CN117946413A - Fibrin gel independent of thrombin and inspired by mussel protein, and preparation method and application thereof - Google Patents
Fibrin gel independent of thrombin and inspired by mussel protein, and preparation method and application thereof Download PDFInfo
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- 230000037314 wound repair Effects 0.000 description 1
Abstract
The invention provides a fibrin gel which is independent of thrombin and inspired by mussel protein, the fibrin gel is formed by crosslinking fibrinogen and a 'knot' mimic peptide-catechol-grafted compound through 'hole-knot interaction', and the catechol structure can enhance the tissue adhesive strength of the fibrin gel. The invention also provides a method for preparing the 'knot' mimic peptide-catechol-grafted compound, a raw material composition of fibrin gel, a preparation method and a kit, and application of the fibrin gel and the kit in preparing in-situ quick-setting hemostatic materials. The fibrin gel can be used for quickly stopping bleeding, is quickly gelled, has strong wet adhesion, and can be widely applied to stopping bleeding of accidental wounds or surgical wounds.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a strong-adhesion fibrin gel adhesive for accidental wounds and surgical bleeding, and a preparation method and application thereof.
Background
Uncontrolled bleeding, which occurs after trauma or during surgery, is a major cause of death worldwide. Uncontrolled bleeding often leads to poor results and increased mortality. Controlling bleeding volume is an important measure to reduce perioperative complications and mortality and improve patient prognosis.
Tissue adhesives have been developed as part of surgical instruments and are widely used as sealants and hemostatic agents to aid in controlling bleeding during surgical procedures or wounds. Fibrin glues and cyanoacrylates are two very representative types of tissue adhesives that are used clinically. Fibrin glue is concentrated fibrinogen, fibrin crosslinking is carried out under the catalysis of thrombin, fibrin clots are formed in situ at bleeding parts, bleeding is blocked, and the fibrin glue has better biocompatibility and biodegradability. However, fibrin glue has a low adhesive strength, and the adhesive strength on wet tissues is further limited, so that the fibrin glue is easily washed away by blood flow under the influence of continuous tissue tension and blood, and the hemostatic performance of the fibrin glue is not exerted. In addition, when fibrin glue is used, thrombin solution can enter the blood circulation through the broken vascular gap with a high risk of thrombus formation. Cyanoacrylate adhesives provide greater tissue adhesion, but such adhesives suffer from severe tissue irritation and inflammation due to the release of heat and degradation to toxic substances (e.g., cyanoacetate, formaldehyde, etc.) through exothermic reactions, and are difficult to remove, which limits their use in wound repair. Therefore, development of a tissue adhesive having excellent wet adhesion and biocompatibility is urgently required.
Fibrin glue is a widely used hemostatic agent in surgery to form fibrin clots at the bleeding site by mimicking the coagulation cascade. In studies of the fibrin polymerization process, it was found that thrombin triggers the release of the fibrinopeptides from the central region of fibrinogen, thereby exposing the functional amino acid sequence, which is the binding site, known as the fibrinogen "knot". The "knot" carries a positive charge, whereas the "pore" structure at both ends of fibrinogen has a strong negative charge, and the "pore" and "knot" specifically bind through strong electrostatic and hydrogen bonding, which specific binding is known as "pore-knot interactions", which are key steps in initiating fibrin polymerization, and further cross-link to form fibrin clots. The study of the "pore-junction interactions" critical to self-assembled cross-linking of fibrin found that the simulated short peptide sequences of fibrinogen "junctions" also specifically bound to the "pores" of fibrinogen, by which the study initiated fibrin polymerization (I.Litvinov,O.V.Gorkun,S.F.Owen,H.Shuman,and J.W.Weisel.Polymerization of fibrin:specificity,strength,and stability of knob-hole interactions studied at the single-molecule level.Blood.2005.106(9)). prior art has devised an engineered fibrin matrix with which therapeutic proteins were polymerized in a fibrin network structure without reliance on thrombin, through this delivery platform of fibrin, achieving a sustained infusion therapeutic function of therapeutic proteins (A.S.Soon, S.E.Stabenfeldt, W.E.Brown, and t.h. barker. Biomaterials.2010.31 (7)). Furthermore, there is a study using "junction" mimetic peptides to design peptide-polyethylene glycol polymers by "pore-junction interactions", incorporating polyethylene glycol (A.S.Soon,C.S.Lee,and T.H.Barker.Modulation of fibrin matrix properties via knob:hole affinity interactions using peptide-PEG conjugates.Biomaterials.2011.32(19)). in fibrin network structure without relying on thrombin this study suggests the possibility that fibrin-polyethylene glycol hybrid hydrogels having utility in tissue engineering can be produced by designing mimetic peptide-polyethylene glycol polymers, but this study does not mention the application of fibrin-polyethylene glycol hybrid hydrogels to the hemostatic field. Huang et al constructed fibrinogen/hyaluronic acid hydrogel for 3D cell engineering in 2017, completed self-assembly crosslinking based on the "pore-junction interaction" between fibrinogen and "junction" mimetic peptide grafted hyaluronic acid, and formed hydrogel (S.Huang,C.Wang,J.Xu,L.Ma,and C.Gao.In situ assembly of fibrinogen/hyaluronic acid hydrogel via knob-hole interaction for 3D cellular engineering.Bioact Mater.2017.2(4)). in situ. However, the hydrogel constructed in this study has weak mechanical properties, high swelling rate, and no adhesive strength, and as such, the study is not mentioned as being applied to the hemostatic field.
The strong adhesion between the tissue adhesive and the tissue interface is important to control bleeding, however the adhesive's adhesive behavior is often impaired by liquids at the tissue surface, reducing interface interactions. Development of adhesives with strong adhesion in humid environments presents challenges. Inspired by catecholamines rich in mussel mucins, catechol-based functionalized adhesive hydrogels developed in recent years have been used in many biomedical fields, such as tissue repair and regeneration, antibacterial applications and drug delivery (W.Zhang,R.Wang,Z.Sun,et al.Catechol-functionalized hydrogels:biomimetic design,adhesion mechanism,and biomedical applications.Chem Soc Rev.2020.49(2)). catechol structures play a key role in wet adhesion, the benzene ring of the catechol group bearing pi bonds, the phenolic hydroxyl group bearing lone pair electrons and hydrogen atoms, making it possible to create covalent bonds (schiff base and michael addition reactions with sulfhydryl and amino groups on tissues) and/or noncovalent bonds (hydrogen bonds, metal complexation, pi-pi and/or cation-pi interactions) with different substrates. The covalent and non-covalent interactions of catechol structures with tissues provide mussels with strong adhesion and can adhere to virtually any substrate. Therefore, the introduction of catechol structures into compounds has become a fundamental idea to improve adhesive strength of adhesives.
The ideal hemostatic material should not depend on the body coagulation mechanism, can play a hemostatic role even when the body is in coagulation disorder, and should not depend on thrombin at the same time, so as to reduce the risk of thrombus, and should have storage stability, guarantee to have excellent hemostatic effect after long-time storage; in addition, it should combine the desired biocompatibility, rapid gelation and better moist tissue adhesion. Therefore, the invention is particularly important to provide a novel hemostatic material which can solve the problems of poor wet tissue adhesion and limited hemostatic effect existing in the existing hemostatic materials.
Disclosure of Invention
To overcome the above-mentioned drawbacks of the prior art, a primary object of the present invention is: a functional cross-linking agent with strong wet adhesion is provided which can cross-link with fibrinogen to form a solid hydrogel and which is capable of providing strong adhesion to any moist tissue substrate.
Another object of the invention is: an adhesive is provided which rapidly stanches, rapidly gels, has strong wet adhesion, and is intended for use in trauma or surgical wounds.
Another object of the invention is: methods of making the adhesive are provided.
Still another object of the present invention is: methods of hemostasis using the adhesives are provided.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides a mussel protein-inspired functional cross-linking agent, which is composed of a high molecular compound skeleton grafted with catechol structure and a 'knot' mimic peptide. This invention refers to the term `node` mimetic peptide-catechol-grafted compound.
In the functional crosslinking agent, the 'knot' mimic peptide and catechol are grafted on the same high molecular compound framework; the 'knot' mimic peptide is used as a cross-linking point and is used for cross-linking with fibrinogen to form solid hydrogel; after forming the solid hydrogel, the catechol structure serves to increase the tissue adhesion strength of the solid hydrogel. In the prior art, the catechol-grafted compound obtained by only grafting catechol on a high molecular compound skeleton cannot be crosslinked with fibrinogen to form solid hydrogel, and the tissue adhesive strength cannot be improved. The 'knot' mimic peptide-catechol-grafted compound of the invention can participate in fibrinogen crosslinking to form solid hydrogel and can improve tissue adhesion strength.
In the functional crosslinking agent, the macromolecular compound skeleton is from a macromolecular compound with a carboxyl and carbon-carbon double bond structure; the preferable high molecular compound has better biocompatibility and certain strength; the most preferred polymer backbone is from either of a methacryloylated gelatin or a carboxylic acid functionalized polyethylene glycol acrylate.
In the functional crosslinking agent, the molecular weight of the macromolecular compound skeleton is 100-600 kDa; preferably 150-400 kDa; the molecular weight of the preferred high molecular weight compound backbone is similar to the molecular weight of fibrinogen (340 kDa), facilitating crosslinking of the functional crosslinking agent and fibrinogen by "pore-junction interactions".
In the functional crosslinking agent, the 'knot' mimic peptide is a short peptide sequence capable of self-assembling with fibrinogen based on 'hole-knot interaction', and is preferably any one of GPRPFPAC or GPRPAAC.
In the functional crosslinking agent, the 'knot' mimic peptide is used for self-assembly with fibrinogen based on 'hole-knot interaction', the catechol structure is used for providing wet adhesion performance, and the macromolecular compound skeleton is used for providing a group or bond capable of connecting the 'knot' mimic peptide and catechol or derivatives thereof. Therefore, the functional crosslinking agent has the property of combining with fibrinogen and has wet tissue adhesiveness, and is very suitable for preparing fibrin gel adhesives. The fibrin gel prepared by the functional crosslinking agent has catechol structure in the structure, and can perform covalent and/or non-covalent interaction with functional groups on tissues, so that the wet adhesive strength of the fibrin gel can be improved.
The functional cross-linking agent can be loaded on a solid hemostatic material; the solid hemostatic material can be in the form of any one of gauze or sponge.
In the functional cross-linking agent, the 'knot' mimic peptide can self-assemble and cross-link with fibrinogen in blood based on 'hole-knot interaction', and a blood clot-like blocking is formed at a wound; the catechol structure and the functional groups on the tissues have covalent and/or non-covalent interactions, so that the wet adhesion performance is improved. Therefore, the functional crosslinking agent is very suitable for being loaded on the solid hemostatic material, so that the functional crosslinking agent has the property of combining with fibrinogen in blood and has wet tissue adhesiveness.
According to the invention, the influence of the catechol content in the functional crosslinking agent on the adhesive strength of the crosslinking agent wet tissue is further observed through experiments, and the fact that the catechol content in the 'knot' mimic peptide-catechol-grafted compound can bring about more ideal adhesive strength of the wet tissue within the range of 10 mu mol/g-100 mu mol/g is found; wherein the catechol content is 50 mu mol/g-100 mu mol/g, the effect is better; the functional crosslinking agent has a wet tissue adhesion strength which is at a further catechol content of 80. Mu. Mol/g to 100. Mu. Mol/g, in particular 100. Mu. Mol/g.
In the preferred functional crosslinking agent, the content of the 'knot' mimic peptide in the 'knot' mimic peptide-catechol-grafted compound is 588 mu mol/g-0.59 mu mol/g; preferably 29.4. Mu. Mol/g to 1.12. Mu. Mol/g; more preferably 11.76. Mu. Mol/g to 2.84. Mu. Mol/g; most preferably 8.82. Mu. Mol/g.
The functional crosslinking agent disclosed by the invention can be prepared by the following method:
1) Taking an amino or alkylamino substituted catechol derivative and a high molecular compound with a carboxyl and carbon-carbon double bond structure in the structure as raw materials, and carrying out condensation reaction on the amino or alkylamino and the carboxyl to obtain a catechol-grafted compound; in this step, in order to avoid and reduce oxidation of catechol as much as possible, it is preferable that the reaction is carried out in an acidic liquid atmosphere having a pH of 5 and in a nitrogen atmosphere;
2) Reacting the catechol-grafted compound obtained in 1) with a 'knot' mimic peptide in a nitrogen atmosphere, namely grafting the 'knot' mimic peptide through a carbon-carbon double bond on the catechol-grafted compound to obtain the 'knot' mimic peptide-catechol-grafted compound, and acidifying the 'knot' mimic peptide-catechol-grafted compound to a pH value of 5-6. The "knob" mimetic peptide is a short peptide sequence capable of self-assembly with fibrinogen based on "pore-knob interactions", preferably either one of GPRPFPAC or GPRPAAC.
In the preparation method of the functional cross-linking agent, the grafting sequence is critical to successful synthesis of the functional cross-linking agent: if the 'knot' mimic peptide is grafted on the high molecular compound with the carboxyl and carbon-carbon double bond structure, and then catechol is grafted, the 'knot' mimic peptide can react with the carboxyl in the high molecular compound to generate condensation reaction of amino and carboxyl besides reacting with the carbon-carbon double bond, so that the carboxyl on the high molecular compound is occupied, the reaction of catechol and the carboxyl on the high molecular compound is reduced, and the grafting of catechol is not facilitated. Therefore, the synthesis sequence of the catechol grafted first and then the 'knot' mimic peptide is grafted in the invention, so that the successful grafting of the catechol and the binding activity of the 'knot' mimic peptide are ensured.
In a second aspect, the present invention provides a fibrin gel, a fibrin adhesive, which is a solid hydrogel of fibrin inspired by mussel proteins and which is obtained by crosslinking a "desmodromic" mimetic peptide-catechol-grafted compound with fibrinogen.
The fibrin gel is a solid hydrogel which completes fibrin crosslinking through 'hole-knot interaction' and has strong wet adhesion function. The completion of fibrin cross-linking by "pore-junction interaction" refers to the process of forming a fibrin solid hydrogel by cross-linking fibrinogen through "pore-junction interaction" by the active amino acid sequences at both ends of fibrinogen and the "junction" mimetic peptides on the "junction" mimetic peptide-catechol-grafted compound without depending on thrombin; meanwhile, the hydrogen bonding effect of the catechol structure on the peptide-catechol-grafted compound and amino, hydroxyl and other groups on the surface of fibrinogen also participates in the formation of solid hydrogel; the strong wet adhesion is achieved by covalent and/or non-covalent interactions of catechol structures in gel structures with functional groups on tissue.
The "knot" mimetic peptide-catechol-grafted compound refers to the functional crosslinking agent of the first aspect of the invention, and is formed by grafting catechol structure and "knot" mimetic peptide on a high molecular compound framework. Wherein the definition and description of the "junction" mimetic peptide and the polymer skeleton are the same as those of the first aspect of the present invention.
The "pore-junction interaction" refers to that in the process of crosslinking fibrin, the active amino acid sequences at two ends of fibrinogen can be called as "pores", the amino acid sequence with binding activity exposed by fibrin after thrombin catalysis is called as "junction", and the "junction" and the "pores" can be combined together through strong non-covalent interactions (electrostatic and hydrogen bonding) to complete the crosslinking of fibrin. The "knob" mimetic short peptide sequences of the prior art also bind specifically to fibrinogen; the "knot" mimetic peptide-grafted compounds, further constructed based on the "knot" mimetic short peptide sequences, can also bind to fibrinogen via "pore-knot interactions" to complete fibrin cross-linking.
The fibrin gel is prepared from a raw material A and a raw material B; the raw material A is fibrinogen, and the raw material B is a 'knot' mimic peptide-catechol-grafted compound. When the raw materials are used for preparing the gel, the raw materials A and B are mixed, and fibrin crosslinking is rapidly completed by 'hole-knot interaction' during mixing, and meanwhile, catechol structures in a gel structure can interact with tissues, so that wet adhesion strength is improved. The fibrinogen may be any one selected from human fibrinogen, bovine fibrinogen or porcine fibrinogen.
The gel time of the fibrin gel of the invention is related to the molar ratio of the "pore" to "knot" mimetic peptide, and as the molar ratio of the "pore" to "knot" increases, the gel time of the fibrin gel shortens, whereas above a certain molar ratio, the gel time of the fibrin gel increases. The wet adhesion strength of the fibrin gel is related to the catechol content of the "knob" mimetic peptide-catechol-grafted compound, and as the catechol content increases, the wet tissue adhesion strength of the fibrin gel also increases. In view of the influence of the molar ratio of the 'holes' to the 'knots' in the raw materials and the catechol content on the overall performance of the gel, the invention further optimizes the molar ratio of the 'holes' to the 'knots' in the raw materials through experiments, wherein the molar ratio of the 'holes' to the 'knots' in the raw materials is 1:10-10:1; preferably 1:5 to 5:1; more preferably 1:2 to 2:1; most preferably 1:1.5. At these preferred "pore" to "knot" molar ratios, fibrinogen cross-linking is rapidly accomplished, forming a solid fibrin gel at a minimum gel time. The invention further optimizes the content of catechol structure through experiments, and discovers that the ideal wet tissue adhesive strength can be brought to gel when the 'knot' mimic peptide-catechol-grafted compound with the catechol content within the range of 10 mu mol/g-100 mu mol/g is used as a raw material; wherein the catechol content is 50 mu mol/g-100 mu mol/g, the effect is better; when the catechol content is further 80 to 100. Mu. Mol/g, especially 100. Mu. Mol/g, the wet tissue adhesion strength of the crosslinked fibrin gel is maximized. In summary, these preferred "pore" to "knot" molar ratios and catechol levels can provide superior hemostatic properties to the fibrin gel of the invention as a whole, especially when the "pore" to "knot" molar ratio in raw material A, B is 1:1.5 and the catechol content in raw material B is 100 μmol/g, the hemostatic properties of the crosslinked gel can be optimized, and the rapidly formed gel can have both enhanced gel strength and strong wet tissue adhesion.
In a third aspect, the present invention also provides a feedstock composition for preparing the fibrin gel of the second aspect of the invention, comprising feedstock a and feedstock B; the raw material A is fibrinogen, and the raw material B is a 'knot' mimic peptide-catechol-grafted compound; the molar ratio of the 'holes' in the raw material A to the 'knots' mimic peptide in the raw material B is 1:10-10:1; preferably 1:5 to 5:1; more preferably 1:2 to 2:1; most preferably 1:1.5.
The "knot" mimetic peptide-catechol-grafted compound refers to the functional crosslinking agent of the first aspect of the invention, and is formed by grafting catechol structure and "knot" mimetic peptide on a high molecular compound framework. Wherein the definition and description of the "junction" mimetic peptide and the polymer skeleton are the same as those of the first aspect of the present invention.
The invention finds through experiments that when the raw material A and the raw material B are both solutions or prepared into solutions, the concentration of the raw material composition is related to the strength of the fibrin gel obtained by crosslinking: when the concentrations of the raw material A and the raw material B are both in the range of 5% (w/v) to 20% (w/v), the strength of the fibrin gel increases and the wound blocking effect increases as the concentrations of the raw material A and the raw material B increase.
In the preferred feedstock composition of the present invention, both feedstock A and feedstock B are in the form of solutions; the concentration of fibrinogen in the raw material A is 5% (w/v) to 20% (w/v); preferably 10% (w/v) to 20% (w/v); more preferably 15% (w/v) to 20% (w/v); most preferably 20% (w/v). The concentration of the 'knot' mimic peptide-catechol-grafted compound in the raw material B is 5% (w/v) to 20% (w/v); preferably 10% (w/v) to 20% (w/v); more preferably 15% (w/v) to 20% (w/v); most preferably 20% (w/v).
The invention finds through experiments that in the raw material composition, the molar ratio of the 'holes' in the raw material A to the 'knots' mimic peptides in the raw material B is related to the gel time of the fibrin gel: when the molar ratio of "pores" to "junctions" is in the range of 1:10 to 1:1.5, the gelation time shortens with increasing molar ratio; and when the molar ratio of "pore" to "junction" is in the range of 1:1.5 to 10:1, the gelation time is prolonged with the increase of the molar ratio. This means that the shortest gel time is achieved with a 1:1.5 molar ratio of "pores" to "knots" which serves to rapidly seal the bleeding wound. Meanwhile, the gel time is shorter when the molar ratio of the pore to the junction is 1:10-10:1; gel time can be further shortened when the molar ratio of "pores" to "junctions" is 1:5 to 5:1; gel times are minimized when the molar ratio of "pores" to "junctions" is 1:2 to 2:1, especially 1:1.5.
Experiments show that in the raw material composition, the catechol content in the raw material B is related to the wet tissue adhesion strength of the fibrin gel: when the catechol content in the raw material B is in the range of 10 mu mol/g to 100 mu mol/g, the wet tissue adhesion strength is enhanced against the increase of the catechol content; when the catechol content is 100 mu mol/g, the strongest wet tissue adhesion strength can be obtained, the fibrin clot is protected from being washed away by blood flow, and the effect of sealing the tissue wound is improved. Therefore, the catechol content in the raw material B is 10 mu mol/g-100 mu mol/g; preferably 50. Mu. Mol/g to 100. Mu. Mol/g; more preferably 80. Mu. Mol/g to 100. Mu. Mol/g; most preferably 100. Mu. Mol/g.
In the raw material composition, the content of the 'knot' mimic peptide in the raw material B in the 'knot' mimic peptide-catechol-grafted compound is 588 mu mol/g-0.59 mu mol/g; preferably 29.4. Mu. Mol/g to 1.12. Mu. Mol/g; more preferably 11.76. Mu. Mol/g to 2.84. Mu. Mol/g; most preferably 8.82. Mu. Mol/g.
In the raw material composition of the present invention, the fibrinogen may be any one selected from human fibrinogen, bovine fibrinogen and porcine fibrinogen.
The raw material composition can be in various specific forms which are pharmaceutically or clinically acceptable, for example, freeze-dried powder, injection, sponge or granule.
In a fourth aspect, the present invention also provides a kit for preparing a fibrin gel according to the second aspect of the invention, comprising a first precursor reagent and a second precursor reagent packaged separately from each other; the first precursor reagent comprises fibrinogen and the second precursor reagent comprises a "knob" mimetic peptide-catechol-grafting compound; the mass ratio of fibrinogen in the first precursor reagent to the conjugated' mimetic peptide-catechol-grafted compound in the second precursor reagent is 1:4.4-22.7:1; preferably 1:2.2 to 11.4:1; more preferably 1:0.9 to 4.5:1; most preferably 1.5:1.
In the kit of the present invention, the fibrinogen contained in the first precursor reagent may be any one selected from human fibrinogen, bovine fibrinogen and porcine fibrinogen.
The "knot" mimetic peptide-catechol-grafted compound refers to the functional crosslinking agent of the first aspect of the invention, and is formed by grafting catechol structure and "knot" mimetic peptide on a high molecular compound framework. Wherein the definition and description of the "junction" mimetic peptide and the macromolecular compound skeleton are the same as those of the first aspect of the invention.
In a preferred kit of the invention, the first precursor reagent and/or the second precursor reagent further comprise auxiliary materials and/or additives. The auxiliary materials are selected from one or more of glycine, arginine hydrochloride, sodium citrate, sucrose and sodium chloride. The additive is one or more than two selected from growth factors, interleukins, vitamins and silver ions; the growth factor can be further selected from one or more of platelet growth factor, epidermal growth factor or fibroblast growth factor; the interleukin may be further selected from one or more of interleukin 2, interleukin 6 or interleukin 8; the vitamin may be further selected from one or more of vitamin B, vitamin C, vitamin E or vitamin K.
In the kit of the invention, the first precursor reagent and/or the second precursor reagent can be various pharmaceutically or clinically acceptable specific dosage forms, such as freeze-dried powder, sponge or particles.
The kit can further comprise an independently packaged solvent for configuration, wherein the solvent for configuration can be any one or a mixture of a plurality of phosphate buffer salt solution, HEPES biological buffer solution, 0.9% sodium chloride solution, calcium chloride solution and deionized water. The formulation of the solvent for formulation is preferably an injection.
In a fifth aspect, the present invention also provides a method of preparing a fibrin gel according to the second aspect of the invention, comprising:
1) Preparing a first precursor solution in which fibrinogen is dissolved in a solvent, and controlling the concentration of the first precursor solution to be 5% (w/v) to 20% (w/v);
2) ① preparing a 'knot' mimic peptide-catechol-grafted compound, carrying out condensation reaction of amino groups and carboxyl groups on dopamine hydrochloride and a high molecular compound with a carboxyl and carbon-carbon double bond structure in the structure, and then carrying out graft polymerization reaction on the dopamine hydrochloride and a 'knot' mimic short peptide sequence to obtain the 'knot' mimic peptide-catechol-grafted compound; controlling the catechol content in the 'knot' mimic peptide-catechol-grafted compound to be 10 mu mol/g-100 mu mol/g; controlling the content of the 'knot' mimic peptide in the 'knot' mimic peptide-catechol-grafted compound to be 588 mu mol/g-0.59 mu mol/g;
② Dissolving the 'knot' mimic peptide-catechol-grafted compound prepared in ① to obtain a second precursor solution, and controlling the concentration of the second precursor solution to be 5-20% (w/v);
3) Mixing the first precursor solution obtained in the step 1) with the second precursor solution obtained in the step 2), and controlling the molar ratio of the 'holes' in the first precursor solution to the 'knot' mimic peptide in the second precursor solution to be 1:10-10:1, so as to obtain the fibrin gel.
In a preferred preparation method of the present invention, the concentration of fibrinogen contained in the first precursor solution in step 1) is 10% (w/v) to 20% (w/v); more preferably 15% (w/v) to 20% (w/v); most preferably 20% (w/v).
In a preferred preparation method of the invention, in ① of the step 2), the catechol content in the "knob" mimetic peptide-catechol-grafted compound is controlled to be 50 mu mol/g to 100 mu mol/g; more preferably 80. Mu. Mol/g to 100. Mu. Mol/g; most preferably 100. Mu. Mol/g; controlling the content of the 'knot' mimic peptide in the 'knot' mimic peptide-catechol-grafted compound to be 29.4 mu mol/g-1.12 mu mol/g; preferably 11.76. Mu. Mol/g to 2.84. Mu. Mol/g; most preferably 8.82. Mu. Mol/g.
In a preferred method of preparation of the invention, the second precursor solution of step 2) contains a concentration of "knob" mimetic peptide-catechol-grafting compound of 10% (w/v) to 20% (w/v); more preferably 15% (w/v) to 20% (w/v); most preferably 20% (w/v).
In a preferred preparation method of the invention, in step 3), the molar ratio of the "pore" in the first precursor solution to the "junction" mimetic peptide in the second precursor solution is controlled to be 1:5-5:1; more preferably 1:2 to 2:1; most preferably 1:1.5.
In a sixth aspect, the invention also provides a method of preparing a kit according to the fourth aspect of the invention, comprising: adding auxiliary materials and/or additives into the first precursor solution and the second precursor solution prepared in the fifth aspect respectively, and freeze-drying to obtain a first precursor reagent and a second precursor reagent; packaging the first precursor reagent and the second precursor reagent independently, and controlling the mass ratio of fibrinogen in the first precursor reagent to the "knot" mimic peptide-catechol-grafted compound in the second precursor reagent to be 1:4.4-22.7:1, preferably 1.5:1; packaging the prepared solvent independently; finally, the first precursor reagent, the second precursor reagent and the configuration solvent which are packaged independently are combined and packaged to obtain the kit according to the fourth aspect of the invention.
In the preparation method of the kit, the first precursor reagent and/or the second precursor reagent are freeze-dried agents, sponges or particles; the preparation solvent is injection solvent.
In the preparation method of the kit, the definition and the preparation method of the first precursor solution and the second precursor solution are the same as those of the fifth aspect of the invention; the definition and preparation of the first precursor reagent and the second precursor reagent are the same as in the fourth aspect of the present invention. The definition and selection of the auxiliary materials, the additive and the solvent for preparation are the same as those of the fourth aspect of the invention.
In a seventh aspect, the invention also provides application of the raw material composition or the kit in preparing an in-situ rapid-setting hemostatic material.
The application of the raw material composition comprises the following steps: the raw materials A and B are respectively prepared into injectable solution, and then are simultaneously and evenly injected or sprayed on the bleeding wound site, so that solid hydrogel can be quickly formed at the bleeding wound site.
The application of the kit comprises: the first precursor reagent and the second precursor reagent are respectively prepared into injectable solution by using a configuration solvent, and then are simultaneously and uniformly injected or sprayed on a bleeding wound site, so that solid hydrogel can be quickly formed in situ on the bleeding wound site.
The bleeding wound comprises organ bleeding caused by accidental wounds or occurring in surgery; the organ may be liver, spleen, kidney, stomach, heart or skin.
In the application of the invention, when the raw materials A and B are co-injected into a bleeding wound, (1) fibrin solid clots can be formed on the surface of the wound instantly (1 s-2 s) to seal the wound; (2) The high-concentration raw material enhances the strength of the fibrin gel, can resist the blood pressure, keeps the integrity of the gel and improves the effect of plugging wounds; (3) Meanwhile, catechol in fibrin gel and tissues form covalent and/or non-covalent interactions, so that the adhesive strength of the tissues is improved, the impact of blood can be resisted, the fibrin gel is protected from being washed away by blood flow, and the effect of plugging wounds is further enhanced.
Aiming at the defects of weak adhesion of fibrin glue, thrombus risk generated by depending on thrombin and the like in the background technology, the invention designs a 'knot' mimic peptide-catechol-grafted compound through an innovative technology, and under the condition of not depending on thrombin, the fibrin glue can be rapidly crosslinked with 'holes' of fibrinogen through 'hole-knot interaction', so as to form solid fibrin gel; meanwhile, catechol in gel and a tissue interface are subjected to covalent and/or non-covalent interaction, so that the adhesive strength of fibrin glue is improved.
Compared with the prior art, the invention has the advantages that: rapid gelation, high gel strength, strong adhesion to wet tissues and good hemostatic effect:
(1) The functional crosslinking agent can crosslink with fibrinogen to form fibrin solid hydrogel, and meanwhile, the catechol structure in the functional crosslinking agent can enhance the tissue adhesive strength of the solid fibrin hydrogel.
(2) The fibrin gel independent of thrombin and inspired by mussel protein can be immediately crosslinked with fibrin (1 s-2 s) through 'hole-junction interaction', so as to form solid fibrin gel and seal wounds.
(3) The fibrin gel which is independent of thrombin and inspired by mussel protein is formed by crosslinking raw materials with high concentration, so that the strength of the fibrin gel is improved, the blood pressure can be resisted, the integrity of the gel is maintained, and the effect of plugging wounds is enhanced.
(4) The catechol in the fibrin gel which is independent of thrombin and inspired by mussel protein can perform covalent and/or non-covalent interactions with tissues, so that the adhesive strength of the tissues is improved, the blood impact can be resisted, the fibrin gel is protected from being washed away by blood flow, and the effect of plugging wounds is further enhanced.
(5) The fibrin gel which is independent of thrombin and inspired by mussel protein is independent of thrombin, reduces the risk of thrombosis and has long-term storage stability.
The fibrin gel which is independent of thrombin and inspired by mussel protein has the advantages of rapid gelation, high gel strength, strong wet tissue adhesion and rapid hemostatic effect, so that the fibrin gel can be used for hemostasis application of liver, spleen, kidney, heart, stomach and skin hemorrhage in accidental wounds or operations.
Drawings
Fig. 1 is an SEM image of fibrin cross-links of comparative example 1.
FIG. 2 is an SEM image of a thrombin-independent and mussel protein-inspired fibrin gel of example 1.
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of "knob" mimetic peptide-catechol-grafted gelatin in example 1.
Fig. 4 shows a comparison of the hemostatic times of example 1, example 9 and example 17 and comparative examples 1 to 5.
Fig. 5 shows a comparison of the blood loss amounts of example 1, example 9, and example 17 and comparative examples 1 to 5.
Detailed Description
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 invention provides fibrin gel inspired by mussel protein and thrombin-independent, which is fibrin solid hydrogel inspired by mussel protein and has a honeycomb-like network structure, wherein the network structure is formed by unordered interconnection of compact lamellar walls; the dense lamellar walls are formed by cross-linking fibrinogen with a "knob" mimetic peptide-catechol-grafting compound. The fibrin gel is formed by taking fibrinogen (two ends of which are provided with 'holes') and 'knot' mimic peptide-catechol-grafted compound as raw materials and completing fibrin crosslinking through 'hole-knot interaction' under the condition of not depending on thrombin. Catechol in can interact with tissues, improving wet adhesion strength of the gel. The mol ratio of the holes to the knots in the raw materials is 1:10-10:1; preferably 1:5 to 5:1; more preferably 1:2 to 2:1; most preferably 1:1.5. The catechol content in the 'knot' mimic peptide-catechol-grafted compound is 10 mu mol/g-100 mu mol/g; preferably 50. Mu. Mol/g to 100. Mu. Mol/g; more preferably 80. Mu. Mol/g to 100. Mu. Mol/g; most preferably 100. Mu. Mol/g.
The strong adhesion properties of the gels of the present invention are achieved by "binding" the catechol structure of the mimetic peptide-catechol-grafted compound to the tissue through covalent and/or non-covalent interactions.
The fibrin gel raw material independent of thrombin and inspired by mussel protein can be prepared according to the following method:
(1) Preparation of raw material A solution: dissolving fibrinogen to obtain a raw material A solution; the concentration of the resulting raw material A solution is controlled to be not less than 5% (w/v), preferably 10% (w/v) to 20% (w/v).
(2) Preparation of raw material B solution: the method comprises the steps of firstly carrying out condensation reaction on amino groups and carboxyl groups on a high molecular compound with a carboxyl group and a carbon-carbon double bond structure in the dopamine hydrochloride and the structure, and then carrying out graft polymerization reaction on the high molecular compound and a 'knot' simulated short peptide sequence to obtain a 'knot' simulated peptide-catechol-grafted compound, wherein the catechol content in the 'knot' simulated peptide-catechol-grafted compound is controlled to be 10 mu mol/g-100 mu mol/g, preferably 50 mu mol/g-100 mu mol/g, more preferably 80 mu mol/g-100 mu mol/g, and most preferably 100 mu mol/g; controlling the content of "binding" mimetic peptide in the "binding" mimetic peptide-catechol-grafted compound to be 588. Mu. Mol/g to 0.59. Mu. Mol/g, preferably 29.4. Mu. Mol/g to 1.12. Mu. Mol/g, more preferably 11.76. Mu. Mol/g to 2.84. Mu. Mol/g, and most preferably 8.82. Mu. Mol/g; the "knob" mimetic peptide-catechol-graft compound is dissolved to obtain a raw material B solution, and the concentration of the obtained raw material B solution is controlled to be 5% (w/v) to 20% (w/v), preferably 10% (w/v) to 20% (w/v), more preferably 15% (w/v) to 20% (w/v), and most preferably 20% (w/v).
(3) The storage method comprises the following steps: and (3) respectively freeze-drying the raw material solution A obtained in the step (1) and the raw material solution B obtained in the step (2) according to the volume ratio of 1:10-10:1 to obtain sponges, and storing.
(4) Preparing fibrin gel by using the sponge obtained by freeze-drying in the step (3): and respectively dissolving the spongy raw material A and the spongy raw material B in a solvent to obtain an injectable raw material A solution and an injectable raw material B solution. The equal volumes of the raw material A solution and the raw material B solution are injected or sprayed on the bleeding part at the same time, so that solid hydrogel can be quickly formed at the bleeding part in situ. Preferably, the injection tool of the raw material solution A and the raw material solution B can be a double syringe, a syringe and a Pasteur pipette when in use.
In the above preparation scheme, the solvent in the step (4) may be any one or a combination of several of phosphate buffer solution, HEPES biological buffer solution, 0.9% sodium chloride solution, calcium chloride solution and deionized water, and the use amount thereof is not particularly limited, and may be formulated according to the actual required concentration.
The present invention is further illustrated by the following examples based on the above embodiments.
Example 1
The preparation method of the fibrin gel independent of thrombin and inspired by mussel protein comprises the following specific raw materials and steps:
(1) Preparation of a component A solution: namely, preparation of fibrinogen solution: 1g of fibrinogen is slowly placed in a preheated 0.9% sodium chloride solution, and after complete dissolution, a component A solution with a mass-volume percentage (w/v) of 20% (w/v) is obtained.
(2) Preparation of the component B solution: namely preparation of "knob" mimetic peptide-catechol-grafted gelatin solution: ① Successively grafting dopamine hydrochloride and a 'knot' mimic short peptide sequence GPRPFPAC (from Genscript company) on the methacryloylated gelatin, and controlling the catechol content in the grafted compound to be 100 mu mol/g and the content of the 'knot' mimic peptide to be 8.82 mu mol/g to obtain the 'knot' mimic peptide-catechol-grafted gelatin; ② The "knob" mimetic peptide-catechol-grafted gelatin was completely dissolved in a pre-heated 0.9% sodium chloride solution to give a 20% (w/v) mass-to-volume percent B-component solution. In the "knot" mimic peptide-catechol-grafted gelatin, the nuclear magnetic characterization of successful grafting of the "knot" mimic peptide and catechol on gelatin is shown in fig. 3: the peak at 6.8ppm of the "knob" mimetic peptide-catechol-grafted gelatin, compared to the methacryloylated gelatin, shows successful grafting of the catechol structure onto the gelatin; the peak intensities (peaks for hydrogen on carbon-carbon double bonds) at 5.63ppm and 5.39ppm were significantly reduced compared to the methacrylated gelatin, showing successful grafting of the "knob" mimetic peptide onto gelatin.
(3) And (3) storing: respectively freeze-drying the obtained A component solution and B component solution according to the volume ratio of 1:1, and storing in a spongy state;
(4) The using method comprises the following steps: the spongy A and B components were dissolved in a solution containing 0.9% sodium chloride in a volume fraction ratio of 1:1, respectively, to give injectable solution of A and B components, each having a concentration of 20% (w/v). The molar ratio of "pores" in the A component to "junctions" in the B component was 1:1.5. The A component solution and the B component solution are filled into a duplex injector in equal volumes, and the A component solution and the B component solution are injected/sprayed on a bleeding part through a nozzle, so that the solid hydrogel can be in situ converted.
(5) The solid hydrogel structure is as described in fig. 2: honeycomb-like interconnect structures having dense sheet-like walls formed by self-assembled cross-linking of fibrinogen and gelatin.
Example 2
The preparation and use methods are generally the same as in example 1, except that: the fibrinogen concentration of the injectable A-component solution was 15% (w/v) and the "knob" mimetic peptide-catechol-grafted gelatin concentration of the B-component solution was 15% (w/v) by adjusting the amount of sodium chloride solution used in step (4) of example 1.
Example 3
The preparation and use methods are generally the same as in example 1, except that: the fibrinogen concentration of the injectable A-component solution was 10% (w/v) and the "knob" mimetic peptide-catechol-grafted gelatin concentration of the B-component solution was 10% (w/v) by adjusting the amount of sodium chloride solution used in step (4) of example 1.
Example 4
The preparation and use methods are generally the same as in example 1, except that: the final equivalent volume of "pore to knot" molar ratio of the A-component solution and the B-component solution loaded into the duplex syringe was 1:1 by adjusting the fibrinogen amount in step (1) of example 1 from 1g to 1.5 g.
Example 5
The preparation and use methods are generally the same as in example 1, except that: the final equivalent volume of "pore-to-knot" molar ratio of the A-component solution and the B-component solution loaded into the duplex syringe was 1:0.5 by adjusting the fibrinogen usage from 1g to 3g in step (1) of example 1.
Example 6
The preparation and use methods are generally the same as in example 1, except that: the resulting equal volumes were loaded into the "wells" of the A-and B-component solutions of the duplex syringe by adjusting the fibrinogen usage from 1g to 0.75g in step (1) of example 1: the junction "molar ratio was 1:2.
Example 7
The preparation and use methods are generally the same as in example 1, except that: the catechol content of the post-grafting compound was adjusted from 100. Mu. Mol/g in step (2) of example 1 to 80. Mu. Mol/g.
Example 8
The preparation and use methods are generally the same as in example 1, except that: the catechol content of the post-grafting compound was adjusted from 100. Mu. Mol/g in step (2) of example 1 to 50. Mu. Mol/g.
Example 9
The preparation method of the fibrin gel independent of thrombin and inspired by mussel protein comprises the following specific raw materials and steps:
(1) Preparation of a component A solution: namely, preparation of fibrinogen solution: 1g of fibrinogen is slowly placed in a preheated 0.9% sodium chloride solution, and after complete dissolution, a component A solution with a mass-volume percentage (w/v) of 20% (w/v) is obtained.
(2) Preparation of the component B solution: namely, the preparation of a solution of the 'knot' mimic peptide-catechol-grafted polyethylene glycol: ① Successively grafting dopamine hydrochloride and a 'knot' mimic short peptide sequence GPRPFPAC (from Genscript company) on carboxylated polyethylene glycol acrylate, controlling the catechol content in the grafted compound to be 100 mu mol/g and the content of the 'knot' mimic peptide GPRPFPAC to be 8.82 mu mol/g, so as to obtain 'knot' mimic peptide-catechol-grafted polyethylene glycol; ② The "knob" mimetic peptide-catechol-grafted polyethylene glycol was completely dissolved in a pre-heated 0.9% sodium chloride solution to give a 20% (w/v) mass-to-volume percent B-component solution.
(3) And (3) storing: respectively freeze-drying the obtained A component solution and B component solution according to the volume ratio of 1:1, and storing in a spongy state;
(4) The using method comprises the following steps: the spongy A and B components were dissolved in a solution containing 0.9% sodium chloride in a volume fraction ratio of 1:1, respectively, to give injectable solution of A and B components, each having a concentration of 20% (w/v). The molar ratio of "pores" in the A component to "junctions" in the B component was 1:1.5. The A component solution and the B component solution are filled into a duplex injector in equal volumes, and the A component solution and the B component solution are injected/sprayed on a bleeding part through a nozzle, so that the solid hydrogel can be in situ converted.
Example 10
The preparation and use methods are generally the same as in example 9, except that: the fibrinogen concentration of the injectable A-component solution was 15% (w/v) and the concentration of the "knob" mimetic peptide-catechol-grafted polyethylene glycol of the B-component solution was 15% (w/v) by adjusting the amount of sodium chloride solution of step (4) of example 9.
Example 11
The preparation and use methods are generally the same as in example 9, except that: the fibrinogen concentration of the injectable A-component solution was 10% (w/v) and the concentration of the "knob" mimetic peptide-catechol-grafted polyethylene glycol of the B-component solution was 10% (w/v) by adjusting the amount of the sodium chloride solution of step (4) of example 9.
Example 12
The preparation and use methods are generally the same as in example 9, except that: the resulting equal volumes were loaded into the "wells" of the A-and B-component solutions of the duplex syringe by adjusting the fibrinogen usage from 1g to 1.5g in step (1) of example 9: the junction "molar ratio was 1:1.
Example 13
The preparation and use methods are generally the same as in example 9, except that: by adjusting the amount of fibrinogen used in step (1) of example 9 from 1g to 3g, the resulting equal volumes were loaded into the "wells" of the A-component solution and B-component solution of the duplex syringe: the junction "molar ratio was 1:0.5.
Example 14
The preparation and use methods are generally the same as in example 9, except that: the resulting equal volumes were loaded into the "wells" of the A-and B-component solutions of the duplex syringe by adjusting the fibrinogen usage from 1g to 0.75g in step (1) of example 1: the junction "molar ratio was 1:2.
Example 15
The preparation and use methods are generally the same as in example 9, except that: the catechol content of the post-grafting compound was adjusted from 100. Mu. Mol/g in step (2) of example 9 to 80. Mu. Mol/g.
Example 16
The preparation and use methods are generally the same as in example 9, except that: the catechol content of the post-grafting compound was adjusted from 100. Mu. Mol/g in step (2) of example 9 to 50. Mu. Mol/g.
Example 17
The preparation method of the solid hemostatic material loaded with the functional crosslinking agent comprises the following specific raw materials and steps:
1) Preparation of "desmoid" mimetic peptide-catechol-grafted gelatin solution: ① Successively grafting dopamine hydrochloride and a 'knot' mimic short peptide sequence GPRPFPAC (from Genscript company) on the methacryloylated gelatin, and controlling the catechol content in the grafted compound to be 100 mu mol/g and the content of the 'knot' mimic peptide GPRPFPAC to be 8.82 mu mol/g to obtain 'knot' mimic peptide-catechol-grafted gelatin; ② The "knob" mimetic peptide-catechol-grafted gelatin was completely dissolved in a pre-heated 0.9% sodium chloride solution, resulting in a mass volume percent (w/v) of 0.4% (w/v).
2) Preparation of a solid hemostatic material loaded with a functional crosslinking agent: ① Completely soaking gelatin sponge in the 0.4% (w/v) "knot" mimetic peptide-catechol-grafted gelatin solution obtained in step 1); ② After 6 hours, the gelatin sponge loaded with the "knob" mimetic peptide-catechol-grafted gelatin in step ① was removed, freeze-dried, and stored as a sponge.
3) The using method comprises the following steps: the spongy gelatin sponge loaded with the "knot" mimic peptide-catechol-grafted gelatin is pressed on a bleeding part, so that a blood clot can be formed on the surface of a wound.
Comparative example 1
Topical lyophilized fibrin adhesives (Protect Laishi, available from Shanghai Laishi) include enzymatic and fibrinogen reagents. The enzyme reagent and the fibrinogen reagent are respectively prepared into solutions according to the specification, and the enzyme crosslinking is completed after about 1s of mixing to obtain the fibrin adhesive, and the microstructure of the adhesive is shown in figure 1.
Comparative example 2
Thrombin solutions and 20% (w/v) fibrinogen solutions were prepared separately, and the methods of preparation and use were similar to the procedure of comparative example 1, except that: the fibrinogen solution concentration was 20% (w/v).
Comparative example 3
20% (W/v) fibrinogen solution and 20% (w/v) catechol-grafted gelatin were prepared separately, and the preparation method and use method were similar to the procedure of example 1 except that: in the preparation of the component B solution, the methacryloylated gelatin does not react with the 'knot' mimic short peptide sequence GPRPFPAC after reacting with dopamine hydrochloride, and the obtained grafted compound is catechol-grafted gelatin.
Comparative example 4
20% (W/v) fibrinogen solution and 20% (w/v) "knot" mimetic peptide-grafted gelatin were prepared separately, and the preparation method and use method were similar to the procedure of example 1 except that: in the preparation of the component B solution, the methacryloylated gelatin does not react with dopamine hydrochloride and directly reacts with the 'knot' mimic short peptide sequence GPRPFPAC, and the obtained grafted compound is 'knot' mimic peptide-grafted gelatin.
Comparative example 5
The preparation of 20% (w/v) fibrinogen solution and 20% (w/v) catechol- "knot" mimetic peptide-grafted gelatin, respectively, was similar to the procedure of example 1, except that the preparation procedure of the B component was different in the grafting sequence, the "knot" mimetic peptide was grafted first, and then catechol was grafted, and the content of "knot" mimetic peptide "was not controlled to be 8.82. Mu. Mol/g because it reacted with the carbon-carbon double bond and carboxyl group in the methacryloylated gelatin, and the amount of free carboxyl group was reduced because the carboxyl group in the methacryloylated gelatin reacted with the" knot "mimetic peptide, resulting in a grafted catechol content of less than 100. Mu. Mol/g.
Comparative example 6
An absorbent gelatin sponge (xiang en) was used according to the instructions.
Performance testing
To verify the properties of the self-assembled fibrin gels obtained in examples 1 to 17 and comparative examples 1 to 6, gel time property tests, gel strength tests, adhesive strength tests and animal hemostasis experiments were performed, respectively, as follows.
Gel time test
Detecting an object:
Inventive examples 1 to 16, as well as comparative examples 1 to 5;
The detection method comprises the following steps:
Rheological analyses were performed on examples 1 to 16 and comparative examples 1 to 5 to compare the gel times, and the results are shown in Table 1. The specific operation method comprises the following steps: dynamic rheology experiments were performed at 37 ℃ using a HAAKE RS6000 rheometer with parallel plate (P20 TiL,20-mm diameter) geometry. The time-sweep oscillation test of the hydrogels of examples 1 to 16 and comparative examples 1 to 5 was performed at a frequency of 1Hz for 300 seconds at a strain of 5%. The pre-gel solution was strain scanned to verify the linear response. The gel point is determined when the torsional modulus (G ') exceeds the loss modulus (G').
Gel strength test
Detecting an object:
Inventive examples 1 to 16, as well as comparative examples 1 to 5;
The detection method comprises the following steps:
Rheological analyses were performed on examples 1 to 16 and comparative examples 1 to 5 to compare the gel strengths, and the results are shown in Table 1. The specific procedure is the same as the gel time test, and the gel strength is recorded as the final torsional modulus G' (Pa).
Adhesion Strength test
Detecting an object:
Inventive examples 1 to 17, as well as comparative examples 1 to 6;
The detection method comprises the following steps:
The specific operation is as follows: pig skin was cut into a rectangle of 40 mm by 20mm, and two pieces of pig skin were bonded together with 500. Mu.l of each of examples 1 to 17 and comparative examples 1 to 6. The adhesive strength was then tested at a strain rate of 4 mm/min. The reading of the gel when it was detached from the pigskin was recorded as the adhesive strength (Kpa). The detection results are shown in Table 1.
Hemostatic Effect test
Detecting an object:
Inventive examples 1 to 17, as well as comparative examples 1 to 6;
The detection method comprises the following steps:
SD rat liver partial cut bleeding model: after SD rats are anesthetized, the abdomen is exposed, the abdomen is fixed on an operation table, the middle incision of the abdomen is performed, the liver is exposed, and a3 cm-0.5 cm liver part incision bleeding model is formed on the liver by surgical scissors; the bleeding site was covered with the weighed filter paper, the mixed solution of the a-and B-components of the present invention examples 1 and 9, the sponge of example 17, the precursor solutions of comparative examples 1 to 5, and the sponge of comparative example 6, respectively, as hemostatic materials until the bleeding stopped, and the bleeding time and the blood loss were recorded, and the results are shown in table 1, fig. 3, and fig. 4.
TABLE 1
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The values of the hemostatic time and the blood loss are expressed as (mean ± standard deviation).
Analysis of results:
As can be seen from Table 1, examples 1 to 16 have a gel time ranging from 1 to 3s, and in the case where the kind and concentration of the "junction" mimetic peptide-catechol-graft compound are the same, the gel time of the gel follows the "pore: the junction "molar ratio increases and decreases, followed by" pore: the junction "continued increase in molar ratio prolonged, at" pore: junction "molar ratio reaches 1: at 1.5, the gel time was at a minimum of 1s. The gel times of examples 1 to 16 were all comparable to comparative example 1 (2 s), comparative example 2 (1 s), comparative example 4 (2 s) and comparative example 5 (1 s), and were all significantly lower than comparative example 3 (32 s). Although the gel times of the inventive examples were comparable to comparative examples 1,2, 4, 5, the corresponding adhesive strengths were significantly higher than comparative examples 1,2, 4, 5.
As can be seen from Table 1, examples 1 to 16 have a gel strength G' ranging from 680 to 1200Pa, a type of mimetic peptide-catechol-graft compound at the "junction" and "pore: the gel strength increased with increasing fibrinogen concentration and concentration of the "knot" mimetic peptide-catechol-graft compound species at the same "molar ratio", and at concentrations of 20% (w/v) fibrinogen and 20% (w/v) "knot" mimetic peptide-catechol-graft compound, the gel strength was maximum and the gel strength was higher than that of each of comparative examples 1,2, and 3 (gel strength of comparative example 1 was 360Pa, gel strength of comparative example 2 was 580Pa, and gel strength of comparative example 3 was 60 Pa), although the gel strength of some examples of the present invention may be comparable to that of comparative examples 4 (720 Pa) and comparative example 5 (1190 Pa), but the corresponding adhesive strength was significantly higher than that of comparative examples 4 and 5.
As is clear from Table 1, examples 1 to 16 have adhesion strengths ranging from 27 to 44kPa, and were found to simulate the type and concentration of the peptide-catechol-graft compound at the "junction", the "pore: the gel adhesive strength increased with increasing catechol content at the same junction "molar ratio, and at a content of 100. Mu. Mol/g, the gel adhesive strength was highest and was higher than that of each of comparative examples 1 to 5 (adhesive strength of comparative example 2: 2kPa, adhesive strength of comparative example 2: 3kPa, adhesive strength of comparative example 3: 22kPa, adhesive strength of comparative example 4: 14kPa, adhesive strength of comparative example 5: 18 kPa). The adhesion strength of example 17 was 0.7kPa, which is higher than that of comparative example 6 (0 kPa).
As is clear from Table 1, FIG. 4 and FIG. 5, the average hemostatic time of the gels prepared in example 1 and example 9 of the present invention was 15 to 20 seconds, which are significantly lower than the hemostatic time of 41 seconds or more of each comparative example. The average blood loss of the gels prepared in example 1 and example 9 was 68-83 mg, which were significantly lower than the average blood loss of 374-656 mg of the comparative examples. The average hemostasis time of example 17 was 102±10s, significantly lower than the average hemostasis time of comparative example 6 (155±15 s); the average blood loss of example 17 was 652±94s, which was significantly lower than that of comparative example 6 (1103±93 mg).
In a word, the fibrin gel which is independent of thrombin and inspired by mussel protein can be rapidly (about 1 s) crosslinked to form fibrin clots when being applied to bleeding wounds, plays a role in rapidly plugging the wounds and blocks blood outflow; meanwhile, fibrin clots formed by crosslinking the raw materials with high concentration can resist the blood pressure, keep the integrity of gel and enhance the effect of plugging wounds; meanwhile, the catechol structure in the fibrin clot and the tissue form covalent and/or non-covalent interactions, so that the tissue adhesion strength of the clot is improved, the blood impact can be resisted, the fibrin clot is prevented from being washed away by blood flow, and the wound blocking effect is further enhanced. The rapid gelation and catechol structure of fibrin gel inspired by the mussel protein are not depended on thrombin, so that the gel has the functions of rapidly plugging wounds and strongly adhering tissues, thereby achieving excellent hemostatic effect. The functional cross-linking agent is loaded on a solid hemostatic material, and can form blood-like clots on the surface of a wound when applied to the bleeding wound, so as to play a role in plugging the wound; meanwhile, the catechol structure loaded in the solid hemostatic material and the tissue form covalent and/or non-covalent interactions, so that the tissue adhesion strength of the solid hemostatic material is improved, and the wound blocking effect is further enhanced.
The foregoing describes in detail specific embodiments of the present invention. It should be understood that the invention is not limited to the particular embodiments, but is intended to cover any variations or modifications, equivalents, and improvements therein within the spirit and principles of the invention, and not to obscure the true spirit and scope of the invention.
Claims (13)
1. A functional cross-linking agent inspired by mussel protein is a 'knot' mimic peptide-catechol-grafted compound formed by grafting a catechol structure and a 'knot' mimic peptide on a high molecular compound skeleton; the high molecular compound skeleton is from any one of methacrylic acid acylated gelatin or carboxylic acid functionalized polyethylene glycol acrylate, preferably the molecular weight range is 100-600 kDa, more preferably 150-400 kDa; the "knob" mimetic peptide is any one of GPRPFPAC or GPRPAAC; the catechol content of the "tie" mimetic peptide-catechol-grafted compound is in the range of 10 to 100. Mu. Mol/g, preferably 50 to 100. Mu. Mol/g, more preferably 80 to 100. Mu. Mol/g, most preferably 100. Mu. Mol/g; the content of the 'knot' mimic peptide in the 'knot' mimic peptide-catechol-grafted compound is 588 mu mol/g-0.59 mu mol/g; preferably 29.4. Mu. Mol/g to 1.12. Mu. Mol/g; more preferably 11.76. Mu. Mol/g to 2.84. Mu. Mol/g; most preferably 8.82. Mu. Mol/g.
2. A method of preparing the functional crosslinker of claim 1, comprising:
1) Taking an amino or alkylamino substituted catechol derivative and a high molecular compound with a carboxyl and carbon-carbon double bond structure in the structure as raw materials, wherein the skeleton of the high molecular compound is from any one of methacryloylated gelatin or carboxylic acid functionalized polyethylene glycol acrylate, and the molecular weight is preferably in the range of 100-600 kDa, more preferably in the range of 150-400 kDa; the amino or alkylamino and the carboxyl are subjected to condensation reaction to obtain catechol-grafted compound; preferably the reaction is carried out in an acidic liquid environment at a pH of 5 and in a nitrogen atmosphere;
2) And (2) reacting the catechol-grafted compound obtained in the step (1) with a 'knot' mimic peptide in a nitrogen atmosphere, namely grafting the 'knot' mimic peptide through a carbon-carbon double bond on the catechol-grafted compound, wherein the 'knot' mimic peptide is one of GPRPFPAC and GPRPAAC sequences, so as to obtain the 'knot' mimic peptide-catechol-grafted compound, controlling the catechol content in the obtained 'knot' mimic peptide-catechol-grafted compound to be 10-100 mu mol/g and the 'knot' mimic peptide content to be 588-0.59 mu mol/g, and acidifying the obtained 'knot' mimic peptide-catechol-grafted compound to a pH value of 5-6.
3. A hemostatic material, characterized in that: obtained by loading the functional crosslinking agent of claim 1 on a solid hemostatic material; the solid hemostatic material is preferably in the form of any one of gauze or sponge.
4. A raw material composition for preparing thrombin-independent and mussel protein-inspired fibrin gel, characterized in that: comprises a raw material A and a raw material B; the raw material A is fibrinogen, and the raw material B is the 'knot' mimic peptide-catechol-grafted compound in claim 1; the molar ratio of the 'holes' in the raw material A to the 'knots' mimic peptide in the raw material B is 1:10-10:1; preferably 1:5 to 5:1; more preferably 1:2 to 2:1; most preferably 1:1.5.
5. The raw material composition according to claim 4, wherein: the raw material A and the raw material B are solutions; the concentration of fibrinogen in the raw material A is 5% (w/v) to 20% (w/v); preferably 10% (w/v) to 20% (w/v); more preferably 15% (w/v) to 20% (w/v); most preferably 20% (w/v); the concentration of the 'knot' mimic peptide-catechol-grafted compound in the raw material B is 5% (w/v) to 20% (w/v); preferably 10% (w/v) to 20% (w/v); more preferably 15% (w/v) to 20% (w/v); most preferably 20% (w/v).
6. A kit for preparing thrombin-independent and mussel protein-inspired fibrin gel, characterized in that: comprising a first precursor reagent and a second precursor reagent packaged independently of each other; the first precursor reagent comprises fibrinogen and the second precursor reagent comprises a "knob" mimetic peptide-catechol-grafting compound; the mass ratio of fibrinogen in the first precursor reagent to the conjugated' mimetic peptide-catechol-grafted compound in the second precursor reagent is 1:4.4-22.7:1; preferably 1:2.2 to 11.4:1; more preferably 1:0.9 to 4.5:1; most preferably 1.5:1.
7. The kit of any one of claims 6, wherein: the first precursor reagent and/or the second precursor reagent further comprise auxiliary materials and/or additives; the auxiliary materials are selected from one or more than two of glycine, arginine hydrochloride, sodium citrate, sucrose and sodium chloride; the additive is one or more than two selected from growth factors, interleukins, vitamins and silver ions; the growth factor is further selected from one or more of platelet growth factor, epidermal growth factor or fibroblast growth factor; the interleukin is further selected from one or more of interleukin 2, interleukin 6 or interleukin 8; the vitamin is further selected from one or more of vitamin B, vitamin C, vitamin E or vitamin K.
8. The kit of any one of claims 6-7, wherein: the preparation method also comprises an independently packaged preparation solvent, wherein the preparation solvent is any one or a mixture of a plurality of phosphate buffer salt solution, HEPES biological buffer solution, 0.9% sodium chloride solution, calcium chloride solution and deionized water.
9. A method of preparing a thrombin-independent and mussel protein-inspired fibrin gel comprising:
1) Preparing a first precursor solution in which fibrinogen is dissolved in a solvent, and controlling the concentration of fibrinogen in the first precursor solution to be 5% (w/v) to 20% (w/v), preferably 10% (w/v) to 20% (w/v); more preferably 15% (w/v) to 20% (w/v); most preferably 20% (w/v);
2) ① preparing a 'knot' mimic peptide-catechol-grafted compound, carrying out condensation reaction of amino groups and carboxyl groups on dopamine hydrochloride and a high molecular compound with a carboxyl and carbon-carbon double bond structure in the structure, and then carrying out graft polymerization reaction on the dopamine hydrochloride and a 'knot' mimic short peptide sequence to obtain the 'knot' mimic peptide-catechol-grafted compound; the high molecular compound skeleton is from any one of methacrylic acid acylated gelatin or carboxylic acid functionalized polyethylene glycol acrylate, preferably the molecular weight range is 100-600 kDa, more preferably 150-400 kDa; the "knob" mimetic peptide is a short peptide sequence capable of cross-linking with fibrinogen based on "pore-knob interactions", preferably either one of GPRPFPAC or GPRPAAC; controlling the catechol content of the "tie" mimetic peptide-catechol-grafted compound to be between 10 μmol/g and 100 μmol/g, preferably between 50 μmol/g and 100 μmol/g, more preferably between 80 μmol/g and 100 μmol/g, and most preferably 100 μmol/g; controlling the content of the 'knot' mimic peptide in the 'knot' mimic peptide-catechol-grafted compound to be 588-0.59 mu mol/g, preferably 29.4-1.12 mu mol/g; more preferably 11.76. Mu. Mol/g to 2.84. Mu. Mol/g; most preferably 8.82. Mu. Mol/g;
② Dissolving the "knob" mimetic peptide-catechol-graft compound prepared in ① to obtain a second precursor solution, controlling the concentration of the second precursor solution to be 5% (w/v) to 20% (w/v), preferably 10% (w/v) to 20% (w/v), more preferably 15% (w/v) to 20% (w/v), and most preferably 20% (w/v);
3) Mixing the first precursor solution obtained in the step 1) with the second precursor solution obtained in the step 2), and controlling the molar ratio of the 'holes' in the first precursor solution to the 'knots' mimic peptides in the second precursor solution to be 1:10-10:1, preferably 1:5-5:1, more preferably 1:2-2:1, and most preferably 1:1.5, so as to obtain the fibrin gel independent of thrombin and inspired by mussel proteins.
10. A fibrin gel prepared by the raw material composition of any one of claims 4 to 5, or a fibrin gel prepared by the kit of any one of claims 6 to 8, or a fibrin gel prepared by the method of claim 9.
11. Use of a raw material composition according to any one of claims 4-5 or a kit according to any one of claims 6-8 for the preparation of a fast setting hemostatic material in situ.
12. The use according to claim 11, wherein the use of the feedstock composition according to any one of claims 4 to 5 comprises: the raw materials A and B are respectively prepared into injectable solution, and then are simultaneously and evenly injected or sprayed on a bleeding wound site, and solid hydrogel is originally and rapidly formed on the bleeding wound site.
13. The use of claim 11, wherein the use of the kit of any one of claims 6-8 comprises: the first precursor reagent and the second precursor reagent are respectively prepared into injectable solution by using a configuration solvent, and then are simultaneously and uniformly injected or sprayed on a bleeding wound site to form solid hydrogel in situ.
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