CN112979998B

CN112979998B - Anisotropic conductive hydrogel and preparation method and application thereof

Info

Publication number: CN112979998B
Application number: CN202110208875.0A
Authority: CN
Inventors: 白硕; 李洁龄
Original assignee: Zhongke Nanjing Green Manufacturing Industry Innovation Research Institute; Institute of Process Engineering of CAS
Current assignee: Zhongke Nanjing Green Manufacturing Industry Innovation Research Institute; Institute of Process Engineering of CAS
Priority date: 2021-02-24
Filing date: 2021-02-24
Publication date: 2023-09-05
Anticipated expiration: 2041-02-24
Also published as: CN112979998A

Abstract

The invention provides an anisotropic conductive hydrogel, a preparation method and application thereof, wherein the anisotropic conductive hydrogel comprises a conductive supporting material, a conductive active substance, an initiator, a catalyst and water; the mass fraction of the conductive supporting material is 5-30%; the concentration of the conductive active material is 5-25 mg/mL; the concentration of the initiator is 0.5-5 mg/mL; the concentration of the catalyst is 0.5-5 mM. The anisotropic conductive hydrogel provided by the invention has an oriented geometric structure and good oriented conductive capability, and has important significance for repairing damaged tissues through tissue engineering.

Description

Anisotropic conductive hydrogel and preparation method and application thereof

Technical Field

The invention belongs to the field of medical materials, and particularly relates to an anisotropic conductive hydrogel and a preparation method and application thereof, in particular to an anisotropic conductive hydrogel with good conductive effect and a preparation method and application thereof.

Background

Aiming at the problem of limited self-repairing capability of biological tissues after serious injury, tissue engineering rapidly develops. Tissue engineering refers to a technology for collecting seed cells from a patient or a donor, constructing functional tissues which can be implanted into the patient through in-vitro culture or tissue engineering scaffold culture, repairing and replacing defective or pathological tissues and promoting functional recovery. The hydrogel is a soft material with high water content and is formed by a three-dimensional network structure, and is widely used as a tissue engineering scaffold due to the characteristics of good biocompatibility, good response to external stimulus, mechanical properties matched with human tissues and the like.

As a tissue engineering scaffold, the hydrogel not only can meet the requirement that the mechanical property is matched with human tissue, but also can consider the function and structural characteristics of special tissue. For example, natural tissues or organs are generally composed of cells in a certain spatial distribution and arrangement, and the distribution and orientation of the cells within the tissue is critical to achieving a specific function of the tissue. Taking skeletal muscle as an example, in order to achieve high strength stretch and contraction resistance, the muscle cells constituting skeletal muscle are arranged highly in parallel in a bundled manner. When skeletal muscle is damaged, the satellite cells in a silent state are also re-differentiated into highly aligned myotubes along the direction of the myocells under the constraint of the fasciculated muscle cells, so that the self-repair of the muscle tissue function is realized. In addition, the nerve tissue and the muscle tissue with the conductive performance have sensitivity to electric signals, and the recovery of damaged nerve and muscle tissue can be effectively promoted by applying external electric field stimulation. However, there are relatively few studies currently conducted to direct cell directional alignment growth in combination with geometric constraints and electric field stimulation.

CN106111193B discloses a preparation method of silver nanoparticle loaded catalyst hydrogel, which comprises the following steps: mixing and dissolving a high molecular monomer, a cross-linking agent, a photoinitiator and monovalent silver ion salt in water according to a certain proportion, adding the uniformly mixed solution into a quartz mold, and irradiating for a certain time under ultraviolet light to obtain hydrogel loaded with silver nano particles; silver nano particles in the nano composite hydrogel are uniformly distributed, and the average size is 2-3nm; no surfactant is added in the reaction process, and the silver nano particles have clean surfaces, high surface activity and excellent catalytic performance; the silver-loaded nanoparticle catalyst hydrogel has excellent catalytic activity in experiments of degrading organic dyes such as p-nitrophenol, methyl orange or methylene blue by sodium borohydride, and secondary pollution is not caused after the reaction is completed; the catalyst still maintains good catalytic effect after repeated cyclic use; the nano composite hydrogel has few preparation steps, is convenient to store and transport after being dried, and is beneficial to industrial practical application. But it is difficult to apply to the field of tissue repair.

CN105294934B discloses a method for preparing a high-strength antibacterial hydrogel, which comprises the following steps: dissolving maleic acid, acrylamide, carboxymethyl chitosan, a cross-linking agent, an initiator and a catalyst in water solution by using cooled boiled distilled water, rapidly transferring the prepared solution into a glass mold, and placing the sealed mold in an environment of 45-55 ℃ for heat preservation reaction for 2-6 hours; then taking out the hydrogel formed in the mould, soaking the hydrogel in a soluble metal salt solution with the concentration of 0.05-0.25M for 24 hours, and taking out the hydrogel to obtain the high-strength antibacterial hydrogel. The hydrogel provided by the invention has a good antibacterial effect while showing a certain mechanical strength, so as to meet the application requirements of the hydrogel in aspects of cartilage repair, tissue engineering and the like of a human body.

Because the research of guiding the directional arrangement growth of cells by combining geometric constraint and electric field stimulation is relatively few at present, the preparation of the hydrogel with directional arrangement structure and directional electric conduction capability, and the directional arrangement of cells is guided by the synergistic effect of physical constraint and directional electric field stimulation, the hydrogel has important significance for promoting the recovery of muscle tissues and nerve tissues. Therefore, how to provide an anisotropic conductive hydrogel with good conductive effect is a problem to be solved.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide an anisotropic conductive hydrogel and a preparation method and application thereof, in particular to an anisotropic conductive hydrogel with good conductive effect and a preparation method and application thereof. The anisotropic conductive hydrogel provided by the invention has an oriented geometric structure and good oriented conductive capability, and has important significance for repairing damaged tissues through tissue engineering.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

in a first aspect, the present invention provides an anisotropically conductive hydrogel comprising a conductive support material, a conductive active material, an initiator, a catalyst, and water.

The mass fraction of the conductive supporting material is 5-30%.

The concentration of the conductive active material is 5-25 mg/mL.

The concentration of the initiator is 0.5-5 mg/mL.

The concentration of the catalyst is 0.5-5 mM.

The mass fraction of the conductive support material may be 5%, 10%, 15%, 20%, 25% or 30%, the concentration of the conductive active material may be 5mg/mL, 10mg/mL, 15mg/mL, 20mg/mL or 25mg/mL, etc., the concentration of the initiator may be 0.5mg/mL, 1mg/mL, 1.5mg/mL, 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, 4mg/mL, 4.5mg/mL or 5mg/mL, etc., the concentration of the catalyst may be 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM or 5mM, etc., but the concentrations of the initiator are not limited to the values listed above, and other values not listed in the above numerical ranges may be equally applicable.

The anisotropic conductive hydrogel prepared by the specific components has a directional geometric structure and good directional conductive capacity, can guide directional migration, proliferation and differentiation of cells through the synergistic effect of the geometric structure and a directional electric field, and has important significance for repairing damaged tissues through tissue engineering.

Preferably, the conductive active material includes any one or a combination of at least two of silver nanowires (AgNW), carbon nanotubes, polyaniline, polypyrrole, or polythiophene, for example, a combination of silver nanowires and carbon nanotubes, a combination of silver nanowires and polyaniline, or a combination of carbon nanotubes and polyaniline, etc., but not limited to the above-listed combinations, other non-listed combinations within the above-listed combinations are equally applicable, and preferably a combination of carbon nanotubes and polyaniline.

The selection and combination of the specific conductive active substances can obviously improve the conductivity of the anisotropic conductive hydrogel, and the conductivity can be up to 1.05x10 ^-2 S/cm, the directional arrangement of cells can be better guided, and the repair of damaged tissues is realized.

Preferably, the conductive support material includes any one or a combination of at least two of GelMA (methacrylic acid acylated gelatin), acrylamide, polyethylene glycol acrylic acid or acrylic acid, for example, a combination of GelMA and acrylamide, a combination of GelMA and acrylic acid, or a combination of GelMA and polyethylene glycol acrylic acid, etc., but not limited to the above-listed combinations, other non-listed combinations within the above-listed combinations are equally applicable.

Preferably, the initiator comprises ammonium persulfate and/or potassium persulfate.

Preferably, the catalyst comprises N, N-tetramethyl ethylenediamine (TEMED).

In a second aspect, the present invention provides a method for preparing an anisotropic conductive hydrogel as described above, the method comprising the steps of:

(1) Mixing conductive active substances with an aqueous solution of a conductive support material, performing ultrasonic treatment, and then mixing with an initiator and a catalyst to obtain a prepolymer solution;

(2) Transferring the prepolymer solution obtained in the step (1) into a mold, then transferring to the surface of a precooled substrate, and standing to obtain a directionally frozen prepolymer;

(3) And (3) standing and polymerizing the directional frozen prepolymer obtained in the step (2), heating and thawing, and cleaning to obtain the anisotropic conductive hydrogel.

The preparation method can be used for rapidly and conveniently preparing the anisotropic conductive hydrogel to have an oriented geometric structure.

Preferably, the temperature of the pre-cooled substrate in the step (2) is between-80 ℃ and-40 ℃.

Preferably, the time of standing in the step (2) is 1-20min.

Preferably, the substrate of step (2) comprises a copper block.

Preferably, the polymerization temperature in the step (3) is-25 to-15 ℃ and the time is 12-48h.

The temperature of the pre-cooled substrate may be-80 ℃, -70 ℃, -60 ℃, -50 ℃ or-40 ℃, and the like, the standing time may be 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min or 20min, and the like, the polymerization temperature may be-25 ℃, -24 ℃, -23 ℃, -22 ℃, -21 ℃, -20 ℃, -19 ℃, -18 ℃, -17 ℃, -16 ℃ or-15 ℃, and the like, and the standing time may be 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h or 48h, and the like, but the method is not limited to the above-listed values, and other non-listed values within the above range are applicable.

As a preferable technical scheme of the invention, the preparation method comprises the following steps:

(2) Transferring the prepolymer solution obtained in the step (1) into a mold, then transferring to the surface of a precooled substrate, and standing for 1-20min to obtain a directionally frozen prepolymer;

(3) And (3) standing and polymerizing the directionally frozen prepolymer obtained in the step (2) for 12-48 hours at the temperature of minus 25-minus 15 ℃, then heating and thawing, and cleaning to obtain the anisotropic conductive hydrogel.

In a third aspect, the invention also provides the use of an anisotropically conductive hydrogel as described above for the preparation of a repair material for damaged tissue.

Compared with the prior art, the invention has the following beneficial effects:

the invention prepares the anisotropic conductive hydrogel by selecting specific components, which has directional geometry and good directional conductivity, and can be prepared by the synergistic effect of the geometry and the directional electric fieldGuiding directional migration, proliferation and differentiation of cells, and has important significance for realizing the repair of damaged tissues through tissue engineering; the conductivity of the anisotropic conductive hydrogel can be significantly improved by selecting specific conductive active substances and combinations thereof, and the conductivity can reach 1.05X10 at most ^-2 S/cm, the directional arrangement of cells can be better guided, and the repair of damaged tissues is realized.

Drawings

FIG. 1 is a cross-sectional scanning electron microscope picture of the anisotropic conductive hydrogel provided in example 1;

FIG. 2 is a longitudinal section scanning electron microscope image of the anisotropic conductive hydrogel provided in example 1;

FIG. 3 is the conductivity test results of the anisotropic conductive hydrogel provided in example 2;

FIG. 4 is the conductivity test results of the anisotropic conductive hydrogel provided in example 7;

FIG. 5 is the conductivity test results of the anisotropic conductive hydrogel provided in example 8;

FIG. 6 is the conductivity test results of the anisotropic conductive hydrogel provided in example 9;

FIG. 7 is the conductivity test results of the anisotropic conductive hydrogel provided in example 10;

FIG. 8 is the results of conductivity testing of the anisotropic conductive hydrogels provided in example 11;

FIG. 9 is a image of the fluorescence of cells of a control group in a cell induction test;

FIG. 10 is a fluorescence image of cells of example 1 set in a cell induction test.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

In the following test experiments, murine myoblasts were purchased from Shanghai Enzymogen Biotechnology Inc., model CC-Y2024.

Example 1

The embodiment provides an anisotropic conductive hydrogel, which is prepared by the following steps:

(1) Stirring and dissolving GelMA in a water bath at 60 ℃ to obtain a GelMA aqueous solution with the mass fraction of 5 wt%;

(2) Dropwise adding the concentrated AgNW ethanol dispersion liquid into the GelMA aqueous solution in the step (1), wherein each drop of the concentrated AgNW ethanol dispersion liquid is subjected to ultrasonic treatment for 30 seconds, so that the final concentration of the AgNW is 5mg/mL, and continuing ultrasonic treatment for 10 minutes after the drop of the AgNW is completely added, so as to obtain a dispersion liquid;

(3) Adding an initiator Ammonium Persulfate (APS) and a catalyst TEMED into the dispersion liquid obtained in the step (2), and carrying out ultrasonic treatment for 1min to uniformly mix the APS and the TEMED with the solution to obtain a prepolymer solution, wherein the final concentration of the APS is 1mg/mL, and the final concentration of the TEMED is 0.5mg/mL;

(4) Pre-cooling a copper block in liquid nitrogen for 1h, transferring the prepolymer solution obtained in the step (3) into a self-made PTFE (polytetrafluoroethylene) mould, slowly transferring the mould onto the surface of the pre-cooled copper block (-80 ℃), and standing for 2min to obtain a directional frozen product;

(5) And (3) rapidly transferring the directional frozen product obtained in the step (4) into a refrigerator at the temperature of minus 20 ℃, standing for 24 hours, thawing the sample at the temperature of 20 ℃, soaking and washing the sample with deionized water for 3 times, and removing redundant initiator and catalyst to obtain the anisotropic conductive hydrogel, wherein the scanning electron microscope pictures of the cross section and the longitudinal section of the anisotropic conductive hydrogel are respectively shown in figures 1 and 2.

Example 2

(1) Stirring and dissolving GelMA in a water bath at 60 ℃ to obtain a GelMA aqueous solution with the mass fraction of 10 wt%;

(2) Dropwise adding the concentrated AgNW ethanol dispersion liquid into the GelMA aqueous solution in the step (1), wherein each drop of the concentrated AgNW ethanol dispersion liquid is subjected to ultrasonic treatment for 30 seconds, so that the final concentration of the AgNW is 10mg/mL, and continuing ultrasonic treatment for 10 minutes after the drop of the AgNW is completely added, so as to obtain a dispersion liquid;

(3) Adding an initiator Ammonium Persulfate (APS) and a catalyst TEMED into the dispersion liquid obtained in the step (2), and carrying out ultrasonic treatment for 1min to uniformly mix the APS and the TEMED with the solution to obtain a prepolymer solution, wherein the final concentration of the APS is 3mg/mL, and the final concentration of the TEMED is 1mg/mL;

(4) Pre-cooling a copper block in liquid nitrogen for 2 hours, transferring the prepolymer solution obtained in the step (3) into a self-made PTFE (polytetrafluoroethylene) mould, slowly transferring the mould onto the surface of the pre-cooled copper block (-80 ℃), and standing for 1min to obtain a directional frozen product;

(5) And (3) rapidly transferring the directional frozen product obtained in the step (4) into a refrigerator at the temperature of minus 20 ℃, standing for 48 hours, thawing the sample at the temperature of 20 ℃, soaking and washing the sample with deionized water for 3 times, and removing redundant initiator and catalyst to obtain the anisotropic conductive hydrogel.

Example 3

This example provides an anisotropically conductive hydrogel, which was prepared in the same manner as in example 1, except that AgNW was replaced with an equal amount of carbon nanotubes.

Example 4

This example provides an anisotropically conductive hydrogel, which was prepared in the same manner as in example 1, except that AgNW was replaced with an equivalent amount of acrylamide monomer.

Example 5

This example provides an anisotropic conductive hydrogel, which was prepared in the same manner as in example 4, except that the mass fraction of the aqueous acrylamide solution was 15%.

Example 6

This example provides an anisotropic conductive hydrogel, which was prepared in the same manner as in example 4, except that the mass fraction of the aqueous acrylamide solution was 20%.

Example 7

This example provides an anisotropically conductive hydrogel, which was prepared in the same manner as in example 2, except that AgNW was replaced with an equal amount of carbon nanotubes.

Example 8

This example provides an anisotropically conductive hydrogel, which was prepared in the same manner as in example 2, except that AgNW was replaced with an equivalent amount of polyaniline.

Example 9

This example provides an anisotropic conductive hydrogel, which was identical to example 2 except that AgNW was replaced with carbon nanotubes and silver nanowires in the preparation step, and the final concentrations of carbon nanotubes and silver nanowires were 2mg/mL and 8mg/mL, respectively.

Example 10

This example provides an anisotropic conductive hydrogel, which was identical to example 2 except that AgNW was replaced with polyaniline and silver nanowires in the preparation step, and the final concentrations of polyaniline and silver nanowires were 2mg/mL and 8mg/mL, respectively.

Example 11

This example provides an anisotropic conductive hydrogel, which was prepared in the same manner as in example 2, except that AgNW was replaced with polyaniline and carbon nanotubes, and the final concentrations of polyaniline and carbon nanotubes were 2mg/mL and 8mg/mL, respectively.

Comparative example 1

The comparative example provides a conductive hydrogel, which is prepared by the following steps:

(4) And (3) rapidly transferring the prepolymer solution obtained in the step (3) into a refrigerator at the temperature of minus 20 ℃, standing for 24 hours, thawing the sample at the temperature of 20 ℃, soaking and washing the sample with deionized water for 3 times, and removing redundant initiator and catalyst to obtain the conductive hydrogel.

Conductivity test:

the anisotropic conductive hydrogels provided in examples 2, 7-11 were subjected to conductivity testing using an electrochemical workstation, and the results are shown in fig. 3-8 and the following table:

from the figures and tables, it can be seen that within the range of the preferred raw materials of the invention, the conductivity of the anisotropic conductive hydrogel is significantly improved, which indicates that the anisotropic conductive hydrogel can better guide the directional arrangement of cells and realize the repair of damaged tissues.

Cell induction test:

the conductive hydrogel provided in comparative example 1 and the anisotropic conductive hydrogel provided in example 1 were subjected to cell induction test, respectively, in the control group and the example 1 group, as follows:

(1) Soaking the hydrogel in 75% ethanol for 12h and ultraviolet radiation for 2h respectively for sterilization;

(2) Soaking the sterilized hydrogel in a cell culture medium (DMEM) for 12 hours, and changing the liquid for multiple times during the period to ensure that the hydrogel is completely in a fresh culture medium environment;

(3) Inoculating newly digested murine myoblasts on the surface of the hydrogel soaked in the cell culture medium in the step (2), and culturing for 4 hours at 37 ℃;

(4) Electrically stimulating (2V, 3 Hz) myoblasts spread on the surface of the hydrogel in the step (3) by using an electric stimulation device for 1h, and then continuously culturing; repeating the electric stimulation for 1 time every day for two subsequent days, and performing 3 electric stimulation experiments on myoblasts;

(5) The cells treated by the electrical stimulation are fluorescently labeled with a live cell dye, and the stained cells are observed by a confocal microscope to characterize survival and directional growth of the muscle cells under the electrical stimulation.

The results are shown in fig. 9 and 10, wherein fig. 9 is a control group, fig. 10 is a group of example 1, and the arrow in fig. 10 indicates the induction direction.

As can be seen from fig. 9 to 10, the anisotropic conductive hydrogel provided by the invention can significantly induce directional migration and arrangement of cells, and can realize repair of damaged tissues when applied to the damaged tissues.

The applicant states that the anisotropic conductive hydrogel of the present invention, and the preparation method and application thereof, are illustrated by the above examples, but the present invention is not limited to the above examples, i.e., it does not mean that the present invention must be practiced by relying on the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims

1. The anisotropic conductive hydrogel is characterized in that the anisotropic conductive hydrogel is prepared from conductive supporting materials, conductive active substances, an initiator, a catalyst and water;

the mass fraction of the conductive supporting material is 5-30%;

the concentration of the conductive active material is 5-25 mg/mL;

the concentration of the initiator is 0.5-5 mg/mL;

the concentration of the catalyst is 0.5-5 mM;

the conductive active material is a combination of carbon nanotubes and polyaniline;

the conductive supporting material is GelMA;

the anisotropic conductive hydrogel is prepared by a method comprising the following steps:

2. The anisotropic conductive hydrogel of claim 1, wherein the initiator comprises ammonium persulfate and/or potassium persulfate.

3. The anisotropic conductive hydrogel of claim 1, wherein the catalyst comprises N, N-tetramethyl ethylenediamine.

4. A method for preparing an anisotropic conductive hydrogel according to any of claims 1-3, comprising the steps of:

5. The method of preparing an anisotropic conductive hydrogel according to claim 4, wherein the pre-chilled substrate in step (2) has a temperature of-80 to-40 ℃.

6. The method of producing an anisotropic conductive hydrogel according to claim 4, wherein the time of the standing in step (2) is 1 to 20 minutes.

7. The method of preparing an anisotropic conductive hydrogel according to claim 4, wherein the substrate of step (2) comprises copper blocks.

8. The method for preparing anisotropic conductive hydrogel according to claim 4, wherein the polymerization temperature in step (3) is-25 to-15 ℃ and the time is 12 to 48 hours.

9. The method for preparing an anisotropic conductive hydrogel according to claim 4, comprising the steps of:

10. Use of an anisotropically conductive hydrogel according to any one of claims 1-3 for the preparation of a repair material for damaged tissue.