Mussel biomimetic adhesive, preparation method and application

Abstract

This invention provides a mussel-inspired biomimetic adhesive and its preparation method. The adhesive is made by uniformly mixing dopamine-modified hyaluronic acid, polydopamine, and gallium nitrate in a specific ratio. The adhesive prepared by this invention is simple to prepare, injectable, and convenient for practical applications. The adhesive prepared by this invention maintains excellent adhesion performance in humid environments and exhibits excellent adhesion performance in humid environments with a pH range of 1–10. This adhesive is non-cytotoxic, and its various properties can be adjusted according to application requirements. It can be applied not only to the bonding of various materials such as glass, metals and their oxides, plastics, and rubber, but also has broad application prospects in clinical medicine, including the bonding and repair of skin or tissue organs, sealing and hemostasis, cell encapsulation, and delivery of bioactive molecules.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and more specifically to a mussel biomimetic adhesive, its preparation method, and its application. Background Technology

[0002] Wound closure is a crucial factor affecting the efficacy of surgical procedures. Rapid, safe, and effective wound closure techniques can reduce patient suffering. Adhesion is considered an effective wound closure technique with potential applications.

[0003] There are two main types of commercially available tissue adhesives: cyanoacrylate adhesives, but their decomposition products have unavoidable biotoxicity, limiting their application; and fibrin-based adhesives, but these are limited by their high price and relatively weak adhesive properties, preventing widespread use. Furthermore, most adhesive hydrogels fail to achieve effective adhesion under humid conditions because water molecules can form a thin hydration layer on the target substrate surface, hindering close contact between the hydrogel and the substrate. In addition, water molecules disrupt the non-covalent interactions between the adhesive hydrogel and the target substrate.

[0004] To develop tissue adhesives with superior performance, scientists have drawn inspiration from marine adhesive organisms (such as mussels and sandcastle worms) and developed a series of marine-based adhesives. While these adhesives have solved the problems of the two types mentioned above, they generally suffer from cumbersome synthesis steps, slow curing speed, and poor mechanical properties.

[0005] Therefore, it is necessary to innovate wound closure techniques and develop a new generation of clinically significant tissue adhesives. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a mussel-inspired biomimetic adhesive, its preparation method, and its application. This hydrogel adhesive has high biocompatibility, high wet adhesion, and controllable mechanical properties. It is simple to prepare, cures quickly, and is inexpensive.

[0007] According to a first aspect of the present invention, a method for preparing a mussel-inspired adhesive is provided, comprising the following steps:

[0008] Dopamine-modified hyaluronic acid was dissolved in distilled water to obtain the first solution;

[0009] A gallium source solution is added to the first solution, and after mixing thoroughly, a second solution is obtained.

[0010] Add polydopamine solution to the second solution, mix well, and adjust the pH to obtain the desired adhesive.

[0011] The mechanical properties of the adhesive are controlled by adjusting the pH.

[0012] As an optional implementation method, the pH adjustment range is 1 to 10.

[0013] As an optional embodiment, the dopamine-modified hyaluronic acid has a dopamine modification degree of 10-70% and a molecular weight of hyaluronic acid of 10-1500 kDa.

[0014] As an optional implementation, the gallium source is one or more of gallium nitrate, gallium sulfate, gallium chloride, gallium acetate, and gallium bromide.

[0015] As an optional embodiment, the concentration of the polydopamine solution is 0.5 to 3.0 mg / mL, and the particle size of the polydopamine is 20 to 5000 nm.

[0016] As an optional implementation, the molar ratio of dopamine to gallium ions in dopamine-modified hyaluronic acid is between (1:1) and (1:3).

[0017] In a second aspect of the present invention, a mussel biomimetic adhesive prepared by the aforementioned method is provided.

[0018] According to a third aspect of the present invention, the aforementioned mussel biomimetic adhesive is provided for use in the preparation of hydrogels for the adhesion and repair of skin or tissue organs and / or for sealing hemostasis and / or for cell encapsulation.

[0019] According to a fourth aspect of the present invention, the aforementioned mussel-inspired adhesive is provided for use in underwater bonding of materials.

[0020] According to a fourth aspect of the present invention, the application of the aforementioned mussel-inspired adhesive in the delivery of bioactive substances is provided.

[0021] As can be seen from the above technical solution of the present invention, the mussel biomimetic adhesive of the present invention is obtained by modifying the polyphenol groups on hyaluronic acid and the polyphenol hydroxyl groups on polydopamine with dopamine, which are coordinated with gallium ions to form a gel.

[0022] In this process, dopamine is used to modify the polyphenol groups on hyaluronic acid and coordinate them with gallium ions to form permanent crosslinks, thereby increasing the elasticity of the polymer network. At the same time, there are a large number of hydrogen bonds and π-π bonds in the entire system. These weak bonds can be reversibly broken and recombined, thereby increasing the energy dissipation of the network. This combination of strong and weak bonds greatly increases the cohesive force of the polymer, thereby improving the mechanical properties of the material.

[0023] The mussel-inspired adhesive of the present invention uses hydrophobic polydopamine to disrupt the water layer that hinders adhesion, and then the polyphenol groups on the dopamine-modified hyaluronic acid interact with various substrates to generate strong wet adhesion.

[0024] The mussel biomimetic adhesive prepared by this invention has a wide range of adhesive properties. It exhibits excellent underwater adhesive properties in solutions with pH values ​​ranging from 1 to 10, and also has different mechanical properties at different pH values, which can be adjusted according to application requirements. Attached Figure Description

[0025] Figure 1 This is a flowchart illustrating the preparation process of the mussel-inspired biomimetic adhesive of the present invention.

[0026] Figure 2A This is a physical image of the sample in Embodiment 3 of the present invention.

[0027] Figure 2B This is a physical image of the sample in Embodiment 4 of the present invention.

[0028] Figure 3 This is a diagram illustrating the adhesion performance of sample 4-2 in this embodiment of the invention.

[0029] Figure 4 This is a diagram illustrating the underwater adhesion performance of sample 4-2 in this embodiment of the invention.

[0030] Figure 5 This is a diagram illustrating the self-healing performance of sample 4-2 in this embodiment of the invention.

[0031] Figure 6 This is a diagram illustrating the plasticity properties of sample 4-2 in this embodiment of the invention.

[0032] Figure 7 This is a statistical chart comparing the hemostatic performance of sample 4-2 in the embodiments of the present invention.

[0033] Figure 8 This is a test diagram of the encapsulation capacity of sample 4-2 for curcumin in an embodiment of the present invention.

[0034] Figure 9 This is a graph showing the release capacity test of curcumin for sample 4-2 in this embodiment of the invention. Detailed Implementation

[0035] To better understand the technical content of the present invention, specific embodiments are described below in conjunction with the accompanying drawings.

[0036] Various aspects of the invention are described in this disclosure with reference to the accompanying drawings, in which numerous illustrative embodiments are shown. The embodiments of this disclosure are not necessarily intended to encompass all aspects of the invention. It should be understood that the various concepts and embodiments described above, as well as those described below in more detail, can be implemented in any of a number of ways.

[0037] In nature, marine animals such as mussels exhibit strong and repeatable underwater adhesion to various substrates in seawater. Mussel underwater adhesion is achieved by disrupting the hydration layer through the cationic lysine and hydrophobic residues in mussel foot protein (Mfps), thereby allowing catechol groups to bind to the exposed surface.

[0038] Based on this, the present invention develops wet adhesion hydrogels based on hydrophilicity-hydrophobicity balance by carefully designing complex hydrogel networks and carefully selecting their components.

[0039] The technical solution of the present invention is to disperse dopamine-modified hyaluronic acid, polydopamine, and gallium ions in an aqueous solution, mix them, and then gel them. By adjusting the pH, adhesives with different mechanical properties are obtained.

[0040] Combination Figure 1 As shown in the exemplary embodiment of the present invention, a method for preparing a mussel-inspired adhesive is provided, comprising the following steps:

[0041] Dopamine-modified hyaluronic acid was dissolved in distilled water to obtain the first solution;

[0042] A gallium source solution is added to the first solution, and after mixing thoroughly, a second solution is obtained.

[0043] Add polydopamine solution to the second solution, mix well, and adjust the pH to obtain the desired adhesive.

[0044] The mechanical properties of the adhesive are controlled by adjusting the pH.

[0045] As an optional implementation, the pH range is 1 to 10. The higher the pH value, the tighter the coordination of metal-polyphenols and the better the mechanical properties, which increases the time for the adhesive to maintain the gel shape during the adhesion process, especially during underwater adhesion, thereby prolonging the gel adhesion time. However, when the pH exceeds 10, it will destroy the polymer network, thereby affecting the mechanical properties and reducing the gel adhesion time. Therefore, the pH range needs to be 1 to 10.

[0046] Hydrogels with poor mechanical properties swell rapidly in aqueous solutions, disrupting their network and causing gel dissociation, leading to adhesive failure. In contrast, hydrogels with good mechanical properties maintain their basic gel shape, network structure, and gel integrity even after swelling, thus retaining their adhesive properties. Therefore, the mechanical properties of adhesives can be adjusted by regulating the pH value to control the duration of gel adhesion.

[0047] As an optional embodiment, the dopamine-modified hyaluronic acid has a dopamine modification degree of 10-70% and a molecular weight of hyaluronic acid of 10-1500 kDa.

[0048] It is understandable that the preparation process of dopamine-modified hyaluronic acid can use existing technology, and no further limitations are made here.

[0049] As an optional implementation, the gallium source is one or more of gallium nitrate, gallium sulfate, gallium chloride, gallium acetate, and gallium bromide.

[0050] As an optional embodiment, the concentration of the polydopamine solution is 0.5 to 3.0 mg / mL, and the particle size of the polydopamine is 20 to 5000 nm.

[0051] As an optional implementation, the molar ratio of dopamine to gallium ions in dopamine-modified hyaluronic acid is between (1:1) and (1:3).

[0052] In another exemplary embodiment of the present invention, a mussel biomimetic adhesive prepared by the aforementioned method is provided. The mussel biomimetic adhesive achieves underwater adhesion by adjusting the hydrophilic-hydrophobic balance. When the adhesive comes into contact with a wet material, the polydopamine on the adhesive surface will destroy the water film on the material surface due to its hydrophobicity, thereby exposing the dry material surface. Then, the hydrophilic dopamine-modified hyaluronic acid can directly contact the dry material surface and achieve wet adhesion through phenolic hydroxyl linkage.

[0053] In another exemplary embodiment of the present invention, the aforementioned mussel biomimetic adhesive is provided for use in preparing hydrogels for the adhesion and repair of skin or tissue organs and / or sealing and hemostasis and / or cell encapsulation, which can be applied to wounds, especially arteries and internal organs for wet adhesion and rapid hemostasis.

[0054] In other embodiments, the aforementioned mussel-inspired adhesive can also be applied to the bonding of various materials such as glass, metals and their oxides, plastics, and rubber.

[0055] In another exemplary embodiment of the present invention, an application of the aforementioned mussel biomimetic adhesive in underwater bonding of materials is provided, which enables the bonding of materials in a humid environment.

[0056] In another exemplary embodiment of the present invention, the application of the aforementioned mussel biomimetic adhesive in the delivery of bioactive substances, including drugs, bioactive factors, or cells, is provided.

[0057] The following examples illustrate specific implementations of the present invention. However, these examples are merely for illustrative purposes and do not limit the scope of the invention in any way.

[0058] Unless otherwise specified, all reagents used in the following examples are commercially available reagents.

[0059] Example 1

[0060] [Preparation of different dopamine-modified hyaluronic acid derivatives]

[0061] 2g of hyaluronic acid (HA) was dissolved in 200mL of 0.1M MES buffer solution, and the pH of the solution was adjusted to 5.5 using 0.1M HCl. Then, 1.4g of EDC and 0.2g of NHS were added to the hyaluronic acid solution, and the reaction was activated for about 30 minutes. Dopamine was then added to the above reaction mixture and reacted at room temperature in the dark for 12 hours. The resulting reaction solution was dialyzed (dialysis bag molecular weight cutoff: 3500Da), and the pH of the solution was maintained at 5.5 during the purification process.

[0062] After dialysis, the hyaluronic acid derivatives were freeze-dried to obtain different dopamine-modified hyaluronic acid derivatives, as shown in Table 1.

[0063] Table 1. Preparation of different dopamine-modified hyaluronic acid derivatives

[0064]

[0065] The dopamine-modified hyaluronic acid used in the following examples has a molecular weight of 400 kDa and a dopamine modification degree of 35.0%, namely samples 1-3.

[0066] Example 2

[0067] [Preparation of Mussel Bionic Adhesives under Different Types of Gallium Ions]

[0068] 4.00 g of dopamine-modified hyaluronic acid was dissolved in 100 mL of distilled water. 0.75 g of gallium source was added to the solution, followed by polydopamine. The pH of the solution was adjusted to 8, and the mixture was thoroughly mixed to obtain binders with different gallium sources, as shown in Table 2.

[0069] Table 2 Preparation of different gallium source binders

[0070] Sample number Dopamine-modified hyaluronic acid quality Gallium ion type Gallium ion mass 2-1 4.0g Gallium nitrate 0.75g 2-2 4.0g Gallium sulfate 0.75g 2-3 4.0g Gallium chloride 0.75g 2-4 4.0g Gallium acetate 0.75g 2-5 4.0g Gallium bromide 0.75g

[0071] The following examples use gallium nitrate as the gallium source.

[0072] Example 3

[0073] [Preparation of Mussel Bionic Adhesives at Different pH Levels]

[0074] 4.00 g of dopamine-modified hyaluronic acid was dissolved in 100 mL of distilled water. 0.75 g of gallium nitrate was added to adjust the pH of the solution. The solution was mixed thoroughly to obtain adhesives at different pH values, as shown in Table 4.

[0075] Table 3. Preparation of mussel biomimetic adhesives at different pH values.

[0076]

[0077] The following examples use a solution with a pH of 8.

[0078] Example 4

[0079] Preparation of Mussel Biomimetic Adhesives with Different Polydopamine Sizes and Concentrations

[0080] 4.00 g of dopamine-modified hyaluronic acid was dissolved in 100 mL of distilled water, 0.75 g of gallium nitrate was added, and then polydopamine was added to adjust the pH of the solution to 8. The solution was thoroughly mixed to obtain adhesives with different polydopamine sizes and masses, as shown in Table 4.

[0081] Table 4. Preparation of biomimetic mussel adhesives with different sizes and masses of polydopamine.

[0082] Sample number Dopamine hyaluronic acid quality gallium nitrate mass Polydopamine size Polydopamine quality 4-1 4.0g 0.75g 20nm 1.0g 4-2 4.0g 0.75g 200nm 1.0g 4-3 4.0g 0.75g 500nm 1.0g 4-4 4.0g 0.75g 1000nm 1.0g 4-5 4.0g 0.75g 3000nm 1.0g 4-6 4.0g 0.75g 5000nm 1.0g 4-7 4.0g 0.75g 200nm 0.5g 4-8 4.0g 0.75g 200nm 2.0g 4-9 4.0g 0.75g 200nm 3.0g

[0083] The polydopamine used in the following examples has a particle size of 200 nm and a mass of 1.0 g.

[0084] Example 5

[0085] Mechanical properties of mussel-inspired adhesives

[0086] Take photos of samples 3-1, 3-2, 3-3, 3-5, 3-6, and 3-7, as follows: Figure 2A As shown, the gel shape becomes increasingly three-dimensional with increasing pH. This is because, with increasing pH, the coordination of the metal-polyphenol changes from monocoordination at low pH to dicoordination or tricoordination at high pH, ​​resulting in a denser polymer network and improved rheological properties of the gel. Consequently, the hydrogel exhibits a more three-dimensional shape, and the corresponding mechanical properties of the gel gradually improve. This allows the adhesive to maintain the basic gel morphology for a longer period during adhesion, especially underwater, preserving the gel's network structure and integrity, thus extending the gel's adhesion time. Therefore, the mechanical properties of the adhesive can be adjusted by regulating the pH value to control the gel adhesion time.

[0087] Take photos of samples 4-2, 4-8, and 4-9, such as... Figure 2BAs shown in the figure, the shape of the hydrogel becomes more and more three-dimensional with the increase of polydopamine content. This is related to the coordination of polydopamine with free gallium ions after the addition of polydopamine. The addition of polydopamine also greatly increases the cohesive force of the polymer network, thereby enhancing the mechanical properties.

[0088] Example 6

[0089] [Adhesive properties of mussel-inspired adhesives]

[0090] The adhesive prepared in sample 4-2 was subjected to an adhesion experiment. As can be seen from the figure, the adhesive of the present invention exhibits adhesion at the dry interface ( Figure 3 ) and wet interface ( Figure 4 It exhibits excellent adhesion properties, and it can be seen that the prepared hydrogel can achieve adhesion of a variety of materials.

[0091] Example 7

[0092] [Self-healing and plasticity properties of mussel-inspired adhesives]

[0093] Due to the irregularity of the wound, the adhesive should possess both self-healing and plasticity properties. Here, we evaluate the self-healing properties of the adhesive in sample 4-2. Figure 5 ) and plasticity ( Figure 6 ).

[0094] The self-healing property was demonstrated by first preparing a hydrogel into a cylinder with a diameter of 2 cm and a thickness of 1 cm, and then breaking it in the middle. The changes in the hydrogel cylinder were observed without any further treatment.

[0095] Plasticity tests demonstrate the plasticity of hydrogels by molding them into various shapes and transforming them between different shapes.

[0096] As can be seen, the adhesive of the present invention has a certain self-healing ability and plasticity, and can adapt to various irregular wounds.

[0097] Example 8

[0098] Evaluation of the hemostatic properties of mussel-inspired adhesives

[0099] The hydrogels prepared from Sample 4-2 and the control sample were used to conduct a rat tail amputation hemostasis experiment to evaluate the hemostatic performance of the adhesive. SD rats aged 8-10 weeks were randomly divided into groups (3 rats per group) according to their body weight before the experiment: a: blank group, no treatment; b: control group, Samples 3-5; c: experimental group, Sample 4-2. The rats were subjected to tail amputation experiments, and the amount of bleeding in different groups was recorded during the operation.

[0100] from Figure 7 As can be seen, the addition of polydopamine introduces more phenolic hydroxyl groups, which increases the number of adhesion sites, thereby improving the adhesive properties of the hydrogel and giving it better hemostatic properties.

[0101] Example 9

[0102] [Mussel-inspired adhesive used for hemostasis in rat liver]

[0103] SD rats aged 8-10 weeks were randomly divided into groups (n=10 per group) according to body weight before the experiment: a) Sample 4-2 mussel biomimetic adhesive hydrogel group; b) Commercial hemostatic agent; c) Model blank group. During the operation, the amount and time of bleeding were recorded for each group.

[0104] The results showed that the hemostatic effect of commercial hemostatic agents was worse than that of the mussel biomimetic adhesive group of the present invention, indicating that the mussel biomimetic adhesive prepared by the present invention has a better hemostatic effect on wounds.

[0105] Example 10

[0106] [Mussel-inspired adhesives for the encapsulation and release of bioactive substances]

[0107] Taking sample 4-2, which is loaded with curcumin, as an example, the loading and release of the drug in the solution were analyzed by ultraviolet light testing.

[0108] Drug encapsulation experiments:

[0109] Weigh 25.0 mg of Curcumin and dissolve it in anhydrous ethanol, then dilute to 25.00 mL to prepare a 1.000 mg / mL Cur stock solution. Accurately measure an appropriate amount of the stock solution and dilute it with anhydrous ethanol to prepare a series of reference solutions with mass concentrations of 1.0, 2.0, 3.0, 4.0, 5.0, 10, and 20 μg / mL. Using anhydrous ethanol as a blank control solution, measure the absorbance (A) at 430 nm.

[0110] A standard curve was plotted with the mass concentration of Cur (C) on the x-axis and the value of A on the y-axis, yielding a linear regression equation. The results showed that Cur exhibited good linearity in the range of 1.0–20.0 μg / mL, and its encapsulation efficiency and drug loading were calculated.

[0111] Encapsulation efficiency = M1 / M0

[0112] Drug loading capacity = M1 / (M0+M2)

[0113] In the formula: M0: mass of curcumin added; M1: calculated mass of curcumin in samples of Examples 4-5; M2: mass of samples 4-5 added.

[0114] Each sample was measured in parallel three times, and the test results are the mean ± standard deviation (SD).

[0115] Drug release experiment:

[0116] The prepared drug-loaded adhesive was placed in 30 mL of PBS buffer solution with pH = 6.0, 6.8, and 7.2, respectively, and placed in a constant temperature shaking incubator at 37°C (100 rpm) for drug release experiments. At predetermined time intervals, 1 mL of the release solution was taken out and then 1 mL of fresh buffer solution was added to maintain a constant volume.

[0117] The concentration of drug released from the hydrogel was analyzed using a UV spectrophotometer. The maximum absorption peak of curcumin was at 430 nm. Each sample was measured in triplicate, and the test results are the mean ± standard deviation (SD).

[0118] like Figure 8 , 9 As shown, the adhesive of the present invention has a drug loading capacity of 4.0% for curcumin; drug release experiments have demonstrated that the adhesive of the present invention has a better drug release rate, reaching 70%, in a solution with pH=6.0.

[0119] Therefore, the adhesive of the present invention has very good biocompatibility and is particularly suitable as a carrier of bioactive substances. The bioactive substances encapsulated in the adhesive of the present invention achieve the effect of sustained drug release through molecular diffusion and material degradation.

[0120] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for preparing a mussel-inspired adhesive, characterized in that, It consists of the following steps: Dopamine-modified hyaluronic acid was dissolved in distilled water to obtain a first solution; the degree of dopamine modification of the dopamine-modified hyaluronic acid was 10-70%, and the molecular weight of the hyaluronic acid was 10-1500 kDa; A gallium source solution was added to the first solution and mixed thoroughly to obtain the second solution; the molar ratio of dopamine to gallium ions in the dopamine-modified hyaluronic acid was (1:1) to (1:3); Add polydopamine solution to the second solution, mix well, and adjust the pH to obtain the desired adhesive; the concentration of the polydopamine solution is 0.5~3.0 mg / mL, and the particle size of the polydopamine is 20~5000 nm. The mechanical properties of the adhesive are controlled by adjusting the pH. The pH range is 1 to 10. Within this pH range, the higher the pH value, the tighter the coordination of the metal-polyphenol and the better the mechanical properties. This increases the time the adhesive can maintain its gel shape during underwater adhesion, thereby prolonging the gel adhesion time.

2. The method for preparing the mussel-inspired adhesive according to claim 1, characterized in that, The gallium source is one or more of gallium nitrate, gallium sulfate, gallium chloride, gallium acetate, and gallium bromide.

3. A mussel biomimetic adhesive prepared by the method according to any one of claims 1-2.

4. The use of the mussel biomimetic adhesive of claim 3 in the preparation of hydrogels for skin adhesion and repair and / or sealing hemostasis and / or cell encapsulation.

5. The application of the mussel biomimetic adhesive of claim 3 in the preparation of hydrogels for the bonding and repair of tissues and organs and / or sealing and hemostasis and / or cell encapsulation.

6. The use of the mussel biomimetic adhesive of claim 3 in the preparation of underwater bonding products for materials.

7. The use of the mussel biomimetic adhesive of claim 3 in the preparation of a system for delivering bioactive substances.

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