Mytilus edulis bionic adhesive based on polymalic acid and preparation method and application thereof
阅读说明：本技术 一种基于聚苹果酸的贻贝仿生粘合剂及其制备方法和应用 (Mytilus edulis bionic adhesive based on polymalic acid and preparation method and application thereof ) 是由 吴红 唐友红 杨铁虹 乔友备 余喆 于 2019-10-16 设计创作，主要内容包括：本发明公开了一种基于聚苹果酸的贻贝仿生粘合剂及其制备方法应用,属于贻贝仿生高分子生物粘合剂技术领域,本发明公开的贻贝仿生粘合剂以聚苹果酸作为粘合剂的骨架,聚苹果酸分子中含有丰富的悬挂羧基,具有生物降解性好、生物相容性好的优点,加入含有邻苯二酚基团的分子,利用其含有的氨基与聚苹果酸的羧基进行酰胺反应,将具有粘合作用的邻苯二酚结构连接到聚苹果酸上,使制得的贻贝仿生粘合剂具备良好的组织粘合性、生物相容性和可降解性。利用该粘合剂对金属、玻璃、鸡蛋膜这三种不同的材料进行粘性测试,结果表明对这三种材料均有不同的粘合能力。(The invention discloses a mussel bionic adhesive based on polymalic acid and a preparation method and application thereof, belonging to the technical field of mussel bionic high-molecular biological adhesives. The adhesive is used for carrying out adhesion tests on three different materials, namely metal, glass and egg membranes, and the results show that the adhesive has different adhesive capacities on the three materials.)
1. A mussel bionic adhesive based on polymalic acid is characterized in that polymalic acid and molecules containing catechol groups are subjected to amide reaction to prepare the polymalic acid;
wherein, the polymalic acid is used as a skeleton of the mussel bionic adhesive and is used for providing carboxyl groups required by an amide reaction; molecules containing catechol groups for providing amino groups for amide reactions.
2. The polymalic acid-based mussel biomimetic adhesive as claimed in claim 1, wherein the amount of the catechol group-containing molecule is adjusted to be 1-5 times the molar number of carboxyl groups carried by polymalic acid, depending on the number of carboxyl groups in polymalic acid.
3. The polymalic acid-based mussel biomimetic adhesive of claim 1, wherein the polymalic acid has a weight average molecular weight between 800 and 20000.
4. The polymalic acid-based mussel biomimetic adhesive of claim 1, wherein the polymalic acid comprises α -polymalic acid, β -polymalic acid and γ -polymalic acid.
5. The polymalic acid-based mussel biomimetic adhesive as in claim 1, wherein the molecules containing catechol groups comprise dopamine, levodopa, norepinephrine and 3, 4-dimethoxybenzaldehyde.
6. The method for preparing a polymalic acid-based mussel biomimetic adhesive according to any of claims 1-5, comprising the steps of:
1) dissolving polymalic acid in water, adjusting the pH value to 5.5-6.5, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and reacting at room temperature in a dark place until carboxyl groups are activated;
2) adding molecules containing catechol groups into the reaction system obtained in the step 1), reacting at room temperature for 2-4 days in a nitrogen atmosphere, and dialyzing, purifying and freeze-drying the reaction product to obtain the polymalic acid-based mussel bionic adhesive.
7. The method for preparing a polymalic acid-based mussel biomimetic adhesive according to claim 6, wherein in step 1), an alkaline solution is used to adjust the pH value to between 5.5-6.5.
8. The method for preparing a polymalic acid-based mussel biomimetic adhesive as claimed in claim 6, wherein in step 1), 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride and N-hydroxysuccinimide are used in an amount of 1.5 times the amount of the substance of carboxyl groups in the polymalic acid.
9. Use of a polymalic acid-based mussel biomimetic adhesive as claimed in any of claims 1 to 5 as a mussel biomimetic macromolecular bioadhesive.
10. The use of claim 9, wherein the polymalic acid-based mussel biomimetic adhesive has a non-specific adhesive effect and is capable of acting as an adhesive coating, tissue sealant, hemostatic agent and drug carrier.
Technical Field
The invention belongs to the technical field of biological adhesive preparation, and relates to a polymalic acid-based mussel bionic adhesive and a preparation method and application thereof.
Background
The medical adhesive has the advantages of effective hemostasis, avoidance of secondary damage to human tissues caused by acupuncture, convenience in use, no need of dismantling and the like, and is more and more concerned by people. The use of bioadhesives, tissue sealants and hemostats to control blood loss and promote tissue healing has been well utilized in clinical surgery for the past two decades. In the military field, bioadhesives may be used for emergency treatment of wounds.
Fibrin glue and the synthetic class of cyanoacrylate adhesives are two classes of tissue adhesives that have been widely utilized. Fibrin glue has the advantages of rapid curing and biodegradability, but has relatively poor adhesion and tensile strength, and may cause allergic reactions and the like. Cyanoacrylate adhesives have the advantages of strong adhesion, fast set times, instant adhesion to tissue and ease of use. However, since the slow degradation of cyanoacrylates may lead to rejection of the body, the release of heat from the polymerization reaction and the toxicity of the degradation products limit its use. In addition, fibrin glue and cyanoacrylate only work best when applied to the dry surgical field, which greatly limits its application to the wet tissue adhesion and hemostasis fields in many visceral organ surgeries. In addition, fibrin glue and cyanoacrylate only work best when applied to the dry surgical field, which greatly limits its application to the wet tissue adhesion and hemostasis fields in many visceral organ surgeries. To date, there is no tissue adhesive or sealant on the market that can be widely used for both external and internal tissue adhesion and hemostasis.
Mussel bionic adhesive is a new hotspot in the research of people on adhesives at present. Mussels can adhere to various nonspecific interfaces in water, and the strong adhesion capability of mussels is that amino acid containing catechol structure called L-3, 4-dihydroxyphenylalanine (L-DOPA) exists in adhesion protein secreted by feet of mussels. Under oxidizing or alkaline conditions, the oxidation of the hydroxyl group of catechol on DOPA to o-quinone promotes cross-linking of foot-binding proteins, followed by triggering intermolecular cross-linking, resulting in a protein network with cohesive and bulk elastic properties. Recent studies have shown that DOPA in its oxidized state is also capable of promoting its strong adhesion to biological surfaces by forming covalent bonds with nucleophilic groups on biological surfaces such as-
In order to provide the above-mentioned bioadhesive with a stronger binding capacity, it is considered that the content of dopamine in the product should be increased as much as possible. As reported for CA-DA-PEG, citric acid first undergoes an esterification reaction with polyethylene glycol, and then dopamine is attached to the polyethylene glycol backbone by reacting the amino group with the carboxyl group on citric acid. However, polyethylene glycol contains reactive hydroxyl groups only at both ends, resulting in a low content of dopamine in the synthesized product, limiting its adhesive strength.
Polymalic acid (PMLA) is an aliphatic polyester formed by ester bond linkage of malic acid as a unique monomer, and is finally degraded into water and carbon dioxide to be discharged out of a body under the action of in-vivo enzymes, so that no accumulated toxicity exists. Its relatively high solubility, high spontaneous degradation rate and immune inertia under physiological conditions make it a novel drug controlled release carrier material superior to polysaccharides and polypeptide biopolymers. The polymalic acid mainly has three configurations of alpha-, beta-and gamma-, as follows:
the compound has a plurality of active centers (suspension carboxyl groups), can be covalently connected with a plurality of groups with biological functions, and is a high-efficiency carrier for connecting drugs and functional groups. At present, the beta-polymalic acid is mainly found in organisms.
Currently, there is no report of reacting polymalic acid with dopamine and similar molecules containing catechol structures to produce bioadhesives.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a mussel bionic adhesive based on polymalic acid and application of a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a mussel bionic adhesive based on polymalic acid, which is prepared by amide reaction of polymalic acid and molecules containing catechol groups;
wherein, the polymalic acid is used as a skeleton of the mussel bionic adhesive and is used for providing carboxyl groups required by an amide reaction; molecules containing catechol groups for providing amino groups for amide reactions.
Preferably, the amount of the molecules containing catechol groups is adjusted to be 1-5 times of the mole number of the carboxyl groups carried by the polymalic acid according to the number of the carboxyl groups in the polymalic acid.
Preferably, the weight average molecular weight of the polymalic acid is between 800 and 20000.
Preferably, the polymalic acid includes α -polymalic acid, β -polymalic acid and γ -polymalic acid.
Preferably, the molecule containing a catechol group comprises dopamine, levodopa, norepinephrine, and 3, 4-dimethoxybenzaldehyde.
The invention also discloses a preparation method of the polymalic acid-based mussel bionic adhesive, which comprises the following steps:
1) dissolving polymalic acid in water, adjusting the pH value to 5.5-6.5, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and reacting at room temperature in a dark place until carboxyl groups are activated;
2) adding molecules containing catechol groups into the reaction system obtained in the step 1), reacting at room temperature for 2-4 days in a nitrogen atmosphere, and dialyzing, purifying and freeze-drying the reaction product to obtain the polymalic acid-based mussel bionic adhesive.
Preferably, in the step 1), an alkaline solution is adopted to adjust the pH value to be between 5.5 and 6.5.
Preferably, in step 1), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are used in an amount of 1.5 times the amount of the substance of the carboxyl group in the polymalic acid.
The invention also discloses application of the polymalic acid-based mussel bionic adhesive as a mussel bionic high-molecular biological adhesive.
Preferably, the polymalic acid-based mussel biomimetic adhesive has a non-specific adhesive effect and can be used as an adhesive coating, a tissue sealant, a hemostatic agent and a drug carrier.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a mussel bionic adhesive based on polymalic acid, which takes the polymalic acid as a skeleton of the adhesive, the polymalic acid molecules contain rich suspended carboxyl groups and have the advantages of good biodegradability and good biocompatibility. The adhesive is used for carrying out adhesion tests on three different materials, namely metal, glass and egg membranes, and the results show that the adhesive has different adhesive capacities on the three materials.
Therefore, the polymalic acid-based mussel bionic adhesive disclosed by the invention can be used as a biological adhesive with non-specific adhesion effect on various substrates such as metal, glass, egg membranes and the like; can be used as an adhesive coating, a tissue sealant, a hemostatic agent, a drug carrier and the like, so that the mussel bionic macromolecular biological adhesive is suitable for being applied to medical adhesives.
The invention also discloses a preparation method of the polymalic acid-based mussel bionic adhesive, which innovatively provides an optimal synthesis path and conditions suitable for the mussel bionic adhesive, and the method is simple to operate, mild in reaction conditions, low in requirements on reaction equipment and suitable for large-scale production.
Drawings
FIG. 1 is a reaction flow diagram for preparing a polymalic acid-based bioadhesive (PMLA-DA) according to the present invention; wherein, the reaction flows of beta-PMLA-DA and gamma-PMLA-DA are from left to right;
FIG. 2 is a nuclear magnetic spectrum of the product (β -PMLA-DA, β -PMLA, γ -PMLA-DA, γ -PMLA);
FIG. 3 is a photograph showing the adhesion of beta-PMLA-DA to three materials of metallic iron, glass and egg membrane; wherein (a) is metallic iron; (b) is glass; (c) is egg membrane;
FIG. 4 is a bar graph of the bond strength of β -PMLA-DA to metallic iron, glass and eggshell membrane (from left to right);
FIG. 5 is a graph showing the results of MTT assay for the cytotoxicity of polymalic acid-based bioadhesive β -PMLA-DA;
FIG. 6 is an in vitro degradation curve of PMLA-DA of different compositions.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the accompanying drawings:
the invention discloses a mussel bionic adhesive based on polymalic acid, which comprises polymalic acid and molecules with catechol groups; the adhesive is mussel bionic macromolecular biological adhesive, and has good biodegradability and non-specific adhesiveness.
The polymalic acid is an alpha, beta and gamma configuration polymer with carboxyl.
The molecule with the catechol group is a dopamine-like molecule, and the catechol group achieves the adhesion effect through coordination bonds, covalent bonds or hydrogen bonds and other mechanisms between the catechol group and various materials.
Referring to fig. 1, a reaction flow chart for preparing a polymalic acid-based bioadhesive (PMLA-DA) according to the present invention, the specific preparation method is as follows:
1. preparation of Polymalic acid of two configurations
The preparation of the polymalic acid is carried out by adopting a method disclosed by the prior art, and comprises the following preparation methods reported in the documents:
research on the novel synthesis method of polymalic acid and its benzyl polymalic acid ester as a drug carrier, namely Zhang Yu, Qiaoyou, Zhouqing, Yuji and Wuhong, modern biomedical progress 2018,18(10): 1849-1853.
YB Qiao,X Duan,L Fan,W Li,H Wu*,YK Wang.Synthesis of Controlled Molecular Weight Poly(β-Malic Acid)and Conjugation with HCPT as Polymeric DrugCar rier.J Polym Res(2014)21:397-406
The average molecular weight of the prepared beta-polymalic acid product is 1000-20000, and the yield is 4.7%; the average molecular weight of the gamma-polymalic acid is 750-5000, and the yield is 75%.
2. Preparation of PMLA-DA
Respectively weighing 0.20g of beta and gamma PMLA with different configurations, dissolving the PMLA in 10ml of deionized water, adding 0.1mol/L of sodium hydroxide solution to adjust the pH value to 5.5-6.5, adding 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to 1.5 times of the molar weight of carboxyl, reacting for 2 hours at room temperature in a dark place, and activating carboxyl groups; then adding 0.40g of dopamine according to the amount of 1.5 times of the mole number of the carboxyl polymalic acid, vacuumizing, and introducing N2Reacting for 3d at room temperature, dialyzing, lyophilizing and purifying to obtain PMLA-DA. The yield is 76.1%, the substitution degree of dopamine in beta-PMLA-DA is 13.2%, and the substitution degree of dopamine in gamma-PMLA-DA is 7.9%.
3. Viscosity test of PMLA-DA