High-stability injectable conductive nano composite hydrogel and preparation method thereof

Abstract

The invention belongs to the field of injectable conductive hydrogel, and discloses high-stability injectable conductive nano composite hydrogel and a preparation method thereof. The method comprises the following steps: pre-polymerizing a dopamine monomer by adopting a nano clay dispersion liquid in a weakly alkaline oxygen-containing environment to form a polydopamine functionalized PDA-LAP nanosheet, and introducing the polydopamine functionalized PDA-LAP nanosheet into dopamine grafted gelatin and a conductive high-molecular polymer poly (3, 4-ethylenedithiophene) / polystyrene sulfonate; the injectable conductive nano composite hydrogel GPLP is constructed based on the non-covalent bond supramolecular interaction. The GPLP hydrogel prepared by the invention has excellent shear thinning, self-repairing, tissue adhesion, stability, conductivity and biocompatibility, and has a huge application prospect in the fields of electrical stimulation therapy, tissue engineering and regenerative medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of injectable conductive hydrogels, and particularly relates to an injectable conductive nanocomposite hydrogel with high stability and a preparation method thereof. Background Art

[0002] Injectable hydrogels refer to a class of water-swellable polymers that are physically or chemically cross-linked and have a certain fluidity and can be applied by minimally invasive injection. Due to their unique sol-gel transition characteristics, injectable hydrogels can fill irregular geometric lesions in a non-invasive or minimally invasive manner. In recent years, with the development of biomedical technology, conductive hydrogels have attracted great attention from researchers due to their unique electrical properties and biocompatibility. Conductive hydrogels can interact with electrophysiological processes in vivo, providing new possibilities for applications such as nerve regeneration, muscle repair, and electrical stimulation therapy. However, most of the existing injectable conductive hydrogels are composed of a mixture of dynamic reversible networks and conductive fillers. Due to weak intermolecular interactions and uneven electrical permeation, their durability and conductivity are poor. Although the addition of strong irreversible covalent bonds can improve its mechanical stability, it is often at the expense of good injectability. Therefore, developing a hydrogel that is both injectable and conductive and highly stable is of great significance for expanding its application in the biomedical field.

[0003] Gelatin is a natural polymer material obtained by partial hydrolysis of collagen. It has good biocompatibility and can promote cell adhesion and proliferation. Gelatin molecular chains contain primary amine, carboxyl and hydroxyl functional groups, which can be easily modified to prepare hydrogel materials. However, pure gelatin-based hydrogels often lack tissue adhesion, self-healing and electrical conductivity. Polydopamine (PDA) is formed by self-polymerization of dopamine (DA) in an alkaline environment. PDA has a structure similar to mussel foot protein and has high adhesion to various surfaces even in a humid environment. It also has unique electrical properties and can be used as a modifier of conductive materials to improve the conductivity and mechanical properties of composite materials. In addition, its inherent antioxidant and anti-inflammatory properties are beneficial to reduce oxidative stress and inflammation at the site of injury, creating a favorable environment for effective tissue regeneration. However, strong oxidants such as sodium hydroxide or sodium periodate often lead to excessive oxidation or cross-linking of catechol groups, resulting in poor adhesion and limited reusability. Nanoclay ( LAP) is a nanomaterial with a lamellar structure that has a negative surface charge and a positive edge charge. It has a diameter of 25nm and a thickness of about 1nm. The lamellar structure of LAP is easily intercalated by DA small molecules. At the same time, the weakly alkaline oxygen-containing environment of the LAP dispersion can directly polymerize DA to form PDA, and no other strong oxidants need to be introduced during the reaction. DA monomers are adsorbed on LAP nanosheets. After oxidative polymerization, some of the remaining catechol groups can form hydrogen bonds with the functional groups of the polymer macromolecules, or undergo Michael addition, Schiff base reaction, etc. In addition, due to its anisotropic charge, LAP can form a dynamic hydrogel network with polymer molecules through supramolecular interactions. Summary of the invention

[0004] In order to solve the problems of the prior art, the present invention provides an injectable conductive nanocomposite hydrogel with high stability and a preparation method thereof. Dopamine monomer is prepolymerized in a weakly alkaline oxygen-containing environment using nanoclay to obtain polydopamine-functionalized PDA@LAP nanosheets, gelatin is grafted with dopamine and then mixed evenly with the nanosheets and a high molecular conductive polymer PEDOT:PSS, and an injectable conductive nanocomposite hydrogel is constructed based on non-covalent supramolecular interactions.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] A method for preparing an injectable conductive nanocomposite hydrogel with high stability comprises the following steps:

[0007] (1) Add dopamine hydrochloride to the diluted Tris-HCl solution and stir until it is completely dissolved.

[0008] The dilution multiple is 8 to 10 times;

[0009] The stirring condition is 300-500 rpm;

[0010] (2) Add nanoclay to the product and stir at room temperature in the dark, and then perform subsequent treatment to obtain PDA@LAP nanosheets.

[0011] The mass ratio of dopamine hydrochloride to nanoclay is 0.4:1;

[0012] The reaction time is 20 to 25 hours;

[0013] The subsequent treatment is to centrifuge at 10000 rpm for 5 minutes, discard the supernatant, and freeze-dry the precipitate.

[0014] (3) Add gelatin to phosphate buffer, stir until completely dissolved, then add dopamine hydrochloride, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide under gas protection, stir to react, and subsequently treat to obtain gelatin-dopamine.

[0015] The mass ratio of gelatin to dopamine hydrochloride is 1:1;

[0016] The molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 522:521;

[0017] The stirring condition is 300-500 rpm;

[0018] The reaction time is 20 to 25 hours;

[0019] The reaction temperature is 30-40°C;

[0020] The gas is nitrogen, and the reaction is carried out under the protection of nitrogen flow;

[0021] The subsequent treatment is to dialyze the product in deionized water for 7 days and then freeze-dry it;

[0022] The pH value of the solution was maintained at 5.5 during the reaction.

[0023] (4) Add the PDA@LAP nanosheets prepared in step (2) into the PEDOT:PSS dispersion and stir until the reaction is complete to obtain PDA@LAP / PEDOT:PSS.

[0024] The reaction time is 6 to 10 hours;

[0025] The stirring condition is 200-400 rpm;

[0026] (5) Adding the gelatin-dopamine prepared in step (3) to the PDA@LAP / PEDOT:PSS prepared in step (4), stirring the reaction to obtain the injectable conductive nanocomposite hydrogel.

[0027] The reaction time is 20 to 25 hours;

[0028] The reaction temperature is 35-40°C;

[0029] The stirring condition is 100-300 rpm.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] The present invention introduces polydopamine-functionalized PDA@LAP nanosheets into dopamine-grafted gelatin and conductive polymer PEDOT:PSS, and constructs an injectable conductive nanocomposite hydrogel through non-covalent supramolecular interactions. The problems of poor durability and conductivity and lack of biological activity of existing injectable conductive hydrogels are solved. The introduction of non-covalent supramolecular interactions can give the hydrogel excellent injectability, and can fill irregular geometric lesions through minimally invasive injection, avoiding the trauma of traditional implant surgery, and has significant advantages in clinical applications; in addition, the constructed nanocomposite hydrogel has excellent physiological stability, self-healing properties and conductive properties, and can be used in the fields of electrical stimulation therapy, drug delivery, tissue engineering and regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0033] Figure 1 This is a macroscopic photograph of the injectable conductive nanocomposite hydrogel of the present invention;

[0034] Figure 2 This is a SEM photo of the injectable conductive nanocomposite hydrogel provided by the present invention;

[0035] Figure 3 To evaluate the electrical properties of the injectable conductive nanocomposite hydrogel of the present invention;

[0036] Figure 4 To evaluate the shear thinning properties of the injectable conductive nanocomposite hydrogel of the present invention;

[0037] Figure 5 To evaluate the self-healing properties of the injectable conductive nanocomposite hydrogel of the present invention;

[0038] Figure 6 To evaluate the stability of the injectable conductive nanocomposite hydrogel of the present invention;

[0039] Figure 7 To evaluate the tissue adhesion performance of the injectable conductive nanocomposite hydrogel of the present invention;

[0040] Figure 8 The blood compatibility of the injectable conductive nanocomposite hydrogel of the present invention was evaluated. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0042] Example 1

[0043] A method for preparing an injectable conductive nanocomposite hydrogel with high stability comprises the following steps:

[0044] (1) Add 0.4 g dopamine hydrochloride to 200 mL Tris-HCl solution diluted 10 times, and stir at 300 rpm until completely dissolved;

[0045] (2) 1.0 g of nanoclay was added to the reaction solution in step (1), and the mixture was magnetically stirred at 300 rpm for 24 hours at room temperature in a dark place, and then the resulting solution was centrifuged at 10,000 rpm for 5 minutes, the supernatant was discarded, and the resulting product was freeze-dried.

[0046] (3) Add 2.0 g of gelatin into PBS buffer and stir until completely dissolved;

[0047] (4) Weigh 2.0 g dopamine hydrochloride, 1.0 g EDC and 0.6 g NHS, add them to the reaction solution of step (3) under nitrogen protection, and stir at 37° C. for 24 hours;

[0048] (5) dialyzing the solution obtained in step (4) in deionized water for 48 hours and then freeze-drying;

[0049] (6) Weigh 0.04 g of the product obtained in step (2) and add it to 4 mL of PEDOT:PSS and stir for more than 6 hours until it is evenly dispersed;

[0050] (7) Weigh 160 mg of the product of step (5) and add it to step (6). After stirring at 37° C. for 24 hours, the GPLP injectable conductive nanocomposite hydrogel is obtained.

[0051] Example 2

[0052] A method for preparing an injectable conductive nanocomposite hydrogel with high stability comprises the following steps:

[0053] (1) Weigh 0.4 g dopamine hydrochloride and add it to 200 mL Tris-HCl solution diluted 10 times, and stir magnetically at 300 rpm until it is completely dissolved;

[0054] (2) Add 1.0 g of nanoclay to the reaction solution of step (1), stir magnetically at 300 rpm for 24 hours at room temperature in a dark environment, then centrifuge at 10,000 rpm for 5 minutes, discard the supernatant, and freeze-dry the precipitate.

[0055] (3) Add 2.0 g of gelatin into PBS buffer and stir until completely dissolved;

[0056] (4) Weigh 2.0 g dopamine hydrochloride, 1.0 g EDC and 0.6 g NHS, add them to the solution in step (3) under nitrogen protection, and stir magnetically at 37° C. for 24 hours;

[0057] (5) The reaction solution obtained in step (4) was dialyzed in deionized water for 48 hours, and then freeze-dried and stored at -20°C for future use;

[0058] (6) Weigh 0.04 g of the product obtained in step (2) and add it to 4 mL of PEDOT:PSS and stir for more than 6 hours until it is evenly dispersed;

[0059] (7) 240 mg of the product of step (5) was added to step (6), and magnetic stirring was performed at 37° C. for 24 hours to obtain the GPLP injectable conductive nanocomposite hydrogel.

[0060] Example 3

[0061] A method for preparing an injectable conductive nanocomposite hydrogel with high stability comprises the following steps:

[0062] (1) Weigh 0.4 g dopamine hydrochloride and add it to 200 mL Tris-HCl solution diluted 10 times, and stir with a magnetic stirrer at 300 rpm until it is completely dissolved;

[0063] (2) Add 1.0 g of nanoclay to the reaction solution in step (1), stir magnetically at 300 rpm for 24 hours at room temperature in a dark place, centrifuge the resulting solution at 10,000 rpm for 5 minutes, discard the supernatant, and freeze-dry the resulting precipitate and store it at -20°C for later use.

[0064] (3) Add 2.0 g of gelatin into PBS buffer and stir until fully dissolved;

[0065] (4) 2.0 g dopamine hydrochloride, 1.0 g EDC and 0.6 g NHS were weighed and added to the reaction solution of step (3) under nitrogen protection, and magnetically stirred at 37° C. for 24 hours;

[0066] (5) The reaction solution obtained in step (4) was dialyzed in deionized water for 48 hours, and then freeze-dried and stored at -20°C for future use;

[0067] (6) Weigh 0.04 g of the product obtained in step (2) and add it to 4 mL of PEDOT:PSS and stir for more than 6 hours until it is evenly dispersed;

[0068] (7) 320 mg of the product of step (5) was added to step (6), and the mixture was stirred at 37° C. for 24 hours to obtain the GPLP injectable conductive nanocomposite hydrogel.

[0069] The actual image of the injectable conductive nanocomposite hydrogel prepared in this example is as follows Figure 1 As shown in the figure, except for the 1% gelatin-dopamine group, which could not form a gel state, the other groups could return to the gel state after a suitable gelation time and remain stable. Figure 2 As shown in the figure, the microstructure and element distribution show that the hydrogel has a porous structure and the components are dispersed evenly. Figure 3 As shown in the figure, the electrochemical impedance of the hydrogels with different concentrations was obtained by AC impedance measurement. According to the conductivity formula (σ=L / RS), the conductivity of GPLP4, GPLP6 and GPLP8 were 0.52mS / cm, 0.63mS / cm and 0.47mS / cm respectively, indicating that the hydrogels have conductivity. The shear thinning performance of the injectable conductive hydrogel prepared in this example is evaluated as follows: Figure 4 As shown in Figure 2, as the shear rate increases, the viscosity of the conductive hydrogel gradually decreases, indicating that it has excellent injectability. Figure 5 As shown in Figure 2, with repeated mutations in strain, the gel network was destroyed and then restored, indicating that the GPLP injectable conductive nanocomposite hydrogel has self-healing properties. Figure 6 As shown in the figure, the strong interaction between the polydopamine functionalized nanosheets and PEDOT:PSS makes the hydrogel highly stable. The GPLP6 hydrogel prepared in this example was immersed in PBS buffer. After three weeks, the material showed no obvious changes except fading. The tissue adhesion performance of the GPLP prepared in this example was evaluated as follows: Figure 7 As shown in the figure, the GPLP hydrogel adhered to the rat sciatic nerve and showed good adhesion after continuous washing. The adhesion strength of GPLP at different concentrations was tested, and the highest value was 3.5±0.03kPa. Figure 8 As shown, the hemolysis ratios of the GPLP hydrogels of different mass concentrations prepared in this experimental example were all below 5%, indicating that they had excellent blood compatibility.

[0070] Conclusion: A highly stable injectable conductive nanocomposite hydrogel was successfully prepared. The conductive hydrogel has excellent shear thinning, self-repair, tissue adhesion, stability, conductivity and biocompatibility, and can be used in electrical stimulation therapy, tissue engineering and regenerative medicine.

[0071] Finally, it is noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included therein.

Claims

1. A method for preparing an injectable conductive nanocomposite hydrogel with high stability, characterized in that: The following steps are involved: Dopamine hydrochloride was oxidized in the weak alkaline oxygen-containing microenvironment of nanoclay to obtain a polydopamine-functionalized nanosheet, named PDA@LAP nanosheet. Subsequently, gelatin and dopamine were grafted to obtain dopamine-grafted gelatin. PDA@LAP was introduced into the dopamine-grafted gelatin and conductive polymer, which were combined with each other through the supramolecular action of non-covalent bonds to finally form an injectable conductive nanocomposite hydrogel.

2. The method for preparing the highly stable injectable conductive nanocomposite hydrogel according to claim 1, characterized in that: The preparation method of the PDA@LAP nanosheet comprises the following steps: (1) adding dopamine hydrochloride to the diluted Tris-HCl solution and stirring until completely dissolved to obtain solution 1; (2) adding nanoclay to solution 1 of step (1), mechanically stirring at room temperature in the dark for 20 to 25 hours to obtain solution 2; wherein the mass ratio of dopamine hydrochloride to nanoclay is 0.4:1; (3) Centrifuge the solution 2 in step (2) for 5 to 10 minutes, freeze-dry the resulting precipitate, seal it and store it at -15 to -20°C; wherein the centrifugal speed is 8000 to 10000 rpm.

3. The method for preparing the highly stable injectable conductive nanocomposite hydrogel according to claim 1, characterized in that: The preparation method of gelatin-dopamine comprises the following steps: (1) adding gelatin into phosphate buffered saline solution (PBS, pH=7.4, 0.1 M) and stirring until completely dissolved to obtain solution 1; (2) nitrogen is introduced into the solution 1 of step (1), dopamine hydrochloride is added under nitrogen protection, and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added, and the mixture is stirred at 30-40° C. for 20-25 hours to obtain a solution 2; wherein the mass ratio of gelatin to dopamine hydrochloride is 1:1; the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 522:521; during the reaction, the pH value of the solution 2 is maintained at 5.5; (3) The solution 2 in step (2) was dialyzed in deionized water for 40 to 50 hours and then freeze-dried.

4. The method for preparing the highly stable injectable conductive nanocomposite hydrogel according to claim 1, characterized in that: The preparation method of the GPLP hydrogel comprises the following steps: (1) adding the nanosheets prepared in claim 2 into a dispersion of a conductive polymer poly (3,4-ethylenedithiophene) / polystyrene sulfonate (PEDOT:PSS) and stirring for 6 to 10 hours to obtain a solution 1; (2) Adding different concentrations of gelatin-dopamine prepared in claim 3 to solution 1 of step (1), stirring at 35-40° C. for 20-25 hours to obtain GPLP of different concentrations.

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