en) | Silylated biomolecules | |
| CN114209889B (en) | Modified chitosan/functional short peptide composite hydrogel and preparation method and application thereof | |
| CN114381015A (en) | Injectable and self-repairing hydrogel and preparation method and application thereof | |
| CN115645625A (en) | Injectable hydrogel based on silk fibroin/hyaluronic acid and preparation method and application thereof | |
| CN114288469A (en) | Composition, preparation method and application of double-crosslinked collagen filler | |
| CN111528220B (en) | Hydrogel for slowly releasing chlorine dioxide and preparation method thereof | |
| WO2024078310A1 (en) | Collagen hydrogel, preparation method therefor, and use thereof | |
| CN116041727B (en) | Synthesis preparation method and application of carbon-carbon double bond modified keratin injectable hydrogel | |
| CN112126069B (en) | Rapidly curable hydrogel based on collagen and hyaluronic acid, preparation method and application thereof | |
| EP4284338A1 (en) | Compositions and methods for making and using hybrid network hydrogels | |
| CN117582548A (en) | Injectable multi-crosslinking composite hydrogel stent with function of in-situ repair of anterior cruciate ligament and preparation method thereof | |
| CN117487198A (en) | Self-crosslinking sodium hyaluronate-recombinant collagen hydrogel and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |