CN114470316B - Conductive inverse opal film for inducing nerve cells and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a conductive inverse opal film for inducing nerve cells and a preparation method and application thereof, wherein the preparation method of the conductive inverse opal film comprises the following steps: step one, preparing a nano-particle colloidal crystal array template, filling gaps of the template with a hydrogel precursor solution, and removing the template after curing to obtain an inverse opal substrate; stretching the obtained inverse opal substrate along different angles in the linear direction of the single-row holes of the inverse opal substrate to obtain an inverse opal film with anisotropic elliptical porous patterns; and step three, preparing a conductive hydrogel precursor solution, filling the conductive hydrogel precursor solution into the inverse opal film with the anisotropic elliptical porous pattern, and curing the conductive inverse opal film with the anisotropic elliptical porous pattern by using ultraviolet light to obtain the conductive inverse opal film with the anisotropic elliptical porous pattern. The invention has the advantages of simple operation, low cost, good induction effect and the like.
Description
Technical Field
The invention belongs to the field of biological materials, relates to a film material, and particularly relates to a conductive inverse opal film for inducing nerve cells, and a preparation method and application thereof.
Background
The nervous system plays a potential central role in human life, and can properly regulate the physiological functions of the body according to the change of the environment, thereby realizing the unification of the body and the environment. Neuronal damage and degeneration can lead to permanent tissue damage and severe functional impairment, which is unavoidable in humans. However, the inherent self-regeneration ability of nerve tissue is limited, and thus the repair and regeneration of damaged nerve tissue has received a great deal of attention. There have been many studies aimed at solving these problems, in which nerve-oriented induction is considered as an effective method. In this regard, substrate materials with surface topography have great potential in nerve orientation. Despite many advances made by using biomaterials with surface topographies, challenges remain in achieving neuronal induction due to the softness of these biomaterials. In contrast, patterned rigid substrates, while exhibiting directional induction potential, often hinder the survival and proliferation of nerve cells due to inconsistencies between the substrate and the organism in vivo. In addition, the unidirectional induction scheme is mostly not beneficial to the rapid formation of the neural network. Therefore, it would still be desirable to have new methods for designing neural induction to build complex nervous systems.
Disclosure of Invention
The invention provides a conductive inverse opal film for inducing nerve cells, and a preparation method and application thereof, aiming at overcoming the defects of the prior art.
To achieve the above objects, the present invention provides a method for preparing a conductive inverse opal film for inducing nerve cells, having the following features: the method comprises the following steps:
step one, preparing an inverse opal substrate: obtaining a highly ordered self-assembled nano-particle colloidal crystal array template by using a vertical deposition method, filling gaps of the nano-particle colloidal crystal array template with a hydrogel precursor solution, and removing the nano-particle colloidal crystal array template after solidification to obtain an inverse opal substrate;
step two, preparing the inverse opal film with the anisotropic elliptical porous pattern: stretching the obtained inverse opal substrate along different angles in the linear direction of the single-row holes of the inverse opal substrate to obtain an inverse opal film with anisotropic elliptical porous patterns;
step three, preparing the conductive inverse opal film with the anisotropic elliptical porous pattern: preparing a conductive hydrogel precursor solution, filling the inverse opal film with the anisotropic elliptical porous pattern with the conductive hydrogel precursor solution, and curing by using ultraviolet light to obtain the conductive inverse opal film with the anisotropic elliptical porous pattern.
Further, the present invention provides a method for preparing a conductive inverse opal film for inducing nerve cells, which may further have the following characteristics: in the first step, the vertical deposition method comprises the following specific steps: vertically inserting the cleaned glass slide into an ethanol solution of monodisperse nanoparticles; the concentration of the ethanol solution of the monodisperse nano particles is 1-2wt%; under the environment of constant temperature and humidity, obtaining a self-assembled nano-particle colloidal crystal array template with ordered height after the solvent is volatilized; the constant temperature and humidity conditions are that the temperature is 30-40 ℃ and the humidity is 50-60%.
Further, the present invention provides a method for preparing a conductive inverse opal film for inducing nerve cells, which may further have the following characteristics: in the first step, the nanoparticles in the nanoparticle colloidal crystal array template are silicon dioxide.
Further, the present invention provides a method for preparing a conductive inverse opal film for inducing nerve cells, which may further have the following characteristics: in the first step, the hydrogel precursor solution is selected from one of a Polystyrene (PS)/toluene solution or a polyvinylidene fluoride (PVDF)/Dimethylformamide (DMF) solution. The concentration of the hydrogel precursor solution is 10 to 20wt%.
Further, the present invention provides a method for preparing a conductive inverse opal thin film for inducing neural cells, which may further have the following characteristics: wherein, in the second step, the stretching ratio is 3-6 times.
Further, the present invention provides a method for preparing a conductive inverse opal film for inducing nerve cells, which may further have the following characteristics: in the second step, the stretching angle is 15 degrees, 30 degrees, 45 degrees or 60 degrees which is included angle between the stretching angle and the linear direction of the single row of holes.
Further, the present invention provides a method for preparing a conductive inverse opal thin film for inducing neural cells, which may further have the following characteristics: wherein, in the second step, the condition of stretching the inverse opal substrate is heating in water bath at 70-80 ℃.
Further, the present invention provides a method for preparing a conductive inverse opal film for inducing nerve cells, which may further have the following characteristics: in the third step, the conductive hydrogel prepolymer solution comprises 3,4-ethylenedioxythiophene: polystyrene sulfonate (PEDOT: PSS) solution, polyacrylamide (PAAm) prepolymer solution and 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP); poly 3,4-ethylenedioxythiophene: the solid content of the polystyrene sulfonate solution was 1.2%; the polyacrylamide prepolymer solution comprises acrylamide (AAm), N-N ' methylene-bis-acrylamide (MBA) and deionized water, wherein the concentration of the acrylamide is 0.2 g/mL-1,N-N ' methylene-bis-acrylamide, and the mass fraction of the acrylamide relative to the mass fraction of the N-N ' methylene-bis-acrylamide is 3.45wt%; poly 3,4-ethylenedioxythiophene: the volume ratio of the polystyrene sulfonate solution to the polyacrylamide prepolymer solution is 3:2; the dosage of the 2-hydroxy-2-methyl-1-phenyl-1-acetone is 3,4-ethylenedioxythiophene: 1v/v% of the total amount of the polystyrene sulfonate solution and the polyacrylamide prepolymer solution.
The invention also protects the conductive inverse opal film with the anisotropic elliptical porous pattern prepared by the preparation method.
The conductive inverse opal film having the anisotropic elliptical porous pattern is used to induce nerve cells. In particular, the compound is used for inducing the directional growth of nerve cells and promoting the growth and differentiation of the nerve cells.
The invention has the beneficial effects that: the invention provides a conductive inverse opal film with an anisotropic elliptical porous pattern, a preparation method thereof and application of the conductive inverse opal film in inducing directional growth of nerve cells and promoting growth and differentiation of the nerve cells. The conductive inverse opal film has anisotropy, excellent conductivity and biocompatibility, and can well promote the proliferation and differentiation and the directional growth of nerve cells along different directions, thereby being beneficial to forming a neural network and promoting the repair and regeneration of nerve injury. The method has the following advantages:
1. the method is based on inverse opal substrate preparation, is simple and convenient, has lower cost, simple and easy operation, can be used for mass preparation, and has low technical requirement.
2. The invention designs a conductive inverse opal film with an anisotropic elliptical porous pattern, the surface appearance of which can be changed simply by changing the stretching angle, thereby promoting the directional growth of nerve cells. The filled conductive hydrogel endows the conductive hydrogel with excellent conductivity, thereby playing a positive role in the growth and differentiation of nerve cells.
3. The film prepared by the invention can induce nerve cells to be directionally arranged, so that a neural network is formed, the repair and regeneration of nerve injury are facilitated, and the film has a wide application prospect in biomedical application, particularly in tissue engineering.
Drawings
FIG. 1 is a flow chart of the preparation of an inverse opal film having an anisotropic elliptical porous pattern;
FIG. 2 is a schematic representation of cell culture on an inverse opal film with an anisotropic elliptical porous pattern before and after perfusion of a conductive hydrogel;
FIG. 3 is an immunofluorescence and Scanning Electron Microscope (SEM) image of PC12 cells after incubation on anisotropic inverse opal film after stretching at different angles; wherein, a-e are immunofluorescence pictures of PC12 cells cultured on a common glass slide and inverse opal films stretched at 0 degrees, 15 degrees, 30 degrees and 45 degrees respectively, and f-j are corresponding SEM images; all films were stretched 6 times with a 50 μm scale;
FIG. 4 is an immunofluorescence and SEM image of PC12 cells after culturing on an inverse opal film before and after perfusion of a conductive hydrogel; wherein, a-b are immunofluorescence pictures of PC12 cells cultured on a 30-degree stretched inverse opal film and a 30-degree stretched inverse opal film infused with a conductive hydrogel respectively, and c-d are corresponding SEM images; all films were stretched 6 times with a 50 μm scale.
Detailed Description
Preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings so that the advantages and features of the invention may be more readily understood by those skilled in the art. It should be understood that these examples are for illustrative purposes and are not intended to limit the scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention. The conditions not specified in the examples are generally those in routine experiments.
The present embodiment provides a conductive inverse opal thin film with anisotropic elliptical porous pattern for inducing nerve cells, the preparation process is shown in fig. 1, and the preparation method comprises the following steps:
step one, preparing an inverse opal substrate:
the cleaned slide glass was vertically inserted into a deposition flask containing an ethanol solution of 1.5wt% monodisperse silica nanoparticles. And (3) volatilizing the solvent in a thermostatic chamber with the temperature of 45 ℃ and the humidity of 50% to obtain the self-assembled silica nanoparticle colloidal crystal array template with ordered height.
And filling gaps of the colloidal nanoparticle colloidal crystal array template with 10wt% of PS/toluene solution, and volatilizing and curing the solvent at normal temperature to obtain a photonic crystal-hydrogel hybrid system. And corroding the silicon dioxide particles in the photonic crystal-hydrogel hybrid system by using 4wt% of hydrofluoric acid to obtain the PS inverse opal substrate.
In this embodiment, the hydrogel precursor solution may also be a PVDF/DMF solution.
Step two, preparing the inverse opal film with the anisotropic elliptical porous pattern:
and (3) slowly stretching the PS inverse opal substrate to 6 times of the original length in a 70 ℃ water bath by using a vernier caliper, wherein the stretching directions are respectively 0 degree, 15 degrees, 30 degrees and 45 degrees of included angles with the linear direction of the single-row holes, so that four kinds of inverse opal films with anisotropic elliptical porous patterns with different surface morphologies are obtained.
Step three, preparing the conductive inverse opal film with the anisotropic elliptical porous pattern:
preparing a PAAm pre-polymer solution, wherein the PAAm pre-polymer solution comprises AAm, MBA and deionized water, and the concentration of AAm is 0.2g mL -1 The mass fraction of MBA to AAm was 3.45wt%. And then mixing the PEDOT (PSS solution with the solid content of 1.2%) and the PAAm prepolymer solution according to the volume ratio of 3:2, and adding 1v/v% of HMPP to obtain the conductive hydrogel prepolymer solution.
And (3) pouring the conductive hydrogel precursor solution onto the inverse opal film with the anisotropic elliptical porous pattern, and irradiating the inverse opal film with ultraviolet light for 120 seconds for curing to obtain the conductive inverse opal film with the anisotropic elliptical porous pattern.
The conductive inverse opal film with the anisotropic elliptical porous pattern provided in this example was applied to induce nerve cells.
1. Inverse opal films with anisotropic elliptical porous patterns are used for nerve cell culture:
preparing materials: setting a control group and an experimental group: the control group is a glass slide, and the experimental groups are inverse opal films (inverse opal films obtained in the second step) with the stretching times of 6 times and the stretching angles of 0 degrees, 15 degrees, 30 degrees and 45 degrees respectively. Each group had 3 identical materials. The above materials were placed in 15 wells of a 24-well plate, irradiated with ultraviolet light, and sterilized overnight.
Cell inoculation: the cells used in the experiment were PC12 cells derived from murine adrenal pheochromocytoma, and the digested cells were seeded into 15 wells of a 24-well plate, the number of cells per well being 5X 10 4 Cells/ml were cultured in RPMI1640 medium containing 1% penicillin-streptomycin and 10% fetal bovine serum for 24h.
Cell induction culture: after 24h post-inoculation culture, the medium was changed to 1% horse serum, 1% penicillin-streptomycin and Nerve Growth Factor (NGF) (50. Mu.g mL) -1 ) Composed RPMI1640 culture medium, cultured for 7d.
And (3) immunofluorescence staining: the medium was discarded and the substrates were fixed with the cells using 4% paraformaldehyde for 30 minutes at room temperature. Then, the cell membrane was permeabilized and the protein was fixed using Phosphate Buffered Saline (PBS) and 0.1% Triton-X100-th solution at room temperature for 1 hour. Next, the fixed samples were stained with mouse anti- β III-tubulin primary antibody (1. Then, the cells were washed 3 times with PBS and a 0.1 triton-X100-equivalent solution, and counterstained with a mixture of a secondary antibody (1. Finally, DAKO fluorescent fixative was used to protect the sample from quenching prior to coverslipping. Confocal microscopy pictures were taken using a laser scanning microscope. As shown in fig. 3a-e, PC12 cells were aligned in a specific orientation on the stretched film compared to cells grown on a conventional glass slide showing disordered microfilaments (fig. 3 a). Specifically, when the film is stretched at 0 °, cells tend to grow in the direction of stretching (fig. 3 b), whereas extension of neurons has two growth directions on 15 °, 30 ° and 45 ° stretched films. Except for one along the direction of stretching, the other direction almost coincides with the direction of the ridges, thus forming an interdigitated neural network on the 15 °, 30 ° and 45 ° stretched films.
Scanning Electron Microscope (SEM) pre-processing before shooting: the substrate with the cells was first washed 3 times in PBS and fixed in a 2.5wt% glutaraldehyde solution overnight at 4 ℃. Then washed 3 times with deionized water to wash out residual glutaraldehyde solution. Next, they were dehydrated for 10 minutes by ethanol with concentration gradients of 30%, 50%, 70% and 90%, respectively. Finally, they were dehydrated 3 times with 100% alcohol before taking SEM images to ensure that the samples could be completely dehydrated. Images were taken using a scanning electron microscope. Similar to the immunofluorescence results, the SEM images (FIGS. 3 f-j) more clearly show the relationship between the surface topography of the stretched film and the direction of neuronal growth. These features indicate that stretched inverse opal films with anisotropic elliptical porous patterns can effectively induce PC12 cell growth in a specific direction.
2. Conductive inverse opal films with anisotropic elliptical porous patterns are used for neural cell culture:
preparing materials: setting a control group and an experimental group: the control group is a glass slide, and the experimental group is an inverse opal film (the inverse opal film obtained in the second step) and a conductive inverse opal film (the conductive inverse opal film obtained in the third step) with the stretching ratio of 6 times and the stretching angle of 30 degrees respectively. Each group had 3 identical materials. The above materials were placed in 9 wells of a 24-well plate, irradiated with uv light, and sterilized overnight.
Cell culture: the procedure was as above, and the results of observing the culture of PC12 cells on the inverse opal film before and after the perfusion of the conductive hydrogel are shown in FIG. 4. From the immunofluorescence images (fig. 4 a-b), it was shown that PC12 cells on the conductive hydrogel infused stretched inverse opal film showed better distribution and had longer neurites than the bare stretched inverse opal film, indicating tight junctions between cells and membrane. SEM images revealed the relationship between substrate type and PC12 cell growth (FIGS. 4 c-d). It can be seen that through the introduction of the conductive hydrogel, the vertical neural network is relatively more complete and has more sufficient interlocking on the composite film, which is beneficial to the connection and conduction of neural signals.
The above examples are only for illustrating the technical idea and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the content of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (8)
1. A method for preparing a conductive inverse opal film for inducing nerve cells, which is characterized by comprising the following steps:
the method comprises the following steps:
step one, preparing an inverse opal substrate: obtaining a highly ordered self-assembled nano-particle colloidal crystal array template by using a vertical deposition method, filling gaps of the nano-particle colloidal crystal array template with a hydrogel precursor solution, and removing the nano-particle colloidal crystal array template after solidification to obtain an inverse opal substrate;
step two, preparing the inverse opal film with the anisotropic elliptical porous pattern: stretching the obtained inverse opal substrate along different angles in the linear direction of the single-row holes of the inverse opal substrate to obtain an inverse opal film with anisotropic elliptical porous patterns; the stretching angle is 15 degrees, 30 degrees, 45 degrees or 60 degrees of an included angle in the linear direction of the single row of holes;
step three, preparing the conductive inverse opal film with the anisotropic elliptical porous pattern: preparing a conductive hydrogel precursor solution, filling the conductive hydrogel precursor solution into an inverse opal film with an anisotropic elliptical porous pattern, and curing by using ultraviolet light to obtain the conductive inverse opal film with the anisotropic elliptical porous pattern;
the conductive hydrogel prepolymer solution comprises 3,4-ethylenedioxythiophene: polystyrene sulfonate solution, polyacrylamide prepolymer solution and 2-hydroxy-2-methyl-1-phenyl-1-propanone;
poly 3,4-ethylenedioxythiophene: the solid content of the polystyrene sulfonate solution was 1.2%;
the polyacrylamide prepolymer solution comprises acrylamide, N-N' methylene-bis-acrylamide and deionized water, and the concentration of the acrylamide is 0.2g mL -1 The mass fraction of N-N' methylene bisacrylamide relative to acrylamide is 3.45wt%;
poly 3,4-ethylenedioxythiophene: the volume ratio of the polystyrene sulfonate solution to the polyacrylamide prepolymer solution is 3:2;
the dosage of the 2-hydroxy-2-methyl-1-phenyl-1-acetone is 3,4-ethylenedioxythiophene: 1v/v% of the total amount of polystyrene sulfonate solution and polyacrylamide prepolymer solution.
2. The method of preparing a conductive inverse opal film for inducing neural cells according to claim 1, wherein:
in the first step, the vertical deposition method comprises the following specific steps:
vertically inserting the cleaned glass slide into an ethanol solution of monodisperse nanoparticles;
and obtaining the self-assembled nano-particle colloidal crystal array template with ordered height after the solvent is volatilized in the environment of constant temperature and humidity.
3. The method of preparing a conductive inverse opal film for inducing neural cells according to claim 1, wherein:
in the first step, the nanoparticles in the nanoparticle colloidal crystal array template are silicon dioxide.
4. The method of preparing a conductive inverse opal film for inducing neural cells according to claim 1, wherein:
in the first step, the hydrogel precursor solution is selected from one of a polystyrene/toluene solution or a polyvinylidene fluoride/dimethylformamide solution.
5. The method of preparing a conductive inverse opal film for inducing neural cells according to claim 1, wherein:
wherein, in the second step, the stretching ratio is 3-6 times.
6. The method of preparing a conductive inverse opal film for inducing neural cells according to claim 1, wherein:
wherein, in the second step, the condition of stretching the inverse opal substrate is heating in water bath at 70-80 ℃.
7. The conductive inverse opal film having an anisotropic elliptical porous pattern manufactured by the manufacturing method of any one of claims 1 to 6.
8. Use of the conductive inverse opal film having an anisotropic elliptical porous pattern of claim 7 for inducing neural cells.
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