CN116531551A - Fibrinogen-based patch and preparation method and application thereof - Google Patents
Fibrinogen-based patch and preparation method and application thereof Download PDFInfo
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- CN116531551A CN116531551A CN202310515045.1A CN202310515045A CN116531551A CN 116531551 A CN116531551 A CN 116531551A CN 202310515045 A CN202310515045 A CN 202310515045A CN 116531551 A CN116531551 A CN 116531551A
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- 108010049003 Fibrinogen Proteins 0.000 title claims abstract description 210
- 102000008946 Fibrinogen Human genes 0.000 title claims abstract description 210
- 229940012952 fibrinogen Drugs 0.000 title claims abstract description 208
- 238000002360 preparation method Methods 0.000 title abstract description 12
- 125000001165 hydrophobic group Chemical group 0.000 claims abstract description 108
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 21
- 230000004048 modification Effects 0.000 claims abstract description 19
- 238000012986 modification Methods 0.000 claims abstract description 19
- 239000003381 stabilizer Substances 0.000 claims abstract description 19
- 238000004108 freeze drying Methods 0.000 claims abstract description 16
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 16
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 12
- 230000000694 effects Effects 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 91
- 238000003756 stirring Methods 0.000 claims description 23
- 150000003141 primary amines Chemical class 0.000 claims description 13
- 230000023597 hemostasis Effects 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical group O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 claims description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 8
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- -1 amide bond Chemical group 0.000 claims description 6
- 125000004185 ester group Chemical group 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 235000002639 sodium chloride Nutrition 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 4
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 claims description 4
- 208000031737 Tissue Adhesions Diseases 0.000 claims description 4
- 239000004480 active ingredient Substances 0.000 claims description 4
- 229960003589 arginine hydrochloride Drugs 0.000 claims description 4
- 239000011203 carbon fibre reinforced carbon Chemical group 0.000 claims description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 4
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 4
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 4
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 235000011147 magnesium chloride Nutrition 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 235000011083 sodium citrates Nutrition 0.000 claims description 3
- 235000011148 calcium chloride Nutrition 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 238000009210 therapy by ultrasound Methods 0.000 claims description 2
- 125000001183 hydrocarbyl group Chemical group 0.000 claims 1
- 230000002439 hemostatic effect Effects 0.000 abstract description 21
- 238000006243 chemical reaction Methods 0.000 abstract description 14
- 239000003106 tissue adhesive Substances 0.000 abstract description 12
- 108010010803 Gelatin Proteins 0.000 description 36
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- 235000011852 gelatine desserts Nutrition 0.000 description 36
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- 108010080379 Fibrin Tissue Adhesive Proteins 0.000 description 22
- 208000027418 Wounds and injury Diseases 0.000 description 21
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- 108090000190 Thrombin Proteins 0.000 description 16
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- 239000007853 buffer solution Substances 0.000 description 12
- 238000002156 mixing Methods 0.000 description 12
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 10
- 208000032843 Hemorrhage Diseases 0.000 description 10
- 238000007710 freezing Methods 0.000 description 10
- 230000008014 freezing Effects 0.000 description 10
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- 108010073385 Fibrin Proteins 0.000 description 9
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- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 8
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- HNYAWMSQSBERBE-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) hexanoate Chemical compound CCCCCC(=O)ON1C(=O)CCC1=O HNYAWMSQSBERBE-UHFFFAOYSA-N 0.000 description 5
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- 238000000338 in vitro Methods 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 229920001661 Chitosan Polymers 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 3
- 108010035532 Collagen Proteins 0.000 description 3
- 108010059814 TachoSil Proteins 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 229920001436 collagen Polymers 0.000 description 3
- 239000002874 hemostatic agent Substances 0.000 description 3
- 208000014674 injury Diseases 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 239000000565 sealant Substances 0.000 description 3
- 229940030990 tachosil Drugs 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
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- 238000001356 surgical procedure Methods 0.000 description 2
- LRKGVVJOUNKTGG-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) dodecanoate Chemical compound CCCCCCCCCCCC(=O)ON1C(=O)CCC1=O LRKGVVJOUNKTGG-UHFFFAOYSA-N 0.000 description 1
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- IUILQPUHQXTHQD-UHFFFAOYSA-N 1-hydroxypyrrolidine-2,5-dione;2-methylprop-2-enoic acid Chemical compound CC(=C)C(O)=O.ON1C(=O)CCC1=O IUILQPUHQXTHQD-UHFFFAOYSA-N 0.000 description 1
- KOVPXZDUVJGGFU-UHFFFAOYSA-N 8-methoxy-8-oxooctanoic acid Chemical compound COC(=O)CCCCCCC(O)=O KOVPXZDUVJGGFU-UHFFFAOYSA-N 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 241000352333 Amegilla alpha Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 206010008164 Cerebrospinal fluid leakage Diseases 0.000 description 1
- 208000034656 Contusions Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241001197082 Knodus beta Species 0.000 description 1
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- 206010067125 Liver injury Diseases 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 229920002201 Oxidized cellulose Polymers 0.000 description 1
- 229940122791 Plasmin inhibitor Drugs 0.000 description 1
- 208000002847 Surgical Wound Diseases 0.000 description 1
- 206010051373 Wound haemorrhage Diseases 0.000 description 1
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- 150000001408 amides Chemical class 0.000 description 1
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- 239000000945 filler Substances 0.000 description 1
- 210000003736 gastrointestinal content Anatomy 0.000 description 1
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- 231100000753 hepatic injury Toxicity 0.000 description 1
- 229940106780 human fibrinogen Drugs 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
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- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
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- 235000018977 lysine Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
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- 230000000877 morphologic effect Effects 0.000 description 1
- 239000004745 nonwoven fabric Substances 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
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- 229910052708 sodium Inorganic materials 0.000 description 1
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- 238000006467 substitution reaction Methods 0.000 description 1
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- 125000001273 sulfonato group Chemical group [O-]S(*)(=O)=O 0.000 description 1
- 239000003894 surgical glue Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229960003766 thrombin (human) Drugs 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 229940033618 tisseel Drugs 0.000 description 1
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- 230000008733 trauma Effects 0.000 description 1
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Classifications
-
- 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
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/106—Fibrin; Fibrinogen
-
- 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
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/02—Surgical adhesives or cements; Adhesives for colostomy devices containing inorganic materials
-
- 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
- A61L2400/04—Materials for stopping bleeding
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Surgery (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Inorganic Chemistry (AREA)
- Materials For Medical Uses (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a fibrinogen-based patch, a preparation method and application thereof, wherein the patch comprises fibrinogen and a stabilizer, and part of lysine residues of the fibrinogen contain hydrophobic groups; the stabilizer is used for maintaining the activity of fibrinogen. The preparation method comprises the following steps: and (3) dropwise adding a hydrophobic modification reagent into the fibrinogen solution, dialyzing after the reaction, and then freeze-drying to obtain the patch. The patch prepared by the method has excellent mechanical strength and adhesion performance, and the performance under the same condition is obviously superior to that of the existing surgical adhesive product and the emergency hemostatic product. The patch has good biocompatibility, can be absorbed by human body, and has great potential in clinical application.
Description
Technical Field
The invention relates to a fibrinogen-based patch, and a preparation method and application thereof, and belongs to the field of medical tissue engineering.
Background
Uncontrolled bleeding after trauma or during surgery is a major cause of death worldwide, resulting in death in more than 200 tens of thousands of people per year. It is necessary and critical to minimize blood loss, seal tissue and organ structures during surgical procedures, reduce post-surgical complications, and reduce operating time in the operating room. Wound dressings and tissue adhesives are devices used to prevent leakage of blood and other body fluids.
Surgical hemostatic agents commonly used today include fibrin glue, gelatin, and oxidized cellulose. Among them, fibrin glue is one of the most widely used hemostatic agents because of its good procoagulant properties and biocompatibility. The mechanism of action is the last enzyme cascade reaction of the simulated human body autologous coagulation reaction process, and the thrombin is utilized to convert fibrinogen into fibrin monomers, and the fibrin monomers are crosslinked into fibrin glue networks to seal wounds, thereby playing a role in hemostasis. The fibrin glue has poor adhesion performance of mechanical strength, and cannot realize high-efficiency adhesion and sealing of wound wet tissues. Although fibrin glue has a high procoagulant performance, the hemostatic effect is not very desirable. How to enhance the mechanical strength of fibrin gel is then a key challenge to solve the weak hemostatic properties.
The physical and mechanical strength of fibrin glue depends on the structure of the fibrin monomers, the crosslinked network of fibrin glue, and the degree of crosslinking. Fibrinogen is an oligosaccharide protein of molecular weight 340kDa, consisting of two symmetrical halves, each half containing three polypeptide chains of A.alpha., B.beta.and gamma. Fibrinogen is sheared by thrombin to release peptide chains FpA and FpB, the amino acid residues exposed by the formed fibrin monomers are complemented with another fibrin monomer to form a "button-hole interaction", and finally the fibrin glue cross-linked network is formed by extension, alternation and overlapping. At present, means for improving the mechanical strength of fibrin glue mainly comprise: regulating fibrinogen and thrombin concentration, and introducing filled protein. First, it is reported in the literature that the concentration of thrombin affects the thickness of fibrin formed filaments, which is related to the mechanical strength of fibrin glue. Initially, researchers improved mechanical properties by adjusting the concentration of fibrinogen and thrombin in fibrin glue. Thus, the fibrinogen and thrombin concentrations of the currently commercial fibrin glues (Tisseel, evicel, hemaseel, etc.) are different, but the mechanical properties of adhesion are not significantly improved. Second, scientists have improved the overall mechanical properties of hydrogels by introducing proteins into the gel network. Researchers have improved mechanical strength but still have limited improvement by adding collagen or gelatin (such as CoStasis et al) as a filler for fibrin glue network structures. In addition, there are also carriers to which fibrinogen and thrombin are immobilized to improve mechanical properties. TachoSil Fibrin Sealant Patch is a sealant for supporting human fibrinogen and thrombin on a horse collagen sponge. When TachoSil is applied to a bleeding or other body fluid area, moisture causes thrombin to cleave fibrinogen, rapidly forming a clot of fibrin glue, adhering to the tissue surface for hemostasis and wound sealing. However, the absorption of moisture by the sponge for dissolving fibrinogen is a relatively time consuming process, which is detrimental to the rapid occlusion of blood-containing wounds; likewise, its performance is still largely dependent on the mechanical properties of the fibrin glue itself, with limited improvement in overall hemostatic and sealing properties. According to literature reports, after pressing the wound with a TachoSil patch for 3min in a pig liver injury bleeding model (10 mm diameter, 10mm deep gap), the wound was not successfully plugged and blood still continued to flow out (Nature Biomedical Engineering, 1131-1142 (2021)). Thus, fibrinogen/gelatin, while having excellent procoagulant and biocompatible properties, does not provide a significant improvement in mechanical strength (bulk strength and adhesion properties) over prior art improvements.
In order to realize the breakthrough of the hemostatic performance of fibrinogen/gelatin, the key point is that: how to control the composition and structure of fibrinogen/gelatin itself to improve its mechanical strength and adhesion properties?
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a fibrinogen-based patch, and a preparation method and application thereof.
The invention adopts the following technical method:
a fibrinogen-based patch comprising fibrinogen and a stabilizing agent, a portion of the lysine residues of the fibrinogen containing hydrophobic groups; the stabilizer is used for maintaining the activity of fibrinogen.
In the above technical solution, the stabilizer is used for maintaining the activity of fibrinogen, which means not only maintaining the spatial structure and biological activity of fibrinogen; also included is maintaining fibrinogen stability in the patch state and during the patch manufacturing process. The stabilizer may be one or more of sodium chloride, calcium chloride, potassium chloride, magnesium chloride, sodium citrate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, arginine hydrochloride, glycine.
The hydrophobic group is a hydrocarbon group containing one or more groups of carbonyl, carbon-carbon double bond, ester group, amide bond and phenyl.
Preferably, the hydrophobic group is-CO- (CH) 2 ) n CH 3 N=1-19; more preferably, the hydrophobic group is-CO- (CH) 2 ) n CH 3 N=2-10; most preferably, the hydrophobic group is-CO- (CH) 2 ) n CH 3 ，n＝3-6。
Preferably, the hydrophobic group forms an amide bond (-NH-CO-) with a primary amine of a fibrinogen lysine residue.
The lysine residues containing hydrophobic groups on the fibrinogen account for 2% -70% of the total number of lysine residues, and the lysine residues are calculated by number; the total number of lysine residues is the sum of lysine residues containing hydrophobic groups and unmodified lysine residues.
Preferably, the lysine residues on fibrinogen containing hydrophobic groups comprise 5% to 40% of the total number of lysine residues; more preferably, the lysine residues on fibrinogen containing hydrophobic groups comprise 10% to 30% of the total number of lysine residues.
The proportion of lysine residues containing hydrophobic groups on fibrinogen to the total number of lysine residues needs to be within a specific range to ensure that fibrinogen molecules containing hydrophobic groups on a microscopic level are entangled with each other, so that the mechanical properties of the fibrinogen patch are reflected on a macroscopic level. If the proportion of lysine residues containing hydrophobic groups on fibrinogen to the total number of lysine residues is too low, e.g. below 2%, the mechanical properties of the fibrinogen patch on a macroscopic level are not significantly changed compared to the unmodified fibrinogen patch. If the proportion of lysine residues containing hydrophobic groups on fibrinogen is higher than 70% of the total number of lysine residues, the fibrinogen protein is denatured and the spatial structure is unstable due to substitution of a large number of basic amino acids (lysines) on the surface of the protein, and finally the mechanical properties of the patch are deteriorated, even inferior to those of original unmodified fibrinogen. Therefore, the degree of modification of lysine residues in a specific ratio is beneficial to realizing the high mechanical properties of modified fibrinogen.
The patch contains not less than 2mg fibrinogen containing hydrophobic groups per square centimeter. Preferably, the patch contains 4-100 mg fibrinogen containing hydrophobic groups per square centimeter; preferably, the patch contains 10-50 mg fibrinogen containing hydrophobic groups per square centimeter; more preferably, the patch contains 20 to 30mg of fibrinogen containing hydrophobic groups per square centimeter.
The patch must have at least a certain amount of fibrinogen containing hydrophobic groups per square centimeter to ensure that the patch has continuous and uniform fibrinogen to achieve a certain strength of mechanical properties.
The fibrinogen may be derived from any one or more of human, bovine, porcine and recombinant sources.
The fibrinogen-based patch may contain a pharmaceutically active ingredient which is a substance having medical utility or physiological activity and which may be used to treat, alleviate, or ameliorate the symptoms of a disease. The pharmaceutically active ingredient comprises a drug for targeted or controlled release. Preferably, the pharmaceutically active ingredient comprises a plasmin inhibitor.
Preferably, the surgical patch further comprises one or more of collagen, albumin and hyaluronic acid. The collagen is derived from any one or more of human source, bovine source, porcine source, equine source and recombinant.
The invention also provides a preparation method of the fibrinogen-based patch, which comprises the following specific steps:
(1) carrying out ultrasonic treatment on the mixed solution of fibrinogen and a stabilizer, wherein the mass fraction of the obtained fibrinogen solution is 5-200 mg/mL;
(2) dropwise adding a hydrophobic modification reagent into the fibrinogen solution under the stirring condition, and reacting for 0.5-5 h at 37 ℃ to obtain the fibrinogen solution containing hydrophobic groups;
(3) dialyzing the obtained fibrinogen solution containing the hydrophobic group for 5-72 h at the temperature of 4-37 ℃;
(4) concentrating the obtained fibrinogen solution containing the hydrophobic group to 50-200 mg/mL, wherein the concentration temperature is 4-37 ℃; and freeze-drying at-85 to-35 ℃ to obtain the fibrinogen-based patch.
In the above technical scheme, the hydrophobic modification reagent contains a hydrophobic group, and the hydrophobic group is used for modifying fibrinogen lysine residue; the hydrophobic modification reagent also comprises a succinimide group, and the succinimide group is used for reacting with primary amine of lysine residue to form stable amide bond.
The composition of the hydrophobic modification reagent comprises two groups, and one group is used for modifying fibrinogen lysine residues and is a hydrophobic group. Another part of the groups is used for reaction with primary amines on fibrinogen lysine residues,finally take off. For example, the succinimide groups described herein react with primary amines on lysine residues to form stable amide linkages, releasing N-hydroxysuccinimide (NHS).
Preferably, the succinimide group contains a sulfonate (-SO) 3 - ). Sulfonate groups can increase the water solubility of the hydrophobically modified agent and more importantly increase the efficiency of the reaction of the hydrophobically modified agent with fibrinogen.
When the hydrophobic modification reagent contains a succinimide group and a hydrophobic group, the reactive group (succinimide group) on the hydrophobic modification reagent can reduce or prevent the hydrophobic modification reagent from inducing protein denaturation of fibrinogen during the reaction process. The hydrophobic modification reagent provided by the invention cannot be anhydride with a hydrophobic group, the anhydride with the hydrophobic group is very easy to denature fibrinogen, the denatured fibrinogen is easy to precipitate out of solution, the hydrophobic group cannot be modified on the fibrinogen, and the fibrinogen-based patch provided by the invention cannot be formed.
The hydrophobic group is a hydrocarbon group containing one or more groups of carbonyl, carbon-carbon double bond, ester group, amide bond and phenyl.
Preferably, the hydrophobic group is-CO- (CH) 2 ) n CH 3 N=1-19; more preferably, the hydrophobic group is-CO- (CH) 2 ) n CH 3 N=2-10; most preferably, the hydrophobic group is-CO- (CH) 2 ) n CH 3 ，n＝3-6。
Preferably, the hydrophobic group forms an amide bond (-NH-CO-) with a primary amine of a fibrinogen lysine residue.
The invention also provides the application of the fibrinogen-based patch, and the patch can be used for tissue adhesion.
The patch is used for stopping bleeding; the hemostasis can be used for hemostasis in cardiovascular, liver, pancreas, thyroid operations; the patch may undergo a coagulation reaction upon contact with tissue and/or blood to subsequently form a clot, and have strong binding energy to nearby tissue based on strong interactions between fibrinogen molecules, which may act synergistically to effectively prevent, reduce or stop bleeding.
The patch is used for jointing structures and/or sealing physiological leakage; for leakage of cerebrospinal fluid, lymph, bile, gastrointestinal contents, and for air leakage of the lungs.
The patch is used for repair of damaged tissue, which refers to the disruption of the normal continuity of tissue structure, such as incisions caused by surgical, biological, or chemical means; also included are contusion tissue, as well as cuts, punctures, lacerations, open injuries, penetrating injuries caused by, for example, tearing, pressure and biting.
Compared with the prior art, the invention has the following advantages:
1. the invention unexpectedly prepares a brand new fibrinogen patch for the first time, wherein the patch comprises fibrinogen containing hydrophobic groups, the fibrinogen containing the hydrophobic groups has strong intermolecular interaction relative to wild type (unmodified) fibrinogen, and the hydrophobic groups promote mutual entanglement among fibrinogen protein molecules, so that the patch has super-strong mechanical property and adhesion property.
2. The fibrinogen patch provided by the invention is self-formed without depending on the action of externally added thrombin. The fibrinogen/gelatin in the prior art is based on the reaction of fibrinogen and thrombin in the application of tissue sealing and hemostasis, and the relevant effect of tissue sealing can be exerted without single fibrinogen. The method does not depend on additional thrombin, can reduce the cost of materials and avoid the problem of difficult enzyme storage. The prior art relies on dissolving fibrinogen and thrombin to form a solution, or absorbing water to dissolve, and the dissolution process is generally time consuming and complex and requires preparation in advance. The patch provided by the invention can be taken at any time in the practical application process.
3. The fibrinogen patch based on the hydrophobic group has good biological activity, can promote proliferation and differentiation of cells, and is beneficial to wound healing and defect tissue recovery; the degradation speed of the patch can be regulated by changing the components of the hydrophobic groups, so that the regeneration speed of the self tissue organ can be met.
4. The preparation method used by the invention has convenient operation and simple process; the obtained patch is convenient to use and can be cut according to the size of a wound.
Drawings
FIG. 1 scanning electron microscope image of the fibrinogen patch of example 1;
FIG. 2 molecular dynamics mimics unmodified fibrinogen molecular interactions, hydrophobic group-containing fibrinogen molecular interactions of the present invention, comparing to see that strong interactions of hydrophobic group-containing fibrinogen molecules occur, inducing entanglement of molecules;
FIG. 3 time for unmodified fibrinogen and fibrinogen containing hydrophobic groups of example 1 of the present invention to be recognized by thrombin to form fibrin gel;
FIG. 4 pH and in vitro cytotoxicity corresponding to the Surgicel Fibrillar products of example 1, comparative example 1 and comparative example 4;
FIG. 5 shear strength of 3 surgical adhesive products (Fibrin Glue, surgicel Fibrillar, gelatin spring) of example 1, comparative example 1 and comparative example 4;
FIG. 6 adhesive energy of 3 surgical adhesive products (Fibrin Glue, surgicel Fibrillar, gelatin spring) of example 1, comparative example 1 and comparative example 4;
FIG. 7 New Zealand white rabbits of the surgical adhesive products (Surgicel Fibrillar, gelatin Spongge) of example 1 and comparative example 4 for liver hemostasis time and blood loss;
FIG. 8 New Zealand white rabbits (Combat Gauze, hemCon, celox, chitoSAM) for 4 emergency hemostatic products of example 1 and comparative example 4 for liver hemostasis time and blood loss;
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The technical problems, technical schemes and beneficial effects to be solved by the invention are described in detail below with reference to specific embodiments. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that several variations and modifications can be made by those skilled in the art without departing from the precursors of the inventive concept. These are all within the scope of the present invention.
The present invention provides a fibrinogen patch having excellent mechanical properties and adhesive properties, the patch comprising fibrinogen and a stabilizer, a part of lysine residues of the fibrinogen containing a hydrophobic group. The hydrophobic groups of lysine residues on the surface of fibrinogen are modified, and in a solution state, most of van der Waals interactions of the hydrophobic groups are destroyed due to participation of water molecules (electrostatic action, hydrogen bonds, etc.). During the removal of water to form the patch, van der Waals interactions between the hydrophobic chains are enhanced by close proximity interactions, resulting in a fibrinogen patch with strong mechanical and adhesive properties. The stabilizer is one or more of sodium chloride, calcium chloride, sodium citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, arginine hydrochloride and glycine.
The stabilizer is used for maintaining the spatial structure and biological activity of fibrinogen. The stabilizer is used for maintaining the stability of fibrinogen in the patch state and also for maintaining the stability of fibrinogen in the patch preparation process. The stabilizer is introduced when preparing fibrinogen solution in the patch.
Shear strength test: reference is made to american society for testing and materials standards (ASTM F2255-05). The fibrinogen patch is added to the surface of fresh pigskin, the area of the patch is 10×20mm, and the other pigskin is placed on the surface of the patch for adhesion, so that the two pigskins are kept overlapped at the patch part. The shear strength of the samples was tested using a universal material tester at a strain rate of 5 mm/min.
Adhesion energy test: reference is made to american society for testing and materials standards (ASTM F2256-05). The fibrinogen patch was added to the surface of fresh pigskin with an area of 15 x 35mm, and the other pigskin was placed in alignment on the surface of the patch. The adhesion properties of the samples were tested using a universal material tester at a strain rate of 5 mm/min.
Liver hemostasis experiment of New Zealand white rabbits: liver leaves of New Zealand white rabbits (2.5-3 kg) were used to make a 10mm long incision 5mm deep on the liver surface, and the pre-bleeding amount was recorded as free bleeding for 10 s. The patch was applied to the liver incision surface and pressed for 15s. The wound closure status, hemostatic time and blood loss for wound bleeding were evaluated.
Example 1
(1) Taking a fibrinogen solution (PBS buffer solution) with the mass fraction of 100mg/mL, stirring and uniformly dispersing by ultrasonic waves;
(2) under the condition of avoiding light, 200 mu L of 0.4M N- (caproyl oxy) succinimide is dropwise added into the fibrinogen solution under the condition of stirring, dispersion is carried out under the triple mixing process of vortex 5min and ultrasonic 5min, and the fibrinogen solution containing hydrophobic groups is obtained after reaction for 4h under the condition of 37 ℃;
(3) dialyzing the obtained fibrinogen solution containing the hydrophobic group at 4 ℃ for 72 hours;
(4) freezing the obtained fibrinogen solution containing the hydrophobic group, and concentrating to 100mg/mL at 37 ℃; and freeze-drying to obtain fibrinogen patch.
The hydrophobic group-containing fibrinogen solution is formed by reacting a hydrophobic modifying agent (N- (caproyloxy) succinimide) with a primary amine of a fibrinogen surface lysine residue to form a stable amide bond, and the stable amide bond is bonded to fibrinogen molecules.
The microstructure of the patch prepared in this example is shown in fig. 1, and the surface of the patch has a porous structure. The hydrophobic group of fibrinogen in the patch is-CO- (CH) 2 ) 4 CH 3 Reacts with primary amines of lysine residues on the fibrinogen surface to form amide linkages (-NH-CO-). Van der Waals interactions between hydrophobic chains enhance the induction of protein intermolecular entanglement due to close proximity interactions (FIG. 2), resulting in patches with strong mechanical and adhesive properties. The stabilizer of the patch in this example is Na 2 HPO 4 、KH 2 PO 4 NaCl and KCl. To facilitate comparison of the mechanical properties of the different examples with the comparative examples, the stabilizers of the following preparation process remain the same. Meanwhile, the stabilizer in the invention can also be sodium chloride, and the sodium chlorideOne or more of calcium, potassium chloride, magnesium chloride, sodium citrate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, arginine hydrochloride, and glycine. The patch prepared in this example contains 20mg fibrinogen containing hydrophobic groups per square centimeter.
Example 1 adhesive energy of the patch was 160J/m 2 The shear strength was 8750Pa. In addition, the time for the unmodified fibrinogen and the fibrinogen containing hydrophobic groups of the invention to be catalyzed by thrombin to form fibrin gel is similar, indicating that the modified fibrinogen retains the ability to be recognized by thrombin (FIG. 3). The patches have a suitable pH and low cytotoxicity (fig. 4); after 28 days of implantation into the rat liver wound area, the wound healed well.
Example 2
(1) Taking a fibrinogen solution (PBS buffer solution) with the mass fraction of 100mg/mL, stirring and uniformly dispersing by ultrasonic waves;
(2) under the condition of avoiding light, 200 mu L of 0.4M 2,5-dioxopyrrolidin-1-yl dodecanoate is dropwise added into the fibrinogen solution under the condition of stirring, dispersion is carried out under the triple mixing process of vortex 5min and ultrasonic 5min, and the fibrinogen solution containing hydrophobic groups is obtained after reaction for 5h under the condition of 37 ℃;
(3) dialyzing the obtained fibrinogen solution containing the hydrophobic group at 4 ℃ for 72 hours;
(4) freezing the obtained fibrinogen solution containing the hydrophobic group, and concentrating to 100mg/mL at 37 ℃; and freeze-drying to obtain fibrinogen patch.
The hydrophobic group of fibrinogen in the patch of this example is-CO- (CH) 2 ) 10 CH 3 Reacts with primary amines of lysine residues on the fibrinogen surface to form amide linkages (-NH-CO-). The patch prepared in this example contains 20mg fibrinogen containing hydrophobic groups per square centimeter. Adhesion energy of 175J/m 2 The shear strength was 10320Pa. The patch has a suitable pH and low cytotoxicity; after 28 days of implantation into the rat liver wound area, the wound healed well.
Example 3
(1) Taking a fibrinogen solution (PBS buffer solution) with the mass fraction of 100mg/mL, stirring and uniformly dispersing by ultrasonic waves;
(2) under the condition of avoiding light, 200 mu L of 0.4M N-hydroxysuccinimide methacrylate is dropwise added into the fibrinogen solution under the condition of stirring, dispersion is carried out under the triple mixing process of vortex 5min and ultrasonic 5min, and the fibrinogen solution containing hydrophobic groups is obtained after reaction for 3h under the condition of 37 ℃;
(3) dialyzing the obtained fibrinogen solution containing the hydrophobic group at 25 ℃ for 24 hours;
(4) freezing the obtained fibrinogen solution containing the hydrophobic group, and concentrating to 100mg/mL at 4 ℃; and freeze-drying to obtain fibrinogen patch.
The hydrophobic group of fibrinogen in the patch of this example is-CO-CH (CH) 3 )＝CH 2 Containing a carbon-carbon double bond, reacts with a primary amine of a fibrinogen surface lysine residue to form an amide bond (-NH-CO-). The patch prepared in this example contains 20mg fibrinogen containing hydrophobic groups per square centimeter. Adhesive energy of 146J/m 2 The shear strength was 7320Pa. The patch has a suitable pH and low cytotoxicity; after 28 days of implantation into the rat liver wound area, the wound healed well.
Example 4
(1) Taking a fibrinogen solution (PBS buffer solution) with the mass fraction of 100mg/mL, stirring and uniformly dispersing by ultrasonic waves;
(2) under the condition of avoiding light, 200 mu L of 0.4M 2, 5-dioxarrolide-1-yl methyl glutarate (2, 5-Dioxopyrrolidin-1-yl methyl glutarate) is dropwise added into the fibrinogen solution under the condition of stirring, dispersion is carried out under the triple mixing process of vortex 5min and ultrasonic 5min, and the fibrinogen solution containing hydrophobic groups is obtained after reaction for 3h under the condition of 37 ℃;
(3) dialyzing the obtained fibrinogen solution containing the hydrophobic group at 25 ℃ for 24 hours;
(4) freezing the obtained fibrinogen solution containing the hydrophobic group, and concentrating to 100mg/mL at 37 ℃; and freeze-drying to obtain fibrinogen patch.
The hydrophobic group of fibrinogen in the patch of this example is-CO- (CH) 2 ) 3 -COOCH 3 Containing ester groups, react with primary amines of lysine residues on the fibrinogen surface to form amide linkages (-NH-CO-). The patch prepared in this example contains 20mg fibrinogen containing hydrophobic groups per square centimeter. Adhesive energy of 156J/m 2 The shear strength was 8568Pa. The patch has a suitable pH and low cytotoxicity; after 28 days of implantation into the rat liver wound area, the wound healed well.
Example 5
(1) Taking a fibrinogen solution (PBS buffer solution) with the mass fraction of 100mg/mL, stirring and uniformly dispersing by ultrasonic waves;
(2) 200 mu L of 0.4M 1- (2, 5-dioxarrolidin-1-yl) 8-methyl octanedioate (1- (2, 5-Dioxopyrrolidin-1-yl) 8-methyl suberate) is dropwise added into the fibrinogen solution under the light-shielding condition, dispersion is carried out under the triple mixing process of vortex 5min and ultrasonic 5min, and the reaction is carried out for 3h under the condition of 37 ℃ to obtain the fibrinogen solution containing hydrophobic groups;
(3) dialyzing the obtained fibrinogen solution containing the hydrophobic group at 25 ℃ for 24 hours;
(4) freezing the obtained fibrinogen solution containing the hydrophobic group, and concentrating to 100mg/mL at 37 ℃; and freeze-drying to obtain fibrinogen patch.
The hydrophobic group of fibrinogen in the patch of this example is-CO- (CH) 2 ) 5 -COOCH 3 Containing ester groups, react with primary amines of lysine residues on the fibrinogen surface to form amide linkages (-NH-CO-). The patch prepared in this example contains 20mg fibrinogen containing hydrophobic groups per square centimeter. Adhesive energy of 159J/m 2 The shear strength was 8850Pa. The patch has a suitable pH and low cytotoxicity; after 28 days of implantation into the rat liver wound area, the wound healed well.
Comparative example 1
(1) Taking a fibrinogen solution (PBS buffer solution) with the mass fraction of 100mg/mL, stirring and uniformly dispersing by ultrasonic waves;
(2) dropwise adding 200 mu L of PBS solution into the fibrinogen solution under the condition of light shielding, dispersing under the triple mixing process of vortex 5min and ultrasonic 5min, and reacting for 4 hours at 37 ℃ to obtain an unmodified fibrinogen solution;
(3) the resulting unmodified fibrinogen solution was dialyzed for 72h at 4 ℃.
(4) Freezing the obtained unmodified fibrinogen solution, and concentrating to 100mg/mL at 37 ℃; and then freeze-drying to obtain unmodified fibrinogen patch (unmodified fibrinogen has relatively weak intermolecular interaction during lyophilization, and the formed patch is not formed).
Comparative example 1 differs relatively from example 1 in that there is no modification of the hydrophobic group. The patch prepared in this example contained 20mg fibrinogen per square centimeter.
Adhesive energy of 10J/m 2 The shear strength was 1233Pa. The mechanical and adhesive properties of the unmodified fibrinogen patch were much lower than those of the patch of the present invention (fig. 5 and 6). Thus, it is demonstrated that modification of the hydrophobic groups is a key factor in the improvement of the fibrinogen patch performance of the present invention.
Comparative example 2
(1) Fibrinogen solution containing hydrophobic groups:
(1) taking a fibrinogen solution (PBS buffer solution) with the mass fraction of 100mg/mL, stirring and uniformly dispersing by ultrasonic waves;
(2) under the condition of avoiding light, 200 mu L of 0.4M N- (caproyl oxy) succinimide is dropwise added into the fibrinogen solution under the condition of stirring, dispersion is carried out under the triple mixing process of vortex 5min and ultrasonic 5min, and the fibrinogen solution containing hydrophobic groups is obtained after reaction for 4h under the condition of 37 ℃;
(3) the resulting fibrinogen solution containing hydrophobic groups was dialyzed for 72h at 4 ℃.
(2) Unmodified fibrinogen solution:
(1) taking a fibrinogen solution (PBS buffer solution) with the mass fraction of 100mg/mL, stirring and uniformly dispersing by ultrasonic waves;
(2) dropwise adding 200 mu L of PBS solution into the fibrinogen solution under the condition of avoiding light, dispersing the fibrinogen solution under the triple mixing process of vortex 5min and ultrasonic 5min, and reacting the fibrinogen solution for 4 hours at 37 ℃ to obtain an unmodified fibrinogen solution;
(3) the resulting unmodified fibrinogen solution was dialyzed for 72h at 4 ℃.
The binding energy of the fibrinogen solution containing hydrophobic groups was 7.5J/m 2 The shear strength was 800Pa. The mechanical properties of the patch of the embodiment 1 of the invention are far better than those of the fibrinogen solution containing the hydrophobic groups, which shows that the invention can achieve excellent mechanical effects only by forming the patch. In one aspect, the patch of the present invention may increase adhesion to tissue by absorbing water; on the other hand, during the water removal to form the patch preparation, van der Waals interactions between hydrophobic chains are enhanced due to close proximity interactions, contributing to the strong mechanical properties of the fibrinogen patch. The patch form of the present invention has an important role in its overall performance.
The binding energy of the unmodified fibrinogen solution was 6.5J/m 2 The shear strength was 750Pa. It can be seen that the fibrinogen solution containing hydrophobic groups is not significantly different from the unmodified fibrinogen solution in terms of solution viscosity and mechanical properties.
Comparative example 3
(1) Gelatin patch modified by hydrophobic group and unmodified gelatin patch
Gelatin patches modified with hydrophobic groups:
(1) taking gelatin solution (PBS buffer solution) with the mass fraction of 100mg/mL, stirring and uniformly dispersing by ultrasonic;
(2) under the condition of avoiding light, 200 mu L of 0.4M N- (caproyloxy) succinimide is dropwise added into the gelatin solution under the condition of stirring, the gelatin solution is dispersed under the triple mixing process of vortex 5min and ultrasonic 5min, and the gelatin solution containing hydrophobic groups is obtained after reaction for 4h under the condition of 37 ℃;
(3) dialyzing the obtained gelatin solution containing hydrophobic group at 37deg.C for 72 hr;
(4) freezing the obtained gelatin solution containing hydrophobic groups, and concentrating to 100mg/mL at 37deg.C; and freeze-drying to obtain the gelatin patch modified by the hydrophobic group. The patch prepared in this example contained 20mg of modified gelatin per square centimeter.
Unmodified gelatin patch:
(1) taking gelatin solution (PBS buffer solution) with the mass fraction of 100mg/mL, stirring and uniformly dispersing by ultrasonic;
(2) under the condition of avoiding light, 200 mu L of PBS solution is dropwise added into the gelatin solution under the condition of stirring, dispersion is carried out under the triple mixing process of vortex 5min and ultrasonic 5min, and the mixture is reacted for 4 hours at 37 ℃ to obtain unmodified gelatin solution;
(3) dialyzing the unmodified gelatin solution at 37deg.C for 72 hr;
(4) freezing the obtained unmodified gelatin solution, concentrating to 100mg/mL, and concentrating at 37deg.C; and freeze-drying to obtain unmodified gelatin patch. The patch prepared in this example contained 20mg of gelatin per square centimeter.
(2) Albumin patch modified with hydrophobic group and unmodified albumin patch
Albumin patch modified by hydrophobic group:
(1) taking a bovine serum albumin solution (PBS buffer solution) with the mass fraction of 100mg/mL, stirring and uniformly dispersing by ultrasonic;
(2) under the condition of avoiding light, dropwise adding 200 mu L0.4M of N- (caproyloxy) succinimide into the bovine serum albumin solution under the condition of stirring, dispersing the solution under the triple mixing process of vortex 5min and ultrasonic 5min, and reacting the solution for 4 hours at 37 ℃ to obtain an albumin solution containing a hydrophobic group;
(3) dialyzing the obtained albumin solution containing the hydrophobic group for 72 hours at the temperature of 4 ℃;
(4) freezing the obtained albumin solution containing the hydrophobic group, and concentrating to 100mg/mL at 37 ℃; and then freeze-drying to obtain the albumin patch modified by the hydrophobic group. The patch prepared in this example contained 20mg of modified albumin per square centimeter.
Unmodified albumin patch:
(1) taking a bovine serum albumin solution (PBS buffer solution) with the mass fraction of 100mg/mL, stirring and uniformly dispersing by ultrasonic;
(2) under the condition of avoiding light, 200 mu L of PBS solution is dropwise added into the bovine serum albumin solution under the condition of stirring, dispersion is carried out under the triple mixing process of vortex 5min and ultrasonic 5min, and the mixture is reacted for 4 hours at 37 ℃ to obtain an unmodified albumin solution;
(3) dialyzing the obtained unmodified albumin solution at 4 ℃ for 72 hours;
(4) freezing the obtained unmodified albumin solution, and concentrating to 100mg/mL at 37 ℃; and freeze-drying to obtain the unmodified albumin patch. The patch prepared in this example contained 20mg albumin per square centimeter.
The hydrophobic group-containing modified gelatin patch and the unmodified gelatin patch of this comparative example were not significantly changed in mechanical properties and tissue adhesion properties of the patches before and after the hydrophobic group modification from the hydrophobic group-containing modified albumin patch and the unmodified albumin patch. In addition, the gelatin and albumin before modification or after modification do not actually form a patch structure after freeze-drying, but are in powder form, and are not patches according to the invention.
This demonstrates that hydrophobic groups are modified onto fibrinogen molecules, that they form patches after lyophilization, rather than as powders, and that the lyophilized fibrinogen has strong mechanical properties. Under the same conditions, gelatin and albumin do not produce the corresponding phenomena. We speculate that the morphological and mechanical property enhancement of fibrinogen patches is related to its own compositional characteristics. Fibrinogen consists of two symmetrical halves, each half containing three polypeptide chains of a, B and γ, a linear structure of protein molecules. Due to the special composition and structure, the mechanical properties are unexpectedly improved after being modified by hydrophobic groups.
Comparative example 4
Commercially available 3 surgical adhesive products (absorbable): strong life Surgicel Fibrillar, shanghai Laiyi Fibrin Glue and Xiang En Gelatin Sponge Gelatin Sponge;
commercially available 4 emergency hemostatic products (non-absorbable, in vitro use): quikClot Combat Gauze (kaolin impregnated to Gauze), celox Gauze hemostat (chitosan laminated to Gauze), chitoSAM 100 (non-woven fabric spun with chitosan), hemCon ChitoGauze (chitosan loaded to Gauze).
The 3 surgical adhesive products selected in the comparative example are surgical adhesives which are widely applied in the prior art and have better performance in the prior similar products; the 4 emergency hemostatic products selected are the main representatives for major bleeding in daily accidents, war and natural disasters. Whether existing surgical adhesive products or emergency hemostatic materials, their tissue adhesion properties and hemostatic properties are still further improved.
Comparing the 3 surgical adhesive products (absorbable, in vitro use; fibrin Glue, surgicel Fibrillar, gelatin spring) of example 1, comparative example 1 and comparative example 4, it was found that the fibrinogen patch of the present invention was significantly higher in shear strength and adhesive energy than the 3 surgical adhesive products of comparative example 1 and comparative example 4 (fig. 5 and 6). Meanwhile, the patch provided by the invention is also obviously superior to commercial Fibrin Glue (Fibrin Glue) in mechanical property, and is expected to become a novel tissue sealant.
The hemostatic function of the same/similar form (patch/sponge) of sealant was compared in a new zealand white rabbit liver hemostasis experiment (surgical wound model). The fibrinogen patch of the present invention (example 1) exhibited a shorter hemostatic time and less blood loss compared to the surgical adhesive products of comparative example 1 (unmodified fibrinogen patch) and comparative example 4 (Surgicel Fibrillar, gelatin Sponge) because the fibrinogen patch of the present invention has excellent mechanical strength and adhesive property, and exhibited excellent hemostatic performance (fig. 7).
The 4 emergency hemostatic products (non-absorbable, in vitro use; combat Gauze, hemCon, celox, chitoSAM) of example 1 and comparative example 4 were compared. These 4 kinds of urgent hemostatic products are all recommended for hemostasis in the case of massive hemorrhage, and the principle mainly depends on the procoagulant property of the material itself, but the hemostatic efficiency needs to be further improved. The fibrinogen patch provided by the invention has excellent mechanical strength and adhesion performance, and can have good adhesion effect on bleeding injury parts, so that the hemostatic performance is obviously superior to that of urgent hemostatic products Combat Gauze, hemCon, celox and ChitoSAM (figure 8).
In conclusion, the fibrinogen patch provided by the invention is obviously superior to surgical adhesive products and emergency hemostatic products in the prior art in both adhesion performance and hemostatic performance. Meanwhile, the patch obtained by the unexpected invention has good biocompatibility and can be absorbed by human bodies. The patch provided by the invention has excellent performance and safety, and has great potential in clinical application.
Claims (10)
1. A fibrinogen-based patch, characterized in that the patch comprises fibrinogen and a stabilizer, a portion of the lysine residues of the fibrinogen containing hydrophobic groups; the stabilizer is used for maintaining the activity of fibrinogen.
2. The fibrinogen-based patch of claim 1, wherein the hydrophobic group is a hydrocarbon group comprising one or more of carbonyl, carbon-carbon double bond, ester group, amide bond, phenyl group.
3. The fibrinogen-based patch of claim 1, wherein the hydrophobic group forms an amide bond (-NH-CO-) with a primary amine of a fibrinogen lysine residue.
4. The fibrinogen-based patch of claim 1, wherein the fibrinogen contains 2% -70% of lysine residues with hydrophobic groups.
5. The fibrinogen-based patch of claim 1, wherein the patch contains no less than 2mg of fibrinogen containing hydrophobic groups per square centimeter.
6. The fibrinogen-based patch of claim 1, wherein the stabilizing agent is one or more of sodium chloride, calcium chloride, potassium chloride, magnesium chloride, sodium citrate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, arginine hydrochloride, glycine.
7. The fibrinogen-based patch of claim 1, wherein the patch comprises a pharmaceutically active ingredient.
8. The method for preparing the fibrinogen-based patch of claim 1, comprising the specific steps of:
(1) carrying out ultrasonic treatment on the mixed solution of fibrinogen and a stabilizer, wherein the mass fraction of the obtained fibrinogen solution is 5-200 mg/mL;
(2) dropwise adding a hydrophobic modification reagent into the fibrinogen solution under the stirring condition, and reacting for 0.5-5 h at 37 ℃ to obtain the fibrinogen solution containing hydrophobic groups;
(3) dialyzing the obtained fibrinogen solution containing the hydrophobic group for 5-72 h at the temperature of 4-37 ℃;
(4) and freeze-drying the obtained fibrinogen solution containing the hydrophobic groups to obtain the fibrinogen-based patch.
9. The method of claim 8, wherein the hydrophobic modification reagent comprises a hydrophobic group, the hydrophobic group being used to modify a fibrinogen lysine residue; the hydrophobic modification reagent also comprises a succinimide group, and the succinimide group is used for reacting with primary amine of lysine residue to form stable amide bond.
10. Use of a fibrinogen-based patch according to claim 1 for haemostasis or tissue adhesion.
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| CN202310515045.1A CN116531551A (en) | 2023-05-09 | 2023-05-09 | Fibrinogen-based patch and preparation method and application thereof |
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