CN117582556B - Method for reducing antigenicity of biomedical material - Google Patents
Method for reducing antigenicity of biomedical material Download PDFInfo
- Publication number
- CN117582556B CN117582556B CN202410077625.1A CN202410077625A CN117582556B CN 117582556 B CN117582556 B CN 117582556B CN 202410077625 A CN202410077625 A CN 202410077625A CN 117582556 B CN117582556 B CN 117582556B
- Authority
- CN
- China
- Prior art keywords
- super
- biomedical
- hydrophilic polypeptide
- aqueous solution
- carbon
- 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.)
- Active
Links
- 239000003519 biomedical and dental material Substances 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 49
- 229920001184 polypeptide Polymers 0.000 claims abstract description 65
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 65
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 65
- 238000011282 treatment Methods 0.000 claims abstract description 56
- 239000000463 material Substances 0.000 claims abstract description 45
- 239000000427 antigen Substances 0.000 claims abstract description 44
- 102000036639 antigens Human genes 0.000 claims abstract description 44
- 108091007433 antigens Proteins 0.000 claims abstract description 44
- 229960003638 dopamine Drugs 0.000 claims abstract description 38
- 238000005507 spraying Methods 0.000 claims abstract description 28
- 239000011203 carbon fibre reinforced carbon Substances 0.000 claims abstract description 26
- 238000000889 atomisation Methods 0.000 claims abstract description 23
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims abstract description 20
- 150000001875 compounds Chemical class 0.000 claims abstract description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 5
- 210000001691 amnion Anatomy 0.000 claims description 56
- 239000007864 aqueous solution Substances 0.000 claims description 33
- 230000005855 radiation Effects 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 10
- 239000003999 initiator Substances 0.000 claims description 9
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000000502 dialysis Methods 0.000 claims description 8
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- 238000006116 polymerization reaction Methods 0.000 claims description 6
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 claims description 6
- YXMISKNUHHOXFT-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) prop-2-enoate Chemical compound C=CC(=O)ON1C(=O)CCC1=O YXMISKNUHHOXFT-UHFFFAOYSA-N 0.000 claims description 5
- 241001465754 Metazoa Species 0.000 claims description 5
- 210000004087 cornea Anatomy 0.000 claims description 5
- 230000005251 gamma ray Effects 0.000 claims description 5
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 4
- 229910021641 deionized water Inorganic materials 0.000 claims description 4
- 210000003516 pericardium Anatomy 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 241000283690 Bos taurus Species 0.000 claims description 2
- 210000004877 mucosa Anatomy 0.000 claims description 2
- 210000002747 omentum Anatomy 0.000 claims description 2
- 210000004303 peritoneum Anatomy 0.000 claims description 2
- 210000004224 pleura Anatomy 0.000 claims description 2
- 210000000813 small intestine Anatomy 0.000 claims description 2
- 239000007921 spray Substances 0.000 claims description 2
- 230000000052 comparative effect Effects 0.000 description 33
- 238000012360 testing method Methods 0.000 description 15
- 239000000243 solution Substances 0.000 description 12
- 239000003599 detergent Substances 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 10
- 238000004132 cross linking Methods 0.000 description 9
- 239000003102 growth factor Substances 0.000 description 9
- 238000002791 soaking Methods 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 102000004142 Trypsin Human genes 0.000 description 8
- 108090000631 Trypsin Proteins 0.000 description 8
- 239000003638 chemical reducing agent Substances 0.000 description 8
- 239000012588 trypsin Substances 0.000 description 8
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 7
- 230000000890 antigenic effect Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 102400001368 Epidermal growth factor Human genes 0.000 description 3
- 101800003838 Epidermal growth factor Proteins 0.000 description 3
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 description 3
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000027455 binding Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 229940116977 epidermal growth factor Drugs 0.000 description 3
- 230000036571 hydration Effects 0.000 description 3
- 238000006703 hydration reaction Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 210000002826 placenta Anatomy 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 2
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 210000001136 chorion Anatomy 0.000 description 2
- 239000013068 control sample Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 229960003964 deoxycholic acid Drugs 0.000 description 2
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 2
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 108010091212 pepstatin Proteins 0.000 description 2
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- 239000012114 Alexa Fluor 647 Substances 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
- 208000010412 Glaucoma Diseases 0.000 description 1
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000002805 bone matrix Anatomy 0.000 description 1
- 108010089934 carbohydrase Proteins 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 210000000795 conjunctiva Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000000819 hypertonic solution Substances 0.000 description 1
- 229940021223 hypertonic solution Drugs 0.000 description 1
- 239000000815 hypotonic solution Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000010526 radical polymerization reaction Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000004289 sodium hydrogen sulphite Substances 0.000 description 1
- FCZYGJBVLGLYQU-UHFFFAOYSA-M sodium;2-[2-[2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy]ethoxy]ethanesulfonate Chemical compound [Na+].CC(C)(C)CC(C)(C)C1=CC=C(OCCOCCOCCS([O-])(=O)=O)C=C1 FCZYGJBVLGLYQU-UHFFFAOYSA-M 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- TUQOTMZNTHZOKS-UHFFFAOYSA-N tributylphosphine Chemical compound CCCCP(CCCC)CCCC TUQOTMZNTHZOKS-UHFFFAOYSA-N 0.000 description 1
- 230000009278 visceral effect Effects 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Abstract
The invention belongs to the technical field of biomedical materials, and discloses a method for reducing antigenicity of a biomedical material, which comprises the steps of preparing an antigen shielding material-dopamine modified super-hydrophilic polypeptide by adopting a carbon-carbon double bond modified compound, super-hydrophilic polypeptide and 3-methacrylic dopamine, and carrying out atomization spraying treatment on the biomedical material, wherein the amino acid sequence of the super-hydrophilic polypeptide is YYTYYSYYT. By the technical scheme, the antigenicity of the biomedical material can be reduced, and the structural integrity and mechanical property of the biomedical material can be maintained.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a method for reducing antigenicity of biomedical materials.
Background
The amniotic membrane is the innermost layer of placenta, is smooth, has no blood vessel, nerve and lymph, has certain elasticity and is about 0.02-0.5 mm thick. Amniotic membrane has a structure similar to that of conjunctival tissue of human eyes, contains substances required for growth of conjunctival cells and corneal epithelial cells, and is widely used for treating diseases in the field of ophthalmology, including conjunctiva, cornea, limbal stem cell reconstruction, glaucoma surgery and the like. The amniotic membrane in clinical use at present mainly comes from human sources, and is used as an allograft implantable medical device, and has certain antigenicity in comparison with an animal-derived implantable medical device although the amniotic membrane has lower immunogenicity.
The existing methods for removing antigenic substances generally adopt the "subtractive principle", such as Chinese patent 202080057956.9, when removing antigenic biomolecules, a reducing agent, a detergent and/or an enzyme is used to dissolve hydrophilic biomolecules in the bioprosthetic tissue, wherein the reducing agent can optionally comprise beta-mercaptoethanol, dithiothreitol and tributylphosphine; the detergent may optionally include a nonionic detergent, a zwitterionic detergent, and an ionic detergent; the enzyme may be selected from the group consisting of nucleases, lipases, carbohydrases and depolymerases. Although the use of detergents, reducing agents and/or enzymes as described above may remove antigens to a large extent from biomedical materials, it is inevitable that the biomedical materials are damaged in terms of structural integrity and mechanical properties, which may directly lead to loss of part of functional components and degradation of mechanical properties of the biomedical materials. For example, when the above-mentioned "reducing agent, detergent and/or enzyme" is used to dissolve hydrophilic biological molecules in bioprosthetic tissue, the amnion material has a relatively low thickness, and therefore has relatively low mechanical properties such as maximum uniaxial tensile fracture stress, which inevitably results in the destruction of structural integrity and mechanical properties, and also causes the loss of functional components (such as growth factors), which affects the use of the amnion biomedical material.
Therefore, there is a need to develop a novel method for reducing antigenicity of biomedical materials, so as to overcome the defects of the prior art and meet clinical demands to a greater extent.
Disclosure of Invention
The invention aims to provide a method for reducing the antigenicity of biomedical materials, which is characterized in that compared with the existing method for reducing the antigenicity of biomedical materials by adopting the subtraction principle, the method is realized by the atomization spraying treatment of an antigen shielding material-dopamine modified super-hydrophilic polypeptide, thereby realizing the effects of reducing the antigenicity of biomedical materials and maintaining the structural integrity and mechanical properties of the biomedical materials.
In order to solve the technical problems, the invention provides a method for reducing antigenicity of biomedical materials, which aims to overcome the defects of the prior art and meet clinical requirements to a greater extent. Unlike the solution (i.e., using detergents, reducing agents and/or enzymes to remove antigens from biomedical materials), the present invention provides a "non-subtractive principle" solution, thereby achieving both reduced antigenicity of biomedical materials and maintaining the structural integrity and mechanical properties of biomedical materials themselves.
The invention is realized by the following technical scheme: a method for reducing antigenicity of biomedical material comprises preparing antigen shielding material-dopamine modified super-hydrophilic polypeptide from carbon-carbon double bond modified compound, super-hydrophilic polypeptide and 3-methyl acrylamide, atomizing and spraying biomedical material,
the amino acid sequence of the super-hydrophilic polypeptide is YYTYYTYT, and the structural formula is as follows:
。
the biomedical material comprises an allogenic material and an animal source material, wherein the allogenic material comprises human amniotic membrane, cornea and pericardium; the animal source material comprises amniotic membrane, cornea, pericardium, small intestine mucosa, pleura, peritoneum and fat omentum of pig or cattle.
Before atomization spraying treatment, the biomedical material is subjected to radiation treatment, wherein the radiation treatment is to carry out gamma ray radiation on the biomedical material in an air atmosphere, and the radiation dosage is controlled to be 5-50 kGy.
Before atomization spraying treatment, the biomedical material is subjected to ultraviolet irradiation treatment, wherein the ultraviolet irradiation treatment is to irradiate the biomedical material for 60-120 min by adopting an ultraviolet curing lamp with the wavelength of 50-280 w and 365nm in an air atmosphere.
The preparation method of the antigen shielding material-dopamine modified super-hydrophilic polypeptide comprises the following steps:
a. preparing an aqueous solution of the super-hydrophilic polypeptide with the mass concentration of 5% and an aqueous solution of the carbon-carbon double bond modified compound with the mass concentration of 2.5%, respectively, and reacting for 22-26 hours at the temperature of 20-37 ℃ according to the volume ratio of the aqueous solution of the super-hydrophilic polypeptide to the aqueous solution of the carbon-carbon double bond modified compound being 0.6-1.2:1 to prepare the carbon-carbon double bond modified super-hydrophilic polypeptide;
b. preparing an aqueous solution of the super-hydrophilic polypeptide modified by the carbon-carbon double bond with the mass concentration of 5%, adding an aqueous solution of 3-methacryloyl dopamine with the mass concentration of 2.5%, carrying out polymerization reaction for 1-24 h at the temperature of 4-40 ℃ in the presence of an initiator, and carrying out dialysis purification and freeze-drying to obtain the polypeptide.
The carbon-carbon double bond modified compound is N-acryloyloxy succinimide.
The initiator is a mixture of ammonium persulfate and sodium bisulfite.
The dialysis purification is to soak the materials in deionized water for 3 to 5 days at room temperature by adopting a dialysis bag with the molecular weight cut-off of 2000 to 3000.
The atomization spraying treatment is to spray the surface of the biomedical material by adopting an atomization spraying machine.
Compared with the prior art, the invention has the following advantages:
(1) In the existing method for reducing the antigenicity of the biomedical material based on the subtraction principle, the structural integrity and mechanical properties of the biomedical material are affected by removing the antigen from the biomedical material by using a detergent, a reducing agent and/or an enzyme, in particular to the amniotic material with a thinner thickness. Based on the above, the invention provides a method for reducing antigenicity by adopting a non-subtractive principle, namely adopting an antigen shielding material-dopamine modified super-hydrophilic polypeptide to carry out atomization spraying treatment on a biomedical material, or adopting irradiation crosslinking or ultraviolet irradiation crosslinking to carry out crosslinking treatment on the biomedical material before the atomization spraying treatment, thereby realizing the function of reducing antigenicity of the biomedical material and keeping the component integrity and mechanical property of the biomedical material. Both treatments are based on the "non-subtractive principle" and may be used together or one of them may be used alone.
(2) When the biomedical material is processed based on the non-subtraction principle, the process of soaking treatment by the aqueous solution does not exist, so that the growth factors contained in the amniotic membrane can be effectively reserved, and the loss of effective functional components in the biomedical material can be effectively reduced compared with the conventional method of soaking treatment by the aqueous solution. For example, growth factors in the amniotic membrane for ophthalmic use are important functional components for promoting ocular surface regeneration, and the conventional way of reducing antigenicity by soaking in aqueous solution inevitably causes the loss of water-soluble growth factors.
(3) The invention adopts the special super-hydrophilic polypeptide for the first time, and the 3-methacryloyl dopamine can be combined with the antigenic determinant by the synergistic effect with the 3-methacryloyl dopamine, and the super-hydrophilic polypeptide can form a hydration layer on the surface of the antigenic determinant, thereby inhibiting antigen-antibody combination and achieving the aim of reducing the antigenicity of the biomedical material.
(4) The antigen shielding material-dopamine modified super-hydrophilic polypeptide is prepared by a carbon-carbon double bond polymerization reaction, namely, the first step is to modify the carbon-carbon double bond of the super-hydrophilic polypeptide to obtain the carbon-carbon double bond modified super-hydrophilic polypeptide so as to endow the super-hydrophilic polypeptide with a carbon-carbon double bond functional group, thereby enabling the super-hydrophilic polypeptide to have a free radical polymerization chemical reaction with 3-methacryloyl dopamine; the second step is that the super-hydrophilic polypeptide modified by carbon-carbon double bonds and 3-methyl acrylamide are polymerized together, and the chemical co-polymerization mode can ensure the stable combination of the super-hydrophilic polypeptide and dopamine, thereby being beneficial to the stable combination of the super-hydrophilic polypeptide and biomedical materials and playing the antigen shielding role.
Drawings
FIG. 1 is a schematic diagram of a chemical reaction for preparing an antigen shielding material-dopamine modified super-hydrophilic polypeptide according to the present invention;
FIG. 2 is a schematic diagram of the principle of reducing antigenicity of biomedical materials by adopting an antigen shielding material-dopamine modified super-hydrophilic polypeptide in the invention;
FIG. 3 is a graph showing the results of antigenicity test of the materials of examples 2 to 6 and comparative examples 1 to 7 according to the present invention.
Detailed Description
The objects, technical solutions and advantageous effects of the present invention will be described in further detail below.
It is noted that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed, and unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention aims at providing a method for reducing antigenicity of biomedical materials, which at least comprises the following schemes:
(1) The method comprises the steps of adopting an antigen shielding material-dopamine modified super-hydrophilic polypeptide to carry out atomization spraying treatment on a biomedical material;
(2) After the biomedical material is subjected to radiation treatment, the biomedical material is treated in the mode (1);
(3) After the biomedical material is subjected to ultraviolet irradiation treatment, the biomedical material is treated in the mode of (1).
In the treatment mode, the configuration transformation of the protein type antigen in the biomedical material can be realized through radiation or ultraviolet irradiation treatment, so that the antigenicity of the biomedical material is reduced. The principle of reducing antigenicity of biomedical materials by adopting the antigen shielding material-dopamine modified super-hydrophilic polypeptide is as follows: the dopamine is combined with the antigenic determinant, so that the superhydrophilic polypeptide part forms a hydration layer on the surface of the antigenic determinant, and the hydration layer is used for inhibiting antigen-antibody combination, thereby achieving the antigen shielding effect on the biomedical material and achieving the aim of reducing the antigenicity of the biomedical material.
It should be noted that, the term "simultaneously" as used herein means: the treatment processes for biomedical materials are sequentially performed in sequence, but do not mean that the two treatment processes occur simultaneously in time. Namely: firstly radiating or irradiating with ultraviolet light, and then atomizing and spraying by adopting the original shielding material-dopamine modified super-hydrophilic polypeptide.
Specifically, when the radiation treatment is performed, the biomedical material is subjected to gamma ray radiation in an air atmosphere, and the radiation dose is controlled to be 5-50 kGy. When the ultraviolet irradiation treatment is carried out, the biomedical material is irradiated for 60-120 min by an ultraviolet curing lamp with the wavelength of 50-280 w and 365nm in the air atmosphere.
The preparation method for preparing the antigen shielding material-dopamine modified super-hydrophilic polypeptide comprises the following steps:
1) Preparation of carbon-carbon double bond modified super hydrophilic polypeptide
Preparing an aqueous solution of the super-hydrophilic polypeptide with the mass concentration of 5% and an aqueous solution of the carbon-carbon double bond modified compound (namely N-acryloyloxy succinimide) with the mass concentration of 2.5%, respectively, reacting for 22-26 hours at 20-37 ℃ according to the volume ratio of the aqueous solution of the super-hydrophilic polypeptide to the aqueous solution of the carbon-carbon double bond modified compound of 0.6-1.2:1, and freeze-drying to obtain the carbon-carbon double bond modified super-hydrophilic polypeptide;
2) Preparation of antigen shielding material-dopamine modified super-hydrophilic polypeptide
Preparing the super-hydrophilic polypeptide modified by the carbon-carbon double bond into an aqueous solution with the mass concentration of 5%, adding an aqueous solution of 3-methacrylic dopamine with the mass concentration of 2.5%, adding an initiator, carrying out polymerization reaction for 1-24 h at the temperature of 4-40 ℃, then soaking in deionized water for 3-5 days at room temperature by adopting a dialysis bag with the molecular weight cutoff of 2000-3000, and freeze-drying to obtain the polypeptide.
The amino acid sequence of the super-hydrophilic polypeptide is YYTYYTYT, and the amino acid sequence corresponding to three letters is Tyr-Tyr-Thr-Ser-Tyr-Tyr-Thr, and the structural formula is as follows:
。
further, the chemical reaction process of the prepared antigen shielding material-dopamine modified super-hydrophilic polypeptide is shown in fig. 1, and the schematic diagram for reducing the antigenicity of the biomedical material is shown in fig. 2.
The initiator added in the invention is a mixture of ammonium persulfate and sodium bisulphite, and the mass concentration of the mixture is in the range of 0.1-1%. Preferably, the mass ratio of the ammonium sulfate to the sodium bisulfite in the initiator is 1:1, and can be used for initiating the polymerization reaction between carbon-carbon double bonds.
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. In the following embodiments, the super-hydrophilic polypeptide is purchased from Shanghai Yao Biotechnology Co., ltd; the model of the atomizing applicator was FS1006, available from Panxoenergy ultrasonic technology Co., ltd; the human amniotic membrane is obtained from legal and regular hospitals after the procedures of ethical examination, informed consent of a donor and the like, and the obtaining way is legal and compliant.
Example 1:
the embodiment is a preparation method of an antigen shielding material-dopamine modified super-hydrophilic polypeptide, which comprises the following steps:
preparing 5% by mass concentration of super-hydrophilic polypeptide (YYTYYTYT) aqueous solution, adding 2.5% by mass concentration of carbon-carbon double bond modified compound, namely N-acryloyloxy succinimide aqueous solution, reacting for 24 hours at 25 ℃ according to the volume ratio of the super-hydrophilic polypeptide aqueous solution to the N-acryloyloxy succinimide aqueous solution of 1:1, and freeze-drying to obtain the carbon-carbon double bond modified super-hydrophilic polypeptide.
Preparing the prepared super-hydrophilic polypeptide modified by the carbon-carbon double bond into an aqueous solution with the mass concentration of 5%, adding an aqueous solution of 3-methacryloyl dopamine with the mass concentration of 2.5%, adding an initiator to initiate polymerization for 24 hours at 37 ℃, soaking the solution in deionized water for 3 days at room temperature by adopting a dialysis bag with the molecular weight cut-off of 2000, and freeze-drying to obtain the polypeptide.
The initiator added in this example was a mixture of ammonium persulfate and sodium bisulfite at a mass concentration of 0.5%, wherein the mass concentration ratio of ammonium sulfate to sodium bisulfite was 1:1.
Example 2:
the method for carrying out atomization spraying treatment on biomedical materials by adopting the antigen shielding material-dopamine modified super-hydrophilic polypeptide prepared in the embodiment 1 comprises the following treatment steps:
preparing the freeze-dried antigen shielding material-dopamine modified super-hydrophilic polypeptide into an aqueous solution with the mass solubility of 1%, and spraying the surface of the human amniotic membrane by adopting an atomization spraying machine.
Example 3:
the embodiment is a method for treating biomedical materials by adopting radiation to realize the configuration transformation of protein type antigens in the biomedical materials so as to reduce antigenicity.
Specifically, gamma ray radiation is carried out on human amniotic membrane in an air atmosphere, and the radiation dosage is controlled at 25kGy.
Example 4:
the embodiment is a method for processing biomedical materials by ultraviolet irradiation to realize the configuration transformation of protein type antigens in the biomedical materials so as to reduce antigenicity.
Specifically, human amniotic membrane is subjected to ultraviolet irradiation treatment in an air atmosphere, and irradiation is performed for 60min by using an ultraviolet curing lamp with the wavelength of 85w and 365 nm.
Example 5:
the method for carrying out atomization spraying treatment by adopting radiation treatment and antigen shielding material-dopamine modified super-hydrophilic polypeptide simultaneously comprises the following steps:
gamma-ray radiation is carried out on human amniotic membrane in an air atmosphere, and the radiation dose is 25kGy. Then, the antigen shielding material-dopamine modified super-hydrophilic polypeptide prepared in the embodiment 1 is adopted to carry out atomization spraying treatment on the human amniotic membrane subjected to the radiation treatment by adopting an atomization spraying machine. When in spraying, the mass solubility of the adopted antigen shielding material-dopamine modified super-hydrophilic polypeptide aqueous solution is 1 percent.
Example 6:
the method for carrying out atomization spraying treatment by adopting ultraviolet irradiation treatment and antigen shielding material-dopamine modified super-hydrophilic polypeptide simultaneously comprises the following steps:
ultraviolet irradiation treatment is carried out on human amniotic membrane in an air atmosphere, and irradiation is carried out for 60min by using an ultraviolet curing lamp with the wavelength of 85w and 365 nm. Then, the antigen shielding material-dopamine modified super-hydrophilic polypeptide prepared in the embodiment 1 is adopted to carry out atomization spraying treatment on the human amniotic membrane subjected to ultraviolet irradiation treatment by adopting an atomization spraying machine. When in spraying, the mass solubility of the adopted antigen shielding material-dopamine modified super-hydrophilic polypeptide aqueous solution is 1 percent.
Comparative example 1:
comparative example 1 is fresh human amniotic membrane.
Comparative example 2:
comparative example 2 is a human amniotic membrane prepared by antigen removal using Triton X-100, DNAse I and RNAse in the reference paper "preparation of decellularized natural bone matrix for removal of antigen component of transplantation immune response" ("Chinese tissue engineering research and clinical rehabilitation", 2011,15 (08): 1355-1359 ").
The method comprises the following specific steps:
fresh human amniotic membrane was prepared, repeatedly rinsed with sterile distilled water, and placed in a jar containing protease inhibitor (0.1 mg/L Laprotin, 0.5 mg/L pepstatin A, 0.6 mg/L pepstatin A) and 0.05 mol/L Tris-HCI (pH 7.4) buffer, and then shaken at constant temperature of 4℃for 5 d. Then the buffer solution in the wide-mouth bottle is replaced by Tris-HCI (pH 7.4) buffer solution containing 3 percent TritonX-100, the protease inhibitor is also added, and the sterile distilled water is used for washing after the constant temperature oscillation is carried out for 7 d at 4C; then adding DNAseI and RNAse mixture, digesting 24h at room temperature, adding Tris-HCI (pH 7.4) buffer containing 3% Triton X-100 into the bottle again, and washing with sterile distilled water after blocking at constant temperature of 7 d at 4 ℃.
Comparative example 3:
comparative example 3 is a human amniotic membrane prepared by the antigen removal method using sodium dodecyl sulfate, trypsin, and sodium deoxycholate in the reference patent "a low antigenicity amniotic membrane tissue and its treatment method" (application No. 202010180761.5)).
The method comprises the following specific steps:
taking out the dehydrated amniotic membrane, putting back into a rotary drum, adding 500 parts by weight of sodium dodecyl sulfate solution with the mass fraction of 0.1% and the temperature of 20 ℃, treating for 30min in the rotary drum, drying the waste liquid, adding 500 parts by weight of water with the temperature of 20 ℃, cleaning for 60min, repeating for 5 times, and dehydrating by a dehydrator;
taking out the dehydrated amniotic membrane, putting back into a rotary drum, adding 500 parts by weight of trypsin solution with pH of 8 and temperature of 20 ℃, treating for 60min in the rotary drum, controlling the temperature to be in waste liquid, adding 500 parts by weight of water with temperature of 20 ℃, cleaning for 60min, repeating for 5 times, and dehydrating by a dehydrator;
taking out the dehydrated amniotic membrane, putting back into a rotary drum, adding 500 parts by weight of 1% sodium deoxycholate solution with the mass fraction of 20 ℃, treating for 120min in the rotary drum, drying the waste liquid, adding 500 parts by weight of water with the temperature of 20 ℃, cleaning for 60min, repeating for 5 times, and dehydrating by a dehydrator.
Comparative example 4:
comparative example 4 is a human amniotic membrane prepared by the method of "an absorbable medical biofilm and its preparation method" (application number 201610839636.4) in which a hypotonic and hypertonic solution interlacing treatment is used for preliminary treatment, and then the material after the preliminary treatment is placed in a surfactant solution for continuous treatment for antigen removal.
The method comprises the following specific steps:
placing fresh amniotic membrane in pure water, vibrating at room temperature 22C for 2 hours at 100rpm, then placing in 10% sodium chloride solution, vibrating at room temperature 22C at 100rpm for 2 hours, taking the amniotic membrane as a cycle, and carrying out co-cycle treatment for 1-3 times; then, the mixture was placed in 1.0% Sodium Dodecyl Sulfate (SDS), and the mixture was subjected to shaking treatment at 100rpm for 10 hours, and finally, the mixture was placed in 1.0% Triton X-200, and the mixture was subjected to shaking treatment at 100rpm for 2 hours.
Comparative example 5:
comparative example 5 is a human amniotic membrane prepared by the method of antigen removal using trypsin, disodium ethylenediamine tetraacetate and dnase in reference patent "a tissue patch and preparation method thereof" (application No. 200810150793. X).
The method comprises the following specific steps:
the preparation of decellularized amniotic membrane comprises taking placenta of healthy puerpera, peeling off the amniotic membrane under aseptic operation, washing with physiological saline, separating the amniotic membrane and chorion to obtain amniotic membrane, soaking in a mixed digestion solution containing 1-4 g/L trypsin and 0.1-0.4 g/L disodium ethylenediamine tetraacetate for at least 1 hr, washing with Phosphate Buffer (PBS), soaking in a DNase solution containing 30-50U/m 1 for more than 25 min, removing residual DNA components, reducing immunogenicity, and rinsing with PBS for later use.
Comparative example 6:
comparative example 6 is a human amniotic membrane prepared by the method of antigen removal using trypsin in the reference patent "amniotic membrane graft or covering for preventing visceral adhesion or bleeding" (application No. 95100812.9).
The method comprises the following specific steps:
the amniotic membrane was taken from a fresh human placenta taken at the time of caesarean section. The amniotic membrane was manually separated from the chorion and washed with distilled water. Clean membranes were first soaked in 10% trypsin solution for 3 hours.
Comparative example 7:
comparative example 7 is a human amniotic membrane prepared by the method of antigen removal using sodium dodecyl sulfate and trypsin in the reference patent "bio-derived amniotic membrane, composite bio-derived amniotic membrane and method of preparation thereof" (application No. 200410036792.4).
The method comprises the following specific steps:
taking fresh healthy human amniotic membrane, rinsing with normal saline for 3 times, degreasing chloroform/methanol (1:1, v/v) to obtain clear supernatant, and rinsing with normal saline for 3 times, each time for 10 minutes; 0.5% w/v SDS (sodium dodecyl sulfate) for 4 hours, and physiological saline was rinsed 3 times for 10 minutes each; 0.25% w/v trypsin was digested for 8 hours, and rinsed 3 times with physiological saline for 10 minutes each.
To further illustrate the substantial features and advantages of the present invention, the following test examples were implemented.
Test example 1: amniotic antigenicity test (human serum lgM and IgG binding test)
The test included the following materials: the amniotic membrane prepared in examples 2-6 and comparative examples 1-7.
The testing method comprises the following steps: lgM and IgG binding assays were performed with human serum. Human serum was obtained from healthy human volunteers (n=5, including all ABO blood groups) and pooled into one single human serum reagent. Fresh human amniotic membrane was used for the control sample. The slides were then washed with PBS and samples were blocked with 10% goat serum for 30 minutes at room temperature. Biotin-bound goat-derived anti-human IgM or lgG was allowed to act at room temperature (1:100 concentration) for 30 minutes to detect IgM or IgG binding. Alexa Fluor 647-conjugated streptavidin was applied as secondary antibody at room temperature and allowed to act for 30 min. Finally, images were taken by a confocal laser microscope and fluorescence intensity was calculated by Image J software. The calculation formula is as follows:
fluorescence relative Intensity = sample fluorescence Intensity (unit: intensity)/control sample (i.e., fresh human amniotic membrane) fluorescence Intensity (unit: intensity)
The experimental results are shown in table 1 and fig. 3.
TABLE 1
Human IgM binding fluorescence relative intensity (unit: Intensity/ Intensity) | human IgG binds to fluorescent relative intensities (units: Intensity/ Intensity) | |
example 2 | 0.12 | 0.09 |
Example 3 | 0.24 | 0.18 |
Example 4 | 0.26 | 0.2 |
Example 5 | 0.08 | 0.04 |
Example 6 | 0.09 | 0.05 |
Comparative example 1 | 1 | 1 |
Comparative example 2 | 0.36 | 0.19 |
Comparative example 3 | 0.38 | 0.21 |
Comparative example 4 | 0.22 | 0.1 |
Comparative example 5 | 0.24 | 0.15 |
Comparative example 6 | 0.42 | 0.25 |
Comparative example 7 | 0.26 | 0.17 |
The experimental results (Table 1 and FIG. 3) show that examples 2-6, compared to comparative example 1 (fresh human amniotic membrane), all have low specific binding fluorescence intensity of human IgM/IgG, i.e. low antigenicity, and in particular, examples 5 and 6, all have lower antigenicity than comparative examples 2-7. Namely, the method shows that: compared with the existing 'subtraction principle' technical scheme for reducing the antigenicity of the biomedical material (namely, removing antigens from the biomedical material by using a detergent, a reducing agent and/or enzyme), the 'non-subtraction principle' technical scheme provided by the invention is that the biomedical material is crosslinked by adopting irradiation crosslinking or ultraviolet irradiation crosslinking, and/or the biomedical material is subjected to atomization spraying treatment by using the antigen shielding material-dopamine modified super-hydrophilic polypeptide, so that the technical effect of reducing the antigenicity of the biomedical material is realized, wherein the effect of reducing the antigenicity of the biomedical material is better when the two technologies are used in combination.
Test example 2: amnion mechanical strength test-maximum uniaxial tensile breaking stress
The test included the following materials: the amniotic membrane prepared in examples 2-6 and comparative examples 1-7.
The testing method comprises the following steps: and (5) testing mechanical properties of the material by using an Instron 5967 universal tester. Samples were cut into 2.5cm x 1cm rectangles, n=6 for each group, and soaked with PBS for 2 hours at 37 ℃ prior to testing. The thickness was measured at 3 positions randomly with a thickness gauge, and the average value of the thickness was taken for stress calculation. Thereafter mounted on a jig of a universal tester and preloaded with a force of 0.1N, uniaxially stretched at a constant speed of 12 mm/min until fracture failure. At the same time, stress-strain curves were recorded and the maximum uniaxial tensile break stress was calculated.
The experimental results are shown in table 2.
TABLE 2
The experimental results (table 2) show that examples 2-6 have near maximum uniaxial tensile stress at break, i.e. better mechanical strength, than comparative example 1 (fresh human amniotic membrane). In addition, the maximum uniaxial tensile breaking stress of comparative examples 2 to 7 was reduced to some extent as compared with comparative example 1. Namely, the method shows that: compared with the existing 'subtraction principle' technical scheme for reducing the antigenicity of the biomedical material (namely, removing antigens from the biomedical material by using a detergent, a reducing agent and/or enzyme), the 'non-subtraction principle' technical scheme provided by the invention is that the biomedical material is crosslinked by irradiation crosslinking or ultraviolet irradiation crosslinking, and/or the biomedical material is subjected to atomization spraying treatment by using the antigen shielding material-dopamine modified super-hydrophilic polypeptide, so that the characteristic of maintaining the self mechanical property of the biomedical material is realized.
Test example 3: measurement of functional component (growth factor) content in amniotic membrane
The test included the following materials: the amniotic membrane prepared in examples 2-6 and comparative examples 1-7.
The testing method comprises the following steps: the amniotic membrane was freeze-dried, weighed, placed in a refiner for pulverization treatment, and centrifuged at 10000g at 4℃for 15 minutes, and the supernatant was separated to measure the growth factor concentration. The two growth factor contents were analyzed using an epidermal growth factor (Epidermal growth factor, EGF) and hepatocyte growth factor (hepatocyte growth factor, HGF) ELISA kit.
The experimental results are shown in tables 3 and 4.
TABLE 3 Table 3
TABLE 4 Table 4
The results of the experiments (tables 3 and 4) show that examples 2-6 have similar concentrations of epidermal growth factor and hepatocyte growth factor compared to comparative example 1 (fresh human amniotic membrane). In addition, comparative examples 2 to 7 have a somewhat reduced concentration of both growth factors as compared to comparative example 1. Namely, the method shows that: compared with the conventional aqueous solution soaking treatment mode, the method provided by the invention effectively reduces the loss of effective functional components in the biomedical material. According to the invention, the biomedical material is crosslinked by irradiation crosslinking or ultraviolet irradiation crosslinking, and/or the antigen shielding material-dopamine modified super-hydrophilic polypeptide is subjected to the non-subtractive principle of atomization spraying treatment on the biomedical material, so that the process of soaking treatment by an aqueous solution does not exist, and therefore, the growth factors contained in the amniotic membrane can be effectively reserved.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent variation, etc. of the above embodiment according to the technical matter of the present invention fall within the scope of the present invention.
Claims (6)
1. A method of reducing antigenicity of a biomedical material, characterized by: the method comprises the following steps:
(a) Preparing an aqueous solution of the super-hydrophilic polypeptide with the mass concentration of 5% and an aqueous solution of the carbon-carbon double bond modified compound with the mass concentration of 2.5%, respectively, and reacting for 22-26 hours at the temperature of 20-37 ℃ according to the volume ratio of the aqueous solution of the super-hydrophilic polypeptide to the aqueous solution of the carbon-carbon double bond modified compound being 0.6-1.2:1 to prepare the carbon-carbon double bond modified super-hydrophilic polypeptide;
(b) Preparing an aqueous solution of the super-hydrophilic polypeptide modified by carbon-carbon double bonds, wherein the mass concentration of the aqueous solution is 5%, adding an aqueous solution of 3-methacryloyl dopamine, the mass concentration of which is 2.5%, carrying out polymerization reaction for 1-24 hours at the temperature of 4-40 ℃ in the presence of an initiator, and carrying out dialysis purification and freeze-drying to prepare the antigen shielding material-dopamine modified super-hydrophilic polypeptide;
(c) After the biomedical material is subjected to radiation treatment or ultraviolet irradiation treatment, the biomedical material is subjected to atomization spraying treatment by adopting an antigen shielding material-dopamine modified super-hydrophilic polypeptide,
the amino acid sequence of the super-hydrophilic polypeptide is YYTYYTYT, and the structural formula is as follows:
,
the radiation treatment is to carry out gamma ray radiation on biomedical materials in an air atmosphere, the radiation dosage is controlled to be 5-50 kGy,
the ultraviolet irradiation treatment is to irradiate biomedical materials for 60-120 min by adopting an ultraviolet curing lamp with the wavelength of 50-280 w and 365nm in an air atmosphere.
2. The method according to claim 1, characterized in that: the biomedical material comprises an allogenic material and an animal source material, wherein the allogenic material comprises human amniotic membrane, cornea and pericardium; the animal source material comprises amniotic membrane, cornea, pericardium, small intestine mucosa, pleura, peritoneum and fat omentum of pig or cattle.
3. The method according to claim 1, characterized in that: the carbon-carbon double bond modified compound is N-acryloyloxy succinimide.
4. The method according to claim 1, characterized in that: the initiator is a mixture of ammonium persulfate and sodium bisulfite.
5. The method according to claim 1, characterized in that: the dialysis purification is to soak the materials in deionized water for 3 to 5 days at room temperature by adopting a dialysis bag with the molecular weight cut-off of 2000 to 3000.
6. The method according to claim 1, characterized in that: the atomization spraying treatment is to spray the surface of the biomedical material by adopting an atomization spraying machine.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202410077625.1A CN117582556B (en) | 2024-01-19 | 2024-01-19 | Method for reducing antigenicity of biomedical material |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202410077625.1A CN117582556B (en) | 2024-01-19 | 2024-01-19 | Method for reducing antigenicity of biomedical material |
Publications (2)
Publication Number | Publication Date |
---|---|
CN117582556A CN117582556A (en) | 2024-02-23 |
CN117582556B true CN117582556B (en) | 2024-03-19 |
Family
ID=89920534
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202410077625.1A Active CN117582556B (en) | 2024-01-19 | 2024-01-19 | Method for reducing antigenicity of biomedical material |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117582556B (en) |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1115673A (en) * | 1994-02-24 | 1996-01-31 | 研究发展基金会 | Aminotic membrane graft or wrap to prevent adhesions or bleeding of internal organs |
CN110156931A (en) * | 2019-04-22 | 2019-08-23 | 皖西学院 | Pectinate hydrophilic class peptide polymer, preparation method and application |
CN110237298A (en) * | 2019-04-26 | 2019-09-17 | 重庆科技学院 | Carbon/carbon compound material, biological planting body and its biological functional method of modifying |
CN110545754A (en) * | 2017-04-13 | 2019-12-06 | 祥丰医疗私人有限公司 | Medical device coated with polydopamine and antibody |
CN110684078A (en) * | 2019-10-21 | 2020-01-14 | 华中科技大学 | Cationic antibacterial peptide modified by dopamine or derivatives thereof, and preparation and application thereof |
CN113499484A (en) * | 2021-07-08 | 2021-10-15 | 深圳先进技术研究院 | Surface hydrophilic layer modification method for implantable medical device and application |
CN114258310A (en) * | 2019-12-09 | 2022-03-29 | 爱德华兹生命科学公司 | Bioprosthetic tissue preparation |
CN116196487A (en) * | 2022-12-30 | 2023-06-02 | 广东博迈医疗科技股份有限公司 | Coating and bracket with same, and preparation method and application thereof |
-
2024
- 2024-01-19 CN CN202410077625.1A patent/CN117582556B/en active Active
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1115673A (en) * | 1994-02-24 | 1996-01-31 | 研究发展基金会 | Aminotic membrane graft or wrap to prevent adhesions or bleeding of internal organs |
CN110545754A (en) * | 2017-04-13 | 2019-12-06 | 祥丰医疗私人有限公司 | Medical device coated with polydopamine and antibody |
CN110156931A (en) * | 2019-04-22 | 2019-08-23 | 皖西学院 | Pectinate hydrophilic class peptide polymer, preparation method and application |
CN110237298A (en) * | 2019-04-26 | 2019-09-17 | 重庆科技学院 | Carbon/carbon compound material, biological planting body and its biological functional method of modifying |
CN110684078A (en) * | 2019-10-21 | 2020-01-14 | 华中科技大学 | Cationic antibacterial peptide modified by dopamine or derivatives thereof, and preparation and application thereof |
CN114258310A (en) * | 2019-12-09 | 2022-03-29 | 爱德华兹生命科学公司 | Bioprosthetic tissue preparation |
CN113499484A (en) * | 2021-07-08 | 2021-10-15 | 深圳先进技术研究院 | Surface hydrophilic layer modification method for implantable medical device and application |
WO2023279663A1 (en) * | 2021-07-08 | 2023-01-12 | 深圳先进技术研究院 | Surface hydrophilic layer modification method for implantable medical device and application |
CN116196487A (en) * | 2022-12-30 | 2023-06-02 | 广东博迈医疗科技股份有限公司 | Coating and bracket with same, and preparation method and application thereof |
Non-Patent Citations (1)
Title |
---|
聚多巴胺在生物材料表面改性中的应用;刘宗光等;化学进展;20151231;第27卷;第212-219页 * |
Also Published As
Publication number | Publication date |
---|---|
CN117582556A (en) | 2024-02-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5916266A (en) | Raw membranous material for medical materials and manufacturing methods thereof | |
KR100587868B1 (en) | Chemical cleaning of biological material | |
AU2018236910A1 (en) | Methods of removing alpha-galactose | |
WO2022057841A1 (en) | Prosthetic heart valve and preparation method therefor | |
JP2017525732A (en) | Extracellular matrix composition | |
US20210402057A1 (en) | Process for obtaining decellularized extracellular matrix, decellularized extracellular matrix, use thereof and kit | |
WO2023115912A1 (en) | Preparation method for decellularized matrix biomaterial | |
EP3572103B1 (en) | Biological tissue matrix material, preparation method therefor and use thereof in otological repair material | |
EP4108265A1 (en) | Functionalized biological matrix material, preparation method therefor and use thereof | |
CN115087470A (en) | Functionalized biological matrix material and preparation method and application thereof | |
Erten et al. | Detergent-free decellularization of bovine costal cartilage for chondrogenic differentiation of human adipose mesenchymal stem cells in vitro | |
JP2019162440A (en) | Tissue product enzyme treatment method | |
RU2342162C1 (en) | Method for bone tissue biomaterial obtaining, and material for osteoplasty and tissue engineering obtained by such method | |
JP2008228744A (en) | Treatment method for preventing transplantation tissue with biological origin from calcification and tissue treated thereby | |
CN117582556B (en) | Method for reducing antigenicity of biomedical material | |
CN109550082A (en) | A kind of preparation method of acellular matrix gel | |
EP3188596B1 (en) | Human dermis, preparation and use thereof | |
Kumaresan et al. | Development of Human Umbilical cord based scaffold for tissue engineering application | |
CN107551314A (en) | A kind of E7 collagem membranes for promoting mesenchymal stem cells MSCs adhesion and preparation method thereof | |
JP2007124901A (en) | Method for extracting collagen | |
Shchotkina et al. | The Effect of sterilization on the bovine pericardium scaffold decellularized by the glutaraldehyde-free technology | |
CN114848912B (en) | Acellular dermis and preparation method thereof | |
AU774997B2 (en) | Chemical cleaning of biological material | |
CN117018280A (en) | Periosteum material for promoting local endogenous bone regeneration and repair 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 |